WO2006032102A1

WO2006032102A1 - Method of producing fragrance by inactivation or reduction of a functional protein with betaine aldehyde dehydrogenase (badh) activity

Info

Publication number: WO2006032102A1
Application number: PCT/AU2005/001458
Authority: WO
Inventors: Robert James Henry; Qinsheng Jin; Daniel Lex Ean Waters; Louis Michael Bradbury; Timothy Liam Fitzgerald
Original assignee: Grain Foods CRC Ltd
Current assignee: Grain Foods CRC Ltd
Priority date: 2004-09-22
Filing date: 2005-09-22
Publication date: 2006-03-30
Anticipated expiration: 2007-03-22
Also published as: CN101061219A

Abstract

The invention relates to methods of increasing production of fragrance by an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, comprising reducing or eliminating the activity of the functional protein in the organism. The invention also relates to methods of establishing whether an organism is capable of producing a fragrance, and to methods of producing organisms which produce fragrance.

Description

Method of producing fragrance by inactivation or reduction of a functional protein with Betaine Aldehyde Dehydrogenase (BADH) activity

Field of the Invention

5 The invention relates to methods for., increasing _.-fragrance production in an organism, methods for identifying organisms that are capable of producing fragrance, and to organisms that produce fragrance.

10 Background of the Invention

It is to be understood that a reference herein to a prior art publication does not constitute an admission that the publication forms a part of the common general knowledge 15 in the art in Australia, or any other country.

Fragrance is a desirable characteristic of many foods. For example, the fragrance associated with rice varieties such as Basmati 370, Khao Dawk Mali 105, Kyeema, Dumsorhk,

20 Dellmont, Amber and Goolarah rice varieties make these particular rice varieties very popular, and valuable, food resources. Fragrance has also been associated with varieties of other cereal crops such as oats, barley, wheat, as well as non-cereal plants such as the leaves of

25 pandan (Pandanus amaryllifolius) . However, many plants and other organisms do not produce fragrance, or produce very low amounts of fragrance. These non-fragrant organisms are often genetically very closely related to fragrant organisms, but lack the ability to produce

30 sufficient amounts of fragrance to be desirable products.

The number of organisms that produce fragrance is limited, and many organisms which do not .produce fragrance have other desirable qualities that make them attractive for the food industry. For example, organisms in addition to plants such as fungi, yeast and bacteria are used extensively in the food industry for the manufacture of cheese, bread, yoghurt, fermented beverages including beer and wine, and other food products. The ability to impart fragrance to these food products would be a desirable attribute of such organisms.

It would therefore be desirable to be able to impart on non-fragrant organisms the ability to produce fragrance and/or to be able to identify organisms that are capable of producing fragrance. However, the biological mechanisms that leads to the production of fragrance is not known. Although fragrance has been associated with the production of volatile chemicals such as 2-acetyl-l- pyrroline, the biological processes' leading to the production and breakdown of these volatile chemicals is not known.

Further, the molecular genetics of fragrance production has not been characterised in any organisms, and prior to the present invention no genes associated with the trait have yet been identified.

Given the limited knowledge of the biochemistry and genetics associated with production of fragrance, it has not previously been possible to manipulate non-fragrant organisms to produce fragrance, or to effectively identify those organisms that produce fragrance, or are at least capable of producing fragrance. Accordingly,, it would be desirable to provide improved • methods for imparting on organisms the ability to produce fragrance, and methods for detection of organisms that are capable of producing fragrance.

Summary of the Invention

In a first aspect, the invention provides a method of increasing production of fragrance by an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, the method comprising reducing or eliminating the activity of the functional protein in the organism.

In a second aspect, the invention provides a method of ^• producing an organism which produces a fragrance, the method comprising the steps of:

(a) providing an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1;

(b) reducing or eliminating the activity of the functional protein in the organism.

In one embodiment, the activity of the functional protein is reduced or eliminated by inhibiting the activity of the functional protein.

In another embodiment, the activity of the functional protein is reduced or eliminated by reducing or eliminating the ability of the organism to express the functional protein. In one embodiment, the functional protein comprises the amino acid sequence EG(C or G)RLG(S or P)V(V or I)S.

In another embodiment, the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P. Typically, the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC, wherein X may be any amino acid and n is an integer from 25 to 30 (ie. 25, 26, 27, 28, 29 or 30) .

The functional protein may comprise the amino acid sequences (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC and EG(C or G)RLG(S or P)V(V or I)S, wherein n is an integer from 25 to 30 (ie. 25, 26, 27, 28, 29 or 30).

In various embodiments, the functional protein has an amino acid sequence that is:

(a) at least 35% identical to the amino acid sequence of SEQ ID NO: 1;

(b) at least 37% identical to the amino acid sequence of SEQ ID NO: 1; (c) at least ,38% identical to the amino acid sequence of

SEQ ID NO: 1; (d) at least 39% identical to the amino acid sequence of

SEQ ID NO: 1;

(e^*) at least 40% identical to the amino acid sequence of SEQ ID NO: 1;

(f) at least 41% identical to the amino acid sequence of SEQ ID NO: 1; (g) at least 44% identical to the amino acid sequence of^'

SEQ ID NO: 1; (h) at least 45% identical to the amino acid sequence of

SEQ ID NO: 1; (i) at least 50% identical to the amino acid sequence of

SEQ ID NO: 1; (j) at least 60% identical to the amino acid sequence of

SEQ ID NO: 1; (k) at least 70% identical to the amino acid sequence of SEQ ID NO: 1;

(1) at least 75% identical to the amino acid sequence of

SEQ ID NO: 1; (m) at least 80% identical to the amino acid sequence of

SEQ ID NO: 1; (n) at least 85% identical to the amino acid sequence of

SEQ ID NO: 1; (o) at least 87% identical to the amino acid sequence of

SEQ ID NO: 1;

(p) at least 90% identical to the amino acid sequence of SEQ ID NO: 1;

(q) at least 95% identical to the amino acid sequence of

SEQ ID NO: 1/ or (r) at least 99% identical to the amino .acid sequence of

SEQ ID NO: 1.

The functional protein may be encoded by any gene that . encodes for a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1. ^' • ^■

In various embodiments, the coding sequence of the gene encoding the functional protein comprises a nucleotide sequence that is: (a) at least 65% identical (for example, at least 70%, 75%, 80%, -85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 30; (b) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 4;

(c) _ at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least

95% identical, to the nucleotide sequence of SEQ ID NO: 6;

(d) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 8;

(e) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 10;

(f) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 12; (g) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 14; (h) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 16; (i) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 18; (j) at least 65% identical (for example, at least 70%, 75%,. 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 20;

(k) at least 65% identical (for example, at least 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 22; (1) at least 65% identical^' (for example, at least 70%,

75%, 80%,.85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 24.

In various embodiments the gene encoding the functional protein comprises a nucleotide sequence that is at least: (a) 65% identical to SEQ ID NO: 2

(b) 70% identical to. SEQ ID NO: 2

(c) 75% identical to SEQ ID NO: 2

(d) 80% identical to SEQ ID NO: 2;

(e) 85% identical to SEQ ID NO: 2; (f) ^"90% identical to SEQ ID NO: 2;

(g) 95% identical to SEQ ID NO: 2; or (h) 99% identical to SEQ ID NO: 2.

The organism may be any organism capable of producing a fragrance when activity of the functional protein is reduced or eliminated. For example, the organism may be selected from the group consisting of plants, algae, fungi, yeast and bacteria. In one embodiment, the organism is a plant. The plant may¬ be any plant capable of producing a fragrance when the activity of the functional protein is reduced or eliminated. The plant may be monocotyledonous or dicotyledonous. The plant may be a cereal crop plant. Examples of suitable cereal crop plants include rice, oats, barley, sorghum, maize, wheat, rye, amaranth, rape and spelt. The plant may be a legume. Examples of legumes include alfafa, beans, broom, carob, clover, cowpea, lupine, mung bean, mimosa, peas, peanuts, soybeans, tamarind and vetch. The plant may be an oilseed producing plant. Examples of oilseed producing plants include rape, c^'anola, hemp, linseed, sunflower, safflower and cotton.

In another embodiment, the organism is a fungus. The fungus may be any fungus capable of producing fragrance when the activity of the functional protein is reduced or eliminated. Examples of suitable fungi include

Aspergillus sp. , Penicillium sp. and Roquefort sp,

In another embodiment, the organism is. a yeast. The yeast- may be any yeast capable of producing fragrance when the activity of the functional protein is reduced or eliminated. Examples of suitable yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe and Yarrawia lipolytics.

In yet another embodiment, the organism is a bacteria. The bacteria may be any bacteria capable of producing fragrance when the activity of the functional protein is reduced or eliminated. Examples of suitable bacteria include Staphylococcus xylosus, Escherichia coli, Bacillus subtilus, Bacillus cereus, Lactococcus delbrueckii, Lactococcus lactis, Lactobacillus casei, Lactobacillus delbrueckii and Leuconostoc.

The activity of the functional protein may be reduced or eliminated using any methods known in the art for reducing or eliminating the activity of a protein in an organism. The activity of the functional protein may be^'redu'ced or eliminated by inhibiting the activity of the functional protein, or by reducing or eliminating the ability of the organism to express the functional protein.

The activity of the functional protein may be inhibited by introducing into the cells of the organism an inhibitor of the functional protein. The inhibitor may be, for example, a protein inhibitor which inhibits, degrades or cleaves the functional protein, or the inhibitor may be a chemical inhibitor of the functional protein such as pyridoxal 5' -phosphate.

The ability of the organism to express the functional protein may be reduced or eliminated using any methods which result in a reduction or elimination of expression of the functional protein sufficient to result in an increase in the production of fragrance by the organism.

In one embodiment, the ability of the organism to express the functional protein is reduced or eliminated by introducing into the cells of the organism a nucleic acid molecule that is capable of reducing or eliminating expression of the functional protein. The nucleic acid molecule is typically complementary to at least a portion of the gene that encodes the functional protein. The nucleic acid molecule may be ssDNA, ssRNA, dsDNA, dsRNA, or a ribozyme. The molecule may be an anti-sense molecule, a co-suppressor molecule (positive sense suppression) , or any other molecule that is capable of reducing or eliminating expression of -the functional protein. The anti-sense molecule may be, for example, an anti-sense RNA, anti-sense DNA, interference RNA (dsRNA, iRNA, siRNA, hpRNA or ihpRNA) or a ribozyme.

In yet another embodiment, the ability of the organism to express the functional protein is reduced or eliminated by ^• introducing into one or more genes encoding the functional protein a mutation which reduces or eliminates expression of the functional protein. The mutation may be any mutation which reduces or eliminates the ability of the organism to express the functional protein. The mutation may be a deletion, an insertion, or- a substitution of one or more base pairs in the gene encoding the functional protein.

The mutation may be in any portion of the gene encoding the functional protein which results in a reduction or elimination of expression of the functional protein.

In one embodiment, the mutation is in the coding sequence of the gene.

In another embodiment, the mutation is in the non-coding sequence of the gene. The non-coding sequence of the gene may be 5' non-coding sequence, or 3' non-coding sequence. Suitably, the 5^"' non-coding sequence is promoter sequence. The non-coding sequence may be in an intron- of the gene, or at the boundary of an intron or an exon.

The mutation may be introduced by any methods known in the art for introducing a mutation into a gene, including site-specific recombination, homologous recombination, transposon or retrotransposon mutagenesis, chemical mutagenesis, mutagenesis by radiation, etc.

Organisms in which the gene encoding the functional protein has been mutated may be identified using any methods known in the art. For example, methods such as -PCR, RT-PCR, TILLING, southern or northern hybridisation, etc. may be employed to identify mutants.

In one embodiment, reducing or eliminating the ability of the organism to express the functional protein results in ^• increased production by the organism of one or more compounds selected from the group consisting of 2-acetyl- 1-pyrroline, 2-^"(l-ethoxyethenyl) -1-pyrroline, 2-acetyl-

1, 4, 5, 6-tetrahydropyridine, and those fragrant compounds . described in Widjaja et al. (1996) Comparative Studies on Volatile Components, J. Sci. Food Agric. 70: 151-161. Typically, reducing or eliminating the ability of the organism to express the functional protein results in increased production by the organism of 2-acetyl-l- pyrroline.

In a third aspect, the invention provides a method of establishing whether an organism is capable of producing fragrance, comprising determining whether an organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

The organism may be identified as capable of producing fragrance if the organism is not capable of expressing a functional protein that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, or if the organism expresses levels of a functional protein that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 that are not sufficient to reduce or eliminate the production of fragrance. ^"The organism may be identified as not capable of producing fragrance if the organism is capable of expressing a functional protein that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 to a level that is sufficient to reduce or eliminate the production of fragrance.

The capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined using any methods known in the art for determining the capability of an organism to express a protein.

In one embodiment, the capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by detecting the functional protein. Methods for detecting the functional protein may include immunological methods such as ELISA and immunoblotting, protein analysis methods and proteomics such as SDS-PAGE electrophoresis, 2D gel electrophoresis, mass spectrometry methods such as MALDI- TOF or SELDI-TOF and enzyme assay for the functional protein.

In another embodiment, the capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by methods for detecting mRNA transcripts of the gene encoding the functional protein. Examples of suitable methods for detecting RNA transcripts include northern blot analysis, dot blot analysis, RT-PCR and micro-array analysis.

In yet another embodiment, the capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by methods which detect mutations in the gene encoding the functional protein that result in production of a protein that does not reduce or eliminate the ability of the organism to produce fragrance (a "non-functional protein") or no expression of a functional protein. For example, the ability of the organism to produce a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by identifying whether the gene encoding the functional protein in other individuals of the same species contains mutations such as single nucleotide polymorphisms (SNP' s), nucleotide insertions or deletions, or simple repeat sequences (SSR' s) or microsatellites, which result in expression of a non-functional protein, or no expression of a protein from the gene. Examples of methods that are suitable for detecting polymorphisms in the gene include PCR, RT-PCR, sequencing, restriction length polymorphism (RFLP) , microarray analysis, TILLING, temperature gradient gel electrophoresis and HPLC.

Typically, determining whether the organism is. capable of expressing a functional protein comprises determining^' whether the organism is homozygous for a mutant fgr gene.

In a fourth aspect, the invention provides a method of' producing an organism which produces fragrance, the method comprising the steps of:

(a) identifying one or more parent organisms which comprise at least one mutant fgr gene;

(b) culturing at least two of the one or more parent organisms under conditions which permit mating of the organisms to produce progeny;

(c) selecting one or more progeny that are homozygous for the mutant fgr gene to thereby provide an organism which produces fragrance.

Typically, the organism is a plant. In one embodiment, the method comprises: (a) identifying one or more parent plants comprising at least one mutant fgr gene; (b) crossing two of the one or more parent plants to produce progeny plants; (c) selecting one or more progeny plants that are homozygous for the mutant fgr gene to thereby provide a plant which produces fragrance.

The plant may be any plant which is capable of producing a fragrance when expression of the functional. protein is reduced or eliminated. The plant may be monocotyledonous or dicotyledonous. The plant may be a cereal crop plant. Examples of suitable cereal crop plants include rice, oats, barley, sorghum/ maize, wheat, rye, amaranth, rape, and spelt. The plant may be a legume. Examples of legumes include alfafa, beans, broom, carob, clover, cowpea, lupine, mun_.g bean, mimosa, peas, peanuts, soybeans, tamarind and vetch. The plant may be oilseed. Examples of oilseeds include rape, canola, hemp, linseed, sunflower, and safflower. The plant may be^' bamboo.

The one or more parent organisms may be heterozygous for the mutant fgr gene.

In a fifth aspect, the invention provides an organism produced by the method of the first, second or fourth aspect.

In a sixth aspect, the invention^' provides a method for producing fragrance comprising incubating an organism of ^• the fifth aspect under conditions which permit production of fragrance.

In a seventh aspect, the invention provides the use of a gene that encodes a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, for producing an organism that produces fragrance.

In an eighth aspect, the invention provides the use of a mutant fgr gene, for producing an organism that is capable of producing fragrance. ■ In a ninth aspect, the invention provides a nucleic acid molecule capable of reducing or- eliminating expression of a functional protein in an organism, the functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the ninth aspect, the nucleic acid molecule comprises a nucleic acid molecule which is complementary to at least a portion of the gene in the organism encoding the functional protein. Suitably, the nucleic acid molecule is an anti-sense molecule, an anti- sense vector encoding an anti-sense molecule, a co- suppressor molecule, or a co-suppressor vector encoding a co-suppressor molecule. Suitably, the anti-sense molecule is an oligonucleotide.

In a tenth aspect, the invention provides a fragrant molecule produced by the method of the sixth aspect.

In an eleventh aspect, the invention provides 2-acetyl-l- pyrroline produced by reducing or eliminating in an organism the activity of a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO:1. _ .

In a twelfth aspect, the invention provides the use of a nucleic acid molecule capable of hybridising to an fgr gene or a mutant fgr gene of an organism for determining whether the organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1. In a thirteenth aspect, the invention provides a nucleic acid molecule capable of hybridising to an fgr gene or a mutant fgr gene of an organism when used for determining whether the organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

In a fourteenth aspect, the invention provides a nucleic acid molecule capable of distinguishing between an fgr gene and a mutant fgr gene of an organism.

It will be appreciated by persons skilled in the art that the nucleic acid molecule may distinguish an fgr gene from^' a mutant fgr gene by: (a) hybridising to a mutant fgr gene but not an fgr gene;

(b) hybridising to an fgr gene but not a mutant fgr gene;

(c) hybridising to an fgr gene to form a hybrid with a melting temperature higher than that of a hybrid

'^' formed with a mutant fgr gene; or

(d) hybridising to a mutant fgr gene to form a hybrid with a melting temperature higher than that of a hybrid formed with an fgr gene.

In one embodiment of the twelfth to fourteenth aspect, the nucleic acid molecule is capable of hybridising to the fgr gene but not to a mutant fgr gene. An example of a nucleic acid molecule that is capable of hybridising to the fgr gene of rice but not a mutant fgr gene of rice is a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 27. In another embodiment of the twelfth to fourteenth aspect, the nucleic acid molecule is capable of hybridising to a mutant fgr gene but not the fgr gene of an organism. An example of a nucleic acid molecule that is capable of hybridising to a mutant fgr gene of rice but not the fgr gene of rice is a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 26.

It will be appreciated by persons skilled in the art that the nucleic acid molecule capable of distinguishing between an fgr gene and a mutant fgr gene of an organism may be a probe or a primer. The probe or primer may be any length provided they are capable of distinguishing between a mutant fgr gene and an fgr gene of an organism. Suitable probes or primers may be readily determined using methods known in the art and the sequences described herein.

BRIEF DESCRIPTION OP THE DRAWINGS

Figure 1 illustrates the position of the rice fgr gene on that portion of chromosome 8 of the rice genome bounded by markers SSRJ22 and SSRJ07.

Figure 2 illustrates the position of BAC clones on chromosome 8 of the rice genome in the region of fgr.

Figure 3 illustrates a map of the location of genes on the rice BAC clone AP004463.

Figure 4 illustrates an alignment of nucleotide sequence of part of the rice fgr gene from non-fragrant rice phenotypes with the mutant fgr gene from fragrant rice. AP004463 is Nipponbare (non-fragrant) sequence, R07 and F07 are sequences from Kyeema (fragrant) .

Figure 5 illustrates the rice fgr gene (SEQ ID NO: 2) of Nipponbare, showing the nucleotide sequence of the coding (in bold) and non-coding (in plain text) portion of the fgr gene. The start codon is underlined. Exons (15 in total) are in bold.

Figure 6 is (A) the amino acid sequence of the functional protein (SEQ ID NO: 1) encoded by the fgr gene on chromosome 8 of the rice variety Nipponbare; (B) the amino acid sequence of a protein encoded by a mutant fgr gene of the rice variety Kyeema, and (C) the amino acid sequence of the protein encoded by the betaine aldehyde dehydrogenase gene (BAD) on chromosome 4 of the rice variety Nipponbare.

Figure 7 illustrates an alignment using Clustal W of the nucleotide sequence of cDNA from the fgr gene from a non- fragrant rice variety (rice_fgr_BAD2) (top) with that of cDNA from a mutant fgr gene from a fragrant rice variety (Rice_truncated_BAD2) .

Figure 8 illustrates (A) the amino acid sequence (SEQ ID NO: 1) of a functional protein from the non-fragrant rice variety Nipponbare and (B) nucleotide sequence (SEQ ID NO: 30) encoding the functional protein.

Figure 9 illustrates (A) the amino acid sequence (SEQ ID NO: 3) of a functional protein from non-fragrant wheat and (B) nucleotide sequence (SEQ ID NO: 4) encoding the functional protein. Figure 10 illustrates (A) the amino acid sequence (SEQ ID NO: 5) of a functional protein from non-fragrant barley and (B) nucleotide sequence (SEQ ID NO: 6) encoding the functional protein.

Figure 11 illustrates (A) the amino acid sequence (SEQ ID NO: 7) of a functional protein from non-fragrant sorghum and (B) nucleotide sequence (SEQ ID NO: 8) encoding the functional protein.

Figure 12 illustrates (A) the amino acid sequence (SEQ ID NO: 9) of a functional protein from non-fragrant Zea mays and (B) nucleotide sequence (SEQ ID NO: 10) encoding the functional protein.

Figure 13 illustrates (A) the amino acid sequence (SEQ ID NO: 11) of a functional protein from non-fragrant Z. tenuifolia and (B) nucleotide sequence (SEQ ID NO: 12) encoding the functional protein.

Figure 14 illustrates (A) the amino acid sequence (SEQ ID NO: 13) of a functional protein from non-fragrant Scizosaccharomyces pombe and (B) nucleotide sequence (SEQ ID NO: 14) encoding the functional protein.

Figure 15 illustrates (A) the amino acid sequence (SEQ ID NO: 15) of a functional protein from non-fragrant Saccharomyces cerevisiae and (B) nucleotide sequence (SEQ ID NO: 16) encoding the functional protein.

Figure 16 illustrates (A) the amino acid sequence (SEQ ID NO: 17) of a functional protein from non-fragrant Yarrowia lipolytica and (B) nucleotide sequence (SEQ ID NO: 18) encoding the functional protein.

Figure 17 illustrates (A) the amino acid sequence (SEQ ID NO: 19) of a functional protein from non-fragrant Staphylococcus xylosus and (B) nucleotide sequence (SEQ ID NO: 20) encoding the functional protein.

Figure 18 illustrates (A) the amino acid sequence (SEQ ID NO: 21) of a functional protein from non-fragrant

Bacillus subtilus and (B) nucleotide sequence (SEQ ID NO: 22) encoding the functional protein.

Figure 19 illustrates (A) the amino acid sequence (SEQ ID NO: 23) of a functional protein from non-fragrant E. coli and (B) nucleotide sequence (SEQ ID NO: 24) encoding the functional protein.

Figure 20 illustrates (A) an alignment and (B) a score table indicating the percent identity between the amino acid sequence -of a functional protein from wheat, barley, rice and Z. tenuifolia. Both (A) and (B) were generated using Clustal W with default parameters.

. Figure 21 illustrates an alignment, using Clustal W, of the amino acid sequence of a functional protein (BAD2) from wheat, barley, rice, Z. tenuifolia, sorghum and Staphylococcus xylosus, and the betaine aldehyde dehydrogenase protein from rice. ^■

Figure 22 illustrates an alignment, using Clustal W, of the coding region of the gene encoding a functional protein from wheat,, barley, rice, Z. tenui_.folia, sorghum, S. xylosus, and that of BADl from rice.

Figure 23 illustrates the percent identity calculated using Clustal W (with default parameters) between functional proteins from the organisms listed. The percentage identity between each pair of organisms is listed under Score.

Figure 24 illustrates the primer positions and PCR fragments that are generated using an embodiment of the method of the invention to detect whether a plant is capable of producing fragrance.'

Figure 25 illustrates the result of gel electrophoresis of PCR products amplified using primers SEQ ID NO: 25, 26, 27 and 28 to amplify fgr sequence from a non-fragrant rice variety (Nipponbare) (lane 2) , a fragrant rice variety (Kyeema) (lane 3) , a heterozygous rice variety (Kyeema/Gulfmont) (lane 4), a negative control (water) (lane 5), and Roche DNA ladder XIV (lOObp) standard markers (lanes 1 and 6) .

Figure 26 illustrates the results of gel electrophoresis of PCR product from 96 individuals from an unselected F2 rice population segregating for fragrance and amplified using primers SEQ ID Nos: 25, 26, 27 and 28. Molecular markers (Roche DNA ladder XIV (lOObp) ) are marked S. The band of approximately 580 bp corresponds to the positive control amplified by both external primers (ESP and EAP) . The 355 bp band corresponds to a PCR product amplified from the non-fragrant allele by the internal non-fragrant sense primer (INSP) and the external antisense primer (EAP) . The 257 bp band corresponds to a PCR product amplified from the fragrant allele by the internal fragrant antisense primer (IFAP) and the external sense primer (ESP) .

Figure 27 illustrates the nucleotide sequence (SEQ ID NO: 29) of the BAD gene from Saccharomyces cerevisiae.'

Figure 28 illustrates an alignment, using ClustalW, of the region of the nucleotide sequence of BADl and BAD2 of wheat used for design of BAD2 RNAi. The region illustrated has 76.8% identity between BADl and BAD2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the production and identification of organisms that are capable of producing fragrance.

As used herein, the term "fragrance" refers to the aroma and flavour resulting from the production of one or more of the fragrant molecules that are produced by fragrant varieties of rice such as Basmati 370 or Kyeema, but which are not produced or produced in non-detectable amounts to the human senses, by non-fragrant varieties of rice such as Nipponbare. It is well known that this same aroma and flavour is produced by other fragrant organisms, and is associated with foods such as popcorn, corn tortillas, baguettes, ham, cheese, mung bean, green tea, wine and other fragrant rice varieties.

The fragrance may result from the production of one or more fragrant molecules such as, for example, 2-acetyl-l- pyrroline, 2- (1-ethoxyethenyl) -1-pyrroline or 2-acetyl- 1, 4,5, ^β-tetrahydropyridine or those fragrant molecules described in Widjaja et al. (1996), J. Sci. Food Agric. 70:151-161. Typically, the fragrance results from the production of 2-acetyl-l-pyrroline. It will be appreciated by persons skilled in the art that an organism capable of producing fragrance will have the biological requirements to produce fragrance, even if the organism does not produce fragrance under all conditions, or in all parts of the organism. For example, while an organism may have the genes necessary for producing fragrance, and is therefore capable of producing fragrance, it may produce fragrance only under certain conditions, or in particular parts of the organism. The conditions may be conditions such as temperature, growth medium and light, ectopic application of chemicals etc., and can readily be determined by persons skilled in the art. In multicellular organisms such as plants, fragrance may be produced in one or more parts of the plant such as, for example, seeds, leaves, pollen, flowers, roots, stems or fruit, or throughout the entire organism.

It will be appreciated by those skilled in the art that aroma may result from secretion or release of fragrant molecules from cells of the organism. It will also be appreciated by those skilled in the art that flavour may result from intracellular accumulation of fragrant molecules, as well as secretion or release of fragrant molecules.

In one aspect, the invention provides a method of increasing production of fragrance by an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, the method comprising reducing or eliminating the activity of the functional protein in the organism.

It will be understood by those skilled in the art that an ^' increase in production of fragrance by an organism is an ^• increase relative to the fragrance produced by the organism in which the activity of the functional protein has not been reduced or eliminated. As used herein, a reference to the production of fragrance by an organism refers to at least some cells of the organism secreting, releasing and/or accumulating a sufficient amount of fragrant molecules to be detectable to senses such as smell and/or taste. It will be appreciated by persons skilled in the art that an increase in production of fragrance by an organism may be due to any one or more of the following: (a) increased synthesis of the fragrant molecules; (b) increased accumulation of the fragrant molecules;

(c) increased release of the fragrant molecules from the cells of the¹ organism. ^■

As used herein, a "functional protein" is a protein which when expressed in an organism, reduces or eliminates the ability of that organism to produce fragrance. The inventors have found that reducing the activity of a functional protein having amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 in an organism increases the production of fragrance by the organism. The inventors have found that mutations in the gene which encodes a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 reduce or eliminate expression of the functional protein, and therefore reduce or eliminate the activity of the functional protein, and that such mutations result in a fragrant phenotype.

It will be understood by those skilled in the art that "% identical" refers to the percent of identical amino acids that align in an alignment of at least two amino acid sequences, or the percent of identical nucleotides that align in an alignment of at least two nucleotide sequences. The alignment may, for example, be performed, and the percent identity determined, using any of the following: (a) BLAST program using default parameters (Word size 3, Blosum 62 matrix, Gap costs: Existence II Extension 1) ;

(b) ALIGN program in the GCG software package (Devereux et al (1984) Nuclei Acid Research, 12: 387) using default parameters;

(c) GAP alignment program in the GCG software package

(Devereux et al .(1984) Nuclei Acid Research, 12: 387) using a gap weight of 5.0 and a length weight of 0.3;

(d) CLUSTALW using default parameters (the default parameters listed by the European Bioinformatics

Institute at http://www.ebi.ac.uk/clustalW/ are DNA Gap Open Penalty = 15.0, DNA Gap Extension Penalty = 6.66, DNA Matrix: Identity, Protein Gap Open Penalty = 10.0, Protein Gap Extension Penalty = 0.2, Protein Matrix = Gonnet, Protein/DNA END Gap = -1, Protein/DNA GAPDIST = 4) .

Typically, the percent identity is determined using

CLUSTALW using the default parameters. As used herein, the expression "fgr gene" refers to a gene from any organism which encodes a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1. It will be appreciated by those skilled in the art that the fgr gene may be known by different names in different organisms, such as, for example, BAD2, BADH2, BADH15, BADHl, BAD, BADl, BBD, BBDl, BBD2, Betaine aldehyde dehydrogenase gene, and that these genes are included within the scope of the expression "fgr gene". Accordingly, the BAD2 gene, the BADH2 gene, the BADH15 gene, the BADHl gene, the BAD gene, the BADl gene, the BBD gene, the BBDl gene, and the BBD2 gene are examples of an "fgr gene". As used herein, a "mutant fgr gene" is a mutant allele of an fgr gene that does not encode a functional protein.

The nucleotide sequence of the fgr gene of rice variety Nipponbare is provided in SEQ ID NO: 2. It will be- appreciated by persons skilled in the art that the fgr gene sequence may vary between rice varieties due to the degeneracy of the genetic code. Accordingly, it is envisaged that the fgr gene of a rice variety may be at least 65% identical, (for example, at least 70%, 75%, 80%, 85% or 90% identical) typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the nucleotide sequence of the coding region of the fgr gene of:

(a) a variety of rice is at least 65% identical (for ^' example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 30;

(b) a variety of wheat is at least 65% identical (for example, 70%, 75%-, 8.0%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 4;

(c) a variety of barley is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 6;

(d) a variety of sorghum is at least 65% identical (for example, 70%, 75%, ^'80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 8; (e) a variety of Zea mays is at least 65% identical

(for example, 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 10;

(f) a variety of Z. tenuifolia is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 12;

(g) a strain of Schizosaccharomyces pombe is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 14; (h) a strain of Saccharomyces cerevisiae is at least 65% identical, (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 16;

(i) a strain of Yarrowia lipolytica is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 18; (j) a strain of Staphylococcus xylosus is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 20; (k) a strain of Bacillus subtilus is at least.65% identical (for example, 70%, 75%, 80%, 85% or 90% identical) , typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 22;

(1) a strain of E. coli is at least 65% identical (for example, 70%, 75%, 80%, 85% or 90% identical), typically at least 95% identical, to the nucleotide sequence of SEQ ID NO: 24.

The inventors have found that organisms which express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 are either unable to produce fragrance, or produce a low amount of fragrance. As used herein, the term ^λλnon-fragrant" refers to an organism that is not capable of producing detectable fragrance, or produces low amounts of fragrance. As described herein, fragrant rice varieties were found to carry mutations in the fgr gene which eliminated the ability of the fragrant rice to express the functional protein from the fgr gene. Non- fragrant rice were found to have at least one fgr gene that was not mutated and therefore were capable of expressing the functional protein.. Without wishing to be bound by theory, the inventors believe that the functional protein is involved in the metabolism of 2-acetyl-l- pyrroline, and consequently reduces or prevents accumulation of 2-acetyl-l-pyrroline by the organism. The inventors have further found that proteins having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 are widespread throughout many different organisms. As described herein, the inventors have identified proteins having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 in various plants, including wheat, barley, rice, lawn grass, Sorghum and Zea mays, yeast including Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytics, and bacteria including Staphylococcus xylosus, Escherichia coli and Bacillus subtilis. Accordingly, the inventors anticipate that fragrance production may be increased in these, and many other different organisms, by reducing or eliminating the activity of the protein in those organisms.

A protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 from many organisms may be readily identified using standard methods known in the art. For example, proteins with amino acid sequences having at least 30% identity with SEQ ID NO: 1 may be identified by comparison of SEQ ID NO: 1 with sequence databases for organisms such as plants, fungi, yeast and bacteria. Examples of such databases include the nucleotide and protein databases at National Centre for Biotechnological Information (NCBI), Genbank, European Molecular Biology Laboratory (EMBL) , DNA Data Bank of Japan (DDBJ) , The Institute for Genomic Research (TIGR), Plant Genome Database (PlantGDB) , etc. The comparisons are typically conducted using computer- based sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool) (see, for example, Altschul et al. (1996) Methods in Enzymology 266: 260)., the GCG Package (Devereux et al. (1984) Nucleic Acids Research, 12: 387), FASTA (Altshul et al. (1990) J. MoI. Biol. 215: 403), ClUSTALW (Thompson et al (1994) Nucleic Acids Research 22(22) : 4673-4680) . An alignment of the amino acid sequence of functional protein ^• from non-fragrant rice, wheat, barley, Z.tenuifolia, sorghum, Staphylococcus xylosus and Schizosaccharomyces pombe is shown in Figure 21 by way of example to illustrate the diverse species of organisms which express a protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ- I-D NO. 1. It will therefore be appreciated that fragrance production may be increased in any of those organisms which carry one or more genes encoding a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1.

The inventors have further found that the region corresponding to position 158 to position 480 of SEQ ID NO: 1 is more highly conserved in the functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 expressed by diverse organisms. Accordingly, in one embodiment, the functional protein comprises an amino acid sequence that is at least 45% identical to the amino acid sequence from position 158 to position 480 of SEQ ID NO: 1.

The inventors have also identified the following conserved amino acid sequences among proteins having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 from different organisms: ( a) EG ( C or G) RLG ( S or P) V (V or I ) S ;

(b) (V or I or L) (S or T or A)LELGGK(S_. or N)P, typically (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC, wherein X may be any amino acid and n is an integer from 25 to 30.

Accordingly, in one embodiment, the functional protein comprises the amino acid sequence EG(C or G)RLG(S or P)V(V or I)S. In another embodiment, the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P. In a further embodiment, the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC, wherein X may be any amino acid and n is an integer from 25 to 30. In yet another embodiment, the functional protein comprises the amino acid sequences (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC and EG(C or G)RLG(S or P)V(V or I)S, wherein X is any amino acid and n is an integer from 25 to 30.

The organism may be any organism capable of expression of a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 and which would be capable of producing fragrance if expression of the functional protein were reduced or eliminated. It will be appreciated by persons skilled in the art that an organism capable of expression of a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO. 1 will have the biological requirements to produce the functional protein, even if the organism does not produce the functional protein in all conditions or all parts of the organism.

In a preferred embodiment, the invention provides a method of increasing production of fragrance in: (a) a plant which is capable of expressing a functional protein having an amino acid sequence that is at least 60% identical (for example, at^' least 70%, 75%, 80%, 85%, 87% or 90% identical) to the amino acid sequence of SEQ ID NO: 1; ■

(b) a fungi which is capable of expressing a functional protein having an amino acid sequence that is at least 38% identical (for example, at least 39% identical) to the amino acid sequence of SEQ ID NO: 1;

(c) a yeast which is capable of expressing a functional protein having an amino acid sequence that is at least 38% identical (for example, at least 40% or 41% identical) to the amino acid sequence of SEQ ID NO: 1; or

(d) a bacteria which is capable of expressing a functional protein having an amino acid sequence that is at least 35% identical (for example, at least 37%, 44% or 45% identical) to the amino acid sequence of SEQ ID NO: 1; the method comprising reducing or eliminating the activity of the functional protein of the plant, fungi, yeast or bacteria.

Suitably, when the organism is:

^■(1) a plant, the functional protein has an amino acid sequence that is at least 75% identical to SEQ ID NO:

1;

(2) a yeast, the functional protein has an amino acid sequence that is at least 38% identical to SEQ ID NO: 1; or (3) a bacterium, the functional protein has an amino acid sequence that is at least 37% identical to SEQ ID NO: 1.

5 In one embodiment, the organism is a plant. Suitable plants include monocotyledonous or dicotyledonous plants. Examples of monocotyledonous plants include asparagus, bamboo, corn, barley, wheat, rice, sorghum, onion, pearl millet, rye, oats, rape, Zoysia tenuifolia (lawn grass),

^•10 Musa acuminata, Pandan. Examples of- dicotyledonous plants include tomato, beans, ^'soybeans, peppers, lettuce, .peas, alfalfa, cabbage, broccoli, cauliflower, brussel sprouts, raddish, carrot, beets, eggplant, spinach, cucumber, squash, sunflowers. In a preferred embodiment, the plants

15 are selected from the group consisting of wheat, rice, barley, oats, maize, sorghum and Zoysia tenuifolia. In .various embodiments:

(1) when the plant is non-fragrant rice, the functional protein has an amino acid sequence that is at least

20 75% identical (for example, at least 80%, 85%, 90% or 95% identical), typically at least 99% identical, to SEQ ID NO: 1;

(2) when the plant is non-fragrant wheat, the functional protein has an amino acid sequence that is at least

25 60% identical (for example, at least 65%, 70%, 75%, 80%, 85%, 90% or 95% identical), typically at least 99% identical, to SEQ ID NO: 3;

(3) when the plant is non-fragrant barley, the functional protein has an amino acid sequence that is at least

30 ^' 70% identical (for example, at least 70%, 75%, 80%, 85%, 90% or 95%), typically at least 99% identical, to SEQ ID NO: 5; when the plant is non-fragrant sorghum, the functional protein has an amino acid sequence that is at least 60% identical (for example, at least 65%, 70%, 75%, 80%, 90% or 95% identical), typically at least 99% identical, to SEQ ID NO: 7;

(5; when the plant is non-fragrant corn, the functional protein has an amino acid sequence that is at least 60% identical (for example, at least 65%, 70%, 75%, 80%, 90% or 95% identical), typically at least 99% identical, to SEQ ID NO: 9.

Examples of the amino acid sequence of the functional protein and DNA encoding the functional protein from various plants are as follows:

Plant SEQ ID NO SEQ ID NO.

Nucleotide AMINO ACID

Rice SEQ ID NO: 2 or SEQ ID NO: 1

30

Wheat SEQ ID NO: 4 SEQ ID NO: 3

Barley SEQ ID NO: .6 SEQ ID NO: 5

Sorghum SEQ ID NO: 8 SEQ ID NO: 7

Zea mays SEQ ID NO: 10 SEQ ID NO: 9

z! Tenuifolia SEQ ID NO: •12 SEQ ID NO: 11

In a preferred embodiment, the plant is rice. The species or variety of rice may be any species or variety of rice that expresses a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1. Examples of suitable species include Oryza sativa, Oryza australiensis, and Oryza- rufipogon. Examples of rice species or varieties which are non-fragrant include Nipponbare, Akitamachi, Amaroo 1, Rexmont, Sakha 101, Gulfmϋnt, Rufipogon, Vialone Nano, Koshihikara, Calrose, M202 and Shimizi Mochi.

In another embodiment, the organism is a yeast. Suitable yeasts include species selected from the group consisting of Saccharomyces cerevisiae (as defined by Barnett et al. (1990) "Yeasts Characteristics and Identification", 2^nd Edition, Cambridge University Press) , Saccharomyces bayanus, Sacccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces servazzii, Saccharomyces unisporus, Saccharomyces kluyveri, Saccharomyces dairiensis, Saccharomyces exiguus, Saccharomyces catellii, Candida spp such as Candida utilis, Candida paraffinica, Candida lipolytica, Rhodotorula species, Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces fragilis, Kloeckera spp such as Kloeckera apiculata, Pichia spp such as Pichia angusta, Pichia pastoris, Hansenula spp. such as Hansenula polymorpha, Torulopsis spp., Zygosaccharomyces rouxii, Schizosacchάromyces pombe, Torulaspora delbrueckii_r Yarrowia lipolytica etc. In various embodiments:

(1) when the yeast cell is Saccharomyces cerevisiae, the functional protein has an amino acid sequence that is at least 35% identical, typically 38% identical, to SEQ ID NO: 1;

(2) when the yeast cell is Schizosaccharomyces pombe, the functional protein has an amino acid sequence that is at least 40% identical, typically 41% identical, to SEQ ID NO: 1; (3) when the yeast cell is Yarrawia lipolytica, the functional protein has an amino acid sequence that is at least 35% identical, typically 39% identical, to SEQ ID NO: 1.

Examples of the amino acid sequence of the functional protein and DNA encoding the functional protein from various yeast is as follows:

Yeast SEQ ID NO SEQ ID NO.

Nucleotide .AMINO ACID

Schizosaccharomyces SEQ ID NO: 14 SEQ ID NO: pombe 13

Saccharomyces SEQ ID NO: 16 SEQ ID NO: cerevisiae 15

Yarrowia lipolytica SEQ ID NO: 18 SEQ ID NO:

17

In yet another embodiment, the organism is a bacterium. Suitable bacteria include Lactobacillus spp., Lactococcus sp., Bacillus subtilis, Bacillus cereus, Escherichia coli, Staphylococcus xylosus . In various embodiments:

1) when the bacterium is Staphylococcus xylosus, the functional protein has an amino acid sequence that is at least 40% identical, typically 45% identical, to the amino acid sequence of SEQ ID NO: 1;

2) when the bacterium is Bacillus subtilus, the functional protein has an amino acid sequence that is 40% identical, typically 44% identical, to the amino acid sequence of SEQ ID NO: 1; 3) when the bacterium is E. coli, the functional protein has an amino acid sequence that is at least 30% identical, typically 37% identical, to the amino acid sequence of SEQ ID NO: 1

Examples of the amino acid sequence of the functional protein and DNA encoding the functional protein from various bacteria is as follows :

Bacteria SEQ ID NO: SEQ ID NO.

DNA AMINO ACID

Staphylococcus SEQ ID NO : 20 SEQ ID NO: 19 xylosus

Bacillus SEQ ID NO : 22 SEQ ID NO: 21 subtilis

Escherichia SEQ ID NO : 24 SEQ ID NO: 23 coli

It will be appreciated by those skilled in the art that the fragrance may be produced by the entire organism, or by a part thereof. For example, in the case of plants, a part thereof may include parts such as leaves, skin, pollen, seeds, fruit, roots, embryo, bracts, kernel, ovum, stem or flowers.

The method of the first aspect of the present invention comprises reducing or eliminating the activity of the functional protein. As used herein, the expression "reducing or eliminating the activity of the functional protein" refers to reducing or eliminating the ability of the functional protein to reduce or eliminate the ability of an organism to produce fragrance. The activity of the functional protein may be reduced or eliminated by any methods known in the art for reducing or eliminating activity of a protein. For example, the activity of the functional protein may be reduced or eliminated by inhibiting the enzymatic activity of the functional protein, by degrading the functional protein, or by reducing or eliminating the ability of the organism to express the functional protein.

In one embodiment, the activity of the functional protein is reduced or eliminated by inhibiting the enzymatic activity of the functional protein. The enzymatic activity of the functional protein may be inhibited by, for example, introducing into the cell one or more enzymatic inhibitors of the functional protein, or by introducing into the cell a protein which interacts with the functional protein in a manner which blocks or prevents the functional protein from reducing or eliminating fragrance. An example of an inhibitor of the functional protein is pyridoxal 5'-phosphate, which may be ^'introduced into the cells of the organism using methods known in the art. An example of a protein which interacts with the functional protein in a manner which blocks or prevents the functional protein from reducing or eliminating fragrance is a protein which specifically cleaves or degrades the functional protein.

In another embodiment, the activity of the functional ^' protein may be reduced or eliminated by reducing or eliminating the ability of the organism to express the functional protein. As used herein, the term "express" refers to the production by the organism of a protein. The ability of the organism to express the_. functional protein may be reduced or eliminated by any methods known in the art for reducing or eliminating expression of a protein. In some embodiments, the amount of RNA transcribed from the gene encoding the functional protein is reduced or eliminated. In other embodiments, the ability to translate protein from the RNA transcripts of the functional protein is reduced or eliminated.

The ability of an organism to.express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid, sequence of SEQ ID NO. 1 may be reduced or eliminated by introducing into cells of the organism a nucleic acid molecule which reduces or eliminates expression of the functional protein.

In one embodiment, the nucleic acid molecule which reduces or eliminates expression of the functional 'protein is an antisense molecule. As used herein, an "anti-sense molecule" is a nucleic acid molecule comprising a sequence that is complementary to a specific DNA or RNA target sequence and is capable of hybridising to the target sequence to reduce or eliminate transcription or translation of the target sequence. The term "hybridise" will be understood by those skilled in the art to refer to a process by which a nucleic acid strand anneals with a substantially complementary strand through base pairing. Examples of anti-sense molecules include: anti-sense nucleic acid, including single stranded or double stranded anti-sense DNA or RNA, co-suppressor DNA or RNA, interference RNA (including RNAi, siRNA, hpRNA, ihpRNA) , ribozymes . The anti-sense molecule may be an anti-sense RNA. As used herein, an anti-sense RNA refers to an RNA molecule that is complementary to, or at least partially complementary to, and therefore capable of forming a duplex with, a target RNA molecule to thereby reduce or ^• eliminate translation from the target RNA molecule. The anti-sense RNA molecule may be complementary, or partially complementary, to a coding or non-coding region of the target RNA molecule. The anti-sense RNA molecule may be any length which reduces or eliminates expression of the functional protein. Methods for the use of anti-sense RNA for reducing or eliminating expression of a gene are known and are .described in, for example, US Patent No.

5,107,065; Smith et al. , Nature 334: 724-726 (1988); Van der Krol et al. , Nature 333: 866-869 (1988); Rothstein et al., Proc. Natl. Acad. Sci . USA 84: 8439-8443 (1987); Bird et al., Bio/Technology 9: 635-639 (1991); Bartley et al. , Biol. Chem. 267: 5036-5039 (1992); Gray et al . , Plant MoI. Bio. 19: 69-87 (1992) . The anti-sense molecule may be interference RNA (including RNAi, siRNA, hpRNA and ihpRNA) . As used herein, interference RNA refers to dsRNA-mediated interference of gene expression in which double stranded RNA that is complementary to a target nucleic acid sequence is used to selectively reduce or eliminate expression of the target gene. Methods for the production and use of RNAi are known in the art and are described in, for example, CP. Hunter, Current Biology (1999) 9:R440-442; Hamilton et al. (1999) Science 286:

950-952; Ding, Current Opinion in Biotechnology (2000) 11: 152-156.

The anti-sense molecule may be a ribozyme. As used herein, the term "ribozyme" refers to an RNA molecule comprising sequence complementary to a target RNA sequence when the complementary sequence hybridises with the target sequence. Methods for the production and use of ribozymes for reducing or eliminating expression of genes is known and described in, for example, Kim and Cech, (1987) Proc. Natl. Acad. Sci. USA, 84: 8788-8792; Reinhold-Hurek and Shub (1992) nature 357: 173-176; US Pat No. 5,254,678; Methods in Molecular Biology (1997) vol. 74, Chapter 43

"Expressing Ribozymes in Plants" (Turner and Humana Press, Totowa, N.J. Eds. ) .

In another embodiment, the nucleic acid molecule which reduces or eliminates expression of the functional protein is a co-suppressor RNA molecule. A co-suppressor RNA molecule is homologous to at least a portion of the RNA transcript of the gene to be suppressed. Methods for reducing or eliminating gene expression using co- suppressor RNA are known and are described in, for example, US Patent No. 5,231,020; Krol et al. Biotechniques 6: 958-976 (1988); MoI et al. , FEBS Lett. 268: 427-430 (1990); Grierson et al. , Trends ^■ in Biotech 9: 122-123 (1991); Krol et al (1990) The Plant Cell 2, 291- 299; Napoli et al. (1990) The Plant Cell 2: 279-289; US Pat. No. 5,231,020; WO95/34668; Angell and Baulcombe (1997) The EMBO Journal 16,12: 3675-3684.

The sequence of the nucleic acid molecules which reduce or eliminate expression of the functional protein can be readily determined using the sequence of the fgr gene, or the coding sequence of the fgr gene (cDNA sequence) described herein, and the methods provided herein.

Typically, the nucleic acid molecule which reduces or eliminates expression of the function protein is an oligonucleotide, suitably an anti-sense oligonucleotide. Antisense oligonucleotides may be any length that is sufficient to reduce or eliminate expression of the fgr gene. Suitably, the anti-sense oligonucleotides are greater than lObp in length. More suitably, the anti- sense oligonucleotides are between 10 and 100 bp in length, more typically between 12 and 50 bp in length. The anti-sense oligonucleotides may be any of the abovementioned antisense molecules. The oligonucleotides may be synthesised manually or by an automated synthesiser using methods known in the art (see, for example, Oligonucleotide Synthesis: Methods and Applications

(Methods in Molecular Biology), Pict Herdewijin (ed.) Humana Press (2004)) . The anti-sense oligonucleotide typically comprises non-phosphodiester internucleotide linkages such as alkylphosphonates, phosphorothioates, phosphate esters, alkylphosphonothiates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters (as described in, for example, Brown, Meth. MoI. Bio. 20, 1-8 m(1994); Sonveaux, meth. MoI. Biol. 26, 1-72 (1994); Uhlmann et al . , Chem Rev. 90, 543-583 (1990) .

The nucleic acid molecule which reduces or eliminates expression of the functional protein may be part of a vector. Typically, the vector is an expression vector.

As used herein, an "expression vector" refers to a nucleic acid .construct in which a nucleic acid molecule which reduces or eliminates expression of the functional protein is operably linked to a vector whereby the vector sequence specifies expression of nucleic acid molecules from the expression vector when the vector is introduced into cells of an organism. Typically, the nucleic acid molecules are anti-sense molecules or co-suppressor molecules. Suitable vectors for the expression of nucleic acid molecules in organisms are known and include any vectors that are ■ suitable for expression of RNA in that organism. For example, plasmid vectors such as the pUC-derived series of vectors (such as pUC8, pUC9, pUC18, pUC19, pUC23, pSK- derived, p-GEM derived,. pSP-derived, or pBS-derived) vectors are suitable for use in bacteria. Ti and Ri plasmid derived vectors for use with Agrobacterium tumefaciens are suitable vectors for plants. Suitable Ti and Ri plasmid derived vectors include those disclosed in US Pat. No. 4,440,838; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. D T. Dennis, D H Turpin, D D Lefebrve, D B Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997) ; Barton and Chilton (1983) Agrobacterium Ti plasmid vectors for plant genetic engineering, Methods in Enzymology 101: 527-539.

It is contemplated that replication deficient viral vectors may be employed for expression of RNA in an organism. Such vectors include, for example, in plants wheat dwarf virus (WDV) shuttle vectors such as aspWl-Il and PWl-GUS _.(see Ugaki et al. (1991) Nucleic Acids Res. 19:371-377) .

The nucleic acid molecule which reduces or eliminates expression of the functional protein may be introduced into the cells by any methods known in the art, such as ^■ those described in, for example, Hannon (2002) RNA Interference, Nature 418: 244-251; Bernstein et al (2002) The rest is silence. RNA 7: 1509-1521; Hutvagner et al. RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp (2002) A system for stable expression of short interferring RNAs in mammalian cells. Science 296: 550-553. Methods for introduction of nucleic acid molecules which reduce or eliminate expression of the functional protein into cells of the organism include transfection, transformation, electroporation, Agrobacterium tumefaciens-mediated transformation, microprojectile-mediated transformation (see, for example, Glick and Thompson (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, FIa.: CRC Press (1993); Sambrook et al. (eds.), Molecular^" Cloning: A Laboratory Manual (Second Edition), Plainvi-ew, N.Y.: Cold Spring Harbor Laboratory Press (1989); Duan et al. (1996) Nature Biotech. 14: 494-498) .

In another embodiment, expression of the functional protein is reduced or eliminated by mutating the gene encoding the functional protein (the fgr gene) such that the mutated gene does not express the functional protein. The fgr gene may be mutated by any method which results in reduction or elimination of expression of the functional protein. Sequences of the gene encoding a functional protein are described herein and may be used to mutate the gene using the methods provided herein. It will be understood by those skilled in the art that in some cases, a protein may still be expressed by the mutated gene, but the expressed protein will not be functional. For example, when the mutation is a mutation which results in formation of a stop codon, a truncated protein that is not a functional protein may be produced. The gene encoding the functional protein may be mutated by inserting at least one additional base pair into the gene. The insertion may create a frame shift which results in expression of a truncated non-functional protein, or no protein expression". The insertion may comprise translation and/or transcription stop signals. The insertion may be a single base pair, or a plurality of base pairs. For example, the' insertion may be a gene which encodes a selectable marker. As used herein, "selectable marker" refers to a gene or nucleic acid sequence encoding a trait or phenotype which can be selected or screened for in an organism. Examples of selectable markers include antibiotic resistance genes, carbon source utilisation genes, amino acid production genes etc. Selectable markers for use in plants are well known in the art and are described in, for example, Ziemienowizc A. (2001) Plant selectable markers and reporter genes. Acta Physiologiae Plantarum. 23(3) :363- 374. Selectable markers for use with yeast are known in the art and are described in, for example, Rothstein (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods in Enzymology, 194: 281-301; Sherman et al . "Methods in Yeast Genetics" (1981) Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, New York; Guthrie'and Fink (1991) "Guide to Yeast Genetics and Molecular Biology", Methods in Enzymology VoI 194, Academic Press. Selectable markers for use with bacteria are described in, for ^■ example, Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) ; Molecular Cloning a Laboratory Manual, second edition. (Cold Spring Harbor: Cold Spring Harbor Laboratory Press) ; Ausubel, F. M., Brent, R., Kingston, R., Moore, D., Smith, J.. , . Seidman, J., and Struhl, K., eds . (1987); and Current Protocols in Molecular Biology. (New York: John Wiley and Sons) . Methods for mutating genes in plants by introducing insertions into genes of the plant are described in, for example, Krysan et al. OMICS 6:163-174; Greco et al . Plant Physiology 125:1175- 1177; Gelvin, Annu. Rev. Plant. Physiol. Plant. MoI. Biol. 51:223-256; Hirochika, Plant Molecular Biology 54(3) :325- 334; Henikoff^, and Comai . Annu. Rev. Plant Biol. 54:375- 401. Methods for mutating genes of yeast by introducing insertions into yeast genes are known and are described in, for example, Rothstein (1991); Johnston, Riles and hegemann (2002), Gene Disruption, Methods in Enzymology, 350: 290-315; Sherman et al. "Methods in Yeast Genetics" (1981) Cold -Spring Harbor Laboratory Manual, Cold Spring Harbor, New York; Guthrie and Fink (1991) "Guide to Yeast Genetics and Molecular Biology", Methods in Enzymology VoI 194, Academic Press. Methods for mutating genes in bacteria by inserting sequence into the gene are known and are described in, for example, Hayes, Annual Review of Genetics 37:3-29.

An insertion may be made in a gene using, for example, transposon mutagenesis, homologous recombination or site specific recombination. An example of site-specific recombination is the cre-lox recombination system of bacteriophage Pl (see Abremski et al. (1983) Cell 32(4) .1301-1311; Sternberg et al . (1981) J.MoI.Biol ^' 150(4)487-507; J: MoI.Biol 150(4) : 467-487; J.MoI. Biol. 150 (4) : 603-608) , which has been used to promote recombination of specific locations on the genome of plant cells .(see, for example, US Pat. No. 5,658,772), animal cells (US Pat. No. 5,801,030) . A further example of site- specific recombination is the FLP recombinase system of Saccharomyces cerevisiae (see, for example, US Pat.. No. 5,654,182) . Activity of the FLP recombinase has been demonstrated in plants (see Lyznik et al. 1996; Lue et al. 2000) in addition to yeast.

The gene encoding the functional protein may be disrupted by introducing an insertion by homologous recombination as described in, for example, US Pat. No. 6,750,379.

The gene encoding the functional protein may be mutated by using transposon mutagenesis. Use of transposons to mutate genes in bacteria, yeast and plants is known in the art. Transposons, retrotransposons and methods for the mutagenesis of genes using transposons and retrotransposons in plants is described in, for example, Bennetzen (1996) Trends Microbiol. 4:347-353; Voytas (1996) Genetics 142:569-578; Hiroshik et al. (1996) PNAS 93:7783-7788; US Pat. No. 6,720,479. Transposons and methods for the mutagenesis of genes of yeast using transposons is described in, for example, Kumar et al (2002) .Insertional mutagenesis: transposon-insertion libraries as mutagens in yeast. Methods in Enzymology 350: 219-229. Transposons and methods for the mutagenesis of genes in bacteria using transposons are described in, for example, Kwon et al. (2002) Functional screening of bacterial genome for virulence genes by transposon footprinting. Methods Enzymol, 358:141-52; Burne et al (1994) Methods in Enzymology 235: 405 - 426; de Lorenzo and Timmis (1994) Methods in Enzymology, 235: 386-405.

The gene encoding the functional protein may be mutated by deleting at least one base pair from the gene that results in a reduction or elimination of expression of the functional protein from that gene. The deletion may be any size, and in any location in the gene encoding the functional protein, provided the deletion results in a reduction or elimination of expression of the functional protein by the gene. The deletion may be in the coding sequence. The deletion may be in the 5' non-coding region, such as the promoter, which prevents production of a transcript. The deletion may be in an intron or at an intron/exon boundary. The deletion may be in the 3' coding region. The deletion may be a substantial portion of the gene, or the entire gene.

Methods for the production of deletion mutations in plants are described in Li, Plant Journal 27 (3) :235-242/ Henikoff and Comai . Annu. Rev. Plant Biol. 54:375-401; Rice et al . Plant Physiol. 123:427-438. Methods for the production of deletion mutations in yeast are described in Kumar and Snyder, Nature Genetics 2 (4) : 302-312. Methods for the production of deletion mutations in bacteria are described in, for example, Miller, J. H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York (1972); Miller, J. H., A Short Course in Bacterial Genetics, Cold Spring Harbor Press, New York (1995); Sambrook et al. , Molecular Cloning: H Laboratory Manual 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor (1989) , and

Current Protocols in Molecular Biology, Ausubel et al. eds . , John Wiley and Sons (1995) .

The gene encoding the functional protein may be mutated by substituting at least one base pair of the gene so that the gene no longer encodes a functional protein. The substitution may be in the coding or non-coding portion of the gene. The substitution may result in formation of a stop codon (TGA, TAG, TAA) , or an amino acid substitution that results in loss of function of the functional protein. The substition may be in a non-coding portion of the gene which results in reduction or elimination in production of RNA transcript. The substitution may be introduced into the gene using methods known in the art.

The gene encoding the functional protein may be mutated by other methods known in the art. For example, the gene may be mutated by exposing the organism or parts thereof to mutagens such as ionising radiation, UV radiation, chemical mutagens, etc. Examples of ionising radiation include beta, gamma or X-ray radiation. Examples of chemical mutagens include ethyl methyl sulfonate, methyl N-nitrosoguanidine, N-nitroso-N-ethylurea, N-nitroso-N- methylurea, ethidium bromide, diepoxybutane. The time and dosage for exposure of the organism or parts thereof .to ■ the mutagen will vary depending on the organism arid the mutagen that is used, and can be readily determined by the person skilled in the art.

The gene may be mutated using recombinant DNA technology to delete, insert or alter the sequence of the gene. For example, the gene may be mutated by inserting a nucleic acid sequence into the gene such that the gene is no longer capable of expressing a functional protein. The nucleic acid sequence may be any nucleic acid sequence that disrupts expression of the gene. For example, the nucleic acid sequence that is inserted may be a selectable marker. Methods for inserting nucleic acid molecules into genes to inactivate the genes are known in the art. For example, methods for inserting nucleic acid molecules into the genes of plants are described in, for example, Transgenic Plants: Fundamental and Applications, Andrew Hiatt (Ed) (1993) . Methods for inserting nucleic acid molecules into the genes of yeast are described in Sherman et al. "Methods in Yeast Genetics" (1981) Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, New York. Methods for inserting nucleic acid molecules into the genes of bacteria are described in Sambrook, J., Fritsch, E. F., and Maniatis, T.- (1989); Molecular Cloning a Laboratory Manual, second edition. (Cold Spring Harbor: Cold Spring Harbor Laboratory Press); Ausubel, F. M., Brent, R., Kingston, R., Moore, D., Smith, J., Seidman, J., and Struhl, K.,' eds. (1987); and Current Protocols in Molecular Biology.

Mutants generated by any of the above methods, or naturally occurring mutants, may be screened by any methods known in the art. For example, mutants may be identified using TILLING (Target Induced Local Lesion IN ^• Genomes) . Typically, in TILLING the fgr gene of one or more organisms to be screened is amplified arid annealed with the amplified wild type fgr gene, and heteroduplexes are detected to determine whether the fgr gene has been mutated. Methods for TILLING are described in, for example, McCallum et al. (2000) Nature Biotechnology 18:455-457. Typically, TILLING is carried out following mutagenesis. However, it will be_. appreciated by those skilled in the art that TILLING may also be employed to identify organisms in the wild with naturally occurring mutations in the fgr gene.

The invention also provides a method of establishing whether an organism is capable of producing fragrance. The method thereby permits a person skilled in the art to identify those organisms that are capable of producing fragrance, and those organisms that are not capable of producing fragrance. This permits, for example, screening of mutant organisms for the ability to produce fragrance.

In one embodiment, the method- comprises the steps of:

(a) obtaining a sample of an organism;

(b) determining from the sample whether the organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

The sample may be any sample of the organism. However, it will be appreciated by persons skilled in the art that some parts of an organism, for example some parts of a plant, may be more suitable for determining the capability of the organism to produce fragrance than others. For example, in rice plants, a typical sample would be the rice grains. It will also be appreciated that the type of sample used will depend on the method for determining whether the organism is capable of expressing the functional protein and can readily be determined by the person skilled in the art.

The capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by any method known in the. art for determining the capability of an organism to express a protein. In one embodiment, the capability of the organism to express a functional protein having an amino acid sequence that is at least 30% identical, to the amino acid sequence of SEQ ID NO: 1 is determined by detecting mRNA • transcripts of the gene encoding the functional protein from a sample of the organism. Typically, the RNA transcripts are of the fgr gene. Preferably, RNA is extracted from a sample of the organism using methods known in the art, such as those described in Ausubel, F. et al., 1989-1999, "Current Protocols in Molecular Biology" (Green Publishing, New York) ; . In detecting mRNA transcripts of the gene that encodes the functional protein, total RNA may be used, or mRNA may be isolated from the total RNA and used subsequent to isolation. Once the RNA or mRNA is obtained, mRNA transcripts may be detected by a number of methods known in the art.

For example, mRNA transcripts of the gene encoding the functional protein may be detected by RT-PCR in which the mRNA transcripts are amplified by extension of primer pairs complementary to cDNA synthesised from the mRNA . transcripts. As used herein, the term "primer" refers to a short-length, single stranded polydeoxynucleotide that is chemically synthesised by known methods (involving, for example, triester, phosphoramidite, or phosphonate chemistry), such as described by Engels, et al. , Agnew. Chem. Int. Ed. Engl. 28: 716-734 (1989) . They are then purified, for example, by polyacrylamide gel electrophoresis. The sequence of the primer may be selected such that the primer is substantially complementary to^' a target sequence and therefore capable of hybridising to the target. Once the primer is hybridised to the target it may be extended by the addition of deoxyribonucleotides to the 3' end of the primer using a DNA polymerase, or by the addition of ribonucleotides using an RNA polymerase. As used herein, "primer pairs" will be understood by those skilled in the art to refer to a pair of primers, one of which is capable of hybridising to a first strand of a double stranded nucleic acid molecule (for example, a cDNA molecule or cDNA:mRNA hybrid) , and the other of which is capable of hybridising to the second strand of the double stranded nucleic acid molecule to permit amplification of sequence corresponding to, and located between, the primer pairs by PCR (eg. the .cDNA molecule or cDNA:mRNA hybrid) . It will be understood by persons skilled in the art that cDNA refers to the DNA molecule generated by using mRNA as a template to synthesise a DNA molecule having a sequence- complementary to the mRNA sequence. Preferably, the mRNA transcripts are detected using reverse transcriptase to synthesise a cDNA strand followed by amplification of the cDNA sequence by polymerase chain reaction (RT-PCR) . As used herein, reverse transcriptase is an enzyme which synthesises a cDNA strand from a mRNA template. "Polymerase chain reaction," or "PCR," as used herein generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described in U.S. Patent No. 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using a first and second primer capable of hybridizing preferentially to .a target nucleic acid. Typically, the primers used in the PCR method will be complementary to nucleotide sequences within the template at both ends of the nucleotide sequence to be amplified, although primers complementary to the nucleotide sequence to be amplified also may be used. Wang, et al. , in PCR Protocols, pp.70- 75 (Academic Press, 1990); Ochman, et al. , in PCR Protocols, pp. 219-227; Triglia, et al., Nucl. Acids Res. 16:8186 (1988) .

The reaction conditions for the extension reaction such as annealing time and temperature and extension time and temperature will vary depending on the sequence of the primer and the nature of the polymerase ^' used in the extension reaction. The appropriate reaction conditions to be used may be determined as described in Wang, et al., in PCR Protocols, pp.70-75 (Academic Press, 1990); Ochman, et al., in PCR Protocols, pp. 219-227.

In another approach, mRNA transcripts of the gene which encodes a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be detected using nucleic acid hybridisation techniques. In using such techniques, RNA or mRNA that has been extracted from a sample of the organism is hybridised with a probe that comprises sequence that is complementary to the gene encoding the functional protein. As used herein, the term "probe" refers to a nucleic acid molecule having a nucleotide sequence that is substantially complementary to its target nucleic acid sequence to be able to form a detectable hybrid probe:target duplex under high stringency hybridisation conditions. A "probe:target' duplex" is a structure that is a double-stranded structure formed between two complementary nucleic acid molecules . The structure is sufficiently stable to withstand wash conditions following hybridisation and to be detected by means of radioisotopes, chemiluminescent molecules, fluorophores or other fluorescent tags, enzymes that may be bound to the probe such as digoxigenin, luciferase, alkaline phosphatase or haptens. As used herein, "peptide-nucleic acids" (PNAs) are compounds comprising ligands linked to a peptide backbone rather than to a phosphodiester backbone. Representative ligands include either the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5- methylcytosine or thiouracil) or artificial bases (e.g., bromothymine, azaadenines or azaguanines, etc.) attached to a peptide backbone through a suitable linker. The PNAs are able to bind complementary ssDNA and RNA strands. ^■Methods for making and using PNAs are disclosed in U.S. Pat. No. 5,539,082 and, for example, Basile A, Giuliani A, Pirri G and Chiari M, Electrophoresis, 2002 Mar;23 (6) : 926- 9.

The RNA or mRNA isolated from the sample of the organism, or the cDNA synthesised from the mRNA, may be immobilised on a solid support prior to hybridisation with probe. The solid support may be, for example, a hybridisation membrane such as nylon or nitrocellulose, a glass slide or microchip. Hybridisation of the probe with the immobilised RNA, mRNA or cDNA may be by northern hybridisation, dot-blot hybridisation or any other hybridisation techniques known in the art. Alternatively, the probe may be immobilised on a solid support such as nylon or nitrocellulose, a glass slide or microchip. In this approach, the RNA, mRNA or cDNA extracted from the tissue sample is labelled to permit detection of hybridisation to the probe. The term "hybridization" refers to a well known method whereby under sufficiently stringent hybridization conditions, a nucleic acid hybridizes specifically only to substantially complementary sequences. As used herein, a nucleic acid sequence is "substantially complementary" to another nucleic acid sequence if greater than 85% of the sequence is capable of forming Watson-Crick base pairing with the other sequence, preferably 90% of the sequence, more preferably 95% of the sequence and even more preferably 100% of the sequence. A substantially complementary sequence may contain mismatches in the sequence,. or may comprise ends such as primer ends which are outside the sequence between the translation start sites, or ends which are added to assist in, for example, cloning of the probe or detection of hybridisation of the probe. Sequences that are substantially complementary will hybridise under stringent conditions as defined for a particular system. Defining appropriate hybridization conditions is within the skill of the art. See eg.

Sambrook et al . , DNA Cloning, • vols . I, II and III. Nucleic Acid Hybri'dization. However, ordinarily, "stringent conditions" for hybridization or annealing of nucleic acid molecules are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 5O⁰C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylρyrrolidone/50mM sodium phosphate buffer at pH

6.5 with 75OmM NaCl, 75mM sodium citrate at 42⁰C.

Another example is use of 50% formamide, 5 X SSC (0.75M NaCl, 0.075M sodium citrate),. 5OmM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 X Denhardt' s solution, sonicated salmon sperm DNA (50μg/mL) , 0.1% SDS, and 10% dextran sulfate at 42⁰C, with washes at 42⁰C in 0.2 X SSC and 0.1% SDS.

Following washing of the hybridisation complexes, the hybridisation complexes are detected according to well known techniques . Labelled nucleic acid probes capable of specifically hybridizing to a target or labelled_ RNA, mRNA or cDNA can be labelled by any one of several methods typically used to detect the presence of hybridized nucleic acids. One common method of detection is the use of autoradiography using nucleic acid labeled with 3H, 1251, 35S, 14C, or 32P, or the like. The choice of radioactive isotope depends on research preferences due to ease of synthesis, stability, and half lives of the selected isotopes. Other labels include compounds (e.g., biotin and digoxigenin) , which bind to antiligands or antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.

The probe may be of any length that Is sufficient to permit the probe to hybridise specifically to the mRNA transcripts of, or cDNA synthesised from the mRNA transcripts' of, the gene which encodes the functional protein. Preferably, the probe comprises at least 15 base pairs. More preferably, the probe comprises at least 50 base pairs. Even more preferably, the probe comprises at least 300 base pairs.

Probe sequences may be determined using methods known in the art and the nucleotide sequences described herein.

Following RT-PCR or hybridisation, labelled nucleic acids may be detected by means known in the art. For ^• example, radioactively labelled molecules may be detected Using photographic film, phosphoimagers, scintillation counters, fluorescently labelled molecules may be detected using a photodetector, enzymatic labels may be detected by providing to the enzyme a substrate and detecting the reaction. Expression of a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be detected by detecting the functional protein or fragments thereof using^" antibody specific to the functional protein. Antibody to the functional protein may be produced by methods known in the art. Firstly, the* functional protein must be produced and isolated. The functional protein may be produced by any methods known in the art for production and isolation of proteins. For example, the cDNA molecules synthesised from mRNA transcripts may be cloned using recombinant DNA techniques in a manner to permit overexpression of the first and second gene using vectors and techniques known in the art. The cloned genes may be expressed from eukaryotic cell lines, plant, yeast, fungal or bacterial cells. The gene products may then be purified using well known methods such as precipitation with ammonium sulphate, PEG precipitation, isoelectric focusing, gel electrophoresis, gel filtration chromatography such as ion exchange, reverse phase, hydroxyappetite, affinity and combinations thereof.

Once the gene products are isolated, antibodies specific to the protein may be raised against the protein products using methods well known in the art (see for example Antibodies: A Laboratory Manual, CoLd Spring

Harbour Laboratory, 1988) . The antibodies may then be employed using immunodetection methods to detect expression, or lack of expression, of the ^■ functional protein. Immunodetection methods involve obtaining a sample that may contain the protein, contacting the sample with an antibody raised against the protein and detecting binding of the antibody to the protein.

Typically, a sample of the organism is incubated with an antibody raised against the functional protein for sufficient time and under conditions sufficient to permit formation of immune complexes between antibody and protein. Examples of methods in which antibodies and samples may be incubated include immunohistochemistry (see for example, Diagnostic immunopathology, 2^nd Edition, Colvin,R.B., Bhan, A.K., McCluskey. Eds, Raven Press, New York, 1995), ELISA plate, dot blot, western blot and FACS analysis. Generally, the complexes are washed to remove unbound antibodies and the immune complexes detected.

Antibodies to the function protein can be employed in the detection of expression of the functional protein in tissue sections of the organism, as well as fixed cells by immunohistochemical or immunopathological analysis. Cytochemical analysis wherein these antibodies are labelled directly (with, for example, with fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or are labelled by using secondary labelled anti-species antibodies (with various labels as exemplified herein) to track the histopathology of disease also are within the scope of the present invention.

Expression of the functional protein may be detected using an ELISA assay in which antibody specific to the functional protein is immobilised on a solid support and subsequently incubated with a sample of the organism for a period of time readily determined by those skilled in ^'the art. Following incubation, the immune complexes are washed to remove unbound protein and the complexes incubated with a second labelled antibody that is specific to the protein to form a "sandwich" immune complex. The functional protein can thereafter be detected in the sample by detecting the presence of bound labelled antibody. It will be appreciated by those skilled in the art, however, that ELISA assays may be carried out in many different known ways depending on, for example, the sample to be tested, the type of antibody used and the method of detection employed.

Detection of immune complex formation is well known in the art and may be achieved through the application of a number of approaches. Preferably, the antibodies are labelled with a detectable marker such as, for example, a radioactive label, a fluorescent label, a biological or enzymatic tag or other labels known in the art. Secondary binding ligands such as secondary antibodies may also be employed.

The capability of the organism .to express a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1 may be determined by amplifying a portion of the genome corresponding to the gene encoding the functional protein and sequencing the amplified portion to determine whether the amplified portion comprises a gene that encodes a functional protein. Methods for sequencing of amplified nucleic acid are known in the art and are described in, for example, Sambrook et al . (1989) .

Mutations in the gene encoding the functional protein may be detected using primer or probe sequences to known polymorphisms associated with a fragrant phenotype. For example, primer or probe sequences complementary to polymorphic regions of the fgr gene in the fragrant rice variety Kyeema (see Figure 4) may be used to determine whether other varieties of rice or other organisms have such polymorphisms. The sequence of primer pairs or probes which are capable of hybridising to polymorphic regions, and are therefore capable of detecting varieties having polymorphisms, can be readily determined by those skilled in the art following sequencing of the mutant fgr gene from fragrant organisms. Hybridisation (or lack thereof) of the primers or .the probes to the target nucleic acid may be detected by methods well known in the art. For example, the primers or probe may be labelled as described above. Hybridisation of the primers may be detected using PCR (as described above) . In one form, real-time PCR may be used to detect hybridisation of the primers to the gene encoding the functional protein.

Methods for detecting mutations using real-time PCR are described in, for example, Germer et al. (2000) Genome Res. 10(2) : 258-266;Shwartz et al (2004) 32(3) : e24; Tapp et al. (2000) Biotechniques 28(4) : 732-738. The primer sequences may be determined using methods known in the art and the nucleotide sequences described herein.

Primer sequences may be between lObp and 50bp in length (for example, between 10 and 40 bp, 10 and 30 bp, 12 and 30 bp) , typically 12 and 25 bp in length. The primer may be used alone in a primer extension reaction, or the primer may be one primer of a primer pair for use, in PCR. Methods for PCR are known in the art and are described herein. -The primer may be labelled using methods known in the art and described herein for use as a probe.

The invention also provides a method for producing an organism which produces fragrance. The method comprises identifying one or more parent organisms which comprise at least one mutant fgr gene that is not capable of expressing a functional protein. Any of the abovementioned methods may be used to identify the parent organisms. It is also envisaged that organisms produced by embodiments of the second aspect in which the fgr gene is mutated may be used as the parent organism.

Once the parent organisms have been identified, then at least two of the parent organisms may then be cultured under conditions which permit mating between the organisms to produce progeny.^' As used herein, the term "mating" refers to any process in which DNA exchange occurs between the parent organisms. Mechanisms of DNA exchange include conjugation, phage-mediated transduction, protoplast fusion, sexual recombination, etc. Following mating of the parent organisms, the resulting progeny are selected for those that are homozygous for the mutant fgr gene.

It will be apparent to persons skilled in the art that an organism which produces fragrance may produce fragrance in all or part of the organism.

In one embodiment, the organism is a plant. When the organism is a plant, the method typically comprises:

(a) identifying a first parent plant and a second parent plant, wherein the first and second parent plants •comprise at least one mutant fgr gene, and wherein the first and second plants are capable of cross- pollination;

(b) pollinating the first parent plant with pollen from the second parent plant, or pollinating the second parent plant with pollen from the first parent plant;

(c) culturing the pollinated plant under conditions to produce progeny plants;

(d) • selecting progeny plants that are homozygous for the mutant fgr gene. Methods for selecting homozygous progeny include any of the above methods for determining whether an organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1. It will be understood by those skilled in the art that as the fragrance phenotype is a recessive phenotype, it is preferred that the fragrant organism be homozygous.

In another embodiment, the organism is a yeast. Yeast cells may be cultured under conditions which permit mating by: (a) pooling the population of genetically diverse yeast cells; (b) sporulating the pooled cells and germinating the spores to produce haploid cells;

(c) hybridisation of compatible mating types of the haploid cells to produce hybrid yeast cells .

Methods for sporulation, obtaining haploids and hybridisation of the haploids to produce hybrid yeast cells are known in the art and are described in, for example, Chapter 7, "Sporulation and Hybridisation of Yeast" by R.R. Fowell, A.H. Rose and J.S. Harrison, 1969, Academic Press; EP 0 511 108 B. Methods for the generation of intraspecific or interspecific hybrids using cell fusion techniques are described in, for example, (Spencer et al. (1990) in, Yeast Technology, Spencer JFT and Spencer DM (eds.), Springer Verlag New York) .

It will be appreciated by those skilled^' in the art that once organisms have been obtained which are heterozygous or homozygous for the mutant fgr gene, those heterozygous or homozygous organisms may be used in breeding programmes or mating procedures to transfer the ability to produce fragrance to non-fragrant organisms.

The fragrant organisms identified or produced by the methods of the invention may be used to produce any food product for which that organism is suitable. For example, cereal crops may be used to produce rice, flour and grains for use in the production of food products such as, for example, bread, beer and other fermented and non-fermented beverages. Yeast may be used in the production of, for example, bread, beer and wine. Fungi and bacteria may be used in the production of, for example, cheeses and fermented dairy products such as yoghurt.

The invention will now be illustrated by way of reference only to the following non-limiting example.

EXAMPLES

Example 1

Introduction

To identify the chromosomal location of the gene encoding the fragrance phenotype in rice, segregating populations were mapped using simple sequence repeat (SSR) or microsatellite² and single nucleotide polymorphism (SNP)³ markers. The availability of a rice genome sequence¹⁴ permitted comparison of the sequences of fragrant and non- fragrant genotypes. This allowed targeted re-sequencing of genes and genomic sequences in a fragrant genotype to the most likely parts of chromosome 8. Materials and Methods

Plant materials

A population of 168 field grown F₂ individuals derived from a cross between Kyeema (Pelde//Della/Kulu) (tall, jasmine- style, aromatic, long-grain, Australian cultivar) and Gulfmont (Lebonnet//CI9881/PI 331581) (early-maturing, semi-dwarf, non-aromatic, long-grain, USA cultivar) supplied by Yanco Agricultural Institute, NSW Agriculture were used as the mapping population in this study.

Following genetic mapping, candidate genes were identified in the region between flanking markers in the published genome sequence and re-sequenced in the fragrant rice variety Kyeema. Polymorphisms were identified by a visual inspection of alignments of sequence derived from Kyeema with the published genome sequence of Nipponbare, a non- fragrant variety. Polymorphisms that were considered a possible cause of fragrance were genotyped in 12 fragrant and 67 non-fragrant varieties.

Genetic mapping Fragrance was^' evaluated according to Berner and Hoff¹⁹. The phenotype of F₂ individuals were classified as fragrant, segregating or non-fragrant by tasting dehulled F₃ seed. At least 12 F₃ seeds from individual F₂ plants were chewed individually. F₂ plants were rated homozygous fragrant or non-fragrant if all 12 ■ F₃ seeds were fragrant or non- fragrant, respectively. F₃ seeds from heterozygous F₂ plants were expected to contain both fragrant and non- fragrant seeds, therefore if the sample from a single F2 plant was a mixture of fragrant and non-fragrant, the F₂ plant was considered heterozygous. The observed segregation ratio of fragrant:segregating:non-fragrant was tested by χ² analysis against the expected ratio for a single gene.

Following identification, SSRs were assessed for polymorphism by comparison of parental alleles. Polymorphic SSRs were genotyped in F₂ individuals from the mapping population. The genetic distance between fgr and the polymorphic SSRs were estimated using _.MAPMAKER V.3.0 and determined as the percentage of recombinant chromosomes (cM) .

Bioinformatics and computer programs

The major gene controlling the grain fragrance in rice has been located between the RGl and RG28 RFLP markers (Lorieux et al. 1996) . Fourteen BAC clones were selected based on their proximity between RFLP markers Rl and RG28 and the sequences of BAC were obtained ^"from GenBank

(http://rgp.dna.affrc.go.jp) . SSRs were identified by using Simple Sequence Repeat Identification Tool (SSRIT) from http://www.gramene.org/db/searches/ssrtool. In addition, nine already-mapped microsatellite markers (S.R. McCouch et al, 2002) between RGl and RG28 were selected for assessment of polymorphism. The primer sequences for these markers are available from http: //www.gramene.org/microsat/ssr.html. ■

Oligonucleotide primers were designed using Primer Premier Version 5.0 (Premier Biosoft International, Palo Alto, CA) . Sequence was aligned using ChromasPro version 1.15 Technelysium Pty Ltd (www.technelysium.com.au/ChromasPro.html) . BAC AP004463 sequence was obtained from the NCBI web site (www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val =24460082) . EST sequences were obtained from the Knowledge-based Oryza Molecular biological Encyclopedia (KOME) web site (http://cdna01.dna.affrc.go.jp/cDNA/) using the search term AP004463 and were selected based on their proximity to microsatellite marker SSR-J02 and SNP marker RSP04, and on their predicted functions.

DNA extraction, PCR, genotyping and sequence analysis

Genomic DNA was extracted using a Qiagen Dneasy^® 96 Plant Kit (Qiagen GMbH, Germany) . DNA preparations were diluted with TE buffer to a final concentration of approximately 10 ng per μl. Oligonucleotide primers were synthesised by Proligo Australia Pty Ltd. PCR was performed using a Perkin Elmer, Gene Amp PCR system 9700. The reaction volume was 25μl containing 20ng of extracted genomic DNA, 2.5mM MgCl₂, 200μM total dNTPs, 1 unit of Platinum® Taq DNA Polymerase (Gibco BRL®) , lxGibco® PCR Buffer (minus MgCl₂) and 0.2μM of each forward and reverse primer. Cycling conditions were 94°C for 2 minutes followed by 30 cycles of 94⁰C for 30 s, 55⁰C for 30 s and 72⁰C for 1 minute followed by a final extension of 72⁰C for 7 minutes. SSRs were amplified by PCR and analysed by electrophoresis in either ethidium bromide stained (0.5ug/ml-l) 2.0% agarose or using a Corbett Robotics Gel-Scan 2000™. A 100 bp ladder molecular weight standard (Roche) was used to estimate PCR fragment size. Prior to sequencing, PCR products were purified using a montage PCR filter device, Millipore Corporation. Sequence reactions were performed using BigDye Terminator version 3.1, Applied Biosystems, and the completed reactions purified by ethanol precipitation. The reaction products were analysed on an Applied Biosystems 3730 Genetic Analyser. 5

Results

Genetic mapping of molecular markers and the fragrance phenotype demonstrated that the markers RM515 and SSR-J07

10 flanked fgr (Fig.l) . The physical distance between RM515 and SSRJ07 is 386 591 bp. The data suggested that fgr was closer to RM515 than to SSRJ07. Sequencing was initially focused on a one Mbp region including both these markers but careful examination of mapping data suggested that one

15 BAG (clone AP004463, Fig. 2) was most likely to contain the gene. Sequencing of 17 genes in this BAG revealed significant sequence variations in only one. Other genes in this region showed very little polymorphism.

20. The 17 cDNAs (Table 1) were selected based on their position in the AP004463 BAG clone (Fig. 3) and on the assigned putative function of the predicted product. Primers were designed for the regions in the AP004463 BAC clone corresponding to the genes that produce these cDNAs

25 and these regions were subsequently amplified and sequenced from the fragrant rice cultivar Kyeema. This analysis revealed 6 polymorphisms, 3 of which were in exons, including a large polymorphism within an exon of one candidate gene, listed on KOME as cDNA clone^'

30 J023088C02. This large polymorphism contained a total of 6 SNP'-s and 8 deletions within a 25bp region (Fig 4) . To aid ^■ confirmation that this polymorphism was the cause of the fragrance phenotype, sequence analysis of this exon region was carried out on 12 diverse fragrant and 61 non-fragrant rice varieties. The 12 fragrant varieties showed the sequence polymorphism .observed in Kyeema while the 61 non- fragrant varieties showed sequence identical to the published Nipponbare sequence. Sequence analysis of the other 3 exon polymorphisms and 1 intron polymorphism, from the candidate gene, was undertaken in 4 other fragrant varieties, these polymorphisms were present in all of these varieties. The full length DNA sequence of the fgr gene from non- fragrant rice variety Nipponbare is shown in Figure 5 (SEQ ID NO: 2) . .

An alignment of the cDNA sequence from fragrant and non- fragrant phenotypes is shown in Figure 7.

Other Organisms

Amino acid and nucleotide sequences of the coding region of the functional protein from rice (Nipponbare) , wheat, ^' barley, sorghum, Zea mays, Z. tenvifolia, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Yarrowia lipolytica, Staphylococcus xylosus, Bacillus subtilis and E.coli is shown Figures 8 to 19. An alignment of the amino acid sequence of the functional protein from wheat, barley, rice and Z. tenvifolia using ClustalW is shown in Figure 20, together with the pariwise % identity between the sequences (bottom under Score) . An^' alignment of the amino acid sequence of the BADH2 protein from various organisms (as indicated) using ClustalW is shown in Figure 21. An alignment of the amino acid sequence. of the coding region of the BAD2 gene from various organisms, and BADl from rice, using ClustalW is shown in Figure 22. The percent identity of the amino acid sequence of protein encoded by the fgr gene for various organisms (as indicated) using ClustalW is shown in Figure 23. The parwis_.e percent identity is given under Score.

Table 1. cDNA' s listed on BAC clone AP004463 and polymorphisms found in Kyeema.

Discussion

The predicted amino acid sequence for the protein encoded by the fgr gene from Nipponbare and the mutant fgr gene from Kyeema is shown in Fig. 6. A peptide sequence (VTLELGGKSP) and a cysteine residue (28 amino acid residues away in both BADl and BAD2), found in the genes from non fragrant rice, is highly conserved in aldehyde dehydrogenases¹⁵. These conserved elements are lost in the shorter protein that would be encoded by the gene in fragrant varieties. BAD genes also contain the conserved peptide EGCRLGSVVS found in the gene from non-fragrant varieties.

The fgr gene from Nipponbare encodes a protein with high similarity to Betaine aldehyde dehydrogenase (BAD) . BAD from oats has been shown to have wide substrate specificity for amino aldehydes and related compounds. BAD in rice is encoded by a gene on chromosome 4. Barley has been shown to contain two BAD isozymes, probably with different substrate specificities¹⁷. The fgr gene from Nipponbare corresponds to the BAD2 gene from barley. The production of two different subunits in the same subcellular compartment allows for the possible formation of heterodimers of the two subunits .

Although the biochemical pathway leading to fragrance in rice has not been established, L-proline has been shown to be a precursor of aroma in rice¹⁸. Without wishing to be bound by theory, the inventors believe that the fgr gene may encode a protein that either catalyses the formation or the removal of 2-acetyl-l-pyrroline or precursors of 2-acetyl-l-pyrroline. However, fragrance is a recessive trait suggesting a loss of function is responsible for the accumulation of 2-acetyl-l-pyrroline while the truncated version of the protein that is encoded by the fragrant genotypes is not functional and favours the later hypotheses.

Example 2

For any individual analyst, the ability to distinguish between fragrant and non-fragrant samples diminishes with each successive analysis because the senses become saturated or physical damage occurs from abrasions to the tongue which often result from chewing the hard grain.

Described below is the development of a PCR assay for fragrance genotyping in rice.

Plant Materials

All rice samples were supplied by Yanco Agricultural Institute, NSW Agriculture. A diverse collection of 14 fragrant and 74 non-fragrant varieties in addition to a population of 168 field grown F₂ individuals derived from a cross between Kyeema (Pelde/Della/Kulu) (tall, Jasmine- style, long-grain, Australian cultivar) and Gulfmont (Lebonnet//CI9881/PI 331581) (early-maturing, semi-dwarf, non-aromatic long-grain USA cultivar) was used to validate the marker.

Genetic Mapping

Genetic mapping was carried out as described in Example 1.

Primer Design

Oligonucleotide primers were designed, using Primer Premier- Version 5.0 (Premier Biosoft International, Palo

Alto, CA) . For non-fragrant varieties the sequence of the gene encoding functional protein was obtained from the NCBI website (www.ncbi .nlm.nih.gov) ^• Gen Bank Accession number - AP004463 and for fragrant varieties the sequence of the fgr gene was used (Figure 7 - Rice truncated, BAD2) .

DNA extraction, PCR and genotyping

Genomic DNA was extracted from leaf material using a Qiagen DNeasy^® 96 Plant Kit (Qiagen GMbH, Germany) and from whole seeds as described by Bergman et al. (2001) Cereal Chemistry 78:257-260. Rough leaf DNA extractions were performed by boiling 0.1 g of leaf material in 50 μl 1OX PCR Buffer (Gibco BRL^®) for 10 min. Oligonucleotide primers were synthesised by Proligo Australia Pty Ltd.

PCR was performed using 0.2 μl Platinum^® Taq DNA Polymerase

(Gibco BRL^®), 1 μl of genomic DNA 10 ng μl^"1, 2.5 μl of 1OX buffer (Gibco BRL^®) , 1 μl of 50 mM MgCl₂ (Gibco BRL^®) , 1 μl of dNTPs [5 mM] , 2.5 μl of each primer (ESP, IFAP, INSP and EAP Table X) [2 μM] , in a total volume of 25 μl. PCR was performed using a Perkin Elmer, Gene Amp PCR system 9700. Cycling conditions were an initial denaturation of 94⁰C for 2 min followed by 30 cycles of 5 s at 94°C_r 5 s at 58°C, 5 s at 72°C; concluding with a final extension of 72⁰C for 5 min.

Table 2

PCR products were analysed by electrophoresis in ethidium bromide stained (0.5 μg ml ) 1.0% agarose gels. A 100 bp ladder molecular weight standard (Roche) was used to estimate PCR fragment size.

Results

Development of a single tube Allele Specific PCR fragrance assay

Four primers, two that anneal to sequences common to both fragrant and non-fragrant varieties and external to the area where the mutation occurs and two that are specific to one of the^' two possible alleles were designed and synthesised (Figure 24) . The two external primers were designed to act as an internal positive control amplifying a region of approximately 580 bp in both fragrant (577 bp) and non-fragrant (585 bp) genotypes. Individually, these external primers also pair with internal sequences to give products of varying size, depending upon the genotype of the DNA sample. The internal primers, IFAP and INSP (Table 2), will anneal only to their specified genotype producing DNA fragments with their corresponding external primer pair, ESP and EAP respectively. Using these four primers in a PCR results in three possible outcomes. In all cases a positive control band of approximately 580 bp is produced. In the first case a band of 355 bp is produced indicating a variety or individual is homozygous non-fragrant. In the second case a band of 257 bp is produced indicating a variety or individual is homozygous fragrant. In the third case both bands of sizes 355 bp and 257 bp are produced indicating an individual is heterozygous non-fragrant.

Determination of plant genotype using single tube ASA PCR fragrance assay

PCR products were easily separated on an agarose gel. The PCR product of approximately 580 bp serves as a positive control and is present in every sample. Fragrant individuals have a second product of 257 bp in size while non-fragrant individuals give a product of 355 bp in size, heterozygotes can also be discriminated by the presence of all three PCR products (Figure 25) . The assay predicted the phenotype of 168 F₂ progeny segregating for fragrance with 100% accuracy (46 homozygous fragrant, 80 heterozygotes, 42 homozygous non- fragrant) . (Figure 26) . The assay also allows discrimination between fragrant and non-fragrant grains using DNA derived from rice grains using a simple NaOH extraction protocol (Bergman et al._r 2001) and leaves using a simple 10 min boiling protocol. Further evaluation demonstrated the capacity of the assay to work on a broad range of fragrant varieties such as Basmati 370, Kyeema, Khao Dwak Mali' 105 and Moosa Tarom.

The results illustrate a specific PCR assay which allows determination of the genotypic status of an individual rice plant, either homozygous fragrant, homozygous non- fragrant or heterozygous non-fragrant. The assay is a simple robust- method for screening rice to determine its fragrance status across a wide range of rice varieties and within segregating populations using DNA isolated from rice following simple, inexpensive and rapid extraction protocols. The PCR products can be analyzed easily and inexpensively on agarose gel or alternatively using more sophisticated high throughput equipment, making the assay a very versatile tool.

The following Examples are prophetic examples of .,. embodiments of the invention.

Prophetic Example 1.

Transposon mutagenesis of maize to produce fragrant maize due to elevated concentrations of 2-acetyl-l-pyrroline Transposon mutant lines may be generated and screened according to the method of

Robert J. Bensen, Gurmukh S. Johal, Virginia C. Crane, John T. Tossberg, Patrick S. Schnable, Robert B. Meeley, and Steven P. Briggs . (1995) Cloning and Characterization of the Maize AnI Gene. The Plant Cell, Vol. 7, 75-84.

A population of Mu-containing Fl maize families (20,000 - 30,000 individuals) would be generated and subsequently screened using polymerase chain reaction (PCR) to detect the presence of Mo insertional alleles in the gene which encodes BAD2. Genomic DNA would be isolated from leaf material using Qiagen^® MagAttract™ 96 chemistry applied to the MWG Biotech TheOnyx liquid handling robot.

A forward primer, 5'-ATGGCCTCGCAAGCGAT^{^}-S' (.SEQ ID NO:

31) ; and a reverse primer,

5'- TCCACCTCTTATAATGGCACAGTT -3' (SEQ ID NO: 32); would be used to anneal to the 5' end of the BAD2 coding sequence and the 3' UTR of the BAD2 mRNA respectively. A primer 5'- CCCTGAGCTCTTCGTC (CT)ATAATGGCAATTATCTC-3' (SEQ ID NO: 33) would be used to anneal to the distal portion of the terminal inverted repeat common to all functional Mu elements. Primers would be synthesised by methods known in the art. PCR would be performed with a Corbett Rotor- Gene™ and PCR products detected by Sybr-Green. When PCR products are detected with Sybr-Green, the result would first be confirmed by agarose- gel electrophoresis of an aliquot of the reaction followed by DNA sequencing. The PCR volume would be 10 μl containing 10 ng^' of extracted genomic DNA, 1.5 mM 200 μM total dNTPs, 0.5 unit of Platinum^® Taq DNA Polymerase (Gibco BRL^®) , Ix Gibco^® PCR Buffer (minus MgCl₂) and 0.2 μM each of the forward and reverse primer. Cycling conditions would be 5 min at 94⁰C followed by 35 cycles of 94⁰C for 30 s, 55⁰C for 30 s and 72 ⁰C for 30 s followed by a final extension of ^'12 ⁰C for 7 min. PCR products would be sequenced by using BigDye Terminator cycle sequencing (Perkin-Elmer Applied Biosystems, Forster City, CA) and the products analysed by an Applied Biosystems 3730 DNA Analyzer (Perkin-Elmer Applied Biosystems, Forster City, CA) .

Fi individuals containing insertions in the BAD2 encoding gene would be identified by their production of PCR products using either the forward or reverse primers which anneal to the BAD2 encoding gene paired with the Mu- specific primer. F₂ seed from plants producing PCR products would then be planted in the greenhouse and scored for the fragrance phenotype.

The concentration of 2-acetyl-l-pyrroline (2AP) in maize leaves would be determined by grinding approximately 5 g of leaf material under nitrogen followed by incubation in 20 ml pure ethanol (99.9%) for 24 h at room^" temperature [Natta Laohakunjit and Athapol Noomhorm (2004) Flavour and Fragrance Journal 19: 251-259. Supercritical carbon dioxide extraction of 2-acetyl-l-pyrroline and volatile, components from pandan leaves] . The supernatant would be filtered in preparation for GC-MS analysis and extracts analysed in duplicate, using a HP 5890 'Series II GC/HP 5972 mass selective detector (MSD) (Hewlett-Packard, California, US.) fitted with a capillary column (Innowax, 25 m x 0.2 mm i.d., 0.4 μm film thickness; Agilent Technologies, CA) . A 2 μl solution of the extract would be injected for analysis. Oven temperature would be held at 50 ⁰C for 2 min, then programmed to increase from 50 ⁰C to 170 ⁰C at 7 °C/min and would be held at 170 ⁰C for 5 min. Other operating conditions would be as follows: injector temperature, 170 °C; carrier gas, helium at a flow rate of 0.6 ml/min; ion source temperature, 230 ⁰C; electron multiplier voltage, 260.0 V. The samples would be injected in the splitless mode. Compounds would be tentatively identified by matching their mass spectrometric data with those obtained from the same equipment. Quantitative determination of 2AP in extracts performed by using measurements ^•of peak area of m/z (mass/charge) 41(50), 43(100), 55(2), 67(0.2), 68(8), 83(11), 111(5), with the aid of the instrument's digital integrator. Correlating peak areas with concentrations would be performed by means of a standard calibration curve obtained between 10 and

200 ng/injection. To minimize errors an external standard would be used, with 2, 4, 6-trimethylpyridine (TMP) (Sigma Aldrich, St. Louis, MO) as the external standard in ^• quantitative analysis of 2AP.

Prophetic Example 2

Method for UV mutagenesis of yeast to produce increased levels of 2AP in yeast

Strains and media

In this example, Saccharomyces cerevisiae (bakers yeast) , supplied by Fa. Wieninger, Passau, Germany, is used^' to isolate strains of yeast with non functional BAD genes. The yeast would be grown on YPD medium (1% yeast extract, 2% polypeptone, 2% glucose and if necessary, 2% agar) at 28°C. Mutagenesis and mutant isolation

Cells grown for 24 hours at 28°C in liquid YPD media would be spread on YPD medium plates (20μl per plate) . The plates would be placed under a UV lamp (Toshiba GL15) at a distance of 35cm and irradiated for 15 seconds. Following irradiation, the plates would be incubated at 28°C and after 24 hours individual colonies picked and subcultured ^■into individual flasks containing 1 ml of liquid YPD media. Flasks would be briefly vortexed and further incubated for 2 hours, 2μl of each would then be used in corresponding PCR screens.

PCR, genotyping and sequence analysis

Oligonucleotide primers would be synthesised by methods known in the art. PCR would be performed using 0.2μL

Platinum® Taq DNA Polymerase (Gibco BRL®) , 2μL of yeast culture (DNA), 2.5μL of 1OX buffer (Gibco BRL®), lμL of 50 mM MgCl₂ (Gibco BRL®), lμL of dNTPs (5mM) , 2.5μL of each primer pairs - (Table 1) [2mM] , made up to 25μL. PCR would be performed using a Perkin Elmer, Gene Amp PCR system

9700. Cycling conditions would be an initial denaturation of 94⁰C for 2 min followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C; concluding with a final extension of 72°C for 5 min. Three PCR reactions would be . performed on each sample, each reaction using a different primer pair (Table 1) .

PCR products would be analysed by electrophoresis in ethidium bromide stained (0.5ug ml^"1) 1.0% agarose gels. A 100 bp ladder molecular weight standard (Roche) would be used to estimate PCR fragment size.

PCR products would be purified using a montage PCR filter •device (Millipore Corporation) . Sequence reactions would be performed using BigDye Terminator version 3.1 (Applied Biosystems) , and the completed reactions purified by ethanol precipitation. The reaction products would be analysed on an Applied Biosystems 3730 Genetic Analyser. Sequence alignment would be performed using ChromasPro version 1.15 (Technelysium Pty Ltd, www.technelysium.com.au/ChromasPro.html)> the sequence in Figure 27 would be used as the standard wild type sequence.

Primers and primer design

Oligonucleotide primers would be designed using Primer Premier Version 5.0 (Premier Biosoft International, Palo Alto, CA) .

Examples of the primers that could be used to sequence across the entire BAD gene in Saccharomyces cerevisiae are shown in Table 3 below.

Table 3. primers for PCR and sequencing BAD gene in Saccharomyces cerevisiae.

Analysis of 2AP by Gas Chromatography

Isolation and analysis of 2AP by Gas Chromatography would be performed according to the methods of Munch, P and Schieberle (1998) Quantitative Studies on the Formation of Key Odorants in Thermally Treated Yeast Extracts Using Stable Isotope Dilution Assays. Journal of Agricultural Food Chemistry. 46, 4695-4701.

Prophetic Example 3

Materials and Methods for the Production of Transgenic Wheat with BAD2 RNAi

Design of RNAi insert

The RNAi insert would come from the 5' end of the wheat BAD2 cDNA. This region shows 76.8 percent homology to the same region in the BADl gene homologue. In this example, the RNAi insert is designed such that the transgenic plants show specific interference of BAD2 without interference of the BADl homologue. (Figure 28)

PCR, genotyping and sequence analysis

Oligonucleotide primers would be synthesised by methods known in the art. PCR would be performed using 0.2μL Platinum® Taq DNA Polymerase (Gibco BRL®) , 2μL cDNA , 2.5μL of 1OX buffer (Gibco BRL®), lμL of 50 mM MgCl₂ (Gibco BRL®), lμL of dNTPs, (5mM) , 2.5μL of each primer pairs - (Table 1) [2mM] , made up to 25μL. PCR would be performed using a Perkin Elmer, Gene Amp PCR system 9700. Cycling' conditions would be an initial denaturation of 94°C for 2 min followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C; concluding with a final extension of 72°C for 5 min. Three PCR reactions would be performed on each sample, each reaction using a different primer pair.

PCR products would be purified using a montage PCR filter device (Millipore Corporation) . Sequence reactions would be performed using BigDye Terminator version 3.1 (Applied Biosystems), and the completed reactions purified by ethanol precipitation. The reaction products would be analysed on an Applied Biosystems 3730 Genetic Analyser. Sequence alignment would be performed using ChromasPro version 1.15 (Technelysium Pty Ltd, www.technelysium.com.au/ChromasPro.html), the sequence in Figure 28 would be used as the standard wild type sequence.

KNA extraction and cDNA synthesis

RNA would be extracted using a Qiagen RNeasy extraction kit and cDNA synthesised using Roche cDNA synthesis kit and a anchored poly T primer.

BNAi Transgenic Plants Method

(The following process is based on the work by Loukoianov et al. ^' ^Regulation of VRN-I Vernalization Genes in Normal and Transgenic Polyploid Wheat' Plant Physiol..2005; 138: 2364-2373.)

The RNAi construct would be made in the binary vector pMCGlβl. This vector contains a cassette designed for making inverted repeat transcripts of a gene, flanking a loop, which should efficiently produce a double-stranded RNA. Expression of the transgene is driven by the 35S promoter followed by the Adhl intron. A 245-bp segment from BAD2 would be cloned in the sense orientation between restriction sites Ascl-Avrll and in antisense orientation between restriction sites SgfI-Spel . Immature embryos of Wheat variety ^Banks' would be transformed .with the vector/RNAi. construct by microprojectile bombardment as described in Okubara et al. , (2002) Theor. Appl. Genet. 106 (1) : 74-83. 3 mg/L bialaphos would be added to shoot regeneration and rooting media to select the transformants .

Positive transgenic plants would be confirmed by PCR of genomic DNA using primers Fvectorl and Rvectorl designed from the vector sequence flanking the sense and antisense insertions (Yan et al. , 2004b) and by Southern blot using a probe for the 35S promoter. Transcription of the transgene in the selected Tl plants and positive T2 progeny would be confirmed by RT-PCR using primers OCS- PolyA_F and Ri_AntiS_R for the transcribed region of the OCTOPINE SYNTHETASE PoIyA region of the pMCGlβl vector (Yan et al, 2004b) . Transcription levels of the endogenous BAD2 would be investigated using RT-PCR with primers FBAD2RNAU (5' CACATCAATGGAGATTTGGAGGGA 3') (SEQ ID NO: 40) and RBAD2RNAU (5' AAAGCCGCTGCGCTTGTTCC 3') (SEQ ID NO: 41) .

Assay for 2AP in Wheat Plants

The concentration of 2-acetyl-l-pyrroline (2AP) in wheat leaves would be determined by grinding approximately 5 g of leaf material under nitrogen followed by incubation in 20 ml pure ethanol (99.9%) for 24 h at room temperature [Natta Laohakunjit and Athapol Noomhorm (2004) Flavour and Fragrance Journal 19: 251-259 Supercritical carbon dioxide extraction of 2-acetyl-l-pyrroline and volatile components from pandan leaves] .

The supernatant would be filtered in preparation for GC-MS analysis. Extracts would be analysed in duplicate, using a HP 5890 Series II GC/HP 5972 mass selective detector (MSD) (Hewlett-Packard, California, US.) fitted with a capillary column (Innowax, 25 m x 0.2 mm i.d., 0.4 μm film thickness; Agilent Technologies, CA) . A 2 μl solution of the extract would be injected for analysis. Oven temperature would be held at 50 ⁰C for 2 min, then programmed to increase from 50 ⁰C to 170 °C at 7 °C/min and would be held at 170 ⁰C for 5 min. Other operating conditions would be as follows: injector temperature, 170 ⁰C; carrier gas, helium at a flow rate of 0.6 ml/min; ion source temperature, 230 ⁰C; electron multiplier voltage, 2600 V. The samples would be injected in the splitless mode. Compounds would be tentatively identified by matching their mass spectrometric data with those obtained from the same equipment. Quantitative determination of 2AP in extracts performed by using measurements of peak area of m/z (mass/charge) 41(50), 43(100), 55(2), 67(0.2), 68(8), 83(11), 111(5), with the aid of the instrument's digital . integrator. Correlating peak areas with concentrations would be performed by means of a standard calibration curve obtained between 10 and 200 ng/injection. To minimize errors an external standard would be used, with 2, 4, 6-trimethylpyridine (TMP) (Sigma Aldrich, St. Louis, MO) as the external standard in quantitative analysis of 2AP.

Prophetic Example 4

Method for mutagenesis of rice and detection by TILLING to produce fragrant rice due to elevated concentrations of 2- aσetyl-1-pyrroline

A population of Ml rice families (5000 -10,000 individuals) would be generated by ethylmethanesulfonate (EMS) mutagenesis. Seeds would be mutageneised by- immersion in a 20 mM EMS solution for 18 hours. The Ml plants would be self fertilized to produce an M2 population of 5000 -10,000 individuals. Genomic DNA would be isolated from the leaf material derived ^'from bulks of five M2 individuals using Qiagen^® MagAttract™ 96 chemistry applied to the MWG Biotech TheOnyx liquid handling robot. Exons of the BAD2 encoding gene would be amplified by polymerase chain reaction (PCR) using the 5' FAM labeled primers;

Fl, 5' TTGATTGTGGGAAGCC 3' (SEQ ID NO: 42), Rl, 5'

GCATTAACACGGAGGAG 3' (SEQ ID NO: 43) F2, 5' TTTTGATGTGCCCTCT 3', R2 (SEQ ID NO: 44), 5'

ACCAGTTTCATAACTCCC 3' (SEQ ID NO: 45)

F3, 5' GGTGCTCCTTTGTCATC 3' (SEQ ID NO: 46) R3, 5'

TCCTAACTGCCTTCCTT 3' (SEQ ID NO: 47)

F4, 5' TGCCAACTGAGTAAAGAA 3' (SEQ ID NO: 48) R4, 5' TGGTCAGGAGCAAGAA 3¹ (SEQ ID NO: 49)

F5, 5' TTGCACAGAGCGAATA 3' (SEQ ID NO: 50) R5, 5^?

GACAAGATAAACCTACGG 3' (SEQ ID NO: 51) F6, 5' TAGTCGGTGTATGCTCTT 3' (SEQ ID NO: 52) R6, 5' AAACAATGCCAACCC- 3' (SEQ ID NO: 53)

F7, 5' CTGGTGCTGTGCTTTC 3' (SEQ ID NO: 54) R7, 5' AGTCCATCCCGTCATAC 3' (SEQ ID NO: 55) F8, 5' TCCAAGCTGTAATGTAAT 3' (SEQ ID NO: 56) R8, 5' TAACCAATGCCGATG 3' (SEQ ID NO: 57)

Primers would be synthesised by methods known in the art.

PCR would be performed with a Perkin Elmer, Gene Amp PCR system 9700. The PCR volume would be 10 μl containing 10

^• ng of extracted genomic DNA, 1.5 mM 200 μM total dNTPs,

0.5 unit of Platinum^® Taq DNA. Polymerase (Gibco BRL^®) , Ix

Gibco^® PCR Buffer (minus MgCl₂) and 0.2 μM each of the forward and reverse primer. Cycling conditions would be 5 min at 94⁰C followed by 35 cycles of 94⁰C for 30 s, 55⁰C for 30 s and 72 ⁰C for 30 s followed by a final extension of 72 ⁰C for 7 min.

Post PCR, PCR products would be incubated for 15 minutes at 45 ⁰C following the addition of 20 μl CEL I reaction mix (2.4 ml water, 420 μl 10x CEL I buffer (100 mM MgSO₄, 100 m M 4- (2hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , pH 7.5, 100 mM KCl, 0.2% Triton^® X-100, 2 μg/mL bovine serum albumin), 36 μl CEL I extract) [Till, B.J., Colbert, T., Tompa, R., Enns, L.C, Codomo, CA. , Johnson, J.E., Reynolds, S.H., Henikoff, J.G,, Greene, E.A., Steine, M.N., Comai, L., Henikoff, S. (2003) High- Throughput TILLING for Functional Genomics. Methods in Molecular Biology, Vol. 236: Plant Functional Genomics: Methods and Protocols. p205-220] The reaction would be terminated by the addition of 5 μL 150 mM EDTA, pH 8.0, and loaded on an Applied Biosystems 3730 DNA Analyzer (Perkin-Elmer Applied Biosystems, Forster City, CA) . Mutants would be identified by the appearance of peaks in the chromatogram that corresponded to cleaved PCR products. Upon identification of mutant pools, genomic DNA from individuals within the pool would be PCR amplified and sequenced using BigDye Terminator cycle sequencing, (Perkin-Elmer Applied Biosystems, Forster City, CA) and the products analysed by a Applied Biosystems 3730 DNA Analyzer (Perkin-Elmer Applied Biosystems, Forster City, CA) .

Homozygous M2 mutant individuals would be grown to maturity and leaf material collected for determination of 2AP concentration. Heterozygous M2 mutant individuals would be grown to maturity, and the M3 seed collected. The M3 seeds would be germinated and homozygous mutant individuals identified by sequencing. Upon identification of homozygous mutant M3 individuals, leaf material would be collected for determination of 2AP concentration.

The concentration of 2-acetyl-l-pyrroline (2AP) in rice leaves would be determined by grinding approximately 5 g of leaf material under nitrogen followed by incubation in 20 ml pure ethanol (99.9%) for 24 h at room temperature ^• [Natta Laohakunjit and Athapol Noomhorm (2004) Flavour and Fragrance Journal 19: 251-259

Supercritical carbon dioxide extraction of 2-acetyl-l- pyrroline and volatile components from pandan leaves] . The supernatant would be filtered in preparation for GC-MS analysis. Extracts would be analysed in duplicate, using a HP 5890 Series II GC/HP 5972 mass selective detector (MSD) (Hewlett-Packard, California, US.) fitted with a capillary column (Innowax, 25 m * 0.2 mm i.d., 0.4 μm film thickness; Agilent Technologies, CA) . A 2 μl solution of the extract would be injected for analysis. Oven temperature would be held at 50 ⁰C for 2 min, then programmed to increase from 50 ⁰C to 170 ⁰C at 7 °C/min and held at 170 ⁰C for 5 min. Other operating conditions would be as follows: injector temperature, 170 ⁰C; carrier gas, helium at a flow rate of 0.6 ml/min; ion source temperature, 230 °C; electron multiplier voltage, 2600 V. The samples would be injected in the splitless mode. Compounds would be tentatively identified by matching their mass s'pectrometric data with those obtained from the same equipment. Quantitative determination of 2AP in extracts performed by using measurements of peak area of m/z (mass/charge) 41(50), 43(100), 55(2), 67(0.2), 68(8), 83 (11) , 111 (5) , with the aid of the instrument's digital integrator. Correlating peak areas with concentrations would be performed by means of a standard calibration curve obtained between 10 and 200 ng/injection. To minimize errors an external standard would be used, with 2, 4, 6-trimethylpyridine (TMP) (Sigma Aldrich, St. Louis/ MO) as the external standard in quantitative analysis of 2AP.

REFERENCES

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14. Goff, S.A., Ricke, D., Lan, T.H. et al A draft sequence of the rice genome (Oryza sativa L ssp japonica) Science 296, 92-100 (2002) . 15. Li, Q.-L., Gao, X.-R., Yu, X.-H., Wang, X.-Z. & An, L.J. Molecular cloning and characterization of betaine aldehyde dehydrogenase from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco. Biotechnology Letters 25, 1431-1436 (2003) .

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Claims

CLAIMS :

1. A method of increasing production of fragrance by an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ^' ID NO: 1, the method comprising reducing or eliminating the activity of the functional protein in the organism.

2. A method of producing an organism which produces a fragrance, the method comprising the steps of:

(a) providing an organism capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1;_. (b) reducing or eliminating the activity of the functional protein in the organism.

3. The method of claim 1 or 2 wherein the functional protein comprises the amino acid sequence EG(C or G)RLG(S or P)V(V or I)S.

4. The method of any one of claims 1 to 3 wherein the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P.

5. The method of any one of claims 1 to 4 wherein the functional protein comprises the amino acid sequence (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC, wherein X is any amino acid and n is an integer from 25 to 30.

6. The method of any one of claims 1 to 5 wherein the functional protein comprises the amino acid sequences (V or I or L) (S or T or A)LELGGK(S or N)P(X)_nC and EG(C or G)RLG(S or P)V(V or I)S, wherein n is an integer from 25 to 30.

7. The method of any one of claims 1 to 6 wherein the functional protein has an amino acid sequence that is at least 40% identical to the amino acid sequence of SEQ ID NO: 1,

8. The method of any one of claims 1 to 7 wherein the functional protein has an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1.

9. The method of any one of claims 1 to 8 wherein the functional protein has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1.

10. The method of any one of claims 1 to 9 wherein the functional protein has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

11. The method of any one of claims 1 to 10 wherein the functional protein has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

12. The method of any one of claims 1 to 11 wherein the functional protein is the protein encoded by the BAD2,

BADH2, BADH15, BADHl, BAD, BADl, BBD, BBDl or BBD2 gene.

13. The method of any one of claims 1 to 12 wherein the organism is selected from the group consisting of plants, fungi, yeast and bacteria.

14. The method of any one of claims 1 to 13 wherein the organism is a plant.

15. The method of claim 14 wherein the plant is selected from the group consisting of cereal crop plant, legume, and oilseed.

16. The method of any one of claims 1 to 13 wherein the organism is a fungus.

17. The method of claim 16 wherein the fungus is selected from the group consisting^' of Aspergillus sp., Penicillium sp. and Roquefort sp.

18. The method of any one of claims 1 to 13 wherein the organism is a yeast..

19. The method of claim 18 wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Yarrawia lipolytica.

20. The method of any one of claims 1 to 13, wherein the organism is a bacteria.

21. The method of claim 20 wherein the bacteria is selected from the group consisting of Staphylococcus xylosus, Escherichia coli, Bacillus subtilus, Bacillus cereus, Lactococcus delbrueckii, Lactococcus lactis, Lactobacillus casei, Lactobacillus delbrueckii and Leuconostoc sp.

22. The method of any one of claims 1 to 21 wherein the activity of the functional protein is reduced or eliminated by inhibiting the activity of the functional protein.

23. The method of any one of claims 1 to 21 wherein the activity of the functional protein is reduced or

^■eliminated by reducing or eliminating the ability of the organism to express the functional protein.

2'4. The method of claim 23 wherein the ability of the organism to express the functional protein is reduced or eliminated by introducing into the cells of the organism a nucleic acid molecule that is capable of reducing or eliminating expression of the functional protein.

25. The method of claim 24 wherein the nucleic acid molecule is selected from the .group consisting of ssDNA, ssRNA, dsDNA, dsRNA and ribozyme.

26. The method of claim 25 wherein the nucleic acid molecule is an anti-sense molecule or a co-suppressor molecule.

27. The method of claim 26 wherein the anti-sense molecule is selected from the group consisting of anti-sense RNA, anti-sense DNA, interference RNA (dsRNA, iRNA, siRNA, hpRNA or ihpRNA) and ribozyme.

28. The method of claim 23 wherein the ability of the organism to express .the functional protein is reduced or eliminated by introducing into one or more genes encoding the functional protein a mutation which reduces or eliminates expression of the functional protein.

29. The method of claim 28 wherein the mutation is selected from the group consisting of an insertion, a deletion and a substitution.

30. The method of claim 28 or 29 wherein the mutation is in the coding sequence of the gene.

31. The method of claim 28 or 29 wherein the mutation is in the non-coding sequence of the gene.

32. The method of any one of claims 1 to 31 wherein reducing or eliminating the ability of the organism to express the functional protein results in increased production by the organism of one or more compounds selected from the group consisting of 2-acetyl-l- pyrroline, 2- (1-ethoxyethenyl) -1-pyrroline and 2-acetyl- 1,4,5, 6-tetrahydropyridine.

33. A method of establishing whether an organism is capable of producing fragrance, comprising determining whether an organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

34. A method of producing an organism which produces fragrance, which comprises the steps of: (a) identifying one or more parent organisms which comprise at least one mutant fgr gene;

35. The method of claim 34 wherein the organisms are plants.

36. A method of producing a plant which produces fragrance, which comprises the steps of: (a) identifying one or more parent plants comprising at least one mutant fgr gene;

(b) crossing two of the at least one parent plants to produce progeny plants;

(c) selecting one or more progeny plants that are homozygous for the mutant fgr gene to thereby provide a plant which produces fragrance.

37. The method of claim 36 wherein the- one or more parent organisms may be heterozygous for the mutant fgr gene.

38. An organism produced by the method of any one of claims 2 to 32 or 34 to 37.

39. A method for producing fragrance comprising incubating an organism of claim 38 under conditions which permit production^' of the fragrance.

40. The use of a gene that encodes a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1, for producing an organism that produces fragrance.

41. The use of a mutant fgr gene, for producing an organism that is capable of producing fragrance.

42. A nucleic acid molecule capable of reducing or eliminating expression of a functional protein in an organism, the functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

43. A fragrant molecule produced by the method of claim 39.

44. The fragrant molecule of claim 43, which is 2-acetyl- 1-pyrroline.

45. Use of a nucleic acid molecule capable of hybridising to an fgr gene or a mutant fgr gene of an organism for determining whether the organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

46. A nucleic acid molecule capable of hybridising to an fgr gene or a mutant fgr gene of an organism when used for determining whether the organism is capable of expressing a functional protein having an amino acid sequence that is at least 30% identical to the amino acid sequence of SEQ ID NO: 1.

47. A nucle^'ic acid molecule capable of distinguishing between an fgr gene and a mutant fgr gene of an organism.

48. The nucleic acid molecule of claim 47, wherein the nucleic acid molecule is capable of hybridising to the fgr gene but not to a mutant fgr gene.

49-. The, nucleic acid molecule of claim 47, wherein the nucleic acid molecule is capable of hybridising to a mutant fgr gene but not the fgr gene of an organism.