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WO2008074069A1 - Pigmentation du mouton - Google Patents

Pigmentation du mouton Download PDF

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Publication number
WO2008074069A1
WO2008074069A1 PCT/AU2007/001954 AU2007001954W WO2008074069A1 WO 2008074069 A1 WO2008074069 A1 WO 2008074069A1 AU 2007001954 W AU2007001954 W AU 2007001954W WO 2008074069 A1 WO2008074069 A1 WO 2008074069A1
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Prior art keywords
agouti
ovine
animal
sheep
gene
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Inventor
Belinda Jane Norris
Vicki Anne Whan
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Commonwealth Scientific and Industrial Research Organization CSIRO
Australian Wool Innovation Ltd
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Commonwealth Scientific and Industrial Research Organization CSIRO
Australian Wool Innovation Ltd
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Priority claimed from AU2006907055A external-priority patent/AU2006907055A0/en
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Publication of WO2008074069A1 publication Critical patent/WO2008074069A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to sheep pigmentation and, in particular, to methods of identifying sheep that are heterozygous carriers of the undesirable, recessive self-colour black, badgerface and reverse badgerface, otherwise known as "black and tan", alleles.
  • the agouti signalling protein encoded by the agouti gene is expressed by epithelial keratinocytes and binds to the melanocortin 1 receptor (MClR) on the surface of pigment producing melanocytes in the skin and hair/wool follicle.
  • the agouti protein has been demonstrated to be a competitive antagonist of MClR, inhibiting the binding of melanocyte stimulating hormone ( ⁇ MSH) and causing a switch from production of eumelanin (black/brown) to phaeomelanin (yellow/red) pigment.
  • agouti alleles have spontaneously arisen in mice with mutations in both the coding_and regulatory ⁇ regions ' affecting agouti expression and function and subsequent coat pigmentation patterns.
  • the agouti gene has also been linked to coat colour in other mammalian species including domestic dogs, cats and horses as well as the rat and the fox.
  • horse, rat and fox recessive black coat colour alleles have been identified as nucleotide deletions, creating a truncated or predicting a non-functional agouti protein (Eizirik et al 2003, Rieder et al 2001, Kuramoto et al 2001, Vage et al 1997) .
  • In recessive non-agouti black German Shepherd dogs a SNP causing an Arg-to-Cys amino acid substitution in the agouti protein is responsible for the phenotype (Kerns et al 2004) .
  • agouti protein In mice, pleiotropic effects have implicated the agouti protein as having an important biological role in obesity, diabetes, tumor growth and embryonic lethality.
  • Wild sheep similar to other wild mammalian species are generally tan-bodied with pale bellies. Darker areas, present as dorsal body, head and leg stripes, are usually more common in males and become more extensive with age. This wild-type colour is very rare in domestic sheep.
  • the dominant white/tan (A wt ) agouti allele is responsible for the white wool phenotype in modern sheep breeds while the most recessive allele, non-agouti (A a ) results in eumalanic (black/brown) wool.
  • a b Another recessive agouti allele badgerface (A b ) is characterised by a pale dorsal, phaeomelanic and darker ventral eumelanic pattern and is dominant to A a .
  • the frequency of the recessive non- agouti also known as self-colour
  • a a gene in the Australian wool flock has been estimated at 0.03 (Hayman and Cooper 1965) . With ⁇ 6% of the flock expected in this case to be carriers this is a relatively high frequency of such an undesirable allele.
  • the inventors To identify the genetic cause of white coat colour in the domestic sheep the inventors have characterised the ovine agouti gene structure.
  • the inventors' analysis of the ovine agouti locus of domestic sheep shows its organisation to be different to that in other domestic species such as mouse, rat, dog, cow and pig, with a duplication of a large portion of the ovine genome responsible for the dominant white A wt allele.
  • the duplication occurs 5' of the ASIP gene coding sequence within the predicted promoter region and involves about 190Kb of DNA sequence encompassing the ASIP and AHCY coding sequences and the promoter region of the ovine ITCH gene.
  • Ubiquitous expression of an ASIP coding sequence in one copy is regulated by a duplicated ITCH promoter.
  • the inventors have found that a single copy ASIP gene with a silenced ASIP promoter has occurred in recessive black sheep.
  • the inventors have further found that white Merinos have at least one duplicated agouti allele while all of the recessive black Merinos only contain single copy agouti alleles.
  • the inventors' data indicates that all ASIP expression in the white sheep was driven from the duplicated copy of an ITCH promoter positioned upstream of the duplicated ASIP gene.
  • the inventors have identified a number of dominant white alleles at the ovine ASIP locus .
  • the inventors have analysed the ovine gene sequence and determined that a unique sequence exists at the boundary between the duplicated copies and termed this sequence the ' junction point' . This sequence is present in alleles containing two, three, four or more copies of ASIP. This sequence can thus be used as a DNA marker to identify animals that contain alleles which have multiple copies of ASIP.
  • the present invention provides a method of identifying a heterozygous carrier of a recessive black allele in an ovine animal whose white colouration is a result of ubiquitous, dominant expression of a duplicate agouti gene under the control of the itch promoter, comprising the steps of:
  • the present invention provides a method of reducing undesirable pigmentation characteristics in a flock of sheep comprising identifying animals that are heterozygous carriers of a single copy recessive black allele and removing the heterozygous carriers from the flock, comprising the steps of:
  • the invention provides a method of identifying a heterozygous carrier of a single copy recessive black allele in an ovine animal whose white colouration is a result of ubiquitous, dominant expression of a duplicate agouti gene under the control of the itch promoter, comprising the steps of:
  • the present invention provides a method of reducing undesirable pigmentation characteristics in a flock of sheep comprising identifying animals that are heterozygous carriers of a single copy recessive black allele and removing the heterozyjgous carriers from the flock, comprising the steps of:
  • the present invention provides a method of identifying a heterozygous carrier of a single copy recessive black allele in an ovine animal whose white colouration is a result of ubiquitous, dominant expression of a duplicate agouti gene under the control of the itch promoter, comprising the steps of:
  • the present invention provides a method of reducing undesirable pigmentation characteristics in a flock of sheep comprising identifying animals that are heterozygous carriers of a single copy recessive black allele and removing the heterozygous carriers from the flock, comprising the steps of:
  • the present invention provides a method of identifying a single copy recessive black allele in an ovine animal, comprising measuring the level of agouti expression in the ovine animal and correlating the level of expression to phenotype .
  • the present invention provides a method of identifying a single copy recessive black allele in an ovine animal, comprising measuring the level of agouti expression in the ovine animal and correlating the level of expression to genotype (i.e. number and arrangements of Junction points) .
  • the present invention provides a method of identifying an ovine animal which expresses an agouti gene, comprising the steps of:
  • the present invention provides a method of identifying an ovine animal which expresses an agouti protein, comprising the steps of: 1. providing a nucleic acid from the ovine animal; and
  • the presence or absence of duplicate agouti genes is established by determining the presence or absence of duplicate agouti alleles.
  • the nucleic acid sample is derived from a diploid cell of the ovine animal.
  • the nucleic acid sample is derived from a haploid cell of the ovine animal.
  • sperm or ova of a single sperm typically 10 to 100, are isolated and an ovine animal without a single duplicate in about 50% of the single sperm or ova tested is identified as a heterozygous carrier .
  • invention ⁇ provides Ii method of identifying an ovine animal which expresses an agouti protein, comprising the steps of: 1. providing a nucleic acid from the ovine animal; and
  • the presence or absence of one or more agouti genes under the control of the ITCH promoter is established by determining whether the genome of the animal comprises one or more duplicate agouti genes, whereby an ovine animal with one or more duplicate agouti genes is identified as an animal which expresses the agouti protein.
  • an isolated nucleic acid molecule selected from the group consisting of sequences set forth in SEQ ID NOs: 21 to 31, 35, 36 and 38 to 41.
  • nucleotide sequence is as set forth for BAC clone CH243-373J16 (SEQ ID NO: 21 or 26), CH243- 45504 (SEQ ID NO: 22 or 27) or CH243-160L8 (SEQ ID NO: 23 or 28) in Fig 9 or Fig 10, the 143 bp sequence highlighted therein (SEQ ID NO: 29, 30 or 31) and set forth in Fig 11 and polymorphic forms of said sequences.
  • the present invention relates to polymorphisms within 2000bp of the 143 bp highlighted in Fig 9 and Fig 10 and set forth in Fig 11.
  • a method of establishing a relationship between a polymorphism and/or haplotype and the genotype of ovines at the agouti locus comprising the steps of:
  • the method further comprises genotyping each ovine animal in the agouti locus. Thereafter it comprises deducing a relationship between a polymorphism and/or haplotype and copy number of the agouti gene which, in an embodiment, itself be used as the basis of a test for identifying heterozygous carriers.
  • Fig 1 Schematic figure of alleles proposed at the agouti locus.
  • the wild type ancestral ASIP allele is believed to be single copy with an ancestral functional ASIP promoter controlling expression (A+) .
  • A+ ancestral functional ASIP promoter controlling expression
  • Dominant white (A wt ) has three alleles proposed, all with a duplication and with or without polymorphisms in one ASJP copy proposed to be the non-functional copy because of either the polymorphisms in the coding sequence (D5 and A) , and/or mutation (s) in the ASIP promoter.
  • agouti alleles were identified for each of the recessive black pigmentation phenotypes of self-colour black (A a ) and badgerface (A b ) . Except for the wild type allele (A+) , the agouti promoter is proposed to be non- functional .
  • FIG. 1 Schematic figure summarising the interpretation of the BAC clones sequence data.
  • the proposed wild type (A+) single dose agouti allele is shown in (i) .
  • the approximate site of the 5 1 and 3 1 duplication breakpoints are shown by arrows.
  • the insertion site (junction point) of the duplicate copy could be proximal, at the 5 1 breakpoint (ii) OR distal at the 3" breakpoint (iii) , indicated by dashed arrows.
  • An enlarged 190kb duplicated region (iv) shows the approxijrnate_pojfitions__oJL "the ASIP (open boxes IB - IE) and ITCH (open boxes It, It' and IA) non-coding exons.
  • Fig 3 Schematic figure showing the structure of the duplicated ASIP allele and the position of Ovine BAC clones used to characterise the ASIP allele.
  • Clones #164- INRA, #218-INRA, and #229-INRA were from the INRA Romanov Sheep BAC library and clones CH243-160L8, CH243-234K21, CH243-45504, CH243-373J16, CH243-489F15 from the CHORI BACPAC Texel sheep BAC library.
  • the INRA BAC clones did not span the Junction, 5' or 3' breakpoints.
  • Clones CH243- 45504 and CH243-489F15 spanned the junction between DNA copies; clone CH243-160L8 spanned the 3' breakpoint and clone CH243-373J16 the 5' breakpoint.
  • Fig 4 (a) . Sequenom Assay Primers Agt309, Agt307, Agt308 and the Extension primer are used in the Sequenom iPLEX allelotyping assay and the amount of C (Junction point) or A (5 1 Breakpoint) is determined. b. ABI3130xl Assay Primers Agt309, Agt307gtg and
  • Agt308Fam are used for capillary electrophoresis on ABI 3130x1.
  • Agt309 and Agt308Fam span the 5 1 breakpoint and produce a 238bp product; and Agt307gtg and Agt308Fam span the junction point and produce a 242bp product.
  • the area under each peak is calculated using GeneMapper software (Liz 600 as size standard) .
  • the quantification of junction points for both assays is calculated as the ratio of junction point products to 5 1 breakpoints products.
  • the forward primers (Agt307, Agt307gtg and Agt309) are used in 100 fold excess to the competitively shared common primer Agt308(for Sequenom) or Agt308Fam (for the ABI 3130x1 assay)
  • Fig 7. Gene expression transcripts for agouti and ITCH exons in sheep skin and internal tissue samples.
  • Fig 10 Extension of sequence shown in Fig 9 from Ovine BAC clones CH243-373J16 (SEQ ID NO: 26), CH243-45504 (SEQ ID NO: 27) and CH342-160L8 (SEQ ID NO: 28) . Junction and breakpoint sequences span ⁇ 1000bp either side of the 'common' ⁇ 143bp sequence identified in Fig 9. These sequences have been aligned in Fig 11 to show 15 polymorphisms identified from these three BAC clones sequences .
  • SNP polymorphisms
  • FIG. 12 Schematic diagram showing, possible ⁇ ,
  • Homozygous white genotypes and C: Heterozygous carrier genotypes. The ratio of junction point to 5 1 breakpoint + junction point is shown on the right for each genotype.
  • Fig. 13A is a graph showing the results of asymmetric competitive PCR using an ABI-3130x1 Genotyping assay on known Merino heterozygous carriers of self-colour black or badgerface (Lanes 1 to 8) and random white Merinos of unknown carrier status (Lanes 9 to 16) .
  • Fig. 13B is a graph showing the results of asymmetric competitive PCR using a Sequenom iPlex assay on known Merino heterozygous carriers of self-colour black or badgerface (Lanes 1 to 8) and random white Merinos of unknown carrier status (Lanes 9 to 16) .
  • Fig. 13C is a graph showing the results of asymmetric competitive PCR using TaqMan Gene Copy assay on known Merino heterozygous carriers of self-colour black or badgerface (Lanes 1 to 8) and random white Merinos of unknown carrier status (Lanes 9 to 16) .
  • Fig. 14 is a graph showing the results of screening of commercial sheep for the presence of 1, 2, 3, or 4 junction points. The number of junction points is indicated on the X-axis.
  • Fig. 15 depicts an alignment of DNA sequence of the junction point from CH243-45504 (SEQ ID NO: 38), the 5' breakpoint from CH243-373J16 (SEQ ID NO: 39) and the 3' breakpoint from CH243-373J16 (SEQ ID NO: 40) illustrating the difference at the junction point, 5' breakpoint and 3' breakpoint. The beginning and end of the ⁇ 143bp region is marked with arrows. The flanking sequences that are unique to the 5' breakpoint and 3' breakpoint are in bold.
  • the " desirable dominant white A wt allele has been identified as a tandem duplication on sheep chromosome 13 affecting the agouti (ASIP) , AHCY and ITCH gene regions .
  • the duplication results in the presence of two copies of the ASIP and AHCY gene coding sequences and places a second copy of the ITCH promoter at the 5 ' end of a ASIP gene coding sequence.
  • the gene duplication therefore puts a functional agouti gene under the control of a ITCH gene promoter in domestic white sheep. This gene duplication does not affect the ITCH gene expression but leads to ubiquitous expression of the agouti gene resulting in the dominant white, A wt phenotype.
  • the inventors believe that at least one duplicated copy agouti allele with a functional ASIP gene under the control of a functional ITCH promoter is required for the white sheep phenotype.
  • the recessive A a and A b alleles occur when there is only a single non-functional agouti gene copy.
  • These single copy recessive black alleles could result from non-allelic pairing and gene recombination between duplicated copy alleles.
  • Single copy recessive black alleles include self-colour black, badgerface and reverse badgerface alleles.
  • the present inventors have identified the gene duplication breakpoints, junction points and mutations within the agouti gene coding sequence are expected to be responsible for the recessive self-colour black phenotypes of Merino and other domestic sheep breeds.
  • the high sequence identity between the two agouti copies is consistent with the duplication being a recent event following sheep domestication.
  • the identification of two non-synonymous mutations in the coding sequence; a 5bp deletion and an A/T SNP, and a third potential functional mutation; a 9bp deletion removing three amino acids of the signal sequence suggests a more ancient duplication.
  • a single gene copy is present in cattle # therefore the duplication has occurred following_the split of the Caprinae and the Bovinae.
  • the presence or absence of a junction point and/or the copy number is measured by determining the number of junction point PCR products to
  • 5' breakpoint products 5' breakpoint products. Suitable methods for measurement of the number of junction and break point products are well known to the person skilled in the art. In an embodiment an asymmetric competitive PCR method and detection with ABI3130xl or sequenom iPLEX or Taqman syber green or probe assays is used.
  • a sample is obtained from a white sheep and DNA extracted and a determination made of the: a) presence or absence of a junction point b) number of copies of the junction point and, optionally. c) differences between 5 1 breakpoint sequence and junction point sequence as set forth in Fig 11 and ratio of same; d) differences between 3 ' breakpoint sequence and junction point sequence as set forth in Fig 11 and ratio of same; e) presence or absence of the A substitution for T (A/T SNP) in exon 4 f) number of copies of the A and T alleles in exon 4 g) presence or absence of a 5bp deletion h) number of copies of the 5bp deletion i) presence or absence of a 9bp deletion j ) number of copies of the 9bp deletion.
  • the sample may be any sample from the sheep from which genomic DNA may be obtained.
  • suitable samples include blood, sperm, oocyte or skin.
  • a Taqman assay may be used for genotyping SNP such as the A/T SNP that will determine both presence and relative dose.
  • SNP genotyping
  • Sequenom iPLEX Sequenom iPLEX
  • pyroseguencing etc technology
  • the presence or absence of the changes which alter the size of the molecule such as the 5bp deletion can be determined by PCR and the size of the band on an capilliary sequencer or acrylamide gel or by sequence analysis of the PCR product. In an embodiment this is assayed quantitatively using a standard PCR genotyping assay.
  • the relative dose of a deletion such as the 5bp or the 9bp (D 9 ) deletion is determined from the area under the normal allele and D 5 or D 9 allele peaks using Genemapper.
  • Other methods for measuring presence/absence and dose of the 5bp deletion include genotyping, Sequenom iPLEX and pyrosequencing.
  • the presence of a junction point is determined by detecting amplification of a sequence comprising the sequence represented by SEQ ID NO: 30 using primers which hybridise to sequence which flank the sequence from position 991 to 1133 of SEQ ID NO: 27 ( ⁇ 143bp region of CH243 -45504, Figure 10) .
  • the junction point may be detected using primers Agt307 (or Agt307gtg) and Agt308 (or Agt308Fam) in a PCR reaction and detecting the amplification of a product (see Figs. 4 a and b and 13A) .
  • the presence of a junction point is determined by detecting the presence of a sequence comprising a sequence selected from the group consisting of SEQ ID NO: 22, 24, 27, 30, 35 and 38.
  • the copy number of the agouti gene is established through a PCR-based analysis. Quantification of the copy number may be achieved by amplification of the junction point and a corresponding non-duplicated sequence (or the 5 1 or 3 1 breakpoints) in a competitive PCR assay.
  • This approach relies on the fact that a tandem duplication has one unique DNA sequence not present on non-duplicated alleles, namely the junction point, which is also referred to herein as the duplication break point.
  • the sequences around the junction point and the 5' and 3' break points for the agouti duplication in domestic sheep is shown in Figs 9 10 and 11, and based on this primers have been developed for a junction point copy number assay.
  • the copy number of the junction points may be established by determining the relative proportion of junction points to 5' breakpoints.
  • the relative proportion of junction points to 5' breakpoints may be determined, for example, by determining the proportion of PCR product amplified using primers specific for the junction point (eg. Agt307gtg and Agt308Fam) relative to the PCR product amplified using primers specific for the 5' breakpoint (eg. Agt309 and Agt308Fam) .
  • a common forward or reverse primer is used in conjunction with a specific opposite primer for either the normal 5' or 3' breakpoint copy or the junction point copy and a quantitative assay is then used to quantify the two copies based on a relative proportion of diagnostic nucleotides within the break point and junction point products.
  • the copy number of the junction point can be determined by detecting the incorporation of C relative to A onto a primer having sequence 5 ' GAGTCGGACACAACAGAG 3 1 (SEQ ID NO: 41) (complement of SEQ ID NO: 18) (see Fig. 4a) .
  • the proportion of C to A will give the proportion of junction points to 5 1 breakpoints.
  • the quantitative assay is pyrosequencing, in which a shared sequencing primer initiates DNA synthesis and the relative quantities of the different nucleotides added at a point of difference between the two sequences is measured.
  • an Sequenom iPLEX assay may be employed to determine copy number, the Sequenom iPLEX assay differs from the existing hME genotyping assay in that all reactions for the Sequenom iPLEX assay are terminated after a single base extension and these/base extension products are detected using mass spectrometry based upon mass differences between the different bases added jin the normal, and
  • the quantitative assay may also be minisequencing, taqman PCR, invader, illumina, radioisotope incorporation and gel based densitometry.
  • the ⁇ 143 bp sequences in common across the junction and breakpoints is highlighted in bold in Fig 9 & 10, and listed again in Fig 11.
  • the sequence of the ⁇ 143bp region of: CH243-160L8 is represented in SEQ ID NO: 31; CH243-373J16 is represented in SEQ ID NO: 29; CH243-45504 is represented in SEQ ID NO: 30.
  • this sequence is in common, there are polymorphisms in this and the flanking sequences that we have identified and further analysis may also identify new polymorphisms. Any polymorphisms including SNP in the x common 7 sequence could be used in an assay to determine the presence of gene copy of this sequence. Any polymorphisms or alternate primer sequences in the flanking sequences could also be used in an assay to determine the presence or copy number of the flanking sequence.
  • an animal having a single duplicate of the agouti gene is established by determining that the genome of the animal has a single junction point.
  • an animal having a non- duplicated allele of the agouti gene is established by determining that the genome of the animal has a single duplicate of the agouti gene.
  • an animal having a non-duplicated allele of the agouti gene is established by determining that the genome of the animal has a single junction point.
  • an animal is determined to have a single junction point by determining the proportion of junction points to 5 1 breakpoints, whereby the genome of the animal has a single junction point if the proportion of junction points to 5' breakpoints is about 0.5 or less.
  • the proportion of junction points to 5 ' breakpoints is determined using a TaqMan_jLSsay,.
  • an animal is determined to have a single junction point by determining the proportion of junction points to junction points and 5 1 break points, whereby the animal has a single junction point if the proportion of junction points to junction points and 5 ' break points is greater than 0 and less than 0.5, typically less than 0.4, and still more typically about one third.
  • the proportion of junction points to junction points and 5 ' breakpoints is determined using a genotyping or Sequenom iPLEX assay.
  • An animal which expresses the agouti gene may be identified by establishing whether the genome of the animal comprises an agouti gene under the control of the ITCH promoter.
  • the presence of an agouti gene under control of the ITCH promoter is established by determining the presence of a duplicate of the agouti gene. Typically, by determining the presence of one or more junction points of the agouti gene.
  • the number of duplicate agouti genes is established.
  • the number of duplicate agouti genes may be determined by determining the number of junction points of the agouti gene.
  • the number of junction points will equal the number of duplicates of the agouti gene.
  • an animal having two duplicates of the agouti gene will have two junction points.
  • an animal is determined to have one or more junction points by determining the proportion of junction points to 5' breakpoints, whereby the genome of the animal has one or more junction points if the proportion of junction points to 5 ' breakpoints is greater than zero, typically 0.5 or more.
  • the proportion of junction points to 5 ' breakpoints is determined using a TaqMan assay.
  • an animal is determined to have one or more junction points by determining the . proportion of junction points to 5" breakpoints, whereby the genome of the animal has two or more junction points if the proportion of junction points to 5 1 breakpoints is about 1.0 or more.
  • the proportion of junction points to 5 1 breakpoints is determined using a TaqMan assay.
  • an animal is determined to have one or more junction points by determining the proportion of junction points to 5" breakpoints, whereby the genome of the animal has three or more junction points if the proportion of junction points to 5" breakpoints is about 1.5 or more.
  • the proportion of junction points to 5' breakpoints is determined using a TaqMan assay.
  • an animal is determined to have one or more junction points by determining the proportion of junction points to junction points and 5 ' breakpoints, whereby the genome of the animal has one or more junction points if the proportion of junction points to junction points and 5 ' breakpoints is greater than zero, typically 0.3 or more.
  • the proportion of junction points to junction points and 5' breakpoints is determined using a genotyping or Sequenenom iPLEX assay.
  • an animal is determined to have one or more junction points by determining the proportion of junction points to junction points and 5' breakpoints, whereby the genome of the animal has two or more junction points if the proportion of junction points to junction points to 5' breakpoints is 0.5 or more.
  • the proportion of junction points to junction points and 5' breakpoints is determined using a genotyping or Sequenenom iPL ⁇ X assay.
  • an animal is determined to have one or more junction points by determining the proportion of junction points to junction points and 5 # breakpoints, whereby the genome of the animal has three or more junction points if the proportion of junction points to junction TM points and 5" breakpoints is 0.6 or more.
  • # the proportion of junction points to junction points and 5' breakpoints is 0.6 or more as determined using a genotyping or Sequenenom iPLEX assay.
  • the ovine animal is a domestic sheep ⁇ Ovis Aries) , more particularly, one of the wool breeds of which the major breed is Merino or one of the meat breeds of which the major breeds are Dorset, Border Leicester, Coopworth, Suffolk, White Suffolk, Southdown Texel, Romanov, Wiltshire Horn, Wiltipoll or one of the meat and wool breeds of which the major breeds are the Leicester Long wool Perendale, Polwarth, Romney, Coopworth and Drysdale.
  • the DNA panel consisted of samples from 94 baldy self- colour black sheep (from 28 different producers) ; 48 spotted self-colour black sheep (from 16 producers) ; 48 badgerface animals (from 12 producers) ; 87 white carrier animals (57 confirmed to be carriers by commercial pedigree testing and 30 suspected carriers) from a total of 20 different producers; and 96 random white animals (of unknown carrier status) from 22 producers.
  • RNA was prepared from the postnatal and adult skin of white and recessive black Merino sheep using Trizol (Invitrogen) in accordance with the manufacturer's recommendation. In each extraction, 5 ml of Trizol was used to " extract 200-500 mg of skin. To ensure removal of contaminating DNA, total RNA was treated with Dnasel (Ambion DNA-free Austin, TX, USA) . KNA quality was visually assessed using agarose gel electrophoresis and a UV transilluminator and quantified by spectrophotometry.
  • PCR primers for the amplification of intron sequences from genomic DNA templates were designed from sheep ASIP EST sequence. PCR primers used are shown in Table 4. PCR primers were designed to amplify intron 1 regions between non-coding exon IE and coding exon 2 (Agtl and Agt2) , intron 2 regions between exon 2 and exon 3 (Agt3 and Agt4) and intron 3 regions between exons 3 and 4 (Agt5 and Agt ⁇ ) .
  • PCR reactions (20 ⁇ l) contained 50 ng gDNA, 200 ⁇ M dNTP's, 10 pmole of each primer, I x Q solution, 1.5 mM MgCl 2 0.5 units Taq polymerase (Qiagen) and 1 x reaction buffer (Qiagen) .
  • PCR conditions were 94 0 C for 3 min; (94 0 C for 30 s, 57 0 C for 1 min and 72 0 C for 2 min) for 35 cycles; and 72 0 C for 5 min.
  • PCR products were amplified from a white merino ram and a self-colour black merino lamb. PCR products were cloned using a TOPO TA Cloning Kit (Invitrogen) and sequenced using Big Dye
  • Terminator 3.1 reaction mix PCR products spanning exons 2
  • the three INRA Romanov Sheep BAC library clones (Vaiman et al . ) were obtained from the BAC-YAC Resource centre of the Animal Genetics Department of INRA.
  • Shotgun plasmid libraries were prepared by the Australian Genome Research Facility (AGRF, http://www.agrf.org.au/) from each of three INRA ovine BAC clones INRA-164H8, INRA-218G7 and INRA-229C6 DNA which had tested positive for ASIP coding sequence by PCR.
  • Clones INRA-164H8 and INRA-218G7 were sequenced to 8x coverage and clone INRA-229C6 to 2x coverage by the AGRF. Sequence data was imported into Sequencher v4.2 (Gene Codes) fo . r_ analysis. " Clones ⁇ NRA-218G7 ( ⁇ 75kb) and INRA-229C6
  • Clone CH243-373J16 was predicted to provide sequence data further 5' and clone CH243-160L8 further 3 1 of the INRA-164H8 sequence (Fig. 3).
  • Clones CH243-234K21 and CH243-373J16 contained the putative nonfunctional x A f ASIP allele.
  • These five clones of the 29 CHORI Texel sheep BAC clones - CH243-455O4, CH243-489F15, CH243-373J16, CH243-160L8 and CH243-234K21 were selected for further analysis.
  • the ovine ASIP gene By sequencing the ASIP gene from Merino sheep, we determined the genomic organisation of the ovine ASIP coding exons 2, 3 & 4 and introns 2 and 3 to be similar to that reported for the bovine, human and mouse genes (see Figure 2) . Each coding exon was flanked by a consensus splice donor and acceptor site.
  • the ovine ASIP gene encodes a putative 133 amino acid protein which is 98%, 76% and 74% identical to the bovine (133aa) , mouse (131aa) and human (132aa) proteins, respectively.
  • Primer Agt9 (positioned in exon It) and primer Agt6 (in the 3' UTR of the agouti gene) were used to amplify cDNA derived from the skin of white sheep. These products were cloned and sequenced and seven alternative transcripts were obtained. Significant differences were evident for the sequences of the first ovine__nc>n ⁇ codiiig_ ⁇ ex ⁇ ns compared to those reported for cow, mouse and pig. In addition to one or more non-coding exons designated IA to IE (Fig. 3), every ovine transcript also had two non- coding exons which we have labelled It and It ' .
  • the non- coding exons It and It 1 were also part of the 5 1 UTR of the neighbouring downstream ITCH gene (Fig. 3) .
  • a terminal G nucleotide on the sequence of ASIP transcripts derived by 5" RACE determined exon It to be the most 5' non-coding exon.
  • the D 5 deletion would result in a premature stop codon 60 amino acids downstream of the start site, truncating the agouti protein before the functionally important cysteine signalling domain (amino acids 91-130) .
  • the T to A SNP would predict a change of cysteine (amino acid 123) to serine which would disrupt the highly conserved signalling region of the protein.
  • the D 9 deletion would result in the loss of an SRL tripeptide which may affect the function of the ASIP transport leader sequence but not the remainder of the protein, while the synonymous G5060C SNP would not be expected to disrupt ASIP function.
  • Haplotypes 1 and 2 were determined by cloning and sequencing PCR products from the genomic DNA of a white (A wt ) and a self-colour black (A a ) merino sheep. The white animal was heterozygous for haplotypes 1 and 2 and the self-colour black animal was homozygous for haplotype 2.
  • Ovine ASIP haplotypes 3 and 4 were determined by sequencing of INRA BAC clones INRA- 218G7 and INRA-164H8 from Romanov sheep.
  • Haplotypes 1, 2, 3 and 4 are also designated N 9 N 5 T (non-deleted and T allele), N 9 D 5 T (5bp deletion and T allele) D 9 N 5 T (9bp deletion and T allele) and N 9 N 5 A (non-deleted and A allele) respectively.
  • Transcripts of both N 9 N 5 T and D 9 N 5 T haplotypes could be predicted to produce functional agouti protein while N 9 D 5 T and N 9 N 5 A haplotype transcripts would be predicted to produce non-functional agouti protein. Using haplotype 1 (Table.
  • haplotype 4 (N 9 N 5 A) with a nucleotide change every 117 bp is quite different from haplotypes 2 and 3 that differ once every 1076 bp and 1793 bp respectively.
  • ⁇ 9 bp INDEL may affect the function of the ASIP transport leader sequence
  • Table 5 Merino, Romanov, and Texel sheep haplotypes for a 5379 bp sequenced region of the ovine ASIP gene spanning from Exon 2 to the 3' UTR. Sequence identity to the master sequence (haplotype 1) is shown with a dash; a colon represents a deletion.
  • the Merino sheep haplotypes were determined from the PCR, cloning and DNA sequencing of genomic DNA from a white and a recessive self-colour black Merino sheep.
  • the Romanov sheep haplotypes were determined from the DNA sequencing of three BAC clones; INRA-218G7, INRA-229C6 and INRA-164H8.
  • the Texel sheep haplotypes were determined from the DNA sequences of five BAC clones; CH243-234K21, CH243-373J16, CH243- 160L8CH243-489F15, and CH243-455O4.
  • the 5 bp INDEL was identified previously in white but not black- faced sheep breeds (Smit et a.1. ) .
  • the T5181A SNP of the Romanov and Texel haplotypes 4 and 5 was genotyped in a coat colour DNA panel of 373 Merino sheep. x Association analysis of the D 5 INDEL and A/T SNP with recessive black Merino phenotypes
  • Sequence data from clones CH243-455O4, CH243-234K21 and CH243-489F15 spanned the junction between the tandem duplicated copies (Fig. 3).
  • BAC clone CH243-160L8 spanned the 3 1 breakpoint and clone CH243-373J16 spanned the 5' breakpoint.
  • Haplotypes 5 and 7 in Table. 5 were identified from the Texel sheep BAC clone sequences.
  • Haplotype 5 was designated ASIP copy 1 as it was positioned 5' to haplotype 6 and haplotype 7 both designated ASIP copy 2 (Fig. 3) .
  • Haplotypes 6 and 7 were identified from the Texel sheep BAC clone sequences.
  • Haplotype 5 was designated ASIP copy 1 as it was positioned 5' to haplotype 6 and haplotype 7 both designated ASIP copy 2 (Fig. 3) .
  • Haplotype 5 (ASIP copy 1) could be derived from recombination and/or gene conversion between Texel sheep equivalents of haplotypes 1 and 4. Both of the Texel sheep haplotype 6 clones (ASIP copy 2) shared two nucleotides (at positions 4133 bp and 5060 bp) with haplotype 5 (ASIP copy 1 ) but were otherwise identical to haplotype 7 (ASIP copy 2) . Haplotype 5 also had an A nucleotide at position 3096 bp not present in any other haplotypes (Table 5) .
  • ASIP geii ⁇ expression was assessed in various tissues of a white and a recessive black merino sheep.
  • RNA quality was visually assessed using agarose gel electrophoresis and a UV transilluminator and quantified by spectrophotometry.
  • RT-PCR analysis of skin and internal tissues from a white and a recessive black Merino sheep are shown in Fig. 7.
  • the same alternatively spliced forms of ovine ASIP were expressed in liver, kidney, heart, spleen and skin tissues tested from white Merinos but were not amplified from any tissues of a self-colour black Merino (Fig.7 a-f) .
  • ASIP ubiquitous expression has also been reported in humans
  • D 9 alleles were preferentially expressed in Merino skin as visualised by the band intensities (Fig. 15) .
  • the D 5 allele was not amplified by competitive RT-PCR and was considered comparatively lowly expressed or not expressed at all (Fig. 15 Lanes 1-9) .
  • ASIP expression was detected in all tissues examined from white sheep with multiple transcripts identified from both the skin and internal tissues. Expression was not detected from any tissues of recessive self-colour black sheep. Further, all transcripts identified from white sheep skin began with the ITCH non- coding exons It and It 1 and none contained the 5bp or T5173A predictive non-functional mutations. This data indicated that all expression in the white sheep was driven from the duplicated copy of an ITCH promoter positioned upstream of the ASIP gene and that the identified functional mutations are not present in these expressed ASIP gene copies. The data also indicated that ther ⁇ progenitor ASIP gene promoter was probably not functional. Because ASIP expression was not detected in the single copy alleles of the recessive black sheep, the likely causative mutation of the recessive black phenotype is an as yet unidentified regulatory mutation of the progenitor gene promoter region.
  • the expressed (copy 2) ASIP functional coding sequence under strong selection pressure, the two ASIP regions subsequently diverged in sequence generating functional and non- functional haplotype clusters.
  • assays were developed for determining the presence of the gene duplication and for determining relative copy numbers of the gene duplications.
  • a 5 ⁇ l PCR reaction volume was established containing 5-50ng ovine gDNA template, 0.5 mmol/L dNTPs, 10 pmole of Forward primers Agt309 and Agt307gtg, 0.1 pmole of common reverse primer Agt308Fam, 3 mmol/L MgCl 2 and 0.625 ⁇ l of 1OX reaction buffer (Qiagen Hot Star Taq) .
  • PCR conditions were 95°C for 15 min, 40 cycles of 95°C for 30s, 60 0 C for 30s and 72°C for lmin and 1 cycle of 72°C for 5 min.
  • junction point the unique point where the 5' end of one copy meets the 3' end of the second copy
  • 5 1 breakpoint were cloned into pCR2.1- TOPO ® .
  • a dilution series of plasmid DNA containing the junction point from 0% to 100% mixed with decreasing
  • junction point and A (5' breakpoint) are incorporated in an iPLEX extension reaction and the ratio C/C+A is used to calculate the number of ASIP copies in the genome ( Figure 5B) .
  • the sequence of the PCR product generated from amplification of the junction point in clone CH243 -45504 using primers Agt307 and Agt308 is represented in SEQ ID NO: 35.
  • the sequence of the PCR product generated from amplification of the 5' breakpoint in clone CH243-373J16 using primers Agt309 and Agt308 is represented in SEQ ID NO: 36.
  • a TaqMan Gene Copy assay can be used to quantify the number of copies of a DNA sequence relative to a reference DNA sequence using a standard curve method (Applied Systems Bulletin #2) .
  • a dilution series of a single calibrator DNA sample was used to produce standard curves for each PCR product of the junction point and a single copy reference sequence using primers Agt308 and Agt307 (junction points) and primers Agt308 and Agt309 (5' break point) .
  • a white sheep DNA sample independently determined to have two junction
  • the calibrator sample was used as the calibrator sample.
  • the 5' breakpoint sequence was assumed to be single copy in the ovine genome and was used as the reference (two alleles) sequence.
  • Another single copy ovine gene, WARS (Tryptophanyl-tRNA synthetase) was also used to validate the assay. All assays were performed in quadruplicate.
  • the standard curve (amount of genomic DNA (ng) vs C ⁇ value) was used to determine the amount of junction point and 5' breakpoint DNA in 16 test samples.
  • All Merino sheep have two 5' breakpoint sequences in their genomes, one on each chromosome. Sheep will cluster into groups depending on the ratio of 5' breakpoints to junction points in their genomes. For example, sheep with no ASIP genomic duplications will have two 5' breakpoints, no junction points and will cluster at 0% (diamonds in Fig. 6 and Fig. 12A) . Sheep with two 5' breakpoints and one junction point will cluster in the 33% group (represented by the triangles in Fig. 6 and Fig. 12C) ; and those sheep with two 5' breakpoints and two junction points will cluster in the 50% group (Fig. 12B and C) .
  • the DNA sequence at the 5 ' breakpoint was used in the three assays described to represent a single copy product at the ASIP locus.
  • the DNA sequence at the 3 1 breakpoint of the ASIP locus or of any known ovine single copy gene sequence could also be used to represent a single copy product in similar assays designed to measure gene copy number.
  • the abovementioned three assays were used to assess DNA samples from sixteen white Merino sheep (Fig. 13A, 13B and 13C) . Eight of the sheep were confirmed heterozygous carriers of self-colour black or badgerface alleles via pedigree analysis and the other eight Merino sheep were randomly sampled white animals DNA samples.
  • Fig. 13A, 13B and 13C Results of the analysis of the DNA sample from the 16 sheep using the three assay systems referred to above are shown in Fig. 13A, 13B and 13C. All three assays gave the same estimates of the gene copy number for each animal sample. Six (grey columns) of the sixteen animals were confirmed as carriers with one duplicate allele (3 gene copies) . All three assays also showed that five of the eight known carriers (by pedigree analysis) had 2 junction points (see Fig. 12C, 50% junction points) .
  • Example 4 ABI 3130x1 Junction point copy number analysis of commercial sheep breeds. i Preparation of gDNA
  • Samples of blood were collected from Australian sheep including; 86 white carriers (i.e white sheep with one black allele) Dams or Sires of Merino recessive black lambs (confirmed by pedigree analysis) ,
  • DNA from whole blood was visually assessed using agarose gel electrophoresis and a UV transilluminator and quantified by spectrophotometry.
  • the ABI 3130x1 assay was used to assess the number of junction points per genotype of the DNA samples from the white sheep of unknown carrier status and the 86 confirmed white carriers of self-colour black or badgerface alleles.
  • the proportion of sheep with one, two three or four junction points and thus correspondingly 3, 4, 5 or 6 copies of the 190 Kb region bound by the duplication breakpoints (Fig 2 & 3) are shown in Fig. 14.
  • Approximately 55% of the known carriers were confirmed as heterozygous carriers of a single duplicated allele (Fig 14, 1 junction point) .
  • Carrier sheep with a two (or more) junction point alleles are not distinguished from homozygous white animals which may also have two or more junction points ( Figure 14 and see also Fig 12 carrier types with two or three junction points) .
  • the copy number assay identifies single copy junction point carrier sheep and further can determine the total number of copies of the 190kb region per genotype (Fig 14) .
  • the assay has been successfully used to confirm the presence of at least one dominant white duplicated allele in a wide range of commercial white sheep breeds including; Merino, Dorset, Border Leicester, Coopworth, Suffolk, White Suffolk, Southdown, Texel, Romanov cross, Wiltshire Horn, Whiltipoll, Leicester long wool, Perendale, Polwarth, Romney, Coopworth and Drysdale.
  • the test is thus expected to identify single junction point carriers of any commercial white sheep breed.
  • the test can be applied to the ram lamb portion of a stud flock to identify potential future carrier sires. Alternatively the test can be applied to stud flock sires and dams and the results used to make mating decisions to help avoid producing multiple junction point carriers, single junction point carriers or black lambs.
  • Incorporating a targeted strategy of genotyping into the breeding strategy together with progeny testing will help reduce carriers.
  • a breeding company can screen the alleles of each generation of animals to ensure that those selected to breed contain the required genotypes. Such a screening program will reduce the frequency of undesirable pigmentation causing alleles and thus reduce the incidence of pigmented and carrier lambs born.
  • the assay allows for the screening of a haploid cells; individual sperm or ovum.
  • Semen can be collected either by ejaculation or surgically and single sperm, 20-100 cells, can be collected from fresh or " frozen samples by catapulting single sperm heads or other appropriate isolation methods would include smears or isolation and purification of single sperm from semen using density gradient separation, and expertise to pick up individual sperm by micromanipulation under a phase-contrast microscope.
  • Each individual sperm is delivered into a PCR tube containing lysis solution and after incubation PCR buffer added.
  • PCR assays are performed for the junction point and 5' breakpoint sequences in a single or nested PCR reaction and a carrier animal is indicated if approximately 50% of the single sperm samples which amplify a 5' breakpoint PCR product do not also amplify a junction point PCR product.
  • isolation of ⁇ 10-20 individual ova, lysis and junction point and 5' breakpoint PCR assays indicate a carrier ewe if approximately 50% of the single ova samples which amplify a 5' breakpoint PCR product do not also amplify a junction point PCR product.
  • the assay can be conducted at or soon after birth enabling the detection of germ-line recombination events that generate undesirable single copy recessive black alleles.
  • genotyping of offspring of a white sheep with a negative test result is performed, particularly for a high value animal such as a stud ram.
  • Carrier sheep with a two (or more) junction point alleles are not distinguished from homozygous white animals which may also have two or more junction points. Therefore, for example, a triplicated agouti gene in a suspect ram may be detected and distinguished from an animal homozygous for the agouti duplication by creating embryos with semen from the ram and, for example, a homozygous black ewe. The embryos are genotyped and, if the sire is a homozygote for
  • the result will be that 50% of the embryos are carriers (Aa) and 50% are white (AA) .
  • At least two super- ovulated ewes are used to produce around 18 embryos when artificially inseminated. The embryos are flushed from the ewe at between 6-10 days gestation and the DNA extracted from individual embryos for genotyping.
  • Table 4 PCR primers used for characterising the ASIP locus and for analysis of expression of the ASIP and ITCHgenes.
  • the itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a 18H mice. Nat. Genet. 18: 143-146.

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Abstract

L'invention concerne un procédé d'identification d'un vecteur hétérozygote d'un allèle noir récessif chez un animal ovin dont la coloration blanche est une conséquence de l'expression dominante ubiquitaire d'un gène agouti double sous le contrôle du promoteur de la gale, comprenant les étapes consistant à : 1. fournir un échantillon d'acide nucléique d'un animal ovin; et 2. établir le nombre de doubles du gène agouti chez l'animal ovin par analyse de l'échantillon d'acide nucléique, moyennant quoi un animal ovin présentant un double unique du gène agouti est identifié comme vecteur hétérozygote.
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CN113278712A (zh) * 2021-07-23 2021-08-20 中国农业大学 分析绵羊毛色的基因芯片、分子探针组合、试剂盒及应用
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CN117721215A (zh) * 2023-12-19 2024-03-19 中国科学院西北高原生物研究所 一种用于选育青海藏羊毛色品系的试剂盒和方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103740825A (zh) * 2014-01-08 2014-04-23 中国农业科学院兰州畜牧与兽药研究所 一种控制绵羊毛色Agouti基因拷贝变异检测试剂盒
CN113278712A (zh) * 2021-07-23 2021-08-20 中国农业大学 分析绵羊毛色的基因芯片、分子探针组合、试剂盒及应用
CN113278712B (zh) * 2021-07-23 2021-11-09 中国农业大学 分析绵羊毛色的基因芯片、分子探针组合、试剂盒及应用
CN115851983A (zh) * 2022-11-24 2023-03-28 山东省土地发展集团有限公司 基因芯片及绵羊的snp位点组合在分析绵羊生长相关性状中的应用
CN117721215A (zh) * 2023-12-19 2024-03-19 中国科学院西北高原生物研究所 一种用于选育青海藏羊毛色品系的试剂盒和方法

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