CN102870670B - Universal type breeding method for rice engineering maintainer line, and application thereof in propagation of ordinary nucleic male sterility lines of rice - Google Patents

Abstract

The invention discloses a method for cultivating a universal engineering maintainer line of rice, which comprises the following steps: constructing a binary expression carrier of a color marker gene C; adopting a transgenic method to transfer the carrier into a variety whose genotype is SS to obtain a transgenic line; and then Obtain homozygous lines by selfing; analyze the insertion site of gene C in each homozygous line, and screen and construct a candidate library for engineering maintainer lines; combine the rice common male sterile line with genotype ss with the candidate The homozygous lines in the library were crossed to obtain F ₁ , and F ₁ was self-propagated to obtain F ₂ , and the uncolored seeds in F ₂ were cultivated, and the number of sterile plants in each group of F ₂ lines after cultivation was investigated. The F ₁ seeds from F ₂ with the number of sterile plants accounting for more than 98% were screened out as engineering maintainer lines. The cultivation method of the invention has the advantages of short period, strong versatility, high efficiency, etc., and the cultivated engineering maintainer line can be applied in the mechanized and large-scale propagation of common nuclear male sterile lines of rice.

Description

A kind of breeding method of general-purpose rice engineering maintainer line and its application in the propagation of rice general male sterile line

technical field

The invention belongs to the technical field of reproduction of crop sterile lines, in particular to a method for the propagation of male sterile lines of self-pollination crops (such as rice, wheat, etc.).

Background technique

Using the male sterility of plants to breed male sterile lines, and then use this genetic tool to produce hybrid seeds in large quantities, can make the heterosis of many crops, especially self-pollination crops, be used in production.

According to the three-type theory, male sterility in plants is divided into three genetic types: cytoplasmic male sterility, nuclear male sterility and cytoplasmic male sterility.

Cytoplasmic male sterility means that the sterility traits are only controlled by cytoplasmic genes and have nothing to do with the nucleus. The sterility traits purely controlled by cytoplasmic genes have not been applied in production because there is no restorer line.

Nuclear male sterility means that the sterility traits are only controlled by nuclear genes and have nothing to do with cytoplasm. It is divided into dominant nuclear sterility and recessive nuclear sterility. Most of the nuclear sterility that has been found so far is recessive sterility.

Cytoplasmic-nuclear interaction male sterility is jointly controlled by cytoplasmic genes and nuclear genes. It not only maintains the sterility of the maintainer line, but also restores the fertility of the F1 hybrid with the help of the restorer line. This type of sterility The breeding line, the maintainer line and the restorer line form a three-line set, which is the most classic and mature method in production at present, that is, the three-line method. However, the breeding procedures and production links of the three-line method are relatively complicated, so that the cycle of breeding new combinations is long, the efficiency is low, the promotion links are many, and the speed is slow. At the same time, the cost of seeds is high and expensive.

Among the types of nuclear male sterility, recessive genic sterility is divided into environment-sensitive genic sterility and common genic sterility according to whether the sterility is sensitive to environmental factors.

Environmentally sensitive genic male sterility mainly refers to photothermosensitive genic male sterility. Its fertility is not only controlled by the genic male sterile gene, but also regulated by the length of light and temperature outside. Fertility, short days and low temperature lead to fertility, with the characteristics of fertility conversion, one line can be used for two purposes, and one line is reduced compared to three lines, which is often called the two-line method. In addition to not requiring a maintainer line, the photothermosensitive male sterile line is easy to breed and has higher efficiency. It also has: 1) a wide recovery spectrum, and almost all normal varieties of the same subspecies can restore their fertility to normal; 2) genetic behavior Simple, sterility is controlled by 1 to 2 pairs of recessive genes, easy to transfer and stable, and conducive to the cultivation of various types of sterile lines. However, it should also be pointed out that since the fertility of the photothermosensitive sterile line is regulated by external light and temperature, if the light and temperature changes are not within the control range during the breeding process of the photothermosensitive male sterile line, it will lead to a reduction in the production of the photothermosensitive male sterile line or even fail.

Ordinary nuclear sterility is generally not affected by external environmental factors. No matter how the environmental conditions change, it always shows sterility. This type of sterility is relatively common in nature. Compared with plasmogenetic-nuclear interactive sterility and light-temperature-sensitive genic male sterility, common genic male sterile has the following characteristics: 1) Since it is only controlled by a pair of recessive nuclear genes, and the fertility is not affected by the environment, it is easy to breed The sterile rate and sterile plants are 100% common GMS lines; 2) The varieties with normal fertility are its restorer lines, the restoration spectrum is extremely wide, and the freedom of combination makes the probability of breeding excellent combinations great 3) It can keep up with the pace of conventional rice breeding, open up a new field of heterosis between indica and japonica subspecies, and make rice yield higher than that of existing hybrid rice. Therefore, common GMS lines are rich in sources, relatively simple in breeding, stable in fertility, and easy to restore. They are ideal sterile materials. Fertility conversion and reproduction are very difficult, which has become a key factor restricting its large-scale production and utilization.

At present, the hotspots of studying common GMS lines are focused on the location and cloning of GMS genes. For example, CN1884541A Chinese Patent Document (Application No. 200610027399.8) discloses a "protein code for controlling the degradation of rice tapetum". Sequence", in which Zhang Dabing et al. obtained a common nuclear sterility gene TDR of rice through map-based cloning, and found that the protein encoded by the gene controls the fertility of pollen by controlling the degradation of the rice tapetum through functional analysis. There are few reports in the existing literature on the large-scale propagation and application of common GMS lines to actual production. At present, common GMS lines mainly use backcrossing and asexual reproduction to reproduce offspring. For example, the Chinese patent document No. CN102121052A records In order to create and propagate recessive genetic male sterile lines in rice by backcrossing using molecular marker assisted selection. Chinese patent document No. CN101946715A records the use of microspore culture and asexual cloning techniques to select and propagate a recessive male sterile line of Brassica napus. In addition, it has been reported in the literature that common GMS lines of rice can also be propagated through regeneration, and common GMS lines of cotton can be propagated by cuttings. Through the above method, scientific researchers can breed a small amount of common CMS lines for scientific research and experiments, but the number of common CMS lines obtained by breeding is rare and requires a lot of manpower and material resources. If the problem of large-scale reproduction of common GMS can be solved, the large-scale application of common GMS to hybrid seed production will greatly release the utilization potential of heterosis.

As more and more research institutions and scientific researchers realize the huge utilization value and potential of the common GMS line, many research institutions have recently increased their research investment and research on the large-scale reproduction of the common GMS line. efforts, and has made gratifying progress. For example, the Hunan Hybrid Rice Research Center has achieved success by locating and cloning the sterile gene s and the fertile gene S to construct an engineering maintainer line to propagate the common GMS line, but this method requires cloning The sterile gene s and the fertile gene S increase the difficulty of the construction of the engineering maintainer line and greatly prolong the construction progress of the engineering maintainer line. Therefore, it is of great significance to explore a simple and versatile common GMS line breeding and creation method, which will promote the large-scale application of common GMS lines in production and further release the utilization potential of heterosis.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, provide a method for cultivating general-purpose rice engineering maintainers with simple steps, convenient operation, short cycle and high efficiency, and correspondingly provide a general-purpose rice engineering maintainer The application of the line in the propagation of the common genetic male sterile line of rice, which can realize the mechanized and large-scale breeding and production of the common genetic male sterile line of rice, and can greatly speed up the process from the selection of the common genetic male sterile line to the realization of the application .

In order to solve the above-mentioned technical problems, the technical solution proposed by the present invention is a method for cultivating a general-purpose rice engineering maintainer line, comprising the following steps:

(1) Prepare a rice general male sterile line whose genotype is ss, and s represents the sterility gene controlling the sterility traits of the rice general male sterile line;

(2) Construct the binary expression vector pC of the color marker gene C;

(3) Using existing transgenic methods (such as Agrobacterium-mediated method or biolistic transformation method) to transfer the binary expression vector pC into the source parent or fertility of the above-mentioned rice common male sterile line with genotype SS In normal varieties (S indicates the fertility gene S that is dominant to the sterile gene s), primers were designed according to the color marker gene, and T ₀ generation transgenic lines with the color marker gene C were obtained by PCR screening; then T ₀ The first generation of transgenic lines obtained homozygous lines with a single copy of the color marker gene through continuous self-propagation, and its genotype was SSCC (the genotype was verified by test crossing);

(4) Analysis of the insertion site of the color marker gene C in the rice genome in each homozygous line obtained in step (3) (the insertion site analysis method generally uses the conventional linker method or TAIL-PCR method), And according to the insertion position of the color marker gene C in the rice genome, a series of single-copy homozygous strain repertoires are screened out, so that the rice genome in the homozygous strain repertoire has a color marker gene C every 0~2cM , the homozygous line library constitutes an engineering maintainer candidate library;

(5) Cross the rice general male sterile line (ss) in step (1) with each homozygous line (SSCC) in the engineered maintainer candidate pool in step (4) to obtain _F1 heterozygous seeds , F ₁ heterozygous seeds were self-propagated in one generation to obtain F ₂ seeds of various genotypes, and then the seeds with the color marker gene C in the F ₂ seeds were first eliminated through the color sorting process, and the remaining seeds without the color marker gene C were removed. Group _F2 seeds were cultivated respectively to obtain corresponding _F2 strains of each group, the number of sterile plants in the aforementioned _F2 strains of each group was investigated, and the _F2 strain groups with the number of sterile plants accounting for ≥98% were screened out, and The F ₁ heterozygous seeds derived from the F ₂ line group were used as the engineered maintainer line (genotype s ^c S ^C ) of the common GMS line of rice.

In the above-mentioned cultivation method of the present invention, the engineering maintainer heterozygous seeds whose genotype is s ^c S ^C are confirmed according to the genetic linkage analysis of the color marker gene C and the fertility gene S in the _F2 generation, which is based on the following basic principles : Rice general male sterile line (ss) is crossed with each line in the engineering maintainer candidate library to obtain F ₁ heterozygous seeds, and F ₁ heterozygous seeds are self-propagated for one generation to obtain F ₂ seeds. After the _F2 seeds carrying the color marker gene are planted, the _F2 population is obtained, and the number of sterile plants in the _F2 population is investigated. If the number of sterile plants accounts for about 1/4, the analysis determines that the color marker C and the fertile gene S are located at different locations. On the chromosome; if the proportion of sterile plants is greater than 1/4 and less than 98%, the color marker C and the fertile gene S are located on the same chromosome, and the recombination rate between the color marker C and the fertile gene S is between 1% and 50% %; if the proportion of sterile plants is above 98%, it can be determined that the color marker C and the fertile gene S are located on the same chromosome, and the recombination rate between the color marker C and the fertile gene S is below 1%. Therefore, The F ₁ seeds from F ₂ with the number of retained sterile plants accounting for more than 98% are the engineered maintainer lines obtained in the present invention.

In the above-mentioned breeding method, the common genetic male sterile line of rice can be obtained through genetic selection, physical and chemical mutagenesis, genetic engineering and other various means or formal ways, preferably, the common genetic male sterile line of rice The sterility trait of is a recessive nuclear sterility trait controlled by a single gene, the fertility gene S is dominant to the sterility gene s, and the sterility trait is not affected by external environmental conditions. In the preferred scheme, since the sterility traits are only controlled by a pair of recessive nuclear genes, and the fertility is not affected by the environment, it is easier to select and breed common karyophytes with 100% sterile rate and sterile plants. education system.

In the above cultivation method, in the step (2), the binary expression vector pC is specifically preferably any one of pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301, pBI121 (especially preferably pCAMBIA1300 or pCAMBIA1390).

In the above breeding method, in the step (2), the color marker gene C can be expressed in the seeds, so that the seeds are marked with a color, so that the seeds with the color marker gene can be color-selected in subsequent applications. The color marker gene is preferably the red fluorescent protein gene DsRed , the red fluorescent protein gene RFP , the green fluorescent protein gene GFP , the green fluorescent protein gene EGFP or the blue fluorescent protein gene EBFP , wherein it is more preferably the red fluorescent protein gene DsRed or green Fluorescent protein gene EGFP . According to the expression site of the color marker gene C in the seed, an appropriate specific expression promoter can be selected. For example, the color marker gene C is expressed in the endosperm and the endosperm-specific expression promoter is selected. The endosperm-specific promoter has: P _Gt1 (GenBank: EU264103.1) and P _GluB1 (GenBank: AY427569.1 or GenBank: JN389781.1).

In the above breeding method, the sterile gene s is preferably msp1 , pair1 , pair2 , zep1 , mel1 , pss1 , tdr , udt1 , gamyb4 , ptc1 , api5 , wda1 , cyp704B2 , dpw , mads3 , osc6 , rip1 , csa or aid1 , the fertility gene S is the corresponding rice wild-type gene.

As a general technical concept, the present invention also provides an application of the general-purpose rice engineering maintainer line cultivated by the above method in the propagation of the common GMS line of rice. The engineering maintainer line is color-marked gene C and genotype It is a heterozygous seed of s ^c S ^C. The genetic background of the engineered maintainer line is the same as that of the common nuclear male sterile line (ss) of rice. The only difference is that the genome of the engineered maintainer line has the color marker gene C and the fertility gene S. In addition, the color marker gene C in the engineering maintainer line is closely linked with the fertile gene S (the genotype is verified by test crossing), and when the breeding application is performed, the engineering maintainer line is first combined with the rice common rice whose genotype is ss. The genetic male sterile line is crossed, and the seeds of the engineering maintainer line and the ordinary rice genetic male sterile line are obtained in the hybrid offspring (the ratio of the two is generally 1:1). At this time, the color marker gene C is specific to the engineering maintainer line seeds. Driven by a specific expression promoter, it is specifically expressed in the seeds of the engineered maintainer line, and the seeds of the engineered maintainer line can be sorted out by a color sorter, and the genotype of the engineered maintainer line selected by color sorting is s ^c S ^C The seeds of the rice line can continue to be hybridized with the common GMS line of rice whose genotype is ss, and the hybrid seeds whose genotype is s ^c S ^C and the common GMS seeds whose genotype is ss are propagated; the remaining seeds are Ordinary GMS seeds of rice with genotype ss do not contain genetically modified components, and can be crossed with restorer lines for hybrid seed production, and can also be crossed with heterozygous seeds with genotype s ^c S ^C for the production of their own materials reproduce and perpetuate.

Compared with the prior art, the present invention has the advantages of:

(1) Create an engineering maintainer line by transgenic means to propagate the common genetic male sterile line of rice, and the common genetic male sterile line of rice obtained by breeding does not contain genetically modified components; and the varieties with normal fertility are the recovery of the common genetic male sterile line of rice line, the recovery spectrum is extremely wide, and the combination is free, which greatly increases the probability of breeding excellent combinations; it can keep up with the pace of conventional rice breeding, open up a new field of heterosis between indica and japonica subspecies, and make rice yields higher than existing hybrid rice Based on the realization of higher production goals;

(2) The reproduction process is simple and the reproduction efficiency is high: the reproduction of the sterile line is not restricted by environmental conditions, and the seed purity is high; although there is one more engineered maintainer line than the two-line method, the engineered maintainer line does not require additional reproduction. At the same time as the common nuclear sterile line, the engineering maintainer line can be selected by machine; more importantly, there is no need to clone the sterile gene s and the fertile gene S, which greatly simplifies and speeds up the construction of the engineering maintainer line; The body has also been further optimized, from the transformation of the sterile line recipient to the transformation of the fertile line recipient to construct the engineering maintainer line, which further reduces the difficulty of transformation and the construction cost of the engineering maintainer line;

(3) The versatility is greatly improved: for any common sterility line at any sterility site, you can find a line from the engineering maintainer candidate library to make the color marker gene C correspond to the fertile site of the sterility site The genetic distance≤1cM, therefore, the method of the present invention greatly improves the versatility of engineering maintainer lines;

(4) Intelligent sorting, easy to operate: through machine intelligent sorting instead of manual control, it provides the possibility for large-scale mechanized seed production.

Description of drawings

Fig. 1 is the process flow chart of common GMS line breeding and seed production in the embodiment of the present invention.

Fig. 2 is the endosperm-specific green fluorescent protein expression vector pEGt constructed in the embodiment of the present invention.

Fig. 3 is a flow chart of screening engineering maintainer lines in the present invention.

Fig. 4 is an analysis diagram of the influence of gene recombination in the hybrid seed production of the engineered maintainer line and the common GMS line in the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

Example 1:

An application of the general-purpose rice engineering maintainer line of the present invention in the breeding and seed production of common rice male sterile lines, the specific object of its application is the common nuclear male sterile line of rice T98B, which specifically includes the following steps (for the process flow, see figure 1):

1. Breeding of common nuclear sterile lines: ⁶⁰ Co-γ-ray mutagenesis, a rice sterile mutant was obtained from rice T98B, and genetic analysis showed that the sterility trait was caused by a recessive single gene (ie, the sterility gene ) control, which belongs to a common GMS line of rice (genotype is ss ). We can also purchase common nuclear sterile mutants of rice from the mutant library RiceGE, website: http://signal.salk.edu/cgi-bin/RiceGE; common nuclear sterile genes applicable to the method of the present invention are as follows 1.

Table 1: List of rice general sterility mutants

nuclear sterile gene Proteins encoded by fertility genes corresponding fertility gene function NCBI accession number RiceGE mutant order number msp1 LRR receptor kinase LRRkinas early microspore development AB103395.1 PFG_3A-07135.R pair1 Coiled-coil domain protein homologous chromosome synapsis AB158462.1 FL009505 pair2 HORMA domain protein homologous chromosome synapsis AB109238.1 RMD_TTL-04Z11KJ91 zep1 Coiled-coil domain protein Synaptonemal complex formation during meiosis GU479042.1 PFG_1B-18520.U mel1 ARGONAUTE (AGO) family protein germ cell division before meiosis AB297928.1 RMD_03Z11CW42-2 pss1 Kinesin family protein Dynamic Changes of Male Gamete Meiosis BK007977.1 T07702T tdr bHLH transcription factor tapetum degradation AK106761.1 PFG_3A-08673.R udt1 bHLH transcription factor tapetum degradation AY953870.1 PFG_3D-00330.R gamyb4 MYB transcription factor Aleurone and anther development NM_001051127.1 PFG_1E-03950.R ptc1 PHD-finger transcription factor Tapetum and pollen grain development GU597363.1 RMD_02Z15CL49 api5 antiapoptotic protein 5 Delayed tapetum degradation NM_001053191.1 PFG_2C-50137.R wda1 carbon lyase Lipid synthesis and pollen grain exine formation AK100751.1 PFG_2D-01704.R cyp704B2 Cytochrome P450 gene family Anther sac and pollen exine development NM_001055627.2 T07707T dpw fatty acid reductase Anther sac and pollen exine development NM_001055618.1 RMD_05NPBMB39 mads3 homeotic class C transcription factors Late anther development and pollen development AK108568.1 FL059810 osc6 lipid transfer family protein Liposome and pollen exine development NM_001074690.1 M0033824 rip1 WD40 domain protein Pollen maturation and germination DQ491004.1 PFG_1D-01918.L csa MYB transcription factor Distribution of pollen and anther sac sugars NM_001049255 PFG_K-05711. aid1 MYB transcription factor Anther sac dehiscence AY429017.1 T25683T

2. Construction of a binary expression vector: Using pCAMBIA1300 as the backbone vector, a binary expression vector of the color marker gene EGFP was constructed. The color marker gene EGFP is driven by the endosperm-specific promoter _PGt1 (see Figure 2).

3. Transform the source parent of the common GMS line: the above-mentioned binary expression vector was transferred into the genome of the source parent rice T98B of the above-mentioned common GMS line whose genotype was SS by using the Agrobacterium-mediated method. The total genetic distance of the genome is P. According to the recombination linkage law, when the recombination rate is 1%, the genetic distance is 1cM. Therefore, the color marker gene EGFP is inserted 1cM to the left or 1cM to the right of the fertile gene S, and the recombination rate of the two genes can be obtained. 1% of the strains, so theoretically only need P/2 single-copy transformants of the T ₀ generation to obtain a strain with a linkage frequency of 99% between the color marker gene EGFP and the fertility gene S; The distance is about 1750cM. If a strain whose linkage frequency between the color marker gene EGFP and the fertility gene S is 99% is to be obtained, theoretically 875 transformants of a single copy of the _T0 generation are required; in this embodiment, a total of Obtained 3133 T ₀ generation transgenic lines with the color marker gene EGFP ; after harvesting the seeds, 1546 homozygous lines with a single copy color marker gene were obtained by selfing for 6 generations, and the genotype was SS/ EGFPEGFP .

4. Insertion site analysis: According to the insertion sites of the 1546 homozygous lines with a single copy of the color marker gene obtained in step 3 of flanking sequence analysis, the insertion sites of these 1546 homozygous lines are more evenly distributed In the rice genome, the genetic distance between the insertion sites in the rice genome is 0.1cM-1.93cM, that is, the genetic distance between the single-copy color marker gene EGFP is 0.1cM-1.93cM, which is in line with the engineering maintainer line candidate library. There is a standard for color-marked genes at intervals of 0-2.0 cM.

5. Genetic linkage analysis: cross the above-mentioned common genital sterile line (ss) of rice T98B with 1546 homozygous lines (SS/ EGFPEGFP ) with color marker genes in step 4, and obtain 1433 F ₁ heterozygous seeds , heterozygous seeds are self-propagated for one generation to obtain _F2 seeds, and the seeds carrying the fluorescent marker gene EGFP in _F2 are selected by color sorting, and about 50 seeds are selected from the remaining _F2 seeds that do not carry the fluorescent marker gene EGFP . After planting, 1433 F ₂ populations were obtained, and the number of sterile plants in F ₂ populations was investigated. Among the 1433 F ₂ populations, the proportion of sterile plants in two populations is close to 98% (only one population is greater than 98%, and the other is 96.08%), which are 98.04% and 96.08% respectively, expanding these two F ₂ populations to 1000 strains and investigate the proportion of sterile strains, the proportion of sterile strains is still 98% and 96% respectively, wherein the proportion of sterile strains is 98% in F ₁ heterozygous strains derived from the F ₂ population with the color marker gene and The linkage frequency of the fertile gene was greater than 99%, which met the requirements for the construction of the engineering maintainer line, and was selected as the engineering maintainer line, and the genotype was recorded as s ^egfp S ^EGFP .

In step 5 of this embodiment, the screening process of the engineering maintainer line is shown in Figure 3. Firstly, the common nuclear sterile line (ss) is crossed with each strain in the engineering maintainer line candidate library to obtain _F1 hybrid seeds (s ^egfp S ^EGFP ), self-propagation of heterozygous seeds to obtain F ₂ seeds in one generation, there are nine genotypes in the obtained F ₂ seeds, which are SSCC, SsCc, SSCc, SsCC, ssCc, ssCC, sscc, Sscc, SScc (where C refers to EGFP ) (see Figure 3), the F ₂ seeds of the first six genotypes can be sorted and eliminated by color sorting (that is, the part selected by the black box in Figure 3), and the remaining three genotypes are not After the F ₂ seeds with the color-marked gene are planted, the F ₂ populations _of each group are obtained, and the proportion of the number of sterile plants in the F ₂ population of each group is investigated. The color marker EGFP and the fertile gene S of the zygotic seeds are located on different chromosomes; if the proportion of sterile plants is between 25% and 98%, the color marker EGFP and the fertile gene of the F ₁ heterozygous seeds from the corresponding source are determined S is located on the same chromosome, and the recombination rate between the color-marked EGFP and the fertile gene S is between 1% and 50%; if the proportion of sterile plants is above 98%, the F ₁ heterozygous seed of its corresponding source is determined to be The color marker EGFP and the fertility gene S are located on the same chromosome, and the recombination rate between the color marker EGFP and the fertility gene S is less than 1%. Therefore, the F ₁ heterozygous seeds from which the F ₂ whose number of sterile plants accounted for more than 98% was selected was the engineered maintainer line required in this embodiment. It can be seen from the above that the heterozygous seeds whose genotype is s ^egfp S ^EGFP in the present invention are obtained according to the genetic linkage analysis of the color marker gene EGFP and the fertility gene S in the F ₂ generation.

6. Propagation application: the engineering maintainer heterozygous seed whose genotype is s ^egfp S ^EGFP in step 5 is crossed with the above-mentioned rice T98B common nuclear male sterile line whose genotype is ss . The genetic distance of the fertility gene S is ≤1cM, so there is a recombination probability of ≤1%. Both were below 0.5%, while the engineering maintainer line with genotype s ^egfp S ^EGFP and the common GMS line with genotype ss were both above 49.5%. The recombinants with genotype ssCc and the engineered maintainer line with genotype ^segfp S ^EGFP were sorted out by color sorting, and the common nuclear male sterile line with genotype ss and the recombinants with genotype Sscc were left to see the reproduction Obtain the purity ≥ 99% of common GMS line (according to the purity requirements stipulated in the hybrid rice seed quality classification national standard GB4404.1-1996: the original species level parent ≥ 99.9%, the fine-bred level parent ≥ 99.0%, therefore P in the present invention /2 T ₀ generation transformants can obtain an ideal engineering maintainer line, and the sterile line can be propagated through the engineered maintainer line, and its purity can reach the purity requirements of the fine-bred parent), and the genotype can be further recombined into Sscc by removing impurities child removal. However, the genotype of the recombinants eliminated by color selection is ssCc. Since the recombinants are sterile, they cannot produce fruit when crossed with the common nuclear sterile line ss. Therefore, the recombinants will not affect the engineering maintainer line ^segfp S ^EGFP to continue to be used Propagation of common GMS lines.

In this example, the engineered maintainer line whose genotype is ^segfp S ^EGFP was crossed with 3 common GMS lines whose genotype was ss, all the 3 common GMS lines could set seeds normally, with an average seed setting rate of 95.3%. Harvest the seeds of 3 ordinary GMS lines by individual plants, and sort the engineering maintainer seeds whose genotype is ^segfp S ^EGFP by the color sorter, and the remaining seeds are the GMS lines whose genotype is ss For seeds, the ratio of labeled fluorescent seeds to non-labeled fluorescent seeds was 1:1 (see Table 2 below) through Chi-square analysis, and the non-fluorescent seeds were common GMS seeds to be propagated.

Table 2: Seed fluorescence statistical analysis results

Sterile strain number Effective number of grains per plant (grains) Fluorescence (granule) Non-fluorescing (grains) ^X2 (1:1) T98BS1 884 451 433 0.327 T98BS2 841 428 413 0.233 T98BS3 793 407 386 0.504

X ² <P=3.841, in line with the ratio of 1:1.

Example 2:

An application of the general-purpose rice engineering maintainer line of the present invention in the breeding and seed production of rice common genetic male sterile lines, the specific object of its application is the common nuclear sterile line of rice CSA gene mutation, specifically comprising the following steps (technical process See Figure 1):

1. Purchase common nuclear sterile lines: The CSA gene encodes a MYB transcription factor expressed in tapetum cells, which is a key regulatory transcription factor for rice pollen development. The mutant type is common nuclear sterile, and the genotype is recorded as csa / csa . Purchase rice Nipponbare csa gene mutants from the mutant library RiceGE, website: http://signal.salk.edu/cgi-bin/RiceGE, see Table 1 above for order numbers.

2. Construction of a binary expression vector: Using pCAMBIA1300 as the backbone vector, a binary expression vector of the color marker gene EGFP was constructed. The color marker gene EGFP is driven by the endosperm-specific promoter _PGt1 (see Figure 2).

3. Transformation of the source parent of the common GMS line: using the Agrobacterium-mediated method to transfer the above-mentioned binary expression vector into the genome of the parent rice Nipponbare of the above-mentioned common GMS line whose genotype is CSA / CSA , Suppose the total genetic distance of the genome is P, according to the recombination linkage rule, when the recombination rate is 1%, the genetic distance is 1cM, so the color marker gene EGFP is inserted 1cM to the left or 1cM to the right of the fertile gene CSA , and both genes can be recombined Therefore, in theory, only P/2 single-copy transformants of the T ₀ generation are needed to obtain a strain with a linkage frequency of 99% between the color marker gene EGFP and the fertility gene CSA ; The total genetic distance is about 1750cM. If a strain whose linkage frequency between the color marker gene EGFP and the fertility gene CSA is to be 99% is to be obtained, theoretically 875 transformants of single copy of the T _generation are required; in this embodiment , a total of 3631 transgenic lines of the T ₀ generation with the color marker gene EGFP were obtained; after harvesting the seeds, 1732 homozygous lines with a single copy of the color marker gene were obtained through self-propagation for 6 generations, and the genotype was CSACSA / EGFPEGFP .

4. Insertion site analysis: According to the insertion sites of the 1546 homozygous lines with a single copy of the color marker gene obtained in step 3 of flanking sequence analysis, the insertion sites of these 1732 homozygous lines are more evenly distributed In the rice genome, the genetic distance between the insertion sites in the rice genome is 0.1cM-1.96cM, that is, the genetic distance between the single-copy color marker gene EGFP is 0.1cM-1.96cM, which is in line with the engineering maintainer line candidate library. There is a standard for color-marked genes every 0-2cM.

5. Genetic linkage analysis: Cross the above-mentioned Nipponbare common nuclear sterile line ( csa / csa ) with the 1732 homozygous lines ( CSACSA / EGFPEGFP ) with the color marker gene in step 4, and obtain 1546 F ₁ heterozygous lines Hybrid seeds and heterozygous seeds are self-propagated in one generation to obtain _F2 seeds, and the seeds carrying the fluorescent marker gene EGFP in _F2 are selected by color sorting, and about 50 seeds are selected from the remaining _F2 seeds that do not carry the fluorescent marker gene EGFP 1546 F ₂ populations were obtained after the seeds were planted, and the number of sterile plants in the F ₂ population was investigated. Among the 1546 F ₂ populations, there is a population whose proportion of sterile plants is greater than 98%, which is 98.69%. Expand this F ₂ population to 1000 plants and investigate the proportion of sterile plants. The proportion of sterile plants is still greater than 98%, indicating that The linkage frequency of the color marker gene and the fertility gene in the _F1 heterozygous line derived from the _F2 population is greater than 99%, which meets the requirements for the construction of the engineering maintainer line, and is selected as the engineering maintainer line, and the genotype is recorded as csa ^egfp CSA ^EGFP .

In step 5 of this example, the screening process of the engineering maintainer line is shown in Figure 3. First, the common nuclear sterile line ( csa / csa ) is crossed with each strain in the engineering maintainer line candidate library to obtain the F ₁ hybrid zygotic seeds ( csa ^egfp CSA ^EGFP ), self-propagation of heterozygous seeds to obtain F ₂ seeds in one generation, there are nine genotypes in the obtained F ₂ seeds, which are SSCC, SsCc, SSCc, SsCC, ssCc, ssCC, sscc, Sscc , SScc (where s refers to csa , S refers to CSA , c refers to egfp , C refers to EGFP ) (see Figure 3), the _F2 seeds of the first six genotypes can be sorted and eliminated by color sorting (that is, The part selected by the black box in 3), the remaining three genotypes of F ₂ seeds without color marker genes were planted to obtain F ₂ populations in each group, and the proportion of sterile plants in F ₂ populations in each group was investigated. If the number of sterile plants If the ratio is close to 1/4, it is judged that the color marker EGFP and the fertile gene S of the F ₁ heterozygous seeds of the corresponding source are located on different chromosomes; if the proportion of sterile plants is between 25% and 98%, it is judged that the The color marker EGFP and the fertile gene CSA of the F ₁ heterozygous seeds from the corresponding source are located on the same chromosome, and the recombination rate of the color marker EGFP and the fertile gene CSA is between 1% and 50%; if the proportion of sterile plants is between More than 98%, it is determined that the color marker EGFP and the fertility gene CSA of the corresponding source _F1 heterozygous seeds are located on the same chromosome, and the recombination rate of the color marker EGFP and the fertility gene CSA is below 1%. Therefore, the F ₁ heterozygous seeds from which the F ₂ whose number of sterile plants accounted for more than 98% was selected was the engineered maintainer line required in this embodiment. It can be seen from the above that the heterozygous seeds whose genotype is csa ^egfp CSA ^EGFP in the present invention are obtained according to the genetic linkage analysis of the color marker gene EGFP and the fertility gene CSA in the _F2 generation.

6. Propagation application: the engineering maintainer heterozygous seed whose genotype is csa ^egfp CSA ^EGFP in step 5 is crossed with the above-mentioned rice Nipponbare common nuclear male sterile line whose genotype is csa / csa , because the color marker gene EGFP in the engineering maintainer line The genetic distance from the fertile gene CSA is ≤1cM, so there is a recombination probability of ≤1%. As shown in Figure 4, the offspring will produce two types of recombinants, the genotypes are ssCc and Sscc (where s refers to csa , S refers to CSA , c refers to egfp , C refers to EGFP ), and their proportions are all below 0.5%, while the proportion of engineering maintainer lines with genotype csa ^egfp CSA ^EGFP and common nuclear sterile lines with genotype csa / csa Both are above 49.5%. The recombinants whose genotype is ssCc and the engineering maintainer line whose genotype is csa ^egfp CSA ^EGFP are sorted out by color sorting, leaving the common nuclear sterile line whose genotype is csa / csa and the recombinants whose genotype is Sscc, Visible propagation obtains the purity >=99% of common GMS line (according to the purity requirements stipulated in the hybrid rice seed quality classification national standard GB4404.1-1996: original seed level parent >=99.9%, improved seed level parent >=99.0%, so the present invention In P/2 T ₀ generation transformants, an ideal engineering maintainer line can be obtained, and the sterile line can be propagated through the engineered maintainer line, and its purity can reach the purity requirement of the fine-bred parent), and the genotype can be further divided into Sscc recombinants were removed. The genotype of the recombinants eliminated by color selection is ssCc. Since the recombinants are sterile, they cannot produce fruit when crossed with the common nuclear sterile line ss. Therefore, the recombinants will not affect the engineering maintainer csa ^egfp CSA ^EGFP to continue to be used Propagation of the common GMS line csa / csa .

In this example, the engineered maintainer line whose genotype is csa ^egfp CSA ^EGFP is crossed with 6 common GMS lines whose genotype is csa / csa , all 6 common GMS lines can set fruit normally, with an average seed setting rate of 96.8 %. Harvest the seeds of 6 common GMS lines by individual plants, and sort the engineering maintainer seeds with genotype csa ^egfp CSA ^EGFP by the color sorting machine, and the remaining seeds are the genotype csa / csa GMS For breeding line seeds, the ratio of labeled fluorescent seeds to non-labeled fluorescent seeds is 1:1 (see Table 3 below) through Chi-square analysis, and the non-fluorescent seeds are the common GMS seeds to be propagated .

Table 3: Seed fluorescence statistical analysis results

Sterile strain number Effective number of grains per plant (grains) Fluorescence (granule) Non-fluorescing (grains) ^X2 (1:1) cas1 910 463 447 0.247 cas2 894 458 436 0.493 cas3 923 469 454 0.212 cas4 887 457 430 0.762 cas5 903 464 439 0.637 cas6 878 446 432 0.192

X ² <P=3.841, in line with the ratio of 1:1.

Claims (8)

1. the method for cultivation of a universal paddy rice engineering maintenance line may further comprise the steps:

(1) preparing genotype is the common line with genic sterile of paddy rice of ss, and s represents to control the sterile gene of the sterile proterties of the common line with genic sterile of paddy rice;

(2) binary expression vector of structure color mark gene C;

(3) adopt among the source parent or the normal kind of fertility of existing transgenic method with the described binary expression vector transgene type common line with genic sterile of above-mentioned paddy rice that is SS, screening obtains having the T of color mark gene C ₀For transgenic line; Again with T ₀Obtain to have single homozygous lines that copies the color mark gene for transgenic line by continuous self propagated, its genotype is SSCC;

(4) the insertion site of color mark gene C in rice genome in each homozygous lines of obtaining of analytical procedure (3), and according to the on position of color mark gene C at rice genome, filter out the homozygous lines group storehouse of a series of single copies, make the rice genome in the homozygous lines group storehouse every 0 ~ 2cM a color mark gene C just be arranged, this homozygous lines group storehouse has namely constituted an engineering maintenance line candidate storehouse;

(5) each homozygous lines in the engineering maintenance line candidate storehouse in the common line with genic sterile of paddy rice in the step (1) and the step (4) is hybridized, obtain F ₁The heterozygosis seed, F ₁A heterozygosis seed self propagated generation obtains the F of several genes type ₂Seed selects technology to reject F earlier by look then ₂The seed that has the color mark gene C in the seed is not respectively organized F with the color mark gene C with residue ₂Seed is cultivated respectively, obtains the corresponding F that respectively organizes ₂The aforementioned F that respectively organizes investigates in strain system ₂Sterile strain number in the strain system filters out the F that accounting 〉=98% is counted in sterile strain ₂Strain system group, and with this F ₂The F in strain system group source ₁The heterozygosis seed is as the engineering maintenance line of the common line with genic sterile of described paddy rice.

2. method of cultivation according to claim 1, it is characterized in that: the sterile proterties of the common line with genic sterile of described paddy rice is the recessive cytoblast sterile proterties by single-gene control, can educate the sterile gene s of gene S is dominance, and sterile proterties is not influenced by external environmental condition.

3. method of cultivation according to claim 1 and 2, it is characterized in that: in the described step (2), described binary expression vector is specially pCAMBIA1300, pCAMBIA1301, pCAMBIA1390, pCAMBIA3301 or pBI121.

4. method of cultivation according to claim 3, it is characterized in that: described binary expression vector is pCAMBIA1300 or pCAMBIA1390.

5. method of cultivation according to claim 1 and 2, it is characterized in that: in the described step (2), described color mark gene is the red fluorescence marker gene DsRed, the red fluorescence marker gene RFP, green fluorescence protein gene GFP, green fluorescence protein gene EGFPOr blue fluorescent protein gene EBFP

6. method of cultivation according to claim 5, it is characterized in that: described color mark gene is the red fluorescent protein gene DsRedOr green fluorescence protein gene EGFP

7. method of cultivation according to claim 1 and 2, it is characterized in that: sterile gene s is Msp1, Pair1, Pair2, Zep1, Mel1, Pss1, Tdr, Udt1, Gamyb4, Ptc1, Api5, Wda1, Cyp704B2, Dpw, Mads3, Osc6, Rip1, CsaOr Aid1, can educate gene S and be corresponding paddy rice wild type gene.

8. one kind as the application of universal paddy rice engineering maintenance line in the common line with genic sterile breeding of this paddy rice that method of cultivation obtains as described in each in the claim 1～7, it is characterized in that: it is to have the color mark gene C and genotype is s that described engineering keeps ^cS ^CThe heterozygosis seed, in the described engineering maintenance line color mark gene C with can educate gene S close linkage, when breeding application, the common line with genic sterile of described paddy rice that earlier with described engineering maintenance line and genotype is ss is hybridized, in filial generation, obtain the common line with genic sterile seed of engineering maintenance line seed and paddy rice simultaneously, by color selector described engineering maintenance line seed separation is come out, remaining seed is the common line with genic sterile seed of paddy rice that genotype is ss again.

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