CN112251525B

CN112251525B - Molecular marker for gene assisted breeding related to rape dominant genic male sterility and application thereof

Info

Publication number: CN112251525B
Application number: CN202011022677.7A
Authority: CN
Inventors: 曾新华; 闫晓红; 吴刚; 袁荣; 王淼; 罗军玲; 刘芳; 朱莉; 李晓飞; 李均
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2022-07-15
Anticipated expiration: 2040-09-25
Also published as: CN112251525A

Abstract

The invention discloses a molecular marker of gene assisted breeding related to rape dominant genic male sterility and application thereof, relating to the technical field of rape molecular breeding. The invention provides a molecular marker, an identification primer and a kit for rape dominant karyon sterility related gene assisted breeding and application in rape and related crop dominant karyon male sterility heterosis utilization assisted breeding. The invention has the advantages of simple operation, low cost, short period and the like, and the marker has good stability and is not influenced by environmental factors, and can carry out molecular marker auxiliary selection in the early generation, shorten the breeding period and improve the breeding efficiency. The molecular marker gene sequence information can be used for realizing the auxiliary selective breeding of dominant karyon male sterility molecular markers of rape and related crops; can realize three-line hybrid seed production of dominant cell nucleuses of rape and related crops; can realize the identification and selection of rape and related crops in recurrent group.

Description

Molecular marker for gene assisted breeding related to rape dominant genic male sterility and application thereof

Technical Field

The invention belongs to the technical field of rape molecular breeding, and particularly relates to a molecular marker for gene assisted breeding related to rape dominant genic male sterility and application thereof.

Background

Male sterility is the most cost-effective way to utilize crop heterosis. At present, the male sterility types mainly utilized in the international rape heterosis utilization are as follows: MSL (Europe and North America), PGS (North America), OGURA-INRA (North America and Europe), Borima CMS (China). MSL and PGS both belong to nuclear male sterility, the hybrids of the MSL system account for 70-80% of the European hybrids, and the hybrids of the PGS system account for 40% of the Canadian market (2006). Nuclear male sterility has many advantages in heterosis utilization: the sterility is thorough and stable, only two lines are matched, the breeding period is greatly shortened, the method is not limited by the restoring and maintaining relation, the strong vigor combination is easily obtained, the negative effect of the sterile cytoplasm and the potential risk of cytoplasm simplification do not exist, and the method has great potential in heterosis utilization at present. Especially, recessive genic male sterility has wide restoration spectrum, stable fertility and easy transformation, thereby providing wide prospect for the utilization of heterosis. However, recessive genic male sterility also has obvious defects: it is difficult to obtain the complete sterile population, and 50% of fertile plants in the sterile line must be pulled out manually during propagation and seed production. Therefore, how to remove 50% of fertile plants in the maternal line by an economic and effective method is a main problem to be solved by widely applying the nuclear sterility; meanwhile, the transformation difficulty of the 8029AB nucleus male sterile line is higher.

8029AB is a new type of dominant nucleus male sterile material, whose fertility is subject to 1 pair of multiple alleles (BnMS 5)^a/BnMS5^c/BnMS5^e) And (5) controlling. When BnMS5^cThe gene is homozygous, BnMS5^aThe fertility is normal when the gene is homozygous or heterozygous. Thus, the genotype is BnMS5^aAnd BnMS5^cBnMS5^cThe individual of (a) shows fertility and the genotype is BnMS5^eBnMS5^eAnd BnMS5^cBnMS5^eThe individual plant of (2) shows sterility. Therefore, the material can simulate cytoplasmic male sterility to realize three-line seed production (figure 1): two-type sterile line (AB line, genotype: BnMS 5)^eBnMS5^eAnd BnMS5^aBnMS5^e) Temporary maintainer line (genotype: BnMS5^cBnMS5^c) Restorer line (genotype: BnMS5^aBnMS5^a). Namely, 100% of sterile population is generated by hybridizing a sterile line and a temporary maintainer line, and then the full sterile population and a restorer line are used for producing hybrid seeds, thereby solving the problem that 50% of fertile plants of a parent line need to be pulled out in hybrid seed production of dominant genic male sterility.

Disclosure of Invention

In view of the above, the present invention aims to provide a molecular marker for assisted breeding of a gene related to rape dominant nuclear sterility and an application thereof, which can accelerate the breeding of rape crop dominant nuclear male sterility three-line method, thereby obtaining 100% sterile population during breeding and seed production.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a molecular marker for gene assisted breeding related to rape dominant genic male sterility, which comprises the following components: and BnMS5^aSNP marker SNPMS5 for gene coseparation^a-5, and BnMS5^cSNP marker SNPMS5 for gene coseparation^c-3 and BnMS5^eSNP marker SNPMS5 with co-separated genes^e-1；

Wherein the marker SNPMS5^a-5 comprises the nucleotide sequence shown in SEQ ID NO.1Shown upstream primer SNPMS5^a-5F and a downstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.2^a-5R；

Marker SNPMS5^c-3 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.3^c-3F and downstream primer SNPMS5 with nucleotide sequence shown as SEQ ID NO.4^c-3R；

Marker SNPMS5^e-1 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.5^e-1F and a downstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.6^e-1R。

The invention also provides a primer group for amplifying the molecular marker, wherein the primer group comprises the following primers: SNPMS5^a-5F、SNPMS5^a-5R、SNPMS5^c-3F、SNPMS5^c-3R、SNPMS5^e-1F and SNPMS5^e-1R。

The invention also provides application of the molecular marker in identifying rape dominant genic male sterility related genes, wherein the rape dominant genic male sterility related genes comprise BnMS5^a、BnMS5^cAnd BnMS5^e。

The invention also provides application of the primer group in identifying rape dominant genic male sterility related genes, wherein the rape dominant genic male sterility related genes comprise BnMS5^a、BnMS5^cAnd BnMS5^e。

The invention provides a kit for identifying a rape dominant genic male sterility related gene, which comprises the molecular marker or the primer group.

The invention also provides application of the molecular marker in breeding three-line rape, wherein the three-line rape comprises: rape dominant cell nuclear sterile line, rape dominant cell nuclear temporary maintainer line and rape dominant cell nuclear restoring line.

Preferably, when the rape dominant karyocyte sterile line is bred, the rape dominant karyocyte sterile gene BnMS5 is obtained^eThe sterile material of (A) is used as female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (A) is a male parent crossing, through backcrossing, crossing or tissue culture methodMethod, selecting a composition having SNPMS5^eProgeny of-1 molecular marker with genotype BnMS5^eBnMS5^eOr BnMS5^cBnMS5^eThe sterile line of (1).

Preferably, when the rape dominant nucleus temporary maintainer line is selected, the rape dominant nucleus fertile gene BnMS5 is possessed^cThe fertile material of (2) is female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (2) is a male parent for hybridization, and the SNPMS5 is selected by a backcross, hybridization or tissue culture method^c-3 progeny of the molecular marker, the genotype being BnMS5^cBnMS5^cThe temporary protection system of (1).

Preferably, when the rape dominant karyon restorer line is selected, the rape dominant karyon restorer gene BnMS5 is possessed^aThe fertile material of (2) is female parent, and the genotype is BnMS5^cBnMS5^cThe material is used as male parent for hybridization, and the method of backcross, hybridization or tissue culture is adopted to select the material with SNPMS5^a-5 progeny of the molecular marker, the genotype of which is BnMS5^aBnMS5^aOr BnMS5^aBnMS5^cThe recovery system of (1).

The invention also provides application of the molecular marker or the primer group in rape recurrent selective breeding.

The invention provides a molecular marker, a primer group, a kit and application of gene assisted breeding related to rape dominant nuclear sterility, wherein the molecular marker and a male nuclear sterility gene BnMS5 are used^eThe nuclear sterile gene BnMS5 has close linkage and accurate and reliable identification result^eThe application in breeding provides a good tool; the molecular marker of the invention is used for carrying male nuclear sterile gene BnMS5^eThe material identification method is simple, can be carried out in the seedling stage, can meet the requirements of breeding methods such as recurrent selection and the like which need to carry out a large number of hybrids, greatly reduces the labor cost, improves the breeding efficiency, and has great promotion effect on expanding the germplasm basis of rape breeding; the molecular marker of the invention also aims at cloning the nuclear sterile gene BnMS5^eHas important significance.

The invention also provides application of the molecular marker in rape and related crop dominant nuclear male sterility heterosis utilization auxiliary breeding. The invention has the advantages of simple operation, low cost, short period and the like, and the marker has good stability and is not influenced by environmental factors, and can carry out molecular marker auxiliary selection in the early generation, shorten the breeding period and improve the breeding efficiency. The molecular marker gene sequence information can be used for realizing the auxiliary selective breeding of dominant karyon male sterility molecular markers of rape and related crops; can realize dominant cell nucleus three-line hybrid seed production of rape and related crops; can realize the identification and selection of rape and related crops in recurrent group.

Biological preservation information

Brassica napus (Brassica napus L.)8029A, which is preserved in China center for type culture Collection in 8 months and 17 days of 2020, and the specific address is university of Wuhan, China with the preservation number of CCTCC NO: p202006;

cabbage type rape (Brassica napus L.)8029B, which is preserved in China center for type culture Collection in 8 months and 17 days of 2020, and the specific address is Wuhan university, Wuhan China, the preservation number is CCTCC NO: p202007.

Drawings

FIG. 1 is a process chart of three-line hybrid seed production of nuclear male sterile line;

FIG. 2 is a 8029AB sterile and fertile flower androecium phenotype, wherein A is a flower of sterile material 8029A and B is a flower of fertile material 8029B;

FIG. 3 is a diagram of the tag SNPMS5^a-5、SNPMS5^c-3、SNPMS5^e-1 amplification effect maps in the restorer line, the maintainer line and the sterile line, respectively, wherein M is Marker; 1-8 are marked SNPMS5^a-5 amplification in a restorer line; 9-16 are marked SNPMS5^e-1 amplification in sterile lines; 17-24 are marked SNPMS5^c-3 amplification in a maintainer line.

Detailed Description

The invention provides a molecular marker for gene assisted breeding related to rape dominant genic male sterility, which comprises the following components: and BnMS5^aSNP marker SNPMS5 with co-separated genes^a-5, and BnMS5^cGene co-segregating SNP markersSNPMS5^c-3 and BnMS5^eSNP marker SNPMS5 for gene coseparation^e-1；

Wherein the marker SNPMS5^a-5 comprises an upstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.1^a-5F and downstream primer SNPMS5 with nucleotide sequence shown in SEQ ID NO.2^a-5R；

Marker SNPMS5^e-1 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.5^e-1F and downstream primer SNPMS5 with nucleotide sequence shown in SEQ ID NO.6^e-1R。

The invention preferably takes the rape dominant male sterility amphimorphic line 8029AB and the rape restorer line 6449 as materials to construct the near isogenic line 8030AC, and obtains the restorer gene BnMS5 by screening SNP markers^aLinked SNP molecular marker SNPMS5^a-5; and maintenance gene BnMS5^cLinked SNP marker SNPMS5^c-3; and sterile gene BnMS5^eLinked SNP marker SNPMS5^e-1. In the invention, the 8029AB is a novel dominant nuclear male sterile material, and the fertility of the material is subjected to 1 pair of multiple alleles (BnMS 5)^a/BnMS5^c/BnMS5^e) And (5) controlling. When BnMS5^cThe gene is homozygous, BnMS5^aThe fertility is normal when the gene is homozygous or heterozygous. Thus, the genotype is BnMS5^aAnd BnMS5^cBnMS5^cThe individual of (A) shows fertility, and the genotype is BnMS5^eBnMS5^eAnd BnMS5^cBnMS5^eThe individual plant of (2) shows sterility. Therefore, the material can simulate cytoplasmic male sterility to realize three-line seed production (figure 1): two-type sterile line (AB line, genotype: BnMS 5)^eBnMS5^eAnd BnMS5^aBnMS5^e) Temporary maintainer line (genotype: BnMS5^cBnMS5^c) Restorer line (genotype: BnMS5^aBnMS5^a). Namely, the sterile line and temporary maintainer line are crossed to generate 100 percent of sterile group, and then the whole sterile group is utilizedThe hybrid seed is produced by the hybrid line and the restoring line, thereby solving the problem that 50 percent of fertile plants in the female parent row need to be pulled out in the hybrid seed production of the dominant genic male sterility.

In the present invention, the label SNPMS5^aThe size of-5 (KX223882) is preferably 200bp, and the sequences of the primer pairs are as follows: SNPMS5^a-5F：5’-CATTCTTTAACAGAGATAGGTGTC-3’(SEQ ID NO.1)；

SNPMS5^a-5R：5’-AGACTCTTGAGTACAGTCTCACG-3’(SEQ ID NO.2)；

Marker SNPMS5^cThe size of-3 (KX223881) is preferably 140bp, and the sequences of the primer pairs are respectively as follows:

SNPMS5^c-3F：5’-ATTGTCCCCTTGTAGCGCGG-3’(SEQ ID NO.3)；

SNPMS5^c-3R：5’-ATTTCGTGAAGATGCAAGTTCG-3’(SEQ ID NO.4)；

marker SNPMS5^eThe size of-1 (SEQ ID NO.7) is preferably 150bp, and the sequences of the primer pairs are respectively as follows:

SNPMS5^e-1F：5’-TCCTCCTTTGTTGTTTTCGAG-3’(SEQ ID NO.5)；

SNPMS5^e-1R：5’-TACATTAATCCATTTATTATAGTTT-3’(SEQ ID NO.6)。

the invention also provides a primer group for amplifying the molecular marker, which comprises the following primers: SNPMS5^a-5F、SNPMS5^a-5R、SNPMS5^c-3F、SNPMS5^c-3R、SNPMS5^e-1F and SNPMS5^e-1R。

The sequences of the primers in the primer set of the present invention are the same as the sequences of the primers described above, and are not described herein again.

The rape dominant genic male sterility related gene is preferably identified by PCR (polymerase chain reaction), and the PCR program preferably comprises the following steps: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 5 min.

The invention also provides application of the primer group in identifying rape dominant genic male sterility related genes, wherein the rape dominant genic male sterility related genes comprise BnMS5^a、BnMS5^cAnd BnMS5^e. The preferred PCR method for identifying the rape dominant genic male sterility related gene by using the primer group is the same as the scheme, and the details are not repeated.

Preferably, the kit of the present invention further comprises Taq DNApolymerase, 10 XTAQuffer, 2.5mM dNTP mix and ddH₂And O. In the invention, when the kit is used for identifying the rape dominant genic male sterility related gene, the same PCR program as the scheme is preferably used, and the PCR system is preferably 15 muL: taq DNApolymerase 5U, 10 XTaq Buffer 2.4. mu.L, 2.5mM dNTP mix 0.8. mu.L, 10uM forward and reverse primers 0.2. mu.L each, 2.5U/. mu.L DNA Polymerase 0.2. mu.L, template DNA50ng, and complement ddH₂0 to 15. mu.L.

In the invention, preferably, the near isogenic line 8030AC is constructed by using materials based on the rape dominant male sterility amphimorphic line 8029AB and the rape restorer 6449, so that the cytoplasmic male sterility shown in figure 1 is realized to realize three-line seed production: two-type sterile line (AB line, genotype: BnMS 5)^eBnMS5^eAnd BnMS5^aBnMS5^e) Temporary maintainer line (genotype: BnMS5^cBnMS5^c) Recovery line (genotype: BnMS5^aBnMS5^a). Namely, the sterile line and the temporary maintainer line are hybridized to generate 100 percent of sterile population, and then the full sterile population and the restorer line are utilized to produce hybrid seeds, thereby solving the problem that 50 percent of fertile plants of the female parent line are pulled out in the hybrid seed production of the dominant genic male sterility.

In the present invention, when the rape dominant genic male sterile line is selected, it is preferable to have rape dominant genic male sterile gene BnMS5^eThe sterile material is used as female parent, other materials are used as male parent for hybridization, and the method of backcross, hybridization or tissue culture is adopted to select the material with SNPMS5^eProgeny of-1 molecular marker with genotype of BnMS5^eBnMS5^eOr BnMS5^cBnMS5^eThe sterile line of (1).

The preferred male parent of the invention is genotype BnMS5^cBnMS5^cWhen breeding the rape dominant cell genic male sterile line, the preferred selection specifically comprises: (1) has rape dominant karyon sterile gene BnMS5^eThe sterile material of (2) is female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (A) is hybridized with a male parent, and seeds on the sterile plant are collected to obtain F₁Generation;

(2) screening F₁The mark SNPMS5 can be used in generation^e-1, using sterile single plant of DNA molecule with product size of 150bp amplified as female parent, backcrossing with said male parent in step (1) to obtain BC₁F₁Seed generation;

(3) screening of BC₁F₁The mark SNPMS5 can be used in generation seed^e-1, using sterile single plant of DNA molecule with product size of 150bp amplified as female parent, backcrossing with male parent in step (1) again to obtain BC₂F₁Seed generation;

(4) to BC₂F₁And (3) carrying out continuous backcross on the generation seeds for 2-5 times to obtain sterile plants with the characteristics similar to those of the male parent material in the step (1).

In the present invention, the marker SNPMS5 is used^e-1 the system and procedure for amplification of product size 150bp is preferably the same as in the above protocol and will not be described further here.

In the present invention, a sterile plant similar to the paternal material in step (1) is obtained with genotype BnMS5^eBnMS5^eOr BnMS5^cBnMS5^e。

In the present invention, when a dominant nuclear temporary maintainer line of rape is selected, it is preferable to have the dominant nucleus of rapeSex nucleus fertile gene BnMS5^cThe fertile material of (2) is used as female parent, other materials are used as male parent for hybridization, and the method of backcross, hybridization or tissue culture is adopted to select the material with SNPMS5^c-3 progeny of the molecular marker, the genotype of which is BnMS5^cBnMS5^cThe temporary protection series of (1).

The preferred male parent of the invention is the genotype BnMS5^cBnMS5^cWhen breeding the rape dominant nucleus temporary maintainer line, the optimization specifically comprises: 1) has rape dominant nucleus fertile gene BnMS5^cThe fertile material of (2) is female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (A) is hybridized with a male parent, and seeds on the sterile plant are collected to obtain F₁Generation;

2) screening F₁The mark SNPMS5 can be used in generation^a-5 backcrossing the single plant capable of amplifying the DNA molecule with the product size of 200bp as a female parent with the male parent in the step 1) to obtain BC₁F₁Seed generation;

3) screening of BC₁F₁The marker SNPMS5 can be utilized in the generation seed^a-5 backcrossing the single plant capable of amplifying the DNA molecule with the product size of 200bp as a female parent with the male parent in the step 1) to obtain BC₂F₁Seed generation;

4) to BC₂F₁Carrying out continuous 1-4 times of backcross on the generation seeds, selecting a single plant capable of amplifying DNA molecules with the product size of 200bp to carry out listing marking, and bagging for selfing;

5) hybridizing the sterile line obtained by the scheme with the female parent and the inbred progeny obtained in the step 4) as the male parent to obtain a hybrid F₁Generation;

6) screening for hybrids F₁In generation using marker SNPMS5^a-5 and SNPNS5^eObtaining 200bp and 150bp single plants respectively for selfing and obtaining filial generation from crossing;

7) screening selfed progeny by using marker SNPMS5^a-5 and SNPNS5^eThe-1 can respectively obtain 200bp and 150bp single plants, namely temporary maintainer lines, and can only obtain 150bp single plants, namely homozygous sterile lines.

In the present invention, the breeding oil is selectedWhen the vegetable dominant karyon restorer is used, it is preferable that the vegetable dominant karyon restorer has rape dominant karyon restorer gene BnMS5^aThe fertile material of (1) is used as a female parent, other materials are used as male parents for hybridization, and the method of backcross, hybridization or tissue culture is adopted to select the material with SNPMS5^a-5 progeny of the molecular marker, genotype of BnMS5^aBnMS5^aOr BnMS5^aBnMS5^cThe recovery system of (1).

When the dominant cell nucleus restorer line of the rape is bred, the method preferably comprises the following steps: the gene BnMS5 is used as a restoring gene^aThe individual plant of (1) is used as female parent, and the genotype is BnMS5^cBnMS5^cThe material of (b) is a male parent, and artificial hybridization is carried out to obtain a hybrid F₁Generation;

② screening of hybrids F₁The mark SNPMS5 can be utilized in generation^a-5 amplifying to obtain a single plant with a product size of 200bp as a female parent, and backcrossing by taking the male parent material in the step I as a male parent to obtain BC₁F₁Seed generation;

(iii) screening of BC₁F₁The mark SNPMS5 can be utilized in offspring^a-5 amplifying to obtain a single plant with a product size of 200bp as a female parent, and backcrossing by taking the male parent material in the step I as a male parent to obtain BC₂F₁Seed generation;

fourthly, to BC₂F₁Carrying out continuous backcross on the seeds for 1-4 times, and screening out an available marker SNPMS5^a-5 amplifying a single plant with a product size of 200bp fragment to perform bagging selfing or microspore culture;

fifthly, utilizing the single plant or microspore seedling harvested in the step IV to screen the available marker SNPMS5^a-5 amplified product with size of 200bp and cannot utilize SNPMS5^c-3 amplifying the single strain of the fragment, namely the single strain with the genotype BnMS5^aBnMS5^aThe recovery system of (1).

The invention also provides application of the molecular marker or the primer group in rape recurrent selection breeding.

The molecular markers for assisted breeding of genes related to rape dominant genic male sterility and the application thereof provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Analysis of rape cell nucleus dominant sterile material 8029AB genetic rule

1. Experimental Material

The material used in the experiment is rape nucleus dominant three-line 8029AB, wherein the sterile strain is 8029A, the fertile strain is 8029B, and the restoration line is 6449, and the material is from the institute of oil crops of Chinese academy of agricultural sciences.

The rape cell nucleus dominant three-type line 8029AB material source: obtaining 3 sterile single plants in the rape inbred line 8029, hybridizing the sterile single plants with the double 11 in the rape inbred line, hybridizing the first filial generation F1, and backcrossing by using the double 11 as recurrent parents to obtain the near-isogenic line.

2. Genetic rule of cell nucleus dominant sterile material

Adopting a classical genetics method, taking sterile material 8029A as a female parent to hybridize with double No. 11 in the fertility normal rape to obtain F1 and backcross segregation population for analysis. The results of the test showed (table 1): the sterile character of sterile material 8029A is controlled by a pair of dominant nuclear genes.

TABLE 1 genetic analysis of dominant nuclear male sterile line 8029A

Example 2

Genetic rule analysis of rape cell nucleus dominant sterile material 8029A restorer line

Sterile material 8029A is used as a female parent to be hybridized with a restorer line 6449 to obtain F1, F2 and a backcross segregation population for analysis. The test results showed (table 2): the restorer line of sterile material 8029A is controlled by a pair of nuclear genes and is tightly interlocked with the sterile genes.

TABLE 2 genetic analysis of the 8029A restorer line of dominant nuclear male sterile material

Example 3

Acquisition of rape cell nucleus dominant three-line 8029AB assisted breeding molecular marker

1. Primer design was according to BnMS5^a(KX223882)、BnMS5^c(KX223881) and BnMS5^e(SEQ ID NO: 7) Gene sequence and SNP sites on both sides of the vicinity thereof were mined. And (3) selecting SNP sites, and designing primers of the SNP sites by using primer design software, wherein each group of the primers is provided with two primers. The primers were synthesized by the Onychoma corporation.

2. Detection of PCR reaction

DNA of BC1 segregation population partial sterile strain and fertile strain in example 1 and example 2 are used as templates, and primer SNPMS5 is used^a-5F/R、SNPMS5^c-3F/R、SNPMS5^ePCR amplification was performed at-1F/R.

The reaction system of PCR is: taq DNApolymerase 5U, 10 XTaq buffer 2.4. mu.L, 2.5mM dNTP mix 0.8. mu.L, 10uM forward and reverse primers 0.2. mu.L each, 2.5U/. mu.L DNA Polymerase 0.2. mu.L, template DNA50ng, and complement ddH₂0 to 15 μ L;

the reaction conditions of PCR were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 32 times; fully extend the temperature of 72 ℃ for 5 min. Finally, the mixture is stored at 4 ℃.

The results are shown in FIG. 3, where the primer combination SNPMS5^e1F/R amplified only one band of 150bp in the sterile individuals in example 1, but not amplified in the fertile individuals; primer SNPMS5^e1F/R can amplify a 150bp band in both sterile and fertile individuals in example 2; explanation primer SNPMS5^eThe DNA fragment obtained by amplification of the-1F/R is closely linked with the sterile gene and can be used as the nuclear sterile gene BnMS5^eThe molecular marker of (1). Primer combination SNPMS5^c3F/R can amplify a 140bp band in both sterile and fertile individuals in example 1; primer SNPMS5^c1F/R failed to amplify a band in both the sterile and fertile individuals of example 2; explanation primer SNPMS5^c-3F/R amplification of the resulting DNA fragment withKeeps the gene linkage tight and can be used as nuclear sterility to keep BnMS5^cThe molecular marker of (1). SNPMS5^a5F/R failed to amplify a band in both sterile and fertile individuals in example 1; primer SNPMS5^a1F/R can not amplify a band in the sterile single plant in the example 2 and can amplify a band of 200bp in the fertile single plant; illustrative primer SNPMS5^aThe DNA fragment obtained by the amplification of the-5F/R is closely linked with a restorer gene and can be used as a nuclear sterility restorer gene BnMS5^aThe molecular marker of (1).

Example 4

Molecular marker verification test of rape cell nucleus dominant three-line 8029AB

1. Test method

(1) Population construction

Hybridizing sterile material 8029A serving as a female parent with the Zhongshuang 11 of the maintainer line to obtain F1, backcrossing the Zhongshuang 11 serving as a recurrent parent with the sterile plant to finally obtain (8029A x Zhongshuang 11) BC₄Isolating the population.

Hybridizing sterile material 8029A as female parent with restorer line 6449 to obtain F₁Selfing to obtain F₂The fertile plant is used as male parent to cross with sterile brother and sister, and through multi-generation crossing, the separated colony of 8029A x 6449 is obtained.

(2) Identification of fertility genotype of segregating population at seedling stage

In the seedling stage, (8029A × Zhongshui 11) BC₄About 5g of fresh leaves are taken from a single plant of the segregating group, and genome DNA is extracted by adopting a CTAB method. Extracting genome DNA as template, and using SNPMS5^e-1F/R and SNPMS5^c3F/R is used as a primer for PCR amplification; and marking the individual plants according to the amplification product result.

In the seedling stage, about 5g of fresh leaves are taken from a single plant of the (8029A multiplied by 6449) separation group, and genome DNA is extracted by adopting a CTAB method. Extracting genome DNA as template, and using SNPMS5^e-1F/R and SNPMS5^aCarrying out PCR amplification by taking-5F/R as a primer; and (4) marking the individual plants according to the amplification product result.

The reaction system of PCR is: taq DNApolymerase 5U, 10 XTAQUFU2.4. mu.L, 2.5mM dNTP mix 0.8. mu.L, 10uM forward and reverse primers each 02 uL, 2.5U/. mu.L DNA Polymerase 0.2. mu.L, template DNA50ng, complement ddH₂0 to 15 μ L; the reaction conditions of PCR were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 32 times; fully extend the temperature of 72 ℃ for 5 min. Finally, storing at 4 ℃.

(3) Fertility field survey

In full bloom, on (8029A × Zhongshui 11) BC₄And (8029A X6449) isolation of each individual plant of the population for fertility investigation.

(4) Genotyping of part of Individual plants in segregating populations

Respectively from (8029A × medium double 11) BC₄And (8029A X6449) in the segregating population 100 individuals were each selected using the primer SNPMS5^a-5F/R、SNPMS5^c-3F/R and SNPMS5^ePCR amplification was performed at-1F/R.

(5) As a result, the

For (8029A × middle double 11) BC₄The detection results of 100 isolated plants of the segregating population are as follows: the primer SNPMS5 can be used by 52 individuals^eAmplifying a 150bp band by-1F/R; the primer SNPMS5 can be used for all the individuals (100 strains)^cAmplifying a 140bp band by 3F/R; all individuals could not use the primer SNPMS5^aBands were amplified at-5F/R. The field fertility investigation finds that only the primer SNPMS5 can be used^e52 individuals with a 150bp band amplified by-1F/R are sterile plants, and other individuals are fertile plants.

As a result of examining 100 individuals of the (8029A X6449) isolate group, 47 individuals were able to utilize the primer SNPMS5^aAmplifying a 200bp band by 5F/R; all individuals can use the primer SNPMS5^eAmplifying a 150bp band by-1F/R; all individuals could not use the primer SNPMS5^cBands were amplified at-3F/R. The field fertility investigation finds that only the primer SNPMS5 can be used^a47 individuals with a 200bp band amplified by-5F/R are fertile plants, and other individuals are sterile plants.

The above results show that the molecular marker SNPMS5 of the invention^a-5 is closely linked with a nuclear sterile gene restorer gene, and a molecular marker SNPMS5^c-3 is closely linked with a nuclear sterile gene maintenance gene, and a molecular marker SNPMS5^eThe-1 is closely linked with the nuclear sterile gene, and the molecular marker is used for identifying the fertility genotype of the 8029AB material, is accurate and reliable, and can be used as the molecular marker of the nuclear sterile three-line 8029 AB.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

<120> molecular marker for gene assisted breeding related to rape dominant genic male sterility and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cattctttaa cagagatagg tgtc 24

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agactcttga gtacagtctc acg 23

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attgtcccct tgtagcgcgg 20

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atttcgtgaa gatgcaagtt cg 22

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcctcctttg ttgttttcga g 21

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tacattaatc catttattat agttt 25

<210> 7

<211> 981

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgagtaatt ctgcgcctgg agggtcgatg aagaggaagg ggggtgggcg taagcgtaat 60

ccagatgagc taattgttgt tgattccgag tatcgtgcgc caggacgttc atatcggcgc 120

ttggaaaaga agcgagaggc tcgccgtaag gctcagaggg aggagattta tcgaagagaa 180

gaagagacta ttcgtaaggc cgaggaggtt ttttggcgta aagtggatga aacagatggc 240

ttcgatatcg agatcgaggg tgctccttgt tattttggtg gtatgagtgt ttataaaggt 300

ggagtagatt gtccccttgt agtgaagctt tatgcaacgg tgggacttca tcgttacaat 360

atgctagagg ggaccaactt gtatcttcac aaaatagaga aatacgttgt agtttgcacc 420

ctcatgcctg tctcgtataa cattactttg attgctgagg atccagctac ctcctccttt 480

gttgttttcg agactaatgt tgaccaaaga agtttaggcc agattgattt cacttgttat 540

atctcaagac ctaaagggat caagagtgag atttatccga accagttctt cgatgccaag 600

gacttgcctg acaagtggcc ttcaaaggag gcttttgctg atcaaagtcg atttttgtac 660

aagatgcaga aatcagattg ggaagaacat gactggattc gcttgtatat ggaaatttcg 720

ttctttaaca gacataggtg cctgaatcac aacatgtctg atttaaagat tctggacgta 780

gtggtagaga ctgaggaaaa tgtgccacat gagactgtac tcaagagtct taggaatgtc 840

cttgtctaca taagatatga tcaagacttg ggagcggatg gtgtttgcaa acacatagcc 900

attgttcgaa gaactgtcga gccgacaact cactgcgttt gtctcttggg cgagtctcag 960

cttgtgcccg actctgagta a 981

Claims

1. The primer group for amplifying the molecular marker of gene assisted breeding related to rape dominant genic male sterility is characterized in that the molecular marker comprises: and BnMS5^aSNP marker SNPMS5 for gene coseparation^a-5, and BnMS5^cSNP marker SNPMS5 for gene coseparation^c-3 and BnMS5^eSNP marker SNPMS5 with co-separated genes^e-1；

Marker SNPMS5^e-1 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.5^e-1F and downstream primer SNPMS5 with nucleotide sequence shown in SEQ ID NO.6^e-1R；

The primer group comprises the following primers: SNPMS5^a-5F、SNPMS5^a-5R、SNPMS5^c-3F、SNPMS5^c-3R、SNPMS5^e-1F and SNPMS5^e-1R。

2. The primer group of claim 1 for identifying dominant genic male sterility of rapeThe application of related genes is characterized in that the rape dominant karyocyte sterility related genes comprise BnMS5^a、BnMS5^cAnd BnMS5^e。

3. A kit for identifying a dominant genic male sterility related gene of rape, which is characterized by comprising the primer group of claim 1.

4. Use of the primer set of claim 1 for breeding three line rape comprising: rape dominant cell nuclear sterile line, rape dominant cell nucleus temporary maintainer line and rape dominant cell nucleus restoring line;

when breeding rape dominant cell nucleus sterile line, it uses BnMS5 gene with rape dominant cell nucleus sterile gene^eThe sterile material of (2) is female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (1) is hybridized with a male parent, and the SNPMS5 is selected by a backcross, hybridization or tissue culture method^eProgeny of-1 molecular marker with genotype BnMS5^eBnMS5^eOr BnMS5^cBnMS5^eThe sterile line of (3); marker SNPMS5^e-1 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.5^e-1F and a downstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.6^e-1R；

When the dominant cell nucleus temporary maintainer line of rape is bred, the fertile gene BnMS5 with the dominant cell nucleus of rape is used^cThe fertile material of (A) is used as female parent, and the genotype is BnMS5^cBnMS5^cThe maintainer line of (1) is hybridized with a male parent, and the SNPMS5 is selected by a backcross, hybridization or tissue culture method^c-3 progeny of the molecular marker, the genotype of which is BnMS5^cBnMS5^cThe temporary protection series of (1); marker SNPMS5^c-3 comprises an upstream primer SNPMS5 with a nucleotide sequence shown as SEQ ID NO.3^c-3F and downstream primer SNPMS5 with nucleotide sequence shown in SEQ ID NO.4^c-3R；

When breeding rape dominant karyon restorer, rape dominant karyon restorer gene BnMS5 is obtained^aOfUsing breeding material as female parent and using genotype as BnMS5^cBnMS5^cThe material is used as male parent for hybridization, and the method of backcross, hybridization or tissue culture is adopted to select the material with SNPMS5^a-5 progeny of the molecular marker, the genotype of which is BnMS5^aBnMS5^aOr BnMS5^aBnMS5^cThe recovery line of (3); marker SNPMS5^a-5 comprises an upstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.1^a-5F and a downstream primer SNPMS5 with the nucleotide sequence shown as SEQ ID NO.2^a-5R。

5. The use of the primer set of claim 1 for recurrent selective breeding of oilseed rape.