CN114561485B - Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof - Google Patents
Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof Download PDFInfo
- Publication number
- CN114561485B CN114561485B CN202210273636.8A CN202210273636A CN114561485B CN 114561485 B CN114561485 B CN 114561485B CN 202210273636 A CN202210273636 A CN 202210273636A CN 114561485 B CN114561485 B CN 114561485B
- Authority
- CN
- China
- Prior art keywords
- seq
- sequence
- type
- line
- amplified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
-
- 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
- C12Q2600/13—Plant traits
Abstract
The invention belongs to the field of molecular identification methods, and relates to a molecular marker group for identifying a brassica napus cytoplasm three-line, a method and application thereof, wherein the molecular marker group comprises molecular markers of S-quality, N-quality, R-genes and R-genes, primers for identifying the molecular markers of the S-quality and the N-quality are SN-F, N-R and S-R, primers for identifying the molecular markers of the R-genes are R-F and Rr-R, and primers for identifying the molecular markers of the R-genes are R-F and Rr-R. The molecular marker group in the invention can accurately identify rape pol CMS and Shaan 2A cytoplasmic male sterile line, maintainer line, restorer line and hybrid thereof.
Description
Technical Field
The invention belongs to the field of molecular identification methods, and relates to a molecular marker group for identifying and identifying brassica napus cytoplasm three lines, a method and application thereof.
Background
The cytoplasmic male sterile line (Cytoplasmic male sterility, CMS) is a core sterile line for utilizing rape hybrid vigor, opens a new era of utilizing rape hybrid vigor, and has a milestone significance for the development of rape in the world. More than 10 types of cytoplasmic male sterility are found or created at home and abroad, and the types of cytoplasmic male sterility comprise pol CMS, shan 2A CMS, ogu CMS, nap CMS, tour CMS, nca CMS, hau CMS, woad CMS and the like, wherein shan 2A CMS and pol CMS belong to the same type of male sterility. The first internationally useful male sterile line pol CMS of canola was found in 1972, fu Tingdong. 1985 Li Dianrong the first large-area three-line hybrid brassica napus variety Qin oil No.2 used for production in the world was bred using the Shaanxi 2A CMS male sterile line. Through the sustainable development of 50 years, the rape in China comprehensively realizes variety hybridization, wherein pol A and Shaan 2A sterile lines play a vital role in the cultivation of hybrid seeds, and the rape sterile lines still serve as backbone sterile lines for utilizing rape heterosis for a long time in the future, thereby playing an important role in the hybrid utilization of the rape continuously. The identification of the cytoplasmic type of the restorer line needs to take the restorer line as a female parent, the restorer line is pollinated by the maintainer line, if the F2 generation plants are all normal fertile plants, the restorer line is N-quality, if 1/4 sterile plants exist in the population, the restorer line is S-quality, so that the restoration and maintenance relationship of the identification of a rape material takes 1-3 years, the labor and the time are wasted, and the cultivation of new varieties is seriously influenced. Therefore, the development of molecular markers has great significance for the cultivation of new varieties of rape.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art, and provides a molecular marker group for identifying three cytoplasmic lines of brassica napus, a method and application thereof, wherein the molecular marker group can be used for rapidly identifying fertility cytoplasm N, sterile cytoplasm S, fertility gene R and sterile gene R, can be used for accurately identifying male sterile lines, maintainer lines, restorer lines and hybrid seeds of cytoplasmic male sterile lines of brassica napus pol CMS and shan 2A, can be applied to identification of restoring and maintaining relations of rape materials, and can be used for rapidly identifying seed purity of three cytoplasmic hybrid seeds.
The invention provides a molecular marker group for identifying a brassica napus cytoplasm three-line, which comprises molecular markers of S-mass, N-mass, R-gene and R-gene;
identifying the primers of the molecular markers of the S quality and the N quality as SN-F, N-R and S-R;
the primers for identifying the molecular markers of the R genes are R-F and Rr-R;
the primers for identifying the molecular markers of the R gene are R-F and Rr-R;
the sequence of the primer SN-F gene is shown as SEQ ID NO.1, the sequence of the primer N-R gene is shown as SEQ ID NO.2, and the sequence of the primer S-R gene is shown as SEQ ID NO. 3;
the primer R-F gene sequence is shown as SEQ ID NO.4, the primer R-F gene sequence is shown as SEQ ID NO.5, and the primer Rr-R gene sequence is shown as SEQ ID NO. 6.
Further, when the primers SN-F, N-R and S-R are used for amplification in a mixed mode, S can amplify a clear band with the length of 1287bp shown in SEQ ID NO.7 or can amplify two bands with the length of 1287bp shown in SEQ ID NO.7 and 655bp shown in SEQ ID NO.8, wherein the band with the length of 1287bp is thick and clear, the band with the length of 655bp is thin and lighter, and N can amplify the clear band with the length of 655bp shown in SEQ ID NO. 8.
Further, the S-mass gene sequence is shown as SEQ ID NO.7, and the N-mass gene sequence is shown as SEQ ID NO. 8.
Further, the sequence of the R gene is shown as SEQ ID NO. 9.
Further, the R band amplified by the primers R-F and Rr-R is formed by mixing an R1 segment and an R2 segment with the sizes of 734bp, wherein the sequence of R1 is shown as SEQ ID NO.10, and the sequence of R2 is shown as SEQ ID NO. 11.
The invention also provides application of the molecular marker group in three-line identification of cabbage type rape cytoplasm.
Further, the three lines are a sterile line, a maintainer line and a restorer line.
The invention also provides a method for identifying the cytoplasmic genotype of the brassica napus, which comprises the following steps:
the rape DNA is used as a template, three sets of primers of SN-F/N-R/S-R, R-F/Rr-R and R-F/Rr-R are respectively amplified, then the result is judged, and the result identification process is as follows:
the cytoplasmic type of the sterile line is identified as S quality, R bands cannot be amplified, only 1R band of 734bp can be amplified, and the identification result is S (R);
the cytoplasm type of the maintainer line is identified as N, R bands cannot be amplified, only 1R band 734bp can be amplified, and the identification result is N (R);
the restorer line identifies 2 cytoplasmic types, one is S type and the other is N type, R bands cannot be amplified, only 1R band 734bp can be amplified, and the restorer line identification results comprise S (R R) and N (R R) 2 types;
the cytoplasm type of the hybrid is identified as S-shaped, 1R band of 734bp and 1R band of 734bp can be amplified simultaneously, and the identification result is S (R).
Furthermore, the molecular marker set is used for purity identification of Pol A and shan 2A cytoplasmic three-line type hybrid.
Further, the hybrid judgment criteria were: r bands are detected in the sample, the restorer plants are judged, only R bands are detected in the sample, the sterile plants are judged, R and R bands are detected in the sample, and the hybrid seeds are judged.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention develops a set of molecular markers for identifying the fertility cytoplasm N, the sterility cytoplasm S, the fertility gene R and the sterility gene R by utilizing the difference of gene sequences among the cytoplasmic sterile lines, the maintenance lines and the restoring lines of the cabbage type rape, and can accurately identify the cytoplasmic male sterile lines, the maintenance lines, the restoring lines and the hybrid seeds of the rape pol CMS and the Shaan 2A by combining the molecular marker sets, thereby being applicable to the identification of the restoring and maintaining relationship of rape materials and simultaneously being used for the rapid identification of the seed purity of the cytoplasmic three-line hybrid seeds.
2. The invention provides a molecular marker group for identifying cytoplasmic male sterility of brassica napus, which can rapidly identify genotypes of a sterile line S (R, R), a maintainer line N (R, R), a restorer line S/N (R, R) and a hybrid S (R, R) in cytoplasmic male sterility application of the brassica napus;
can rapidly determine and identify cytoplasmic male sterile lines, maintainer lines, restorer lines and hybrid seeds of rape pol A and Shaan 2A types; can simply, quickly and accurately identify the recovery and conservation relation of rape germplasm resources and the purity of rape hybrid seeds of pol A and Shaan 2A types, and has good production applicability.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows molecular marker identification of sterile cytoplasm (S) and fertile cytoplasm (N) of rape according to the present invention;
wherein, figure A shows the S-quality and N-quality primer design schematic diagram;
the diagram B shows the identification result of S-mass and N-mass molecular markers;
FIG. 2 shows the identification result of the molecular marker set of the invention on the cytoplasmic genotype of brassica napus;
FIG. 3 shows the result of rapid identification of rape germplasm resources by a molecular marker group in the invention;
wherein 3, 14 are restorer lines; 6, 11 are sterile lines; the rest is hybrid seeds; m is 2000marker;
FIG. 4 shows the results of purity identification of the molecular marker sets of the three-line type of Pol A and shan 2A cytoplasmic hybrid according to the present invention.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1
1. Design of three-line molecular marker of cabbage type rape cytoplasm
Sequence analysis was performed on cytoplasmic genes of the rape cytoplasmic male sterile line, and 3 primers, SN-F, N-R, S-R (Table 1), were designed based on the in-del differences on the S-cytoplasmic and N-cytoplasmic mitochondrial DNA. SN-F and N-R primer pairs are used for N quality identification, and the amplification band is 655bp; the primer pair SN-F and S-R is used for S quality identification, and 1287bp of the band is amplified. Because the difference between the amplified fragments of N and S is large, 3 primers are mixed into the same reaction system for PCR amplification, so that the S and N quality can be clearly separated. PCR reaction System (16 ul): DNA template, 1ul;2×taq Mix (century well), 8ul; 0.5ul each of the SN-F, N-R primer and the S-R primer; ddH 2 0，5.5ul。
Sequence analysis was performed on the cytoplasmic male sterile nuclear gene R of rape and the restorer gene R thereof, and 3 primers, namely, R-F, R-F and Rr-R (Table 1), were designed based on the SNP difference between the two genes. R-F and Rr-R primer pairs for R-gene identification, and R-F and Rr-R primer pairs for R-and R-amplification, the PCR amplification and electrophoresis are separately performed because the R and R amplified bands are the same size. PCR reaction System (16 ul): DNA template, 1ul;2×Taq Mix,8ul; primer 1 and primer 2 each 0.5ul; ddH 2 0，6ul。
PCR procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 40s, annealing at 60℃for 40s, extension at 72℃for 40s, cycle number 30, extension at 72℃for 10min. The PCR product was detected by electrophoresis on a 1% agarose gel at 140V for 15min. The N/S and R identified Tm values were 60℃and R identified Tm values were 61 ℃.
TABLE 1 rape cytoplasmic male sterile three-line molecular marker identification primer list
The gene sequence of the amplified S substance (1287 bp) in the table 1 is shown as SEQ ID NO.7, the gene sequence of the N substance (655 bp) is shown as SEQ ID NO.8, the gene sequence of the amplified R gene (734 bp) is shown as SEQ ID NO.9, the band of the amplified R gene (734 bp) is formed by mixing R1 and R2, the gene sequence of the R1 band is shown as SEQ ID NO.10, and the gene sequence of the R2 band is shown as SEQ ID NO. 11.
2. Molecular marker identification of cabbage type rape cytoplasm type
PCR amplification is carried out by taking N-substance and S-substance rape DNA as templates, and the result shows that: the three references SN-F, S-R and N-R are mixed and used, so that N quality and S quality can be clearly distinguished. The N rape can amplify a 655bp clear band (FIG. 1B, N), the S rape can amplify a 1287bp clear band (FIG. 1B, S), and the two bands can accurately distinguish the N from the S. In addition, S-type rape has 2 types of bands: only 1287bp clear band (FIG. 1B, S-type I), which was small in the identified sample; another type has two bands of 1287bp and 655bp, the 1287bp band is thick and clear, the 655bp band is thin and pale (FIG. 1B, S matter II), and the majority of the identified rape germplasm belongs to this type.
3. Identification of sterile line, maintainer line, restorer line and hybrid thereof by cytoplasmic three-line molecular markers
The cytoplasmic male sterile line, the maintainer line, the restorer line and the hybrid thereof of the rape are identified by the combined use of S/N, R, r molecular markers, and the results are shown in figure 2: the cytoplasmic type of the sterile line is S-shaped, R bands are not contained, only R bands are included, the identification result is S (R R), wherein the R bands are 2 mixed bands of R1 and R2 which are identical in size and 734 bp; the cytoplasm type of the maintainer line is N, does not contain R bands, only has R bands, and the identification result is N (R); the restorer line has two cytoplasmic types, one is an S-type and one is an N-type, does not contain R bands and only has R bands, so that the restorer line identification result has S (R R) and N (R R) 2 types; the hybrid cytoplasm type is S-type, contains R and R bands, and the identification result is S (R). The identification result shows that the CMS male sterile line, the maintainer line, the restorer line and the hybrid thereof can be accurately identified by the molecular marker combination of S/N, R, r.
4. Rapid identification of rape germplasm resources by cytoplasmic three-line molecular markers
In order to verify the accuracy of the cytoplasmic male sterile three-line molecular markers, sterile lines (No. 4 and No.6 samples), maintainer lines (No. 2 samples) and restorer line materials (No. 1, no.3 and No.5 samples) are respectively selected in the flowering phase of rape, and PCR identification is carried out by using S/N, R and r molecular markers, wherein the results are shown in FIG. 3, the No.4 and No.6 samples only have r bands, the No.4 sample is an S quality I type band, the No.6 sample is an S quality II type band, and the identification results are all sterile lines S (r r); sample No.2 also had only r bands, but exhibited an N-band, identified as maintainer line N (r); the samples No.1, no.2 and No.5 all have R bands, the sample No.1 presents N-type band, the sample No.3 is S-type II band, the sample No.5 is S-type I band, the identification result No.1 is N (R R) restorer line, and the samples No.3 and No.5 are S (R R) restorer line. The identification results are actually consistent, the identities of the sterile line, the maintainer line and the restorer line of the detected materials are accurately determined, and the S or N quality type is accurately identified for the restorer line, so that the set of molecular markers can be used for rapidly and accurately identifying the three-line materials of the cytoplasm of the rape, thereby replacing the traditional measuring and restoring link and shortening the cultivation period of new varieties of the rape.
5. Rapid identification of purity of rape hybrid by using cytoplasmic three-line molecular markers
The method can accurately and rapidly identify the purity of the rape hybrid seeds, and has very important significance for reducing seed quality risks of seed enterprises, ensuring seed quality safety and maintaining peasant rights and interests. At present, the main body of rape hybrid in China is still a cytoplasmic three-line hybrid of Pol A and Shaan 2A types. The brassica napus cytoplasmic three-line molecular marker can be used as a universal marker for purity identification of Pol A and shan 2A cytoplasmic three-line type hybrid.
Hybrid species are typically composed of 3 types of seeds: class 1 is hybrid seed; the 2 nd class is sterile line seeds; class 3 is the seed of the male restorer. In purity identification, we generally only use R and R molecular markers for purity identification for cost saving. Extracting DNA from rape hybrid seedlings sprouting for 5 days, and respectively carrying out PCR amplification on detection samples by using R and R molecular markers, wherein the results show that the detection samples have R strips only in the samples No.3 and No. 14, and the detection samples are judged to be restorer plants; the samples 6 and 11 had only R bands, and were judged as sterile line plants, while the remaining 11 samples had both R and R bands, and were judged as hybrid (fig. 4), with a hybrid ratio of 73.3%, a sterile line ratio of 13.3%, and a restorer line ratio of 13.3%. Therefore, the set of cytoplasmic three-line molecular markers can be used as universal markers for rapidly identifying the seed purity of the Pol A and shan 2A type three-line hybrid seeds, and the identification period is less than 1 week, so that the seed purity identification time is greatly shortened, and the quality safety of rape seeds can be effectively ensured.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> Shaanxi province hybrid rape research center
<120> molecular marker group for identifying brassica napus cytoplasmic three lines, method and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Synthesis
<400> 1
ttcatacggc gagagtcatt g 21
<210> 2
<211> 25
<212> DNA
<213> Synthesis
<400> 2
caagaccata gaataggaga accac 25
<210> 3
<211> 22
<212> DNA
<213> Synthesis
<400> 3
gctcgttcgg ctacttatct tg 22
<210> 4
<211> 21
<212> DNA
<213> Synthesis
<400> 4
gggatgcgat cctgatattt g 21
<210> 5
<211> 20
<212> DNA
<213> Synthesis
<400> 5
gggatgcgat cctgatatcg 20
<210> 6
<211> 21
<212> DNA
<213> Synthesis
<400> 6
ctccaaaagg accagaaagc a 21
<210> 7
<211> 1287
<212> DNA
<213> Synthesis
<400> 7
ttcatacggc gagagtcatt ggttcaaatc caatagtagg taaaaccggc cgaaacccct 60
gctttttcca gcatgacagc aagcacggac tggcagcaag gatgaaactg aatgaccaaa 120
gcatcggaaa agcagaggag tgtgctgact tcagtagcta gtcaaccaac ttctgctcaa 180
ccctaggaac aaggaaagta tcagccgata actctgttca cttggtgtgg gattcctaaa 240
agtttactat tatcgcacgc caaagagtgt gctgaaaagg gaatagttgg ttaactaccg 300
aagctagaaa caaatacctg ctaaaaccaa gggaagcaag agtaggaact acccacccag 360
gaatcagcat tctaaactgc agcacaagca caaccctgct tttccattcc ttcatagccc 420
acctcttcat tggccataaa gccttgattt ccttggacca cgtacactga tgattggtcg 480
ggctgggcta ctctatgggc gtatgcgcct aaaccaggct caaagcgatg agacttctcg 540
gttagactca aacaatagat caggtcagtt ttgaggagct ttatttgact taagaagaaa 600
gggcagccct ctgatatatc tttcgacttc accctcggcc aaagcacagt aagttagaga 660
ctcctagtct ctactaaatc aatctatagc ttcaacactc tcctagcgca gcaaccttct 720
ctctttctcc cttacttgcc tcccagtctt gagcagcaat catctttgct tatgcggacg 780
aaagtctttc cacagcactc cgacagaagt ctcatatgat gactctgttt actcaagagc 840
cttctttctc gattaaatgc ctgcccacta ccccaagcaa gccgcatagg tctttgttag 900
cagctaggct tctgtttctt tccataagcg acttctcctg tagatgccca aaataaagct 960
ttcgctcaag ggatatcgct tcatttactg cgttagggtg gtgccaagcc cttcatctct 1020
ggctaaggct cagtcagata ggctcaatat cgggcttatc cggcgaagtc tcaaactctg 1080
ttgtgccagg gatatcaggc ttgagttggg acaggtgctt atttcgatac cgcttctgtt 1140
tgttgttttt gtgatgctag tgatcacatt tacagttaga agtaggccat gattgattag 1200
ctagagtaga gctaggttgg tgctgctttg cttggcaatc cttgatctaa ttgaatcaga 1260
atccgcaaga taagtagccg aacgagc 1287
<210> 8
<211> 655
<212> DNA
<213> Synthesis
<400> 8
ttcatacggc gagagtcatt ggttcaaatc caatagtagg taaaaccggc cgaaacccct 60
gctttttcca gcatgacagc aagcacggac tggcagcaag gatgaaactg aatgaccaaa 120
gcatcggttg cttccacttg tggccttctt cgaccgcctt gacaccttac ttaatatcaa 180
ggcagcccac atacatgaag agaagaattg ggtttagcgg taatggcata agccaaagct 240
agatcaccgg agtttaggca aatgggtacg agagaaaaag gttttgaaat gcatcttccg 300
aaaccagata atcaaccgaa ggagattccc ctgcaaaaag gagatggctc tagtttcata 360
ctaatcttcg atcttcgaga ttcccccacg gagcggtaag gcatcccaag gagcgaagcg 420
gctcaaagga ttggcaatga agctcagtct ggtctgaggg gatgggctag gctactctcc 480
ccatacatag gaagaggggg aagtcaagta cctagatcca agatcacgat atccgctttg 540
gtggtaatat ccggcataaa agaaagcacc aaagcaaaca aagtcgattc tttgttgagt 600
caactggcaa ctgggattgc agcggatcta gtggttctcc tattctatgg tcttg 655
<210> 9
<211> 734
<212> DNA
<213> Synthesis
<400> 9
gggatgcgat cctgatattt ggacgtataa tatccttata aacggatttt gtaaggctaa 60
acaggttgat gatggtatga gactattccg aaagatgtct ctgagaggaa tgattgcaga 120
tacagtgact tatagcactc tcatccaagg gttttgtcaa tcaagaaaac ttattgtcgc 180
caaaaaagtc ttccaagaga tggtctctca aggtgttcat cctggtatta tgacttatgc 240
tattttgctg gatgggttgt gtgacaatgg cgaactagaa gaggctttgg gaatacttga 300
tcaaatgcac aagtgtaaga tggaacttga tattggtata tataatatca tcattcacgg 360
gatgtgcaat gcaaataagg tcgatgatgc ttggagtttg ttctgtagcc tctctctcaa 420
aggagtgaag cgtgatattc agtcatacaa cataatgttg tcaggattat gtaagaggag 480
ctcattgtct gaagcggatg cattgtttag aaaaatgaag gaagatgggt atgagccaga 540
tggttgtacg tacaatacac ttatcagagc acatcttcga ggtaatgaca taacaacttc 600
agttcaactc attgaagaaa tgaagaggtg tgggttctct tcagatgctt ccaccgtaaa 660
gattgttatg gatatgttat cgagtggtga attggacaaa agctttctaa atatgctttc 720
tggtcctttt ggag 734
<210> 10
<211> 734
<212> DNA
<213> Synthesis
<400> 10
gggatgcgat cctgatatcg tgacgtttaa tgtccttata aacggatttt gtaaggctaa 60
acaggttgat gttggtatga gactattccg aaagatgtct ctgagaggag tgattgcaga 120
tacagtgact tatagcactc tcatccaagg gttttgtcaa tcaagagaac ttattgtcgc 180
caaagaagtc ttccaagaga tggtctctca aggtgttcat cctggtatta tgacttatgg 240
tattttgctg gatgggttgt gtgacaatgg cgaactagaa gaggctttgg gaatacttga 300
tcaaatgcac aagtgtaaga tggaacttga tattggtata tatagtatca tcattcatgg 360
gttgtgcaat gcaagtaaga tcgatgatgc ttgggatcta ttctgtagcc tctctctcaa 420
aggagtgaag cgtgatattc agtcatacaa cataatgttg tcaggattat gtaaaaggag 480
ctcattgtct gaagcggatg cattgtttag aaaaatgaag gaagatgggt atgagccaga 540
tggttgtacg tacaatacac ttatcagagc acatcttcga ggtagtgaca taacaacttc 600
agttcaactc attgaagaaa tgaagaggtg tgggttctct tcagatgctt ccaccgtaaa 660
gattgttatg gatatgctat caagtggtga attggacaaa agctttctaa atatgctttc 720
tggtcctttt ggag 734
<210> 11
<211> 734
<212> DNA
<213> Synthesis
<400> 11
gggatgcgat cctgatatcg tgacttttaa tgtccttata aacggatttt gtaaggctaa 60
acaggttgat gatggtatga gactattccg aaagatgtct ctgagaggaa tgattgcaga 120
tacagtgact tatagcactc tcatccaagg gttttgtcaa tcaagaaaac ttattgtcgc 180
caaaaaagtc ttccaagaga tggtctctca aggtgttcat cctggtatta tgacttatgc 240
tattttgctg gatgggttgt gtgacaatgg cgaactagaa gaggctttgg gaatacttga 300
tcaaatgcac aagtgtaaga tggaacttga tattggtata tataatatca tcattcacgg 360
gatgtgcaat gcaaataagg tcgatgatgc ttggagtttg ttctgtagcc taccttcgaa 420
aggagtgaag cgtgatattc agtcatacaa cataatgttg tcaggattat gtaagaggag 480
ctcattgtct gaagcggatg cattgtttag aaaaatgaag gaagatgggt atgagccaga 540
tggttgtacg tacaatacac ttatcagagc acatcttcga ggtaatgaca taacaacttc 600
agttcaactc attgaagaaa tgaagaggtg tgggttctct tcagatgctt ccaccgtaaa 660
gattgttatg gatatgttat cgagtggtga attggacaaa agctttctaa atatgctttc 720
tggtcctttt ggag 734
Claims (1)
1. The application of the primer for detecting the molecular marker group in identifying cytoplasmic male sterile lines, maintainers, restorer lines and hybrid seeds of rape pol A and shan 2A types is characterized in that the molecular marker group comprises molecular markers of S-type, N-type, R-type and R-type genes;
identifying the primers of the molecular markers of the S quality and the N quality as SN-F, N-R and S-R;
the primers for identifying the molecular markers of the R genes are R-F and Rr-R;
the primers for identifying the molecular markers of the R gene are R-F and Rr-R;
the sequence of the primer SN-F is shown as SEQ ID NO.1, the sequence of the primer N-R is shown as SEQ ID NO.2, and the sequence of the primer S-R is shown as SEQ ID NO. 3;
the sequence of the primer R-F is shown as SEQ ID NO.4, the sequence of the primer R-F is shown as SEQ ID NO.5, and the sequence of the primer Rr-R is shown as SEQ ID NO. 6;
when the primers SN-F, N-R and S-R are mixed for amplification, the S mass amplifies a clear band with 1287bp length, the gene sequence of which is shown as SEQ ID NO.7, or amplifies two bands with 1287bp and 655bp, the gene sequence of which is shown as SEQ ID NO.7, the gene sequence of which is shown as SEQ ID NO.8, and the N mass amplifies a clear band with 655bp length, the gene sequence of which is shown as SEQ ID NO. 8;
the sequence of the R gene is shown as SEQ ID NO. 9;
the primer R-F and the R-R amplified R band are formed by mixing R1 fragments and R2 fragments with the sizes of 734bp, wherein the sequence of R1 is shown as SEQ ID NO.10, and the sequence of R2 is shown as SEQ ID NO. 11;
the rape DNA is used as a template, three sets of primers of SN-F, N-R, S-R, R-F, rr-R and R-F, rr-R are respectively amplified, then the result is judged, and the result identification process is as follows:
the cytoplasmic type of the sterile line is identified as S-quality, R bands cannot be amplified, and only 1R band of 734bp can be amplified;
the cytoplasm type of the maintainer line is identified as N, R bands cannot be amplified, and only 1R band 734bp can be amplified;
the restorer line identifies 2 cytoplasmic types, one is S type and the other is N type, R bands cannot be amplified, and only 1R band of 734bp can be amplified;
the cytoplasm type of the hybrid is identified as S-shaped, and 1R band of 734bp can be amplified simultaneously.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210273636.8A CN114561485B (en) | 2022-03-19 | 2022-03-19 | Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210273636.8A CN114561485B (en) | 2022-03-19 | 2022-03-19 | Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114561485A CN114561485A (en) | 2022-05-31 |
| CN114561485B true CN114561485B (en) | 2023-09-29 |
Family
ID=81719651
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210273636.8A Active CN114561485B (en) | 2022-03-19 | 2022-03-19 | Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561485B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011020698A1 (en) * | 2009-08-03 | 2011-02-24 | Nickerson International Research | Brassica plant for restoring fertility in an ogura cytoplasmic male-sterility system, method for producing same, and use of said plant |
| CN105002290A (en) * | 2015-08-06 | 2015-10-28 | 华中农业大学 | Molecular detection kit and application for brassica plant cytoplasmic type |
| CN107129991A (en) * | 2016-02-29 | 2017-09-05 | 华中农业大学 | Cabbage type rape pol cytoplasmic male sterility (pol cms) restoring gene Rfp and its application |
-
2022
- 2022-03-19 CN CN202210273636.8A patent/CN114561485B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011020698A1 (en) * | 2009-08-03 | 2011-02-24 | Nickerson International Research | Brassica plant for restoring fertility in an ogura cytoplasmic male-sterility system, method for producing same, and use of said plant |
| CN105002290A (en) * | 2015-08-06 | 2015-10-28 | 华中农业大学 | Molecular detection kit and application for brassica plant cytoplasmic type |
| CN107129991A (en) * | 2016-02-29 | 2017-09-05 | 华中农业大学 | Cabbage type rape pol cytoplasmic male sterility (pol cms) restoring gene Rfp and its application |
Non-Patent Citations (1)
| Title |
|---|
| 甘蓝型油菜陕2A细胞质雄性不育的遗传及RAPD标记;张明龙等;《核农学报》;20041231(第06期);第427-430页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561485A (en) | 2022-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106967803B (en) | High-throughput molecular marker for detecting fertility restorer gene of radish Ogura-CMS (fertility restorer gene) and application of high-throughput molecular marker | |
| CN107988420B (en) | Molecular marker of maize male nuclear sterility gene ms39 and application thereof | |
| WO2021017608A1 (en) | Indel marker for simultaneously identifying cotton cytoplasmic male sterility restoring genes rf1 and rf2 | |
| CN112725503B (en) | Molecular marker closely linked with hypocotyl color related gene of American pumpkin and application | |
| CN106868135B (en) | Primer and method for identifying soybean male sterile line | |
| CN104805212B (en) | PCR label for screening fertility restorer gene Rfo of Ogura cytoplasmic sterile line of Brassica oleracea var.capitata L. | |
| CN105256031B (en) | Utilize the method and its primer special of high-throughput molecular labeling transformation muskmelon female series | |
| CN113637789B (en) | Wheat stripe rust resistance gene YrTD121 linked KASP molecular marker, primer, kit and application | |
| CN103160599A (en) | Method for identifying soybean cytoplasmic male sterile line seed purity through molecular marker | |
| CN114561485B (en) | Molecular marker group for identifying brassica napus cytoplasm three lines, and method and application thereof | |
| Gong et al. | Development and identification of a novel wheat–Thinopyrum scirpeum 4E (4D) chromosomal substitution line with stripe rust and powdery mildew resistance | |
| CN108157165A (en) | A kind of method using Rice Genomics technology fast accurate selection and breeding three lines paddy sterile line | |
| CN110331222B (en) | Molecular marker related to cotton fertility restoration and application thereof | |
| CN102690812A (en) | Molecular marker SIsv0067 closely linked with Setaria italica L. Beauv. heading stage gene | |
| CN116814673A (en) | Genomic structural variation for regulating and controlling content of soluble solids in tomato fruits, related products and application | |
| CN114752702B (en) | Molecular marker BnCa-2C2 closely linked with rape calcium content trait QTL and application thereof | |
| CN117025812A (en) | Primer for amplifying molecular marker closely linked with wheat powdery mildew resistance gene PmCG15-009 and application thereof | |
| CN110468225A (en) | Capsicum cytoplasmic male sterility restores the relevant SNP marker of character and its specific primer and application | |
| CN113151543B (en) | Primer group, method and kit for rapidly identifying calla seedling types by using SSR markers and application of primer group, method and kit | |
| CN105986019B (en) | A kind of the rice extensively identification of affine recessive gms line and the method for utilizing | |
| CN110373487B (en) | InDel marker related to peppery taste character of pepper and application thereof | |
| CN108950050B (en) | Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)10 of Dongfu-type rice, application and detection method | |
| CN108707612B (en) | Gene related to radish late bolting character and application thereof | |
| Du et al. | A novel SCAR marker for detecting Psathyrostachys huashanica Keng chromatin introduced in wheat | |
| CN102154284B (en) | Molecular marker SIsv0408 capable of being closely linked to Setaria italica pollen color gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |