CN103981177B - The molecular marker of cabbage cytoplasm male sterility restoring gene and application - Google Patents
The molecular marker of cabbage cytoplasm male sterility restoring gene and application Download PDFInfo
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Abstract
The invention discloses one and can educate the closely linked molecular marker of gene and application thereof with cabbage cytoplasm male sterility, the closely linked molecular marker of gene be should can educate with cabbage cytoplasm male sterility and Chinese cabbage CMS7311 restoring gene both sides InDel78 labelling closely linked with it and restorer sequence SCAR42 labelling were in, wherein molecular marker InDel78 and SCAR42 is respectively 0.91cM and 0.92cM with the genetic distance of restoring gene, is defined in by restoring gene in the range of one 337kb of Chinese cabbage A09 chromosome.The two molecular marker can be applied not only to the molecular marker assisted selection of Chinese cabbage CMS7311 restoring gene, improves efficiency of selection, accelerates breeding process；Also the molecular mechanism for map based cloning, announcement cytoplasmic male sterility and the fertility restorer thereof of further restoring gene lays the foundation.
Description
Technical field
The invention belongs to the plant molecular marker assistant breeding field of agricultural, relate to male not with a kind of cabbage cytoplasm
Educate and can educate the closely linked molecular marker of gene and application.
Background technology
Chinese cabbage (Brassica campestris L.ssp.pekinensis (Lour.) Olsson) is Cruciferae rue
A kind of sedge belongs to one of most important vegetable crop in Chinese cabbage kind, originates in China, is China or even worldwide bulk vegetable.As far back as last century
The nineties, Academy of Agricultural Sciences of Shaanxi Province vegetable or flower institute just by intervarietal hybridization and baclccrossing techniques cabbage type rape C MS
Middle transformation to Chinese cabbage is bred as CMS7311 (Zhang Lugang, Hao Dongfang, Chinese cabbage temperature sensitive cells matter male sterility line
The research of CMS7311 sterile changeover, Botany Gazette, 2001,43 (11): 1123-1128), research confirms that this CMS also contains further
There is abnormal open reading frame ORF224, and with part temperature-sensing property；And the recovery that can recover CMS fertility is found in Chinese cabbage
System, determines that Restore gene is monogenic inheritance.Owing to traditional fertility Phenotypic Selection to wait until to bloom, directly observed by field
Complete.For carrying the excellent material of Restore gene, be sterile line by its transformation, it is necessary to through hybridization, selfing, simultaneously with not
Educating is that test cross judges the processes such as its genotype, and operating wastes time and energy, and the cycle is long, and breeding process is slow.In order to accelerate Chinese cabbage
Cytoplasmic male sterilty breeding speed, uses modern biotechnology, carries out molecular mark the most urgent.
Summary of the invention
It is an object of the invention to, it is provided that a kind of cabbage cytoplasm male sterility can educate gene closely linked molecule mark
Note, wherein:
(1) InDel labelling and each 1 of the SCAR mark of Chinese cabbage CMS7311 restoring gene are provided；
(2) InDel labelling and the PCR primer sequence of SCAR mark are provided；
(3) to above-mentioned during offer carries out assisted Selection to cabbage cytoplasm male sterility and restoring gene thereof
The method that labelling carries out applying.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of and cabbage cytoplasm male sterility can educate the closely linked molecular marker of gene, it is characterised in that described
Can to educate the closely linked molecular marker of gene with cabbage cytoplasm male sterility be with to be positioned at Chinese cabbage CMS7311 fertility extensive
Multiple genes both sides InDel78 labelling closely linked with it and restorer sequence SCAR42 labelling, wherein, InDel78 labelling
It is made up of restorer sequence InDel78F and sterile line sequence InDel78S；
Described restorer sequence InDel78F size is 1146bp, and particular sequence is as follows:
5-ATCATATCCTGTCTATTTCGTGCTTCTCCTAAAAGAGTGATCTTAAGACCCAATCATCATGAGAATG
TTTCTAGCTCTCTTCCTTCTCTTGGCCTTGACCACATCTTCAAGTAAGTACCTTTCTTCTCTTCTTGGTATACGGTC
CAATAACAGTATTTAAGATTTTATTTCTATGTCTTTCTTTCTCTCTAGATGCAACTTACTGCCTCTGCAAAGATGGG
ACAGAAGACAACGCACTTCAAGCATCAATAGACTATGTTTGTGGAAAATTAGATTGCAACCCAATCCTTGACAAGGG
TGCTTGTTATCAACCAAACACCATCAAAAGCCACTGTGATTGGGCTGTTAACAGCTACTTCCAGAATGTAGCTCAAG
CCCCTGGAAGCTGCGACTTCTCTGGAACTGCCACTACCAGTCAAAACCCACCTSCATGTAAGTAAAAAAGTTTACAT
TTTGATGCGAAAAGTTGTGGTCTTGATGAGGTTCTTTTGTGCAGATTTGGTTACCGGATGTGTCTATCCTTCAAGCG
CTAGGTAACTAATGATCACTAACACAAACCTAAGCCTAACTCATTTACATGGATATGACTTATAAAAAGTTTGGTGT
GTGTATTAACAGCTCTCCAGGGTCGCTTCCTTCAACTACACCACCACCGGGAACAAAGCAGACAAACGGAACCGTTA
CTCCAACCAACGGTGCTTCTGTTTATCAACACTAGCGCTTCTGATGTAACGTTCGTATGGAGACAAAGACATCCACA
TCGGTCCGTCTTTGGTTCCCTTTTGGGTAGAAAAGGAAGCTTCAGTCGTCATTGTGGTTTATATTTCTGTTGTTGCA
TTTATGTGAAAGTTGCTCTAATTAGAATCTTGAGTAAATAAATAAAATAAAAACAGAGCATTATGATAAAGACTTGT
CAATAAAAAGATACCATATTTTTATAATTTCTAATGATCAATCATATAGACTAAAGCAAAATTTATTCACCATTTTA
GTAGCTTCAACATCCAATTTAGATTATTGTTCTACAACTTCAACAACGCAACTGCCACCAGCATCATTTTGTAGTAA
TCTAATCTACCAGATGTTATTCAGTTAAAAAAAATATACTGTGGCAAAGAAAGAGAAACCTATGTGCCTCGTTATAA C-3；
Described sterile line sequence InDel78S size is 881bp, and particular sequence is as follows:
5-ATCATATCCTGTCTATTTCGTGCTTCTCCTAAAAGAGTGATCTTAAGACCCAATCATCATGAGAATG
TTTCTAGCTCTCTTGCTTCTCTTGGCCTTGACCACATCTTCAAGTAAGTCTTTCTTCTCTTCTTCCTAAAGTCTACA
TTTTGATGAAAAAAGTTGCGGTCTTGATGAGGTCTTTTGTGCAGRTTTGGTTACCGGATGTGTCTATCCTTCAAGCG
CTAGGTAACTAATGATCACTAACACAAACCTAAGCCTAACTCATTTACATGGATATGACTTATAAAAAGTTTGGTGT
GTGTATTAACAGCTCTCCAGGGTCGCTTCCTTCAACTACACCATCACCGGGAACAAACAAGACAAACGGTGCTTCCA
GTTTGGTCATTTCCCCTGCTTTCGCAATCTGTTTATCAACACTAGCGCTTCTGATGTAACGTTCGTGTGGAGACAAA
GACATCCACATGGGTCCGTCTTTGGTTCCCTTTTGGGTGGAAAAGGAAGCTTCAGTGGTCATTGTGGTTTATATTTC
TGTTGTTGCATTTATGTGAAAGTTGCTCTAATTAGAATCTTGAGTAAATAAATAAAATCAAAACAGAGCATTATGAT
AAAGACTTGTCAATAAAAAGATACCATATTATTATAATTTCTAATGATCAATCATATAGACTAAAAATTTATTCACC
ATTTTAGTAGCTTCAACATCCAATTTAGATTATTGTTCTACAACTTCAACAACGCAAGTGCCACCAGACATCATTTT
GTAGTAATCTACCAGTTGTTATTCAGTTAAAACTAAGGTTTCTCTCTTTAGGTGTTAGTTATTCAGTTAAAAGAATA
TACTATGTGGCAAAGAAAGAGAAACCTATGTGCCCGTTATAACA-3
Described restorer sequence SCAR42 mark size is length 458bp, and particular sequence is as follows:
5-GGACCAACTATACTTATCCTGTTACTAATCCTGTTGCCGAGCCTAGATTCTCTTTCTCCTCAAAGAA
ATCTATGAAGGCCGTTCGTTCTCCTATACCGTCACCTCTGAAGATGAACACACAGCCTCTCTTATGGTATTCCTCTC
ACGAAACAGCGACTAACGGCTCCTCTTCTCCTTCTTGCTCCTTGACAAAAACGGCAAGCATTTCCTCTTCCGCTGAT
GAAAACTACACGGAGTTTTTCCCGCAAGAACATTCTGATTCTGGTCTCTTGCAAGATATCGTTCAAGAGTTCTTGAA
GAAGAAACGCAACCAGCCTCCGCCACTGATACCACCACCACCACCACCGCCATCTCCACCGATGGTTGGACATCTTG
AAAACTTCCGTGAATTCTCCGCCAACAGTTTGTTTCAACCGATGGTGGAGACATCAAAATTAGATTGCTACGGAAAC ATTAGT-3。
Described restorer sequence InDel78F, sterile line sequence InDel78S and restorer sequence SCAR42 labelling
The sequence of PCR specificity amplification primer is as follows:
ID78F:5-ATCATATCCTGTCTATTTCGTGCT-3；
ID78R:5-GTTATAACGAGGCACATAGGTTTC-3；
SC42F1:5-GGACCAACTATACTTATCCTGTTACT-3；
P42R:5-ACTAATGTTTCCGTAGCAATCT-3。
Above-mentioned cabbage cytoplasm male sterility can educate the closely linked molecular marker of gene answering in assisted Selection
With, concrete grammar is:
A, use CTAB method extract detected materials genomic DNA, referring in particular to the method for (2006) such as Wang Qi；
B, PCR expand: wherein reaction system is 25ul system, and the content of each component materials is respectively as follows: 20ng template DNA；
2.5ul10×buffer；1.8mM MgCl2；0.25mmol/L dNTP；1pmol/L primer: 1.5U Taq archaeal dna polymerase；
ddH2O polishing, to 25ul, mixing, is centrifuged；Amplification program: 94 DEG C/5min of denaturation, then 94 DEG C/60s, 56 DEG C/45s, 72
DEG C/1min, after 35 circulations, 72 DEG C extend 5min；
C, electrophoresis: take 6 × loading buffer mixing of 6ul pcr amplification product and 2ul, click and enter the agar of 0.8%
In sugar gel, electrophoresis 25min under 150V voltage, observes in gel imaging instrument after EB dyes and takes pictures；
D, judgement:
At the purpose band using primer I D78F/ID78R to amplify 1146bp, then this material is with Chinese cabbage CMS fertility
The probability of Restore gene is 99.09%；
At the purpose band using primer SC42F1/R to amplify 458bp, then this material is with Chinese cabbage CMS fertility restorer
The probability of gene is more than 99.08%；
If two pairs of primers amplify purpose band the most simultaneously, then detected materials has Chinese cabbage CMS fertility restorer base
The probability of cause is 100%.
The present invention can educate the closely linked molecular marker of gene, wherein molecular marker with cabbage cytoplasm male sterility
InDel78 and SCAR42 is respectively 0.91cM and 0.92cM with the genetic distance of restoring gene, is limited by restoring gene
In the range of one 337kb of Chinese cabbage A09 chromosome.Can be applied not only to Chinese cabbage CMS7311 restoring gene
Molecular marker assisted selection, improves efficiency of selection, accelerates breeding process, is also the figure position gram of further restoring gene
Grand, to disclose cytoplasmic male sterility and fertility restorer thereof molecular mechanism lays the foundation.
Accompanying drawing explanation
Fig. 1 is that InDel78 is marked at F2Amplification on individuality；
Fig. 2 is qualification and the sterile plant checking of SCAR42 labelling.
In Fig. 1 and 2, symbol is expressed as:
F: individual plant can be educated；S: sterile individual plant；M:DL2000DNA Marker；Asterisk * represents marker detection result and field
Fertility phenotype is inconsistent.
Fig. 3 is the linkage inheritance figure between restoring gene BcRfp and two labellings.This figure be three-primer (SC42F1,
P42F and P42R) PCR amplification；Wherein, the amplification that purpose fragment is SC42F1 and P42R of 458bp；As comparison,
The larger piece section of about 750bp is P42F and P42R amplification, can be used to indicate the presence or absence of template DNA during PCR.
In figure, F: individual plant can be educated；S: sterile individual plant；M:DL2000DNA Marker；Asterisk represent marker detection result and
Field fertility phenotype is inconsistent.
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Detailed description of the invention
Embodiment 1: the InDels labelling chain with Chinese cabbage CMS7311 restoring gene BcRfp is excavated
(1) structure of segregating population
With by 83～2 Chinese cabbages (see to flat, 1996, " rice field kind autumn vegetable combination pattern ", vegetable, 01:28 page) selfing 5
The Chinese cabbage male sterile restoring line 01S325 that generation is bred as, as male parent, pollinates to male sterility line CMS7311, and obtained F1 plants
Strain all shows as educating.F is built by individual plant selfing2For segregating population, in 258 strain individualities of sowing, field fertility is adjusted
Looking into display, 186 strains are male-fertile, and 72 strains are male sterility individual plant, show that fertility separating ratio meets 3:1 (x through x2 test0 2
=1.013, x0.05,1 2=3.841) fertility restorer dominant gene by Dominant gene of this male sterility line, is shown.
(2) DNA extraction and label screening
(1) detected materials genomic DNA is extracted, referring in particular to the method for (2006) such as Wang Qi by CTAB method；
(2) design of primers: restoring gene has been positioned Chinese cabbage genome A09 dyeing by laboratory early-stage Study result
On body in the physical distance of about 1M.This research, based on existing positioning result and relevant Chinese cabbage genome sequence, designs and synthesizes
A series of primers, are expanded by sterile and Fertile material PCR, check order and identify, find a series of InDel labelling, be respectively as follows:
InDel37, InDel55, InDel78, InDel42, InDel55, InDel60 etc..Genetic analysis shows, two labellings
InDel78 and InDel42 is distributed in fertility restorer gene Rf two ends, and the genetic distance is respectively 0.91cM and 0.92cM, and
Genes of interest is limited in the range of about 337Kb.Wherein InDel78 purpose clip size phase between sterile and Fertile material
Difference about about 265bp, uses 0.8% common agarose gel electrophoresis can separate completely, therefore can be directly used for fertility extensive
The molecular marker of multiple genes.Wherein InDel78 and InDel42 the primer sequence is as follows:
ID78F:5-ATCATATCCTGTCTATTTCGTGCT-3；
ID78R:5-GTTATAACGAGGCACATAGGTTTC-3；
P42F:5-ATGGAAGAAGCCCTAAGAAAGT-3；
P42R:5-ACTAATGTTTCCGTAGCAATCT-3.
Embodiment 2: the SCAR of labelling InDel42 converts and identifies
(1) conversion of SCAR mark
Owing to labelling InDel42 PCR primer in sterile material lacks 12bp than Fertile material, use 0.8% agar
Sugar gel electrophoresis is difficult to efficiently separate both, this labelling can only be converted into SCAR42 labelling, can make a distinction.Can educate and plant
Sequence in strain is following (dashed part is sterile material deletion sequence):
ATGGAAGAAGCCCTAAGAAAGTTCAACGAATCTACCTACTCCTTCATACCCGGTTACGAACCCGACCCGATTCCTCT
AACGAGAATCTTTGCCAATGATGCAAACTCCCCGCAGGTTAACAACACACCGTCTTCAAAGGAGGCAATAGTGACCA
TCACCGGATCTGGCAGGACAAGGTACCGTGGCGTTCGCCGAAGGCCGTGGGGACGTTACGCGGCGGAGATACGTGAT
CCCACGTCGAAGGAGAGACGTTGGCTCGGAACGTTCGATACGGCGGAGCAAGCCGCTTGTGCTTACGACTGTGCAGC
TCGTGAGTTTCGTGGATCTAAGGCTCGGACCAACTATACTTATCCTGTTACTAATCCTGTTGCCGAGCCTAGATTCT
CTTTCTCCTCAAAGAAATCTATGAAGGCCGTTCGTTCTCCTATACCGTCACCTCTGAAGATGAACACACAGCCTCTC
TTATGGTATTCCTCTCACGAAACAGCGACTAACGGCTCCTCTTCTCCTTCTTGCTCCTTGACAAAAACGGCAAGCAT
TTCCTCTTCCGCTGATGAAAACTACACGGAGTTTTTCCCGCAAGAACATTCTGATTCTGGTCTCTTGCAAGATATCG
TTCAAGAGTTCTTGAAGAAGAAACGCAACCAGCCTCCGCCACTGATACCACCACCACCACCACCGCCATCTCCACCG
ATGGTTGGACATCTTGAAAACTTCCGTGAATTCTCCGCCAACAGTTTGTTTCAACCGATGGTGGAGACATCAAAATTAGATTGCTACGGAAACATTAGT。
Based on above deletion sequence, redesign SCAR primer SC42F1, for expanding with original P42R primer combination
Purpose band.
SC42F1 primer sequence is: 5-GGACCAACTATACTTATCCTGTTACT-3.
(2) qualification of SCAR mark
In order to verify the reliability of this SCAR42 labelling, applicant is to F2In generation, separates group's individual plant and is expanded.Amplification knot
Fruit shows, it can not amplify band in sterile, and in Fertile material, is no matter heterozygosis Fertile material or pure and mild educates
Material, the most amplifiable go out purpose band.
It is auxiliary that embodiment 3:InDel78 and SCAR42 two are marked at Chinese cabbage CMS7311 restoring gene
Help the application in selection
(1) extracting detected materials genomic DNA by CTAB method, concrete grammar is the same；
(2) PCR reaction
(1) PCR reaction system: 25ul system, the content of each component materials is respectively as follows: 20ng template DNA；2.5ul10×
buffer；1.8mM MgCl2；0.25mmol/L dNTP；1pmol/L primer: 1.5U Taq archaeal dna polymerase；ddH2O polishing is extremely
25ul, mixing, centrifugal；
(2) PCR amplification program: 94 DEG C/5min of denaturation, then 94 DEG C/60s, 56 DEG C/45s, 72 DEG C/1min, 35 are followed
After ring, 72 DEG C extend 5min；
(3) electrophoresis: take 6 × loading buffer mixing of 6ul pcr amplification product and 2ul, click and enter the agar of 0.8%
In sugar gel, electrophoresis 25min under 150V voltage, observes in gel imaging instrument after EB dyes and takes pictures；
(4) interpretation of result:
At the purpose band using primer I D78F/R to amplify 1146bp, then this material is with Chinese cabbage CMS fertility restorer
The probability of gene is 99.09%；At the purpose band using primer SC42F1/R to amplify about 458bp, then this material is with greatly
The probability of Chinese cabbage CMS restoring gene is more than 99.08%；If two pairs of primers amplify purpose band, then the most simultaneously
It is 100% that detected materials has the probability of Chinese cabbage CMS restoring gene.
Claims (3)
1. can educate the closely linked molecular marker of gene with cabbage cytoplasm male sterility for one kind, it is characterised in that described
The closely linked molecular marker of gene can be educated with cabbage cytoplasm male sterility be and be positioned at Chinese cabbage CMS7311 fertility restorer
Gene both sides InDel78 labelling closely linked with it and restorer sequence SCAR42 labelling, wherein, InDel78 labelling by
Restorer sequence InDel78F and sterile line sequence InDel78S composition；
Described restorer sequence InDel78F size is 1146bp, and particular sequence is as follows:
5-ATCATATCCTGTCTATTTCGTGCTTCTCCTAAAAGAGTGATCTTAAGACCCAATCATCATGAGAATGTTTC
TAGCTCTCTTCCTTCTCTTGGCCTTGACCACATCTTCAAGTAAGTACCTTTCTTCTCTTCTTGGTATACGGTCCAAT
AACAGTATTTAAGATTTTATTTCTATGTCTTTCTTTCTCTCTAGATGCAACTTACTGCCTCTGCAAAGATGGGACAG
AAGACAACGCACTTCAAGCATCAATAGACTATGTTTGTGGAAAATTAGATTGCAACCCAATCCTTGACAAGGGTGCT
TGTTATCAACCAAACACCATCAAAAGCCACTGTGATTGGGCTGTTAACAGCTACTTCCAGAATGTAGCTCAAGCCCC
TGGAAGCTGCGACTTCTCTGGAACTGCCACTACCAGTCAAAACCCACCTSCATGTAAGTAAAAAAGTTTACATTTTG
ATGCGAAAAGTTGTGGTCTTGATGAGGTTCTTTTGTGCAGATTTGGTTACCGGATGTGTCTATCCTTCAAGCGCTAG
GTAACTAATGATCACTAACACAAACCTAAGCCTAACTCATTTACATGGATATGACTTATAAAAAGTTTGGTGTGTGT
ATTAACAGCTCTCCAGGGTCGCTTCCTTCAACTACACCACCACCGGGAACAAAGCAGACAAACGGAACCGTTACTCC
AACCAACGGTGCTTCTGTTTATCAACACTAGCGCTTCTGATGTAACGTTCGTATGGAGACAAAGACATCCACATCGG
TCCGTCTTTGGTTCCCTTTTGGGTAGAAAAGGAAGCTTCAGTCGTCATTGTGGTTTATATTTCTGTTGTTGCATTTA
TGTGAAAGTTGCTCTAATTAGAATCTTGAGTAAATAAATAAAATAAAAACAGAGCATTATGATAAAGACTTGTCAAT
AAAAAGATACCATATTTTTATAATTTCTAATGATCAATCATATAGACTAAAGCAAAATTTATTCACCATTTTAGTAG
CTTCAACATCCAATTTAGATTATTGTTCTACAACTTCAACAACGCAACTGCCACCAGCATCATTTTGTAGTAATCTA
ATCTACCAGATGTTATTCAGTTAAAAAAAATATACTGTGGCAAAGAAAGAGAAACCTATGTGCCTCGTTATAAC-3；
Described sterile line sequence InDel78S size is 881bp, and particular sequence is as follows:
5-ATCATATCCTGTCTATTTCGTGCTTCTCCTAAAAGAGTGATCTTAAGACCCAATCATCATGAGAATGTTTC
TAGCTCTCTTGCTTCTCTTGGCCTTGACCACATCTTCAAGTAAGTCTTTCTTCTCTTCTTCCTAAAGTCTACATTTT
GATGAAAAAAGTTGCGGTCTTGATGAGGTCTTTTGTGCAGRTTTGGTTACCGGATGTGTCTATCCTTCAAGCGCTAG
GTAACTAATGATCACTAACACAAACCTAAGCCTAACTCATTTACATGGATATGACTTATAAAAAGTTTGGTGTGTGT
ATTAACAGCTCTCCAGGGTCGCTTCCTTCAACTACACCATCACCGGGAACAAACAAGACAAACGGTGCTTCCAGTTT
GGTCATTTCCCCTGCTTTCGCAATCTGTTTATCAACACTAGCGCTTCTGATGTAACGTTCGTGTGGAGACAAAGACA
TCCACATGGGTCCGTCTTTGGTTCCCTTTTGGGTGGAAAAGGAAGCTTCAGTGGTCATTGTGGTTTATATTTCTGTT
GTTGCATTTATGTGAAAGTTGCTCTAATTAGAATCTTGAGTAAATAAATAAAATCAAAACAGAGCATTATGATAAAG
ACTTGTCAATAAAAAGATACCATATTATTATAATTTCTAATGATCAATCATATAGACTAAAAATTTATTCACCATTT
TAGTAGCTTCAACATCCAATTTAGATTATTGTTCTACAACTTCAACAACGCAAGTGCCACCAGACATCATTTTGTAG
TAATCTACCAGTTGTTATTCAGTTAAAACTAAGGTTTCTCTCTTTAGGTGTTAGTTATTCAGTTAAAAGAATATACT
ATGTGGCAAAGAAAGAGAAACCTATGTGCCCGTTATAACA-3；
Described restorer sequence SCAR42 mark size is length 458bp, and particular sequence is as follows:
5-GGACCAACTATACTTATCCTGTTACTAATCCTGTTGCCGAGCCTAGATTCTCTTTCTCCTCAAAGAAATCT
ATGAAGGCCGTTCGTTCTCCTATACCGTCACCTCTGAAGATGAACACACAGCCTCTCTTATGGTATTCCTCTCACGA
AACAGCGACTAACGGCTCCTCTTCTCCTTCTTGCTCCTTGACAAAAACGGCAAGCATTTCCTCTTCCGCTGATGAAA
ACTACACGGAGTTTTTCCCGCAAGAACATTCTGATTCTGGTCTCTTGCAAGATATCGTTCAAGAGTTCTTGAAGAAG
AAACGCAACCAGCCTCCGCCACTGATACCACCACCACCACCACCGCCATCTCCACCGATGGTTGGACATCTTGAAAA
CTTCCGTGAATTCTCCGCCAACAGTTTGTTTCAACCGATGGTGGAGACATCAAAATTAGATTGCTACGGAAACATTA GT-3。
2. can to educate the closely linked molecular marker of gene thin in Chinese cabbage for the cabbage cytoplasm male sterility described in claim 1
Application in the assisted Selection of cytoplasmic male sterilty.
Apply the most as claimed in claim 2, it is characterised in that concrete grammar is:
(1) detected materials genomic DNA is extracted by CTAB method；
(2) PCR amplification:
A, reaction system are 25ul system, and the content of each component materials is respectively as follows: 20ng template DNA；2.5ul 10×buffer；
1.8mM MgCl2；0.25mmol/L dNTP；1pmol/L primer: 1.5U Taq archaeal dna polymerase；ddH2O polishing is to 25ul, mixed
Even, centrifugal；
B, PCR amplification program: 94 DEG C/5min of denaturation, then 94 DEG C/60s, 56 DEG C/45s, 72 DEG C/1min, 35 circulation after,
72 DEG C extend 5min；
C, electrophoresis: taking the pcr amplification product of 6ul and 6 × loading buffer mixing of 2ul, the agarose clicking and entering 0.8% coagulates
In glue, electrophoresis 25min under 150V voltage, observes in gel imaging instrument after EB dyes and takes pictures；
D, judgement:
At the purpose band using primer I D78F/ID78R to amplify 1146bp, then this material is with Chinese cabbage CMS fertility restorer
The probability of gene is 99.09%；
At the purpose band using primer SC42F1/P42R to amplify 458bp, then this material is with Chinese cabbage CMS fertility restorer
The probability of gene is more than 99.08%；
If two pairs of primers amplify purpose band the most simultaneously, then detected materials has Chinese cabbage CMS restoring gene
Probability is 100%；
ID78F:5-ATCATATCCTGTCTATTTCGTGCT-3；
ID78R:5-GTTATAACGAGGCACATAGGTTTC-3；
SC42F1:5-GGACCAACTATACTTATCCTGTTACT-3；
P42R:5-ACTAATGTTTCCGTAGCAATCT-3.
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| CN106434696B (en) * | 2016-11-19 | 2022-02-08 | 西北农林科技大学 | Chinese cabbage polCMS fertility restorer gene and detection method and application thereof |
| CN107312870B (en) * | 2017-09-04 | 2021-01-08 | 河南省农业科学院园艺研究所 | Molecular marker closely linked with pepper sterility restoring gene, method and application |
| CN108913800B (en) * | 2018-07-24 | 2021-12-28 | 信阳师范学院 | Chinese cabbage hau CMS sterile cytoplasm specific molecular marker and application thereof |
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| CN1849881A (en) * | 2006-06-05 | 2006-10-25 | 华中农业大学 | Selective breeding method for cabbage cytoplasm male sterile line |
| CN101575603A (en) * | 2009-03-31 | 2009-11-11 | 沈阳农业大学 | SCAR marks of genetic sterile multiple allele Ms of celery cabbage and application thereof |
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| CN1849881A (en) * | 2006-06-05 | 2006-10-25 | 华中农业大学 | Selective breeding method for cabbage cytoplasm male sterile line |
| CN101575603A (en) * | 2009-03-31 | 2009-11-11 | 沈阳农业大学 | SCAR marks of genetic sterile multiple allele Ms of celery cabbage and application thereof |
Non-Patent Citations (2)
| Title |
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| investigation on the sterility changeover of male sterility line CMS7311 in heading chinese Cabbage;zhanglu gang，et al;《Acta botanica sinica》;20011130;第43卷(第11期);1123-1128 * |
| 大白菜CMS7311不育发生相关基因表达研究及育性恢复基因定位;许小勇;《中国博士学位论文全文数据库 农业科技辑》;20140515(第5期);全文 * |
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