CN105420357A - Universal SSR marker tightly linked with dominant genic male sterility genes in broccoli - Google Patents
Universal SSR marker tightly linked with dominant genic male sterility genes in broccoli Download PDFInfo
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Abstract
The invention relates to a universal SSR molecular marker SSR10312a tightly linked with dominant genic male sterility genes in broccoli. A primer pair for amplifying the molecular marker SSR10312a by PCR is as shown in Seq ID No.1-2. The invention further provides application of the SSR molecular marker in a broccoli germplasm resource having dominant genic male sterility genes. The SSR molecular marker can be used for performing assistant selection on a broccoli candidate material in the process of transferring the dominant genic male sterility genes at any period of the broccoli candidate material, has the advantages of high efficiency, few limiting factors and the like, and can be used for improving the efficiency of selecting dominant genic male sterility genes of broccoli and shortening the breeding cycle.
Description
Technical field
The present invention relates to biology field, to particularly relate in a kind of broccoli with dominant genic male sterility gene closely linked versatility SSR marker and identifying the application in broccoli dominant genic male sterility gene.
Background technology
Broccoli (Brassicaoleraceavar.italica) has another name called Caulis et Folium Brassicae capitatae, Italian cabbage mustard, that in Cruciferae Brassica genus, with green bouquet, to be product a kind of is in great demand in the valuable feature vegetables of world market, there is abundant nutrition and unique anticancer function, the dark favor by consumers in general.In recent years, broccoli constantly increases in the cultivated area of China and area, has become the main cultivation vegetables in the provinces such as Zhejiang, Gansu, Yunnan, Hebei and important export vegetable.
While broccoli product demand increases, domestic broccoli seeds market is faced with stern challenge, and this is because China starts late to the research of broccoli, and domestic improved seeds are few, and in production, the kind of application mostly is the F of import
1for hybrid, seed price is expensive, seriously governs the development of China's broccoli industry.
Broccoli hybrid vigour is obvious, utilizes male sterile line preparing hybrid kind and carries out the important channel that cenospecies production is heterosis utilization.At present, in broccoli breeding, mainly use both at home and abroad radish cytoplasmic male sterility (OguraCMS) and male parent to produce cross-fertilize seed, but this cytoplasmic male sterile line exist that nectar amount is few, easy problems such as extremely flower bud, seed production be low under low temperature and cloudy condition when producing cenospecies.
Wild cabbage broccoli seminar of Vegetable & Flower Inst., Chinese Academy of Agriculture Science and Vegetable Research center, Beijing found dominant genic male sterility source DGMS79-399-3 in 1979 from wild cabbage, and obtain national inventing patent, through studying for many years, this sterile source has been used successfully in the breeding of wild cabbage hybrid new breed.For expanding the range of application of DGMS79-399-3 sterile source, the nectar amount that solution OguraCMS material exists in broccoli breeding is few, low temperature is dead flower bud easily, the problems such as seed production is low, the broccoli self-mating system that existing research and utilization is excellent is at present as the male parent that backcrosses, be original sterile source with DGMS79-399-3, backcrossed and directive breeding by many generations, be bred as multiple broccoli dominant genic male sterility system, utilize these sterile lines produce cenospecies time sterile line bloom normally, nectar amount is many, low temperature is dead flower bud not easily, seed production is high, overcome the problems that OguraCMS exists.But dominant genic male sterility ties up in backcross transformation process must carry out the selection of sterile individual plant at the florescence according to fertility, time and effort consuming, greatly reduces the efficiency of breeding.And filter out and will contribute to improving breeding efficiency with the cress molecule marker that especially broccoli fertile gene is chain.
At present, with the chain molecule marker of cress fertile gene at turnip type rape (Miao Ying etc., " application bulked-DNA finds the RAPD mark of turnip type rape Male sterile gene ", Xiamen University's journal (natural science edition), 39 (5): 682-685, 2000), Chinese cabbage (Deng Xiaohui etc., " the RAPD analysis of Chinese cabbage Pol cytoplasm male sterility line and its maintenance line and the screening of molecule marker ", Agricultural University Of Nanjing's journal, 1st phase, 2007), (landblink is far away for swede type rape, " molecule marker of brassica napus dominant genic male sterile gene and suppressor gene and application thereof ", Hua Zhong Agriculture University's master thesis, 2004) etc. have been reported in various crop.RAPD (the Wang Xiaowu etc. with wild cabbage dominant genic male sterility material DGMS79-399-3 gene linkage have been obtained in wild cabbage, " one RAPD mark " chain with wild cabbage Dominant Male Sterility Gene, gardening journal, 2nd phase, 1998), SCAR (Wang Xiaowu etc., " SCAR mark for wild cabbage Dominant Male Sterility Gene transformation assisted Selection ", gardening journal, 2nd phase, 2000), RFLP (Liu Yumei etc., " one RFLP mark " chain with wild cabbage Dominant Male Sterility Gene, gardening journal, 5th phase, 2003) mark such as.
But these mark versatilities above-mentioned are all poor, and be only applicable to the individual groups of research separately, and these marks are all single flanking markers, genetic distance is comparatively large, is difficult to carry out the assignment of genes gene mapping.The cabbage mustard BC that Zhang Xinmei etc. obtain through backcross transformation with this dominant sterile source
4segregating population is material, screen 8 SRAP and 1 SSR marker and this dominant male sterile gene close linkage, the linkage distance of nearest double side wings mark ENA14F-CoEm7R and 8C0909 is respectively 0.53cM and 2.55cM (Zhang Xinmei etc., " the closely linked molecule marker of wild cabbage Dominant Male Sterility Gene CDMs399-3 ", Scientia Agricultura Sinica, o. 11th, 2009), but these marks are without the checking of other colonies, also its suitability cannot be judged, and have no report about in broccoli with the closely linked molecule marker of dominant genic male sterility gene.
Therefore, in broccoli, develop molecule marker closely linked with dominant genic male sterility gene, with improve the excellent broccoli dominant genic male sterility system's quality of seed selection and accelerate breeding process, improve breeding efficiency become current problem demanding prompt solution.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides the SSR molecular marker with dominant genic male sterility gene linkage in a kind of broccoli, and provide it and selecting containing the application on dominant genic male sterility gene broccoli plant, for the Fine Mapping of dominant genic male sterility gene in broccoli and molecular cloning are laid a good foundation; Because this mark can to carry out the screening of dominant genic male sterility material in any stage of broccoli candidate material to it, have that efficiency is high, suitability be strong, restriction less, the advantage such as accurate, to provide simply for utilizing molecular marker assisted selection broccoli dominant genic male sterility material, practical and efficient approach.
For reaching this object, present invention employs following technical scheme:
To the invention provides in a kind of broccoli the closely linked versatility SSR molecular marker SSR10312a with dominant genic male sterility gene, the primer pair (sequence is as shown in SeqIDNo.1-2) for this molecule marker of pcr amplification SSR10312a is:
Upstream primer SSR10312a-F:5 ＇-ATCAAGACCTCGGCTGCTTA-3 ＇ and
Downstream primer SSR10312a-R:5 ＇-CCGGCTTTTGCTCTTCTTCT-3 ＇.
Dominant genic male sterility gene described in the present invention can be dominant genic male sterility gene BDMs, the process that this gene specifically obtains is: the present invention is original sterile source with wild cabbage DGMS79-399-3, with 4 broccoli Elite inbred 8554, 90196, 93219 and 94174 is the male parent that backcrosses, backcrossed and directive breeding by many generations (n >=9 generation), obtain broccoli Dominant male sterile line DGMS8554, DGMS90196, DGMS93219 and DGMS94174, wherein DGMS8554 is 10 generations that backcrossed, DGMS90196 is 11 generations that backcrossed, DGMS93219 is 9 generations that backcrossed, DGMS94174 is 9 generations that backcrossed, in flowering period, fertility investigation is carried out to plant, in conjunction with investigation result, obtain the dominant genic male sterility of a pair dominant nuclear gene control broccoli, be BDMs by this unnamed gene, male sterile Ms is dominant to male-fertile ms.
Utilize these colonies in conjunction with RAPD, SSR, SRAP, SCAR equimolecular labeling technique and clusteranalysis (BSA) simultaneously, chain 20 of all and Dominant Male Sterility Gene developed forefathers are marked at broccoli high generation and backcross in segregating population and verify, and adopting SSR molecular marker technology screening to go out a highest combination of primers of identification efficiency, its primer sequence is as shown in SeqIDNo.1-2.
Present invention also offers for the identification of the primer pair containing broccoli dominant genic male sterility gene, described primer pair is that the concrete sequence of described primer pair is foregoing for the primer pair of pcr amplification molecule marker SSR10312a:
Upstream primer SSR10312a-F:5 ＇-ATCAAGACCTCGGCTGCTTA-3 ＇ and
Downstream primer SSR10312a-R:5 ＇-CCGGCTTTTGCTCTTCTTCT-3 ＇.
Present invention also offers the test kit for containing dominant genic male sterility gene in Rapid identification broccoli containing above-mentioned primer pair.
Wherein, described test kit also comprises dNTPs, Taq DNA polymerase, Mg
2+, PCR reaction buffer and standard positive template.
Present invention also offers SSR molecular marker SSR10312a as previously mentioned and identify the application had in the Broccoli Resources resource of dominant genic male sterility gene.
Present invention also offers a kind of method utilizing SSR molecular marker to identify broccoli dominant genic male sterility gene, comprise the following steps:
(1) genomic dna treating measuring plants is extracted;
(2) to treat that the genome of measuring plants is for template, primer pair is utilized to carry out pcr amplification; The concrete sequence of described primer pair is:
Upstream primer SSR10312a-F:5 ＇-ATCAAGACCTCGGCTGCTTA-3 ＇ and
Downstream primer SSR10312a-R:5 ＇-CCGGCTTTTGCTCTTCTTCT-3 ＇;
(3) pcr amplification product is detected.
Wherein, in step (2), PCR reaction system is: 10pmol/ μ L upstream and downstream primer each 0.5 μ L, 2.5U/ μ LTaqDNA polysaccharase 0.2 μ L, 10mol/LdNTPs0.8 μ L, 10 × PCR reaction buffer 1 μ L, 30ng/ μ L template 1 μ L, uses ddH
2o complements to 10 μ L.
In step (2), pcr amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, circulate 35 times altogether, and last 72 DEG C extend 7min, 10 DEG C of preservations.
Be detected as in step (3) and adopt polyacrylamide gel electrophoresis to detect, described polyacrylamide gel electrophoresis refers to the non-denaturing polyacrylamide gel of employing 8%, 160V invariable power electrophoretic separation, the colour developing of last silver dye.
Compared with prior art, the present invention at least has following beneficial effect:
1, the SSR molecular marker utilizing the present invention to obtain, the assisted Selection in dominant genic male sterility gene transformation process can be carried out to it in any period of broccoli candidate material, there is the advantages such as efficiency is high, limiting factor is few, improve the efficiency of seed selection broccoli dominant genic male sterility system, shorten breeding cycle;
2, SSR molecular marker SSR10312a provided by the invention is closely linked versatility SSR molecular marker with dominant genic male sterility gene, empirical tests, its minimum genetic distance in broccoli colony is 0.532cM, by adopting other dominant genic male sterility material to verify, the accuracy rate of this mark is 100%.
3, SSR molecular marker SSR10312a provided by the invention is not only the Fine Mapping of dominant genic male sterility gene in broccoli and clone has established good basis, simultaneously also for utilizing molecular marking supplementary breeding excellent broccoli dominant genic male sterility system to provide theoretical foundation.
Accompanying drawing explanation
Fig. 1 is that the SSR marker SSR10312a of embodiment 1 is to the BC of broccoli DGMS90196
11the polyacrylamide gel electrophoresis figure of the genome amplification result of the random individual plant of male parent 90196, BS and BF and colony that backcrosses of segregating population; Wherein, the 1st swimming lane is DNA standard molecular weight (M); 2nd swimming lane is negative control (CK); 3rd swimming lane is sterile pond (BS); 4th swimming lane is for can educate pond (BF); 5-24 swimming lane is random individual plant.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) or according to the condition of manufacturer's specification sheets suggestion.
In embodiment 1 broccoli with dominant genic male sterility gene closely linked versatility SSR molecular marker SSR10312a screening and utilize SSR molecular marker SSR10312a to carry out the qualification of broccoli sterile gene
The present embodiment test materials used is broccoli self-mating system 8554,90196,93219 and 94174, and corresponding Dominant male sterile line DGMS8554, DGMS90196, DGMS93219 and DGMS94174.These 4 test materialss of broccoli self-mating system 8554,90196,93219 and 94174 are height that wild cabbage broccoli seminar of Vegetable & Flower Inst., Chinese Academy of Agriculture Science is bred as through inbreeding of more generation directive breeding for self-mating system; DGMS8554, DGMS90196, DGMS93219 and DGMS94174 are the broccoli Dominant male sterile line be bred as through too high generation (n >=9 generation) backcross transformation after dominant genic male sterility material and corresponding hybridization between selfed lines; It is peculiar that these germ plasm resources are this seminar, ensures to provide for confirmatory experiment to the public in Two decades years from the applying date.
The qualification of step 1. broccoli plant fertility
For examination material be wild cabbage broccoli seminar of Vegetable & Flower Inst., Chinese Academy of Agriculture Science with wild cabbage dominant genic male sterility material DGMS79-399-3 for sterile source, to carry out with it hybridizing by excellent broccoli self-mating system 8554,90196,93219 and 94174 and many for backcross transformation, 4 broccolis be bred as are high for backcrossing segregating population, wherein DGMS8554 is 10 generations that backcrossed, containing 747 strains, DGMS90196 is 11 generations that backcrossed, containing 338 strains, DGMS93219 is 9 generations that backcrossed, containing 277 strains, DGMS94174 is 9 generations that backcrossed, containing 56 individual plants.All for trying material all in sowing on July 18th, 2011, August 22 was colonizated in greenhouse, spacing in the rows 20cm, line-spacing 30cm, field unified management.
In in January ,-2012 in December, 2011, at initial bloom stage, full-bloom stage and Flowers ending, by strain, fertility investigation is carried out to 4 backcross populations respectively.Start to investigate 9 times altogether to Flowers ending from initial bloom stage, wherein the normal individual plant of pollen fertility is designated as and can educates individual plant, other individual plant with various sterile proterties (stamen is degenerated completely, the atrophy of stamen part, pollen non-activity etc.) is designated as sterile strain, segregation ratio is calculated according to investigation result, MicrosoftExcel2003 software carries out data statistic analysis, uses SAS8.0 to carry out Chi-square test to result.
Result shows: through field investigation, the BC of DGMS8554
10in segregating population 747 strain individual plant, sterile strain is 374 strains, and fertile plant is 373 strains, through card square test, meets the theoretical ratio (χ of 1: 1
2 c=0.0013 < χ
2 0.05,1=3.84); The BC of DGMS90196
11in segregating population 338 strain individual plant, sterile strain is 159 strains, and fertile plant is 179 strains; Through card square test, meet the theoretical ratio (χ of 1: 1
2 c=1.1834 < χ
2 0.05,1=3.84); The BC of DGMS93219
9in segregating population 277 strain individual plant, sterile strain is 126 strains, and fertile plant is 151 strains; Card square test, meets the theoretical ratio (χ of 1: 1
2 c=2.2563 < χ
2 0.05,1=3.84).
Above result shows that this male sterility gene all meets Mendelian's segregation ratio in broccoli 3 segregating populations that backcross, it is mainly a pair dominant nuclear gene and controls, consistent with the result of study of (1997), Liu Yumei etc. (2003) such as Fang Zhiyuan, be BDMs by this unnamed gene in broccoli, male sterile Ms is dominant to male-fertile ms.
Step 2.DNA extracts and ssr analysis
Get the tender leaf of broccoli plant, extract the genomic dna of each individual plant of male parent and colony that backcrosses by CTAB (cetyl trimethylammonium bromide) method of improvement.
Amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, circulate 35 times altogether, and last 72 DEG C extend 7min, 10 DEG C of preservations.Amplified production 8% non-denaturing polyacrylamide gel is separated, and electrophoretic buffer is 0.5 × TBE, 160V constant voltage electrophoretic separation 1.5h, and after electrophoresis, the colour developing of silver dye, shown in its result Fig. 1, and adds up banding pattern.
Step 3.SSR label screening, data statistics and genetic distance calculate
Mainly comprise 4 steps: (1) backcrosses segregating population respectively from DGMS8554, DGMS90196, DGMS93219 and DGMS94174, the DNA of random selecting 10 sterile strains and 10 fertile plants, 4 pairs of DNA near isogene ponds are built according to BSA method, namely 4 to educating pond (BF) and sterile pond (BS), and each pond template DNA concentration is about 30ng/ μ L; (2) with 4 pairs of near isogene pond screening polymorphism primers; (3) genotype of polymorphism primer analysis 4 each individual plants of the segregating population that backcrosses obtained is utilized; (4) JoinMap4.0 software is utilized to carry out data analysis, genetic distance converts recombination value to genetic distance (centimorgan according to Kosambi (1944) function, cM), the linkage distance between linked marker and phenotypic character is calculated.
Data statistical approach: during statistic data, according to the requirement of mapping software, mark using the presence or absence of band as difference different genotype, phenotype according to being marked in BF and BS two pond is sorted out, be designated as " a " by the banding pattern presented in BS pond, and be designated as " h " without band in BF pond, the disappearance caused due to a variety of causes or fuzzy banding pattern are designated as " u ", after having added up banding pattern, result is inputted in MicrosoftExcel2003 software.
Result shows: screen respectively with 2570 pairs of scaffoldSSR primer pairs, 4 pairs of BF and the BS ponds designed according to wild cabbage genome sequencing, wherein filter out respectively in BF and the BS pond of DGMS8554, DGMS90196, DGMS93219 and DGMS94174 3 to, 9 to, 4 to and 2 stablize the primer of polymorphism to existing between two ponds, wherein SSR10312a shows polymorphism in 4 pairs of fertility ponds simultaneously.Primer pair (sequence is as shown in SeqIDNo.1-2) for pcr amplification molecule marker SSR10312a is:
Upstream primer SSR10312a-F:5 ＇-ATCAAGACCTCGGCTGCTTA-3 ＇ and
Downstream primer SSR10312a-R:5 ＇-CCGGCTTTTGCTCTTCTTCT-3 ＇.
The mark SSR10312a simultaneously showing polymorphism in BF and the BS pond of DGMS8554, DGMS90196, DGMS93219 and DGMS94174 is utilized to analyze the individual plant of the height of DGMS8554, DGMS90196, DGMS93219 and DGMS94174 for the segregating population that backcrosses, utilize JoinMap4.0 computed in software genetic distance in conjunction with enquiry data, show that BDMs is 0.532cM with the minimum genetic distance of mark SSR10312a in 4 colonies.
The checking of embodiment 2.BDMs gene flanking marker
10 parts of mark SSR10312a that are that obtained embodiment 1 by the dominant genic male sterility material obtained containing BDMs gene and different broccoli backcross transformation male parent and BDMs gene linkage are utilized to verify, to determine to mark the accuracy for molecule assisted Selection.Through compared with the variable rate technology type of selected materials, this phenotypic data being marked at the banding pattern reflection in 10 parts of materials is consistent with field investigation result, and accuracy rate is 100%.
The BC of genetic distance at DGMS8554 of closely linked versatility SSR marker (SSR10312a) and BDMs with dominant genic male sterility gene in broccoli is obtained in the present embodiment
10the BC of (containing 747 individual plants) and DGMS90196
11genetic distance in (containing 338 individual plants) segregating population is respectively 0.563cM and 0.532cM, this is that molecule marker assisting sifting broccoli dominant genic male sterility material is had laid a good foundation with the closely linked SSR marker of dominant genic male sterility gene in broccoli, for set up a set of fast and the method for precise Identification broccoli dominant genic male sterility kind matter provides theoretical foundation, simultaneously because this is labeled as versatility mark, and it is very near with the genetic distance of target gene in large group, the further Fine Mapping of this gene and clone are laid a good foundation.
Applicant states, the present invention illustrates detailed process equipment and process flow process of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned detailed process equipment and process flow process, namely do not mean that the present invention must rely on above-mentioned detailed process equipment and process flow process and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of ancillary component, the concrete way choice etc. of each raw material of product of the present invention, all drops within protection scope of the present invention and open scope.
Claims (9)
1. in broccoli with dominant genic male sterility gene a closely linked versatility SSR molecular marker SSR10312a, it is characterized in that, for the primer pair of molecule marker SSR10312a described in pcr amplification be:
Upstream primer SSR10312a-F:5 ＇-ATCAAGACCTCGGCTGCTTA-3 ＇ and
Downstream primer SSR10312a-R:5 ＇-CCGGCTTTTGCTCTTCTTCT-3 ＇.
2. for the identification of the primer pair containing broccoli dominant genic male sterility gene, it is characterized in that, described primer pair is the primer pair for pcr amplification molecule marker SSR10312a described in claim 1.
3. containing primer pair described in claim 2 for the identification of the test kit containing broccoli dominant genic male sterility gene.
4. test kit according to claim 3, is characterized in that, described test kit can also comprise dNTPs, Taq DNA polymerase, Mg
2+, PCR reaction buffer and standard positive template.
5. SSR molecular marker SSR10312a according to claim 1 is identifying the application had in the Broccoli Resources resource of dominant genic male sterility gene.
6. utilize SSR molecular marker to identify a method for broccoli dominant genic male sterility gene, it is characterized in that, comprise the following steps:
(1) genomic dna treating measuring plants is extracted;
(2) to treat that the genome of measuring plants is for template, the primer pair for pcr amplification molecule marker SSR10312a described in claim 1 is utilized to carry out pcr amplification;
(3) pcr amplification product is detected.
7. method according to claim 6, it is characterized in that, in step (2), PCR reaction system is: each 0.5 μ L of 10pmol/ μ L upstream and downstream primer, 2.5U/ μ LTaqDNA polysaccharase 0.2 μ L, 10mol/LdNTPs0.8 μ L, 10 × PCR reaction buffer 1 μ L, 30ng/ μ L template 1 μ L, uses ddH
2o complements to 10 μ L.
8. the method according to claim 6 or 7, is characterized in that, in step (2), pcr amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 35 times altogether, last 72 DEG C extend 7min, 10 DEG C of preservations.
9. the method according to any one of claim 6-8, is characterized in that, is detected as and adopts polyacrylamide gel electrophoresis to detect in step (3);
Preferably, described polyacrylamide gel electrophoresis refers to the non-denaturing polyacrylamide gel of employing 8%, 160V invariable power electrophoretic separation, the colour developing of last silver dye.
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