CN102766623B - Drought-enduring maize germplasm identification and screening CAPS (cleaved amplified polymorphic sequence) marker and test method and application thereof - Google Patents
Drought-enduring maize germplasm identification and screening CAPS (cleaved amplified polymorphic sequence) marker and test method and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker for drought-enduring maize germplasm identification, in particular to a drought-enduring maize germplasm identification and screening CAPS marker. The molecular marker includes dhnC397 and rspC1090, wherein the nucleotide sequence of the molecular marker dhnC397 is shown as SEQ.ID No.1, and the nucleotide sequence of the molecular marker rspC1090 is shown as SEQ.ID No.2. The molecular marker and a test method thereof, which are provided by the invention, are used for drought-enduring marker-assisted selection, and thus the application of the drought-enduring molecular marker in the breeding work is pioneered. Meanwhile, the molecular marker provided by the invention can also be used for greatly increasing the drought-enduring selection efficiency in breeding, and has great application potential and high economic value.
Description
Technical field
The invention belongs to biological technical field, particularly the CAPS(C1eaved Amplified Polymorphic Sequences of Maize Idioplasm identification and screening) mark and detection method and application.
Background technology
Corn has become the cereal crop of the world and China's sown area maximum at present.Along with global livestock industry develops and apply the rapidly soaring of corn processed liquid fuel ethanol industry rapidly, the demand of corn is increased considerably.Yet global climate change causes that arid generating period is shorter and shorter, degree is more and more heavier, and grain-production is constituted a serious threat.According to statistics, annual because arid causes the corn underproduction approximately 20%～30%, arid has become the critical limitation factor (Su Zhijun etc., 2010) of corn yield.Although traditional breeding technology has been obtained prominent achievement aspect crop genetic improvement, because its efficiency of selection is lower, the cycle is longer, can not meet the demand of current corn production to improved seeds.Utilize the newest research results of genomics and information biology, carry out the breeding research of corn molecule marker, build practical, economic, efficient molecular breeding technical system, breeding high-yield high-quality multi-resistant corn new variety, become the important research direction (Li Jiansheng etc., 2007) of corn breeding.
Drought tolerance is a very complicated quantitative character.The development of molecular quantitative has promoted the renewal of molecule marker and the structure of high-density genetic linkage maps, excavate out a large amount of QTL(Quantitative Trait Locis relevant to quantitative character), also promoted the application of MAS (Marker-assisted selection) on quantitative character is selected simultaneously.MAS carries out by utilizing the GENERALIZATION OF MODERN BREEDING TECHNIQUE of indirectly selecting to objective trait with the closely linked DNA molecular marker of objective trait, can make breeding man just can follow the trail of the genetic locus of the drought-enduring proterties of regulation and control without measuring phenotype, can remove a large amount of field test work of multiple years from, improve efficiency of selection.Bernier etc. (2009) point out that it is to have limited two important factors that QTL applies on MAS that the QTL effect lacks repeatability in different colonies and environment.In addition, verify drought-enduring gene and in gene the exploitation effective mark very difficult.Therefore, utilizing on the one hand the drought-enduring QTL of consistence and potential gene thereof is to solve a kind of effective way of assistant breeding (Hao et al., 2010).Use on the other hand functional gene inside to cause that the polymorphism motif of phenotypic character variation develops the more effectively excellent allelotrope in fixed group of functional label, and the functional label of exploitation thus will contribute to the proterties of different background genetic stocks is selected to (Hao et al., 2011) in breeding.
Enzyme is cut amplification polymorphism sequence (C1eavedAmplified Polymorphic Sequences, CAPS) be called again the RFLP-PCR technology, according to design special primers such as EST or the gene orders delivered, specific PCR combined with restriction enzyme digestion and detect a kind of technology of polymorphism, belonging to and take the codominant molecule marker of PCR as basis.Its ultimate principle is first with the DNA sequence dna of known site, to remove to design a set of specific PCR primer (19 ~ 27bp).Then apply a certain DNA fragmentation that these primers go to increase on this site; Then with a kind of narrow spectrum restriction enzyme, cut the amplified band of gained and carry out rflp analysis.With take hybridization and compare as basic RFLP, it has following advantage: (1) primer and Restriction enzyme combination are very many, have increased the chance that discloses polymorphism, and easy and simple to handle, available agarose gel electrophoresis analysis; (2) in eukaryote, the CAPS mark is codominance, can distinguish homozygous genotype and heterozygous genes type; (3) required DNA amount is few; (4) result is reliable and stable; (5) swift to operate, level of automation is high.
The CAPS labeling technique because thering is codominance, locus specificity, simple to operate, cost is low, required DNA sample size is few and, to the purity requirement of DNA advantages of higher not, therefore be subject to scientific research personnel's attention.Become at present a very important molecular marking technique of modern biology research, obtained applying quite widely in fields such as Idioplasm identification, assistant breeding, gene identification and map constructions.
Summary of the invention
In order to address the above problem, the object of the invention is to filter out the CAPS mark of Maize Idioplasm identification and screening.
Another object of the present invention is to provide the detection method of above-mentioned molecule marker.
An also purpose of the present invention is to provide the application of above-mentioned molecule marker in drought-enduring gene test and marker-assisted breeding.
In order to realize the object of the invention, the invention provides a kind of molecule marker of Maize Idioplasm identification, described molecule marker comprises dhnC397 and rspC1090, the nucleotide sequence of described molecule marker dhnC397 is as shown in SEQ.ID No1, and the nucleotide sequence of described molecule marker rspC1090 is as shown in SEQ.ID No2.
Two marks of dhnC397 and rspC1090 are based on Maize gene dhn1 and the exploitation of rsp41 gene pleiomorphism motif.Wherein, dhnC397 is based on G/A base mutation in the dhn1 gene and a kind of functional molecular marker of developing.According to dhn1 gene order information design Auele Specific Primer, amplify the fragment that a segment length is 164bp.In Fig. 1, the variation (CCAAGG/CCAAAG) of G and A has occurred in gene order 397 site, restriction enzyme StyI can identify CCAAGG.The PCR product is after the StyI enzyme is cut, and two fragments of the digested one-tenth of the purpose fragment that contains G site 131bp and 33bp, and the purpose fragment that contains the A site is cut recognition site owing to lacking the StyI enzyme, therefore can not be digested.Wherein, rspC1090 is based on G/A base mutation in the rsp41 gene and a kind of functional molecular marker of developing.According to rsp41 gene order information design Auele Specific Primer, amplify the fragment that a segment length is 286bp.In Fig. 2, the variation (CCGG/CCAG) of G and A has occurred in gene order 1090 site, restriction enzyme HpaII can identify the CCGG site.The PCR product is after the HpaII enzyme is cut, and two fragments of the digested one-tenth of the purpose fragment that contains G site 225bp and 61bp, and also have the purpose fragment in A site to cut recognition site owing to lacking the HpaII enzyme, therefore can not be digested.
The present invention also provides the above-mentioned primer of stating molecule marker that increases.
The primer sequence of described dhnC397 is as shown in SEQ.ID No3 and SEQ.ID No4, and the primer sequence of described rspC1090 is as shown in SEQ.ID No5 and SEQ.ID No6.
The present invention also provides the carrier that contains described molecule marker.Described carrier is plant, tissue or cell etc.
A kind of detection method of utilizing molecule marker to carry out Maize Idioplasm identification and screening, the method molecule marker used is dhnC397 and rspC1090.
Concrete, the step of above-mentioned detection method is: the primer amplification target plant genomic dna that utilizes described molecule marker, obtain the key band of this molecule marker in target plant, the described measured nucleotides sequence of plant bands of a spectrum that carries the target allelotype is classified the nucleotide sequence of described molecule marker as.Described plant optimization is corn, and described target plant genomic dna is preferably drought-enduring and drought tolerant corn genomic dna not.
Preferably, the step of above-mentioned detection method is: the drought-enduring and not drought-enduring genomic dna of primer amplification that utilizes above-mentioned molecule marker, obtain the key band of this molecule marker in corn, this key band is divided into the plant bands of a spectrum of drought-enduring excellent allelotype and the plant bands of a spectrum of drought-enduring allelotype not, and the described resulting nucleotides sequence of plant bands of a spectrum that carries drought-enduring excellent allelotype is classified the nucleotide sequence of the molecule marker of above-mentioned molecule marker as.
In aforesaid method, PCR reaction amplification system is:
20 μ L reaction systems comprise each 0.5 μ M of forward and reverse primer, 0.2mM dNTPs, 2.5mM MgCl
2, 1X Taq buffer, 0.5units Taq polysaccharase, DNA profiling 100ng.
In aforesaid method, the pcr amplification program is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃ or 60 ℃ of annealing 45s, 72 ℃ are extended 30-60s, 37 circulations; 72 ℃ are extended 5-10min.
Sty I endonuclease reaction system:
Hpa II endonuclease reaction system:
37 ℃ of reaction 12-16h, then carry out polyacrylamide gel electrophoresis (8%).
The present invention also provides the application of described molecule marker in Maize Idioplasm identification and screening and molecular mark
The present invention also provides the application of described detection method in Maize Idioplasm identification and screening and molecular mark.
The beneficial effect that the present invention possesses is:
1) molecule marker of the present invention can be used for drought tolerant germplasm detection and marker-assisted breeding.Molecular Marker Information provided by the invention is mainly derived from the general drought-enduring QTL of consistence or drought-enduring functional gene, and reliability is strong.Be applicable to very much the realization of modern agriculture molecular breeding.
2) functional label of the present invention's exploitation, relevant to drought-enduring gene function pleomorphism site, is directly used in drought-enduring molecular mark, and the development and application that the present invention is complicated quantitative character functional label provides important theoretical basis and practical experience.
3) molecule marker provided of the present invention is for drought-enduring marker assisted selection, and the material that collects all mark characteristics of drought tolerance bands of a spectrum will be drought-enduring material, for drought tolerance molecule marker primer is applied in breeding work open up the beginning.And will greatly improve efficiency of selection drought-enduring in breeding, there is very large application potential and higher economic worth.
The accompanying drawing explanation
Fig. 1 is dhnC397 functional label expansion schematic diagram of the present invention.
Fig. 2 is rspC1090 functional label expansion schematic diagram of the present invention.
Fig. 3 is the polyacrylate hydrogel electrophorogram that the dhnC397 functional label increases in self-mating system.
Fig. 4 is the polyacrylate hydrogel electrophorogram that the rspC1090 functional label increases in self-mating system.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to protection scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1 utilizes functional gene polymorphism motif design and development CAPS functional label
Material: the self-mating system commonly used (Hao et al., 2011) in 210 parts of China's Maize Production and breeding is selected in test.These self-mating systems derive from respectively different corns ecotope, belong to different hybrid vigour subgroups, have hereditary basis widely.These self-mating systems derive from BSSS, PA, PB, Lan(Lancaster), the red bone in LRC(Luda) and SPT(Siping City head) these six subgroups.
Maize whole-genome association: according to biochip technology, utilize 1536SNP(Single Nucleotide Polymorphism) polymorphism information.Adopt mixed linear model (Mixed Linear Model, MLM) method in TASSEL software to analyze discovery to associated data, on 10 karyomit(e)s of corn, 121 polymorphism SNPs sites are associated with drought-enduring correlated character.In these remarkable associated sites, 29 SNP sites are considered to drought-enduring relevant.Therefore, they are considered to have potential drought-enduring function variation.In this research, choose 2 and the closely-related SNP of a plurality of drought-enduring proterties site, be respectively PZA0355.1 and PZA01671.1.PZA0355.1 contains the A/G Mutation, is positioned at karyomit(e) No. 6, and relative gene is dhn1; PZA01671.1 is positioned at karyomit(e) No. 5, contains the A/G base mutation, relevant with the rsp41 gene.These two genes all are proved relevant with the drought tolerance of crop (Hao et al., 2011).
To existing SNP correlation ID sequence information, according to the requirement of oneself, utilize online tool (http://helix.wustl.edu/dcaps/dcaps.html) to analyze best restriction enzyme site and selectional restriction restriction endonuclease.Then utilize NCBI(http: //blast.ncbi.nlm.nih.gov/Blast.cgi) the BLAST function find the highest sequence information of homologous information.Then with DNAMAN software, the sequence of download is opened, and compared with SNP ID sequence (100bp), find out near extension base sequence sudden change both sides, SNP site, the about 500bp of total length.Recycling Primer5.0 software, fixedly the primer of SNP site one end (band changes the restriction enzyme site of base), make first base that mutational site is the pcr amplification extension, then with this software, need look for the primer that the other end is suitable; Requirement, annealing temperature (Tm) is worth approximately 60 ℃, upstream and downstream primer TM value is adjusted to close, and the product size is about 90-140bp.The primer of design is downloaded, and, in a primer strand of fixing therein, according to selected restriction enzyme, changed a corresponding 1-2 base, synthetic primer after check.The CAPS labeled primer can directly be used Primer5.0 software design primer according to sequence information.
Table 1 is according to amplified fragments size in two functional labels, restriction endonuclease, primer sequence and Maize genomes of Maize gene dhn1 and the exploitation of rsp41 gene pleiomorphism motif.
For designing synthetic primer, cut and grope analysis through a series of PCR checking and enzyme.At first, with chip material, synthetic primer is carried out to pre-expansion product compliance test result, judge that whether the amplified band size is consistent with the purpose band of design, whether the expanding effect of simultaneous verification design of primers (band sharpness and stability) is desirable, for undesirable Primer redesign and checking; The second, select good primer amplification chip material band, carry out corresponding restriction enzyme digestion enzyme reaction, Polyacrylamide Gel Electrophoresis, judge whether to distinguish clearly the SNP site.The 3rd, the mono-clonal of mark and identification and analysis, verified the PCR product by order-checking.
Embodiment 2 utilizes the CAPS functional label to carry out drought-enduring detection and application
DhnC397CAPS functional label drought tolerance identification and analysis: by being that test materials carries out the genotype checking to 210 parts of corn inbred lines, except M14, B104, newly from 218,2002F22, DP62-7 and lucky 495, remaining 204 parts of self-mating system can amplify size for the 164bp fragment in the dhnI gene.Fig. 3 has shown the polyacrylate hydrogel electrophorogram that the dhnC397 functional label increases in self-mating system.204 parts of self-mating systems are carried out to the drought-enduring evaluation in field, there are 8 parts of self-mating systems not obtain field data (N28, N528-1, golden yellow 59, 8-3 early, C28, T8, newly from 153-2 with from 495) according to the comprehensive selection standard value, we are divided into 5 grades by the drought-enduring degree of 196 parts of self-mating systems, be respectively the high drought-enduring highly drought tolerance of HR(), the drought-enduring drought tolerance of R(), the drought-enduring middle drought of M(moderate tolerance), the responsive drought susceptible of S(drought), the high sense of HS(highly drought susceptible).To 196 parts of self-mating systems with the drought-enduring phenotypic data in field after the StyI enzyme is cut, wherein 55 parts of two of digested one-tenth of self-mating system are respectively 131bp and 33bp fragment, be determined to be the G allelic variation, simultaneously 141 parts of self-mating systems are cut recognition site and are not cut open owing to lacking enzyme, are determined to be the A allelic variation.Find out bands of a spectrum from amplification, contain 7 parts of drought-enduring self-mating systems (HR+R) and 24 parts of responsive self-mating systems of drought (HS+S) account for respectively 12.7% and 43.6% of overall proportion in G allelic variation self-mating system; Be respectively 24.1% and 37.6% containing 34 parts of drought-enduring self-mating systems and 53 parts of responsive self-mating system proportions of drought in A allelic variation self-mating system.Find out thus the drought tolerance (12.7%) (table 2) higher than G allelic variation self-mating system containing the drought tolerance (24.1%) of A allelic variation self-mating system.Contain the 0.27(table 4 of the drought-enduring selectivity index of A allelic variation self-mating system (0.01) higher than non-irrigated G allelic variation self-mating system (0.26)).
The self-mating system drought tolerance that table 2 corn dhnC397CAPS functional label detects is estimated
RspC1090CAPS functional label drought tolerance identification and analysis: by being that test materials carries out the genotype checking to 210 parts of corn inbred lines, except long by 3, iron 7922, carbuncle rice, DP62-7 and 5022 (B), it is the purpose fragment of 286bp that remaining 205 parts of self-mating system can amplify size in the rsp41 gene.205 parts of self-mating systems are carried out to the drought-enduring phenotypic evaluation in field, remove 9 parts of self-mating systems that do not obtain field data (N28, N528-1,2002F22, golden yellow 59, early 8-3, C28, T8, newly from 153-2 with from 495), remaining 196 parts of self-mating systems are after the HpaII enzyme is cut, 108 parts of two of digested one-tenth of self-mating system are respectively 225bp and 61bp fragment, and simultaneously 88 parts of self-mating systems are cut recognition site and are not cut open owing to lacking enzyme.The polyacrylate hydrogel electrophorogram that Fig. 4 rspC1090 functional label increases in self-mating system.Contain 30 parts of drought-enduring self-mating systems and 31 parts of responsive self-mating systems of drought account for respectively 27.8% and 28.7% of overall proportion in G allelic variation self-mating system; Be respectively 11.4% and 53.4% containing 10 parts of drought-enduring self-mating systems and 47 parts of responsive self-mating system proportions of drought in A allelic variation self-mating system.Find out thus containing the drought tolerance of A allelic variation self-mating system with containing the comparing of G allelic variation self-mating system, its more not drought-enduring (53.4% > 28.7%) (table 4).Contain the drought-enduring selectivity index of G allelic variation self-mating system (0.19) higher than containing 0.56 of A allelic variation self-mating system (0.37).And while selecting the advantage allelic variation of two marks, the drought-enduring selectivity index of self-mating system will be brought up to 0.83(table 6 simultaneously).
The self-mating system drought tolerance that table 3 corn rspC1090CAPS functional label detects is estimated
Table 4210 part corn inbred line functional label detection case and drought tolerance evaluation
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. a detection method of utilizing molecule marker to carry out Maize Idioplasm identification and screening, it is characterized in that, concrete steps are: utilize the primer amplification target plant corn gene group DNA that molecule marker is dhnC397 and rspC1090, obtain the key band of this molecule marker in the target plant corn, the measured nucleotide sequence of the described corn bands of a spectrum that carry the target allelotype is as shown in SEQ.ID No1 and SEQ.ID No2; Wherein, the nucleotide sequence of described molecule marker dhnC397 is as shown in SEQ.ID No1, and the nucleotide sequence of described molecule marker rspC1090 is as shown in SEQ.ID No2; The primer sequence of described dhnC397 is as shown in SEQ.ID No3 and SEQ.ID No4, and the primer sequence of described rspC1090 is as shown in SEQ.ID No5 and SEQ.ID No6; The pcr amplification system of described molecule marker is as follows: 20 μ L reaction systems comprise each 0.5 μ M of forward and reverse primer, 0.2mM dNTPs, 2.5mM MgCl
2, 1X Taq buffer, 0.5units Taq polysaccharase, DNA profiling 100ng; The program of described pcr amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 55 ℃-60 ℃ annealing 45s, 72 ℃ are extended 30-60s, 37 circulations; 72 ℃ are extended 5-10min; The DNA sequence dna obtained after pcr amplification is obtainable described key band with 8% polyacrylamide gel electrophoresis.
2. the application of the described method of claim 1 in Maize Idioplasm identification and screening and molecular mark.
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| CN106967725A (en) * | 2017-03-31 | 2017-07-21 | 中国水稻研究所 | Rice ear sprouting period related gene, functional label and application |
| CN107419025B (en) * | 2017-08-29 | 2020-06-16 | 中国农业科学院作物科学研究所 | Application of genetic variation in maize SNAC gene in maize drought tolerance detection |
| CN109338000A (en) * | 2018-10-31 | 2019-02-15 | 中国农业科学院蔬菜花卉研究所 | A kind of CAPS labeling method of stearoyl carrier protein desaturase enzyme gene (SAD) |
| CN110637552A (en) * | 2019-09-23 | 2020-01-03 | 四川育良生物科技有限公司 | Quality detection method for drought-resistant corn seeds |
| CN110628932A (en) * | 2019-10-18 | 2019-12-31 | 上海市农业科学院 | CAPS marker for identifying maize germplasm folate genotype and application thereof |
| CN112877456B (en) * | 2021-02-02 | 2022-06-03 | 中国科学院植物研究所 | Molecular marker for drought-enduring green-keeping and efficient phosphorus remobilization capacity of corn and application thereof |
| CN114150082A (en) * | 2021-12-02 | 2022-03-08 | 连云港市农业科学院 | Development and application of KASP molecular marker of corn drought-tolerant gene |
| CN118064638B (en) * | 2024-04-18 | 2024-07-26 | 中国农业大学三亚研究院 | SNP molecular marker locus related to drought tolerance of corn and application thereof |
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