CN107058494B - Method for simplifying purity identification of common vetch variety by adopting SCoT molecular marker - Google Patents
Method for simplifying purity identification of common vetch variety by adopting SCoT molecular marker Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural forage grass breeding and application, and discloses a method for efficiently and scientifically identifying germplasm resource purity of common vetch by SCoT molecular markers. The method for identifying the purity of common vetch variety by adopting SCoT molecular marker is mainly characterized by comprising the following steps: (1) extracting DNA from common vetch germplasm; (2) performing PCR amplification on the DNA of the common vetch germplasm in the step (1) by using an SCoT primer; (3) carrying out agarose gel electrophoresis photographing on the PCR amplification product; (4) and comparing the differences of the common vetch germplasm polymorphism sites to determine the common vetch variety purity identification result. The detection method can finish the identification work of the seed variety purity within 6h, and has the advantages of science, high efficiency, low price, simple operation and the like. Can provide powerful technical support for the purity identification of common vetch varieties, the breeding of new varieties and the identification of the authenticity, stability and specificity of the varieties.
Description
Technical Field
The invention belongs to the technical field of agricultural forage grass breeding and application, and discloses a method for efficiently and scientifically identifying germplasm resource purity of common vetch by adopting molecular markers.
Background
In recent years, along with the development of pasture germplasm resources, pasture breeding is more and more emphasized by scholars, and the cultivation and popularization of new pasture varieties is an important direction for the development of pasture breeding in China. At present, pasture breeding modes mainly comprise two modes of field breeding and molecular breeding, and mainly comprise molecular breeding; however, due to the variation of molecular breeding techniques, the quality of the bred grass germplasm is not good and good, and the purity of germplasm resources cannot be ensured. Therefore, the identification of the purity of the new variety of the pasture is of great importance, and the method is not only the protection and the approval of the new variety, but also an important means for distinguishing fake seeds and maintaining the independent intellectual property of a breeder. Therefore, the research and development of a scientific and efficient method for identifying the purity of the pasture variety is a research subject of major attention of enterprises in colleges and universities.
Common vetch is annual leguminous closed-flower pollination forage, is widely planted in northwest and north China, has strong cold and drought resistance, wide regional adaptability and higher nutritional value, can be used as silage in plateau areas, is a forage crop with excellent quality in forage resources in China, plays an important role in grassland agricultural systems in China, and has wide breeding prospect. The pasture grass germplasm resources are various, and the purity difference among different germplasms directly influences the use and protection of the germplasm resources, so the identification work of the germplasm purity is particularly important, and meanwhile, the registration of new germplasm resources and the analysis of purity and genetic characteristics need a molecular-level germplasm purity identification method which is more accurate, more efficient, lower in cost and simpler than field tests.
In recent years, with the development of research in molecular biology and functional genetics, functional molecular markers have been increasingly emphasized by researchers, and since they may contain a part of a target gene or be closely interlocked with a target gene, a trait can be selected by selecting a certain molecular marker, thereby accelerating the progress of germplasm breeding. Nowadays, a variety of molecular marking methods such as SNP, RAPD, SSR, ISSR, and AFLP have been developed and developed, and these molecular marking techniques have been widely applied to species genetic diversity analysis, germplasm resource identification, genetic map construction, QTL mapping, gene mapping cloning, transgenic plant identification, and molecular assisted breeding.
SCoT molecular marker, namely target codon target polymorphism (Start codon target polymorphism) marker. The molecular marker can obtain a required single primer by utilizing the sequence conservation of the ATG translation initiation site flanking in a plant gene, amplify a corresponding target genome and generate a dominant polymorphic marker, compared with other molecular markers, the SCoT molecular marker has the advantages of simplicity and convenience in operation, low price, good repeatability, rich polymorphism and the like, and is successfully applied to Gaomagmai (Schauer, 2015), soybean (Liqiang, 2013), longan (old tiger, 2010), orange (Korea Hui, 2011), medicinal chrysanthemum (Zhaozhen, 2009) at present, and is widely applied to the aspects of species construction of genetic maps, specific character markers, genomics comparison and the like. However, the application of the molecular marker technology in the purity identification of the common vetch variety is not reported.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provides a simplified common vetch variety purity identification primer adopting SCoT molecular markers.
The invention aims to provide a simplified common vetch variety purity identification kit adopting SCoT molecular markers.
The invention further aims to provide a method for simplifying the purity identification of common vetch varieties by adopting SCoT molecular markers.
With this to common vetch germplasm purity carry out high-efficient accurate appraisal. The molecular marking method is scientific, efficient, convenient and fast, and can be completed within 6 hours under laboratory conditions. In the invention, the 4 common vetch germplasms used in the experiment are all applicable, which shows that the method has very strong practicability and application value, is suitable for popularization and application, and is beneficial to rapid and accurate identification of germplasm purity by a wide variety of inspectors. The invention has wide application prospect in the development of the pasture industry in China, provides important guarantee for the safety of the pasture germplasm in China, ensures the personal interest of culturists and provides powerful technical support for the development of pasture germplasm resources.
The invention aims to develop a detection technology of SCoT molecular markers based on the latest research results of the molecular markers, identify a plurality of germplasms of common vetch, and discloses a method for simplifying identification of common vetch varieties by adopting the SCoT molecular markers.
In order to achieve the above purpose, the invention provides the following technical scheme: the utility model provides an adopt SCoT molecular marker to simplify common vetch variety purity appraisal primer, its key feature lies in that the primer is:
scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively;
scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively;
scot38 primer: 5'-ACGACATGGCGACCACCG-3' are provided.
The utility model provides an adopt SCoT molecular marker to simplify common vetch variety purity appraisal kit, its main characteristics lie in PCR amplification system 2 x Power Taq PCR Master Mix, and the SCoT primer includes the Scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively; scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively; scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively; scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively; scot38 primer: 5' -ACGACATGGCGACCACCG-3.
The method for identifying the purity of common vetch variety by adopting SCoT molecular marker is mainly characterized by comprising the following steps:
(1) common vetch germplasm DNA extraction: randomly sampling, and extracting the genome DNA of the germplasm to be detected for later use by adopting an improved CTAB method (Chenkun pine, 2004);
(2) performing PCR amplification on the common vetch germplasm DNA obtained in the step (1) by adopting an SCoT primer, wherein the total volume of PCR amplification reaction is 10 mu l, and the amplification reaction system is as follows: 2 × Power Taq PCR Master Mix 5.0 μ l, SCoT primers 1 μ l each, including Scot28 primer: 5'-CCATGGCTACCACCGCCA-3' is 1 μ l;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3' is 1 μ l;
scot36 primer: 5'-GCAACAATGGCTACCACC-3' is 1 μ l;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3' is 1 μ l;
scot38 primer: 5' -ACGACATGGCGACCACCG-3 is 1 mu l, the DNA of the common vetch germplasm in the step (1) is 50 ng/mu l, 2.0 mu l and ddH202 mu l; the PCR amplification reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
(3) carrying out agarose gel electrophoresis on the PCR amplification product, wherein the agarose gel concentration is 1.2-1.4%, the gel components comprise 6.1-7.1g of agarose, 20450 ddH20450ml, 50ml of 10 xTBE and 9-12 mu l of nucleic acid dye; the gel running voltage is 129-132V, the electrophoresis time is 2h15min-2h30min, the power supply is closed, and a chemical gel imager is used for photographing;
(4) compare the difference of common vetch germplasm polymorphism site, confirm common vetch variety purity appraisal result, common vetch germplasm average polymorphism site is 21.4-27.4, through statistics 0,1 matrix, statistics allelic factor, calculate and observe the heterozygosity, observe heterozygosity (Ho) and obtain miscellaneous and individual number/sample individual number total number, Ho is 100%, the statistics site is the polymorphism site, allele retention ratio is greater than 85% and can represent this germplasm majority genetic information, variety purity is qualified.
The identification method for simplifying the purity of the common vetch variety by adopting the SCoT molecular marker comprises the following steps of (4) determining the allelic base factors of the primers, counting a 0 and 1 matrix according to the size of the PCR amplification product fragment of the corresponding variety to be detected to obtain the corresponding allelic base factors, calculating the polymorphic information content PIC according to the counted allelic gene and a calculation formula,
wherein: n is the number of alleles; piAnd PjFrequency of the ith and jth alleles, respectively; carry out "0, 1" statistics on the same migration position, construct SCoT analysis band data of common vetch idioplasm, whether carry out complete differentiation to common vetch idioplasm through the used primer of data analysis.
The method for identifying the variety purity of common vetch by adopting SCoT molecular markers is simplified, and the method further comprises that the optimal sampling number of each common vetch germplasm is 10 single plants.
According to the method, DNA extraction, PCR amplification and SCoT analysis are carried out on the common vetch to be tested, polymorphism expression of the technology among common vetch germplasms is confirmed, and common vetch germplasm purity can be efficiently and accurately identified.
Compared with the prior art, the identification method for simplifying the purity of common vetch variety by adopting SCoT molecular marker has the advantages that: the method overcomes the defect of long detection period of the original identification method, can obtain a plurality of polymorphic markers through one-time amplification reaction to identify the germplasm purity of common vetch, has the advantages of being scientific and accurate, improves the detection efficiency, reduces the test cost, and is convenient and fast to operate.
Drawings
FIG. 1 shows an electrophoresis analysis spectrum of an amplification product when the SCoT primer is used for randomly sampling 10 strains of various germplasms of common vetch;
in the figure: from a left side to the right side be in proper order for orchid arrow No. 1, arrow pea No. 17, orchid arrow No. 3, arrow common vetch No. 33.
FIG. 2 is a SCoT band analysis clustering chart when the SCoT primer is used for randomly sampling 10 strains of various germplasms of common vetch;
in the figure: 4 single trunk of common vetch random sampling all gather together according to germplasm, from the top down be in proper order that the arrow is 3, common vetch 33, arrow 1, common vetch 17.
FIG. 3 is a SCoT band analysis clustering chart when the SCoT primer is used for randomly sampling 60 strains of various germplasms of common vetch;
in the figure: 4 single trunk of common vetch random sampling all gather together according to germplasm, from the top down be in proper order that the common vetch is No. 1, common vetch is No. 17, common vetch is No. 3, common vetch 33.
Detailed description of the invention
To illustrate the method of the present invention in more detail, the following description will be made of the test method of the present invention, the sequences of the primers of the present invention are shown in Table 1, and other reagents used in the test are commercially available. The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention.
Example 1: the utility model provides an adopt SCoT molecular marker to simplify common vetch variety purity appraisal primer, its key feature lies in that the primer is:
scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively;
scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively;
scot38 primer: 5'-ACGACATGGCGACCACCG-3' are provided.
Example 2: the utility model provides an adopt SCoT molecular marker to simplify common vetch variety purity appraisal kit, its main characteristics lie in PCR amplification system 2 x Power Taq PCR Master Mix, and the SCoT primer includes the Scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively; scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively; scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively; scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively; scot38 primer: 5' -ACGACATGGCGACCACCG-3.
Example 3: the method for identifying the purity of common vetch variety by adopting SCoT molecular marker is mainly characterized by comprising the following steps:
the method for sampling and extracting DNA from the germplasm of common vetch with 4 common vetch comprises the following steps: randomly sampling, collecting single plant from each germplasm according to 11 gradients of 1, 2, 3, 5, 8, 10, 20, 30, 40, 50 and 60, grinding tender leaf part in 50-100mg mortar, extracting DNA (Chenkunyong, 2004) by modified CTAB method, and uniformly diluting the concentration of extracted DNA to 50 + -1 ng/μ l (Nanodrop Products, Wilmington, DE, USA).
The DNA extraction method may be carried out in a commercially available kit.
The PCR method for the germplasm of common vetch to be tested comprises the following steps:
(1) a primer sequence that is used for 4 vicia sativa germplasm appraisals:
scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively;
scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively;
scot38 primer: 5'-ACGACATGGCGACCACCG-3' are provided.
(2) The SCoT primer is adopted to carry out PCR amplification on the DNA of the test variety, wherein the total volume of PCR amplification reaction is 10 mu l, and the amplification reaction system comprises 2 × Power Taq PCR Master Mix 5.0 mu l, 1 mu l SCoT primer, 50 ng/mu l of DNA of the vetch germplasm for test, 2.0 mu l ddH202 mu l; the PCR reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
(3) carrying out agarose gel electrophoresis on the PCR amplification product, wherein the agarose gel concentration is 1.4%, the gel components comprise 6.4g of agarose, 20450ml of ddH, 50ml of 10 × TBE and 12 μ l of nucleic acid dye; running the gel at 129V for 2h and 15min, turning off the power supply, and taking pictures by using a chemical gel imager.
(4) Through SCoT result analysis, comparing differences of the polymorphic sites of the germplasm of the common vetch with 4, and determining the allelic base factor and the polymorphic information content of the primers; carry out "0, 1" statistics on the same migration position, construct SCoT analysis band data of 4 common vetch germplasms, whether distinguish 4 common vetch germplasms completely through data analysis primer, construct the cluster map of 4 common vetch germplasms simultaneously and find the best number of samplings that the identification germplasm purity needs according to allele retention ratio. The allele retention ratio of more than 85 percent can represent most genetic information of the germplasm, and the purity of the variety is qualified.
On the premise of not calculating the time consumption of DNA extraction, the PCR of the identification method takes 2h45min, SCoT analysis takes 2h15min, and the identification work of the common vetch germplasm can be completed within 6 h. A plurality of polymorphic markers can be obtained through one-time amplification to identify the germplasm of common vetch, so that the test efficiency is improved, the test cost is reduced, and the operation method is convenient and fast. The identification method provided by the invention can be used for screening the purity of common vetch seeds, plays a role in protecting forage grass germplasm resources and preventing fake germplasm from flowing into the market, and also provides technical support for breeding new varieties of forage grass and analyzing germplasm genetic diversity.
Example 4: the identification method for simplifying the purity of the common vetch variety by adopting the SCoT molecular marker comprises the following steps of (4) determining the allelic base factors of the primers, counting a 0 and 1 matrix according to the size of the PCR amplification product fragment of the corresponding variety to be detected to obtain the corresponding allelic base factors, calculating the polymorphic information content PIC according to the counted allelic gene and a calculation formula,
wherein: n is the number of alleles; piAnd PjFrequency of the ith and jth alleles, respectively; carry out "0, 1" at the same migration position"make statistics of, construct SCoT analysis band data of common vetch germplasm, whether used primer carries out complete differentiation to common vetch germplasm through data analysis. The remaining procedure was the same as in example 3.
Example 5: the method for identifying the variety purity of common vetch by adopting SCoT molecular markers is simplified, and the method further comprises that the optimal sampling number of each common vetch germplasm is 10 single plants. The remaining procedure was the same as in example 3.
Example 6: the method for simplifying the identification of the variety purity of common vetch by adopting SCoT molecular markers comprises the following steps of (3) carrying out agarose gel electrophoresis on a PCR amplification product, wherein the agarose gel concentration is 1.2%, the gel components are 6.1g of agarose, 20450ml of ddH, 50ml of 10 xTBE and 9 mu l of nucleic acid dye; running the gel at 129V for 2h and 15min, turning off the power supply, and taking pictures by using a chemical gel imager; the remaining procedure was the same as in example 3.
Example 7: the method for simplifying the identification of the variety purity of common vetch by adopting SCoT molecular markers comprises the following steps of (3) carrying out agarose gel electrophoresis on a PCR amplification product, wherein the agarose gel concentration is 1.4%, the gel components are 7.1g of agarose, 20450ml of ddH, 50ml of 10 xTBE and 12 mu l of nucleic acid dye; the running voltage is 132V, the electrophoresis time is 2h and 30min, the power supply is turned off, and a chemical gel imager is used for photographing; the remaining procedure was the same as in example 3.
Test example 1:
(1) test samples: quality of orchid arrow No. 1 (Lanzhou), quality of orchid arrow No. 3 (Lanzhou), quality of common vetch No. 17 (introduced by Israel), quality of common vetch No. 33 (introduced by Belgium);
(2) test reagents: 2 XPower Taq PCR Master Mix solution from Beijing Soilebao; 5 SCoT primers synthesized by Shanghai;
(3) the sequence table of the SCoT primer is as follows:
(4) common vetch sampling and DNA extraction common vetch germplasm sampling and DNA extraction method is as follows: sampling randomly, sampling 60 strains of each germplasm, grinding tender leaf parts in a mortar of 50-100mg, extracting DNA by using a modified CTAB method (Chenkun pine, 2004), and uniformly diluting the concentration of the extracted DNA to 50 +/-1 ng/mu l (Nanodrop Products, Wilmington, DE, USA).
Note: the DNA extraction method may be carried out in a commercially available kit.
(5) PCR amplification reaction
The test variety DNA was PCR amplified with the SCoT primers shown in Table 1, wherein the total volume of PCR amplification reaction was 10. mu.l, and the amplification reaction system was 2 × Power Taq PCR Master Mix 5.0. mu.l, 1. mu.l SCoT primers (1. mu.l each of Scot28 primer, Scot35 primer, Scot36 primer, Scot37 primer, and Scot38 primer), 50 ng/. mu.l, 2.0. mu.l, ddH germplasm DNA of vetch to be tested, and the like202 mu l; the PCR reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
(6) agarose gel electrophoresis
Subjecting the PCR amplification product to agarose gel electrophoresis, wherein the agarose gel concentration is 1.4%, the gel components are agarose 6.4g and ddH20450ml, 10 × TBE 50ml, nucleic acid dye 12. mu.l, running voltage 129V, electrophoresis time 2h15min, power off, and taking pictures with chemical gel imager (see FIG. 1)
(7) SCoT band analysis
Effective amplification is carried out on 4 common vetch germplasms by utilizing 5 SCoT primers, marking 1, 0 is carried out on the position with the same mobility, the amplified strip is marked as 1, the non-amplified strip is marked as 0, and SCoT analysis band data of the 4 common vetch germplasms with the 5 SCoT primers are constructed. From the obtained data, 5 SCoT primers can completely distinguish 4 common vetch germplasms, and the allele retention ratio of 10 randomly sampled plants is found to be greater than or equal to 90% by calculating the allele retention ratio, and generally, the retention ratio of greater than 85% can represent most of genetic information of the germplasms. This shows that only 10 strains of each germplasm need to be randomly sampled to complete the identification of the germplasm purity. By utilizing the set of scientific, efficient and convenient common vetch purity identification method, the purity of common vetch seeds can be evaluated and identified, the effect of protecting forage grass germplasm resources and preventing fake germplasm from flowing into the market is achieved, and meanwhile, technical support is provided for breeding new forage grass varieties and analyzing germplasm genetic diversity.
FIG. 3 is SCoT band analysis cluster map when SCoT primers are used for randomly sampling 60 strains of various germplasms of common vetch.
In order to more clearly explain the identification method of the present invention, the test method of the present invention will be described in detail below.
1. Principle of
SCoT molecular marker, namely target codon target polymorphism (Start codon target polymorphism) marker. Belonging to single primer amplification, the molecular marker designs a primer according to a conserved initiation sequence, since the initiation region of translation has an initiation codon of ATG (+1, +2 and +3) sequence, the 5' end of the gene is taken as the starting point, the positions of G (+4), +7, +8 and +9 are A, C, C respectively, the 7 nucleotides are fixed, the rest positions are complementary sequences, and the diversity of the molecular marker appears in the sequence. The amplified region is 2 intergenic region, the total length of primer is 18 bp. The universal primer sequence is utilized to synthesize a primer, the PCR amplification and the agarose gel electrophoresis are carried out on the researched material, whether the germplasm of the common vetch can be distinguished or not is determined through the statistical analysis of the specific strip, and the optimal sampling number required for identifying the purity of different germplasms is found.
2. Primer design
20 primers are synthesized by Shanghai, and 5 primers capable of clearly and efficiently identifying the purity of common vetch variety are finally obtained through optimization screening.
The primers were synthesized by Shanghai bioengineering.
Table 1 primer sequence table is as follows:
3. common vetch sampling and DNA extraction
The method for sampling and extracting DNA from the germplasm of common vetch with 4 common vetch comprises the following steps: sampling randomly, adopting single plant according to 11 gradients of 1, 2, 3, 5, 8, 10, 20, 30, 40, 50 and 60 for each germplasm, taking 50-100mg of tender leaf parts, grinding in a mortar, extracting DNA by using an improved CTAB method, and uniformly diluting the concentration of the extracted DNA to 50 +/-1 ng/microliter (Nanodrop Products, Wilmington, DE, USA).
Note: the DNA extraction method may be carried out in a commercially available kit.
4. PCR amplification reaction
The total volume of PCR amplification reaction is 10 mul, the amplification reaction system is 2 × Power Taq PCR Master Mix 5.0 mul, SCoT primer 1 mul, DNA of common vetch germplasm for testing 50 ng/mul, 2.0 mul, ddH202 mu l; the PCR reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
5. agarose gel electrophoresis
Subjecting the PCR amplification product to agarose gel electrophoresis, wherein the agarose gel concentration is 1.4%, the gel components are agarose 6.4g and ddH20450ml, 10 × TBE 50ml, nucleic acid dye 12 μ l, running voltage 129V, electrophoresis time 2h15min, power off, and taking pictures with chemical gel imager.
6. SCoT band analysis
Through SCoT result analysis, comparing differences of the polymorphic sites of the germplasm of the common vetch with 4, and determining the allelic base factor and the polymorphic information content of the primers; carry out "0, 1" statistics on same migration position, construct SCoT analysis band data of 4 common vetch germplasms, whether distinguish 4 common vetch germplasms completely through data analysis primer, construct the cluster map of 4 common vetch germplasms simultaneously and find the best number of taking a sample that appraises the germplasm purity according to allele retention ratio. From the obtained data, 5 SCoT primers can completely distinguish 4 common vetch germplasms, and the allele retention ratio of 10 randomly sampled plants is found to be greater than or equal to 90% by calculating the allele retention ratio, and generally, the retention ratio of greater than 85% can represent most of genetic information of the germplasms. This shows that only 10 strains of each germplasm need to be randomly sampled to complete the identification of the germplasm purity.
Table 2.4 table of variation of vetch germplasm according to gradient random sampling allelic base factor and retention ratio.
In the table: when the random sampling numbers of 4 germplasms are respectively 10 strains, the retention ratio is more than or equal to 90 percent.
Test example 2: an identification method for simplifying the purity of common vetch variety by adopting SCoT molecular markers. According to the method, single-plant genome DNA of 4 common vetch varieties is used as a template, and 20 SCoT primers are used as candidate primers.
20 primers are synthesized by Shanghai, and 5 primers capable of clearly and efficiently identifying the purity of common vetch variety are finally obtained through optimization screening.
The primers were synthesized by Shanghai bioengineering.
20 primers
The steps are the same as the experimental steps of 5 primers, the purpose is to run electrophoresis completely, and the primers with clear bands, good repeatability and obvious polymorphism difference are obtained by checking according to an electrophoretogram. Through optimization, the following 5 primer strips are clear and have high polymorphism, and the genetic difference of the germplasm of 4 vicia sativa can be identified.
Scot28 primer: 5'-CCATGGCTACCACCGCCA-3', respectively;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3', respectively;
scot36 primer: 5'-GCAACAATGGCTACCACC-3', respectively;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3', respectively;
scot38 primer: 5'-ACGACATGGCGACCACCG-3' are provided.
Test example 3: (1) test samples: quality of orchid arrow No. 1 (Lanzhou), quality of orchid arrow No. 3 (Lanzhou), quality of common vetch No. 17 (introduced by Israel), quality of common vetch No. 33 (introduced by Belgium);
(2) test reagents: 2 XPower Taq PCR Master Mix solution from Beijing Soilebao; 5 SCoT primers synthesized by Shanghai;
(3) the sequence table of the SCoT primer is as follows:
(4) common vetch sampling and DNA extraction common vetch germplasm sampling and DNA extraction method is as follows: sampling randomly, sampling 10 strains of each germplasm, grinding tender leaf parts in 50-100mg mortar, extracting DNA by using a modified CTAB method (Chenkun pine, 2004), and uniformly diluting the concentration of the extracted DNA to 50 +/-1 ng/microliter (Nanodrop Products, Wilmington, DE, USA).
Note: the DNA extraction method may be carried out in a commercially available kit.
(5) PCR amplification reaction
The test variety DNA was PCR amplified with the SCoT primers shown in Table 1, wherein the total volume of PCR amplification reaction was 10. mu.l, and the amplification reaction system was 2 × Power Taq PCR Master Mix 5.0. mu.l, 1. mu.l SCoT primers (1. mu.l each of Scot28 primer, Scot35 primer, Scot36 primer, Scot37 primer, and Scot38 primer), 50 ng/. mu.l, 2.0. mu.l, ddH germplasm DNA of vetch to be tested, and the like202 mu l; the PCR reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
(6) agarose gel electrophoresis
Subjecting the PCR amplification product to agarose gel electrophoresis, wherein the agarose gel concentration is 1.4%, the gel components are agarose 6.4g and ddH20450ml, 10 × TBE 50ml, nucleic acid dye 12 μ l, running voltage 129V, electrophoresis time 2h15min, turning off power supply, and imaging with chemical gelAnd (6) taking pictures.
(7) SCoT band analysis
Effective amplification is carried out on 4 common vetch germplasms by utilizing 5 SCoT primers, marking 1, 0 is carried out on the position with the same mobility, the amplified strip is marked as 1, the non-amplified strip is marked as 0, and SCoT analysis band data of the 4 common vetch germplasms with the 5 SCoT primers are constructed. From the obtained data, 5 SCoT primers can completely distinguish 4 common vetch germplasms, and the allele retention ratio of 10 randomly sampled plants is found to be greater than or equal to 90% by calculating the allele retention ratio, and generally, the retention ratio of greater than 85% can represent most of genetic information of the germplasms. This shows that only 10 strains of each germplasm need to be randomly sampled to complete the identification of the germplasm purity. By utilizing the set of scientific, efficient and convenient common vetch purity identification method, the purity of common vetch seeds can be evaluated and identified, the effect of protecting forage grass germplasm resources and preventing fake germplasm from flowing into the market is achieved, and meanwhile, technical support is provided for breeding new forage grass varieties and analyzing germplasm genetic diversity.
FIG. 2.SCoT band analysis cluster map when SCoT primer is used for random sampling of 10 strains of various germplasms of common vetch
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
1. The identification method for simplifying the purity of common vetch variety by adopting SCoT molecular marker is characterized by comprising the following steps:
(1) common vetch germplasm DNA extraction: randomly sampling, and extracting the genome DNA of the germplasm to be detected by adopting an improved CTAB method for later use;
(2) performing PCR amplification on the common vetch germplasm DNA obtained in the step (1) by adopting an SCoT primer, wherein the total volume of PCR amplification reaction is 10 mu l, and the amplification reaction system is as follows: 2 × Power Taq PCR Master Mix 5.0 μ l, SCoT primers 1 μ l each, including Scot28 primer: 5'-CCATGGCTACCACCGCCA-3' is 1 μ l;
scot35 primer: 5'-CATGGCTACCACCGGCCC-3' is 1. mu.l ';
scot36 primer: 5'-GCAACAATGGCTACCACC-3' is 1 μ l;
scot37 primer: 5'-ACGACATGGCGACCAGCG-3' is 1 μ l;
scot38 primer: 5' -ACGACATGGCGACCACCG-3 is 1 mu l, the DNA of the common vetch germplasm in the step (1) is 50 ng/mu l, 2.0 mu l and ddH202 mu l; the PCR amplification reaction program is pre-denaturation at 94 ℃ for 4 min; 35 amplification cycles: denaturation at 94 deg.C for 1min, annealing for 1min, and extension at 72 deg.C for 2 min; extending for 7min at 72 ℃; storing at 4 deg.C;
(3) carrying out agarose gel electrophoresis on the PCR amplification product, wherein the agarose gel concentration is 1.2-1.4%, the gel components comprise 6.1-7.1g of agarose, 20450 ddH20450ml, 50ml of 10 xTBE and 9-12 mu l of nucleic acid dye; the gel running voltage is 129-132V, the electrophoresis time is 2h15min-2h30min, the power supply is closed, and a chemical gel imager is used for photographing;
(4) compare the difference of common vetch germplasm polymorphism site, confirm common vetch variety purity appraisal result, common vetch germplasm average polymorphism site is 21.4-27.4, through statistics 0,1 matrix, statistics allelic factor, calculate and observe the heterozygosity, observe heterozygosity (Ho) and obtain miscellaneous and individual number/sample individual number total number, Ho is 100%, the statistics site is the polymorphism site, allele retention ratio is greater than 85% and can represent this germplasm majority genetic information, variety purity is qualified.
2. The method for simplifying the identification of the purity of the common vetch variety by using the SCoT molecular marker according to claim 1, wherein the step (4) further comprises the steps of determining the allelic base factors of the primers, counting a 0 and 1 matrix according to the size of the PCR amplification product fragment of the corresponding variety to be detected to obtain the corresponding allelic base factors, calculating the polymorphic information content PIC according to a calculation formula according to the counted allelic genes,
wherein: n is the number of alleles; piAnd PjFrequency of the ith and jth alleles, respectively; carry out "0, 1" statistics on the same migration position, construct SCoT analysis band data of common vetch idioplasm, whether carry out complete differentiation to common vetch idioplasm through the used primer of data analysis.
3. The method for facilitating the identification of the variety purity of common vetch using SCoT molecular markers as claimed in claim 1, further comprising the step of obtaining an optimal sampling number of 10 individuals from each common vetch germplasm.
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