CN106676176B - Method for performing SSR (simple sequence repeat) analysis on tetraploid alfalfa by utilizing multiple PCR (polymerase chain reaction) - Google Patents

Abstract

The invention relates to a method for carrying out SSR analysis on tetraploid alfalfa by utilizing multiplex PCR. Specifically comprises 9 multiple PCR-SSR groups (total 24 pairs of SSR primers) and corresponding PCR reaction systems and reaction programs. The 24 SSR markers are uniformly distributed on the chromosome, and the distance between the markers is not less than 10 Mbp; the marker genotype is easy to identify, and the accuracy of the average genotype is higher than 95%. When the scheme is adopted to carry out genetic diversity analysis, population structure analysis and variety identification on the tetraploid alfalfa, the method has the advantages of short time, low cost and high accuracy, and the total efficiency can be improved by 2-3 times.

Description

Method for performing SSR (simple sequence repeat) analysis on tetraploid alfalfa by utilizing multiple PCR (polymerase chain reaction)

Technical Field

The invention relates to a method for carrying out SSR analysis on tetraploid alfalfa by utilizing multiplex PCR, belonging to the technical field of biology.

Background

Alfalfa (Medicago sativa) is the most important forage crop in temperate regions of the world, and two subspecies (m.sativa subssp.sativa and m.sativa subssp.xvaria) which are mainly planted are both homotetraploids, and are naturally outcrossed and seriously inbred. Genetic diversity is the basis for plant breeding and genetic improvement. As most alfalfa varieties are synthetic varieties, the alfalfa varieties are bred by continuously and randomly mating selected excellent single plants and filial generations thereof for several generations. Therefore, 20-40 individuals are generally required to be taken from one alfalfa variety when genetic diversity analysis is performed. When the variety of the study is large, the number of the analysis units (genotypes) is often very large. Due to the limitation of factors such as actual workload, expenses, time and the like, such research plans are often difficult to implement. The number of analysis units (genotypes) in existing related studies does not generally exceed 1000.

Until now, various molecular marker technologies have been used in the research of alfalfa genetic diversity, such as allelic enzymes, seed storage proteins, RFLP, RAPD, SSR, ISSR, EST-SSR, etc. Among them, SSR (Simple sequence repeat, also called Microsolalite DNA, Microsatellite DNA) is most widely used, mainly because it has many advantages: co-dominant inheritance, which can be used for identifying heterozygotes and homozygotes; the marker chromosomes are definite in location and rich in quantity; the specific primers are used, so that the operation is simple, the repeatability is good, and the reliability is high; the requirements on the amount and purity of DNA are not high. For species that have developed sufficient SSR markers, the main drawback is that only one SSR marker can generally be analyzed per assay to obtain genotype information for one site. This limits the scale of the experiment to a large extent, especially for the outcrossing plant alfalfa. Most of the existing genetic diversity studies use fewer SSR markers (< 50) or (and) analyze fewer genotypes (< 1000). In addition, some developed SSR markers also have the problems of high ratio of invalid genotypes (no bands or difficult determination of allele dose) and difficult interpretation of maps, and the like, which also limits the application of SSR in alfalfa genetic diversity research to a certain extent. Therefore, screening excellent SSR markers and establishing a high-flux analysis method are the premise of obtaining accurate test data and improving test efficiency.

The multiplex PCR (multiplex PCR) technique proposed by Chamberland, 1998, is an improvement over the conventional PCR technique by adding multiple pairs of specific primers to a PCR reaction system to amplify multiple target fragments against multiple DNA templates or different regions of the same template. Compared with PCR of single gene molecular marker, multiple PCR can detect several target genes simultaneously in one PCR, the working efficiency is obviously improved, and the cost is obviously reduced. Due to the advantages of the multiplex PCR technology, the multiplex PCR technology is widely applied to various fields of scientific research, such as virus detection, bacteria detection, avian influenza detection, quality character molecular markers and the like.

At present, the research of the multiplex PCR-SSR technology on plants is not deep enough, and the research contents are mostly focused on the screening of primers and the establishment and optimization of a multiplex PCR system; only a few plants such as beet, cotton, waxberry, pepper and the like are deeply researched. However, the existing research proves that the method can obviously improve the information content of single SSR analysis, reduce the cost when SSR primers are used for large-scale analysis, and shorten the time required by the experiment. At present, no method for analyzing genetic diversity of tetraploid alfalfa by utilizing multiple PCR-SSR exists. The patent aims to screen excellent alfalfa SSR markers and establish a high-throughput analysis method based on multiplex PCR.

Disclosure of Invention

The invention aims to provide a method for carrying out SSR analysis on tetraploid alfalfa by utilizing a multiplex PCR technology aiming at the defects of the prior art.

The technical scheme of the invention is summarized as follows: firstly, screening 24 pairs of SSR primers, and optimally combining into 9 PCR groups; then adding a group of 2-3 pairs of SSR specific primer pairs which belong to the same group and have different concentrations into a PCR reaction tube, and simultaneously amplifying a plurality of target fragments in different areas of the same template by adopting different reaction systems; finally, a plurality of amplification products of different primers are separated by one electrophoresis.

Specifically, the technical scheme of the invention comprises the following steps:

1. screening out 24 pairs of SSR primers

According to the existing literature data, factors such as amplification conditions of PCR reaction, distribution condition of primers on chromosome, size, definition and polymorphism of bands, accuracy and stability of genotype and the like are comprehensively considered, and 24 pairs of SSR primers are screened out. The following table shows the screened SSR primers SEQ ID NO: 1 to SEQ ID NO: 48 (wherein SEQ ID NO: 1/SEQ ID NO: 2 is a pair of forward and reverse primers, and so on).

TABLE 1 SSR primers for alfalfa multiplex PCR

2. Multiplex PCR

And optimally combining 9 PCR groups according to the respective characteristics of the primers. Adding 2-3 pairs of SSR specific primer pairs belonging to the same group into a PCR reaction tube, forming a plurality of different reaction systems aiming at the same DNA template, and completing multiple PCR according to the same PCR amplification program. The grouping of multiplex PCR reactions and primer concentrations are shown in Table 2.

TABLE 2 grouping of multiplex PCRs

Note: the primer concentrations refer to the respective concentrations of the forward and reverse primers

Further, the reaction system comprises the following components: 1.0-2.5 ng/. mu.L of template DNA, 0.03-0.10U/. mu.L of Taq DNA polymerase, 0.20-0.35 mM of dNTPs, 1 XPCR Buffer (containing 1.5mM MgCl2), 0.085-0.285. mu.M of primer concentration and 15. mu.L of deionized water.

Further, the PCR amplification procedure comprises the steps of:

(1) synthesizing 24 pairs of primers in table 1;

(2) set up 9 PCR sets according to Table 2 and perform PCR amplification as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending for 8min at 72 ℃, and storing at 4 ℃.

3. Electrophoretic detection

And detecting the amplification product by adopting conventional denaturing polyacrylamide gel electrophoresis.

The invention has the advantages and beneficial effects that:

1) map data of 24 SSR markers were obtained by 9 PCR reactions. During the analysis, the usage amount of drugs and consumables such as template DNA, TaqDNA polymerase, dNTPs, PCR reaction tubes and the like is reduced to 37.5 percent (9/24) of single-primer PCR on average, and the time from the execution of PCR operation to the electrophoresis sample adding is also reduced to 37.5 percent (9/24) of the single-primer PCR.

2) The accuracy is as follows: the genotype of the marker is easy to identify, and the data reading is fast; the selected SSR markers are uniformly distributed on the chromosome, the accuracy of the average genotype is higher than 95%, and the result of genetic diversity analysis is reliable.

Drawings

FIG. 1: the amplification maps of 12 WL323 alfalfa individuals of 3 primer pairs in the group A; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 2: h group 2 pair primer pair 12 Algang alfalfa single plant amplification maps; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 3: d group 3 pair primer pair 12 alfalfa 1 single plant amplification map; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 4: b, amplifying maps of 12 single strains of alfalfa in pasture are obtained by 3 pairs of primers in group; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 5: c group 3 primer pairs 12 amplification maps of three-Deliver alfalfa single plants; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 6: e group 3 primer pairs are used for amplifying the maps of 12 single strains of the Victoria alfalfa; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 7: f group 3 pairs of primers for 12 reindeer alfalfa single plants amplification maps; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 8: g group 2 pair primer pair 12 amplification maps of single plants of the fixed alfalfa; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

FIG. 9: i, amplifying maps of 12 single strains of the Longdong alfalfa by 2 pairs of primers; wherein: m is Marker, 1-12 are amplification results of different alfalfa individuals.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The alfalfa varieties selected in the following examples are to be understood as exemplary, and the method of the present invention is applicable to all alfalfa varieties currently known.

Example 1:

1. extraction of genomic DNA and primer Synthesis

The method comprises the steps of taking alfalfa variety WL323 as a test material, randomly selecting 12 individual plants, shearing enough young and tender leaves, freezing and grinding by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group A primers BBI131F, GBG230 and E776153 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5 ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers BBI131F, GBG230 and E776153 at concentrations of 0.145. mu.M, 0.170. mu.M and 0.135. mu.M, respectively (same forward and reverse primer concentrations), and deionized water to make up 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending for 8min at 72 ℃, and storing at 4 ℃.

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 1 is an amplification map of group A3 pairs of primers (BBI131, GBG230 and E776153). As can be seen from FIG. 1, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are abundant, and the primers can be easily distinguished from each other. Taking a3 as an example, there are 4 bands from top to bottom, which correspond to 4 alleles and are respectively named as a, b, c and d, and then the genotypes of 12 samples are: bccc, aadd, abcd, cccd, bbdd, aaab, abbd, abcd, accd, bccc, bccd.

Example 2:

1. extraction of genomic DNA and primer Synthesis

The method comprises the steps of taking alfalfa variety Algang gold as a test material, randomly selecting 12 individual plants, shearing enough tender leaves, freezing and grinding the leaves by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group H primers CBF96 and GAW212 were synthesized according to the primer sequences in table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers CBF96 and GAW212 were 0.160. mu.M and 0.140. mu.M, respectively (same forward and reverse primer concentrations), and deionized water was supplemented to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 2 is an amplification map of group H primers CBF96 and GAW 212. As can be seen from FIG. 2, the amplification products of the two pairs of primers are clear, the polymorphic sites are abundant, and the primers can be easily distinguished from each other. Taking h1 as an example, there are 6 bands from top to bottom, which correspond to 6 alleles and are respectively named as a, b, c, d, e and f, and the genotypes of the 12 samples are sequentially: ccce, abee, bbee, eeee, eeef, ade, abede, ddee, aabd, acde, abee, bbdd.

Example 3:

1. extraction of genomic DNA and primer Synthesis

Taking alfalfa No. 1 in the alfalfa variety as a test material, randomly selecting 12 single plants, shearing enough tender leaves, freezing and grinding the leaves by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking the lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group D primers GBF56, CAW306, and BBG28 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers GBF56, CAW306 and BBG28 were 0.285. mu.M, 0.115. mu.M and 0.125. mu.M, respectively (the forward and reverse primer concentrations were the same), and deionized water was added to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 3 is an amplification map of group D3 pair primers GBF56, CAW306 and BBG 28. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking d2 as an example, there are 5 bands from top to bottom, which correspond to 5 alleles and are respectively named as a, b, c, d and e, and then the genotypes of 12 samples are: bcde, acde, bcde, ccdd, abce, dddd, ccde, abbd, cddd, acdd, bdde, bcde.

Example 4:

1. extraction of genomic DNA and primer Synthesis

The method comprises the steps of taking alfalfa variety pasture as a test material, randomly selecting 12 individual plants, shearing enough young and tender leaves, freezing and grinding with liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group B primers DMt1H10, CBF156 and DBI28 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5 ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers DMt1H10, CBF156 and DBI28 were 0.225. mu.M, 0.105. mu.M and 0.120. mu.M, respectively (the forward and reverse primer concentrations were the same), and deionized water was added to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 4 is an amplification map of group B3 pair primers DMt1H10, CBF156 and DBI 28. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking b3 as an example, 8 bands are shared from top to bottom, which correspond to 8 alleles and are respectively named as a, b, c, d, e, f, g and h, and then the genotypes of 12 samples are sequentially: dggg, eeef, eefg, abgh, ccee, ddef, efhh, adde, aabg, eeeh, deeh, bdee.

Example 5:

1. extraction of genomic DNA and primer Synthesis

The method comprises the steps of taking the alfalfa variety Sandeli as a test material, randomly selecting 12 individual plants, shearing enough young and tender leaves, freezing and grinding the leaves by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle et al, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group C primers ABE93, CIC338 and AMTIC95 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers ABE93, CIC338 and AMTIC95 were 0.235. mu.M, 0.150. mu.M and 0.140. mu.M, respectively (the forward and reverse primer concentrations were the same), and deionized water was added to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 5 is an amplification map of group C3 pair primers ABE93, CIC338 and AMTIC 95. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking c3 as an example, there are 5 bands from top to bottom, which correspond to 5 alleles and are respectively named as a, b, c, d and e, and then the genotypes of 12 samples are: bee, aace, aaae, bdde, bcce, bbbe, abee, bccc, ccee, abcd, bcee, ccce.

Example 6:

1. extraction of genomic DNA and primer Synthesis

Taking alfalfa variety Victoria as a test material, randomly selecting 12 individual plants, shearing enough young leaves, freezing and grinding by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group E primers HAW178, AAW365 and HAL82 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers HAW178, AAW365 and HAL82 were 0.205. mu.M, 0.100. mu.M and 0.145. mu.M, respectively (the forward and reverse primer concentrations were the same), and deionized water was added to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 6 is an amplification map of group E3 primers HAW178, AAW365 and HAL 82. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking e1 as an example, there are 7 bands from top to bottom, which correspond to 7 alleles and are respectively named as a, b, c, d, e, f and g, and then the genotypes of 12 samples are: gggg, fgggg, cfgg, bggg, dfgg, aefg, bdff, bdfg, ffgg, bdgg, and bggg.

Example 7:

1. extraction of genomic DNA and primer Synthesis

The method comprises the steps of taking alfalfa variety reindeer as a test material, randomly selecting 12 individual plants, shearing enough young and young leaves, freezing and grinding the leaves by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle and the like, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group F primers DAW289, DBE84 and BBG280 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5 ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers DAW289, DBE84 and BBG280 at the same concentrations of 0.160. mu.M, 0.205. mu.M and 0.160. mu.M forward and reverse primers, respectively), and deionized water to make up to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 7 is an amplification map of group F3 primers DAW289, DBE84 and BBG 280. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking f2 as an example, there are 5 bands from top to bottom, which correspond to 5 alleles and are respectively named as a, b, c, d and e, and then the genotypes of 12 samples are: abbd, aabe, aaae, aaaa, aaad, ccce, accc, aaaa, aaac, aadd, bbcc, aabc.

Example 8:

1. extraction of genomic DNA and primer Synthesis

The alfalfa variety is reserved as a test material, 12 individual plants are randomly selected, enough young leaves are cut, after freezing and grinding by liquid nitrogen, genomic DNA is extracted by adopting a CTAB method of Doyle and the like, and the quality and the quantity of the genomic DNA are subjected to electrophoresis detection by using 0.8% agarose gel by taking lambda DNA as a reference standard.

Group G primers HMt1G03 and FAW115 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5 ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers HMt1G03 and FAW115 were 0.195. mu.M and 0.105. mu.M, respectively (same forward and reverse primer concentrations), and deionized water was added to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 8 is an amplification map of group G2 pair of primers HMt1G03 and FAW 115. As can be seen from the figure, the amplification products of the 3 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking g2 as an example, there are 7 bands from top to bottom, which correspond to 7 alleles and are respectively named as a, b, c, d, e, f and g, and then the genotypes of 12 samples are: ddfg, ccgg, accc, bddd, aade, cccd, cceg, abbd, addd, ddfg, adee, dddd.

Example 9:

1. extraction of genomic DNA and primer Synthesis

Taking the alfalfa variety of Longdong as a test material, randomly selecting 12 individual plants, shearing enough young and tender leaves, freezing and grinding the leaves by liquid nitrogen, extracting genome DNA by adopting a CTAB method of Doyle et al, taking lambda DNA as a reference standard, and carrying out electrophoresis detection on the quality and quantity of the genome DNA by using 0.8% agarose gel.

Group I primers GBG288 and BBG238 were synthesized according to the primer sequences in Table 1.

2. Multiplex PCR

(1) The reaction system comprises the following components:

template DNA1.5ng/. mu.L, Taq DNA polymerase 0.035U/. mu.L, dNTPs 0.25mM, 1 XPCR Buffer (containing 1.5mM MgCl2), primers GBG288 and BBG238 were 0.085. mu.M and 0.215. mu.M, respectively (the forward and reverse primer concentrations were the same), and deionized water was supplemented to 15. mu.L.

(2) PCR amplification was performed as follows:

pre-denaturation at 94 ℃ for 4 min;

denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

extending at 72 deg.C for 8min, and storing at 4 deg.C

3. Electrophoretic detection

The detection is carried out by adopting conventional denaturing polyacrylamide gel electrophoresis. FIG. 9 is an amplification map of group I2 pair primer GBG288 and BBG 238. As can be seen from the figure, the amplification products of the 2 pairs of primers are clear, the polymorphic sites are rich, and the primers can be easily distinguished from each other. Taking i1 as an example, there are 7 bands from top to bottom, which correspond to 7 alleles and are respectively named as a, b, c, d, e, f and g, and then the genotypes of 12 samples are: ccdf, ccef, dddf, deeg, dddd, eeeg, addf, bdef, bcdd, abdd, cdef, deeg.

SEQUENCE LISTING

<110> Tianjin college of agriculture

<120> method for carrying out SSR analysis on tetraploid alfalfa by utilizing multiplex PCR

<130>2017

<160>48

<170>PatentIn version 3.5

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<400>22

gtgttcgtcg catatcacct c 21

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<400>23

gagcaaaggg gtttgtctca 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<400>24

gcaactccag ctgcatcttt 20

<210>25

<211>22

<212>DNA

<213> Artificial sequence

<400>25

ttgcctcaac ctctgctaat tc 22

<210>26

<211>22

<212>DNA

<213> Artificial sequence

<400>26

gccgaagagc ctttgatagt aa 22

<210>27

<211>22

<212>DNA

<213> Artificial sequence

<400>27

caccactatc tcttccctca cc 22

<210>28

<211>22

<212>DNA

<213> Artificial sequence

<400>28

tgttggtaat gttcaagctc ca 22

<210>29

<211>20

<212>DNA

<213> Artificial sequence

<400>29

ccgaatgaga gcaaccattt 20

<210>30

<211>20

<212>DNA

<213> Artificial sequence

<400>30

ttgatcaaca gcgaatcgag 20

<210>31

<211>22

<212>DNA

<213> Artificial sequence

<400>31

acgaggcaca cactctctct ct 22

<210>32

<211>22

<212>DNA

<213> Artificial sequence

<400>32

ggtgctttca ttacatccca ta 22

<210>33

<211>22

<212>DNA

<213> Artificial sequence

<400>33

tccgaaccct acttccaaat ta 22

<210>34

<211>22

<212>DNA

<213> Artificial sequence

<400>34

tgggatactg attttctgct tc 22

<210>35

<211>22

<212>DNA

<213> Artificial sequence

<400>35

tcagcagtta gttttggtat gc 22

<210>36

<211>22

<212>DNA

<213> Artificial sequence

<400>36

tgttgaagtt ggagttttgg tg 22

<210>37

<211>20

<212>DNA

<213> Artificial sequence

<400>37

aaagagattg ggtcggtgaa 20

<210>38

<211>21

<212>DNA

<213> Artificial sequence

<400>38

tggttgatca atgttcctcc t 21

<210>39

<211>20

<212>DNA

<213> Artificial sequence

<400>39

accaaaccca cttcccatct 20

<210>40

<211>20

<212>DNA

<213> Artificial sequence

<400>40

ttgaagttgg tggaacagca 20

<210>41

<211>20

<212>DNA

<213> Artificial sequence

<400>41

cgatctggct tgaggatagg 20

<210>42

<211>20

<212>DNA

<213> Artificial sequence

<400>42

ggcagggcta agcttctcat 20

<210>43

<211>22

<212>DNA

<213> Artificial sequence

<400>43

gtcgaaatgg ttgcttctct tt 22

<210>44

<211>21

<212>DNA

<213> Artificial sequence

<400>44

ggttagggtt ttgggtttga a 21

<210>45

<211>22

<212>DNA

<213> Artificial sequence

<400>45

gaaggagaaa aggtgagggt tt 22

<210>46

<211>22

<212>DNA

<213> Artificial sequence

<400>46

tcggatgatg atgaagaagt gt 22

<210>47

<211>18

<212>DNA

<213> Artificial sequence

<400>47

ccggctcaac gatccagt 18

<210>48

<211>19

<212>DNA

<213> Artificial sequence

<400>48

agtgggaatt ggagggtca 19

Claims (5)

1. A method for carrying out SSR analysis on tetraploid alfalfa by utilizing a multiplex PCR technology is characterized by comprising the following steps:

1) the following 24 pairs of SSR primers were combined into 9 PCR groups as follows:

group A: primer pair BBI131 is shown as SEQ ID NO: 1 and SEQ ID NO: as shown in figure 2, the first and second,

primer pair GBG230 is set forth as SEQ ID NO: 3 and SEQ ID NO: as shown in (4) in the figure,

primer pair E776153 is shown in SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;

group B: primer pair DMt1H10 is shown as SEQ ID NO: 7 and SEQ ID NO: as shown in figure 8, the flow of air,

primer pair CBF156 is as set forth in SEQ ID NO: 9 and SEQ ID NO: as shown in figure 10 of the drawings,

primer pair DBI28 is as set forth in SEQ ID NO: 11 and SEQ ID NO: 12 is shown in the specification;

group C: primer pair ABE93 is set forth in SEQ ID NO: 13 and SEQ ID NO: as shown in figure 14, the first and second,

primer pair CIC338 is as shown in SEQ ID NO: 15 and SEQ ID NO: as shown at 16, the flow of the gas,

primer pair AMTIC95 is shown as SEQ ID NO: 17 and SEQ ID NO: 18 is shown in the figure;

group D: the primer pair GBF56 is shown as SEQ ID NO: 19 and SEQ ID NO: as shown at 20, the flow of the gas,

primer pair CAW306 is shown as SEQ ID NO: 21 and SEQ ID NO: as shown at 22, the flow of air is,

primer pair BBG28 is shown as SEQ ID NO: 23 and SEQ ID NO: shown at 24;

group E: primer pair HAW178 is as shown in SEQ ID NO: 25 and SEQ ID NO: as shown at 26, the flow of the gas,

the primer pair AAW365 is shown as SEQ ID NO: 27 and SEQ ID NO: as shown at 28, the flow of the gas,

primer pair HAL82 is shown as SEQ ID NO: 29 and SEQ ID NO: 30 is shown in the figure;

and F group: primer pair DAW289 is shown as SEQ ID NO: 31 and SEQ ID NO: as shown at 32, the flow of the gas,

primer pair DBE84 is as set forth in SEQ ID NO: 33 and SEQ ID NO: as shown at 34, the first and second side walls of the container,

primer pair BBG280 is shown as SEQ ID NO: 35 and SEQ ID NO: 36 is shown;

group G: primer pair HMt1G03 is set forth as SEQ ID NO: 37 and SEQ ID NO: as shown at 38, the first and second side walls,

primer pair FAW115 is set forth in SEQ ID NO: 39 and SEQ ID NO: 40 is shown in the figure;

group H: primer pair CBF96 is set forth in SEQ ID NO: 41 and SEQ ID NO: as shown at 42, the flow of the gas,

primer pair GAW212 is as set forth in SEQ ID NO: 43 and SEQ ID NO: 44 is shown;

group I: primer pair GBG288 is set forth as SEQ ID NO: 45 and SEQ ID NO: as shown at 46, the flow of gas through the nozzle,

primer pair BBG238 is shown as SEQ ID NO: 47 and SEQ ID NO: 48 is shown;

2) multiplex PCR: adding SSR specific primer pairs belonging to the same group into 9 PCR reaction tubes respectively, forming a plurality of different reaction systems aiming at the same DNA template, and completing multiple PCR according to the same PCR amplification program;

3) and separating the amplification products by one-time conventional denaturing polyacrylamide gel electrophoresis to obtain map data of 24 SSR markers.

2. The method according to claim 1, wherein the reaction system comprises template DNA 1.0-2.5 ng/. mu.L, Taq DNA polymerase 0.03-0.10U/. mu.L, dNTPs 0.20-0.35 mM, and 1 × PCR Buffer, wherein the dNTPs comprise 1.5mM MgCl₂The concentration of each pair of the forward primer and the reverse primer is 0.085-0.285 mu M respectively, and the deionized water is supplemented to 15 mu L.

3. The method for SSR analysis of tetraploid alfalfa according to claim 2, wherein the primer concentrations are as follows:

group A: the concentration of the primer pair BBI131 is 0.145. mu.M, the concentration of the primer pair GBG230 is 0.170. mu.M, and the concentration of the primer pair E776153 is 0.135. mu.M;

group B: the concentration of the primer pair DMt1H10 is 0.225. mu.M, the concentration of the primer pair CBF156 is 0.105. mu.M, and the concentration of the primer pair DBI28 is 0.120. mu.M;

group C: the concentration of the primer pair ABE93 is 0.235 mu M, the concentration of the primer pair CIC338 is 0.150 mu M, and the concentration of the primer pair AMTIC95 is 0.140 mu M;

group D: the concentration of the primer pair GBF56 is 0.285 mu M, the concentration of the primer pair CAW306 is 0.115 mu M, and the concentration of the primer pair BBG28 is 0.125 mu M;

group E: the concentration of the primer pair HAW178 is 0.205. mu.M, the concentration of the primer pair AAW365 is 0.100. mu.M, and the concentration of the primer pair HAL82 is 0.145. mu.M;

and F group: the concentration of the primer pair DAW289 was 0.160. mu.M, the concentration of the primer pair DBE84 was 0.205. mu.M, and the concentration of the primer pair BBG280 was 0.160. mu.M;

group G: the concentration of the primer pair HMt1G03 was 0.195. mu.M, and the concentration of the primer pair FAW115 was 0.105. mu.M;

group H: the concentration of the primer pair CBF96 was 0.160. mu.M, and the concentration of the primer pair GAW212 was 0.140. mu.M;

group I: the concentration of the primer pair GBG288 was 0.085. mu.M, and the concentration of the primer pair BBG238 was 0.215. mu.M;

the concentrations above refer to the respective concentrations of the forward and reverse primers.

4. A method for SSR analysis of tetraploid alfalfa according to claim 1 or 2 or 3, wherein the PCR amplification procedure comprises the following steps:

1) pre-denaturation at 94 ℃ for 4 min;

2) denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 1min for 34 cycles;

3) extending for 8min at 72 ℃, and storing at 4 ℃.

5. Use of the method of any one of claims 1-4 for genetic diversity analysis of tetraploid alfalfa.

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