CN106636417B

CN106636417B - Method for constructing pea SSR fingerprint

Info

Publication number: CN106636417B
Application number: CN201611247537.3A
Authority: CN
Inventors: 宗绪晓; 张红岩; 杨涛; 刘荣
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2020-02-11
Anticipated expiration: 2036-12-29
Also published as: CN106636417A

Abstract

The invention discloses a method for constructing a pea SSR fingerprint. The invention provides a marker combination consisting of 31 pairs of pea SSR primers, the nucleotide sequences of which are respectively shown in SEQ ID No.1-62, and the pea strain or the wild kindred species of the pea are identified by utilizing a PCR amplification technology, a non-denaturing polyacrylamide gel electrophoresis or a fluorescence PCR capillary electrophoresis detection technology, so that the identification and the genetic diversity evaluation of the pea strain or the wild kindred species of the pea can be completed in a short time, and the marker combination has the advantages of high efficiency, accuracy, convenient operation, difficult environmental influence on the identification result and the like. The method can effectively monitor the authenticity of the pea seeds, reveal the genetic variation and genetic relationship of the variety from the DNA level, protect the crop variety, prevent counterfeit and shoddy varieties from entering the market, provide technical support for reasonable utilization of excellent germplasm in the process of breeding the pea variety, and have good application prospect.

Description

Method for constructing pea SSR fingerprint

Technical Field

The invention relates to the technical field of molecular biology and plant variety identification, in particular to a method for constructing a pea SSR fingerprint, a method for identifying pea strains or wild allied species thereof by using the constructed pea SSR fingerprint and application.

Background

Pea (Pisum sativum L.) belongs to one of the Vicia (Pipilionoideae) family of Papilionoideae (Vicieae) family of Papilionaceae (Leguminosae) family of Papilionaceae (Pisum) in cultivation, and belongs to an annual (spring sowing) or perennial (autumn sowing) climbing rock-climbing herbaceous self-pollinating plant. Peas are the fourth edible legume crop in the world, and over 60 countries produce dry peas globally; china is the world's principal pea producing country, and the total area and the total yield are the first world. Peas are suitable for cold and cool climates, various lands and drought environments, have the characteristics of high protein content, abundant vitamins, easy digestion and absorption, dual purposes of grains, vegetables, feeds and fertilizers and value increase of deep processing, are important crops for intercropping, interplanting, crop rotation and land cultivation in the structure adjustment of the planting industry, and have important influence on the sustainable agricultural development of China and the food structure of people in China. Therefore, the effective screening of pea varieties is very important.

At present, the main method for identifying the pea variety in China is to carry out field determination according to NY/T2436-2013 'guide pea for testing the specificity, consistency and stability of new plant variety'. The method is simple, convenient and economical, but is limited by factors such as long identification period of a plurality of morphological traits, easy influence of environment and the like, so that identification basis cannot be accurately and quickly provided for new species infringement cases, and meanwhile, the problem that the judgment result is inconsistent due to deviation of the cognition of different testers on certain traits often occurs. Currently, with the centralized utilization of breeding backbone parents, traditional morphological tests cannot meet the requirements of variety identification and purity analysis.

With the rapid development of biotechnology, a DNA fingerprint (fingerprint) established based on a molecular marker technology plays an important role in variety and strain identification. The construction of the variety DNA fingerprint database has important application value in the aspects of pea variety purity and authenticity identification, seed quality standardization, variety approval and property right registration protection, true and false species identification, variety disputes and the like, and plays a significant role in the aspects of researching genetic relationship of pea germplasm resources in China, heterosis group division, germplasm resource innovation utilization, new variety breeding and the like. The current marking technology for constructing the DNA fingerprint of the crop mainly comprises RFLP, RAPD, AFLP, EST, ISSR, SSR and the like. Simple Sequence Repeat (SSR), a second generation of molecular marker technology based on PCR technology, has the advantages of abundant polymorphism, abundant genetic information, convenient operation, co-dominance, high repeatability, etc., and has been widely used in plant resource genetic research and molecular assisted breeding practice.

DNA fingerprinting enables identification of differences or genetic similarities between varieties at the DNA level. Microsatellites (also known as simple repeats, SSRs) are DNA fragments consisting of a short sequence of 1-6 nucleotides repeated in tandem. Since the number of repetitions of the repeating unit in the microsatellite is different, the length of the amplified microsatellite sequence shows polymorphism. The microsatellite sequence has the characteristics of higher polymorphism, good repeatability and stability and the like, is a very effective molecular marker, and is widely applied to the fields of variety identification, fingerprint map construction and the like. In order to effectively protect the intellectual property of pea varieties in China, development of microsatellite marker development in China is necessary, and the microsatellite marker development is applied to research on construction of 'molecular identity cards' of excellent pea varieties.

Disclosure of Invention

The invention aims to provide a method for constructing a pea SSR fingerprint, and the other aim of the invention is to provide a method for identifying pea strains or wild allied species thereof by using the pea SSR fingerprint with high efficiency, accuracy and low cost. The invention also aims to provide an application of the method.

In order to achieve the purpose, 49 parts of representative pea varieties and 32 parts of pea germplasm resource genome DNA are used as templates, and a core genome SSR (simple sequence repeat) and EST-SSR (expressed sequence tag) primer is synthesized by analyzing a pea genome sequence and an EST (expressed sequence tag) sequence. Through screening, the molecular marker combination consisting of 19 pairs of genome SSR primers and 12 pairs of EST-SSR primers can completely identify the difference between 49 parts of pea varieties and 32 parts of pea germplasm resources. The 81 representative reference pea materials are shown in table 1. In table 1, 49 pea cultivars (W7-W55, 6 foreign pea resources (W1-W6), 18 pea core germplasm resources (W56-W73), 73 pea materials in total, collectively referred to as "pea lines", and the other 8 wild pea resources (W74-W81) in table 1 are wild kindred species of peas.

Table 181 parts of pea lines or wild kindred species thereof

After multiple screening, the SSR primers which are uniformly distributed on the linkage groups, have higher polymorphism and more stable PCR amplification, meet two technical platforms of conventional non-denaturing gel electrophoresis detection and capillary fluorescence electrophoresis detection, and are the highest in polymorphic information content value, the most in number of allelic sites, good in repeatability, high in amplification band type definition and uniformly distributed in the linkage groups are finally screened out and used as core primers for constructing the SSR fingerprint spectrum library of the peas.

Based on the screening method, the invention provides a molecular marker combination for identifying pea strains or wild relatives thereof, which is characterized by comprising 19 genome SSR molecular markers and 12 EST-SSR molecular markers, namely SSR4825, SSR4696, SSR28304, SSR24036, B83, EST671, SSR27844, SSR25888, SSR24882, SSR23759, SSR21491, EST619, SSR24112, EST599, SSR22754, P14, SSR24731, SSR25334, EST876, SSR28967, SSR21399, SSR21405, P282, EST, SSR25799, SSR25965, P291, P292, SSR22052, P344 and EST 617;

the molecular markers are obtained by amplifying the following primer pairs respectively: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52, SEQ ID NO.53-54, Primers shown in SEQ ID NO.55-56, SEQ ID NO.57-58, SEQ ID NO.59-60 and SEQ ID NO. 61-62.

The invention provides a primer combination, which contains the following 31 pairs of specific primer pairs, and the nucleotide sequences of the specific primer pairs are respectively shown as SEQID NO. 1-62.

The invention provides a kit containing the primer combination.

The invention provides application of the molecular marker combination or the primer combination in identifying pea strains or wild kindred species thereof.

The invention provides application of the molecular marker combination or the primer combination in improvement of pea germplasm resources.

The invention provides application of the molecular marker combination or the primer combination in constructing a pea SSR plant map.

The invention also provides a construction method of the pea SSR fingerprint, which comprises the following steps of carrying out PCR amplification on genome DNA of different pea varieties and germplasm resources by using the primer combination containing the 31 pairs of primers, carrying out non-denaturing polyacrylamide gel electrophoresis on a PCR product for detection or carrying out fluorescence capillary electrophoresis on the PCR product for detection:

the data of the size of the allelic variation of the homozygous locus is recorded as X/X, wherein X is the size of the allelic variation of the locus;

allelic variation data for a heterozygous locus is reported as X/Y, where X, Y is the two different allelic variations at that locus;

the size of the null allelic variation was recorded as 0/0;

the front of the above "/" is a small segment, and the back of the "/" is a large segment;

and integrating data of different sites to form SSR fingerprints of different pea materials.

The PCR amplification method comprises the following steps:

the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 35s, annealing at 54 ℃ for 40s, and extension at 72 ℃ for 45s for 35 cycles; extending for 5min at 72 ℃, and keeping the temperature for 5min at 10 ℃;

10 μ L of the amplification system was: 2 XTaq PCR Master Mix 5. mu.L, 2. mu.M/. mu.L upstream and downstream primers each 1. mu.L, DNA template 1.5. mu.L, ddH ₂O 2.5μL。

When the non-denatured polyacrylamide gel is used for electrophoresis detection, 8% of the non-denatured polyacrylamide gel is used for electrophoresis, the voltage is 300V, the current is 280mA, and the power is 260W.

When the fluorescence capillary electrophoresis detection is adopted, a fluorescence label is added to the primer, and the details are shown in Table 2.

The invention provides a pea fingerprint spectrum constructed by the construction method.

The pea fingerprints constructed by the invention are shown in tables 5-20.

The invention further provides a method for identifying pea strain or wild kindred species thereof, which comprises the following steps:

(1) extracting DNA of peas to be detected, and performing PCR amplification on the DNA of the peas to be detected by using 31 pairs of primers in the primer combination;

(2) detecting the amplification product by non-denaturing polyacrylamide gel electrophoresis, and carrying out silver staining and color development according to the relative position of the amplification product on the electrophoresis gel; or

And (3) carrying out fluorescence capillary electrophoresis detection on the amplification products, wherein according to the relative positions of the amplification products:

the size of the null allelic variation was recorded as 0/0; the front of the above "/" is a small segment, and the back of the "/" is a large segment;

(3) comparing the results with the pea strains or the wild kindred species fingerprint spectrums in the tables 5-20, if the number of the difference sites between the materials is more than or equal to 3, the pea strains or the wild kindred species are different pea strains or different wild kindred species; if the number of the different sites between the materials is less than 3, the pea line or the wild relative species is similar.

The invention provides application of the method in pea germplasm resource improvement.

The method for constructing the pea DNA fingerprint spectrum utilizes SSR primer combination to amplify DNA of a certain pea strain or wild kindred species thereof to obtain a group of specific DNA fingerprints, so that the method adopts a primer combination identification method to analyze and screen the pea strain or the wild kindred species thereof, and each pea strain or the wild kindred species thereof finds the specific DNA fingerprint thereof. The amplified products of the genome SSR and EST-SSR primers have obvious size difference between band types, and different pea varieties or pea materials can be well distinguished. The banding patterns of the amplification products among individuals in the same variety are consistent. The detection method can finish the identification and genetic diversity evaluation work of the pea strain or the wild kindred species thereof in a short time, and has the advantages of high efficiency, accuracy, low cost, simple and convenient operation and the like. Meanwhile, the detection method can effectively monitor the authenticity of the pea seeds, reveal the genetic variation and genetic relationship of the variety from the DNA level, protect the crop variety, prevent counterfeit and shoddy varieties from entering the market, and provide technical reference for reasonable utilization of excellent germplasm in the process of breeding the pea variety.

Drawings

FIG. 1 is a graph showing the results of cluster analysis of 81 pea lines or their wild kindred species.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all reagents used in the examples are commercially available unless otherwise specified.

The principle of variety judgment of the invention is carried out according to the following national standard, the general rule of DNA fingerprint method for identifying plant varieties NY/T2594-; NY/T2436-2013 "pea for testing specificity, consistency and stability of new plant variety"; the general guidelines for the specificity, consistency and stability of new varieties of GB/T19557.1 plants.

Example 1 screening of genomic SSR and EST-EST primers for the identification of pea lines or wild relatives thereof

In the embodiment, 49 pea varieties which are typically popularized in the main pea producing area of China and 32 pea germplasm resources (shown in table 1) genome DNA are used as templates, and core genome SSR and EST-SSR primers are synthesized by analyzing pea genome sequences and EST sequences. Through screening, the molecular marker combination consisting of 19 pairs of genome SSR primers and 12 pairs of EST-SSR primers can completely identify the difference between 49 parts of pea varieties and 32 parts of pea germplasm resources.

And finally, obtaining 19 pairs of genome SSR molecular markers and 12 pairs of EST-SSR molecular markers by screening.

TABLE 231 pairs of SSR core primers

The primers in Table 2 above correspond to SEQ ID NO.1-62 in the sequence Listing, respectively.

Example 2 construction of characteristic fingerprint of pea and establishment of identification method of pea strain or wild kindred species thereof

1. Pea Total DNA extraction

Extracting DNA of a sample to be tested by adopting an improved CTAB method, taking about 2g of fresh tender leaves, grinding the fresh tender leaves into powder in liquid nitrogen, putting the powder into a 2.0ml centrifuge tube, putting the centrifuge tube into a-80 ℃ refrigerator for storage for standby use, adding β -mercaptoethanol into 2 xCTAB extract preheated to 65 ℃ according to the proportion accounting for 1% of the volume of the solution, fully and uniformly mixing, taking out the ground leaf tissues, adding 800 mu L of preheated CTAB extract into each sample, whirling for 1-2min, carrying out water bath at 65 ℃ for 1h (turning slightly up and down every 15min, fully and uniformly mixing), adding 800 mu L of chloroform/isoamyl alcohol (24: 1) solution into the centrifuge tube after heating in the water bath, mixing for 15-20min, then centrifuging for 15min at 10000rpm, sucking about 600 mu L of supernatant to a new centrifuge tube (the amount can be properly reduced, but contact protein is cut), then adding 95% of ethanol, shaking uniformly, placing the centrifuge tube at-20 ℃ for 2h or 4 ℃ after 2min, fully precipitating at 12000rpm, washing the supernatant for standby use, washing the supernatant for later, adding 2O% of pure DNA, carrying out centrifugation at room temperature, and carrying out air drying to obtain a 500 mu L of pure DNA, and carrying out detection by using an equal volume of 10 mu L of normal volume of 10 mu L of pure ethanol, and carefully carrying out centrifugation, and carrying out.

2. 49 pea varieties and 32 pea germplasm resource genome DNAs (deoxyribonucleic acid) described in example 1 are subjected to PCR amplification by using the primers for amplifying 31 molecular markers obtained by screening in example 1 according to 81 parts of pea reference materials described in example 1, wherein the reaction procedures are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 35s, annealing at 54 ℃ for 40s, and extension at 72 ℃ for 45s for 35 cycles; 7Extending for 5min at 2 ℃, and keeping the temperature for 5min at 10 ℃; 10 μ L of the amplification system was: 2 XTaq PCR Master Mix 5. mu.L, 2. mu.M/. mu.L upstream and downstream primers each 1. mu.L, DNA template 1.5. mu.L, ddH ₂O 2.5μL。

3. Performing gel electrophoresis and silver staining coloration on PCR amplification products: and detecting the amplification product by 8% non-denaturing polyacrylamide gel electrophoresis with an electrophoresis buffer solution of 0.5 xTBE and 200V stable pressure electrophoresis for 2-2.5h, and ending electrophoresis when the sample adding buffer solution is moved to the bottom of the gel. Silver staining for color development, and photographic recording.

(1) Cleaning the glass plate, fully cleaning the flat glass plate and the concave glass plate by using clean water and a detergent, washing by using distilled water, and placing on a glass plate frame for airing; in order to prevent bubbles from being generated during glue pouring, the glass plate is scrubbed by alcohol before glue pouring, and is dried for standby; the assembled glass plates are horizontally aligned and fixed by a clamp.

(2) Pouring glue, adding TEMED 45 μ L and 10% ammonium persulfate 450 μ L TEMED (note: the usage amount of TEMED and ammonium persulfate should be adjusted according to the ambient temperature) into 45ml 8% PAGE glue, mixing quickly and pouring glue. When the interlayer of the glass plate is filled with the glue solution, a comb is lightly inserted into the upper part of the glass plate to polymerize the glue solution for about 25min (note that the specific polymerization time is correspondingly adjusted according to different environmental temperatures). During the glue filling process, the generation of air bubbles should be prevented.

(3) PCR amplification product pretreatment 2.5. mu.L of 6 × Loading buffer was added to 10. mu.L of PCR amplification product, and mixed for 30 s.

(4) And a pipette for electrophoresis is used for blowing and sucking the sample adding groove to remove impurities and bubbles. And (3) dropping 1.5 mu L of product into each sample adding hole for detection, and carrying out electrophoresis for 1-1.5 hours (voltage 300V, current 280mA and power 260W), wherein the electrophoresis time can be properly adjusted according to the size of the target fragment and the actual power. After the upper indicator tape (xylene green) reached the bottom of the gel plate and electrophoresis was complete, the polyacrylamide gel was carefully peeled off and allowed to stain.

(5) Silver staining and rinsing: the polyacrylamide gel was rinsed rapidly 1 time with distilled water for no more than 10 seconds. Silver staining: the gel was stained with the following silver stain formulation and then stained for 10min with parallel shaking.

TABLE 3

Cleaning: after dyeing for 10min, the dyeing solution is poured into a waste liquid barrel, and the rinsing is continued for 2 times with distilled water, wherein each time does not exceed 15 s.

Color development: adding proper developing solution into a plastic box according to the following ratio of developing solution, slightly mixing uniformly, and placing in a parallel oscillator to enable the gel and the developing solution to fully act until the strips are clear.

TABLE 4

Washing: pouring off the developing solution and washing with distilled water for 2-3 times, each time not more than 15 s.

And (3) storage: after cleaning, the gel was laid flat on a glass plate, sealed with a preservative film, the strip results were read and statistically recorded, and then photographed for storage.

And (4) recording the result: the data of the size of the allelic variation of the homozygous locus is recorded as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for heterozygous loci were recorded as X/Y, where X, Y is two different allelic variations at the locus (small before and large after); the size of the null allelic variation was recorded as 0/0. The constructed fingerprint of 81 parts of pea material is shown in table 5-table 20.

TABLE 5

TABLE 6

SSR24112

B83

P291

SSR24036

SSR28304

SSR22754

SSR25799

SSR21405

W1

195/199

162/162

220/220

206/220

133/133

173/173

104/104

130/144

W2

195/195

158/162

220/220

194/194

127/127

161/161

102/102

130/157

W3

187/187

159/162

210/219

212/212

125/134

173/173

102/102

130/130

W4

199/199

160/160

201/201

192/192

125/125

173/173

102/102

144/144

W5

205/205

158/158

219/219

194/194

127/127

173/173

102/102

143/143

W6

223/225

161/161

219/219

218/218

128/128

173/173

102/102

157/157

W7

195/195

163/163

201/201

206/206

127/127

161/173

144/144

130/130

W8

195/195

163/163

219/219

206/206

127/127

170/170

134/134

130/130

W9

189/189

156/156

208/208

192/192

134/134

173/173

108/108

159/159

W10

199/199

164/164

219/219

218/218

133/133

173/173

104/104

144/144

W11

187/187

142/142

208/208

192/192

134/134

158/174

102/108

134/159

W12

199/199

156/156

201/201

194/194

133/133

158/158

100/102

159/159

W13

188/188

142/142

219/219

194/194

133/133

170/170

108/108

134/134

W14

189/225

161/161

219/219

193/193

128/128

161/161

151/151

159/159

W15

195/195

155/155

219/219

194/194

134/134

161/161

102/102

158/158

W16

195/195

161/161

219/219

194/194

127/127

158/158

134/134

130/130

W17

195/195

155/161

219/219

194/194

127/127

158/158

134/134

144/160

W18

199/199

161/161

207/207

194/194

127/127

161/173

134/151

144/144

W19

186/186

161/161

219/219

194/194

127/127

161/161

132/132

144/144

W20

195/195

142/142

208/219

193/193

127/127

161/161

151/151

144/144

TABLE 7

TABLE 8

EST643

P344

EST876

P14

SSR25965

SSR27844

SSR21491

W1

139/142

138/145

154/177

181/187

159/159

113/113

158/158

W2

157/157

145/145

154/189

187/190

167/167

114/114

172/172

W3

144/144

145/157

154/164

187/190

159/163

113/113

172/172

W4

142/142

152/157

154/177

187/190

165/165

112/112

175/175

W5

141/141

157/157

154/176

190/190

142/142

115/115

175/175

W6

139/139

145/145

154/188

190/190

147/147

112/112

172/172

W7

142/142

157/157

154/163

187/197

146/146

112/112

172/172

W8

157/157

158/158

154/163

187/196

146/146

112/112

178/178

W9

141/141

138/145

154/154

187/190

159/161

113/113

169/169

W10

139/139

157/157

154/176

181/181

147/147

112/112

172/172

W11

141/141

138/145

168/190

184/187

155/161

115/115

175/175

W12

139/139

144/151

154/191

187/197

165/165

113/113

169/169

W13

141/141

144/144

154/168

187/190

155/155

114/114

178/178

W14

157/157

138/138

154/191

197/197

146/146

112/112

178/178

W15

144/144

138/138

154/191

187/196

161/161

113/113

169/169

W16

157/157

139/139

154/163

187/197

146/146

113/113

175/175

W17

157/157

138/138

154/154

187/187

155/155

113/113

172/172

W18

142/142

138/145

154/176

190/190

146/159

113/113

169/169

W19

157/157

154/176

196/196

146/146

113/113

169/169

W20

157/157

138/145

154/176

187/196

146/146

113/113

172/172

TABLE 9

Watch 10

SSR24112

B83

P291

SSR24036

SSR28304

SSR22754

SSR25799

SSR21405

W21

195/195

159/159

201/201

194/194

133/133

158/158

134/134

126/130

W22

195/195

142/142

219/219

194/194

127/127

161/161

151/151

144/144

W23

195/195

142/142

220/220

194/194

127/127

173/173

151/151

144/144

W24

220/220

161/161

201/201

202/202

133/133

183/183

144/144

130/130

W25

227/227

161/161

201/201

197/197

127/127

173/173

149/149

144/144

W26

199/199

159/161

219/219

194/194

128/128

158/158

134/134

144/144

W27

189/189

161/161

219/219

194/194

128/128

161/161

151/151

144/144

W28

199/199

159/161

219/219

192/206

127/134

173/183

102/151

144/144

W29

199/199

158/158

219/219

220/220

133/133

161/161

102/149

130/143

W30

195/195

161/161

219/219

194/194

128/128

161/161

151/151

130/130

W31

225/225

161/161

201/201

198/220

133/133

173/173

144/144

130/144

W32

225/225

161/161

201/201

220/220

133/133

173/173

144/144

130/130

W33

195/195

161/161

219/219

194/194

128/128

173/173

151/151

144/144

W34

195/195

162/162

220/220

194/194

128/128

173/173

151/151

144/144

W35

195/195

161/161

219/219

193/193

128/128

161/161

151/151

130/130

W36

220/220

163/163

201/201

220/220

127/127

173/173

144/144

157/157

W37

214/214

161/161

201/201

202/202

133/133

173/173

102/102

156/156

W38

201/201

161/161

219/219

202/202

128/128

177/177

102/102

130/130

W39

201/201

161/161

219/219

193/193

127/127

158/158

151/151

130/130

W40

220/220

161/161

201/201

194/194

133/133

173/173

102/102

129/129

TABLE 11

TABLE 12

EST643

P344

EST876

P14

SSR25965

SSR27844

SSR21491

W21

157/157

145/145

154/164

187/190

153/153

113/113

175/175

W22

157/157

138/138

154/177

188/197

147/147

113/113

172/175

W23

157/157

138/138

154/177

187/197

147/147

113/113

175/175

W24

142/142

157/157

154/164

187/190

159/159

115/115

172/172

W25

139/139

158/158

154/177

187/190

167/167

112/112

172/172

W26

157/157

145/145

154/176

187/190

147/147

113/113

175/175

W27

157/157

138/138

154/176

187/190

146/146

112/112

172/172

W28

142/157

145/145

154/176

187/197

165/165

112/112

175/175

W29

142/142

145/157

154/176

187/190

159/169

112/112

169/175

W30

157/157

138/138

154/163

181/187

146/146

112/112

178/178

W31

138/138

157/157

154/163

187/190

167/167

115/115

172/172

W32

139/139

158/158

154/163

190/190

146/146

115/115

172/172

W33

157/157

154/176

187/196

147/147

112/112

178/178

W34

157/157

138/138

154/177

187/197

147/147

112/112

178/178

W35

157/157

138/138

154/163

181/187

146/146

112/112

178/178

W36

139/139

145/145

154/189

190/190

167/167

115/115

172/172

W37

142/145

157/157

154/189

187/190

159/159

112/112

158/158

W38

132/132

145/145

154/163

187/190

146/146

112/112

178/178

W39

157/157

138/138

154/163

181/187

146/146

113/113

172/172

W40

157/157

154/163

187/190

165/165

112/112

172/172

Watch 13

TABLE 14

SSR24112

B83

P291

SSR24036

SSR28304

SSR22754

SSR25799

SSR21405

W41

220/220

161/161

201/201

202/202

133/133

173/173

102/102

143/143

W42

224/224

159/159

219/219

194/194

128/128

161/161

134/134

143/143

W43

227/227

159/159

201/201

204/204

127/127

161/161

151/151

144/144

W44

227/227

159/159

201/201

204/204

127/127

161/161

151/151

144/144

W45

225/225

156/156

201/201

218/218

134/134

183/183

102/102

159/159

W46

199/199

162/162

208/208

194/194

127/127

158/158

134/134

144/161

W47

199/199

162/162

208/208

194/194

127/127

158/158

134/134

161/161

W48

195/195

160/160

219/219

197/197

128/128

173/173

151/151

144/144

W49

225/225

159/159

201/201

194/204

127/127

161/161

151/151

144/144

W50

195/195

161/161

219/219

194/194

133/133

161/161

102/102

144/144

W51

186/186

161/161

219/219

193/193

127/127

161/161

134/134

144/144

W52

199/201

157/161

207/219

218/218

127/127

161/161

102/102

129/145

W53

199/199

157/159

210/210

197/201

133/133

161/161

102/102

130/143

W54

195/195

163/163

219/219

193/193

127/127

173/173

151/151

130/130

W55

188/199

161/163

201/219

192/220

127/133

158/182

102/102

130/130

W56

186/186

161/161

219/219

194/194

127/127

161/161

102/134

130/144

W57

199/199

142/155

201/219

194/194

134/134

170/170

102/108

130/159

W58

197/197

159/159

202/202

194/194

125/125

170/170

103/103

144/144

W59

199/199

156/156

219/219

194/194

134/134

158/170

102/108

144/159

W60

199/199

155/155

201/208

194/194

127/134

173/173

102/102

130/159

Watch 15

TABLE 16

EST643

P344

EST876

P14

SSR25965

SSR27844

SSR21491

W41

153/153

157/157

154/176

187/190

146/146

112/112

172/172

W42

157/157

138/138

154/176

187/196

146/146

113/113

172/172

W43

157/157

138/138

154/176

187/196

147/147

115/115

160/160

W44

157/157

138/138

154/176

196/196

146/146

115/115

160/160

W45

139/139

138/138

154/190

181/187

147/147

115/115

178/178

W46

157/157

138/138

154/154

187/191

147/147

113/113

175/175

W47

157/157

138/138

154/193

187/190

146/146

113/113

175/175

W48

157/157

139/139

154/177

187/197

146/146

113/113

178/178

W49

157/157

145/145

154/177

187/190

146/146

115/115

172/172

W50

157/157

154/176

187/197

167/167

113/113

172/172

W51

157/157

154/176

187/196

146/146

113/113

172/172

W52

144/144

138/138

154/154

181/187

161/161

113/113

172/172

W53

138/138

145/157

154/163

187/190

149/149

112/112

172/172

W54

157/157

154/163

181/187

146/146

112/112

169/169

W55

138/138

138/157

154/163

187/196

161/167

113/115

160/172

W56

157/157

154/176

196/196

146/146

113/113

169/169

W57

141/141

145/145

154/163

190/190

155/155

113/114

178/178

W58

142/142

139/145

154/177

182/184

166/166

107/107

178/178

W59

141/145

138/144

154/154

187/197

161/161

113/113

172/180

W60

142/144

138/145

154/154

187/190

161/161

113/115

172/172

TABLE 17

Watch 18

SSR24112

B83

P291

SSR24036

SSR28304

SSR22754

SSR25799

SSR21405

W61

188/188

157/157

201/201

192/192

134/134

158/158

108/108

134/134

W62

186/186

161/161

219/219

194/194

127/127

173/173

102/102

130/144

W63

189/199

155/155

207/219

193/193

134/134

170/170

102/108

134/134

W64

198/198

158/158

219/219

191/191

134/134

158/158

102/102

143/143

W65

191/191

155/155

201/201

191/191

134/134

158/161

102/102

158/158

W66

184/184

142/142

201/201

192/192

127/127

158/158

108/108

134/134

W67

189/189

142/159

201/219

192/193

128/134

158/176

108/108

130/144

W68

195/195

159/159

219/219

197/197

128/128

173/173

155/155

130/130

W69

195/195

155/155

207/219

192/192

134/134

170/170

102/108

143/143

W70

195/195

156/156

201/201

194/194

134/134

171/171

102/102

144/144

W71

191/191

167/167

208/208

192/192

134/134

158/158

108/108

134/134

W72

195/199

164/179

220/220

206/218

134/134

170/170

105/105

130/130

W73

195/195

159/159

220/220

194/194

125/125

177/177

108/108

144/144

W74

225/225

159/159

220/220

194/194

128/128

161/161

149/149

130/130

W75

201/201

140/140

207/207

210/210

127/127

173/173

104/104

156/156

W76

186/186

161/161

219/219

194/194

125/125

173/173

103/103

144/144

W77

232/232

155/155

204/204

192/192

133/133

158/158

102/102

144/144

W78

188/188

159/159

201/201

212/212

134/134

173/173

102/102

144/144

W79

199/199

161/161

213/213

198/198

125/125

173/173

104/104

144/144

W80

187/187

160/160

201/201

206/206

133/133

158/158

103/110

144/144

W81

199/199

147/147

208/208

187/187

127/127

167/167

108/108

134/134

Watch 19

EST599

SSR24882

SSR24731

EST619

EST671

SSR25888

SSR22052

SSR25334

W61

167/167

152/152

212/212

112/115

131/148

163/163

138/138

141/141

W62

158/170

150/150

193/193

123/127

130/147

163/163

141/141

137/137

W63

146/146

152/171

187/187

115/124

130/143

166/166

141/141

137/137

W64

170/170

171/171

202/202

123/123

143/147

162/162

141/141

136/136

W65

146/146

152/152

193/193

124/124

130/130

166/166

137/137

W66

146/146

152/152

214/214

124/124

129/147

178/178

137/137

W67

146/146

152/152

193/218

115/124

139/146

163/166

137/137

135/137

W68

148/148

152/156

198/198

126/126

129/129

173/173

137/137

136/136

W69

167/175

150/150

187/187

126/126

143/147

166/166

137/141

136/136

W70

169/169

152/152

208/208

115/115

131/148

166/166

138/138

136/136

W71

165/165

168/168

193/193

124/124

143/148

166/166

138/138

128/128

W72

159/159

152/152

193/198

115/115

131/148

166/166

144/144

136/136

W73

175/175

150/150

202/202

126/126

143/148

166/166

144/144

139/139

W74

173/173

150/150

200/202

126/126

141/141

166/166

137/137

136/136

W75

124/124

150/150

190/190

115/115

143/143

166/166

144/144

127/127

W76

146/146

154/154

192/192

115/115

131/147

166/166

144/144

136/136

W77

124/124

152/152

193/193

115/115

130/147

176/176

144/144

130/130

W78

156/156

151/151

193/193

124/124

130/147

163/163

139/139

128/128

W79

156/156

150/150

193/193

126/126

130/147

165/165

144/144

137/137

W80

158/158

154/154

200/200

126/126

140/147

177/177

139/139

132/132

W81

135/135

152/152

190/190

132/132

145/145

168/168

128/128

165/165

Watch 20

4. The detection method parallel to the step 3 can also adopt the fluorescence capillary electrophoresis detection of the PCR amplification product, and comprises the following specific steps:

PCR product sample preparation and electrophoretic analysis results

Diluting FAM (blue) and HEX (green) fluorescence labeled PCR products by 25 times with ultrapure water, respectively taking 2 diluted solutions with equal volumes, mixing to form a mixed solution, and respectively adding 1 mu L of the mixed solution, 0.1 mu L of LIZ500 molecular weight internal standard and 8.9 mu L of deionized formamide into a deep-well plate hole special for a DNA analyzer; then, denaturing the mixture for 5min at 95 ℃ on a PCR instrument, taking out the mixture, immediately placing the mixture on crushed ice, and cooling the mixture for about 15 min; the samples were centrifuged for 10 seconds instantaneously and subjected to capillary electrophoresis detection, image analysis and data acquisition using GENEMAPPER. Taking DNA MarkerI as a DNA molecular weight standard, and recording the size data of the allelic variation of the homozygous locus as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for a heterozygous locus is reported as X/Y, where X, Y is the two different allelic variations at that locus; the size of the null allelic variation was recorded as 0/0 (note: small fragment before and large fragment after). By integrating the data of multiple loci, SSR fingerprints of different pea lines or wild kindred species thereof are finally formed (tables 5-20). Besides the sample to be detected, each SSR locus also comprises amplification products of 3-5 reference materials.

The optimal dilution of the different fluorescently labeled amplification products is best determined by pre-testing.

And (3) judging a detection result: if the number of different sites between the materials is more than or equal to 3, the materials are different pea lines or different wild related species of peas; if the number of the different sites between the materials is less than 3, the pea line or the wild relative species is similar. Comparing the fingerprint data of 81 parts of pea materials, finding that the number of the difference sites between any two materials is more than 3, and showing that the 31 pairs of core primers can effectively identify the pea strains or the wild kindred species thereof; the Power Marker and MEGA software are used for clustering, the genetic relationship between the reference materials is analyzed, and 81 parts of pea materials can be distinguished when the genetic similarity coefficient is about 0.075 from the figure (figure 1).

Example 3 application of the method of the invention for identifying pea lines or their wild kindreds

Extracting leaf DNA of 6 pea samples with known variety names, and respectively carrying out PCR amplification on the DNA of the pea variety to be detected by 31 pairs of primers in the molecular marker combination obtained by screening in the embodiment 1 of the invention; referring to the PCR reaction system, the PCR reaction program, the polyacrylamide gel electrophoresis and the variation site recording method (method 1) in example 2 of the present invention, the fluorescence capillary electrophoresis method and the variation site recording method (method 2) are adopted in the simultaneous repetition experiment, and the obtained fingerprint code is compared with the characteristic fingerprint spectrum (table 5-table 20) of the pea strain or the wild kindred species thereof obtained in the present invention, so as to verify the accuracy of the identification method established in example 2 of the present invention.

TABLE 21

SSR4825

SSR21399

SSR23759

P292

SSR4696

P282

SSR28967

EST617

F1

191/191

141/141

156/156

210/210

212/212

184/184

172/172

125/125

F2

225/225

141/141

156/156

225/225

195/195

184/184

173/173

125/125

F3

205/205

158/158

163/163

225/225

216/216

201/203

172/172

134/134

F4

195/205

141/141

156/156

200/200

216/216

184/184

182/182

128/128

F5

195/224

154/154

163/163

216/216

195/195

186/186

172/172

127/127

F6

191/195

154/154

163/163

216/216

212/212

186/186

172/172

128/128

TABLE 22

SSR24112

B83

P291

SSR24036

SSR28304

SSR22754

SSR25799

SSR21405

F1

189/189

156/156

208/208

192/192

134/134

173/173

108/108

159/159

F2

195/195

155/155

219/219

194/194

134/134

161/161

102/102

158/158

F3

189/189

161/161

219/219

194/194

128/128

161/161

151/151

144/144

F4

225/225

161/161

201/201

220/220

133/133

173/173

144/144

130/130

F5

227/227

159/159

201/201

204/204

127/127

161/161

151/151

144/144

F6

225/225

159/159

201/201

194/204

127/127

161/161

151/151

144/144

TABLE 23

EST599

SSR24882

SSR24731

EST619

EST671

SSR25888

SSR22052

SSR25334

F1

146/146

152/152

187/187

115/115

131/147

163/166

137/137

F2

146/177

150/150

202/202

124/124

141/141

166/166

137/137

F3

158/158

153/153

187/187

126/126

140/187

163/163

141/141

137/137

F4

154/154

150/150

216/216

123/123

140/140

177/177

141/141

136/136

F5

158/158

150/150

202/202

126/126

141/141

177/177

144/144

152/152

F6

158/158

150/150

202/202

126/126

139/139

177/177

144/144

150/150

Watch 24

EST643

P344

EST876

P14

SSR25965

SSR27844

SSR21491

F1

141/141

138/145

154/154

187/190

159/161

113/113

169/169

F2

144/144

138/138

154/191

187/196

161/161

113/113

169/169

F3

157/157

138/138

154/176

187/190

146/146

112/112

172/172

F4

139/139

158/158

154/163

190/190

146/146

115/115

172/172

F5

157/157

138/138

154/176

196/196

146/146

115/115

160/160

F6

157/157

145/145

154/177

187/190

146/146

115/115

172/172

As a result, it was found that: the 31 pairs of primer combinations in the embodiment 1 of the invention are used for carrying out PCR amplification on 6 pea varieties, the variation sites of the 6 pea samples measured by the two methods are consistent, the results are shown in tables 21-24, the number of the variation sites of the target variety of the pea fingerprint spectrum established by the invention is less than 3, F1-F6 is the 6 pea varieties to be measured, F1-F6 are obtained by comparison and identification and respectively correspond to Haimaichua pea (W9), Anhui bean tip No.1 (W15), grassland 276(W27), Kewang bowl No.2 (W32), Chengguan No. 8 (W44) and Kekuai vegetable bowl No.6 (W49), the accuracy is 100 percent, and the known variety names are consistent. The 31 pairs of primer combinations obtained by screening in the embodiment 1 of the invention can be used for better distinguishing different pea varieties. The method for identifying the pea variety provided by the embodiment 2 of the invention has higher accuracy and is suitable for popularization and application.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> institute of crop science of Chinese academy of agricultural sciences

<120> construction method of pea SSR fingerprint

<130>KHP161119131.4

<160>62

<170>PatentIn version 3.5

<210>1

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gcatgtgcat ttaatcgtgc 20

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tgcacacgta cacacaaatc a 21

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<211>20

<212>DNA

<213> Artificial sequence

<400>3

tgcatgtgca tgtaatcgtg 20

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<400>4

tcacacgcac gtacaaatca 20

<210>5

<211>24

<212>DNA

<213> Artificial sequence

<400>5

ttttcagctg atcggatatc taca 24

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

ggcagcatct tgaaaatcgt 20

<210>7

<211>24

<212>DNA

<213> Artificial sequence

<400>7

gaaggaccaa atcaattctc taaa 24

<210>8

<211>20

<212>DNA

<213> Artificial sequence

<400>8

accgacgtca acgactgata 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<400>9

ccctctccca tctcatctca 20

<210>10

<211>25

<212>DNA

<213> Artificial sequence

<400>10

aaagaaagta gagatccagc actga 25

<210>11

<211>25

<212>DNA

<213> Artificial sequence

<400>11

ttgcctcatt tatcattctc ttatg 25

<210>12

<211>26

<212>DNA

<213> Artificial sequence

<400>12

caaaaggtta tctagctacg acttga 26

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<400>13

gcttcaagct accaagtgga 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence

<400>14

cctcacgggc tctaccatac 20

<210>15

<211>20

<212>DNA

<213> Artificial sequence

<400>15

aagggggaga gaggtggtta 20

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<400>16

tcgccttttc tttcttcttc a 21

<210>17

<211>20

<212>DNA

<213> Artificial sequence

<400>17

tttctggaac ctcgcaaaac 20

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<400>18

ttgcctcaat ttggagacct 20

<210>19

<211>21

<212>DNA

<213> Artificial sequence

<400>19

ggggtgacag tgtagggttt t 21

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<400>20

taggcacacg ctttcatgtt 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<400>21

acacgggatc gagctttaga 20

<210>22

<211>25

<212>DNA

<213> Artificial sequence

<400>22

tcctttcctc taacttcttc cttct 25

<210>23

<211>25

<212>DNA

<213> Artificial sequence

<400>23

aagtctctca tacctaacca accac 25

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<400>24

gcagccaaat ttgaggaaga 20

<210>25

<211>20

<212>DNA

<213> Artificial sequence

<400>25

ttgatcatcc tctcgctttt 20

<210>26

<211>22

<212>DNA

<213> Artificial sequence

<400>26

tgttgtcgtc atcaaaacac ag 22

<210>27

<211>22

<212>DNA

<213> Artificial sequence

<400>27

gcaattttct cactccatct cc 22

<210>28

<211>20

<212>DNA

<213> Artificial sequence

<400>28

aaaggaaagc aactcggtga 20

<210>29

<211>20

<212>DNA

<213> Artificial sequence

<400>29

ggaacgacaa cacgaacctc 20

<210>30

<211>20

<212>DNA

<213> Artificial sequence

<400>30

gacacgttat gcgcacactc 20

<210>31

<211>20

<212>DNA

<213> Artificial sequence

<400>31

tccgcaatgt tctctcgaat 20

<210>32

<211>20

<212>DNA

<213> Artificial sequence

<400>32

ggaggtctcc gcattatcaa 20

<210>33

<211>20

<212>DNA

<213> Artificial sequence

<400>33

agaaaatggc ccacgaatta 20

<210>34

<211>20

<212>DNA

<213> Artificial sequence

<400>34

tgcattgcat tgtgtttgtg 20

<210>35

<211>24

<212>DNA

<213> Artificial sequence

<400>35

tgaagatgtg acaaacaaca gaaa 24

<210>36

<211>20

<212>DNA

<213> Artificial sequence

<400>36

tccttctctg ttcccaccac 20

<210>37

<211>22

<212>DNA

<213> Artificial sequence

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gcagattgac tgctcatgat gt 22

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<211>20

<212>DNA

<213> Artificial sequence

<400>38

agctgattat tgggcacctg 20

<210>39

<211>21

<212>DNA

<213> Artificial sequence

<400>39

ctcgtcctca tcgaaaagct a 21

<210>40

<211>20

<212>DNA

<213> Artificial sequence

<400>40

gtgaacaacg caagggtttc 20

<210>41

<211>26

<212>DNA

<213> Artificial sequence

<400>41

tgattctagt tcatttcaca aacaca 26

<210>42

<211>20

<212>DNA

<213> Artificial sequence

<400>42

cgtcctgcac ctagcttctt 20

<210>43

<211>21

<212>DNA

<213> Artificial sequence

<400>43

tccaccatct aatcccctct t 21

<210>44

<211>20

<212>DNA

<213> Artificial sequence

<400>44

agctgattat tgggcacctg 20

<210>45

<211>20

<212>DNA

<213> Artificial sequence

<400>45

ggcaagcata aaagggacac 20

<210>46

<211>20

<212>DNA

<213> Artificial sequence

<400>46

ttcatccaag aaccctcgac 20

<210>47

<211>20

<212>DNA

<213> Artificial sequence

<400>47

caccaccact ctcacaccat 20

<210>48

<211>21

<212>DNA

<213> Artificial sequence

<400>48

tgcattgcga gagtaagaca a 21

<210>49

<211>20

<212>DNA

<213> Artificial sequence

<400>49

ctgcagaagg ccctgttcta 20

<210>50

<211>25

<212>DNA

<213> Artificial sequence

<400>50

gattcttcat tctcaacaca cattg 25

<210>51

<211>20

<212>DNA

<213> Artificial sequence

<400>51

tgattcgtag accccacaca 20

<210>52

<211>26

<212>DNA

<213> Artificial sequence

<400>52

aaggttaatg tcttcttttt gaagtt 26

<210>53

<211>20

<212>DNA

<213> Artificial sequence

<400>53

caagcgtcga agatgaacaa 20

<210>54

<211>20

<212>DNA

<213> Artificial sequence

<400>54

gctggctgca aaagtttacc 20

<210>55

<211>20

<212>DNA

<213> Artificial sequence

<400>55

gcacatgaaa aatgccaaag 20

<210>56

<211>20

<212>DNA

<213> Artificial sequence

<400>56

ctgttgctgt tggtggtgag 20

<210>57

<211>20

<212>DNA

<213> Artificial sequence

<400>57

gttaccgatg gccatgaatc 20

<210>58

<211>20

<212>DNA

<213> Artificial sequence

<400>58

agcgagtgaa gagggaagtg 20

<210>59

<211>18

<212>DNA

<213> Artificial sequence

<400>59

atgcaaccgg cgcagtat 18

<210>60

<211>20

<212>DNA

<213> Artificial sequence

<400>60

ccaccttttc ctcgcttttt 20

<210>61

<211>21

<212>DNA

<213> Artificial sequence

<400>61

gccaaacggc tttaaaactt c 21

<210>62

<211>21

<212>DNA

<213> Artificial sequence

<400>62

tcgctgttgg aaagagaaga a 21

Claims

1. A molecular marker combination for identifying pea strains or wild related species thereof is characterized by comprising 19 genome SSR molecular markers and 12 EST-SSR molecular markers, namely SSR4825, SSR4696, SSR28304, SSR24036, B83, EST671, SSR27844, SSR25888, SSR24882, SSR23759, SSR21491, EST619, SSR24112, EST599, SSR22754, P14, SSR24731, SSR25334, EST876, SSR28967, SSR21399, SSR21405, P282, EST, SSR25799, SSR25965, P291, P292, SSR22052, P344 and EST 617;

2. A primer combination is characterized by comprising the following 31 pairs of specific primer pairs, and the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO. 1-62.

3. A kit comprising the primer combination of claim 2.

4. Use of the primer combination according to claim 2 or the kit according to claim 3 for the identification of pea varieties.

5. Use of the primer combination of claim 2 or the kit of claim 3 for pea-assisted breeding.

6. A pea SSR fingerprint construction method is characterized in that the primer combination of claim 2 is used for carrying out PCR amplification on DNA of different pea strains or wild kindred species thereof, and the PCR product is subjected to non-denaturing polyacrylamide gel electrophoresis for detection or fluorescent capillary electrophoresis for detection:

the size of the null allelic variation was recorded as 0/0;

and integrating data of different sites to form SSR fingerprints of different peas.

7. The method of claim 6, wherein the PCR amplification method comprises:

8. The application of the pea fingerprints obtained by the construction method of claim 6 or 7 in identifying pea products or assisting pea breeding, wherein the pea fingerprints are fingerprints of 81 pea lines or wild allied species thereof, which are respectively as follows:

and

and

and

and

and

and

and

and

and

and

and

and

and

the above W1-W81 refer to 81 pea lines or wild kindred species thereof, respectively: garfield, DDR-14, bulgarian pea, dunne pea, MG101831, russian pea, qin chong 1, liao chong 1, haimen white flower pea, small white flower pea, numb pea, cabbage pea, vegetable pea, podded big vegetable pea 1, alexandrium tamarindus No.1, fixed pea 2, fixed pea 3, fixed pea 4, fixed pea 5, fixed pea 6, fixed pea 7, fixed pea 8, grassland 23, grassland 24, grassland 224, grassland 276, qinghaochi No.1, sweet potato, arisk, pea 1, podocarpus No.2, podocarpus No.5, podocarpus No.6, podocarpus 7, podocarpus 8, podocarpus 1, podocarpus 3, yun 1, yunshi No.4, yun 8, podocarpus 10, podocarpus 7, podocarpus macropyrus 7, podocarpus gravy No.1, podocarpus gravy 1, podocarpus lucidus gravy No.11, podocarpus gravy No.1, podocarpus macropyrus 7, podocarpus macropyrus cucurbiturius 7, podocarpus macropyrus 1, podocarpus macropyrus 7, pod, pea 171, foal 39, grassland No. 22, grassland No.25, medium pea No.4, safflower pea, sweet broad pea, hairy pea, Yuanbaoshan pea, hemp pea, white pea, green skin pea, small green pea, liwei white pea, hemp pea, big white pea, black pea, Hami pea, vegetable pea, AGG1706, AGG116, AGG105, AGG2380, AGG4023, AGG533, AGG866, G5671 are not required.

9. A method of identifying a pea line or a wild kindred species thereof, comprising the steps of:

(1) extracting DNA of peas to be detected, and performing PCR amplification on the DNA of the peas to be detected by using 31 pairs of primer pairs in the primer combination according to claim 2;

(3) comparing the result with the pea fingerprint spectrum obtained by the construction method of claim 6 or 7 and obtained by the pea fingerprint spectrum obtained by the construction method of claim 8, wherein the number of the difference loci among materials is more than or equal to 3, and the difference loci are different pea strains or different wild related species of peas; if the number of the different sites between the materials is less than 3, the pea line or the wild relative species is similar.