Crops ›› 2025, Vol. 41 ›› Issue (6): 19-27.doi: 10.16035/j.issn.1001-7283.2025.06.003

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Screening and Fingerprint Construction of SSR Molecular Markers in Pea

Chen Haonan1(), Zhao Hongyan2,3, Zhang Wenjing2,3, Zhang Qi2,3, Du Jidao2,3, Wang Qirui2,3, Ren Ruoran2,3, Han Yiqiang1,3()   

  1. 1 College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163711, Heilongjiang, China
    2 College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163711, Heilongjiang, China
    3 National Coarse Cereals Engineering Technology Research Center, Daqing 163319, Heilongjiang, China
  • Received:2024-07-08 Revised:2024-10-12 Online:2025-12-15 Published:2025-12-12

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Abstract:

SSRMMD software was used to scan the pea v1a genome to obtain SSR loci and primers. The primer check (e-PCR) in TBtools was used to analyze the pea genomes of four varieties, including pea v1a, Zhongwan No.6, pea 07v1 and pea 87v1, by electronic PCR-SSR molecular marker polymorphism analysis. Conventional PCR and PAGE electrophoresis were used to verify and construct pea fingerprints. The results showed that a total of 311 601 SSR loci were scanned out of the whole pea genome, and 165 288 pairs of SSR markers were obtained, which were evenly distributed on seven chromosomes. Four pea reference genomes were analyzed by e-PCR by using 122 pairs of SSR markers. It was found that there were 46 pairs of differential markers with amplified fragments less than 300 bp, 29 pairs of co-dominant markers, and 17 pairs of dominant markers; there were 12 pairs of markers with no difference in amplified fragments, 11 pairs of markers without amplified fragments, and 53 pairs of primers with amplified fragments greater than 300 bp. The conventional PCR results showed that seven of the 20 pairs of different markers had polymorphism, and only two of the other 30 pairs had polymorphism. These nine pairs of SSR primers were used to successfully construct the SSR fingerprints of 32 pea varieties. This study shows that the application of whole genome scanning combined with e-PCR methods can quickly obtain SSR markers with high polymorphism and applicability for the marking and identification of pea germplasm resources.

Key words: Pea, SSR, Germplasm resource, Electronic PCR, Fingerprint

Table 1

Introduction sites of different varieties of peas"

编号Code 品种Variety 引种地点Introduction site
1 DWD05-01 甘肃
2 成豌11号 江苏
3 c13 重庆
4 方绿豌豆 山东
5 杂粮甜豌豆 江苏
6 zx-wd-0030 甘肃
7 定豌7号 甘肃
8 zx-wd-0007 甘肃
9 zx-wd-0013 甘肃
10 麻豌豆 山东
11 澳洲红玉豌豆 山东
12 S6071 甘肃
13 黑眼豌豆 山东
14 SW1102 甘肃
15 c01 重庆
16 1702 甘肃
17 彩豌豆 山东
18 S5008 甘肃
19 RC09 甘肃
20 食荚型甜豌豆 山东
21 鲜嫩二号豌豆 宁夏
22 美国甜脆豌豆 宁夏
23 豌豆 山东
24 德利荷兰豆 河北
25 zx-wd-0019 甘肃
26 优选法国008香豆 深圳
27 DWD05-03 甘肃
28 猪耳朵 山东
29 zx-wd-0009 甘肃
30 DWD05-10 甘肃
31 WD04-09 甘肃
32 黑豌豆 山东

Table 2

Agronomic traits of different varieties of pea"

编号
Code		 品种
Variety		 花期
Flowering
period		 花荚期
Flowering and
pod-setting stage		 成熟期
Maturity		 荚型
Pod
type		 粒色
Grain
color		 粒形
Grain
shape		 株高
Plant
height (cm)		 节数
Number of
sections		 荚长
Pod length
(cm)		 单荚粒数
Number of
grains per pod		 百粒重
100-grain
weight (g)
1 DWD05-01 06-24 07-08 08-27 硬 黄 皱 86 13 6.30 4.00 22.29
2 成豌11号 06-24 07-08 08-10 硬 黄 圆 50 14 6.60 5.67 22.03
3 c13 07-01 07-15 08-01 硬 黄 皱 95 18 6.57 6.00 21.01
4 方绿豌豆 06-17 07-01 08-01 硬 绿 皱 25 12 6.67 5.33 38.86
5 杂粮甜豌豆 06-10 06-24 08-01 硬 绿 皱 45 13 6.73 5.67 25.17
6 zx-wd-0030 07-01 07-15 08-15 硬 绿 皱 120 21 7.32 5.50 25.76
7 定豌7号 06-24 07-08 08-01 硬 黄 皱 130 16 6.53 6.33 25.31
8 zx-wd-0007 06-03 06-17 07-25 硬 绿 皱 120 14 6.54 4.50 24.70
9 zx-wd-0013 07-08 07-22 08-02 硬 黄 皱 110 13 5.50 4.33 21.52
10 麻豌豆 07-08 07-22 09-01 硬 麻 皱 50 18 5.87 4.33 19.92
11 澳洲红玉豌豆 06-24 07-08 08-05 硬 黄 皱 60 18 6.37 4.33 23.52
12 S6071 06-24 07-08 08-01 硬 黄 皱 55 13 7.23 5.00 27.38
13 黑眼豌豆 06-17 07-01 08-01 硬 黄 皱 185 27 5.60 4.50 17.71
14 SW1102 06-17 07-01 08-01 硬 黄 皱 50 16 5.60 4.00 27.40
15 c01 06-17 07-01 08-01 硬 黄 皱 51 13 6.60 6.00 18.84
16 1702 06-17 07-01 08-05 硬 黄 皱 51 15 5.87 4.00 24.15
17 彩豌豆 06-10 06-24 08-01 硬 黄 皱 60 18 5.27 4.67 21.69
18 S5008 06-24 07-08 08-05 硬 绿 皱 60 20 7.47 5.33 30.69
19 RC09 06-17 07-01 08-01 硬 麻 圆 85 16 6.24 5.62 26.07
20 食荚型甜豌豆 06-17 07-01 08-01 硬 绿 皱 55 14 6.58 4.78 18.71
21 鲜嫩二号豌豆 06-24 07-08 08-17 硬 黄 皱 80 17 7.27 7.33 22.14
22 美国甜脆豌豆 06-24 07-08 07-15 硬 绿 皱 135 28 6.97 6.33 17.86
23 豌豆 06-17 07-01 08-05 硬 绿 皱 110 32 6.57 3.00 21.05
24 德利荷兰豆 06-24 07-08 08-01 硬 绿 皱 55 16 8.07 8.00 22.03
25 zx-wd-0019 07-01 07-15 08-15 硬 黄 皱 61 12 5.90 4.00 24.27
26 优选法国008香豆 06-24 07-08 08-01 硬 绿 皱 70 11 6.67 4.67 26.97
27 DWD05-03 06-17 07-01 08-17 硬 绿 皱 170 34 5.67 3.00 18.31
28 猪耳朵 07-08 07-22 08-25 硬 黄 皱 125 18 5.23 3.00 29.18
29 zx-wd-0009 07-01 07-15 08-15 硬 黄 皱 60 18 5.63 3.67 23.07
30 DWD05-10 07-08 07-22 08-15 硬 绿 皱 95 17 7.97 4.33 29.01
31 WD04-09 06-17 07-01 08-10 硬 黄 皱 41 13 6.07 5.00 30.22
32 黑豌豆 07-01 07-15 08-10 硬 麻 皱 95 15 5.67 6.67 15.91

Fig.1

Distribution characteristics of SSR locus repeat types in whole pea genome (a) the proportion of different types of repeat units in SSR locus, (b) the number distribution of SSR locus for different A/T base combination types, (c) SSR sequence length distribution."

Table 3

Distribution of SSR markers in the pea genome “Pisum sativum v1a”"

染色体
Chromosome		 染色体大小
Chromosome size (Mb)		 标记数量
Number of markers		 标记密度（对/Mb）
Marker density (pair/Mb)		 相邻两个SSR位点的平均距离
Average distance between two adjacent SSR locus (kb)
Ps01 372.17 23 784 63.90 15.65
Ps02 427.60 22 370 52.32 19.11
Ps03 437.56 20 142 46.03 21.72
Ps04 446.35 11 618 26.02 38.43
Ps05 579.30 32 571 56.22 17.79
Ps06 480.42 27 080 56.36 17.74
Ps07 497.38 27 663 55.62 17.98
平均Average 462.97 23 604 50.92 21.20

Fig.2

Analysis of primers with amplification products in four pea genomes and amplification number of amplification products below 300 bp (a) The number of different amplified fragment sizes in four pea genomes, (b) Venn plots of amplification products below 300 bp in four pea genomes, ZW6: Zhongwan No.6."

Fig.3

e-PCR amplification of some SSR primers in four pea genomes"

Fig.4

PCR results of some SSR primers (a) PCR amplification products of some SSR primers, 1: DWD05-01, 2: Cheng Pea No.11, 3: Square green pea, 4: mixed grain sweet peas. (b) electrophoretic detection results of PsSSR-82 on 32 pea varieties."

Table 4

SSR polymorphism detection results"

编号Code 引物类型Primer type 引物总数Total number of primers 多态性Polymorphism 比例Proportion (%)
1 扩增片段均小于300 bp（有差异，共显性标记） 10 6 60
2 扩增片段均小于300 bp（有差异，显性标记） 10 1 10
3 扩增片段均小于300 bp（无差异） 10 2 20
4 扩增片段大于300 bp 10 0 0
5 无扩增 10 0 0

Table 5

Nine pairs of SSR marker names and information"

引物名称Primer name 上游引物Forward primer 下游引物Reverse primer 染色体Chromosome
PsSSR-6 ACACAAATTCCCCACTCCCC TCAACCGGTGGAAGAAACCC Ps01
PsSSR-27 ACTTGTAAGCAGCTGAGCGT GTAGCTGTGACTATCCCGGC Ps02
PsSSR-56 TCCGGGCGAGACTACTTGAT GTGGATTCCCATGGATCGCA Ps04
PsSSR-77 TCCAGGCTTTGCATGATGGT CCAACGACTCCAAGGCTTCA Ps05
PsSSR-80 ATTTTGGCGGGGTTTTGACG GTCCACCTCCGGGATCAAAG Ps05
PsSSR-81 CCCCAATCACTTTCTCGTGC GGAGCACACTCTCAAGACCC Ps05
PsSSR-82 TGAAAGACAGGGATGCGCTT AGGGCATGTACTTGCTTCGA Ps05
PsSSR-92 TGGGTTCGTTCCCCTAATGG CAGGTCAGGAGCAGAAGGTT Ps06
PsSSR-95 CTGGGTGTGGTTCTTCACGA TTGCTTACCTGCTTGCATGC Ps06

Table 6

Fingerprint codes of 32 pea materials SSR markers"

编号Code 品种Variety 指纹代码Fingerprint code 编号Code 品种Variety 指纹代码Fingerprint code
1 DWD05-01 223142132 17 彩豌豆 313243233
2 成豌11号 423141432 18 S5008 223241233
3 C13 323142332 19 RC09 222243233
4 方绿豌豆 323241132 20 食荚型甜豌豆 222243531
5 杂粮甜豌豆 302142532 21 鲜嫩二号豌豆 312343141
6 WD-0030 432233132 22 美国甜脆豌豆 121143341
7 定豌7号 432123441 23 豌豆 223344313
8 WD-0007 332213132 24 德利荷兰豆 221143443
9 WD-0013 122121112 25 WD-0019 121143343
10 麻豌豆 222223112 26 优选法国008香豆 322143344
11 澳洲红玉豌豆 421323431 27 DWD05-03 133243444
12 S6071 221322112 28 猪耳朵 222252444
13 黑眼豌豆 323321132 29 WD-0009 321253434
14 SW1102 223133122 30 DWD05-010 221252444
15 C01 323131233 31 WD04-09 121143443
16 1702 313133133 32 黑豌豆 121243544
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