Crops ›› 2021, Vol. 37 ›› Issue (1): 38-46.doi: 10.16035/j.issn.1001-7283.2021.01.006

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Analysis of SSR Loci in Whole Genome and Development of Molecular Markers in Tartary Buckwheat

Ma Mingchuan(), Liu Longlong, Liu Zhang, Zhou Jianping, Nan Chenghu, Zhang Lijun()   

  1. Center for Agricultural Genetic Resources Research, Shanxi Agricultural University/Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture and Rural Affairs/Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taiyuan 030031, Shanxi, China
  • Received:2020-04-08 Revised:2021-01-06 Online:2021-02-15 Published:2021-02-23
  • Contact: Zhang Lijun E-mail:mamingchuan6@163.com;15034054161@163.com

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Abstract:

The number of polymorphic SSR markers of tartary buckwheat is limited. In this study, SSRs with 1-6 nucleotide repeats were searched from the published whole genome sequences of tartary buckwheat using MISA software and the characterization of SSR sequences and their polymorphisms were analyzed. A total of 1640 SSR loci were detected in whole genome sequences of tartary buckwheat. The content of tri-nucleotide repeat SSRs was the highest, accounting for 63.29%; the content of penta-nucleotide repeat SSRs was the lowest, only 0.12%. The most abundant repeat motifs were AT/TA, AAG/CTT, ACC/GGT and ATC/GAT. The SSR length ranged from 12 to 476bp, with an average of 23.14bp. The ratio of SSR length ranged from 12 to 19bp was 71.71%, and the SSRs (≥20bp) was 28.29%. Based on the type of SSR loci, 479 pairs of primers were designed and synthesised. The 200 pairs of primers were selected for PCR amplification, 56 of them showed polymorphism, 17 pairs showed polymorphism in five accessions of tartary buckwheat, 48 pairs showed polymorphism in three accessions of common buckwheat, and nine pairs showed polymorphism in tartary buckwheat and common buckwheat. SSR markers can be developed in large-scale based on whole genome sequences of buckwheat and a large number of SSR primers also can be identified for genetic diversity analysis, genetic mapping and variety identification.

Key words: Tartary buckwheat, Whole genome, SSR

Table 1

List of accessions used in accessing SSR level in the tartary buckwheat"

序号No. 资源编号Code 来源地Source 籽粒形状Seed shape 籽粒颜色Seed color 籽粒翅刺Prickly seed 花型Flower type
1 KQ1 山西 短锥 黑色 无 雌雄等长
2 KQ2 西藏 短锥 黑色 无 雌雄等长
3 KQ3 甘肃 长锥 灰色 无 雌雄等长
4 KQ4 青海 长锥 灰色 无 雌雄等长
5 KQ5 山西 短锥 深棕色 有 雌雄等长
6 TQ1 山西 三角形 黑色 无 雌雄异长
7 TQ2 贵州 三角形 黑色 无 雌雄等长
8 TQ3 内蒙古 三角形 褐色 无 雌雄异长

Table 2

Repeat type, number and frequency of SSRs in tartary buckwheat"

重复次数
Repeat number		 重复类型 Repeat type 总计
Total		 比例
Ratio (%)
单核苷酸
Mono-nucleotide		 二核苷酸
Di-nucleotide		 三核苷酸
Tri-nucleotide		 四核苷酸
Quad-nucleotide		 五核苷酸
Penta-nucleotide		 六核苷酸
Hexa-nucleotide
4 0 0 0 0 1 48 49 2.99
5 0 0 667 23 1 13 704 42.93
6 0 106 236 13 0 5 360 21.95
7 0 44 80 13 0 1 138 8.41
8 0 54 21 4 0 1 80 4.88
9 0 26 13 6 0 0 45 2.74
10 0 16 5 6 0 0 27 1.65
11 0 12 1 1 0 0 14 0.85
12 18 10 6 2 0 0 36 2.20
13 10 9 3 1 0 1 24 1.46
14 7 5 1 2 0 0 15 0.91
15 1 4 0 2 0 0 7 0.43
16~25 13 29 5 4 0 0 51 3.11
≥26 5 85 0 0 0 0 90 5.49
总计Total 54 400 1 038 77 2 69 1 640
比例Ratio (%) 3.29 24.39 63.29 4.70 0.12 4.21

Table 3

Occurrence frequency of different microsatellites motifs of tartary buckwheat"

重复类型
Repeat type		 重复基序
Repeat motif		 数目
Number		 重复次数
Repeat number		 平均长度
Mean length (bp)		 比例
Ratio (%)
单核苷酸 A/T 43 12~92 18.74 2.62
Mono-nucleotide C/G 11 12~21 16.18 0.67
二核苷酸 AC/GT 37 6~238 36.27 2.26
Di-nucleotide AG/CT 52 6~12 13.38 3.48
AT/TA 310 6~133 38.67 18.90
三核苷酸 AAC/GTT 104 5~8 16.41 6.34
Tri-nucleotide AAG/CTT 277 5~22 17.48 16.89
AAT/ATT 19 5~16 19.42 1.16
ACC/GGT 199 5~13 21.72 12.13
ACG/CGT 16 5~8 16.5 0.98
ACT/AGT 22 5~9 17.45 1.34
AGC/CTG 80 5~10 16.20 4.88
AGG/CCT 120 5~11 16.25 7.32
ATC/GAT 172 5~14 16.97 10.49
CCG/CGG 29 5~7 16.14 1.77
四核苷酸 ACAT/ATGT 65 5~23 33.72 3.96
Quad-nucleotide AAAG/CTTT,AAAT/ATTT,ACCC/GGGT,
ACCT/AGGT,ACGT/ACGT,ATCC/GGAT		 12 5~10 24.00 0.73
五核苷酸
Penta-nucleotide		 ACAAA,TGGGC 2 4~5 22.50 0.12
六核苷酸
Hexa-nucleotide		 AAAAAG/CTTTTT,AAACCC/GGGTTT,…,
AGGCGG/CCGCCT,CCCCGG/CCGGGG（共48种）		 69 4~13 27.39 4.21

Fig.1

Frequency of SSRs with different length in tartary buckwheat genome"

Fig.2

Variation in sequence length of different repeat types of SSR"

Table 4

Polymorphic analysis for PCR amplicons of 56 SSR primer pairs in eight buckwheat genotypes"

引物编号
Primer No.		 引物序列（5′-3′）
Primer sequence		 SSR基元
SSR motif		 产物长度
Length of
product (bp)		 多态性Polymorphism
5份苦荞
Five tarary buckwheats		 3份甜荞
Three common buckwheats
P7 F:CAAGGTTCATGTAGTAACCGGAG
R: CTGCTTGGTTGATGTTGATGATA		 (AAC)5 119 无 有
P13 F: GGATACCTTCATCTCTTTCGACC
R: TAGTTGTTATTGGAGAAGCAGGG		 (AAC)5 111 无 有
P18 F: CTCAAATCCAGAACTCACTTGCT
R: GCCACGACGTCAATATACTCTTC		 (GAT)6 156 无 有
P21 F: CATCACCACCAGTTCATAAGCC
R: TCCCTCCATTAAAGAACACCATA		 (TGC)6 160 有 无
P27 F: ATAGACACATCTCTGACACCACG
R: ATCATCTTCTTCCATCTTGCTGA		 (GAA)7 152 无 有
P29 F: CGGAAACAACAGCACAAGAGTAT
R: AGGCTATCGCTACTGCTGACTC		 (TAG)6 83 有 无
P30 F: ACCAATCAACCATTTCTTACCCT
R: TAGCTTTGGAGACTTCATCTTCG		 (ACA)7 143 无 有
P31 F: TACAATCTTCCTCCTCAAGGTCA
R: ATTGCTTGGGATTGTTGAAGTAG		 (ACC)6 152 有 有
P34 F: TAGGAGGAAGACCGTTTATAGGC
R: TCATTCCAAAGACTGCTTGAATC		 (CCA)9 156 有 有
P37 F: ATCCGAGGATCACAAAAATCATA
R: GGCAAAGAATCATTTAAAGCAAA		 (GAT)5 135 无 有
P39 F: TACTGGACAGATCATTCAGCTTG
R: TGATGATTCTGATTCTGATGGTG		 (TCACCC)4 138 有 有
P43 F: CACATAAGCTGAAGTCTACGCCT
R: GGTGTTAGGTCACCAGATGAAGA		 (CCTCCA)5 136 无 有
P46 F: ACCAACTGCCAGAGACCATACTA
R: GGAGTATGTAAGCCGAATATCCC		 (AAG)5 138 无 有
P54 F: TCATATTTGGAATTGTGGAGAGG
R: CCATTCATCAAAGGATGACCTAC		 (TGG)5 126 无 有
P58 F: TCCTGCTGCTTCTTACCAAACTA
R: GCTTGCCCTACATGCATTAAGTA		 (ACA)5 132 无 有
P59 F: ACTGTCAAATTTCAGGTCGAAAA
R: GTCTCCGGTTCTCTTTCCTAATC		 (TCA)6 142 有 无
P62 F: GCTCCTCTAGCTCTTCTAGCGAT
R: TCAACGACTTCCACTTTCTCTTC		 (AGA)5 151 有 有
P66 F: ATTACTAGTCTTCTTCGTCGCCC
R: ATGTTATTGTCGTCCAGAGAAGG		 (CT)7 143 有 无
P67 F: GGTTGCATTGAAGTCTATTGAGG
R: TCAGATTCTTCTTCCAACACCTT		 (AAG)6 159 无 有
P68 F: AAGAGCAAAAAGAAATTGCAAGA
R: AGCTGGTGTCATCAAGATCAGTT		 (GAT)5 145 无 有
P75 F: CCGAATATCAACAATTTCTCTCG
R: GACGTTGAATATTGGAGTGAAGG		 (TTC)5 137 无 有
P78 F: TCTTCAGCAACATCAAAGATCAA
R: ATCTTGCCCTTCAATATGGATTT		 (ATC)5 123 无 有
P79 F: TTATAAGTCACCACCACCTCCAG
R: TGGGTACTCCCAGTCATAACATC		 (CAC)7 154 无 有
P82 F: TTCAGATGGCACTAATGATGATG
R: GTTGCTTATGTTTCTTTGCATCC		 (ATG)5 134 无 有
P86 F: AAGAAAAGGATTCATGGTTGGAT
R: GAGCATCAGCTATACGCCTCTTA		 (GAA)6 159 无 有
P88 F: CTCTTCCTCTAGGATTTCTCGGA
R: AAGGATTTTCTTCGGTTGTTGAC		 (ACC)6 93 有 无
P94 F: AGCTGAAGCAGTTTCTACACACC
R: AAGCCTCATTCTCATTCTCCTTC		 (AATGAG)5 87 有 有
P105 F: ATTGGGTAAATGTGGTGTTGAAG
R: CTTGGGAGATTCCTTCTTGTTTT		 (GAA)5 128 无 有
P108 F: ATCATTTTGTTGATAGCCCAGAT R: TACTGCAATGGTTGTGTTTGTGT (CAACAC)4 137 无 有
P110 F: CCACACCAAAGTATGAAATGGTT
R: GAAATGATCTCATGCACATACCA		 (GAT)5 157 无 有
P114 F: GATGACGGAGAAGATATCAGCAG
R: TTCTTCATCATAAAAGAGGGCTG		 (GCA)6 156 无 有
P116 F: AGCTACAACAAGGTGGTATCCAA
R: GAGAAGACTGCAACTGAGCTGAT		 (CAA)5 108 无 有
P117 F: ATCTCAATCTTCTCTGCAGCAAC
R: TGGGATATTAGTTTGTTGAGCCA		 (CAA)5 154 有 有
P118 F: ATCCTGGCATAAAAGATTGGATT
R: TCCTTTTCTGGCTTGATGTAAAC		 (AT)6 83 无 有
P121 F: CTCATTCTCCTCTTTTGCCTCTT
R: GTCCTTAGAAGAGGACGTTCCAG		 (GGA)5 156 无 有
P124 F: GTGAGGATTGGTCTGACATTGAT
R: GATCCCAGTTTCACACTATCACC		 (GAT)7 151 无 有
P125 F: GACGAAATTGACCTTCTTCCAC
R: AGAAGTGGACGTTTCTTCATCAC		 (AGG)5 141 无 有
P127 F: GCTCAAATCCACAAACAATTTTC
R: CAGTAACGAGGAACGGAGAATC		 (GAG)6 138 无 有
P130 F: ACACCATCTCCTCCAACAGAAAC
R: CAAGCGTCTAGCTTTAAGGTGTC		 (CCA)5 113 无 有
P135 F: ATGAAGGTAGAACGTGGTATGGA
R: GCTTCTTCAACAATGGAGTCAGT		 (GAT)5 160 无 有
P143 F: TGCTTTGCTTGACTCTGATGATA
R: GTGAGCTTGTAACTTCCTCCTCA		 (TGA)5 125 有 有
P144 F: GCTAAAGACAATCAAGAGACGGA
R: TAGGATTTCAACCCTCATCATGT		 (AAC)5 133 无 有
P145 F: GGGCATAAGCGATCATACAATAG
R: TTGAGTTTCCTTCATGGTTCAAG		 (ATG)5 89 无 有
P149 F: GTAGCTCTACAACAACAGGCACC
R: GCCATGGTGGTTACCAGAAGTAT		 (CAA)5 152 有 有
P158 F: CAACAACTCATGTGATTGGAAAA
R: TTGATCCCTATAAGATCCTGCAA		 (CAA)5 147 无 有
P159 F: TGGCAGTGAGTCACAGAGTAATG
R: CACGCAAGAATTTTTCAATAAGG		 (GAT)7 148 无 有
P160 F: ATCATCCCCACTTGTCAACTCT
R: GTATCATCCTGGGACTGAGACTG		 (TCTCAG)4 93 有 无
P162 F: GAAAAGGCAGCAGTAAATTCTGA
R: AAGCTATTGGATTTTCTACCCCA		 (CAT)5 155 无 有
P163 F: TCTATACTTGGTGAGGAGTCGGA
R: GTCCCTTATAGCCATTTGCTCTT		 (GAT)6 129 有 有
P171 F: GAGAGAAAGGGGAGAGGAAGAGT
R: CAAACATCTTGAACCTCCAAAAG		 (AAG)5 103 无 有
P179 F: GATGTCTCGAATGATTGTGATGA
R: TGTCATCCTGAGTTTTCTTCGTT		 (AAG)5 115 无 有
P183 F: TGTCCATATTAGACCCACCATTC
R: AGATGTTGCTTTGCTCTTTGAAC		 (GAA)6 127 无 有
P192 F: AACCATCACTTCCTGAAAGAACA
R: CTGAGCCTCGAGATAGAATTTCA		 (AGA)5 84 有 无
P198 F: ATCGGACTCTCCTAAGTCATCG
R: GTCATCATCGGTAGATGGACTGT		 (CTC)5 145 无 有
P199 F: TGATCATGGATCTGGTTATTCAA
R: TAAACCCGATTTCCAAACTTCTC		 (GAG)6 114 无 有
P200 F: TTATGGCTCTGAAAGAGAAGGAA
R: CTACGCAAACAAATGTCATTCAG		 (GAA)5 139 有 无

Fig.3

Polymorphisms of primer 34 and primer 39 in five tartary buckwheat germplasms and three common buckwheat germplasms"

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