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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (4): 908-919.doi: 10.3724/SP.J.1006.2022.14034

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于高基元SSR构建黍稷种质资源的分子身份证

陈小红1(), 林元香1, 王倩1, 丁敏1, 王海岗2, 陈凌2, 高志军3, 王瑞云1,2,*(), 乔治军2,*()   

  1. 1山西农业大学农学院, 山西太谷 030801
    2山西农业大学农业基因资源研究中心 / 农业农村部黄土高原作物基因资源与种质创制重点实验室 / 杂粮种质资源发掘与遗传改良山西省重点实验室, 山西太原 030031
    3内蒙古鄂尔多斯市农牧业科学研究院, 内蒙古鄂尔多斯 017000
  • 收稿日期:2021-02-22 接受日期:2021-06-16 出版日期:2022-04-12 网络出版日期:2021-07-12
  • 通讯作者: 王瑞云,乔治军
  • 作者简介:陈小红, E-mail: 13466897634@163.com
  • 基金资助:
    国家现代农业产业技术体系建设专项(CARS-06-13-13.5-A16);国家自然科学基金项目(31271791);国家留学基金(2019)项目(75-201908140133);山西省自然科学基金项目(201901D11126);山西省重点研发计划(一般项目) (农业)(201803D221008-5);山西省研究生教育创新项目资助(2020SY213)

Development of DNA molecular ID card in hog millet germplasm based on high motif SSR

CHEN Xiao-Hong1(), LIN Yuan-Xiang1, WANG Qian1, DING Min1, WANG Hai-Gang2, CHEN Ling2, GAO Zhi-Jun3, WANG Rui-Yun1,2,*(), QIAO Zhi-Jun2,*()   

  1. 1College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2Center 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
    3Ordos Institute of Agriculture and Animal Husbandry, Ordos 017000, Inner Mongolia, China
  • Received:2021-02-22 Accepted:2021-06-16 Published:2022-04-12 Published online:2021-07-12
  • Contact: WANG Rui-Yun,QIAO Zhi-Jun
  • Supported by:
    China Agriculture Research System(CARS-06-13-13.5-A16);National Natural Science Foundation of China(31271791);National Scholarship Fund Program (2019)(75-201908140133);Natural Science Foundation of Shanxi Province(201901D11126);Key Research and Development Program of Shanxi Province (General Project) (Agriculture)(201803D221008-5);Postgraduate Education Innovation Project in Shanxi Province(2020SY213)

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摘要：

为快速鉴定黍稷(Panicum miliaceum)资源, 建立大数据管理平台, 为种质身份标识和溯源管理提供理论依据。本研究以来源于4个生态栽培区的130份资源为材料, 基于35个高基元SSR (四、五和六碱基重复各21、10和4个)构建分子身份证。结果表明, 35个标记中有30个扩增条带稳定, 可用于分子身份证构建。30个标记共检出等位变异(Na) 90个, 平均为30个; 有效等位变异(Ne)为2.3186~2.9982, 平均为2.7607; Shannon多样性指数(I)为0.9158~1.0873, 平均为1.0472; Nei’s基因多样性指数(Nei)为0.5687~0.6665, 平均为0.6360; 多态性信息含量(PIC)为0.5151~0.7898, 平均为0.6966; 观测杂合度(Ho)为0.5000~ -0.8678, 平均为0.7168; 期望观测杂合度(He)为0.5710~0.6691, 平均为0.6386。基于UPGMA聚类将材料划为3个类群(I、II、III), 就山西省材料而言, 地方品种和育成品种分别划归类群I和III, 农家种在3个类群中均有分布。主成分分析将试材归为4类, 聚类结果与其地理来源一致。基于最少标记区分最多种质的原则, 剔除相似系数高的3个标记(RYW23、RYW49和RYW51), 筛选其余27个标记, 发现仅用17个标记组合(RYW35、RYW40、RYW37、RYW18、RYW30、RYW16、RYW20、RYW19、RYW8、RYW5、RYW3、RYW7、RYW1、RYW14、RYW9、RYW6和RYW10)可将全部材料区分。用ID Analysis 4.0、在线条形码生成器和二维码技术(http://barcode.cnaidc.com/app/html/bcgcode128.php和 https://cli.im/)构建了130份资源的字符串、条形码和二维码DNA分子身份证。

关键词: 黍稷, 高基元SSR, 分子身份证, 种质资源

Abstract:

In order to identify hog millet (Panicum miliaceum) germplasm rapidly, to establish a big data management platform, to provide a theoretical basis for germplasm identification and traceability management, 130 germplasms from four ecological cultivation areas were used as materials to construct a molecular ID based on 35 high-motif SSRs (21, 10, and 4 with four-, five-, and six-nucleotide repeats, respectively). The results showed that 30 out of the 35 SSRs could be used as core markers for the construction of molecular ID cards. Ninety allelic variants were detected; effective allelic variants (Ne) ranged from 2.3186 to 2.9982 with an average of 2.7607; Shannon diversity index (I) ranged from 0.9158 to 1.0873 with an average of 1.0472. The observed heterozygosity (Ho) was 0.5000-0.8678 with a mean of 0.7168; the expected observed heterozygosity (He) was 0.5710-0.6691 with a mean of 0.6386; the Nei’s gene diversity index (Nei) was 0.5687-0.6665 with a mean of 0.6360; the polymorphism information content (PIC) was 0.5151-0.7898, with a mean of 0.6966. All accessions were divided into three groups (Group I, II, and III) according to UPGMA analysis. In terms of Shanxi accessions, landraces and breeding varieties were classified into Group I and III, respectively, and farmers materials were distributed into three groups. Based on PCA analysis, all accessions were classified into four clusters, which were related to their geographical origin. As for the rule that the most germplasms were determined using the least markers, 3 markers were excluded due to their high similarity coefficient with others, namely RYW23, RYW49, and RYW51. Screening the remaining 27 markers, the combinations of 17 SSR (RYW35, RYW40, RYW37, RYW18, RYW30, RYW16, RYW20, RYW19, RYW8, RYW5, RYW3, RYW7, RYW1, RYW14, RYW9, RYW6, and RYW10) could identify all hog millet accessions. The DNA molecular identifications of character strings, bar code, and quick response (QR) codes were constructed via ID analysis 4.0, software online of bar code and QR codes technique (http://barcode.cnaidc.com/app/html/bcgcode128.php and https://cli.im/).

Key words: hog millet (Panicum miliaceum), SSR with high motif, molecular identity card, germplasm resources

表1

黍稷资源生态区分布"

生态区 Ecotope area 来源 Origin 数量 Accession number
黄土高原春夏糜子区LPSS
Loess Plateau Spring & Summer Sowing Hog Millet Ecotope		 山西 Shanxi, China
宁夏 Ningxia, China
河北 Hebei, China
甘肃 Gansu, China
青海 Qinghai, China
陕西 Shaanxi, China		 83
3
1
2
1
2
北方春糜子区 NCSP
Northern China Spring Sowing Hog Millet Ecotope		 山西 Shanxi, China
陕西 Shaanxi, China
内蒙古 Inner Mongolia, China
甘肃 Gansu, China
宁夏 Ningxia, China		 22
2
4
4
1
华北夏糜子区 NCSU
North China Summer Sowing Hog Millet Ecotope		 河南 Henan, China
河北 Hebei, China		 1
2
东北春糜子区 NECS
Northeast China Spring Sowing Hog Millet Ecotope		 内蒙古 Inner Mongolia, China
辽宁 Liaoning, China		 1
1
合计 Total 130

表2

6份糜子试材明细表"

编号
Serial number		 生态区
Ecotope		 统一编号
Unicode		 名称
Name		 原产地
Origin		 备注
Remark
1 NCSP 00000832 鸭爪白
Yazhuabai		 山西大同市
Datong, Shanxi, China		 地方品种
Landrace
2 NCSP 00001637 糜子
Meizi		 陕西榆林市
Yulin, Shaanxi, China		 地方品种
Landrace
3 NCSP 00002250 临河黄糜子
Linhehuangmeizi		 内蒙古临河市
Linhe, Inner Mongolia, China		 地方品种
Landrace
4 LPSS 00006780 黑城小黑糜
Heichengxiaoheimei		 宁夏固原市
Guyuan, Ningxia, China		 地方品种
Landrace
5 NCSP 00006573 宁朔紫秆小红糜
Ningshuoziganxiaohongmei		 甘肃
Gansu, China		 地方品种
Landrace
6 NCSU 00007118 糜子
Meizi		 河北省承德市
Chengde, Hebei, China		 地方品种
Landrace

表3

SSR引物序列及退火温度"

位点
Locus		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 退火温度
Tm (℃)		 重复基序
Repeated motif
RYW1 TAACGCTTCACCTTCAGACC TGAGATGGAGTTGGCTGATG 55.7 (TCATCT)6
RYW2 TTAGGGCTCTCCTGCATCC CAGCGAGTTCACCGTCAAG 57.2 (CGAAGC)5
RYW3 GGAGGCGTGACAATAAAAC GGCGTGAGGTGTTGTTTTT 54.3 (CCTTCC)5
RYW4 AATCCACAACGCACACGAC ATTTGCTCCTCTCGTCGGT 56.9 (GTGCCG)5
RYW5 GACGATGCTCTTGACCTTGT CACCGTGAAATGTCTCTGCT 55.2 (CCTTT)5
RYW6 AGCCGATTTGCTGTGGAGT CTGCCTCCGATGAGTTGGT 57.1 (ACACC)5
RYW7 TCCACTCATCCATTGCTCGT GATGGATTCAAAGGGACGCT 58.7 (CGCGC)5
RYW8 GGGTCAGAGAATACACAGCG GTAGGGAAGGAGAAGTGGGT 55.7 (AATAG)5
RYW9 GGACCCTTCCCTCACAGATT TCCAGTTGCTCTTGCCGTT 58.3 (CTAG)6
RYW10 TGGATTGGGTGGTGGTAT AAGGACGGCAGCACAAAT 53.0 (CGAG)5
RYW14 CGCACAACGACCACAAGAG ATACACCAGAGGAGCACGC 56.7 (GGCC)5
RYW16 ATCTCCTCCGCCTTCTAACCC TGGCAATGGTCGTACAAACT 58.4 (GAGC)5
RYW18 CTCCCTCTTTGTCCTCGTT GCTGCCTCTTCGCTATCTT 54.3 (AGTT)6
RYW19 GAATGATAGGTCCGCAAGG CAGCCTTTGTTCAGTTGTCTC 55.1 (TTAT)5
RYW20 ACCTCTTGCCGCACACTAC TTCTACATCCCCGAACCAC 55.4 (TTGG)6
RYW21 CCCTCCTACTGCTCCCTTT ATTACTCGTTCTCGCCTCG 55.6 (CGGA)6
RYW23 AGGAACAGCAGAGAGAGGG CAGAACACCACGAAACACC 53.7 (GGAA)5
RYW26 TAAGGGTGGCGTTGGATAG AACCCAACAGGTCCTCCAT 56.1 (AGGA)6
RYW28 CCAAGGCTGAGCAGAAAGAT ACAAGGTGAAACCCGAAGC 57.2 (AGGC)5
RYW29 CTTGATTTCTCACGCACCG TGTCCAGCAGTAGTCGTTCCT 57.2 (GCAG)5
RYW30 TAGCCTTCTTTGCCACCACT GCCCGTGATGATATTCGAC 56.6 (TTTC)5
RYW31 ACCCAGAGTCCAGAGAAGC GATGTCCTCCTCCTTCTCC 53.0 (AGCG)5
RYW32 CAGGTTATGGGAGGACGAG GGTGCTACGGTTACAGGGT 54.9 (ATCTT)5
RYW33 CGATTCTACACCGACGAGG TGTAGGGTTCCATTCATCTCC 54.6 (CCATC)5
RYW34 TCCCCCGATTAGGAAAGAT CTGGTGAGGTGATGAAGCC 55.8 (CGATT)5
RYW35 ATTAGCATCCCCCTCCAC ATCCGCTTTCCCAACCAC 54.7 (CGTGC)5
RYW36 TATTGTCCTTCCGCTCCC ATGACTACTCTCCCCCCCT 55.0 (GGCTT)5
RYW37 CATTCCGTTCCTTGTCTTCC CAGTCTCACTCCTGCGATGT 55.3 (GCGAT)5
RYW39 GTTGGGCGAGGTCAATCTG TAGGGAGCCGAAGCAGAAG 58.2 (TCCT)5
RYW40 TGCTCTTCGGCTCTTCTCC ATCAGCTCATCGTGACCCC 57.6 (CAGC)6
RYW43 GGAGATGCTTGCTTGGTTG CAGGAATCGCAAGGAACAG 56.2 (GGAG)5
RYW47 TTGTTTTTGCTGCTGCCTC TGCTGGACTTCTTTTTGCC 56.7 (GCCT)5
RYW49 GCTAAATCCGCTGATGAGGT TGTATGTTGCTCCAGCCTTG 57.0 (TATC)6
RYW50 CAAGGCAGATAGGGCAAGT TCGTCTGCTGCTGGTTTGT 56.1 (GGAG)5
RYW51 TATCGCCGCACCTTACAAC TGAGCCTGCTTCCATCTTG 56.8 (CTGC)5

图1

标记RYW37的扩增图谱"

表4

30个SSR的遗传多样性参数"

引物
Locus		 观测等位变异
Na		 有效等位变异
Ne		 Shannon多样性指数 I 观测杂合度
Ho		 期望杂合度
He		 Nei’s基因多样性指数
Nei		 多态性信息含量
PIC
RYW1 3.0000 2.9844 1.0960 0.6614 0.6675 0.6649 0.6635
RYW3 3.0000 2.9982 1.0983 0.6160 0.6691 0.6665 0.7839
RYW5 3.0000 2.9587 1.0917 0.7520 0.6647 0.6620 0.7306
RYW6 3.0000 2.8314 1.0677 0.7059 0.6495 0.6468 0.7451
RYW7 3.0000 2.5901 1.0140 0.7049 0.6164 0.6139 0.7009
RYW8 3.0000 2.9753 1.0944 0.7155 0.6668 0.6639 0.7870
RYW9 3.0000 2.8813 1.0780 0.7661 0.6556 0.6529 0.7372
RYW10 3.0000 2.5276 0.9892 0.7227 0.6069 0.6044 0.6913
RYW14 3.0000 2.8069 1.0613 0.7258 0.6463 0.6437 0.7370
RYW16 3.0000 2.9801 1.0952 0.6239 0.6673 0.6644 0.7775
RYW18 3.0000 2.9499 1.0899 0.5391 0.6636 0.6610 0.8043
RYW19 3.0000 2.9132 1.0830 0.6218 0.6595 0.6567 0.7999
RYW20 3.0000 2.8143 1.0655 0.6822 0.6472 0.6447 0.7056
RYW21 3.0000 2.5147 0.9878 0.7680 0.6048 0.6023 0.5907
RYW23 3.0000 2.5244 0.9886 0.8537 0.6063 0.6039 0.5886
RYW26 3.0000 2.3186 0.9158 0.6829 0.5710 0.5687 0.6302
RYW28 3.0000 2.5846 1.0193 0.5000 0.6155 0.6131 0.7735
RYW29 3.0000 2.6400 1.0228 0.7578 0.6237 0.6212 0.6209
引物
Locus		 观测等位变异
Na		 有效等位变异
Ne		 Shannon多样性指数 I 观测杂合度
Ho		 期望杂合度
He		 Nei’s基因多样性指数
Nei		 多态性信息含量
PIC
RYW30 3.0000 2.9599 1.0918 0.6198 0.6649 0.6621 0.7898
RYW31 3.0000 2.6094 1.0166 0.6532 0.6193 0.6168 0.7237
RYW33 3.0000 2.7255 1.0428 0.7949 0.6358 0.6331 0.6856
RYW35 3.0000 2.9496 1.0902 0.7179 0.6638 0.6610 0.7457
RYW37 3.0000 2.9802 1.0952 0.8374 0.6672 0.6645 0.7301
RYW39 3.0000 2.7768 1.0548 0.6880 0.6424 0.6399 0.6999
RYW40 3.0000 2.8700 1.0757 0.7458 0.6543 0.6516 0.7362
RYW43 3.0000 2.6909 1.0343 0.8678 0.6310 0.6284 0.6592
RYW32 3.0000 2.6620 1.0270 0.8661 0.6268 0.6243 0.5473
RYW47 3.0000 2.6426 1.0256 0.7812 0.6240 0.6216 0.6584
RYW50 3.0000 2.4418 0.9614 0.7563 0.5930 0.5905 0.5151
RYW51 3.0000 2.7418 1.0413 0.7752 0.6347 0.6322 0.5401
Mean 3.0000 2.7607 1.0472 0.7168 0.6386 0.6360 0.6966

图2

130份黍稷种质资源聚类图"

图3

130份黍稷资源遗传多样性的主成分分析"

图4

92份黄土高原春夏糜子区黍稷材料条形码(附字符串)和二维码DNA分子身份证 A: 条形码(附字符串)DNA分子身份证; B: 二维码DNA分子身份证。"

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