用高基元微卫星标记分析中国糜子遗传多样性

doi:10.3864/j.issn.0578-1752.2017.20.002

中国农业科学 ›› 2017, Vol. 50 ›› Issue (20): 3848-3859.doi: 10.3864/j.issn.0578-1752.2017.20.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

用高基元微卫星标记分析中国糜子遗传多样性

王瑞云^1,2，刘笑瑜¹，王海岗²，陆平³，刘敏轩³，陈凌²，乔治军²

¹山西农业大学农学院，山西太谷 030801；²山西省农业科学院农作物品种资源研究所/农业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，太原030031；³中国农业科学院作物科学研究所，北京100081

收稿日期:2017-05-17 出版日期:2017-10-16 发布日期:2017-10-16
通讯作者: 乔治军，Tel：0351-7065530；E-mail：nkypzs@126.com
作者简介:王瑞云，Tel：15234420135；E-mail：wry925@126.com。刘笑瑜，E-mail：251719093@qq.com。王瑞云和刘笑瑜为同等贡献作者。
基金资助:
国家自然科学基金（31271791）、山西省回国留学人员科研资助项目（2016-066）、国家现代农业产业技术体系建设专项（CARS-06-13.5-A16）、山西省重点研发计划（一般项目）（农业）项目（201603D221003-5）

Evaluation of Genetic Diversity of Common Millet (Panicum miliaceum) Germplasm Available in China Using High Motif Nucleotide Repeat SSR Markers

WANG RuiYun^1,2, LIU XiaoYu¹, WANG HaiGang², LU Ping³, LIU MinXuan³, CHEN Ling², QIAO ZhiJun²

¹College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi; ²Institute of Crop Germplasm Resources, Shanxi Academy of Agricultural Sciences/Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture/Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taiyuan 030031;³Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081

Received:2017-05-17 Online:2017-10-16 Published:2017-10-16

摘要/Abstract

摘要： 【目的】开发高基元（4—6）碱基重复微卫星标记，分析种质资源遗传多样性，为糜子遗传和进化研究提供理论基础。【方法】用隶属函数、主成分分析和聚类分析综合评价糜子资源表型多样性，用前期糜子转录组测序获得高基元SSR引物对地理来源差异大的糜子材料进行PCR扩增检测其多态性，用PowerMarker 3.25计算遗传多样性参数，用PopGen 1.32计算Nei’s遗传距离，用MEGA 5.0进行聚类分析，用Structure 2.2鉴定遗传类群。【结果】96份糜子资源株高和穗长变异最丰富，多样性指数分别为2.08和1.91。PCR扩增发现，占56.29%的85对引物具多态性，其中四、五和六碱基重复引物分别为71对（83.53%）、10对（11.76%）和4对（4.7%）。85个标记扩增产物大小分布为100—450 bp，PIC值平均为0.51，Rp值为1.00—5.75，平均为3.15。四、五和六碱基重复SSR的平均Rp值分别为3.15、2.8和4.0。基于Rp值分析SSR的分布频次，发现85个标记分布区间为0—1、1—2、2—3、3—4、4—5和5—6，分别包含1（1.18%）、15（17.65%）、31（36.47%）、20（23.53%）、12（14.12%）和6（7.06%）个标记，60%（51个）的标记分布在区间2—3和3—4。用85个SSR扩增96份糜子资源，共检测到232个等位变异，每个位点检测到等位变异2—3个，平均2.7294个；62个位点产生3个变异，23个位点产生2个变异；多样性指数为0.2842—1.0633，平均为0.7708；PIC值为0.0400—0.7281，平均为0.4723。不同生态区糜子种质间的遗传距离为0.0093—0.5052（平均为0.1798），遗传一致度为0.6034—0.9907（平均为0.8485）。基于UPGMA将96个糜子基因型聚为4个群组，第一群组主要属于北方春糜子区；第二群主要属于东北春糜子区；第三群组主要属于华北夏糜子区；第四群组主要属于黄土高原春夏糜子区。遗传结构分析将96份试材划分为4个类群，分别代表黄土高原、华北、东北和北方基因库。UPGMA聚类分析和遗传结构分析结果基本一致，均与地理起源相关。【结论】在糜子中构建了85个四、五和六碱基重复微卫星标记，这些高基元SSR的引物分辨率（Rp）高，对不同基因型分辨能力强，PCR扩增多态性好；用其评估中国糜子资源的遗传差异发现，黄土高原春夏糜子区和北方春糜子区资源遗传多样性最丰富。

关键词: 糜子, 高基元SSR, 遗传多样性, 聚类分析, 遗传结构

Abstract: 【Objective】The objective of this study to develop SSRs with high motif (tetra-, penta- and hexa-) nucleotide repeat and use them to evaluate the genetic diversity of common millet germplasms, and to provide significant implications for future linkage maps construction and evolution research of common millet.【Method】Comprehensive evaluation of phenotypic diversity was carried out based on membership function, principal component analysis (PCA) and cluster analysis. SSR primers developed in author’s laboratory by high-throughput sequencing were used to identify polymorphisms in different common millet accessions from various geographic origins. PowerMarker 3.25 and PopGen 1.32 were used to calculate the measures of genetic diversity and Nei’s genetic distance, respectively. MEGA 5.0 and Structure 2.2 were used to carry out cluster analysis and identify genetic groups.【Result】A set of 96 common millet accessions were used and seven phenotypic traits were investigated. Results showed that the genetic variation of tiller numbers and plant height were the most abundant. A total of 85 pairs of amplified primers (56.29%) showed high polymorphism among the 96 genotypes. A total of 71 tetra-nucleotide repeat SSRs were detected, accounted for 83.53%. Meanwhile, 10 (11.76%) penta- and 4 (4.7%) hexa- nucleotide repeat SSRs were identified, respectively. The size of 85 SSRs ranged from 100 to 450 bp with an average value of polymorphism information content (PIC) of 0.51. Resolving power (Rp) value varied from 1.00 to 5.75 (mean = 3.15). The average Rp value of tetra-, penta- and hexa- nucleotide repeat SSRs was 3.15, 2.8 and 4.0, respectively. Evaluating the distribution frequency of 85 SSRs based on Rp, it was observed that their ranges were 0-1, 1-2, 2-3, 3-4, 4-5 and 5-6, with 1 (1.18%), 15 (17.65%), 31 (36.47%), 20 (23.53%), 12 (14.12%) and 6 (7.06%) markers at each interval, respectively. Fifty-one markers accounting for 60% distributed at intervals of 2-3 and 3-4. A total of 232 alleles were amplified among 96 accessions by the 85 SSR markers. 2-3 alleles were generated by each locus, with an average of 2.7294 alleles. Sixty-two markers produced 3 alleles, and 23 markers produced 2 alleles. The range of gene diversity was 0.2842-1.0633, with an average of 0.7708. The range of PIC was 0.0400-0.7281, with an average of 0.4723. Genetic distance and genetic identity of common millet resources with different ecotopes were 0.0093-0.5052 (average=0.1798) and 0.6034-0.9907 (average=0.8485). Cluster analysis based on unweighted pair group method of mathematical averages (UPGMA) separated the 96 accessions into four groups (Northeast spring-sowing, Northern spring-sowing, Northern summer-sowing and Loess Plateau spring & summer-sowing ecotopes). A genetic structure assay indicated a close correlation between geographical region and genetic diversity. 【Conclusion】The present work developed a set of 85 tetra-, penta- and hexa- nucleotide repeat SSRs in common millet. These high motif microsatellite markers with high Rp value can distinguish different genotypes, generate high polymorphisms in polymerase chain reaction amplification. Based on the above molecular markers, the genetic diversity of Chinese common millet germplasms was assessed and those accessions from Northern spring-sowing and Loess Plateau spring & summer-sowing ecotopes are highly and genetically diverse.

Key words: common millet (Panicum miliaceum), high motif SSR, genetic diversity, clustering analysis, population structure

王瑞云，刘笑瑜，王海岗，陆平，刘敏轩，陈凌，乔治军. 用高基元微卫星标记分析中国糜子遗传多样性[J]. 中国农业科学, 2017, 50(20): 3848-3859.

WANG RuiYun, LIU XiaoYu, WANG HaiGang, LU Ping, LIU MinXuan, CHEN Ling, QIAO ZhiJun. Evaluation of Genetic Diversity of Common Millet (Panicum miliaceum) Germplasm Available in China Using High Motif Nucleotide Repeat SSR Markers[J]. Scientia Agricultura Sinica, 2017, 50(20): 3848-3859.

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用高基元微卫星标记分析中国糜子遗传多样性

Evaluation of Genetic Diversity of Common Millet (Panicum miliaceum) Germplasm Available in China Using High Motif Nucleotide Repeat SSR Markers

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