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作物学报 ›› 2019, Vol. 45 ›› Issue (8): 1176-1188.doi: 10.3724/SP.J.1006.2019.84155

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

绿豆SSR标记的开发及遗传多样性分析

叶卫军,陈圣男,杨勇,张丽亚,田东丰,张磊,周斌()   

  1. 安徽省农业科学院作物研究所, 安徽合肥 230031
  • 收稿日期:2018-11-21 接受日期:2019-01-19 出版日期:2019-08-12 网络出版日期:2019-03-16
  • 通讯作者: 周斌
  • 作者简介:叶卫军, E-mail: 963472965@163.com|陈圣男, E-mail: chensn1226@163.com
  • 基金资助:
    本研究由安徽省农业科学院科研项目(18T0206);国家现代农业产业技术体系建设专项(CARS-08-Z11);国家重点研发计划项目资助(2016YFE0203800)

Development of SSR markers and genetic diversity analysis in mung bean

YE Wei-Jun,CHEN Sheng-Nan,YANG Yong,ZHANG Li-Ya,TIAN Dong-Feng,ZHANG Lei,ZHOU Bin()   

  1. Crop Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui, China
  • Received:2018-11-21 Accepted:2019-01-19 Published:2019-08-12 Published online:2019-03-16
  • Contact: Bin ZHOU
  • Supported by:
    This study was supported by the Research Program of Anhui Academy of Agricultural Sciences(18T0206);the China Agriculture Research System(CARS-08-Z11);the National Key Research and Development Program(2016YFE0203800)

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摘要：

SSR标记以其数量丰富、多态性好、共显性遗传等优点在基础研究和育种工作中发挥了重要作用, 但目前绿豆基因组中的SSR标记依然较少。本研究将磁珠富集法和测序技术相结合高通量检测绿豆基因组SSR位点, 鉴定出3,275,355个SSR位点, 开发了2742个SSR标记。选取其中157个SSR进行PCR验证, 发现有90个(57.33%)标记在10份材料中表现出多态性。挑选40个条带清晰、多态性高、染色体上均匀分布的标记对90份绿豆资源进行遗传多样性分析, 单个位点检测到的等位变异数为2~8个, 平均为3.0个, 有效等位基因数为1.31~4.21个, 平均为2.16。Nei’s基因多样性指数在0.23~0.76之间, 平均为0.51。多态性信息含量为0.22~0.72, 平均为0.43。聚类分析将90份材料分为2个类群, 包含4个组。第I组主要由北方资源组成, 第II组种质来源较为分散, 第III组主要由山东的资源构成, 第IV组包含多数河北的种质资源。本研究开发的多态性SSR标记不仅可以用于绿豆种质资源的遗传多样性分析, 也将在高密度遗传图谱构建、基因定位和分子标记辅助育种中发挥重要作用。

关键词: 绿豆, 测序, SSR, 引物设计, 遗传多样性

Abstract:

SSR markers play an important role in basic research and crop breeding due to their advantages of large number, high polymorphism and co-dominant inheritance. However, there are still few SSR markers available in mung bean. In this study, the magnetic bead enrichment method and sequencing technology were combined to identify the SSR loci of mung bean in high throughput, a total of 3,275,355 SSR loci were found, and 2742 markers were developed. A total of 157 markers were selected for validation by PCR method, 90 (57.33%) showed polymorphic among 10 mung bean accessions. Forty SSR markers with clear PCR products, high polymorphism and uniform distribution on chromosomes were selected to evaluate the genetic diversity among 90 mung bean accessions. The number of alleles per marker varied from two to eight, with an average of three. The effective number of alleles ranged from 1.31 to 4.21, with a mean value of 2.16. The Nei’s gene diversity was between 0.23 and 0.76, with an average of 0.51. Polymorphism information content was between 0.22 and 0.72, with a mean of 0.43. Cluster analysis distributed 90 materials into two clusters, including four groups. The germplasm of group II came from several areas, while those of groups I and III were mainly from North China and Shandong province, respectively. Most of the gerplasm from Hebei province were clustered in Group IV. These polymorphic SSR markers will be valuable for genetic diversity analysis, high-resolution genetic linkage maps construction, gene mapping and marker assisted selection in mung bean breeding.

Key words: mung bean, sequencing, SSR, primer design, genetic diversity

表1

供试绿豆材料信息"

编号
No.		 名称
Name		 来源地
Origin		 编号
No.		 名称
Name		 来源地
Origin		 编号
No.		 名称
Name		 来源地
Origin
1 安阳01-2 河南 31 科绿1号 内蒙古 61 潍绿11 山东
Anyang 01-2 Henan Kelyu 1 Inner Mongolia Weilyu 11 Shandong
2 白绿10号 吉林 32 辽绿10L708-5 辽宁 62 潍绿12 山东
Bailyu 10 Jilin Liaolyu 10L708-5 Liaoning Weiyu 12 Shandong
3 白绿11号 吉林 33 辽绿10号 辽宁 63 潍绿4号 山东
Bailyu 11 Jilin Liaolyu 10 Liaoning Weilyu 4 Shandong
4 白绿9号 吉林 34 庐绿2号 安徽 64 潍绿5号 山东
Bailyu 9 Jilin Lulyu 2 Anhui Weilyu 5 Shandong
5 宝绿1号 河北 35 嫩绿2号 黑龙江 65 潍绿7号 山东
Baolyu 1 Hebei Neilyu 2 Heilongjiang Weilyu 7 Shandong
6 保942 河北 36 品绿08116 北京 66 潍绿8号 山东
Bao 942 Hebei Pinlyu 08116 Beijing Weilyu 8 Shandong
7 保绿200403 河北 37 品绿2011-06 北京 67 潍绿9号 山东
Baolyu 200403 Hebei Pinlyu 2011-06 Beijing Weilyu 9 Shandong
8 保绿200520 河北 38 品绿2011-12 北京 68 鹦哥绿 河北
Baolyu 200520 Hebei Pinlyu 2011-12 Beijing Yinggelyu Hebei
9 保绿200621 河北 39 品绿21599 北京 69 渝黑绿3号 重庆
Baolyu 200621 Hebei Pinlyu 21599 Beijing Yuheilyu 3 Chongqing
10 保绿200810 河北 40 苏抗4号 江苏 70 渝绿2号 重庆
Baolyu 200810 Hebei Sukang 4 Jiangsu Yulyu 2 Chongqing
11 保绿201012-7 河北 41 苏黑2号 江苏 71 郑绿10号 河南
Baolyu 201012-7 Hebei Suhei 2 Jiangsu Zhenglyu 10 Henan
12 鄂1001 湖北 42 苏抗1号 江苏 72 郑绿8号 河南
E 1001 Hebei Sukang 1 Jiangsu Zhenglyu 8 Henan
13 鄂绿5号 湖北 43 苏抗3号 江苏 73 中绿10号 北京
Elyu 5 Hubei Sukang 3 Jiangsu Zhonglyu 10 Beijing
14 皇藏峪绿豆 安徽 44 苏绿11-4 江苏 74 中绿11号 北京
Huangcangyu mungbean Anhui Sulyu 11-4 Jiangsu Zhonglyu 11 Beijing
15 吉林绿豆 吉林 45 苏绿12-5 江苏 75 中绿12号 北京
Jilin mung bean Jilin Sulyu 12-5 Jiangsu Zhonglyu 12 Beijing
16 吉绿5号 吉林 46 苏绿15-11 江苏 76 中绿14号 北京
Jilyu 5 Jilin Sulyu 15-11 Jiangsu Zhonglyu 14 Beijing
17 吉绿6号 吉林 47 苏绿16-10 江苏 77 中绿1号 北京
Jilyu 6 Jilin Sulyu 16-10 Jiangsu Zhonglyu 1 Beijing
18 冀黑绿45-1 河北 48 苏绿1号 江苏 78 中绿3号 北京
Jiheilyu 45-1 Hebei Sulyu 1 Jiangsu Zhonglyu 3 Beijing
19 冀绿0204 河北 49 苏绿2号 江苏 79 中绿5号 北京
Jilyu 0204 Hebei Sulyu 2 Jiangsu Zhonglyu 5 Beijing
20 冀绿0514 河北 50 苏绿4号 江苏 80 中绿8号 北京
Jilyu 0514 Hebei Sulyu 4 Jiangsu Zhonglyu 8 Beijing
21 冀绿0816 河北 51 太原06-2 山西 81 明绿1号 安徽
Jilyu 0816 Hebei Taiyuan 06-2 Shanxi Minglyu 1 Anhui
22 冀绿10号 河北 52 太原52 山西 82 明绿2号 安徽
Jilyu 10 Hebei Taiyuan 52 Shanxi Minglyu 2 Anhui
23 冀绿11号 河北 53 太原VC3061A 山西 83 明绿3号 安徽
Jilyu 11 Hebei Taiyuan VC3061A Shanxi Minglyu 3 Anhui
24 冀绿7号 河北 54 太原VC4503B 山西 84 明绿4号 安徽
Jilyu 7 Hebei Taiyuan VC4503B Shanxi Minglyu 4 Anhui
25 冀绿8号 河北 55 太原串辐 山西 85 明绿5号 安徽
Jilyu 8 Hebei Taiyuanchuanfu Shanxi Minglyu 5 Anhui
26 冀绿9号 河北 56 太原早-1 山西 86 明绿7号 安徽
Jilyu 9 Hebei Taiyuanzao-1 Shanxi Minglyu 7 Anhui
27 晋绿1号 山西 57 太原早-2 山西 87 明绿8号 安徽
Jinlyu 1 Shanxi Taiyuanzao-2 Shanxi Minglyu 8 Anhui
28 晋绿3号 山西 58 同1188326 山西 88 明绿9号 安徽
Jinlyu 3 Shanxi Tong 1188326 Shanxi Minglyu 9 Anhui
29 晋绿4号 山西 59 宛绿2号 河南 89 皖科绿1号 安徽
Jinlyu 4 Shanxi Wanlyu 2 Henan Wankelyu 1 Anhui
30 晋绿6号 山西 60 潍绿05-8 山东 90 皖科绿3号 安徽
Jinlyu 6 Shanxi Weilyu 05-8 Shandong Wankelyu 3 Anhui

图1

SSR的类型和频率分布 A: 不同类型SSR的分布频率, Momo-、Di-、Tri-、Tetra-、Penta-、Hexa-分别代表单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复; B~G: 单核苷酸(B)、二核苷酸(C)、三核苷酸(D)、四核苷酸(E)、五核苷酸(F)和六核苷酸(G)的重复类型及频率。"

表2

SSR长度多态性评估"

SSR长度多态性
SSR length
polymorphism		 聚类数量
No. of clusters		 比例
Percentage (%)		 设计引物数量
No. of designed primers
1 492,264 92.15 —
2 33,893 6.34 1920
3 5422 1.01 538
4 1604 0.30 172
5 573 0.11 65
6 231 0.04 28
7 101 0.02 10
8 51 0.01 3
9 29 0.01 2
≥10 41 0.01 4
合计Total 534,209 100.00 2742

Table 3

Information of 90 polymorphic SSR markers"

图2

设计引物的验证分析 A: 157对引物PCR结果的统计分析; B: 11条染色体上的引物扩增情况。MSP表示多态性扩增的标记, MSN表示可扩增出目的条带但无多态性的标记, MNN表示非特异性扩增或无扩增产物的标记。"

表4

40对SSR引物的信息"

引物名称
Name		 等位基因数
Allele number		 有效等位基因数
Effective number of allele		 多态性信息含量
Polymorphism information content		 Nei’s基因多样性
Nei’s gene diversity
Vr1-1 2 1.91 0.36 0.48
Vr1-5 4 2.73 0.58 0.64
Vr1-7 3 1.66 0.37 0.41
Vr1-8 3 1.72 0.38 0.43
Vr1-9 3 2.71 0.56 0.63
Vr2-2 3 2.20 0.45 0.54
Vr2-6 3 1.81 0.41 0.46
Vr2-8 3 2.10 0.44 0.52
Vr3-2 5 4.21 0.72 0.76
Vr3-3 3 2.12 0.43 0.53
Vr3-9 3 2.37 0.51 0.58
Vr3-10 3 1.71 0.35 0.40
Vr4-4 3 2.66 0.54 0.62
Vr4-7 2 1.91 0.36 0.48
Vr5-3 3 2.54 0.54 0.61
Vr5-4 4 3.27 0.64 0.69
Vr5-5 2 1.91 0.36 0.47
Vr5-6 3 2.88 0.58 0.65
Vr6-4 3 1.89 0.39 0.46
Vr6-7 2 1.67 0.31 0.39
Vr6-9 2 1.41 0.24 0.28
Vr6-10 2 1.82 0.34 0.44
Vr6-12 2 1.97 0.37 0.49
Vr7-5 2 1.67 0.33 0.41
Vr7-12 2 1.92 0.37 0.48
Vr7-14 3 2.05 0.43 0.51
Vr7-17 2 1.72 0.33 0.41
引物名称
Name		 等位基因数
Allele number		 有效等位基因数
Effective number of allele		 多态性信息含量
Polymorphism information content		 Nei’s基因多样性
Nei’s gene diversity
Vr7-20 2 1.72 0.33 0.41
Vr7-25 2 1.75 0.34 0.43
Vr8-3 3 2.13 0.42 0.53
Vr8-4 3 1.31 0.22 0.23
Vr8-14 3 2.54 0.52 0.60
Vr9-3 2 1.80 0.35 0.45
Vr9-14 4 2.21 0.51 0.55
Vr10-7 5 2.78 0.58 0.64
Vr10-9 4 3.29 0.64 0.69
Vr10-17 3 1.47 0.30 0.34
Vr11-4 8 3.25 0.64 0.69
Vr11-9 3 2.08 0.43 0.51
Vr11-10 3 1.51 0.30 0.35
平均Mean 3 2.16 0.43 0.51

图3

根据Nei’s遗传距离的绿豆资源UPGMA聚类图 1~90为表1中供试绿豆编号。"

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