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作物学报 ›› 2018, Vol. 44 ›› Issue (05): 686-696.doi: 10.3724/SP.J.1006.2018.00686

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于名优谷子品种晋谷21全基因组重测序的分子标记开发

赵庆英1,**, 张瑞娟2,**, 王瑞良2, 高建华2,3, 韩渊怀3,4, 杨致荣1,3,4,*(), 王兴春2,3,4,*()   

  1. 1 山西农业大学文理学院, 山西太谷 030801
    2山西农业大学生命科学学院, 山西太谷 030801
    3山西农业大学农业生物工程研究所, 山西太谷 030801
    4杂粮种质资源发掘与遗传改良山西省重点实验室, 山西太谷 030801
  • 收稿日期:2017-09-11 接受日期:2018-01-08 出版日期:2018-05-20 网络出版日期:2018-01-29
  • 通讯作者: 赵庆英,张瑞娟,杨致荣,王兴春
  • 作者简介:

    第一作者联系方式: E-mail: 192015271@qq.com

  • 基金资助:
    本研究由国家自然科学基金项目(31471502, 31600289, 31371693, 31771810), 山西省自然科学基金项目(201601D011071), 山西省回国留学人员科研资助项目(2015-067)和山西省留学回国人员科技活动择优资助项目(2014-11)资助

Genome-wide Identification of Molecular Markers Based on Genomic Re-sequencing of Foxtail Millet Elite Cultivar Jingu 21

Qing-Ying ZHAO1,**, Rui-Juan ZHANG2,**, Rui-Liang WANG2, Jian-Hua GAO2,3, Yuan-Huai HAN3,4, Zhi-Rong YANG1,3,4,*(), Xing-Chun WANG2,3,4,*()   

  1. 1 College of Arts and Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2 College of Life Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    3 Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    4 Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taigu 030801, Shanxi, China
  • Received:2017-09-11 Accepted:2018-01-08 Published:2018-05-20 Published online:2018-01-29
  • Contact: Qing-Ying ZHAO,Rui-Juan ZHANG,Zhi-Rong YANG,Xing-Chun WANG
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31471502, 31600289, 31371693, 31771810), the Natural Science Foundation of Shanxi Province (201601D011071), the Research Project from Shanxi Scholarship Council of China (2015-067) and the Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (2014-11).

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摘要：

小米因其营养丰富日益受到重视, 而小米的品质是民众选择小米时最为关注的指标。晋谷21米质优异, 但由于缺少基因组信息, 严重阻碍了其优异米质形成机制的研究。本研究利用高通量测序技术, 对晋谷21全基因组进行重测序, 获得了14.95 Gb高质量测序数据。进一步将其与豫谷1号参考基因组比较, 发掘了169 037个InDel位点和1 167 555个SNP位点, 其中长度在13~50 bp之间适于琼脂糖凝胶电泳检测的InDel位点为14 578个。选择其中1个SNP位点和68个InDel位点验证, 表明利用二代测序技术开发的InDel和SNP标记真实可靠。基于名优谷子晋谷21重测序数据开发的InDel和SNP分子标记具有通用性, 可用于其他谷子、狗尾草和谷莠子等种质资源的相关研究。同时, 开发了一个晋谷21特异的InDel标记2G5501976, 利用该标记即可快速鉴定待测材料是否为晋谷21及其衍生品种。本研究初步揭示了晋谷21的基因组特征, 不仅为深入解析其优异米质形成的分子机制奠定了基础, 而且为相关分子标记辅助育种、遗传分析和基因克隆提供了分子标记资源。

关键词: 谷子, 晋谷21, InDel, SNP, 分子标记, 基因组重测序

Abstract:

Foxtail millet becomes more and more popular for its rich nutrients, and the grain quality is the key concern that consumers would consider when selecting millet brand. Jingu 21 is an elite cultivar with high edible quality. However, the lack of genomic information impedes studies on the molecular mechanisms of millet quality formation. Here, we re-sequenced the whole genome of Jingu 21 using high-throughput sequencing technology, and obtained 14.95 Gb high quality data. By comparing sequence of Jingu 21 with the reference genome of Yugu 1, we identified 169 037 InDels and 1 167 555 SNPs. Of these InDels, 14 578 could be detected easily by agarose gel electrophoresis. One SNP and 68 InDel markers were selected to verify the polymorphism between Jingu 21 and Yugu 1, showing that the InDel and SNP markers developed by using next generation sequencing technology were reliable. Although the InDel and SNP markers were generated based on genome re-sequencing data of the elite cultivar Jingu 21, they could also be used for research on other foxtail millet, green foxtail, and giant foxtail. Moreover, a specific InDel marker 2G5501976 for Jingu 21 was developed, which could be used to identify Jingu 21 and its derivative varieties. Taken all together, the genomic characterization of Jingu 21 not only lays a foundation for elucidating the molecular mechanisms of high quality formation, but also provides a large number of molecular markers for marker-assisted selection of high quality millet, genetic analysis and map-based cloning.

Key words: foxtail millet, Jingu 21, InDel, SNP, molecular marker, genome re-sequencing

表1

15份谷子种质资源信息"

编号
Code		 名称
Name		 原产地
Origin		 编号
Code		 名称
Name		 原产地
Origin
1 晋谷21 Jingu 21 山西 Shanxi 9 酒谷 Jiugu 河北 Hebei
2 mop1 山西 Shanxi 10 碱谷 Jiangu 内蒙古 Inner Mongolia
3 mop2 山西 Shanxi 11 菠菜腿 Bocaitui 内蒙古 Inner Mongolia
4 青狗尾草 Green foxtail 山西 Shanxi 12 大青谷 Daqinggu 内蒙古 Inner Mongolia
5 谷莠子 Giant foxtail 山西 Shanxi 13 龙谷 Longgu 辽宁 Liaoning
6 豫谷1号 Yugu 1 河南 Henan 14 谷莠天然杂交种 Guyou natural hybrid 北京 Beijing
7 青谷 Qinggu 河南 Henan 15 黄粟 Huangsu 江西 Jiangxi
8 黄大粒 Huangdali 河北 Hebei

表2

晋谷21全基因组测序数据信息汇总"

基本信息
Basic information		 测序数据
Sequencing data
清理后读段Clean reads 83 051 988 bp
清理后碱基Clean bases 14 949 357 840 bp
GC含量GC content 45.55%
Q30值Q30 92.96%
匹配读段Mapped reads 96.16%
1×覆盖度Coverage ratio 1× 95.34%
5×覆盖度Coverage ratio 5× 93.39%
10×覆盖度Coverage ratio 10× 91.54%

图1

晋谷21基因组测序质量评估 A: 碱基质量分布图; B: 测序深度分布曲线; C: 测序文库插入片段大小分布图。"

图2

晋谷21和豫谷1号基因组间的InDel统计正负数分别表示晋谷21基因组中插入(正数)或者缺失(负数)的碱基数。"

图3

InDel标记在晋谷21和豫谷1号的多态性分析 InDel标记详细信息见附表1, 每个分子标记的第1个样品为晋谷21, 第2个样品为豫谷1号。M: D2000 DNA Marker [天根生化科技(北京)有限公司, #MD114]。1~11代表该标记在附表1中的顺序。"

附表1

基于晋谷21基因组重测序的InDel和dCAPS分子标记及其引物"

表3

SNP注释结果"

类型
Type		 数目
Number		 区域
Region
基因内Intergenic 645599 -
基因间Intragenic 76 -
内含子Intron 83293 -
基因上游Upstream 12191 -
基因下游Downstream 336911 -
5′UTR UTR_5′_Prime 1614 -
3′UTR UTR_3′_Prime 3169 -
剪切受体Splice site acceptor 111 -
剪切供体Splice site donor 88 -
起始密码子获得Start gained 831 -
起始密码子丢失Start lost 62 CDS
非同义起始密码子Non synonymous start 9 CDS
同义突变Synonymous coding 21448 CDS
非同义突变Non synonymous coding 27694 CDS
同义终止密码子Synonymous stop 34 CDS
终止密码子获得Stop gained 426 CDS
终止密码子丢失Stop lost 138 CDS
其他Other 33861 -

图4

CAPS标记3G14938250对晋谷21和豫谷1号的多态性分析1: 晋谷21 PCR 扩增产物; 2: 豫谷1号 PCR 扩增产物; 3: 晋谷21 PCR产物的Nde I酶切结果; 4: 豫谷1号PCR 产物Nde I的酶切结果; M: D2000 DNA marker [天根生化科技(北京)有限公司, #MD114]。"

图5

InDel和CAPS标记在15个谷子种质资源的多态性分析 A: InDel标记在15个谷子种质资源的多态性分析; B: CAPS标记在15个谷子种质资源的多态性分析。1~15为谷子种质资源, 详细信息见表1。"

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