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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (05): 686-696.doi: 10.3724/SP.J.1006.2018.00686

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-05-20 Published:2018-01-29
  • Contact: Qing-Ying ZHAO,Rui-Juan ZHANG,Zhi-Rong YANG,Xing-Chun WANG E-mail:zryangsx@163.com;wxingchun@163.com
  • 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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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

Table 1

Information of 15 foxtail millet germplasm resources"

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

Table 2

Summary of Jingu 21 whole-genome sequencing information"

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%

Fig. 1

Evaluation of genome sequencing quality in Jingu 21 A: base quality distribution; B: sequencing-depth distribution curve; C: insert size distribution of the sequencing library."

Fig. 2

Summary of InDels between Jingu 21 and Yugu 1 genomes The positive and negative numbers indicate the number of insertion (positive) or deletion (negtive) of base in the Jingu 21 genome."

Fig. 3

Polymorphism analysis of InDel markers between Jingu 21 and Yugu 1 The detailed information of the InDel markers are listed in the supplemental table 1. The first and the second lanes of each marker are Jingu 21 and Yugu 1 respectively. M: D2000 DNA Marker [Tiangen Biotech (Beijing) Co. Ltd., #MD114]. 1~11 represent the order of the markers in supplemental Table 1."

Supplementary table 1

Primer sets of InDel markers based on Jingu 21 re-sequencing data"

Table 3

SNP annotation"

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 -

Fig. 4

Polymorphism analysis of CAPS marker 3G14938250 between Jingu 21 and Yugu 11: PCR amplicon of Jingu 21; 2: PCR amplicon of Yugu 1; 3: PCR amplicon of Jingu 21 digested with Nde I; 4: PCR amplicon of Yugu 1 digested with Nde I; M: D2000 DNA marker [Tiangen Biotech (Beijing) Co. Ltd., #MD114]."

Fig. 5

Polymorphic analysis of the InDel and CAPS markers among 15 foxtail millet germplasm resources A: Polymorphic analysis of InDel markers among 15 foxtail millet germplasm resources; B: Polymorphic analysis of CAPS marker among 15 foxtail millet germplasm resources. The foxtail millet germplasm (1-15) is listed in Table 1."

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