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作物学报 ›› 2014, Vol. 40 ›› Issue (02): 191-197.doi: 10.3724/SP.J.1006.2014.00191

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

基于全基因组重测序信息开发玉米H99自交系特异分子标记

吕远大1,**,李坦1,**,石丽2,张晓林1,赵涵1,*   

  1. 1江苏省农业科学院农业生物技术研究所,江苏南京 210014; 2扬州大学生物科学与技术学院,江苏扬州225009
  • 收稿日期:2013-06-03 修回日期:2013-08-30 出版日期:2014-02-12 网络出版日期:2013-11-14
  • 通讯作者: 赵涵, E-mail: zhaohancn@gmail.com
  • 基金资助:

    本研究由江苏省农业科技自主创新资金[cx(10)435]和江苏省“六大人才高峰”B类项目(005085311107)资助。

Next-generation Sequencing for Molecular Marker Development in Maize Inbred H99

LÜ Yuan-Da1,**,LI Tan1,**,SHI Li2,ZHANG Xiao-Lin1,ZHAO Han1,*   

  1. 1 Institute of Agricultural Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China
  • Received:2013-06-03 Revised:2013-08-30 Published:2014-02-12 Published online:2013-11-14
  • Contact: 赵涵, E-mail: zhaohancn@gmail.com

摘要:

随着基因组测序技术和计算生物学的发展,利用生物信息学的方法大规模挖掘基因组特异分子标记已成为可能。玉米自交系H99具有较强的可再生能力,是玉米转基因研究的主要供体材料,但是目前对H99基因组信息了解甚少。本研究利用高通量Illumina测序技术,对H99全基因组进行重测序,利用生物信息学手段大规模挖掘并开发了4043H99特异性的SSR分子标记。随后利用模拟PCR策略,对开发的SSR分子标记进行电子多态性筛选,针对B73×H99Mo17×H99B73×Mo17三个群体亲本组合共开发2699候选的特异多态性SSR分子标记。随机挑选20SSR分子标记对群体亲本B73H99Mo17进行多态性筛选,进一步证实候选的多态性具有95%的准确率。此外,基于B73参考序列对开发的多态性SSR分子标记进行全基因组染色体定位及基因注释,揭示了候选多态性SSR分子标记在全基因组及基因内的分布特征。为玉米自交系H99开发了大量的分子标记资源,同时也为快速开发品种间多态性分子标记提供了一套高效可行的分析方法。

关键词: 玉米H99自交系, 高通量测序, 电子PCR, 多态性SSR

Abstract:

Next Generation Sequencing (NGS) has provided an effective approach to reveal the large scale of DNA polymorphic loci used as molecular markers to distinguish the genetic variations among different genotypes. Maize inbred line H99 is a common transgenic genotype with its desirable regeneration capacity. However, the genome sequence of H99 is unsequenced, which lags the molecular marker development for further maker assisted selection. Here, we used next generation sequencing to resequence H99 whole genome. The contigs assembled with SOAPdenovo2 were further scanned for potential SSR loci by MISA software. Out of 8 268 SSR loci, 4 043 site-specific primers flanking SSR loci were designed and surveyed in silico for locus polymorphism among H99, B73, and Mo17. Around 2 699 SSR loci showed the polymorphism among above three genotypes. Twenty primer pairs from 20 arms of maize chromosomes were selected and validated, and 19 primers amplified the predicted fragments. In addition, we physically mapped and annotated the polymorphic SSR loci, and elucidated the loci distribution in genome. Taken all together, the new developed SSR primers and their information, as a complement to previous ones, were expected to be beneficial to map based cloning and marker-assisted selection.

Key words: Maize inbred line H99, High throughout sequencing, ePCR, Polymorphic SSR

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