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基于SLAF-seq技术的甘薯SNP位点开发
引用本文:苏文瑾,赵宁,雷剑,王连军,柴沙沙,杨新笋.基于SLAF-seq技术的甘薯SNP位点开发[J].中国农业科学,2016,49(1):27-34.
作者姓名:苏文瑾  赵宁  雷剑  王连军  柴沙沙  杨新笋
基金项目:国家现代产业技术体系建设项目(CARS-11-C-15)、美洲甘薯资源引进与利用(2013-Z6)、科技部国际合作专项(2011DFB31620)、湖北省农业科学院青年基金项目(2014NKYJJ34)、湖北省农业科学院甘薯特色学科项目(2014tsxk06)、湖北省农业科技创新中心项目(2007-620-001-03)、公益性科技研究项目(2012DBA53001)
摘    要:【目的】单核苷酸多态性(SNP)是基因组中最普遍的遗传变异,是构建遗传图谱、完成分子标记辅助育种的一种非常重要的遗传标记。新一代高通量测序平台为SNP位点的检测提供了强有力的技术支持。多倍体作物通常表现为基因组序列大且重复比例高,一直以来多倍体作物的SNP位点挖掘面临巨大的挑战。【方法】共收集300份甘薯种质资源,利用SLAF-seq测序技术进行测序。首先以马铃薯基因组为参照,通过生物信息学分析进行实验方案的系统设计,筛选特异长度的DNA片断,构建SLAF-seq文库。后通过高通量测序的方式获得海量序列,进而通过软件分析比对,获得多态性SLAF标签,最后在多态性SLAF标签上开发大量特异性SNP位点。【结果】对照拟南芥的测序数据与其参考基因组进行比对的结果表明,双端比对效率在本试验中为87.71%,酶切效率为93.22%,说明本试验SLAF建库正常。通过测序共产生498.14 Mb的读长数据,测序后各样品所获得的读长数目在441 595—2 731 920范围内,其中,冀薯4号获得的数据量最大,共计获得2 731 920个读长,对照拟南芥数据量最小,共计获得441 595个读长。测序质量值Q30的范围在88.37%—90.67%,样品3043 Q30测序值仅为87.31%,样品鄂紫1号Q30测序值为91.31%,所有样品Q30值均在80%以上。测序获得GC含量的范围在37.23%—38.09%,样品苏薯9号和浙薯2号存在极端值,样品苏薯9号的GC含量在39.80%,样品浙薯2号GC含量在37.10%,所有样品GC含量均值为37.64%,GC含量普遍不高,说明达到测序要求。本研究共计获得597 094个SLAF标签,样品的平均测序深度为11.77×,其中多态性SLAF标签260 000个,占SLAF标签总数的43.54%。根据所获得的260 000个多态性SLAF标签来统计SNP位点信息,共计获得795 794个群体SNP位点。【结论】共获得498.14Mb的读长数据,597 094个高质量的SLAF标签,260 000个多态性SLAF标签,从260 000个多态性SLAF标签上共计开发获得795 794个高质量SNP位点。表明SLAF-seq技术可以很好地应用于甘薯SNP位点的开发,其效率远远高于SSR、AFLP、RAPD等分子标记技术。从全基因组范围获得的SNP位点可以进一步的用来完成后续群体进化分析和特异性SNP标记的开发。

关 键 词:甘薯  SLAF-seq  SNP位点  高通量测序  分子标记
收稿时间:2015-08-07

SNP Sites Developed by Specific Length Amplification Fragment Sequencing (SLAF-seq) in Sweetpotato
SU Wen-jin,ZHAO Ning,LEI Jian,WANG Lian-jun,CHAI Sha-sha,YANG Xin-sun.SNP Sites Developed by Specific Length Amplification Fragment Sequencing (SLAF-seq) in Sweetpotato[J].Scientia Agricultura Sinica,2016,49(1):27-34.
Authors:SU Wen-jin  ZHAO Ning  LEI Jian  WANG Lian-jun  CHAI Sha-sha  YANG Xin-sun
Affiliation:1.Institute of Food Crops, Hubei Academy of Agricultural Sciences, Wuhan 430064;China Agricultural University/Key Laboratory of Crop Heterosis and Utilization, Ministry of Education/Beijing Key Laboratory of Crop Genetic Improvement, Beijing 100193
Abstract:【Objective】 Single nucleotide polymorphisms (SNP) show high diversity in genomes and they are important markers for map construction and marker assistant selection (MAS). The development of high-throughput sequencing technology can provide the capacity to discover massive numbers of SNP sites in the genome. Polyploid crops have large size genomes along with large scale repetitive sequences, as a result, there are many challenges in developing SNP sites in the polyploidy crops.【Method】300 sweetpotato accessions were collected and sequenced by SLAF-seq technology. The scheme of the experiment was designed based on bio-informatics technology. Taking potato as the reference genome, specific size of DNA were chosen to construct the SLAF-seq library. After high-throughput sequencing, a great amount of sequences were obtained and used to obtain the polymorphism SLAF tags by software alignment, then found the distribution of specific SNP sites. 【Result】 The results coming from the alignment between the sequences of CK Arabidopsis and its reference genome indicated that the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching 87.71%, normal digestion ratio reached 93.22%. With the help of sequencing alignment as well as other bio-informatics technologies, a total of 498.14 Mb reads were obtained,the total number of the reads of each sample varied from 441 595 to 2 731 920. Jishu4 had the largest number of reads with 2 731 920 reads. Arabidopsis had the smallest amount of data with 441 595 reads. The quality of Q30 changed from 88.37% to 90.67%, the Q30 of 3043 only reached 87.31%, while the Q30 of Ezi1 reached as high as 91.31%, the average value of Q30 of each sample was above 80%. The GC content of each sample varied from 37.23% to 38.09%; Sushu9 and Zheshu2 had extreme values with Sushu9 having a GC content of 39.80% , while Zheshu2 had 37.10%.The average value of each sample was 37.64% with the average level of GC content low enough to process the sequencing. In the end, 597 094 high quality SLAFs were produced, 260 000 were polymorphisms, accounting for 43.54%. The final results consisted of 795 794 SNP sites obtained based on the analysis of 260 000 polymorphic SLAFs.【Conclusion】A total of 498.14 Mb reads were obtained and produced 597 094 high quality SLAFs, of which 260 000 were polymorphisms. 795 794 SNP sites were developed based on SLAF-seq technology. This paper provided results that SLAF-seq technology can be well applied in developing SNP sites in sweetpotato, much more efficiently than markers like SSR, AFLP, or RAPD. The developed SNP sites can be further used for the analysis of population evolution and developing specific SNP markers.
Keywords:sweetpotato  SLAF-seq  SNP sites  high-throughput sequencing  molecular marker
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