基于SLAF-seq技术的甘薯SNP位点开发

doi:10.3864/j.issn.0578-1752.2016.01.003

Abstract

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.

Key words: sweetpotato, SLAF-seq, SNP sites, high-throughput sequencing, molecular marker

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.

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SNP Sites Developed by Specific Length Amplification Fragment Sequencing (SLAF-seq) in Sweetpotato

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