中国农业科学2017，Vol.50Issue(9)：1655-1665,11.DOI:10.3864/j.issn.0578-1752.2017.09.011
基于高通量测序组装'赤霞珠'叶绿体基因组及其特征分析
Assembling and Characteristic Analysis of the Complete Chloroplast Genome of Vitis vinifera cv. Cabernet Sauvignon from High-Throughput Sequencing Data
摘要
Abstract
[Objective]A method was built to assemble complete chloroplast (cp) genome of Vitis and analyze its characteristics with Vitis vinifera cv. Cabernet Sauvignon, which will provide a methodological guidance for evolution and phylogenetic analysis of Vitis in the future.[Method]Total genomic DNA was extracted from young leaves of Cabernet Sauvignon using plant genomic DNA kit. The small fragments (350 bp) of DNA libraries were constructed according to the manufacturer's manual for the Illumina HiSeq PE150, and the sequencing depth was 10 fold. Grape cp reads were extracted by BLASTN software according to cp genome sequence of Arabidopsis thaliana (NC000932) and Pinot Noir (DQ424856). SOAPdenovo 2.04 assembled the extracted cp reads into complete chloroplast genome of Cabernet Sauvignon. Then its basic characteristics were analyzed using some bioinformatic softwares. [Result] This research obtained total of 5.2 G raw data after high-throughput sequencing. Among them, 0.42 G clean data of grape cp reads were extracted, and it accounted for about 8％. These extracted grape cp reads assembled the complete cp genome successfully. The characteristic analysis of grape cp genome showed that it was a circular molecule of 160676 bp in length with a typical quadripartite structure, including a pair of inverted repeats (IRA and IRB) of 26235 bp that were separated by large and small single copy regions (LSC and SSC) of 89134 bp and 19072 bp, respectively. A total of 154 predicted genes, including 99 protein-coding genes, 47 tRNA genes and 8 rRNA genes were identified. And the GC content of cp genome was 37.43％. Furthermore, the cp genome of Cabernet Sauvignon contained 37 tandem repeat sequences and 53 dispersed repeats. The length of most tandem repeat sequences was 11-42 bp. They accounted for 0.83％ of whole cp genome, and the dispersed repeats accounted for 5.33％. Additionally, fifty short simple repeats (SSRs) loci of cp genome were detected. And most SSR loci were composed of A or T contributing to an obvious bias in base composition. Distribution of cp SSRs was non-uniform because the regions of LSC, SSC, and IR were located by 39, 7, and 4 SSRs, respectively. The codon usage of protein-coding genes was biased to use A/T bases. And among these codons, leucine (L) and cysteine ? were the most and least used amino acids, respectively. The phylogenetic analysis showed that Cabernet Sauvignon had a closer genetic relationship with Pinot Noir, V. aestivalis and V. rotundifolia.[Conclusion]Based on high-throughput sequencing, the complete cp genome of Cabernet Sauvignon was obtained successfully. Cp and cpDNA were not required to isolate and extract in this method which shortened the experiment time, reduced the labor intensity and improved the feasibility. The subsequent characteristic analysis showed that gene structure, gene order, GC content and codon usage were identical with typical angiosperm. This research provided perfect and detailed data for the study of cp genome of Vitis vinifera, which also supplemented many deficiencies of characteristic analysis of cp genome of Vitis, such as repeat sequences, codon bias and SSRs.关键词
'赤霞珠'/叶绿体基因组/高通量测序/特征分析/系统发育分析Key words
Cabernet Sauvignon/chloroplast genome/high-throughput sequencing/characteristic analysis/phylogenetic analysis引用本文复制引用
谢海坤,焦健,樊秀彩,张颖,姜建福,孙海生,刘崇怀..基于高通量测序组装'赤霞珠'叶绿体基因组及其特征分析[J].中国农业科学,2017,50(9):1655-1665,11.基金项目
国家现代农业产业技术体系建设专项资金(CARS-30-yz-1)、中国农业科学院科技创新工程专项(CAAS-ASTIP-2015-ZFRI)、农业部物种保护项目(2130135-34) （CARS-30-yz-1）
