摘要
【目的】基于玉米细胞质雄性不育材料粗制线粒体DNA高通量测序数据,对叶绿体基因组进行组装。【方法】应用Illumina Hiseq 2500平台进行线粒体DNA测序。利用软件Velvet对过滤后的clean reads进行拼接和叶绿体基因组的组装。采用在线注释软件DOGMA(http://dogma.ccbb.utexas.edu/)对叶绿体基因组完整序列进行基因预测和基因功能分析。【结果】成功组装出C48-2和C黄早四2个不育系及48-2和黄早四2个保持系的叶绿体基因组,大小分别为140 473 bp(C48-2)、140 478 bp(C黄早四)、140 458 bp(48-2)、140 448 bp(黄早四),均包含84种编码基因,30种tRNA基因,4种r RNA基因。新组装的4个叶绿体基因组,在基因组大小及所包含的基因种类及数量方面都与叶绿体参考基因组具有较高的相似性,说明粗制线粒体的高通量测序数据可以用来组装叶绿体基因组。叶绿体基因组高度保守,但也存在SNP及InDel,基于不育系与保持系叶绿体DNA(cpDNA)的SNP位点设计特异引物,可用来鉴定区分CMS-C不育胞质与正常胞质。【结论】利用粗制线粒体DNA的高通量测序数据可以完成叶绿体基因组的组装,玉米CMS-C不育系与保持系的叶绿体基因组具有高度的一致性,但也存在一些多态性位点,基于多态性位点成功开发出可区分CMS-C不育胞质与正常胞质的特异标记。
【Objective】The aim of this study was to construct the chloroplast genome based on maize mitochondria DNA high throughput sequencing data of CMS-C male sterile lines and their maintainer lines.【Method】Crude mitochondria DNA was sequenced by next generation sequencing,Hiseq 2500 sequencing system. Chloroplast genomes were de novo assembled by software Velvet. On line soft DOGMA(http://dogma.ccbb.utexas.edu/) was used for gene prediction and function analysis of chloroplast genomes.【Result】Four chloroplast genomes including 2 male sterile lines, C48-2 and Chuangzaosi,and 2 maintainer lines, 48-2 and Huangzaosi were obtained, the chloroplast genome size for C48-2 is140 473 bp, for Chuangzaosi is 140 478 bp, for 48-2 is 140 458 bp, for Huangzaosi is 140 448 bp.All the four genomes contain 84 protein coding genes, 30 tRNA genes and 4 rRNA genes. Both the chloroplast genome size and the genes content or genes type of the 4 newly assembled were highly similar to the reference genome. These results suggested that it was feasible and reliable to assemble the chloroplast genome based on the mitochondrial DNA high throughput sequencing data. Collinearity analysis show that chloroplast genome was highly conserved. But SNP and InDels were detected between the CMS-C male sterile lines and the maintainer lines, new primers were designed according to the common SNP loci between male sterile and maintainer line, which can be used to distinguish maize CMS-C cytoplasm and normal cytoplasm.【Conclusion】Chloroplast genomes could be constructed based on the sequencing data of mitochondria DNA from high throughput sequencing technology. The chloroplast genome size and component between CMS-C cytoplasm and normal cytoplasm were high consistent. But InDels or SNP were also detected between male sterile lines and maintainer lines. Based on the polymorphic site, the specific chloroplast DNA markers were obtained for identifying the CMS-C and normal cytoplasm in maize.
作者
邱涛
刘孝伟
唐津
张鹏
易洪杨
曹墨菊
QIU Tao;LIU Xiaowei;TANG Jin;ZHANG Peng;YI Hongyang;CAO Moju(Key Laboratory of Crop Genetic Resource and Improvement of Ministry of Education,Key Laboratory of Biology andGenetic Improvement of Maize in Southwest Region of Ministry of Agriculture,Maize Research Institute,SichuanAgricultural University,Chengdu 611130,China)
出处
《四川农业大学学报》
CSCD
北大核心
2019年第1期1-7,共7页
Journal of Sichuan Agricultural University
基金
"十三五"国家重点研发计划(2016YFD0101206)
