RNA-seq技术开发油茶目的基因SSR标记

Development of SSR Markers for Target-Genes Derived from Camellia oleifera by RNA-seq Technology

油茶是中国南方重要的木本油料。利用MISA软件从油茶(Camellia oleifera)果实和叶转录组测序获得的25 297条Unigenes中共搜索挖掘出30 899个SSR标记。在这些SSR标记中,除单核酸苷酸(36.02%)外,主要重复类型是二核苷酸重复(45.79%),其次是三核苷酸重复(16.37%),四以上核苷酸重复占1.81%。Unigenes功能注释中,共648条涉及调控油茶油脂合成和脂肪酸形成积累、产量、花芽分化、生长发育及抗病虫害等目的性状,从这些Unigenes中共搜索挖掘到163个SSR标记,以民玉1号油茶基因组DNA为模板对这些SSR标记的有效性进行初步验证,共筛选出56对具有清晰扩增条带的SSR标记。以选取的5份二倍体油茶种质为材料,对初筛的56对SSR引物进行多态性检测,获得48对具有多态性的SSR标记引物。这些基于高通量数据新开发的油茶目的基因SSR标记可为油茶的指纹图谱构建、遗传多样性分析和分子标记辅助选择育种等奠定基础。

Camellia oleifera is an important woody oil plant in the south of China.A total of 30 899 SSR markers were discovered in the 25 297 unigenes by using MISA software,which were from data of RNA-seq of fruits and leaves of C.oleifera.Among these SSR markers,except for mononucleotide repeats（36.02%）,the dominant repeat type was dinucleotide SSR（45.79%）,followed by trinucleotide（16.37%）,and repeat types of tetranucleotide,pentanucleotide and hexanucleotide were accounted for 1.81%.In the function annotation of Unigenes,648 unigenes were annotated the functions regulating oil biosynthesis,fatty acid accumulation,yield,flower bud formation,growth and development,and resistance to disease and insect pests.163 SSR markers were mined in these targeted Unigenes.The genome DNA of ＇Mingyu No.1＇ was used as template to verify the validity of these SSR markers,and 56 pairs of these markers were found to have clear targeted PCR production.Furthermore,5 diploid accessions of C.oleifera were used as materials to carry out polymorphism detection on 56 pairs of SSR primers,and the results showed that 48 pairs of primers were polymorphic.These newly developed SSR markers of the targeted genes of C.oleifera based on high throughput RNA-seq data,which could provide more and efficiencySSR markers for fingerprinting construction of C.oleifera,genetic diversity analysis and molecular mark assisted breeding.