基于转录组测序的油梨EST-SSR引物开发  

Development EST-SSR Markers Based on Transcriptome Sequences in Avocado

作　　者：应东山[1] 唐浩 韩瑞玺 王文林 王明[1] 王琴飞[1] 汤秀华 张如莲[1] YING Dongshan;TANG Hao;HAN Ruixi;WANG Wenlin;WANG Ming;WANG Qinfei;TANG Xiuhua;ZHANG Rulian(Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences /Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China,Ministry of Agriculture and Rural Affairs /Quality Control and Testing Centre for Seeds and Seedlings of Tropical Crops,Ministry of Agriculture and Rural Affairs,Haikou,Hainan 571101,China;Development Center for Science &Technology,Ministry of Agriculture and Rural Affairs,Beijing 100122,China;Guangxi South Subtropical Agricultural Science Research Institute,Longzhou,Guangxi 532415,China)

机构地区：[1]中国热带农业科学院热带作物品种资源研究所/农业农村部华南作物基因资源与种质创制重点实验室/农业农村部热带作物种子种苗质量监督检验测试中心,海南海口571101 [2]农业农村部科技发展中心,北京100122 [3]广西南亚热带农业科学研究所,广西龙州532415

出　　处：《热带作物学报》2018年第12期2446-2451,共6页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金项目(No.20153118)

摘　　要：利用转录测序技术,开发油梨表达序列标签-简单重复序列(EST-SSRs),为SSR标记在油梨种质资源鉴定、品种选育及遗传连锁图谱构建奠定基础。采用Illumina二代测序的技术,共获得37 639条无冗余的序列,对其进行SSR搜索,共获得6 419条简单序列重复(SSR)。利用Primer 3.0软件设计SSR引物,并以11份油梨种质筛选多态性引物。基于转录组序列开发出的EST-SSR的分布频率为17.05%。在油梨EST-SSR中,单核苷、二核苷和三核苷的重复占主导,占总数的99.07%。单、二、三核苷酸重复单元分别占总SSR的37.47%、31.80%和29.80%;出现频率最高的二核苷酸重复基元是AG/CT,占总数的29.15%,出现最高的三核苷酸重复基元为AAG/CTT,占12.01%。随机选择315个SSR位点合成引物,经11份油梨种质筛选鉴定,227对引物可扩增获得产物,有效扩增率为72.06%;其中34对引物表现出良好多态性,占有效引物的12.78%,占总引物的10.79%。在34对多态性引物中,每对引物扩增等位基因数2～12个。利用高通量测序开发SSR引物有较好的实用性,开发获得的34个具有多态性的油梨SSR标记可用于研究油梨及其相关近缘物种的遗传变异。The present study was conducted to characterize and develop EST-SSR markers in avocado based on the transcriptome sequencing in order to lay a foundation for germplasm resources evaluation, linkage mapping and genetic improvement of avocado. The Illumina next-generation sequencing technology was used for isolating a microsatellite-en-riched library avocado transcriptome. A total of 37 639 non-redundant sequences were isolated and 6 419 EST-SSRs were identified. EST-SSR primers were designed using software Primer 3.0. 315 randomly selected EST-SSR markers were designed and 11 avocado accessions were used for polymorphism. The distribution frequency of EST-SSRs was 17.05%, based on the transcriptome sequences. Mononucleotide dinucleotide and trinucleotide repeats was 37.47%, 31.80% and 29.80% of total SSR, respectively. Among the dinucleotide motifs, AG/CT presented the highest frequency, which accounted for 29.15%. AAG/CTT(12.01%) was the most frequent trinucleotide motif. 227 primer pairs were successfully amplified for genome DNA of 11 avocado resources, accounting for 72.06%. 34 pairs of primers presented good polymorphism, accounting for 12.78% of the effective primers and 10.79% of all primers;Two to twelve gene loci were found for 34 pair of primers. It was concluded that the SSR primers developed by high-throughput sequencing technology was of better practicability, and the 34 polymorphic primers of avocado SSRs could be used for studying genetic variation of avocado and its sibling species.

关 键 词：油梨 转录组 EST-SSR 引物 

分 类 号：S31[农业科学—作物栽培与耕作技术]