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

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.