开口箭基因组中微卫星特征分析与分子标记开发

开口箭(Tupistra chinensis Baker)为百合科(Liliaceae)开口箭属(Tupistra Ker-Gawl)多年生草本植物,其野生资源稀少,为中国珍稀药用植物。该研究基于第二代测序技术首次建立了开口箭的基因组文库与微卫星文库,对开口箭的微卫星组成进行了特征分析,开发了开口箭SSR引物一套,并对开口箭及其近缘种进行了PCR扩增与聚丙烯酰胺凝胶电泳检测。结果表明:(1)通过对开口箭基因组测序与拼接得到23 362条基因序列,检测出微卫星位点1 465个,其中单核苷酸重复最多,二核苷酸重复长度变异最大。(2)所设计的52对微卫星引物中有14对扩增出的条带清晰且多态性丰富,每个位点的复等位基因数(Na)在2~4之间,平均为3.00;多态性信息含量(PIC)在0.336 9~0.785 6,平均为0.546 3。(3)用筛选出13对多态性引物对6个开口箭近缘种,长柱开口箭(Tupistra grandistigma)、筒花开口箭(Tupistra delavayi)、弯蕊开口箭(Tupistra wattii)、剑叶开口箭(Tupistra ensifolia)、吉祥草(Reineckia carnea)和万年青(Rohdea japonica)进行通用性检测,结果大部分引物均能在开口箭的近缘种中成功扩增,通用率较高。该研究所开发的开口箭微卫星分子标记为开展开口箭的遗传多样性研究及种质资源鉴定奠定了基础。

Genomic Microsatellite Characteristic Analysis and Molecular Marker Development for Tupistra chinensis Baker

Tupistra chinensis Baker, belonging to Tupistra Ker Gawl, Liliaceae, is a perennial herb plant and a rare medicinal plant in China with sparse wild type resource. In this study, the genome library and microsatellite library of T. chinensis were established for the first time based on the second generation sequencing technology. Then its microsatellite composition was characterized. A set of simple sequence repeat (SSR) primers was developed, and T. chinensis and its close ribs were treated by PCR amplification and polyacrylamide gel electrophoresis detection. It was found that: (1) the genome sequencing and splicing returned 23 362 gene sequences and 1 465 microsatellite loci. In particular, the mononucleotides were the most popular, while the repetition lengths of dinucleotides were the most variable. (2) Fourteen out of 52 pairs of newly designed microsatellite primers were amplified into clear strips with rich polymorphism; the number of multiple alleles per locus (N_a) was 2-4 (mean 3.00). The polymorphism information content (PIC) was 0.336 9-0.785 6 (mean 0.546 3). (3) The 13 pairs of polymorphism primers as screened were used to detect the generality of 6 close sibs of T. chinensis, including T. grandistigma, T. delavayi, T. wattii, T. ensifolia, Reineckia carnea, and Rohdea japonica. It was found the majority of the primers could be successfully amplified to the close ribs of T. chinensis, showing high generality. The microsatellite molecular markers of T. chinensis developed here underlie its genetic diversity research and germplasm resource identification in the future.