中国鲎基因组微卫星富集文库的构建及分析

Construction and identification of enriched microsatellite DNA libraries from Tachypleus tridentatus

本研究旨在从DNA分子水平上研究中国鲎(Tachypleus tridentatus)种群遗传多样性及种群结构等相关信息,以期为保护其野生群体种质资源提供科学依据。本研究通过生物素—磁珠富集法筛选微卫星标记,构建中国鲎基因组微卫星文库。基因组DNA经Fast Digest Tru1I酶切后,选取400 bp^1000 bp片段,用生物素标记的(GT)15、(CT)15混合探针与其杂交,杂交复合物与链霉亲和素磁珠结合,捕获含有重复序列的微卫星片段,纯化后连接PMD19-T载体克隆,构建基因组微卫星文库。从334个阳性克隆中随机选取196个片段大于400 bp的阳性克隆进行测序,共获得127个微卫星序列,其中完美型占69.3%、非完美型占11.0%、复合型占19.7%。除探针使用的GT和CT的重复序列外,还筛选到多碱基GCT、TGG、AAAC、ACAA、GATTT、TTTTA的重复序列。根据微卫星序列设计选择合成40对引物,结果显示其中3对具有较高多态性,可作为进一步评价中国鲎野生种质资源等遗传信息的有效遗传标记。

This study aims to study the population genetic diversity and population structure of Tachypleus tridentatus and other relevant information from the DNA molecular level, to provide a scientific basis for future protection of its wild germplasm populations. The microsatellites DNA library from T. tridentatus genome was constructed by the enrichment of microsatellite DNA with biotin-magnetic beads. The genomic DNA was digested with Fast Digest Tru I enzyme, the digestion fragments approximately 400-1000 bp in size were then hybridized to the biotin-labeled oligonucleotide probes（GT）15 and（CT）15, and the mixture was incubated with Streptavidin-coated Magnetic Sphere Particles. Fragments containing microsatellite repeat sequences were captured. After purification, the products were ligated to p MD-19 T vector and transformed into Escheri-chia coli. Finally, the microsatellite enriched genome library for T.tridentatus was constructed. A total of 334 positive colonies were obtained via the screening of the PCR products. A total of 196 clones with DNA fragments above 400 bp were randomly selected for sequencing. The results showed that through the sequencing of these positive colonies, 178 positive clones were successfully sequenced with 127 microsatellite loci confirmed. These microsatellites, included perfect（69.3%）, imperfect（11.0%） and compound（19.7%） repeat motifs. In addition to the GT and CT repeats, several other types of repeats were also detected, such as GCT, TGG, AAAC, ACAA, GATTT and TTTTA. Forty pairs of primers were composed and screened, and the results showed that three pairs with higher polymorphism can be used as genetic markers to evaluate the effective genetic information of wild resources of T. tridentatus in the further study.