| Silurus lanzhouensis, commonly known as Yellow Catfish, is a large-size aboriginal fish mainly distributed in the upper stream of the Yellow River. In recent years, due to overfishing, water environment deterioration and other factors, the wild populations of S. lanzhouensish have decreased sharply, making it a endangered species focused on by animal specialists. S. lanzhouensish has successfully farmed, which laid foundation for the conservation and utilization of the unique germplasm resources. EST-SSR is an excellent molecular marker, which could both directly mark functional genes, and have high transferability in closely related species. Therefore, it has great significance for developing S. lanzhouensish EST-SSRs markers and studying their transferability, appling in genetic diversity analysis and species evolution, genetic linkage mapping, quantitative trait loci location and molecular mark assisted breeding.The target of this study includes isolated S. lanzhouensish EST-SSRs and researched their transferability in Siluriformes and Cypriniformes fishes. They can be summarized as the following:(1) Magnetic bead enriched method was used to isolate EST-SSR from S. lanzhouensis. The m RNA of S. lanzhouensis brain tissue was reversed transcription into double-strand c DNA, whose ends were filled, and then linked blunt end adaptor. The c DNA fractions containing microsatellite sequences were captured by hybridizating the linked adaptor c DNA fractions with the oligonucleotide probes(AC)12,(AG)12,(CAG)8 and(GATA)6 attached to strepavdin coated magnetic beads. The selected c DNA fragments were ligated into the p MD19-T vector and transformed into competent E. coli DH5α cells to construct an enriched-SSR c DNA library. Using universal M13 primer and probes primer as double primers to PCR screening, 385 positive clones were identified from 972 transformants in the library. Sequence analysis of these positive clones confirmed 294 EST-SSRs(Gen Bank Accession Number: JZ845089-JZ845194; JZ845699-JZ845789; JZ905271- JZ905367). These EST-SSRs were divided into 3 categories: perfect type 143(44.7%), imperfect type 80 IV(28.9%) and compound type 71(26.4%). The efficiency of magnetic beads enrichment and double primer PCR screening were 30.25% and 76.36% respectively. Besides, 55 EST sequences of not containing SSR were gained(Gen Bank Accession Number: JZ845074-JZ845088; JZ892812-JZ892840; JZ905368-JZ905378). Online BLAST found 23 EST sequences have certain homology with known genes, these ESTs could provide basic data for expression and cloning of related genes of S. lanzhouensis. The results shown that the magnetic beads enrichment method is also suitable for the development of EST-SSR. It have some advantages compared with the traditional methods of developing EST-SSR, such as good universality, strong pertinence, high efficiency, etc.(2) According to unique flanking sequences of each motif, 136 pairs EST-SSR primers were designed and synthesized 55 pairs of them to PCR, 23 pairs EST-SSR primers can gain unique target PCR products, 8 polymorphic EST-SSRs were screened out by PAGE-silver staining. The population genetic structures of 30 S. Lanzhouensis individuals were analyzed by using the 8 polymorphic EST-SSR locis; It can find 2 to 5 alleles(avg. 3.00) in per locus. The expected heterozygosity were ranged from 0.1554~0.7206(avg. 0.4316). The polymorphic information content were ranged from 0.1411~0.6458(avg. 0.3666). These marks would supply solid foundation for S. lanzhouensis genetic diversity evaluation, species resources conservation, construction of genetic linkage map. In addition, they will also be useful for molecular mark assisted breeding of S. lanzhouensis.(3) 14 G-SSRs and 21 EST-SSRs of S. lanzhouensis were selected to analyze the transferability on 12 species of Siluriformes and Cypriniformes fishes. The results indicated the cross-orders transferability of 14 Genomic-SSRs and 21 EST-SSRs in Siluriformes and Cypriniformes fishes was 63.10%、32.14 and 61.11%、44.44% respectively. It revealed that the genomic SSRs and EST-SSRs of S. lanzhouensis have high transferability in closely related species, but it decreased with the increase of genetic distance among species. The total transferability of Genomic-SSRs and EST-SSRs of S. lanzhouensis in 12 different fishes was 47.62%、51.81 respectively. It indicated that the transferability of EST-SSRs was slightly more than genomic SSRs. These results could provide the basic data for the transferability of microsatellite ininterspecies and cross-orders, and using microsatellites to carry out the comparativegenomics analysis. |
PDF Full Text Request