| The black rockfish(Sebastes schlegelii) has been important economic marine fish species of deepwater cage farming and stock enhancement in northern China. In recent years, overfishing and water pollution of coastal water has caused a gradually decrease of fishery resources, therefore artificial breeding and selective improvement has important economic and social benefits. Microsatellite DNA is one of the most widely used DNA molecular markers, it can offer technological support to Selective Breeding of Sebastes schlegelii. EST-SSR Markers have many benefits compared to Genomic-SSR Markers, such as lower cost of development, better cross-species amplification between related species, making a new efficient way of Microsatellite DNA development. S schlegelii belongs to ovoviviparous fish and has polyandry system, monitoring its genetic diversity and genetic differentiation of breeding lines has important practical significance. In this study, EST-SSR markers of S schlegelii were first developed from published EST sequences, in order to provide more options for its breeding. The genetic structure of 17 breeding lines was analyzed using 8 high polymorphic microsatellite markers, results will provide support for germplasm resources conservation and breeding. 1. Development of EST-SSR Markers for Sebastes schlegelii and cross-speciesamplification.In 1980 EST sequences downloaded from the NCBI public database, the occurrence rate of EST-SSR was 9.14%.For all EST-SSR loci, Perfect type ratio was 91.52%. Dinucleotide motif had the highest frequency, and TG/CA motif was the richest one. Trinucleotide motif had 23 types, CTG/CAG and GAA/TTC were the richest ones, the ratio reached to 12.50%.We designed 56 pairs of primers, 46 pairs were proved effective, and 18 pairs of primers expressed polymorphism. These loci were applied to analyse 30 Sebastes schlegelii DNA specimens on the population diversity, the total number of alleles was IV70; The observe number of alleles(Na) ranged from 2 to 9 and the average value was 3.89; The average values of observed heterozygosity(Ho) and expected heterozygosity(He) were 0.3037 and 0.3757, respectively; Polymorphic Information Content(PIC) ranged from 0.0323 to 0.7522, and the average value was 0.3419, 5 loci were highly polymorphic(PIC>0.5) and 4 loci were moderately polymorphic(0.25<PIC<0.5), the distribution ratio of highly and moderately polymorphic loci accounts for 50%.; After Bonferroni correction, 3 loci significantly deviated from Hardy-Weinberg Equilibrium(P-value<0.05), but no Linkage Disequilibrium phenomenon existed.The cross-species amplification of Sebastes schlegelii polymorphic EST-SSR markers showed that: In Sebastes koreanus, all loci were amplified effectually except Locus HJ606 and Locus HJ4183, and 6 loci showed polymorphism; In Sebastiscus marmoratus, all loci were amplified effectually and polymorphic except Locus HJ4215 and Locus HJ4286. The cross-species proportions and polymorphic rates of these loci surveyed in Sebastes koreanus and Sebastiscus marmoratus were 88.89%, 100.00% and 33.33%, 88.89%, respectively.In this study, the EST-SSR markers of Sebastes schlegelii developed possess high polymorphism and cross-species amplification ratio, they can facilitate studies about population genetic structure, phylogenetic analysis, and providing more markers for Selective Breeding of Sebastes schlegelii. 2. Genetic structure analysis of 17 breeding lines of Sebastes schlegeliiAlleles were separated by high-resolution capillary electrophoresis technique, and the total number of alleles was181. The genetic diversity anslysis of 17 breeding lines showed that: the average value of observed number of alleles(Na) was 6.0318, the average value of observed heterozygosity(Ho) and expected heterozygosity(He) were 0.8417 and 0.7201, respectively. This result showed that 17 Sebastes schlegelii breeding lines have high genetic diversity, which means they still possess large breeding potential.The genetic structure analysis of 17 breeding lines showed that: the genetic differentiation index(Fst) ranged from 0.0750 to 0.3301, the genetic differentiation between FH(Rizhao) and FB(Qingdao) was the minimum, and the maximum existed between SH(first filial generation of Changdao) and SE(Rongcheng♀×Qingdao♂). The average value of genetic differentiation index(Fst) was 0.2017, which means 20.17% of genetic differentiation exist among breeding lines and 79.83% exist among the individuals, meanwhile, genetic differentiation between arbitrary two breeding lines reached to a very highly significant level. This result can provide theoretical foundation for next parent fish mate strategy of Sebastes schlegelii selective breeding.The genetic distance between breeding lines was 0.0743 to 0.3268 and its average value was 0.1915, and each breeding line was randomly selected 10 individuals to make individuals’ cluster analysis, the results showed that: it all together formed 5 main nodes composed of several breeding lines and only few individuals scattered, others got together respectively.The genetic diversity and genetic structure analysis of 17 breeding lines of Sebastes schlegelii realized the genetic differentiation monitoring for breeding populations, provided data support for following Breeding Plan. |
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