| Hemibagrus guttatus,formerly known as Mystus guttatus,belongs to order Siluriformes,family Bagridae,genus Hemibagrus,mainly distributed in the Pearl River,the Yuanjiang River and the Qiantang River.H.guttatus,which meat quality,delicious taste,high nutritional value,is an economically important catfish in China,and is listed as the top of the four famous fish in Xijiang River.In recent years,due to human activities such as overfishing,exploitation of shipping and construction of hydropower station,the natural resources of H.guttatus are gradually reduced,so it is urgent to investigate and evaluate its germplasm resources.In this study,we studied the genetic diversity and structure of eight populations(TE,DL,DA,LB,WX,GP,PN and WZ)of H.guttatus from Xijiang River in Guangxi,using chromosome karyotype analysis,microsatellite marker and mitochondrial molecular marker technology.The results provide a scientific basis for germplasm identification,natural resources protection,effective management and sustainable utilization of the H.guttatus.The main results are as follows:(1)Karyotype analysisThe chromosome specimens of wild H.guttatus were prepared by in vivo culture of kidney cells.A total of 110 evenly distributed and distinct intermediate phases were obtained by microscopic observation.The chromosome number 2n = 60,including 8 pairs of middle centromere chromosome(m),13 pairs of sub middle centromere chromosome(sm),3 pairs of sub end centromere chromosome(st)and 6 pairs of end centromere chromosome(t).The karyotype formula is 2n = 60 = 16 m + 26 sm + 6st + 12 t,and the NF = 102.This result is different from that of the early studies on the karyotype of H.guttatus,which is speculated to be caused by measurement error,chromosome variation and difference of sampling site.(2)Microsatellite analysisThe microsatellite site information was obtained based on the H.guttatus transcriptome data.According to the primer design principle,289 pairs of microsatellite primers were preliminarily designed.With DA population as DNA template,15 pairs of SSR primers were screened by TD-PCR,TP-PCR and capillary electrophoresis typing.The number of alleles at 15 loci ranged from5.0 to 16.0,with an average of 9.3 alleles.The observed heterozygosity was0.400～0.900,and the average observed heterozygosity was 0.740.The expected heterozygosity was 0.500～0.937,and the average expected heterozygosity was0.811.Polymorphic information content was 0.465～0.907.Except for PM146,the other sites are highly polymorphic,and these microsatellite loci can be used as effective molecular markers for the study of the genetic resources of H.guttatus.The genetic diversity and genetic structure of 8 H.guttatus populations(256 samples in total)from Xijiang River main stream in Guangxi was analyzed with 15 developed microsatellite loci.There was a total of 1404.0 alleles were detected,and the average number of alleles was 11.7;797.4 available alleles,with an average of 6.6 available alleles.The average observed heterozygosity,average expected heterozygosity and Shannon index of each population were between 0.590～0.742,0.811～0.873,and 1.955～2.228,respectively,and the average polymorphism information content was between 0.778～0.845,indicating that each population of the H.guttatus population had more genetic information and the overall genetic diversity was at a high level.The average inbreeding coefficient between populations was 0.118～0.290,and the genetic differentiation coefficient was 0.0346～0.0905,indicating that there was inbreeding within populations,and the genetic differentiation between populations of H.guttatus was small.The AMOVA results show that the genetic differentiation mainly comes from within the population.And the results of cluster analysis show that there is a certain geographical pedigree structure among the populations of H.guttatus.(3)Mitochondrial DNA analysisBy using PCR amplification and second-generation sequencing,the gene sequence of H.guttatus mitochondrial Cytb(1135 bp)was obtained,and the base composition showed obvious A+T bias.A total of 240 samples from 8populations of mitochondrial Cytb sequences detected 29 polymorphic loci,27 haploid genotype,average haploid genotype variety index and the average range of nucleotide variety index were 0.1931～0.8022,and 0.00011～0.00109,the average number of nucleotide difference is 0.576,characterized by high h low πmodel,genetic diversity was of medium level.The ML phylogenetic tree and the haplotype network diagram of H.guttatus have not found obvious lineage structure and geographical distribution pattern.The genetic differentiation index(0.00007～0.00038)and genetic distance(0.00012～0.00091)between populations were small,and most of the gene exchanges between populations were frequent.The result of AMOVA showed that the genetic differentiation mainly came from within the population,which indicated that the overall genetic differentiation of H.guttatus was not obvious.Neutral test and nucleotide mismatch distribution suggested that the populations of Qianjiang River had experienced a population expansion event.Combined with the genetic diversity information and haplotype results,it is speculated that GP population is the ancestor population of H.guttatus of Guangxi Xijiang. |
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