Development Of SNP Markers Based On Transcriptome Sequencing And Analysis Of Population Genetic Diversity In Channa Argus

The northern snakehead(Channa argus)belongs to genus Channa in Channidae of Perciformes,it is distributed in many rivers and lakes in China.It is very important to estimate the genetic diversity and genetic variation of natural populations of the northern snakehead in order to conserve and rationally utilize its germplasm resources in China.In this study,we sequenced and analyzed the transcriptome of C.argus using the Illumina Hi Seq 2000 sequencing platform and related softwares,and developed polymorphic SNP markers by screening the transcriptome database and locus identification.Polymorphic SNP markers were used to analyze the genetic diversity and genetic differentiation of C.argus.The main results were as follows:1.We sequenced the transcriptome of C.argus(mixing sample of spleen,kidney,liver,gonad,gill,brain,pituitary,intestine,heart,muscle tissues)using the Illumina Hi Seq 2000 and performed an analysis on the data.After strict data cleaning and quality testing,38643574 high-quality clean reads were obtained,and they were assembled into 68684 unigenes.By searching against NR,String and KEGG databases using BLAST,43892 unigenes were successfully annotated.In total,23470 unigenes were classified into 60 function categories under the three ontologies of GO,11202 unigenes were assigned to the COG classification,which could be grouped into 25 functional categories,and a total of 22337 unigenes with significant matches in the database were assigned to 123 KEGG pathways.We located a total of 22069 SNPs from 26504 unigenes.Based on the transcriptome data of C.argus,104 primer pairs were designed to develop SNP markers.A total of polymorphic 32 SNPs were isolated and characterized in the Huanghu Lake population by means of HRM analysis.Thr results showed that the observed heterozygosity ranged from 0 to 0.6944,the expected heterozygosity ranged from 0.1064 to 0.5067,and the minor allele frequency ranged from 0.0556 to 0.4861.Only two SNP loci deviated from the Hardy-Wenberg equilibrium(HWE).2.Eighteen polymorphic SNPs loci were selected to evaluate the genetic diversity of nine populations of C.argus using HRM analysis.The nine wild populations of C.argus were collected from the Huanghe River,the Huaihe River and the Yangze River drainages.The minor allele frequency ranged from 0.208 to 0.415,the polymorphic information content ranged from 0.275 to 0.375,the observed heterozygosity ranged from 0.251 to 0.675,and the expected heterozygosity ranged from 0.330 to 0.500.Chi-square test showed that 12 SNP loci were found to be significantly deviated from HWE in a few populations.3.Nei’s unbiased genetic distances among nine populations of C.argus ranged from 0.0187 to 0.3147.Cluster analysis of UPGMA revealed that nine populations were clustered into two branches: one group from the Yangtze River drainage and another group from other river drainages.Molecular variation analysis(AMOVA)on nine populations showed that 20.06% of the genetic variation occurred among populations,indicating a significant genetic differentiation among nine populations(P < 0.001).While two groups were divided,AMOVA showed that 25.74% the genetic variation occurred between two groups(P < 0.05),and 3.31% were among populations within groups(P <0.001),indicating a significant genetic differentiation(P < 0.05)between two groups.Structure analysis results demenstrated that the nine populations of C.argus were classified as two theoretical natural populations,which supported the UPGMA tree and AMOVA results.Therefore,the results suggest that the nine populations of C.argus may be conserved and utilized as two management units.