| Paramisgurnus dabryanus with high nutritional value is an important economical fish of freshwater aquaculture in China.In recent years,Paramisgurnus dabryanus cultivation has been developing rapidly,but it is also faced with some problems,such as serious germplasm degradation,the population gentic diversity decreased,and significant difference between male and female.In order to reveal the genetic difference between different populations of Paramisgurnus dabryanus,find out its sex-related molecular markers,and provide support for the sex-controlled breeding of Paramisgurnus dabryanus,the following studies were conducted: Firstly,this study carried out genetic diversity analysis of four Paramisgurnus dabryanus populations,which provide foundations for rational utilization and protecting of germplasm resources.Besides,this study carried out reduced-representation genome sequencing and microsatellite to screen sex-related molecular markers of Paramisgurnus dabryanus,which laid the foundation for the sex controlling technology of Paramisgurnus dabryanus.The following results were received from this study:1.Genetic diversity analysis of four populations of Paramisgurnus dabryanusFour populations from Dalian,Fanxian,Wuhan and Taiwan were analyzed by sequencing the mitochondrial cytochrome oxidase subunit Ⅰ(COI)genes.The results showed that 642 base pair(bp)fragment was consisted of A,T,C and G base with 23.5%,30.6%,26.7% and 19.2%,respectively,indicating a preference for A and T base.A total of 44 mutations of nucleotide and 23 haplotypes were identified in 4 populations.The haplotype diversity index ranged from 0.424~0.855,and the nucleotide diversity ranged from 0.00084~0.01659,the results indicated that Paramisgurnus dabryanus from Taiwan population had a higher genetic diversity than Fanxian,Dalian and Wuhan population.The genetic differentiation index(Fst)and genetic distance showed that genetic differentiation among Taiwan and other populations was represents extremely significant difference(P<0.01),also had a further genetic distance with other populations.In general,the results showed that there was a certain genetic difference between the different geographical groups,AMOVA analysis revealed that 52.11% of genetic variations derived among populations and 47.89% of genetic variations occured within populations.In terms of the negatively selective neutrality test,the results indicated that a population expansion occurred in the populations of Dalian,Fanxian and Wuhan population.Which provides a reference for protection of the genetic diversity and breeding work of Paramisgurnus dabryanus.2.Screening of sex-related SNP molecular markers in Paramisgurnus dabryanusThe RAD-seq technology was used to sequence the genome of 60 Paramisgurnus dabryanus from Fanxian(30 female and 30 male).In this method,genome-wide SNP molecular markers were developed and population genetics was studied using SNP markers.A total of 179.35 Gb clean data were obtained through sequencing analysis.The sequencing Q30 was 92.13%,and the GC content was 40.74%;A total of 6,699,778 SNP sites were obtained by bioinformatics analysis;Population evolutionary tree analysis was constructed,principal component and population genetic structure were analyzed based on RAD-seq.The results showed that 60 samples of Paramisgurnus dabryanus were divided into two subgroups from two different ancestral populations.Based on the statistic data,we found that: no SNP sites were found when their frequency more than 80% in male population,and less than 20% in female population,or their frequency more than 80% in female population,and less than 20% male population.The results showed that RAD-seq can not screen out sex-related SNP marker.3.Screening of sex-related microsatellite molecular markers in Paramisgurnus dabryanusIn this syudy,71 pairs microsatellite primers with two polymorphic loci were used to screen out sex-related molecular markers of Paramisgurnus dabryanus.Firstly,71 pairs of microsatellite primers were used to scan the gene pools which were constructed with 10 male and 10 female respectively.The result showed that 43 pairs of primers were amplified specific DNA fragments.Then,the obtained primers were used for single individual test in male and female individuals(5 female and 5 male),the results showed that the amplification of primers(P66,P180,P134,p14-102)in male and female individuals were significantly different.Finally,no significant difference was verified in 15 male and 15 female individuals.Above all,no sex-related microsatellite markers were found by this method in Paramisgurnus dabryanus. |
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