| Spottedknifejaw(Oplegnathus punctatus),commonly known as porgy and black gold drum,belongs to the order perciformes,the Oplegnathus and the genus Oplegnathus.By virtue of its excellent meat taste,nutrition and economic value,spotted knifejaw has become an important new aquatic species.However,with the expansion of the breeding scale and the increase of the breeding density,the problem thatspotted knifejaw is susceptible to viral diseases and the high mortality rate is more prominent compared with other cultured fish.Spotted knifejaw has 22 pairs of autosomes and a set of multiple chromosomes(X1,X2 and Y),and its sex determination mechanism is(X1X2Y)type.Its special type of sex determination is of great significance to enrich and improve people's cognition of sex chromosome,sex determination mechanism and sex chromosome evolution mechanism.In this paper,to explore these two questions,the sequencing,assembly and comparative genomics analysis of the genome of spotted knifejaw were completed for the first time.Then the structural characteristics of the genome were analyzed,and the possible genetic basis of its susceptibility to viral diseases was elabo rated.By analyzing the transcriptome of male and female gonadal in different development stages of spotted knifejaw,the dynamic process of gonadal development was analyzed.Through the comparative analysis of genomic data,a male-specific DNA molecular marker was identified,and a genetic sex identification technique was developed forspottedknifejawusing this DNA marker.(1)The spotted knifejaw genome was sequenced and assembled for the first time,and the assembly results were eventually anchored to 24 chromosomes.The number and size of chromosomes were consistent with the karyotype results(R2=0.97).A total of 22,970 genes were annotated.Functional annotation and BUSCO evaluation indicated that the assembly and annotation results were complete and accurate.It laid a foundation for the study of sex determination and immune molecular mechanism on spotted knifejaw.(2)Comparative genomics analysis of spotted knifejaw showed that the gene families in a number of immune-related GO items were under contraction,such as protein antigen binding,MHCI protein complex,immune system processes and immune response,which may be the genetic molecular basis for the susceptibility of spotted knifejaw to viral diseases.(3)A comparative analysis of the gonad trans criptome in different periods of spotted knifejaw revealed that there were a large number of sex preference SNPs between male and female individuals,indicating sexual chromosome gene differentiation.For the first time,two sex chromosomes X1 and X2 in spottedknifejaw were identified usingthe gonad transcriptome data.Moreover,the estrogen pathway gene in male and female individuals of spotted knifejaw at 60 days old has been differentially expressed,and the direction of gonadal differentiation has been determined.(4)To identify the sex-specific markers of spotted knifejaw,we performed a comparative analysis of the female and male genomes.In this study,an 18 bp insertion was identified in the male genome after verification by sequencing depth and PCR.An effective and rapid method based on PCR was then developed to identify the genetic sex.A male-female shared primer pair and a male-specific primer were designed for PCR amplification to avoid false negative phenomena.To examine the primers in practice,we utilized hundreds of spotted knifejaw fish from different groups to identify their genetic sex,and the results were consistent with their phenotypic sex.The male-specific DNA marker would be helpful for artificial breeding,Y chromosome assembly and further study of the sex determination mechanism.This study is the first to identify an effective sex-specific marker in spotted knifejaw. |
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