Development Of Sex-linked Molecular Markers And Identification Of Candidate Gene For Sex Determination In Southern Catfish Based On Whole Genome Sequencing

Southern catfish?Silurus meridionalis Chen?,also known as big mouth catfish,belongs to Siluriformes,Siluridae,and Silurus.It is an important economic fish which is endemic to China and widely distributed in the Yangtze River basin.It has the characteristics of large size,good meat quality,fast growth,strong disease resistance and high economic value.Normal weight of individual is about 2-5 kg,and the maximum weight can be more than 50 kg.Southern catfish attain sexual maturity first at an age of 2-4 years for male and 3-5 years for the female.The growth rate and individual size of Southern catfish display obvious sexual dimorphism,and females grow faster than males.Therefore,the development of all-female stock would be of significant benefit for aquaculture.However,the basic research of the Southern catfish is relatively weak,and the genome sequence for this important species is still missing,hindering the application of genome-assisted breeding in Southern catfish.In this study,YY supermales were generated in our lab by crossing XY male with XY neofemale,which provide a good model for studying sex determination in fish.Firstly,Illumina,Nanopore,Bionano,and Hi-C technologies were used to sequence and obtain the high-quality whole genome sequences of the XX,XY and YY Southern catfish.Secondly,one male pool and one female pool were resequenced by whole genome approach.Lastly,sex-linked molecular markers were screened and candidate sex-determining gene were identified based on whole genome sequencing and resequencing data.These researches laid the foundation for analyzing the genetic basis of economic traits of Southern catfish at the genomic level and carrying out whole-genome selection breeding and sex control breeding.The main results are as follows.1.The chromosome level genome assembly of Southern catfish.To estimate the genome size and heterozygosity of Southern catfish,49.5 Gb XX Illumina clean reads were used for k-mer analysis.The estimated genome size of XX Southern catfish was 712.42 Mb with 39%GC content,and the heterozygosity rate was approximately0.49%.From Nanopore sequencing,a total of 69.7 Gb,69.2 Gb and 77.1 Gb clean data were generated after filtering and quality control from 81.3 Gb,80.3 Gb and 85.7 Gb raw data for XX,XY and YY,respectively.The average read lengths were 24.29 kb,24.15 kb and 24.67 kb,respectively.The sequencing data were assembled into 741.2Mb,727.2 Mb and 750.0 Mb with contig N50 lengths of 13.19 Mb,13.81 Mb and 15.96Mb,respectively.Additionally,116.4 Gb XX and 174.9 Gb Bionano data,and 80.6 Gb and 80.4 Gb Hi-C data were then used to assemble contigs into scaffolds and further into chromosomes,resulting in 738.9 Mb,723.9 Mb and 739.1Mb with 29chromosomes and scaffold N50 lengths of 28.04 Mb,28.08 Mb and 27.22 Mb for XX,XY and YY,respectively.Further,the genome completeness of these assemblies was estimated using BUSCO based on the Actinopterygii database.The assessment demonstrated that the BUSCO value were 92.0%,90.7%and 92.3%in the XX,XY and YY assemblies,respectively,suggesting high quality of the generated assemblies.The XX genome was annotated and contained 40.12%repeat sequences.A total of 22,965protein-coding genes were predicted based on de novo,homology searching and transcript-based methods,and 22,519?98.06%?genes were functionally annotated in at least one of the protein databases.Average gene length and coding sequence?CDS?length were 16,897 and 1,689 bp,respectively.Furthermore,approximately 93.1%complete BUSCO genes were successfully identified.All these results demonstrated that our genome assemblies and annotation are of high integrity and quality for further analyses.2.Location of the sex chromosome and sex determining?SD?region in Southern catfish.A female pool?41 female individuals?and a male pool?110 male individuals?generated using the offspring by crossing XY male with XY neofemale were used for DNA resequencing.After filtering and quality control of the raw data,51.2 Gb and 38.7 Gb of clean data were obtained,and the number of reads were342,503,436 and 270,953,302,respectively.The clean data were mapped to the Southern catfish XX genome with the mapping rates of 95.90%and 98.96%,and the coverage depths of 58.94x and 46.06x,respectively.Based on the allignments,2,421,301 and 2,468,613 SNPs were obtained in female pool and male pool,respectively.Genome-wide F_ST analyses revealed that chr.24 is the Southern catfish sex chromosome.The SD region was from 3.74-5.91 Mb in X chromosome and 3.75Mb-6.13 Mb in Y chromosome.3.Screening of the sex-linked molecular markers in Southern catfish.Firstly,142,759,127 male k-mers-60 were generated and aligned to the female reference genome.Those k-mers-60 which matched to the female reference genome were discarded.The remaining 21,024,303 male k-mers-60 could not map to the female reference genome,indicating that they might be specific for male individual.Secondly,we assembled those unmapped k-mers-60,and obtained 8954 male-specific contigs.Lastly,the clean reads of female-pooled library were aligned to those assembled contigs.Most of the assembled contigs could be mapped by the clean reads of female pooled library.Only 38 contigs were not mapped by any read of female pooled library.Therefore,we hypothesized that these 38 contigs might be the Y-specific fragments.Specific primers were designed either within the Y-specific sequences to amplify Y-specific bands?male specific markers?or within the consensus flanking sequences at the upstream and downstream of candidate Y-specific fragments to amplify different sized X or Y bands?co-dominant markers?.Three rounds of PCR screening were conducted in different populations of Southern catfish.Finally,eight sex-linked molecular markers were obtained.Seven of them were male-specific markers,and one?marker-8?was a co-dominant marker which could simultaneously distinguish XX,XY and YY individuals.Application of these sex-linked molecular markers in genetic sex identification of different populations of Southern catfish resulted in accurate and reliable results.Genomic alignment analyses revealed that the eight sex-linked molecular markers were all located on SD region of chr.24,confirming that chr.24 is the sex chromosome of Southern catfish.Based on these molecular markers,we constructed a marker-assisted selection?MAS?method for the production of XX all females and YY supermales.4.Identification of the candidate sex-determining gene in Southern catfish.By comparing the X and Y chromosomes,we found that there was a Y-specific insertion in the SD region.Annotation based on homologous search showed that this insertion contained a duplicate of amhr2,named amhr2y.The amhr2y gene showed 81%identity with amhr2 in the aligned coding sequences and 70%in the aligned amino acid sequences.Tissue distribution and gonad transcriptome analyses indicated that it was expressed exclusively in the testis but not in the ovary and other tissues.Fluorescence in situ hybridization?FISH?showed that its expression started from the critical period of molecular sex determination.And it was expressed both in somatic and germ cells of the testis at 5,10,30,and 120 dah?day after hatching?.These results suggested that amhr2y might be the candidate sex-determining gene of Southern catfish.In summary,taking advantage of second-and third-generation sequencing technologies,we constructed the high-quality chromosome-level genome assemblies for the XX,XY and YY Southern catfish.Based on whole genome sequencing and resequencing data,we identified the SD region in chr.24,isolated eight sex-linked markers located on the SD region and established the MAS technique.The candidate sex determining gene,amhr2y,was also identified.The results of this study laid the foundation for the genetic analysis of the economic traits,the study of sex control breeding and molecular mechanisms of sex determination in Southern catfish.