| Aquaculture plays a crucial role in the world’s food supply.Sex control breeding based on sexual size dimorphism in fish and hybrid breeding are two important techniques in aquaculture.Since the morphological differences and chromosome morphology between male and female fish or between hybrids and their parents are too small to be distinguished,genetic identification using molecular markers is required for sex control breeding and hybrid breeding.The male Ictalurus punctatus grows faster than the female and has a lower feed conversion ratio.Breeding an all-male strain is beneficial for increasing the overall yield.The most critical step is to identify YY individuals by molecular markers.However,the currently available molecular markers are designed based on male-specific SNPs and small INDEL,which make it hard to distinguish band differences by PCR amplification and electrophoresis on agarose gel.Thus,more widely applicable and convenient molecular markers for sex identification are required.The family Bagridae is widely distributed in natural waters of China.Among them,the hybrids of Pelteobagrus vachelli and Leiocassis longirostris exhibit superior growth performance and have great potential for aquaculture.Currently there are no available molecular markers for genetic identification between hybrids as well as their parents.Therefore,in this study,comparative genomics analysis was performed to identified polymorphic loci on the genome.Based on these loci,we conducted genetic sex identification of Ictalurus punctatus,as well as genetic identification of Pelteobagrus vachelli,Leiocassis longirostris,and their hybrids using a combination of PCR,LAMP,and other molecular biology techniques.The main results of this study were outlined as the following:1.Rapid genetic sex identification in Ictalurus punctatus based on PCR and LAMPBase on the available XX and YY Ictalurus punctatus reference genomes,as well as RAD-Seq sequencing data,we selected 30 raw sequencing data of RAD-Seq for each sex,and mapped them to the XX genome.Using genome-wide association studies(GWAS),we identified sex-linked SNPs that were enriched on chromosome 4,which was subsequently determined as the X chromosome.On the basis of these sex-linked SNPs,we directly compared the regions enriched in sex-linked SNPs(24.45-24.95 Mb)on the Y chromosome with the corresponding regions(19.5-20 Mb)on the X chromosome and detected several large insertions or deletions between them.Multiple pairs of sex identification primers were designed for these INDELs,and three of the most effective primers(ip1,ip2,and ip3)were identified through PCR screening validation.These primers amplified only a single size fragment in females(418 bp,878 bp,and 274 bp,respectively)but two size fragments in males(524 and 418 bp,878 and 187 bp,and 342 and 274 bp,respectively).Therefore,the genetic sex of Ictalurus punctatus can be accurately identified based on the PCR amplification results.After examination by three different populations using electrophoresis on agarose gel,the accuracy rate reached 100%,suggesting that all three markers could be effectively used for genetic sex identification in channel catfish.To further simplify the genetic sex identification method and meet the needs of field or fish farm production environments,we attempted to apply the loop-mediated isothermal amplification(LAMP)technology,widely used in microbial and viral rapid detection,to fish genetic sex identification.We designed LAMP primer sets targeting X-specific and Yspecific insertion fragments in the previously identified sex-linked INDEL-enriched regions,and obtained a set of LAMP-X primers and LAMP-Y primers through multiple rounds of screening.By testing the amplification results of two primer sets on males and females from different populations,we could accurately identify XX individuals(only LAMP-X is positive)and XY individuals(both LAMP-X and LAMP-Y are positive).Based on this,we developed PCR and LAMP primer sets that could efficiently and accurately identify the genetic sex of Ictalurus punctatus in multiple populations.In theory,both molecular markers developed in this study can be used to identify YY individuals.2.Molecular marker development for genetic identification of Pelteobagrus vachelli,Leiocassis longirostris and their hybrids by comparative genomics analysis.The fish genomes of Pelteobagrus vachelli and Leiocassis longirostris are available,providing abundant resources for comparative genomics analysis.Therefore,we first constructed a phylogenetic tree based on comparative genomics analysis to compare their evolutionary relationships,and estimated that the divergence time between Pelteobagrus vachelli and Leiocassis longirostris was 3.7 million years ago.Additionally,a synteny analysis of their genomes revealed that both species have a karyotype of 2n=52,and their chromosomes showed good collinearity without inter-chromosomal fusion/recombination events.Based on the comparative genomics analyses,we located the gene patj,which exhibited significant differences between the two species.We found a 391 bp INDEL in this gene’s sequence and developed a molecular marker,PVLL,based on this difference.PVLL amplified a 339 bp band in P.vachelli,a 730 bp band in L.longirostris,and both bands in their hybrids.The molecular marker can accurately differentiate the three fish species regardless of sex,and we further validated its effectiveness in different populations.Therefore,we developed an efficient and accurate molecular marker for identifying P.vachelli,L.longirostris,and their hybrids,which could greatly enhance the efficiency of hybrid breeding.Furthermore,based on the results of comparative genomics analysis,we infer that P.vachelli and L.longirostris are closely related and have consistent karyotypes,providing a theoretical basis for successful hybridization between the two species. |
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