| The zig-zag eel(Mastacembelus armatus)is a very important economic fish which belonging to the genus Mastacembelus,family Mastacembelidae,and order Symbranchiformes,and its natural distributed in India,Pakistan,Nepal,Western Africa,Southern China,and other parts of South East Asia.In recent decades,sexual dimorphism of zig-zag eel has been gradually concerned during the development of artificial reproduction and breeding technology,and sexual imbalance in the artificial breeding was brought seriously trouble to farms.In addition,the sex determination mechanism of zig-zag eel is still unclear,which has seriously affected the large-scale breeding efficiency of this species.In this study,the third-generation experimental techniques were used for the genome and gonad development physiological mechanisms of zig-zag eel from genomics,transcriptomics,cytology and histomorphology,which provided a theoretical basis for revealing the sex determination mechanism of zig-zag eel.In this study,by means of tissue sections and morphological analysis,the sex differentiation process of cultured zig-zag was tracked and the sex discriminant equation was established;Secondly,the 2b-RAD-seq technology was used to screen the gender-specific fragments of zig-zag eel,and the gender-specific SNPs molecular marker method was established;Then,the haplotype-resolved genome of zig-zag eel was assembled,annotated and analyzed by using the third-generation sequencing and assembly technology,and the sex determination genes and heterochromatin phenomenon in sex linked region(SLR)were studied and analyzed;Finally,the sex determination genes in SLR region were analyzed and verified by RNA-seq,scRNA-seq,in situ hybridization,gene cloning and fluorescence quantitative qpcr.The main results are as follows:(1)In this study,the external morphological characteristics and gonadal histomorphology of zig-zag eel at different gonadal development stages were tracked and analyzed.The intersex population of cultured zig-zeg eel was found for the first time in the world;There was 18 external morphological characteristics and 15 standardized morphological characters obtained from 300 male,female and intersex zig-zag eels with2-year-old.According to these data,sex discriminant equations were established.The comprehensive discriminant accuracy rate reached 97.0% and the accuracy rate increased by 1.4 times,which provided a simple and efficient sex discrimination technology for the breeding and production of zig-zag eel.(2)In this study,two male-specific tags and two SNPs were identified by 2b-RAD sequencing and verified by PCR amplification.Two female specific deletions with 281 bp and 4938 bp and a male-specific deletion with 156 bp were identified by sequence comparison between X-specific and Y-specific sequences obtained by genome walking(Gen Bank ID:GCF＿900324485.2)from two 2b-RAD tags.Two sets of primers including Y-specific and XY-shared primers were designed based on these identified deletion regions to distinguish the genetic sex of zig-zag eel.The specificity of these primers was confirmed in 81 offspring of one F1 family.Furthermore,two male-specific SNP primers,which specifically amplify one band only in the males,were also designed.These data indicate that zig-zag eel possesses a male heterogametic XY sex determination system.The male-specific markers screened are accurate and universal,which will play an important role on the production of all-male populations of zig-zag eel for aquaculture in the future.(3)The third-generation HiFi sequencing and Hi-C assisted assembly technology completed the genome sequencing with chromosome-level assembly,annotation and comparative genomics analysis of wild male zig-zag eel.(1)HiFi data of 39 G was obtained,and the average length of HiFi reads is 13.34 kb,the error rate is only 0.089%,and the average the largest phased blocks span 99.6%.The partitioned haplotype-specific HiFi reads were assembled into two haploid genomes(hap-x and hap-y).The genome sizes of hap-x and hap-y were 582.0 Mb and 585.6 Mb,which were similar to the genome size of diploid(595.7 Mb)respectively.The hap-Y genome seems to have a better quality in which all of the 24 chromosomes have a gap number of less than 10,with a mean gap number of5.5,while in the hap-X genome the mean gap number is 6.4.Therefore,the hap-Y genome was selected as the main object,with a total of 22069 annotated genes;(2)Genome-wide comparison between the haploid genome of zig-zag eel and the genome data of other seven species showed that zig-zag eel had the closest genetic relationship with ricefield eel(Monopterus albus),and the differentiation time of them was estimated to be between 36 million years;(3)The results of chromosome morphological observation and karyotype analysis showed that zig-zag eel is diploid(2n = 48),of which are classified 5 pairs metacentric chromosome,3 pairs submetacentric chromosome and 16 pairs telecentric chromosome;(4)Two satellite sequences of cen-524(524bp)and tel-190(190bp)were found from the annotation of repeat sequences.By using fluorescent in situ hybridization(FISH),they were further determined that Cen-524 was centromeric sequence and tel-190 was telomere sequence;(5)A 7 Mb sequence on the Y chromosome was found to be associated with SLR,which contained 18738 male-specific SNPs.According to the distribution of SNPs,the SLR was divided into two regions:R1 and R2,in which the repeat content of R1 was 20% and R2 was 50%.The results of CUT & Tag showed that there were also significant differences in the H3K9me3 modified and widely distributed chromosome active components between R1 and R2,which was also called heterochromatin.The R1 was near centromere heterochromatin,which drove the differentiation of SLR in the genome;(6)Two candidate SD genes of syce3 and hmgn6 were found through the combined analysis of genome and transcriptome.The syce3 was specifically highly expressed in testis and hmgn6 was highly expressed both in testis and ovotestis;(7)It is found that there is a wide range of chromatin differentiation in the X and Y type chromosomes of zig-zag eel.The heterochromatin region is mainly concentrated around the centromere,and its length is about 4.0 Mb,and has a very low recombination rate.(4)A sex-determined candidate gene dmrtb1 was screened from the gonad RNA-seq of zig-zag eel,which had 1194 bp full-length transcript and 870 bp open reading frame and 289 encod amino acids.The gene structure and expression of dmrtb1 were further verified and analyzed by gene cloning,q PCR and FISH respectively.The results showed that the sequence similarity of DM domain was less than 45 % compared with other DMRT family genes.In the testis,dmrtb1 were specifically high expression in the seminiferous lobules containing cysts with germ cells,especially in spermatids,spermatozoa,spermatogonia,and spermatocytes,but no expression in ovary.The results of scRNA sequencing showed that spermatocyte and oocyte marker genes were both enriched in the same gonadal cell population,indicating this cell population might have the potential of bisexual differentiation.The expression time of dmrtb1 is related to gonadal development,indicating that dmrtb1 plays an important role in the process of sex differentiation of zig-zag eel. |
