Topmouth Culter (Culter Alburnus) All-female Breeding System Establishment And Evaluation

Topmouth culter(Culter alburnus Basilewsky)is one of the most important freshwater fish species cultivated in China,possessing the characteristics of fast growth and tender meat.Since the breakthrough and improvement of artificial reproduction technique in 1999,breeding technology,feed development,and product processing involved in topmouth culter have been rapid development,and forms an industry with an output value of more than 100 million yuan per year in the regions of Zhejiang,Jiangsu and Shanghai.However,at present most topmouth culter seedlings are produced from wild species that not selected,and the advantage of its potential genetic improvement has not yet been fully mined.In addition,the operation blindness of breeding,introduction,hybridization leads to appearance the phenomenon of germplasm decay or confounding,including reduced growth,miniaturization and early sexual maturity.However,the C.alburnus has intolerant to stress,prone to parasitic diseases,and a 2-3 years period of sexual maturity,which makes the breeding cycle long,the cost of breeding high,and the management difficult.As a consequence,the actual breeding progress is quite slow and the effect is not obvious.Moreover,the female topmouth culter grows larger and faster than the males,and monosex cultivation is beneficial to increase the yield and economic benefit.This study aims at the practical problems or demands of topmouth culter breeding,such as the long cycle of breeding and great potential of monosex cultivation,to establish a technique system for allfemale breeding.A series of related studies have been carried out,including induction,identification and sex determination type in gynogenetic offspring,androgen-induced pseudo-male fish preparation and its gonadal development and sperm quality assessment,sex specific markers identification and verification,preparation and verification of all-female progeny of topmouth Culter,cloning and epigenetic modification of sex-related genes.The main results and conclusions are as follows: 1.Induction,identification and sex-determining type analysis of gynogenetic topmouth culterMeiotic gynogenesis in topmouth culter(Culter alburnus)was induced by activation of freshly collected eggs with diluted and UV-irradiated heterologous sperm from common carp(Cyprinus carpio),followed by a cold shock.The morphological characteristics,karyotype,gonadal development and genetic diversity of the gynogenetic population were analyzed with normal diploid population as control.The results showed that the average induction rate and survival rate of gynogenetic topmouth culter was 10.15% and 21.31%,respectively.The offspring from gynogenesis induction were similar to those of the normal fish,and it was difficult to distinguish them by countable and quantitative traits(P>0.05).The karyotype of the offspring was 2n=16 m + 26 sm + 6 st,NF=90,which was consistent with their female parent.12 microsatellite primers were used to analyze genetic diversity on normal population,G1 and G2 gynogenetic population.Our results showed that the average allele number of these three populations were 1.3333,3.9167 and 3.1667,respectively,and the polymorphism of gynogenetic population was significantly lower than that of normal population,suggesting that meiotic gynogenesis was an effective way to promote gene homozygosity.The specific bands from the male parent carp were not found by RAPD analysis,and the order of the genetic distance in the three populations was meio-G1(0.1982)>control group(0.1540)>meio-G2(0).The gonads of 174 gynogenesis individuals among different ages were observed,and all of which were female individuals with normal ovaries,indicating that the sex-determining type of topmouth culter was XX-XY.2.Identification and characterization of reproduction-related genes and pathways in topmouth culter brain and ovary tissues by RNA-seqIn this study,RNA-seq technology was applied to discover reproduction-related genes and pathways in topmouth culter.Brain(including pituitary)and ovary tissues in 10 gynogenesis fish were used to de novo assemble a comprehensive transcriptome.A total of 58,741,192(28,916,206 in brain and 29,824,986 in ovary,respectively)reads with a Q20 percentage average 98.3% were generated.16,268 SSRs and76,795 putative SNPs and were identified from brain and ovary transcriptoms.Functional analysis identified 2479 and 2605 unigenes in brain and ovary were assigned to reproductive process subcategory,in addition,2660 and 2845 unigenes were assigned to reproduction subcategoriy.Significantly,6 reproduction-related pathways(MAPK signaling pathway,Neuroactive ligand-receptor interaction,GnRH signaling pathway,Melanogenesis,Oocyte meiosis,Steroid biosynthesis)with 34 reproduction-related genes were identified.a qPCR assay using 16 candidate and reference genes which was selected through DGE approach was validated for further detailed expression analyses.They were mainly expressed in brain and ovary tissues and these results were in accordance with those from transcriptome analysis.In addition,the accuracy of the transcriptome data was also verified by detecting the differences in the expression of FSH,NPY,3beta-HSD and PGR genes in normal female ovaries,normal gynogenetic ovaries and malformed gynogenetic ovaries.3.The study of 17?-methyltestosterone-induced reversal on gynogenetic topmouth culter17?-methyltestosterone(17?-MT),a major male-inducing hormone,has been successfully transformed a variety of female fish into a fish that the phenotype is male and the heredity is female.In order to illuminate the effects of 17?-MT on physiological changes of gynogenetic topmouth culter,three different experiments were carried out.Five concentrations(0,10,25,40 and 60 ?g/g)were set up in experiment I and three different treatment and recovery methods of above five concentrations were studied in experiment II.In experiment III,fish were fed with increasing concentrations of 17?-MT(40-140 ?g/g)in outdoor pond.Our results showed that higher concentration(40-60 ?g/g)17?-MT inhibited the growth after long-term feeding,and the mRNA expression of FOXl2?CYP19a were decreased while the mRNA expression of AMH,DMRT1 were increased by qRT-PCR.SOD,E2,T,GH and T4 level in 40 ?g/g and 60 ?g/g groups were significantly increased when compared with those in other groups at 40 d after treatment.Primordial gonads were observed by histological section in all 17?-MT treatment groups at 110 d in experiment II,while all detected gonads developed into testis at same day in experiment III.These results showed that induction with 25-40 ?g/g 17?-MT and treatment within 80 days could reduce the inhibition of fish growth during the process of sex reverse.And hormone treatment for 40-60 days(56-76dpf)is the most sensitive period for exogenous hormone response in gonads of gynogenetic topmouth culter.In addition,the high-efficiency induction rate was obtained by using outpond feeding strategy,and the endocrine mechanism of 17-MT induction and recovery was also studied.It would provide the support of reserved parents and theoretical basis for all-female breeding.4.Physiological-male sperm quality evaluation of gynogenetic topmouth culterIn the breeding process of all-female fish,the sperm quality of pseudo-male is especially important.Here,sperm quality evaluation indexes,such as fecundity,physiological characteristics,biochemical characteristics,ultrastructure,were used to assessment pseudo-male sperm quality.Our results showed that pseudo-male sperm had lower fertilization rate,hatching rate,72 h larva survival rate and preservation time at room tempreture,but existed higher malformation rate of larva comparing with common male sperm.The vitality change under different salinity and pH were basically similar between pseudo-male sperm and common fish sperm,which the optimum salinity was 0-4 and the optimum pH was 5.5.There existed a correlation between the content of ATP enzyme and sperm fecundity,and the ratio of cholesterol to phospholipid content(c/p)was also related with sperm fecundity.More phenomenon of mitochondrial damage and mitochondrial membrane expansion were found in the pseudo-male sperm,which resulted in the diffrence in fecundity.5.Identification sex-specific molecular markers in topmouth culterIdentification sex-linked markers of topmouth culter is very important for the development of all-female breeding and the study on the mechanism of sex determination.RAD-seq and AFLP were used to isolate sex-specific markers in topmouth culter.The results showed that the number of male and female specific RADseq loci were 12 and 7,respectively,but no effective loci was found in the subsequent verification process.A sex-specific marker(E1M9),which was present in the males but absent in the females,was identified from 192 AFLP primers combination.A malespecific marker that obtained from AFLP will provide a powerful experimental basis for judgement the sex of topmouth culter seedling.6.Preparation and genetic characteristics analysis of all-female topmouth culterIn this study,we used two generation of gynogenesis G2 as female parent and the pseudo-male fish induced by 17?-MT as male parent to breed all-female topmouth culter.Gonadal development,growth rate,and genetic characteristics were tested between all-female and common topmouth culter.Our results showed that the fertilization rate(68%)and hatching rate(33%)of all-female fish were relatively lower,but embryo development was normal.It was found that the growth rate of all-female fish was 15.57% faster than that of normal topmouth culter,indicating that the growth advantage of all-female fish was obvious.Genetic structure characteristics was analysis between all-female and common fish by SSR,and the examined results of two population showed that the average alleles was 2.583 and 6.917,the average He was 0.820 and 0.664,the average PIC was 0.505 and 0.729,and the genetic similarity was 0.737 and 0.567,which indicated that the polymorphism of all-female population was significantly lower,the genetic similarity was high,and the genotype consistency was good.No testis tissue was found in the tissue sections of all female offspring in all examined groups,and the female rate of all female fish progeny was 100%.The allfemale had the similar result to common female fish by E1M9 amplification of AFLP analysis,but the marker was not successfully converted to the SCAR marker.In addition,the expression levels of female-specific genes(CYP19 and FOXL2)were significantly higher in the all-female population and gynogenetically developed topmouth culter than in the gonads of fish treated with 17?-MT at the same time(P<0.05),while the opposite trend was found in male-specific genes(DMRT1 and AMH).7.Analysis sexual dimorphism of topmouth culter by WGBS and RNA-seqIn order to reveal the mechanism of sex determination and differentiation in topmouth culter and develop the sex-control breeding technology,WGBS on topmouth culter gonadal tissues was performed using the carp genome as the reference genome,and the results showed that the promoter methylation was negatively correlated with the gene expression level.In addition,the role of DMRT1,SOX9,CYP19 a genes in gonadal differentiation were analyzed based on RNA-seq data.The relative expression levels of SOX9 a,DMRT1,CYP19 a mRNA in different tissues were analyzed by quantitative real-time PCR.The results showed that SOX9 a and DMRT1 were highly expressed in testes,while CYP19 a were highly expressed in ovaries.In addition,promoter CpG methylation modification of SOX9 a,DMRT1,CYP19 a were analyzed using bisulfite sequencing in topmouth culter gonad,which observed inverse relationship between methylation levels and their gene expression.These results suggested that promoter CpG methylation can regulate sexual dimorphic expression of sex-related genes,and epigenetic modification may play an important biological role in the gonad development of topmouth culter.In summary,we successfully established an all-female breeding system of topmouth culter using artificial gynogenesis and hormone-induced reversal techniques.Moreover,the all-female offspring had obvious growth advantage and all were female individuals.In addition,we built a sex-identification technique for all-female progeny,discussed the sex-determining types and differentiation mechanism of topmouth culter,and carried out epigenetic regulation of sex-related genes based on the high-throughput sequencing data,such as RAD-seq,RNA-seq and WGBS.These results will provide new clues and ideas for demonstration the mechanism of sex determination and differentiation in fish,and also will provide important technical reference for all-female breeding of topmouth culter in practice.