Analysis Of Fertility From The Autotriploid And Allotriploid Fish

The definition of distant hybridization is let two species with relatively distant relatives to hybridize,usually,which made the hybrid progeny possess fine traits compare with their parents,and also can increase the genetic recombination and mutation frequency of the offspring.In the distant hybridization of red crucian carp(Carassius auratus red var,RCC,♀,2n=100)and blunt snout bream(Megalobrama amblycephala,BSB,♂,2n=48),In which part of the F1 female and male individuals are able to produce a number of diploid eggs(2n=100)and diploid sperm(2n=100),screening out the male and female individuals to self-cross,in F2,bisexual generative autotetraploid fish populations were formed,Breeding and passage of F2 were carried out,and finally we had been obtained autotetraploid hybrid strains with stable genetic characteristics.The male autotetraploid fish(F8,4n=200)were hybridized with female red crucian carp(2n=100),we obtained autotriploid fish(3n=150).At the same time,by using autotetraploid fish(♂,F8,4n=200)and common carp(Cyprinus carpio L,♀2n=100)to hybridized,we obtained the allotriploid fish(3n=150).In the breeding period,the 2-year-old female autotriploid fish could produce mature eggs hybridize with RCC,which could form the offspring of diploid and tetraploid.In addition,the gynogenesis of autotriploid fish had been formed surviving offspring in fish breeding.These evidences confirmed the female autotriploid fish was fertile.Previous studies generally agreed that triploid fish were sterile,because of the homologous chromosomes of the triploid fish had been disordered in the meiosis period,but the female autotriploid fish were fertile,this provided a good material for exploring the fertility of triploid fish.In order to explore the molecular mechanism of triploid fish fertility,in this study,we compared and analyzed the reproductive characteristics of female autotriploid fish and female allotriploid fish.The main contents of this study were:1.Respectively,we selected the female autotriploid and allotriploid fish(It takes two years for their gonadal development to mature),and they were reared 1-2 years.The two groups of fish ovarian tissue for continuous section tissue were observed,the results showed that in the autotriploid and allotriploid fish ovary paraffin tissue slices,almost the oocytes was in stage I in the non-breeding period.In the breeding period,in the autotriploid fish ovarian tissue sections,a large number of oocytes distributed in II,III,IV phase,it showed that oocytes develop normally.However,in the allotriploid fish ovary paraffin tissue slices,almost all of the oocytes remained in the I phase,these cells did not see the formation of yolk,there are also had very few cells that had been degraded in II,III time periods,which these characteristics indicated that allotriploid ovarian development was abnormal.2.Female autotriploid and allotriploid fish ovary tissues were sequenced for transcriptome,which in order to compared and analyzed of their transcript difference.In transcriptome sequencing data,respectively,we received 46,869,254 and 43,654,554 clean reads from autotriploid and allotriploid fish.After the sequence assembled,we obtained 137,083 and 99,836 unigenes,we received 33,692 and 28,755 annotations of genes.Differences in the transcriptome of the two species were analyzed,the results showed we predicted 33,692 genes in the autotriploid,there were 11,556 differentially expressed genes.We predicted 28,755 genes in the allotriploid fish,which had 6,729 specific genes.8,311 genes were highly expressed in autotriploid fish compared with the allotriploid fish,7,382 genes did relatively low expression.At the same time,230 genes were specific-expressed in autotriploid fish,and 65 genes were specific-expressed in allotriploid fish.We performed COG,GO and KEGG annotation analysis,the results showed that there was no significant difference in genes functional classification between the transcriptome of two kinds of the fish,the differences of some genes expressed and the differences of gene structure maybe are the keys to the difference in the reproductive properties of the two species.3.By comparing and screening the transcript sequence database with KEGG,COG,NR database,16 differentially expressed genes(DEGs)associated with ovarian development were selected and 10 differentially expressed genes(DEGs)associated with meiosis were explored.Partial sequences of two genes cDNA were respectively cloned,the structural differences were compared between the base sequence and the amino acid sequence.The result showed that two fragments of fish have structural deletions,single base mutations,which leading to the difference of amino acids translated.These results may be able to certificate the structure and function of the DEGs were associated,we had reason to believe the difference of gene expressed was related to changes in gene structure.The eight representative DEGs related to ovarian development were verified by RT-PCR and q-PCR,we found that PAZ,Lhx8,cdc2,nfr,stAR were up-regulation of expression irn the autotriploid fish,however,C1q,cdc28,pla2 were down-regulation of expression.The function of these genes were analyzed,we found that they related to the development of primary oocytes,the synthesis of steroid hormones,egg formation,promotion of ovulation and so on.Six representative DEGs related to meiosis were validated by fluorescence quantitative PCR,it displayed most of the genes are upregulated in atotriploid fish ovaries.In summary,the molecular mechanism of female fertile autotriploid fish may be related to the up-regulation and down-regulation of expression of some ovarian-related expression genes,meanwhile,because of the genes associated with meiosis were upregulated in ovary of autotriploid fish,we speculated that the up-regulation of gene expression may reduce the effect of homologous chromosomes pairing disorder result in the sterility of fish during meiosis.This research through the organization level and gene level to explored autotriploid fish fertility issue,and that provided a reference and accumulated data for study the molecular mechanism of the fertility of triploid fish,which also laid a theoretical foundation for the breeding and reproduction of fish.