Establishment Of Hybrid Strains Between Blunt Snout Bream And Topmouth Culter And Their Genetic Characteristic Research

Distant hybridization can introduce a whole genome from one species to another,which changes the gene regulation and expression in the hybrid.If distant hybridization generated fertile heterogenous hybrid,it would provide excellent germplasm banks for genetic breeding.Usually,the first generation(F1)fish hybrids between genus or above genus were bisexual infertile or single gender sterile(female or male infertile).Based on the formation of diploid and triploid hybrids of blunt snout bream(?)× topmouth culter(?)and diploid hybrids of topmouth culter(?)× blunt snout bream(?),the gonad tissue slice,scanning electron microscopy for sperm,back crossing and self crossing experiments proved that both the two kinds of the first generation of diploid hybrids of blunt snout bream and topmouth culter were completely bisexual fertile.The second generation was first produced by self-mating of diploid hybrids in the first generation,respectively.It is very interesting that the second generation hybrids are also bisexual fertile.The fertility of the hybrids was confirmed by gonad tissue slice and scanning electron microscopy for sperm.At this point,two heterogenous fertile hybrid strains were obtained.In this study,the main biological traits of the two heterogenous fertile hybrid strains such as appearance,ploidy and fertility were investigated,and the genetic relationship of 5S rDNA and 45 S rDNA between the two strains and their origin parents were also studied.The main research contents were presents as follow:1.By gonad tissue slice and scanning electron microscopy for sperm,bisexual fertile diploid hybrids of blunt snout bream(?,BSB)×topmouth culter(?,TC)(short for 2nBT1)and bisexual fertile diploid hybrids of topmouth culter(?)× blunt snout bream(?)(short for 2nTB1)were found in the first generation(F1)hybrid,the second generation(F2)of 2nBT1(short for 2nBT2)and F2 of 2nTB1(short for 2nTB2)were successfully obtained by artificial reproduction and self crossing during the reproduction seasons,respectively.In addition,a new kind of hybrid(short for 2nBTB)with the hybrid vigor such as faster growth rate,stronger stress resistance and better meat quality were produced by back crossing between female 2nBT1 and male BSB and the main biological traits of 2nBTB were also studied.The formation.of the two generations hybrid provided good germplasm resources for future genetic breeding and the gain of the 2nBTB showed good prospect for production of new kind of strains for aquaculture with the two kinds of hybrid strains.2.The main biological traits of 2nBT2 and 2nTB2 such as appearance,ploidy,fertility were investigated and comparatively studied with original parents,2nBT1 and 2nTB1.The results show that(1)in appearance,the major appearance characteristic of 2nBT2 and 2nTB2 were not significant different with 2nBT1 or 2nTB1,respectively,and keep hybridity compared with BSB and TC;(2)in ploidy,all the detected samples in the 2nBT2 and 2nTB2 were diploid with 48 chromosomes and triploid or tetraploid individuals were not found in the F2 hybrids;(3)in fertility,gonad tissue slice and scanning electron microscopy for sperm showed that both 2nBT2 and 2nTB2 were bisexual.Thereby two heterogenous fertile hybrid strains were obtained.The offspring of distant hybridization between BSB and TC showing low reproductive isolation compared with many other distant hybridization offspring suggested the genetic relationship between BSB and TC were close in evolution.3.The internal transcribed regions(ITS)sequences of 45 S rDNA of bleeker's yellow tail,common carp,red crucian carp,Japanese crucian carp,BSB and TC were cloned and comparatively studied.The results showed that the ITS sequences similarity between BSB and TC were high and the molecular phylogenetics analysis suggested the genetic relationship between BSB and TC were close and the breeding potential of hybrids might be related to the genetic relationship.4.After gaining the ITS sequences of BSB and TC,we found that there was one kind of ITS 1 with the length of(322bp)in BSB,one kind of ITS1(366bp)in TC.In order to find the genetic mark between BSB and TC and their hybrids,a set of primers based on the conservation of 18S and 5.8S in fish(18S P4F,'-AGTCGTAACAAGGTTTCCGTAG-3',P4A-5'-ATC(A/G)ATGTGTCCTGCAATTCAC-3')were designed and synthesized to amplify 45 S rDNA internal transcribed spacer 1(ITS1)directly from genomic DNA.The results showed that the length of PCR fragments between BSB and TC were obvious different in 2%gel electrophoresis map.So it could be used as a simple genetic mark to exam the hybridity of hybrid progeny of BSB and TC.The ITS1 sequences of BSB,TC,2nBT1,triploid hybrid of female BSB and male TC(short for 3nBT1),2nBT2,2nTB1 and 2nTB2 were amplified.All the two kinds of ITS 1 from parents were seen in the F1 hybrids,proving the hybridity in the 2nBT1,3nBT1 and 2nTB1.However,in the F2 hybrids,including 2nTB1 and 2nTB2,most individuals inherited both the ITS1 of BSB and TC while some lost the ITS1 of one progenitor,indicating that the F2 generation may appear genetic mutation,recombination or differentiation.The genetic change in F2 generation expanded the genetic diversity and may enrich the germplasm resources.5.The 18S rDNA sequences were cloned in BSB and TC.The similarity of the 18S rRNA genes sequences between BSB and TC were almost the same,except a single nucleotide polymorphism(SNP)at position 308 is fixed for cytosine in BSB and thymine in TC.Species-specific copies of rDNA and the corresponding rRNA transcripts were distinguished on the basis of the C308T SNP with direct sequencing of PCR products.Based on the SNP site,the expression of 45S rRNA was tested in BSB,TC,2nBT1,3nBT1,2nBT2,2nTB1 and 2nTB2.The result showed that in the first generation hybrids(2nBT1,3nBT1,2nTB1),neither the 45S rRNA genes of blunt snout bream or topmouth culter were repressed.The nucleolar dominance phenomenon happened in most interspecific hybrids were not observed in the first generation hybrids of BSB and TC.While in the second generation,45S rRNA genes of one progenitor were silenced in some individuals,showing nucleolar dominance;some individuals expressed both the parents' 45S rRNA genes with no nucleolar dominance.These results indicated that nucleolar dominance in the progeny of an interspecific hybrid was not established immediately after hybridization in animal;two or more generations were needed for one progenitor rRNA genes to be silenced in aimmal strain.6.Chromosome mapping of 5S rDNA in BSB,TC,2nBT1,2nBT2,2nTB1 and 2nTB2 were investigated by fluorescence in situ hybridization(FISH)with PCR-obtained 5S fluorescence probes.Four signals were observed in each kind of fish,respectively.The results also showed that two signals(one signal means a 5S rDNA.cluster)were located in the short arm of a big submetacentric chromosome pair,while another two were detected near the terminal region of the short arm of a subterminal chromosome pair in all studied species.In summary,two kinds of hybrid strain were obtained by hybridization between BSB and TC in this study.The formation the two kinds of hybrid strain provide good germplasm resources for future genetic breeding.The close genetic relationship between BSB and TC in molecular phylogenesis may be related to the low reproductive isolation in offspring of distant hybridization between BSB and TC compared with many other distant hybridization offspring.The 45 S rRNA expression researches in the F1 and F2 hybirds reveal the uniparental ribosomal RNA gene silencing(nucleolar dominance)in the progeny of hybrids between BSB and TC may be established by cumulative DNA methylation.The formation of the two kinds of fertile hybrid strains provided good materials for nucleolar dominance research in animal hybrids.