| Fish is the most abundant vertebrates and has the most varieties in the world.It was reported that they were involved in several genome duplication and complex hybridization incidences during the evolution process,which leads to the interaction,fusion and duplication of different genomes and enriched their genetic diversity.Distant hybridization of fish could initiate the occurrence of polyploidy,while both hybridization and polyploidization has important significance for the study of genetic breeding and the evolution of fish.Megalobrama amblycephala Yih,(BSB,2n=48)and Xenocypris davidid bleeker(YT,2n=48)which belong to different subfamily were selected as maternals and paternals,respectively,and two groups of distant crosses between subfamilies were conducted.Ploidies,biological characteristics,molecular genetic relationship,as well as the molecular mechanism of gonad development of the hybrids were investigated,and the major contents and results were as follows:1.Both groups could produce surviving offspring BY and YB.Two different ploidy hybrids,diploid hybrids with 48 chromosomes(2nBY and 2nYB,2n=48)and triploid hybrids with 72 chromosomes(3nBY and 3nYB,3n=72)were found in offspring of BSB and YT.Karyotypes of two different diploid hybrids were 18m+26sm+4s,while two triploids were 27m+39sm+6st.Triploids accounted for 18.92%and 9.66%in embryos of BY and YB,separately,While 11.84%and 8.33%in adults of BY and YB.2.Biological characteristics were analyzed in BY and YB.According to PCA analysis base on the statistics of 7 countable traits and measurable traits,body length/body height(BL/BH),body height/head height and anal fin rays could serve as the morphological marker to distinct diploid hybrid,triploid hybrid and parents.There are no significant difference between 2nBY and 2nYB,but the body height of 3nBY were higher than that of 2nBY(P<0.05),which was closer to that of BSB.However,the body height of 3nYB were smaller than those of other hybrid(P<0.05),which resembles its maternal YT.In addition,the volume of erythrocytes in triploid were bigger than that in diploids(P<0.05),and the ratio of abnormal erythrocytes nucleus in triploids was higher than diploids and their parents;Flesh content of BY,70.65%,was improved than that of BSB,52.92%;Growth rate of BY increased by 32.15%than the average value of BSB and YT.3.Histological observation of hybrids was analyzed in hybrids.Gonad developments of hybrids were non-synchronous and exhibited abundant polymorphism.2nBY showed three states of sexual differentiation:36.4%ovaries,52.7%testes and 10.9%intersex.The development of ovaries in 2nBY occurred in three types:Type ??Type??Type ?,the development of testes in 2nBY appeared in four types:Type ?,Type ?,Type ? and Type IV,and the hermaphrodites had two types:Type ? and Type ?.In contrast,3nBY was composed of 35.7%females and 64.3%males.Gonad development of female 3nBY displayed two types:Type I and Type ?,and males had three types.:Type I?Type??Type ?..In contrast,2nBY showed three states of sexual differentiation:36.4%ovaries,52.7%testes and 10.9%hermaphrodites.The development of ovaries in 2nBY occurred in three types,the development of testes in 2nBY appeared in four types,and the hermaphrodites had two types.Gonad development of YB were similar with that of BY.2nYB were consit of 44.8%females?37.9%males and 17.3%intersex,while 3nYB has 33.3%ovaries and 66.7%testis.There were no much difference between gonad differentiation of YB and BY,except that no Type II ovaries were observed in 3nYB,but there are type III ovaries and Type IV testis in 3nYB.4.Based on two types of molecular markers Sox-HMG Box and 5S rDNA,the variances of nuclear genes in hybrids were investigated.Sequencing of Sox-HMG DNA fragments confirms the genetic heterozygosity of BY and YB,and meanwhile,indicated that 710bp fragments from YT encountered base deletion of introns in 2nBY,2nYB and 3nYB;Sequencing of 5S rDNA fragments presented the recombination of different 188bp repeat units inherited from BSB and YT,respectively.In addition,there were two types of base insertion in the non-transcribed segments(NTS)of parts of 188bp repeat unit:18bp"TTCAAAAAAAAAAAAAAA" fragments in BSB,and 18bp"GAAAGAAAGAAAGAAAAA" in YT.Most insertions in the 200bp or so fragements of hybrids were "GAAAGAAAGAAAGAAAAA",and only 200bp or so fragments in 3nBY has two types of insertionS?5.Mitochondrial genome sequences(mtDNA)were obtained from all the hybrids 2nBY,3nBY,2nYB and 3nYB.2nBY and 3nBY shares identical sequence,and compared to the maternal BSB,there were 20 base substitution located in 9 different segments including NADH 2,CO I and so on;47 base substitution between 2nYB and YT dispersed in 17 different segments including 16s rRNA,NADH 1,NADH 5 and so on;37 base substitution between 3nYB and YT dispersed in 16 different segments including D-loop,rRNA,ND1 and so on?Amino acid analyisis indicated that base substitution in BY caused only one amino.acid mutation in CO I and one amino acid mutation in Cytb,base substitution in 2nYB leads to one amino acid mutation in ND5 and base substitution in 3nYB caused 4 amino acid mutation in ND2,COX1,ND5and ND6,seperately.The average value of transition/transversion bias R between BSB and YT was 5.078 and the value of R among all the hybrids and parents was 21.224;The value R of between BSB and BY was 280.716;The value of R between YT and 2nYB was 38.665;The value of R between YT and 3nYB was 14.031;The value of R between 2nYB and 3nYB was 10.531.The average value of transition/transversion between hybrids and their parents were much bigger than the value R of between BSBand BY6.Using Illumina HiSeq 2000 technology,the transciptome of Type IV testes in diploid BY was analyzed by high-throughput method.After assembling,102,298 Unigenes were obtained in the whole transcriptome,which are annotated into 5 protein data banks including NR,Swiss-Prot,COG,GO KEGG,and the nucleic redundance data bank(NR)in NCBI.84,558 of Unigenes,accounting for 82.66%of the total number of 102,298,were annotated,and of which,80.7%were homologous to the genome sequences of zebra fish(Danio Rerio).58,230 of Unigenes were aligned to NR,which was the most and occupied 68.86%of the total protein annotation sequences.The numbers of Unigenes annotated to Swiss-Prot,COG,GO and KEGG were 50,451,15,861,38,322 and 41,359,respectively.According to the COG functional classification,34,690 Unigenes could be divided into 25 groups,of which the most enrichment is the general function group,following by groups of replication,recombination and repair,transcription factors etc.Additionally,about 40.43%,41,359 of all the Unigenes were annotated to 259 KEGG metabolic pathways.Moreover,13,768 simple sequence repeats(SSR)were also obtained from the transcriptome,of which 634 belong to compound type.In total,based on the formation of four different types of hybrid offspring,we explored how the distant hybridization and polyploidization effected on the biological characters,nuclear genomes and mitochondrial genomes of fish,and meantime,provide substantial transcriptome data for the research on molecular mechanism of gonad development of hybrid fish.This study not only could serve as guidance for the future work on fish genetic breeding,but also is of important significance to the evolution research on hybrid fish and polyploid fish. |
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