Investigation On The Formation And Genetic Traits Of Hybrids Derived From Female Blunt Snout Bream(Megalobrama Amblycephala,(?))×Male Rare Minnow(Gobiocypris Rarus,(?))

Distant hybridization can transfer the genome from one species to another and result in changes in the phenotypes and genotypes of the offspring.It plays important roles in genetic breeding and biological evolution.Previously,the offspring derived from distant hybridization mostly had even number of chromosomes,there were few reports on the biological characteristics of fish hybrids with odd number of chromosomes.Blunt snout bream（Megalobrama amblycephala,BSB,2n=48）and rare minnow（Gobiocypris rarus,RM,2n=50）,are two cyprinidaes with relatively distant relationships and differences in biological traits.In the present study,we obtained the hybrid F₁（BR,2n=49）with odd number of chromosomes by the distant hybridization of female blunt snout bream and male rare minnow.The morphological and molecular genetic characteristics of the hybrid F₁ were systematic investigated.The main results are as follows:1.Morphological characteristics,DNA and number of chromosomes were comparative analyzed among two parents and the hybrid F₁.The results showed that the main measurable traits（including caudal peduncle length/caudal peduncle height,head length/head height,body length/body height）and countable traits（including number of fins,lateral scales,upper lateral scales）of F₁ hybrid were between the parents,reflecting the characteristics of hybridization.In addition,some countable characteristics（for example lower lateral scales and number of anal fins）and measurable characteristics（whole length/body length,caudal peduncle length/caudal peduncle height）were more biased towards the paternal parent than those in the maternal parent.Flow cytometry and chromosomes preparing from blood cell culture showed that the number of chromosomes of the hybrid F₁ was 49,indicating the hybrid F₁ was an allodiploid hybrid fish（2n=49）.2.Structure and sequence characteristics of the 5S rDNA gene were comparative analyzed among the blunt snout bream,rare minnow and hybrid F₁.The results showed that three bands of 5S rDNA were amplified in the hybrid F₁ and the sequence lengths were 188bp,220bp,and 433bp,respectively.The type BR-188bp of 5S rDNA was inherited from the maternal parent BSB（188bp）,the similarity was 99.1%between BSB and the hybrid F₁.The type BR-220bp of 5S rDNA was inherited from the paternal parent RM（223bp）,the similarity was 98.2%between RM and the hybrid F₁.The type BR-433bp of 5S rDNA in the hybrid F₁was a chimeric sequence,it is structurally a repeat of the BR-220bp type,the first 220bp sequence was derived from BSB,among them 188bp sequence was entirely derived from the BSB-188bp,28bp sequence more at the end was consistent with the corresponding region of RM-223bp,and the remaining 213bp sequence was derived from RM（223bp）.These results indicated that heritability and variability were co-exist after distance hybridization.3.The whole length sequence of mitochondrial genome in the hybrid F₁ was amplified by PCR,and the structural and phylogenetic evolution were analyzed.The results showed that the total length of mitochondrial DNA in hybrid F₁ was 16624 bp,which contained 22 typical tRNA genes,13 protein-coding genes,2 rRNA genes,and non-coding regions（control region and light chain replication initiation region）.The content of A+T（55.96%）in mitochondrial DNA was higher than G+C（44.04%）,indicating the mitochondrial DNA had AT base preference.Besides,TAS,CSB,AT repeats and other structures were identified in the D-loop region.The sequence identity of mitochondrial DNA between the hybrid F₁ and the maternal parent was 99.7%,which showed a lower similarity（84.7%）between the hybrid F₁ and paternal parent.The results suggested that the mitochondrial genome of hybrid F₁ was maternal inheritance.4.Comparative analysis of orthologous genes from liver transcriptome among the parents BSB,RM and its hybrid BR,the results showed that there obtained 6664 co-expressed unigenes,of which approximately 15.52%were chimerics.Sanger sequencing was performed and approximately 86.67%（13/15）genes were confirmed chimeric,indicating non-homologous recombination was produced during heterologous chromosome combination.In addition,the expression level of two growth related genes igf1 and ghr in 6-month-old parents BSB,RM and its hybrid BR were detected by qRT-PCR,respectively,the results showed igf1 had a significant higher expression level in hybrid than that in two parents（p<0.05）,while ghr showed a significant higher expression level than paternal RM,and a lower level than maternal BSB.In the present study,allodiploid hybrid was successfully obtained from blunt snout bream（（?））×male rare minnow（（?））.There showed a heritability and variability at the molecular level such as 5S rDNA,mitochondrial genome and orthologous in hybrid F₁.In addition,with it odd number of chromosomes,allodiploid hybrid has 49 chromosomes,and the number of chromosomes derived from the paternal parent is one more than that from the maternal parent.Results of this study provided an ideal experimental material for subsequent exploration that the coordination of the chromosome level（parental effect,dose compensation,silence etc.）after distant hybridization.