Analysis Of Whole-genome Sequence From Equus Parent-offspring Trio Provides Insight Into The Genomic Incompatibilities In The Hybrid Mule(Hinny)

Horses and donkeys are two extremely important species of the Equus.Their common ancestors appeared in the Pliocene period about 4-4.5 million years ago.Until 2.3 million years ago,gene flow from horses to donkeys still occurred.The separation of horse and donkey was completed quickly in a very short time,resulting in dramatic structural variations between chromosomes and a highly similarity between genomic sequences.Crossing of horse and donkey can produce mule(hinny),and the female mule(hinny)even can produce foals.These outstanding features make the horse-donkey-mule a valuable model for studying large the genetic basis of mammal heterocross and the molecular mechanisms of the regulation for heterologous genomes incompatibilities.In order to evaluate the influence of hybridization on the survival ability of mule at the genome level and the transcriptome level,and explore the molecular mechanism by which mules coordinate the evoluted incompatibility in mule genome.First,the Illumina platform was used to sequence the whole-genome sequencing of the whole blood sample from a Equus parent-offspring trio.At the genome level using thoroughbred horse genome as a reference and the parental genome sequence as guidance,we analyzed the genetic diversity of the mule SNP.Combining with deep sequencing method,we alse analyzed the genetic diversity of the mule CNV.The related genes were enriched.Second,combining the Illumina and the PacBio sequencing platform were used to sequence the full-length transcriptome of the whole blood samples from three Equus parent-offspring trio.At the transcript level,we analyzed the structural variation such as mutations,intergenic chimerism and interspecie chimerism.The related genes were enriched.Third,RNA-Seq data was sued to compare the gene expression of horses,donkeys and mules.We analyzed the diversity of expression patterns such as mule-specific expression,parental expression level dominant and non-additive expression.The related genes were enriched.Fourth,with the parental genome sequence as guidance,RNA-Seq data was sued to identify the the SNP markers.The homologous expression bias of the mule transcriptome were analyzed.The following results were obtained after research:1.At the genome level,we identified 31108 MIE SNPs and 799 de novo SNPs by comparative genome analysis in pedigree 1.Gene annotation and functional enrichment analysis of 16987 homozygous MIE SNPs heridated horse,which were found to be mainly related to the "rejection" and 14121 homozygous MIE SNPs heridated donkey,which were found to be mainly related to cancer processes.The mutations occured at the mule genome,which can reduce the heterogeneity between the horse and the donkey genome and increase the fitness of mule.2.Using thoroughbred horse genome as a reference,with the SNP marker as guidance,180 CNVs transmitted from parents were identified based on the depth method.The functional enrichment analysis of related genes of these CNVs was found to be mainly related to the smell traits.Compared with cancer and "rejection"processes,the differences in olfactory traits are generally not fatal,so the normal inheritance of these CNVs may only have a dosage impact.3.Integrating PacBio and Illumina sequencing data,and the parental genome sequence as guidance,we analyzed the structural variation at the full-length transcriptome level of the mule and identified 345 intergenic chimeras occured at least one individual,21 interspecie chimeras occured at least one individual and numerable mule-specific mutant genes.Functional enrichment analysis of these genes that intergenic chimeric genes were mainly related to ribosomes,mule-specific mutation genes and interspecie chimeric genes were mainly related to the "rejection" and cancer processes.The transcriptome structural variation can change the original structure of the gene,which can interfer the gene expression and enhance the adaptability of mule.4.Using RNA-Seq data,with thoroughbred horse genome as a reference,we analyzed the expression patterns of the transcriptomes from three Equus parent-offspring trio.666 mule-specific genes were identified which was significantly higher than that of horses(403)and donkeys(187).Functional enrichment analysis of these genes was found to be mainly related to DNA repair and cell life activities.At the same time,we identified 1805,2285,and 350 non-additively expressed genes in the transcriptomes from three Equus parent-offspring trio.Functional enrichment analysis of these genes was found to be directly related to cancer pathways and basic repair processes.The hybridization can damage the gene structure and expression pattern of mule.These damages can trigger the large-scale expression of genes involved in DNA damage repair,basic repair,and cell cycle regulation,as well as complex interactions between these processes,which can repair these injuries that are not benefited for the mule adaptation.5.Using thoroughbred horse genome as a reference and the parental genome sequence information as guidance,we selected 20443,9190,and 7126 HEB(Homoeolog expression bias)SNP markers for 3 pedigrees,using RNA-Seq data.The gene annotation and homologous expression bias analysis were performed using those SNP markers.We identified 1136 genes that are HEBG at least one individual.Functional enrichment analysis of these genes revealed that three significantly enriched pathways are related to the "rejection" pathway.Moreover,these genes significantly reduce the rejection of paternal genetic characteristics(antigen substances)by significantly expression biase to the paternal which can enhance the adaptability of mule.