Comprehensive Analysis Of Allele-specific Expression And Transmission Ratio Distortion In F₁ Hybrids Of Meat-and Egg-type Chickens

In diploid organisms,each gene locus is composed of two parental alleles,which would interact with each other for determining the phenotypic variation.Better understanding of allele-specific functional differences in farm animals is much important for us to explore the genetic basis underlying economically important traits,which have been poorly understood yet.The huge differences of genetic variations and phenotypic traits observed between meat-and egg-type chickens make chickens become an excellent model for this study.In the present study,genome-wide analysis was applied to explore the allele-specific expression(ASE)and transmission ratio distortion(TRD)in F₁ hybrids of chicken.First,meat-and egg-type chickens were selected for producing a full-sib F₁hybrid population(n=57).Then,genome resequencing of two parents and 38 offspring were performed and liver and breast muscle samples(n=38)were subjected to strand-specificRNA sequencing(ss RNA-seq)for ASE detection at 1,28,and 56 days of age,respectively.Moreover,genome resequencing data of parents and offspring were further used to reveal transmission ratio of paternal alleles to offspring in chickens.Besides,based on transcriptome data,we performed transcriptomic profile analysis of breast muscle and liver at different time points in the early stage of hybrids.Finally,we carried out functional verification of the candidate gene in breast muscle by small interfering RNA(si RNA)at the cellular level.The results of this study are as follows:1.Two parents and their offspring(n=38)were individually sequenced with an average sequencing depth of 30×and 5×,respectively.In total,we got 357.35 Gb of high-quality clean data(Q20>95%)after filtering and quality control,resulting in 2.38billion clean reads in 40 chickens.We identified 6.7 million single nucleotide polymorphisms(SNPs)across these samples.2.The parental genomic SNPs in ncRNA and exon regions were selected for informative SNPs identification.After correction by the resequencing data of offspring,we obtained 303,1090 and 3589 high quality informative SNPs in the ncRNA,synonymous and non-synonymous regions,respectively.Then,a total of 465 informative genes were accurately identified for the further ASE detection.3.These 465 information genes accurately identified were applied to identify ASE genes in breast muscle and liver at different time points(D1,D28 and D56)by ss RNA-seq.There were 0.4%-4.1%of informative genes showing ASE,and 57 of them were found across different tissues and time points.Furthermore,5 ASE genes that showed monoallelic expression were speculated to be potential imprinted genes.4.By analyzing the spatiotemporal pattern of ASE in chickens,except one shared ASE gene found at D1,all the other ASE genes were tissue-specific.Besides,no matter what the time-point pattern of the ASE gene,the same parental allele of this gene almost showed consistently higher or lower expression across all the three time points in the same type tissue.5.Based on the resequencing data of parents and offspring,we further performed the TRD analysis.Finally,a total of 2850 TRD SNPs(p-adj<0.05)and their corresponding400 TRD genes were identified.Our results revealed that the number and ratio of these TRD SNPs were unevenly distributed on macrochromosomes and microchromosomes in chickens.By comparing the list of TRD genes with the ASE genes,we found four genes(DDC,CYP2C23a,MYH1F and IQSEC3)showed both TRD and ASE.Among them,the MYH1F might play an essential role in the fast muscle development.6.Functional enrichment analyses of TRD genes revealed that many genes(e.g.,TGFBR2,TGFBR3,NOTCH1,and NCOA1)with TRD were found in the significantly enriched biological process(BP)and Inter Pro terms in relation to embryonic lethality and germline selection.7.Through the transcriptome analysis,we have obtained a large number of differentially expressed transcripts between different comparisons in the breast muscle and liver.In addition,many significantly enriched Gene ontology(GO)terms and Kyoto encyclopedia of genes and genomes(KEGG)signaling pathways related to muscle growth and development in the breast muscle were found,and a large number of key genes related to growth and development were obtained.8.Through functional verification analysis,we found that MYH1F gene was specifically expressed in muscles.The relative expression of Myo D and Myo G m RNA,Western blot(WB)and immunofluorescence of MYHC,and RNA-seq results showed that the MYH1F gene may play an important role in regulating the differentiation of chicken satellite cells.In conclusion,we established a full-sib F₁ population of a cross between meat-and egg-type chickens to analyze the ASE and TRD by whole-genome resequencing and ss RNA-seq.Results of ASE study indicated that most of ASE genes in chickens are tissue-specific and time-dependent.Results of TRD study suggested that TRD is considerably prevalent in chicken and may be affected by multiple biological mechanisms.We also found a large number of differentially expressed genes(DEGs)related to muscle growth and development in the breast muscle.In addition,validation analysis indicated that it may be due to the imbalance in the transmission and expression levels of the paternal and maternal MYH1F alleles and the important role of this gene in the differentiation of chicken satellite cells,which led to huge differences in skeletal muscle development between meat-and egg-type chickens.In the future,the function and association analysis of these identified ASE genes,TRD genes,and DEGs would be of great importance for us to explore the genetic basis underlying economically important traits in cross populations of farm animals.