Excavating Genes Associated With Tibia Breaking Strength In End-of-lay Hens Based On Transcriptomic Sequencing

Avian bones are not only the foundation of life activities,but also closely related to their production performance.The medullary bone is source of calcium for eggshell formation during laying period.With the increasing of age,calcium deposition of medullary bone is insufficient,and cortical bone acts as body's calcium source,causing the decreasing of bone strength and increasing of bone fragility in laying hens,and layer performance decreased.This study aimed to select genes and genetic markers related to tibial breaking strength in laying hen during late period of laying,which will provide theoretical basis for studying bone metabolism,regulatory mechanism of bone quality and breeding.Twenty Hy-Line Grey hens at 79-week-old with similar body weight were selected,and collecting the tibias.Left tibias were uesd for testing bone breaking strength,and right tibias were snapped frozen in liquid nitrogen for further high-throughput sequencing.The breaking strength was measured by texture analyzer.According to the tibia breaking strength,it was divided into High bone breaking strength group and Low bone breaking strength group.Six individuals with similar weight and different tibia breaking strength were identified H group?H1/H2/H3,3?and L group?L1/L2/L3,3?.Constructing cDNA libraries and transcriptome sequencing after extracting RNA,and sequencing data was used for bioinformatics analysis.Total number of reads obtained from HBS group and LBS group was 50,955,168 and 50,772,867,respectively.A total of 84.58%and 85.59%of sequences in H group and L group were effectively aligned to reference genome of Gallus gallus.There were 9 differentially expressed genes,of which 5 genes were up-regulated?OSCAR,PAX5B,and three LincRNAs?and four genes were down-regulated?TIMP4,KLHL14,MPP7 and a unknown gene?,between two groups.GO function annotation revealed that differentially expressed genes are enriched in biological processes?negative regulation of metalloenzyme activity,notch signaling pathway and protein ubiquitination,etc.?,cellular composition?proteinaceous extracellular matrix,extracellular space and nucleus,etc.?and molecular function?metal Endopeptidase inhibitor activity,protease binding and ubiquitin-protein ligase activity,etc.?.A total of 64,941 alternative splicing events was predicted with ASprofile.The GATK software identified 1,093,870 SNPs.Tibias of twenty Taihang?40 and 57 weeks?and eighteen Hy-Line grey hens?79weeks?were collected for RNA extraction and cDNA reverse transcription.7 reference genes?B₂M,LBR,HPRT1,HMBS,RPL32,18S,RPL5?were selected for RT-qPCR primers identification,of which efficiency were all above 95%.The geNorm,NormFinder and BestKeeper three different algorithms were used for analysis of seven reference genes,and comprehensive results analysis showed B₂M and LBR were preferred stable reference genes combination.Tibias of eighteen Hy-Line gray hens at 79 week of age were collected for RNA extraction and cDNA reverse transcription.The mRNA expression level of KLHL14,TIMP4,OPN and ON was detected by RT-qPCR.The results showed TIMP4 and KLHL14expression was higher in H group compared with that in L group?P<0.05?,while OPN expression was higher in LBS group than HBS group?P<0.05?,and ON expression is not significant between two groups?P>0.05?.The level of TIMP4 and KLHL14 in serum was measured by ELISA,the results indicated that there was not significantly correlated with tibia breaking strength?P>0.05?.Sequencing the PCR results,comparing the sequence of Ensembl database,the results showed there were 6 SNPs in ON gene,which were13632066G>A,13631903A>T,13631874C>T,13631916A>G,13631974A>C,13631916A>G,13631974A>C,13632074A>G.However,all of these six SNPs were not significantly correlatd with tibia breaking strength?P>0.05?.In summary,nine differentially expressed genes were found by high-throughput sequencing.The expression of TIMP4 and KLHL14 was significant between H and L group in end-of-lay hens at mRNA level.B₂M and LBR were identified as stable reference genes in tibia of hens.The expression of OPN was lower in HBS group than LBS group in end-of-lay hens at mRNA level.