Identification Of Hub Gene Related To Milk Production Trait And Functional Validation Of TUBA1C In Buffalo On Basis Of Omics Data

Water buffalo?Bubalus bubalis?distributed in Southern China,has the biological characteristics of resistance to rough feeding,excellent milk quality,large draught power,and long productive life.With the rapid improvement of urbanization and mechanization,the use of buffalo in China has gradually evolved from draught power to the milk and meat production.In particular,nutrient-rich buffalo milk and its dairy products are deeply loved by the consumer,resulting in the buffalo industry has broad market prospects.To date,although the milk production level of crossbred buffaloes in China has reached1500¹700 kg,it is still lower than that of the river buffalo breeds?2000²200 kg?.Thus,lower milk production levels have severely hampered the current development of the buffalo dairy industry.Identifying the causal genes associated with buffalo milk production traits is critical to increasing the milk production level on the species.With the rapid development of high-throughput sequencing technology,multi-omics integration analysis strategy has become an essential tool for genetic analysis of important economic traits in livestock animals.In this regard,we performed the evaluation of genomic data,integration analysis of transcriptomic and genomic data,identification of candidate gene family,and functional verification of candidate genes.This study aims to identify candidate genes related to buffalo milk production traits,which is of great significance to conduct molecular breeding research on dairy buffalo in the future.In the present study,four trials were implemented:Trial 1:Here,we evaluated the extent of linkage disequilibrium?LD?and effective population size?Ne?for 411 purebred Mediterranean buffaloes and 45 crossbred buffaloes based on the genome-wide SNP data.The results showed that the average square of correlation coefficient?r²?between adjacent SNP was 0.13±0.19 across all autosomes for purebred and 0.09±0.13 for crossbred,and the most rapid decline in LD was observed over the first 200 kb.Estimated r²?0.2 extended up to?50 kb in crossbred and 170 kb in purebred populations,while average r²?0.3 were respectively observed in the?10 and 60 kb in the crossbred and purebred populations.The largest phase correlation(R_P,C=0.47)was observed at a distance of 100 kb,suggesting that this phase was not actively preserved between the two populations.Estimated Ne for the purebred and crossbred population at the current generation was 387 and 113 individuals,respectively.These findings may provide useful information to guide the GS and GWAS in buffaloes.Trial 2:To characterize buffalo mammary gland transcriptome and identify the valuable candidate genes related to milk production,we performed the transcriptome analysis of mammary gland tissue,the gene-based genome-wide association studies?GWAS?,and their integration analysis using weighted gene co-expression network analysis?WGCNA?.The DEGs analysis showed that a total of 1420 DEGs were detected across different lactation points.In the gene-based analysis,976 genes were found to have a genome-wide association?P?0.05?that could be defined as the nominally significant GWAS geneset?NSGG?,9 of which were suggestively associated with milk yield(P<10^-4).Using the WGCNA analysis,544 and 225 genes associated with milk yield in the turquoise module were identified from DEGs and NSGG datasets,respectively.A total of12 hub genes?including BNIPL,TUBA1C,C2CD4B,DCP1B,MAP3K5,PDCD11,SRGAP1,GDPD5,BARX2,SCARA3,CTU2,and RPL27A?were identified because they were involved in multiple pathways related to milk production.Our findings provide an insight into the dynamic characterization of the buffalo mammary gland transcriptome,and these potential candidate genes may be valuable for future functional characterization of the buffalo mammary gland.Trial 3:Tubulins are the major constituents of microtubules that play vital roles in many cell processes.In the present study,we perform a genome-wide analysis of the identification,evolution,and expression profiling of buffalo tubulin genes.A total of 33buffalo tubulin sequences were identified and grouped into five clusters based on their structural and phylogenetic features.All the identified buffalo tubulins were distributed on 10 chromosomes randomly.Tandem duplication events mainly help in the expansion of buffalo tubulin family.Transcriptome data and real-time quantitative PCR analysis revealed that tubulin genes had distinct expression patterns in various tissues,and most of them were highly expressed in the early lactation.These findings provide useful information for further functional characterization of tubulin genes and identify candidate genes associated with milk production in buffalo.Trial 4:Based on the above results,we explored the putative function of TUBA1C gene in the buffalo mammary epithelial cells?BBMEC?using the RNA interference?RNAi?and overexpression techniques.The results showed that TUBA1C gene treated with RNAi could significantly affect the growth of BBMEC,resulting in the reduction of cell viability and an increase of apoptosis.In contrast,overexpression of TUBA1C had no significant effect on the growth of BBMEC.Moreover,we found that TUBA1C gene treated with RNAi or overexpression can cause the expression of fat metabolism or milk protein metabolism-related genes?11 and 3?,respectively,thereby affecting the secretion of triglyceride and?/?casein in the BBMEC.In summary,according to the principle of system biology,multi-omics data and functional validation assay were used to identify candidate genes related to milk production traits in buffalo,and further preliminarily clarify their mechanism.These identified candidate genes are more reliable and provide some useful molecular evidence for developing the buffalo breeding program and promoting its inheritance in the future.