| Growth and development traits are important economic traits of sheep,including body weight,body height,body length,chest girth,chest depth,chest width,and shin circumference.It is difficult to improve them in a short period of time by traditional breeding methods.Therefore,it has become an important goal in the field of genetic improvement of meat sheep to explore the key molecular markers related to growth and development traits to improve the meat yield performance.Based on previous multi-population and multi-trait GWAS studies in sheep and beef cattles,it has been identified that the QTL region significantly associated with growth and development traits(such as birth weight,body size and bone development)is located in the 37-38 Mb region of chromosome 6(containing LAP3,MED28,NCAPG,LCORL,FAM184B,and DCAF16 genes).Previous eQTL analysis in muscle tissue revealed significant overlap between GWAS loci and eQTL loci,and SNP loci affecting growth and development traits also affected gene expression.Therefore,this study took Suhu sheep breeding population as the research object.Suhu sheep breeding population is a new population including Duper sheep ancestry and Hu sheep ancestry.Through the previous laboratory screening and verification work,LAP3,MED28,and NCAPG located in the 37-38 Mb region of chromosome 6 were finally selected as candidate genes in this study.This study aimed to investigate the spatiotemporal expression characteristics of LAP3,MED28,and NCAPG at tissue and cellular levels,and their potential effects on the proliferation and differentiation of sheep embryonic myoblasts.Meanwhile,we conducted ChIP-seq of histone modification study to identify functional regulatory regions,and analyzed the association between SNP polymorphisms in the functional regulatory regions of key candidate genes and growth and development traits,so as to explore the important molecular markers affecting the growth and development traits in the 37-38 Mb region of chromosome 6 of Suhu sheep breeding population.The main research findings were as follows:1.Spatiotemporal expression of LAP3,MED28,and NCAPG at tissue and cellular levelsIn order to investigate the spatiotemporal expression characteristics of LAP3,MED28,and NCAPG at different growth and development stages,the RT-qPCR method was used to detect their relative expression levels in heart,liver,spleen,lung,kidney,and longissimus dor si tissues of 120-day-old fetal sheep,3-day-old newborn lambs,and 6-month-old sheep in Suhu sheep breeding population.The results indicated that LAP3,MED28,and NCAPG were widely expressed in various tissues of sheep at different growth and development stages.The relative expression levels of LAP 3,MED28,and NCAPG in the longissimus dor si of 120-day-old fetal sheep were significantly higher than those in 3-day-old newborn lambs and 6-month-old sheep.Next,we further detected the relative expression levels of candidate genes during the proliferation phase and on day 1(D1),D3,D5,and D7 of induced differentiation in sheep embryonic myoblasts,and the results revealed that LAP3,MED28 and NCAPG showed a differential expression pattern before and after induced differentiation of sheep embryonic myoblasts.The above results demonstrated that LAP3,MED28,and NCAPG may play important roles in growth and development of sheep embryonic muscle.2.Functional study of LAP3,MED28,and NCAPG in sheep embryonic myoblastsIn order to elucidate the effects of LAP3,MED28,and NCAPG on the proliferation and differentiation of sheep embryonic myoblasts,we first constructed the small interfering RNA(siRNA)and overexpression recombinant plasmids to target their gene expression levels.Then,the effects of interference or overexpression of LAP3,MED28,and NCAPG on the proliferation of sheep embryonic myoblasts were detected using CCK-8,EdU staining,and flow cytometry.Subsequently,RT-qPCR and indirect immunofluorescence were used to detect their effects on induced differentiation of sheep embryonic myoblasts.The results revealed that interference with LAP3 promoted the proliferation of myoblasts,and inhibited myotube fusion at the later stage of myogenic differentiation,while overexpression of LAP3 resulted in the opposite effect;then,interference with MED28 inhibited the proliferation and differentiation of sheep embryonic myoblasts,while overexpression of MED28 promoted the proliferation and inhibited the differentiation;finally,interference with NCAPG inhibited the proliferation and differentiation of sheep embry onic myoblasts.In summary,the above results indicated that alterations in the expression levels of three candidate genes could significantly affect the proliferation and differentiation of sheep embryonic myoblasts.3.ChIP-seq study of H3K27ac in sheep longissimus dorsi tissues at different growth and development stagesTo further identify the functional regions that regulate the expression of the target candidate genes of LAP3,MED28,and NCAPG,we performed histone H3K27ac modification ChIP-seq in longissimus dors i tissues of 120-day-old fetal sheep and 3-day-old lambs.The results showed that H3K27ac reads distributed with high signal intensity within upstream and downstream 2 kb regions of gene TSS in longissimus dorsi tissues of fetal sheep and lambs.H3K27ac modification regions mainly included promoter region,intron region,and distal intergenic region.Further analysis revealed H3K27ac ChIP-seq modification signal was presented in upstream 0-2 kb promoter region of LAP3 TSS in longissimus dorsi tissues of fetal sheep and lambs.These results suggested that the upstream 0-2 kb promoter region of LAP3 TSS may serve as a functional regulatory region for muscle growth and development in Suhu sheep breeding population,which may play a key role in the regulation of LAP3 expression.4.Association analysis between SNPs at promoter region of LAP3 and growth and development traitsIn order to further explore whether SNP polymorphisms in the promoter region of LAP3 were associated with growth and development traits of Suhu sheep breeding population.the DNA pool sequencing was used to scan the potential SNPs at promoter region(2,000 bp)of LAP3,and the iMLDR methods were used for genotyping.We found that 5 SNPs were detected in the promoter region of LAP3.Population genetic parameter analysis showed that 5 SNPs except SNP4 were in Hardy-Weinberg equilibrium,and the polymorphic information contents were 0.37,0.37,0.37,0.30 and 0.34,respectively,which were moderate polymorphisms.Association analysis indicated that SNP 1-3 were significantly correlated with 6-month-old body height and 6-month-old body length;and SNP4 was significantly correlated with birth weight,birth body length,3-month-old shin circumference,6-month-old body weight,6-month-old body length,and 6-month-old shin circumference;and finally,SNP5 was significantly correlated with 1-month-old body height,2-month-old body height,6-month-old body weight,6-month-old body length,and 6-month-old shin circumference.Further linkage disequilibrium analysis showed that 5 SNPs were strongly linked.The results of combined genotypes association analysis showed that CAGTCTGGAC was the dominant genotype combination in body weight,body length,and shin circumference at birth and 6-month-old.Based on the previous GWAS and eQTL study results related to sheep meat traits,this study explored the important SNP molecular markers in the 37-38 Mb region of chromosome 6 affecting the growth and development traits of Suhu sheep breeding population from multiple dimensions through expression abundance and functional studies of candidate genes at tissue and cellular levels,ChIP-seq analysis,and SNP association analysis.It is expected to be used in the breeding of high-yielding Suhu sheep. |
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