Cao Mingyue, Shi Lijun, Peng Peng, Han Bo, Liu Lin, Lv Xiaoqing, Ma Zhu, Zhang Shengli, Sun Dongxiao
Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory for Animal Breeding, China Agricultural University, Beijing, 100193, China.
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
BMC Genomics. 2021 Jul 27;22(1):575. doi: 10.1186/s12864-021-07893-8.
Our previous genome-wide association study (GWAS) on milk fatty acid traits in Chinese Holstein cows revealed, the SNP, BTB-01556197, was significantly associated with C10:0 at genome-wide level (P = 0.0239). It was located in the down-stream of 5-hydroxytryptamine receptor 1B (HTR1B) gene that has been shown to play an important role in the regulation of fatty acid oxidation. Hence, we considered it as a promising candidate gene for milk fatty acids in dairy cattle. In this study, we aimed to investigate whether the HTR1B gene had significant genetic effects on milk fatty acid traits.
We re-sequenced the entire coding region and 3000 bp of 5' and 3' flanking regions of HTR1B gene. A total of 13 SNPs was identified, containing one in 5' flanking region, two in 5' untranslated region (UTR), two in exon 1, five in 3' UTR, and three in 3' flanking region. By performing genotype-phenotype association analysis with SAS9.2 software, we observed that 13 SNPs were significantly associated with medium-chain saturated fatty acids such as C6:0, C8:0 and C10:0 (P < 0.0001 ~ 0.042). With Haploview 4.1 software, linkage disequilibrium (LD) analysis was performed. Two haplotype blocks formed by two and ten SNPs were observed. Haplotype-based association analysis indicated that both haplotype blocks were strongly associated with C6:0, C8:0 and C10:0 as well (P < 0.0001 ~ 0.0071). With regards to the missense mutation in exon 1 (g.17303383G > T) that reduced amino acid change from alanine to serine, we predicted that it altered the secondary structure of HTR1B protein with SOPMA. In addition, we predicted that three SNPs in promoter region, g.17307103A > T, g.17305206 T > G and g.17303761C > T, altered the binding sites of transcription factors (TFs) HMX2, PAX2, FOXP1ES, MIZ1, CUX2, DREAM, and PPAR-RXR by Genomatix. Of them, luciferase assay experiment further confirmed that the allele T of g.17307103A > T significantly increased the transcriptional activity of HTR1B gene than allele A (P = 0.0007).
In conclusion, our findings provided first evidence that the HTR1B gene had significant genetic effects on milk fatty acids in dairy cattle.
我们之前对中国荷斯坦奶牛乳脂肪酸性状进行的全基因组关联研究（GWAS）表明，单核苷酸多态性（SNP）BTB - 01556197在全基因组水平上与C10:0显著相关（P = 0.0239）。它位于5 - 羟色胺受体1B（HTR1B）基因的下游，该基因已被证明在脂肪酸氧化调节中起重要作用。因此，我们认为它是奶牛乳脂肪酸的一个有前景的候选基因。在本研究中，我们旨在调查HTR1B基因是否对乳脂肪酸性状具有显著的遗传效应。
我们对HTR1B基因的整个编码区以及5'和3'侧翼区的3000 bp进行了重测序。共鉴定出13个SNP，其中1个在5'侧翼区，2个在5'非翻译区（UTR），2个在外显子1中，5个在3'UTR中，3个在3'侧翼区。通过使用SAS9.2软件进行基因型 - 表型关联分析，我们观察到13个SNP与中链饱和脂肪酸如C6:0、C8:0和C10:0显著相关（P < 0.0001至0.042）。使用Haploview 4.1软件进行连锁不平衡（LD）分析。观察到由2个和10个SNP形成的两个单倍型块。基于单倍型的关联分析表明，这两个单倍型块也与C6:0、C8:0和C10:0强烈相关（P < 0.0001至0.0071）。关于外显子1中的错义突变（g.17303383G > T），该突变导致氨基酸从丙氨酸变为丝氨酸，我们使用SOPMA预测它改变了HTR1B蛋白的二级结构。此外，我们使用Genomatix预测启动子区域的三个SNP，g.17307103A > T、g.17305206T > G和g.17303761C > T，改变了转录因子（TFs）HMX2、PAX2、FOXP1ES、MIZ1、CUX2、DREAM和PPAR - RXR的结合位点。其中，荧光素酶测定实验进一步证实，g.17307103A > T的等位基因T比等位基因A显著增加了HTR1B基因的转录活性（P = 0.0007）。
总之，我们的研究结果首次证明HTR1B基因对奶牛乳脂肪酸具有显著的遗传效应。
