Liu C X, Huang R H, Zhou W D, Jiang N J, Liu Q, Ma J F, Li P H, Zhao Q B
Institute of Swine Science (Key Laboratory of Pig Genetic Resources Evaluation and Utilization, Ministry of Agriculture and Rural Affairs (Nanjing)), Nanjing Agricultural University, Nanjing 210095, PR China; College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, PR China.
Institute of Swine Science (Key Laboratory of Pig Genetic Resources Evaluation and Utilization, Ministry of Agriculture and Rural Affairs (Nanjing)), Nanjing Agricultural University, Nanjing 210095, PR China.
Animal. 2025 Jul;19(7):101552. doi: 10.1016/j.animal.2025.101552. Epub 2025 May 23.
The teat number trait in pigs has significant economic value, with Meishan and Erhualian pigs having the highest counts globally. To uncover the genetic basis of teat number variation, this study employed genome re-sequencing for a total of 1 119 individuals. Genome-wide association study (GWAS), meta-analysis, and Bayesian fine mapping were conducted. The GWAS from two breeds revealed multiple significant quantitative trait locus (QTL) for total teat number (TNT) mainly on SSC1, SSC5, SSC10, and others. Bayesian fine mapping targeted the most significant QTL in both Meishan pig GWAS and meta-analysis, mapping it to SSC1: 264 609 542 - 266 079 236 bp. Within this QTL, eighteen protein-coding genes were annotated. Bayesian fine mapping was performed for the second most significant QTL in meta-analysis, which also was the most significant QTL in Erhualian pig GWAS, identifying its mapping at SSC5: 58 228 193 - 59 198 414 bp. One protein-coding gene was annotated within this QTL. After determining the anatomical location of the mammary placodes, RNA-seq results from mammary placodes of extremely high and low 26-day-old Erhualian pig embryos were integrated, and found that only OLFML2A showed significant differential expression among these nineteen candidate genes. OLFML2A was primarily enriched in the extracellular matrix and extracellular matrix organisation pathways, which may play a role in the interaction between epithelial and stromal cells in mammary placodes. In addition, the utilisation of PigGTEx data for transcriptome-wide association study revealed a significant association between the expression level of OLFML2A in the placenta and TNT in Meishan pig. The results of phenome-wide association study further validate the significant association of OLFML2A with teat number traits in lean-type commercial pigs. Molecular validation experiments confirmed that OLFML2A specifically expresses mRNA and protein in mammary placodes. Reverse transcription-quantitative PCR results further confirmed significant differences in gene expression in the mammary placodes between extremely high and low 26-day-old Erhualian pig embryos, with the high group showing significantly higher expression levels. Differential expression analysis of mammary placodes from different litters further confirmed that the differential expression of OLFML2A was not caused by the litter effect of the sows. After integrating multiple omics, we have tentatively identified the OLFML2A gene as a potential causal gene responsible for teat number variation in Meishan and Erhualian pigs. This gene could potentially influence the development of mammary placodes in Meishan and Erhualian pigs, consequently influencing the phenotype of teat number.
猪的乳头数性状具有重要的经济价值,其中梅山猪和二花脸猪的乳头数在全球范围内是最多的。为了揭示乳头数变异的遗传基础,本研究对总共1119头个体进行了全基因组重测序。进行了全基因组关联研究(GWAS)、荟萃分析和贝叶斯精细定位。来自两个品种的GWAS揭示了多个主要位于SSC1、SSC5、SSC10等染色体上的总乳头数(TNT)的显著数量性状位点(QTL)。贝叶斯精细定位针对梅山猪GWAS和荟萃分析中最显著的QTL,将其定位到SSC1:264609542 - 266079236 bp。在这个QTL内,注释了18个蛋白质编码基因。对荟萃分析中第二显著的QTL进行了贝叶斯精细定位,该QTL也是二花脸猪GWAS中最显著的QTL,确定其定位在SSC5:58228193 - 59198414 bp。在这个QTL内注释了一个蛋白质编码基因。在确定乳腺基板的解剖位置后,整合了26日龄二花脸猪极高和极低胚胎乳腺基板的RNA-seq结果,发现在这19个候选基因中只有OLFML2A表现出显著差异表达。OLFML2A主要富集在细胞外基质和细胞外基质组织途径中,这可能在乳腺基板上皮细胞和基质细胞之间的相互作用中发挥作用。此外,利用猪GTEx数据进行全转录组关联研究发现,梅山猪胎盘中OLFML2A的表达水平与TNT之间存在显著关联。全表型关联研究结果进一步验证了OLFML2A与瘦肉型商业猪乳头数性状的显著关联。分子验证实验证实OLFML2A在乳腺基板中特异性表达mRNA和蛋白质。逆转录定量PCR结果进一步证实了26日龄二花脸猪极高和极低胚胎乳腺基板中基因表达的显著差异,高组表达水平显著更高。对不同窝的乳腺基板进行差异表达分析进一步证实,OLFML2A的差异表达不是由母猪的窝效应引起的。整合多种组学后,我们初步确定OLFML2A基因是导致梅山猪和二花脸猪乳头数变异的潜在因果基因。该基因可能影响梅山猪和二花脸猪乳腺基板的发育,从而影响乳头数表型。