Table2_Genetic marker identification of SEC13 gene for milk production traits in Chinese holstein.XLSX

SEC13 homolog, nuclear pore and COPII coat complex component (SEC13) is the core component of the cytoplasmic COPII complex, which mediates material transport from the endoplasmic reticulum to the Golgi complex. Our preliminary work found that SEC13 gene was differentially expressed in dairy cows during different stages of lactation, and involved in metabolic pathways of milk synthesis such as citric acid cycle, fatty acid, starch and sucrose metabolisms, so we considered that the SEC13 might be a candidate gene affecting milk production traits. In this study, we detected the polymorphisms of SEC13 gene and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population. By sequencing the whole coding and partial flanking regions of SEC13, we found four single nucleotide polymorphisms (SNPs). Subsequent association analysis showed that these four SNPs were significantly associated with milk yield, fat yield, protein yield or protein percentage in the first and second lactations (p ≤.0351). We also found that two SNPs in SEC13 formed one haplotype block by Haploview4.2, and the block was significantly associated with milk yield, fat yield, fat percentage, protein yield or protein percentage (p ≤ .0373). In addition, we predicted the effect of SNP on 5′region on transcription factor binding sites (TFBSs), and found that the allele A of 22:g.54362761A>G could bind transcription factors (TFs) GATA5, GATA3, HOXD9, HOXA10, CDX1 and Hoxd13; and further dual-luciferase reporter assay verified that the allele A of this SNP inhibited the fluorescence activity. We speculate that the A allele of 22:g.54362761A>G might inhibit the transcriptional activity of SEC13 gene by binding the TFs, which may be a cause mutation affecting the formation of milk production traits in dairy cows. In summary, we proved that SEC13 has a significant genetic effect on milk production traits and the identified significant SNPs could be used as candidate genetic markers for GS SNP chips development; on the other hand, we verified the transcriptional regulation of 22:g.54362761A>G on SEC13 gene, providing research direction for further function validation tests.

SEC13同源物、核孔和COPII包被复合体成分（SEC13）是细胞质COPII复合体的核心组分，介导从内质网至高尔基体的物质转运。我们的初步研究发现，SEC13基因在奶牛泌乳的不同阶段表达存在差异，并参与乳合成代谢途径，如柠檬酸循环、脂肪酸、淀粉和蔗糖代谢，因此我们认为SEC13可能是一个影响产奶性状的候选基因。在本研究中，我们检测了SEC13基因的多态性，并验证了其在中华荷斯坦牛群体中对于产奶量和组成性状的遗传效应。通过对SEC13全编码区和部分侧翼区域进行测序，我们发现了四个单核苷酸多态性（SNPs）。后续关联分析表明，这四个SNPs与第一次和第二次泌乳期的产奶量、脂肪产量、蛋白质产量或蛋白质百分比存在显著关联（p ≤.0351）。我们还发现，SEC13中的两个SNPs通过Haploview4.2形成了一个单倍型块，该块与产奶量、脂肪产量、脂肪百分比、蛋白质产量或蛋白质百分比显著关联（p ≤ .0373）。此外，我们预测了SNP对5'区域的转录因子结合位点（TFBSs）的影响，并发现22:g.54362761A>G的等位基因A可以结合转录因子（TFs）GATA5、GATA3、HOXD9、HOXA10、CDX1和Hoxd13；进一步的双荧光素酶报告基因检测验证了该SNP的等位基因A抑制了荧光活性。我们推测22:g.54362761A>G的A等位基因可能通过结合转录因子抑制SEC13基因的转录活性，这可能是影响奶牛产奶性状形成的突变原因。总之，我们证实了SEC13对产奶性状具有显著的遗传效应，所识别的显著SNPs可作为GS SNP芯片开发的候选遗传标记；另一方面，我们验证了22:g.54362761A>G对SEC13基因的转录调控，为后续功能验证试验提供了研究方向。