Table_1_Identification of hub genes associated with follicle development in multiple births sheep by WGCNA.XLSX

Sheep exhibit a distinct estrous cycle that includes four different phases: proestrus, estrus, late estrus, and luteal phase. As the estrous cycle repeats, follicular development regularly alternates. We thus investigated ovarian transcriptome data from each of the four phases using weighted gene co-expression network analysis (WGCNA) to identify modules, pathways, and genes essential to follicle growth and development. We clustered mRNA and long non-coding RNA (lncRNA) into different modules by WGCNA, and calculated correlation coefficients between genes and Stages of the estrous cycle. Co-expression of the black module (cor = 0.81, P<0.001) and the yellow module (cor = 0.61, P<0.04) was found to be critical for follicle growth and development. A total of 2066 genes comprising the black and yellow modules was used for functional enrichment. The results reveal that these genes are mainly enriched in Cell cycle, PI3K-Akt signaling pathway, Oocyte meiosis, Apoptosis, and other important signaling pathways. We also identified seven hub genes (BUB1B, MAD2L1, ASPM, HSD3B1, WDHD1, CENPA, and MXI1) that may play a role in follicle development. Our study may provide several important new markers allowing in depth exploration of the genetic basis for multiparous reproduction in sheep.

绵羊具有独特的发情周期（estrous cycle），包含四个阶段：发情前期（proestrus）、发情期（estrus）、发情后期（late estrus）以及黄体期（luteal phase）。随着发情周期循环往复，卵泡发育也呈现规律性交替变化。因此本研究采用加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA），对四个周期阶段的卵巢转录组数据开展分析，以筛选与卵泡生长发育密切相关的共表达模块、信号通路及功能基因。研究团队通过WGCNA将信使RNA（mRNA）和长链非编码RNA（long non-coding RNA, lncRNA）聚类为不同共表达模块，并计算了基因与发情周期各阶段的相关系数。分析发现，黑色模块（相关系数cor=0.81，P<0.001）与黄色模块（cor=0.61，P<0.04）的共表达模式对卵泡生长发育至关重要。本研究共选取黑色与黄色模块内的2066个基因进行功能富集分析，富集结果显示，这些基因主要富集于细胞周期（Cell cycle）、PI3K-Akt信号通路（PI3K-Akt signaling pathway）、卵母细胞减数分裂（Oocyte meiosis）、细胞凋亡（Apoptosis）等重要信号通路中。本研究还筛选出7个可能参与卵泡发育的核心基因（hub gene），分别为BUB1B、MAD2L1、ASPM、HSD3B1、WDHD1、CENPA及MXI1。本研究可为深入解析绵羊多胎繁殖的遗传基础提供若干关键新型分子标记。