Identification Of Potential Candidate Genes For Eyelid Deficiency In Developing Wulong Goose Embryos By Transcriptomic Analysis

The wulong goose is a source of good meat, eggs, and geese in China. The eyelid of Wulong goose serves as a distinctive emblem of this species. The mechanism underlying the eyelid deficiency trait of Wulong geese is unclear owing to the constrained related to conventional genetic techniques. Herein, Wulong goose with Huoyan (eyelid deficiency) trait at embryonic stage of 9 days (E9), 12 days (E12), and 14 days (E14) and Wulong goose with normal eyelids were selected for transcriptome sequencing. Differentially expressed gene (DEG) and functional enrichment analyses were performed, and finally eight DEGs were selected to verify the accuracy of qPCR sequencing. Overall, 466, 962, and 550 DEGs were obtained from the three control groups D9 vs N9, D12 vs N12, and D14 vs N14, respectively, by differential analysis (P<0.05). Among them, CDKN1C, CRH, CROCC, and TYSND1 were significantly expressed in the three groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the enrichment of CROCC and TYSND1 in pathways of cell cycle process, endocytosis, microtubule-based process, microtubule organizing center organization, protein processing, and protein maturation. CDKN1C and CRH were enriched in cell cycle and cAMP signaling pathway. In addition, some collagen family genes were detected among the DEGs, including COL3A1, COL4A5, COL4A2, and COL4A1. FREM1 and FREM2 genes were detected in both Huoyan and normal eyelids. Considering the differences in sex and time, FREM1 was significantly (P<0.01) expressed in the two control groups F14D vs F9D and M14D vs M9D. These results reveal the regulatory mechanism underlying Huoyan trait in Wulong geese and provide guidance for designing experiments using DEGs and gene function pathways. This study can provide scientific references for the protection of breed characteristics, purebred breeding, and hybrid utilization.

五龙鹅（Wulong goose）是我国优质肉用、蛋用鹅类遗传资源，其眼睑形态为该品种的标志性特征。目前，五龙鹅豁眼（Huoyan，眼睑缺损）性状的调控机制尚未阐明，这一问题受制于传统遗传技术的研究瓶颈。本研究选取处于胚胎发育第9天（E9）、第12天（E12）及第14天（E14）的豁眼性状五龙鹅，以及正常眼睑五龙鹅开展转录组测序，随后进行差异表达基因（Differentially Expressed Gene, DEG）分析与功能富集分析，并筛选8个差异表达基因进行qPCR验证以确认测序结果的可靠性。通过差异表达分析（P<0.05），分别在D9 vs N9、D12 vs N12、D14 vs N14三组对照中获得466、962和550个差异表达基因。其中，CDKN1C、CRH、CROCC及TYSND1在三组对照中均存在显著差异表达。基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析结果显示，CROCC和TYSND1显著富集于细胞周期过程、内吞作用、微管相关过程、微管组织中心组装、蛋白质加工及蛋白质成熟等通路；CDKN1C与CRH则显著富集于细胞周期及cAMP信号通路。此外，差异表达基因中还包含部分胶原家族基因，如COL3A1、COL4A5、COL4A2及COL4A1。研究在豁眼组与正常组眼睑组织中均检测到FREM1与FREM2基因的表达。考虑到性别与发育时间的差异，FREM1在F14D vs F9D、M14D vs M9D两组对照中均呈现显著差异表达（P<0.01）。上述结果阐明了五龙鹅豁眼性状的调控机制，为基于差异表达基因与基因功能通路的后续实验设计提供了理论指导，同时可为五龙鹅品种特征保护、纯种选育及杂交利用提供科学参考依据。