Identification of candidate genes associated with milk yield trait in buffaloes ( Bubalus bubalis ) by restriction-site-associated DNA sequencing

ABSTRACT The objectives of our present study included the screening of single nucleotide polymorphisms (SNP) that show significant differences in allelic frequencies between two buffalo populations (Egyptian and Chinese buffaloes), categorization of functional genes associated with these SNP by gene ontology, and pathway analyses to further understand their potential values as candidate genes closely associated with milk yield trait in buffaloes. In this study, double digest restriction-site associated DNA sequencing was performed on Illumina HiSeq 2500 platform for 20 and 25 female buffaloes from Egypt and China, respectively. Approximately 118 Gb of sequencing data were obtained, and a total of 110,129 and 150,535 putative SNP were detected in Egyptian and Chinese buffaloes, respectively. Focused only on those SNP that differed significantly in allelic frequencies between the two populations, we found that genes associated with these SNP were significantly over-represented in the ionotropic glutamate receptor pathway, the endothelin signaling pathway, and the gonadotropin-releasing hormone receptor pathway, which contained a total of 29 genes. Of these, nine genes ( ADCY5, CACNA1A, CREB1, INHBA , INHBB, PIK3R1, PLCB1, PRKCE , and SMAD2 ) participating in the hormonal regulation of lactation, were considered to be promising candidate genes worthy of further investigations for favorable alleles associated with milk yield. Our results provide useful information about genetic variations in Egyptian and Chinese buffaloes. The potential influences of nine candidate genes and their associated SNP on milk yield need to be validated in more buffalo populations.

摘要
本研究的目标包括筛选在两个水牛种群（埃及水牛与中国水牛）间等位基因频率存在显著差异的单核苷酸多态性（single nucleotide polymorphisms, SNP）；通过基因本体（gene ontology, GO）注释对这些SNP关联的功能基因进行分类，并开展通路分析以进一步阐明其作为与水牛产奶性状紧密相关的候选基因的潜在价值。
本研究依托Illumina HiSeq 2500测序平台，分别对20头埃及雌性水牛和25头中国雌性水牛开展双酶切限制性位点关联DNA测序（double digest restriction-site associated DNA sequencing）。最终共获得约118 Gb的测序数据，在埃及水牛和中国水牛中分别检测到110129个和150535个推定SNP。
仅聚焦于两个种群间等位基因频率存在显著差异的SNP，研究发现这些SNP关联的基因在离子型谷氨酸受体通路、内皮素信号通路以及促性腺激素释放激素受体通路中显著富集，共包含29个基因。其中，9个参与泌乳激素调控的基因（ADCY5、CACNA1A、CREB1、INHBA、INHBB、PIK3R1、PLCB1、PRKCE及SMAD2）被认定为极具研究潜力的候选基因，可用于后续探究与产奶性状相关的优势等位基因。
本研究结果为埃及水牛与中国水牛的遗传变异研究提供了有益参考。上述9个候选基因及其关联SNP对产奶性状的潜在影响，仍需在更多水牛种群中进行验证。