藍瑞斯母豬早期胚差異性表現基因之單核苷酸多型性與窩仔數之相關

Abstract

母豬繁殖性能為影響養豬產業生產效率之重要因子，其中又以母豬窩仔數為最重要之繁殖性狀，若能改進窩仔數將能提升養豬產業之經濟效益。隨著分子生物技術之發展，藉由候選基因分析方法 (candidate gene approach) 找尋分子遺傳標記，並應用於標記輔助選拔 (marker-assisted selection)，將可助於改善母豬繁殖性狀之選拔效應。而藍瑞斯品種之母豬為台灣育種策略中的重要豬種，但目前仍未出現能夠顯著影響純種藍瑞斯母豬窩仔數的分子遺傳標記。 豬桑椹胚到囊胚時期的發育，為埋殖前胚成功著床以及胎體成功發育之關鍵時期。有鑑於此，本研究藉由桑椹胚發育至早期囊胚階段中，mRNA 表現量顯著增加或降低之基因作為候選基因，並進一步找尋其調控區域之單核苷酸多型性 (single nucleotide polymorphism, SNP) 分子遺傳標記，同時利用大量鑑定 SNP 基因型技術以瞭解其與窩仔數關係，期能篩選出區分藍瑞斯母豬高低窩仔數的標記，以協助遺傳育種改良。 經由 30 頭純種藍瑞斯母豬定序後發現，在 10 個候選基因裡，共有 37 個 SNP發生於調控區域、14 個 SNP 位於 5’ 端未轉譯區域 (untranslated region, UTR)，以及 8 個發生於轉譯區域或內插子 (intron) 的 SNP，合計共有 59 個新開發之 SNP。進一步利用 SNP 基因定型系統鑑定 161 頭純種藍瑞斯母豬之 SNP 基因型，同時分析各 SNP 基因型對於母豬不同胎次之出生仔豬總頭數 (total number of pigs born, TNB) 與存活仔豬頭數 (number born alive, NBA) 之差異。結果發現，位於 ataxia-telangiectasia mutated (ATM) 基因調控區域與 5’UTR、plasma platelet-activating factor acetylhydrolase (plasma PAFAH) 基因調控區域，以及泌乳素 (prolactin, PRL) 基因調控區域等 7 個 SNP，其基因型與藍瑞斯品種母豬之TNB 及 NBA 有顯著關係存在 (P < 0.05)，說明了這些調控區域的 SNP 可能與控制基因表現有密切關係存在，而 ATM 基因與 plasma PAFAH 基因也可能為調控母豬繁殖效能的重要基因。本研究新開發之 SNP 標記，發現為影響純種藍瑞斯母豬窩仔數的分子遺傳標記，未來若能取得更多之藍瑞斯母豬樣本數及生產紀錄，將能更進一步確認這些 SNP 在遺傳育種改良上之價值，對於純種藍瑞斯之選拔將是一大貢獻。

Reproductive traits, especially litter size, are important components for reducing the costs of pig production. DNA molecular markers can be used for marker-assisted selection (MAS) program through candidate gene approach to improve the rate of selection response. Landrace is the major strain of sow used in commercial pig production in Taiwan but there is no available molecular marker associated with purebred Landrace. The survival and proper development of early embryos are important factors affecting the reproductive traits of sows. The purpose of this study is to discover the SNPs located on the regulatory regions of differentially expressed genes between morula and blastocyst. We also characterized the SNP genotypes of these genes and studied the association of litter size with these genotypes in Landrace sows. After cloning and sequencing regulatory region of the 10 candidate genes in 30 Landrace sows, we found 59 novel SNPs. Furthermore, we genotyped the SNP markers within the candidate genes in 161 Landrace sows. The effects of SNP markers on total number of pigs born (TNB) and number born alive (NBA) were tested. Our results showed the SNPs located on ataxia-telangiectasia mutated (ATM) gene, plasma platelet-activating factor acetylhydrolase (plasma PAFAH) gene and prolactin (PRL) gene were associated with TNB and NBA (P < 0.05). As most of these SNPs located on the regulatory sequences of the candidate genes, they may have direct effects on regulating expression levels of those genes. We therefore propose ATM, plasma PAFAH and prolactin, the host genes for litter size related SNPs, are likely to have major effects on litter size in pigs, and these efforts may help us to improve the reproductive efficiency of pigs. We developed an efficient and effective platform based on litter size related SNPs associated with the regulatory sequences of differentially regulated genes from morula to blastocyst preimplantation embryos. Once validated further with larger sample size from multiple farms, this SNP based screening platform will significantly facilitate the selection of best breeding herd of Landrace sow.