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標題: | 臺灣水牛族群遺傳結構與毛色基因之研析 Analysis of population genetic structure and coat color gene of Taiwan swamp buffalo |
作者: | 辛佩蓉 Pei-Jung Hsin |
指導教授: | 王佩華 Pei-Hwa Wang |
共同指導教授: | 張啟聖 Chi-Sheng Chang |
關鍵字: | 臺灣水牛,族群遺傳結構,毛色基因,微衛星標識,基因晶片,全基因組關聯分析, Taiwan swamp buffalo,Population genetic structure,Coat color gene,Microsatellite marker,Genotyping array,Genome-wide association study (GWAS), |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 自臺灣農業機械化以來,臺灣水牛的數量急劇下降,目前只剩下約 2,002 頭。行政院農業委員會畜產試驗所花蓮種畜繁殖場(後簡稱花蓮場)是臺灣水牛的保種基地,飼養著一群灰水牛和白水牛。為了保持臺灣水牛的遺傳多樣性,了解其族群遺傳結構是十分重要的課題。另一方面,臺灣水牛屬於沼澤型水牛,而沼澤型水牛的毛色以灰色為主,偶爾出現全白色毛色個體。先前的一項研究表明,白色沼澤型水牛的 ASIP 基因中的一段 LINE-1 插入序列可能導致白色毛色;然而,臺灣水牛的白色毛色之確切形成原因尚不清楚。
在本研究中,採集了花蓮場的 78 頭灰色臺灣水牛和 16 頭白色臺灣水牛的血液樣本,並利用 15 組微衛星標識和水牛高密度 SNP 位點基因晶片(90K Axiom® Buffalo Genotyping Array)進行族群遺傳結構分析。結果顯示,剔除不具多態性和可能具有無效交替基因的微衛星標識後,以其餘 12 組微衛星標識檢測所得之平均交替基因數(Na)為 4.4、有效交替基因數(Ne)為 2.678、觀測異質度(Ho)為 0.584、期望異質度(He)為 0.581、多態性訊息含量(PIC)為 0.521 及族群近親係數(FIS)為 -0.008;利用基因晶片中的 14,456 個 SNPs 所得之所有 Na 為 2 ,平均 Ne為 1.616、Ho 為 0.372、He 為 0.360、PIC 為 0.282 及 FIS 為 -0.029。以兩種方法繪製所得之類緣關係樹十分相似,但類緣關係樹節點上表示重覆取樣1,000次生成的百分比數值是以在 14,456 個 SNPs 所得的結果中明顯較高。以兩種方法所進行的群集分析結果中(K = 3),均將所有的 16 頭白水牛與部分灰水牛分至一個次族群,而其餘的灰水牛還可以再分為兩個次族群。 本試驗針對臺灣水牛的 ASIP 基因進行基因分型以檢測 LINE-1 插入序列是否存在,但試驗結果中並未發現此插入序列,應代表臺灣水牛白色毛色之成因與先前的研究不同。本研究亦利用水牛高密度 SNP 位點基因晶片中的 14,456 個 SNPs 針對臺灣水牛的灰色與白色毛色進行全基因組關聯分析(GWAS),結果顯示了與白色毛色相關的 17 個最具顯著性的 SNPs 位點(P < 1 x 10-11),並找出了 26 個相關的基因。此外,前兩個最具顯著性的 SNPs 位點(P < 1 x 10-17)位在第 18 號染色體上,且其位置十分接近 MC1R 基因,而 MC1R 基因是已知的重要的色素調控基因。因此,本試驗針對臺灣水牛的 MC1R 基因之 exon 1 進行了定序,檢測到一個臺灣水牛獨有的錯義突變位點(MC1R c.901C>T)並導致氨基酸置換(p.R301C)。接著針對 115 頭灰色和 18 頭白色臺灣水牛進一步進行 MC1R c.901C>T 基因分型。結果顯示,17 頭白水牛為突變型純合子,1 頭白水牛和 37 頭灰水牛為雜合子,78 頭灰水牛為野生型純合子(P < 1 x 10-21)。用於預測 p.R301C對 MC1R 結構或功能的潛在影響的所有八種工具均表明此變異是有害的。以 qPCR 進行的基因表達分析結果顯示,MC1R、ASIP、MITF、TYR、TYRP1 和 DCT 的基因表達在 1 頭白水牛幼年和 2 頭灰水牛幼年耳朵皮膚組織之間無顯著差異。本試驗亦利用組織化學染色(Fontana-Masson)觀察水牛皮膚組織中的黑色素沉積。結果顯示白水牛皮膚組織的黑色素沉積較少,但仍能產生黑色素。 綜上所述,本研究可以為臺灣水牛保種族群的保育及毛色性狀的選育提供參考資訊。本試驗所發現的 MC1R c.901C>T 是一個極有可能的候選變異基因,會損害 MC1R 蛋白質的功能並導致臺灣水牛的白色毛色形成。 The number of Taiwan swamp buffaloes has dropped sharply since the mechanization of agriculture. There were only 2,002 head left. Hualien Animal Propagation Station (HAPS) is the conservation field of the Taiwan swamp buffalo, raising both gray and white buffaloes. In order to maintain the genetic diversity of the Taiwan swamp buffalo, estimating their population genetic structure is important. On the other hand, the coat color of the swamp buffalo is mainly gray, but white individuals appear occasionally. A previous study indicated that the LINE-1 insertion in the ASIP gene of the white swamp buffalo might cause the white coat color. However, the exact mechanism for the white coat color of the Taiwan swamp buffalo still remained unclear. In this study, blood samples were collected from 78 gray and 16 white Taiwan swamp buffaloes in HAPS. Fifteen microsatellite markers and 90K Axiom® Buffalo Genotyping Array were used for population genetic structure analysis. The results showed that excluding the nonpolymorphic marker and the markers with high null allele frequency, the average number of observed alleles per locus (Na) was 4.4, effective alleles per locus (Ne) was 2.678, observed heterozygosity (Ho) was 0.584, expected heterozygosity (He) was 0.581, polymorphic information content (PIC) was 0.521, and Wright’s F-statistics (FIS) was -0.008 among the 12 microsatellite markers. The results based on the 14,456 SNPs from the genotyping array showed that all SNPs had two observed alleles, and the average Ne was 1.616, Ho was 0.372, He was 0.360, PIC was 0.282, and FIS was -0.029. The phylogenetic trees drawn by the two methods were similar, but the numbers on the nodes of the phylogenetic tree indicating the percentage bootstrap values generated from 1,000 resamplings were much higher in the results of the 14,456 SNPs. The genetic structure clustering results (K = 3) of the two methods both clustered all of the 16 white buffaloes into one subpopulation with some gray buffaloes, and the other gray buffaloes could be divided into two subpopulations. The ASIP gene of the Taiwan swamp buffalo was genotyped to detect the LINE-1 insertion, but no such insertion was found. The cause for the white coat color of the Taiwan swamp buffalo should be different from the previous study. The 14,456 SNPs from the genotyping array were also used to conduct a genome-wide association study (GWAS) on the gray and white coat color of the Taiwan swamp buffalo. The results showed 17 most significant SNPs (P < 1 x 10-11) associated with the white coat color, and 26 related genes were identified. In addition, the two most significant SNPs (P < 1 x 10-17) were on the chromosome 18 and were found to be close to the MC1R gene, which is a well-known pigmentation-related gene. Therefore, the coding region of the MC1R gene of the Taiwan swamp buffalo was sequenced. A missense variant (MC1R c.901C>T) causing an amino acid substitution (p.R301C) was detected exclusively in the Taiwan swamp buffalo. MC1R c.901C>T genotyping was further performed on 115 gray and 18 white Taiwan swamp buffaloes. The results showed that 17 white buffaloes were homozygous for the variant, one white and 37 gray buffaloes were heterozygous, and 78 gray buffaloes were homozygous for the wild-type allele (P < 1 x 10-21). All of the eight tools used to predict potential effect of p.R301C on MC1R structure or function indicated the mutation to be deleterious. The gene expression analysis by qPCR showed that MC1R, ASIP, MITF, TYR, TYRP1, and DCT gene expression in the ear skin tissue between one white calf and two gray calves had no significant difference. Histochemical staining (Fontana-Masson) was used to observe deposition of melanin in the skin tissue of the Taiwan swamp buffalo. The results showed that the skin tissue of the white Taiwan swamp buffalo had less melanin deposition, but it could still produce melanin. In conclusion, this study provided information for breeding designation and coat color trait selection of the Taiwan swamp buffalo. The MC1R c.901C>T is a strong candidate that may damage the MC1R protein function and cause the white coat color of the Taiwan swamp buffalo. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90033 |
DOI: | 10.6342/NTU202303872 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 動物科學技術學系 |
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