利用 DNA 分子標識進行臺灣黃牛族群遺傳結構分析與生長性狀相關性之探討

Abstract

近年來臺灣的國產牛肉市場越來越受到重視，其中臺灣黃牛為我國重要的本土肉牛品種，然而其族群數量卻急遽下降，為了維護臺灣黃牛之純種種源與遺傳歧異度，自 1987 年於農業委員會畜產試驗所恆春分所設立保種場。因此本試驗第一個目標為開發牛隻新微衛星標識（microsatellite markers），以分析臺灣黃牛與其他牛隻族群在遺傳結構上的差異。第二，利用單一核苷酸多態性（single nucleotide polymorphism, SNP）標識分析其與臺灣黃牛族群生長性狀之相關性，以利進行未來肉牛的選拔及育種。 本試驗採用選擇性雜合法（selective hybridization），將含有重複序列片段（repeat units）之探針（probes）與一公一母之雜交黃牛基因體 DNA 樣本進行雜合（hybridization），以建立微衛星標識豐富化之基因庫（microsatellite-enriched genomic library）。共開發 15 組新微衛星標識，於 8 個族群共 637 頭牛隻進行檢測，試驗結果顯示， 15 組新微衛星基因座之交替基因數（number of alleles, Na） 與有效交替基因數（number of effective alleles, Ne）之平均值各為 8.2±4.2 和 3.0±1.4；期望異質度（expected heterozygosity, HE）與觀測異質度（observed heterozygosity, HO）之平均值分別為 0.588±0.191 與 0.424±0.164；多態性訊息含量（polymorphic information content, PIC）之平均值為 0.552±0.188，且其中 10 組具有高度多態性（PIC ≥ 0.50）。另外，FIS 之平均值為 0.184，FIT 之平均值為 0.271，而 FST 之平均值為 0.108。全部新微衛星基因座之綜合個體鑑別率（probability of identity, P(ID)）與綜合近親個體鑑別率（probability of identity among sibs, P(ID)sib）分別為 9.8×10-12 與 3.1×10-5。利用鄰位連接（neighbor-joining）法根據 8 個牛隻族群之遺傳距離所繪製的親緣關係樹中可觀察到，此 15 組新微衛星標識可明確的區分臺灣黃牛族群與其他牛隻族群。 在第二部分的試驗中，選擇MYF5 g.1948 A>G、ZBED6 g.680C>G 及 SREBP1 g.10781C>G 此三個基因變異位點，針對 168 頭臺灣黃牛進行生長性狀之相關性分析，所搜集之性狀資料有牛隻的出生重、4、6、8、10 及 12 月齡體重。試驗結果顯示，MYF5 基因型在牛隻的 12 月齡體重有趨勢上的影響 （P = 0.07）， AG 或 GG 基因型的個體相較於 AA 基因型有較重的體重。而在 ZBED6 基因， CG 或 GG 基因型的牛隻相較於 CC 基因型有顯著較重的 10 月齡體重（P < 0.05）。牛隻 SREBP1 基因型則是與出生重有顯著相關（P < 0.05），雜合子個體相較於純合子個體有較重的出生體重。 綜合所述，本試驗所開發之 15 組牛隻新微衛星標識，可供進行國內臺灣黃牛及其他牛隻族群之族群遺傳結構檢測與個體鑑別及產銷履歷之驗證。此外，在本試驗中所使用的三個 SNP 位點有潛力作為肉牛選拔育種之分子標識。 牛短脊椎綜合症（brachyspina syndrome, BS）為一種隱性遺傳致死疾病，本症狀形成的原因為牛隻 FANCI 基因有 3.3 kb 片段的缺失。本試驗第三個目標為分析臺灣地區荷蘭牛、臺灣黃牛及臺灣水牛族群中帶有此症狀之雜合子個體頻率，結果顯示所檢測之臺灣黃牛及水牛族群皆為正常個體，而在 53 頭荷蘭牛隻中有 5 頭為雜合子，故有必要進行大規模之荷蘭牛族群篩檢，並以選擇性配種的方式，逐步篩除此不良基因。

In recent years, great importance of the domestic beef market has been attached. The Taiwan yellow cattle is an important indigenous beef cattle breed in Taiwan. However, the population size of the Taiwan yellow cattle has dropped dramatically. Therefore, to preserve germplasm resources and genetic diversity, the conservation population has been maintained in Hengchung Branch of Livestock Research Institute, C.O.A. since 1987. The first goal of this study was to develop novel microsatellite markers to clarify the difference of genetic structure between the Taiwan yellow cattle and hybrid cattle. Secondly, using single nucleotide polymorphism (SNP) markers in analyzing their association with growth traits in the Taiwan yellow cattle for future beef cattle selection and breeding. A microsatellite-enriched genomic library was constructed from one male and one female hybrid yellow cattle DNA samples using selective hybridization method with mixed probes containing different repeat units. There were 15 sets of novel microsatellite markers were developed and used to analyze 637 cattle sampled from eight populations. The average number of alleles (Na) and effective alleles (Ne) were 8.2±4.2 and 3.0±1.4, respectively. Among these markers, the average expected heterozygosity (HE) and observed heterozygosity (HO) were 0.588±0.191 and 0.424±0.164, respectively. The estimated average polymorphic information content (PIC) was 0.552±0.188, and 10 of these markers were highly polymorphic (PIC ≥ 0.50). The inbreeding coefficient in the subpopulation (FIS) was 0.184. The inbreeding coefficient in the total population (FIT) was 0.271. The average differentiation among populations (FST) was 0.108. The total probability of identity (P(ID)) and the total probability of identity among sibs (P(ID)sib) were 9.8×10-12 and 3.1×10-5, respectively. The neighbor-joining (NJ) trees were constructed among the eight populations on the basis of the genetic distance estimated from the 15 sets of novel microsatellite markers. The results indicated that Taiwan yellow cattle populations could be successfully separated from other cattle populations by using these markers. In the second part of this study, three SNP markers: MYF5 g.1948 A>G, ZBED6 g.680C>G, and SREBP1 g.10781C>G, were selected to examine their association with growth traits in 168 Taiwan yellow cattle. The traits studied were the body weight (BW) at birth, 4, 6, 8, 10, and 12 months. Association of MYF5 genotypes with BW12 were nearly significant (P = 0.07), with the genotype AG or GG might have heavier body weight than genotype AA. Individuals with the genotype CG or GG of ZBED6 gene showed significantly heavier BW10 than genotype CC (P < 0.05). Association of SREBP1 genotypes with BW0 were significant (P < 0.05), with the heterozygous genotype have heavier birth weight than homozygous genotypes. In conclusion, these 15 sets of novel microsatellite markers developed in this study could be applied for monitoring genetic background structure of Taiwan yellow cattle and other cattle populations. Additionally, these three characterized SNPs could be used as potential markers for selection and breeding of beef cattle. Bovine brachyspina syndrome (BS) is a recessive genetic defect, caused by a 3.3 kb DNA deletion in the bovine FANCI gene. The third part of this study was to analyze the frequency of BS carrier in Taiwan Holstein, Taiwan yellow cattle and Taiwan water buffalo population. The result indicated that all of the Taiwan yellow cattle and water buffalo were normal individuals, and 5 of 53 Holstein cattle were BS carriers. This genetic defect should be eliminated gradually by large-scale genotyping and selective mating.