王 曦，張?jiān)獞c*，賀東昌，張喜忠，李 博，王棟才，靳 光，李福星，楊效民，徐 芳

(1.山西省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所，太原 030032； 2.山西省運(yùn)城市黃牛場(chǎng)，運(yùn)城 044500)

晉南牛與部分地方黃牛之間遺傳多樣性分析

王 曦1，張?jiān)獞c1*，賀東昌1，張喜忠1，李 博1，王棟才1，靳 光1，李福星2，楊效民1，徐 芳1

(1.山西省農(nóng)業(yè)科學(xué)院畜牧獸醫(yī)研究所，太原 030032； 2.山西省運(yùn)城市黃牛場(chǎng)，運(yùn)城 044500)

旨在利用微衛(wèi)星技術(shù)檢測(cè)晉南牛、郟縣紅牛、魯西牛和秦川牛四大地方黃牛的牛群遺傳多樣樣及遺傳結(jié)構(gòu)。采用16個(gè)微衛(wèi)星DNA標(biāo)記對(duì)4個(gè)牛群進(jìn)行檢測(cè)分析。16個(gè)微衛(wèi)星DNA標(biāo)記中，除HEL9位點(diǎn)在所有牛群中呈單態(tài)外，其他15個(gè)位點(diǎn)的有效等位基因數(shù)為2～8個(gè)，平均有效等位基因數(shù)為3.067 6個(gè)。BM1818為低度多態(tài)(PIC=0.083 0，PIC<0.25)，其余位點(diǎn)均為高度多態(tài)(PIC>0.5)，其中HUAT24多態(tài)性最大(PIC=0.727 5)。4個(gè)牛群共檢測(cè)到80個(gè)等位基因，其中晉南牛為63個(gè)，郟縣紅牛為65個(gè)，魯西牛65個(gè)，秦川牛68個(gè)。在IDVGA46位點(diǎn)，僅有晉南牛有B等位基因，而在TGLA44位點(diǎn)，僅有晉南牛沒(méi)有B等位基因。4個(gè)牛群的平均表觀雜合度為0.385 2，平均期望雜合度為0.640 3，平均雜合度為0.578 0。晉南牛在NJ樹(shù)上單獨(dú)聚為一支，與魯西黃牛、郟縣紅牛及秦川牛的遺傳距離分別為0.809 3、0.759 8、0.807 1。結(jié)果表明，晉南牛遺傳資源獨(dú)特，群體遺傳多樣性豐富，是一個(gè)生長(zhǎng)進(jìn)化上較為封閉的群體。

微衛(wèi)星；遺傳多樣性；晉南牛

畜禽品種是畜牧業(yè)生產(chǎn)的基礎(chǔ)，地方畜禽品種更是畜牧業(yè)的稀缺財(cái)富。我國(guó)養(yǎng)牛歷史悠久，不僅積累了豐富的飼養(yǎng)管理經(jīng)驗(yàn)，更選育出不少優(yōu)良地方品種，其中，晉南牛、秦川牛、魯西黃牛與郟縣紅牛等地方品種均為我國(guó)著名的黃牛品種。20世紀(jì)70年代以來(lái)，在國(guó)外肉牛品種與市場(chǎng)經(jīng)濟(jì)的沖擊下，我國(guó)地方牛品種群體不斷萎縮，質(zhì)量下降甚至瀕危滅絕，遺傳資源遭受重大損失。

微衛(wèi)星DNA標(biāo)記具有多態(tài)性強(qiáng)，呈孟德?tīng)柟诧@性遺傳，分布廣泛等特征，常用作動(dòng)物遺傳多樣性評(píng)價(jià)、群體遺傳關(guān)系和分析生物系統(tǒng)進(jìn)化等理想的遺傳標(biāo)記[1-2]。本研究使用微衛(wèi)星技術(shù)，以晉南牛、秦川牛、魯西黃牛和郟縣紅牛為研究對(duì)象，旨在闡明這幾個(gè)地方牛品種的群體遺傳結(jié)構(gòu)和遺傳多樣性狀況，并進(jìn)行對(duì)比，為有效地選種、選育及保種提供依據(jù)和指導(dǎo)。

1 材料與方法

1.1 材料

1.1.1 試驗(yàn)對(duì)象 本試驗(yàn)材料分別從保種場(chǎng)的晉南牛、郟縣紅牛、魯西黃牛和秦川牛保種場(chǎng)各取得耳組織樣品100份。

1.1.2 耳組織樣品的采集 用耳組織取樣鉗從牛耳前沿取樣，加入到裝有75%酒精的1.5 mL離心管中，置入冰壺中運(yùn)回實(shí)驗(yàn)室。

1.1.3 引物來(lái)源及微衛(wèi)星位點(diǎn)選擇 微衛(wèi)星位點(diǎn)應(yīng)選取雜合度高、多態(tài)信息含量高、易擴(kuò)增的位點(diǎn)。根據(jù)這些特點(diǎn)，在牛不同染色體上選16個(gè)多態(tài)性適中，擴(kuò)增片段長(zhǎng)度約200 bp的微衛(wèi)星位點(diǎn)。本試驗(yàn)所用的16對(duì)引物是從國(guó)際互聯(lián)網(wǎng)(http：//sol.marc.usda.gov)檢索獲得，而后由華大基因合成。引物信息如表1所示。

1.1.4 PCR擴(kuò)增 在200 μL EP管中加入：10×PCR緩沖液2.0 μL，2.5mmol·L-1dNTP 1.6 μL，TaqDNA聚合酶0.5 μL 1U，上下游引物各30 ng，基因組DNA約100 ng，加超純水至總反應(yīng)體積20 μL。反應(yīng)程序：94 ℃預(yù)變性5 min，35個(gè)循環(huán)(94 ℃變性30 s，55 ℃退火45 s，72 ℃延伸90 s)，72 ℃延伸10 min，4 ℃保存。

1.2 方法

1.2.1 DNA提取及微衛(wèi)星檢測(cè) 采用苯酚-氯仿抽提法進(jìn)行基因組DNA提取，檢測(cè)其濃度、純度合格后，對(duì)基因組DNA適當(dāng)稀釋，進(jìn)行PCR擴(kuò)增，經(jīng)檢測(cè)PCR擴(kuò)增產(chǎn)物為預(yù)計(jì)片段大小，再用聚丙烯酰胺凝膠電泳進(jìn)行各微衛(wèi)星位點(diǎn)的多態(tài)性檢測(cè)。

1.2.2 統(tǒng)計(jì)分析 等位基因數(shù)及各基因頻率，多態(tài)信息含量(Polymorphism information content，PIC)，有效等位基因數(shù)，平均表觀雜合度，平均期望雜合度，平均雜合度等參數(shù)采用Microsatellite Toolkit軟件統(tǒng)計(jì)分析。群體間的Nei’sDA遺傳距離用Dispan軟件計(jì)算，并構(gòu)建NJ樹(shù)。

2 結(jié) 果

2.1 基因組DNA及各微衛(wèi)星位點(diǎn)產(chǎn)物凝膠檢測(cè)

將從牛耳組織中提取的全基因組DNA進(jìn)行瓊脂糖凝膠(濃度為0.8%)電泳(圖1)。由圖1中可以看出，基因組DNA條帶清晰，點(diǎn)樣孔干凈，無(wú)拖尾現(xiàn)象，樣品亮度均一，無(wú)蛋白污染，不存在降解，可用于后續(xù)試驗(yàn)。

M.DNA相對(duì)分子質(zhì)量標(biāo)準(zhǔn)DL2000；1～7.基因組DNAM.DL2000 marker；1-7.Genomic DNA 圖1 ?；蚪MDNA瓊脂糖凝膠檢測(cè)Fig.1 Agarose gel image of genomic DNA

微衛(wèi)星PCR擴(kuò)增產(chǎn)物瓊脂糖凝膠(濃度為1.5%)檢測(cè)結(jié)果如圖2所示，上樣PCR產(chǎn)物量為3 μL，DNA的量約為100 ng，結(jié)果顯示，PCR產(chǎn)物條帶整齊，特異性好，產(chǎn)物亮度表明PCR濃度較高，能夠滿足微衛(wèi)星的基因型檢測(cè)。

微衛(wèi)星PCR產(chǎn)物聚丙烯酰胺凝膠(濃度為8%)檢測(cè)(以微衛(wèi)星位點(diǎn)ETH10為例)如圖3所示，樣品上樣量為6 μL，圖中雖然有影子帶存在，但是不影響基因型判定。

表1 微衛(wèi)星位點(diǎn)、引物序列和退火溫度

Table 1 Microsatellite locus，sequences and annealing temperature

微衛(wèi)星座位Loci染色體Chromosome引物序列(5'-3')Primersequences退火溫度/℃AnnealingtemperatureILSTS00510F:GGAAGCAATGAAATCTATAGCCR:TGTTCTGTGAGTTTGTAAGC57.3BM21132F:GCTGCCTTCTACCAAATACCCR:CTTCCTGAGAGAAGCAACACC59.6IDVGA273F:GGTCAGTCACAGAGAAGCACAGR:GAAAGCCTGGTACTCATGGAATA57.3ETH2259F:GATCACCTTGCCACTATTTCCTR:ACATGACAGCCAGCTGCTACT61.1BM18241F:GAGCAAGGTGTTTTTCCAATCR:CATTCTCCAACTGCTTCCTT54.4HEL521F:GCAGGATCACTTGTTAGGGAR:AGACGTTAGTGTACATTAAC52.5HAUT2422F:GAAGTGACTTAGCAGCAGCAR:GCATGTCTGTAAGAATAGTAAG52.5TGLA442F:AACTGTATATTGAGAGCCTACCATGR:CACAACTTAGCGACTAAACCACCA59.6TGLA532F:GCTTTCAGAAATAGTTTGCATTCAR:ATCTTCACATGATATTACAGCAGA54.4ETH319F:GAACCTGCCTCTCCTGCATTR:GGAAGGGAAGACCAGGTGTG61.5HAUT2726F:GACTGGATTGTTTGCTTGTTGR:AGCCATCATTATCTTTACTCTG52.5BM181823F:AGCTGGGAATATAACCAAAGGR:AGTGCTCAAGGTCCATGC59.6IDVGA4619F:AAATCCTTTCAAGTATGTTTTCAR:ACTCACTCCAGTATTCTTGTCTG52.5BM122520F:TTTCTCAACAGAGGTGTCCACR:ACCCCTATCACCATGCTCTG59.6ETH105F:GTTCAGGACTGGCCCTGCTAACAR:CCTCCAGCCCACTTTCTCTTCTC50.8HEL914F:CCCATTCAGTCTTCAGAGGTR:CACATCCATGTTCTCACCAG57

F.正向引物；R.反向引物

F.Forward primer；R.Reverse primer

2.2 各位點(diǎn)的群體研究

2.2.1 各等位基因及頻率 對(duì)晉南牛、郟縣紅牛、魯西牛和秦川牛群16個(gè)微衛(wèi)星位點(diǎn)不同等位基因的檢測(cè)結(jié)果顯示，除HEL9為單態(tài)位點(diǎn)，其余位點(diǎn)均表現(xiàn)為多態(tài)，等位基因及頻率如表2～6所示，4個(gè)牛群共檢測(cè)到80個(gè)等位基因，其中晉南牛為63個(gè)，郟縣紅牛為65個(gè)，魯西牛65個(gè)，秦川牛68個(gè)。表2～6列出基因頻率及等位基因。試驗(yàn)中檢測(cè)出了在一些品種特有的等位基因，例如：在IDVGA46位點(diǎn)，僅有晉南牛有B等位基因；而在TGLA44位點(diǎn)，只有晉南牛沒(méi)有B等位基因；其中ETH10與BM1225等位基因最多，為8個(gè)，ILSTS005等位基因最少，為2個(gè)。等位基因的大小主要集中于150～200 bp之間，如表6所示，其中BM1818位點(diǎn)的等位基因片段最大為226～246 bp，IDVGA27位點(diǎn)的片段最小為95～118 bp。

M.DNA相對(duì)分子質(zhì)量標(biāo)準(zhǔn)DL2000；1～3.BM1225位點(diǎn)，PCR產(chǎn)物片段長(zhǎng)度約為210 bp；4～6.BM1818位點(diǎn)，PCR產(chǎn)物片段長(zhǎng)度約為250 bpM.DL2000 marker；1-3.PCR products of BM1225 locus about 210 bp；4-6.PCR products of BM1818 locus about 250 bp圖2 微衛(wèi)星位點(diǎn)BM1225和BM1818 PCR產(chǎn)物瓊脂糖檢測(cè)Fig.2 Agarose gel image of detecting PCR products of BM1225 and BM1818 loci

1，2，5，7.AD；3.BC；4，8.DD；6，9，13.AC；10，12，14.CD；11.BD；15.AB；M.Marker，from down to up were 100，200 and 300 bp，respectively圖3 微衛(wèi)星位點(diǎn)ETH10 PCR產(chǎn)物聚丙烯酰胺凝膠電泳結(jié)果Fig.3 PAGE image of PCR product of ETH10 locus

表2 4個(gè)群體等位基因頻率

Table 2 Allele frequency of loci in 4 populations

位點(diǎn)Loci等位基因AlleleABCDEFGHETH100.09600.24750.02530.54040.07830.00760.00250.0025BM12250.01770.03790.60860.13380.09850.09090.00760.0051ETH2250.28280.52530.13640.02530.0303BM18180.01260.04040.11360.35350.46970.0101BM18240.13640.17680.38890.25000.0480BM21130.10610.22980.49240.1717HEL50.29800.26260.18430.19440.0606HUAT240.11110.38380.29800.19700.0101HUAT270.01770.06570.15400.27780.36870.1162IDGVA270.10100.46460.33840.0960IDUGA460.56310.30300.04800.05050.00510.02780.0025ILSTS0050.95450.0455TGLA440.05810.32320.38640.14140.0909TGLA530.09850.36620.29040.19950.0455ETH30.36110.32580.04040.23740.0354

2.2.2 各微衛(wèi)星位點(diǎn)遺傳特性分析 4個(gè)牛群中的多態(tài)信息含量結(jié)果如表7所示，BM1818為低度多態(tài)位點(diǎn)(PIC=0.083 0，PIC<0.25)，其余位點(diǎn)均為高度多態(tài)(PIC>0.5)，其中HUAT24多態(tài)信息含量值最大(PIC=0.727 5)。在晉南牛群中，BM1818為低度多態(tài)位點(diǎn)(PIC<0.25)，BM2113、ETH、HEL5及TGLA44屬于中度多態(tài)位點(diǎn)(0.5>PIC>0.25)，其余位點(diǎn)為高度多態(tài)位點(diǎn)(PIC>0.5)。郟縣紅牛群中，BM1818為低度多態(tài)位點(diǎn)，ETH225、ETH3及HUAT27為中度多態(tài)位點(diǎn)，其余均為高度多態(tài)位點(diǎn)。魯西黃牛中，BM1818為低度多態(tài)位點(diǎn)，BM1824為中度多態(tài)位點(diǎn)，其余為高度多態(tài)位點(diǎn)。秦川牛中，BM1818與ETH10為低度多態(tài)位點(diǎn)，BM2113、IDVGA27、ETH225及HUAT27等為中度多態(tài)位點(diǎn)，其余均為高度多態(tài)位點(diǎn)。

表3 晉南牛群體等位基因頻率

Table 3 Allele frequency of loci in Jinnan cattle

位點(diǎn)Loci等位基因AlleleABCDEFGHETH100.04000.23000.56000.16000.0100BM12250.73000.06000.13000.0800ETH2250.35000.43000.15000.03000.0400BM18180.49000.5100BM18240.44000.31000.23000.0200BM21130.03000.14000.8300HEL50.31000.21000.26000.19000.0300HUAT240.20000.64000.1600HUAT270.03000.04000.21000.35000.32000.0500IDGVA270.16000.54000.3000IDUGA460.29000.51000.08000.08000.02000.01000.0100ILSTS0050.94000.0600TGLA440.24000.21000.39000.1600TGLA530.10000.24000.33000.27000.0600ETH30.29000.49000.06000.1600

表4 郟縣紅牛群體等位基因頻率

Table 4 Allele frequency of loci in Jiaxian Red cattle

位點(diǎn)Loci等位基因AlleleABCDEFGETH100.15000.35000.5000BM12250.03000.04000.58000.23000.08000.0400ETH2250.26000.54000.11000.03000.0600BM18180.39000.57000.0400BM18240.10000.15000.25000.39000.1100BM21130.29000.30000.07000.3400HEL50.37000.24000.18000.18000.0300HUAT240.02000.07000.49000.38000.0400HUAT270.01000.10000.07000.33000.32000.1700IDGVA270.72000.2800IDUGA460.60000.28000.03000.0900ILSTS0050.94000.0600TGLA440.03000.10000.56000.11000.2000TGLA530.09000.49000.28000.09000.0500ETH30.16000.26000.08000.36000.1400

表5 魯西牛群體等位基因頻率

Table 5 Allele frequency of loci in Luxi cattle

位點(diǎn)Loci等位基因AlleleABCDEFGHIETH100.06120.29590.05100.50000.07140.01020.0102BM12250.04080.60200.12240.07140.14290.01020.0102ETH2250.21430.53060.20410.03060.0204BM18180.05100.16330.45920.28570.0408BM18240.02040.66330.27550.0408BM21130.08160.26530.52040.1327HEL50.27550.28570.14290.24490.0510HUAT240.07140.47960.34690.1020HUAT270.05100.18370.14290.50000.1224IDGVA270.26530.57140.1633IDUGA460.52040.40820.03060.0408ILSTS0050.95920.0408TGLA440.08160.51020.34690.0612TGLA530.11220.43880.20410.19390.0510ETH30.08160.47960.02040.4184

表6 秦川牛群體等位基因頻率

Table 6 Allele frequency of loci in Qinchuan cattle

位點(diǎn)Loci等位基因AlleleABCDEFGHETH100.13270.11220.05100.60200.08160.0204BM12250.04080.07140.52040.12240.11220.10200.02040.0102ETH2250.30610.60200.08160.0102BM18180.24490.7551BM18240.22450.41840.31630.0408BM21130.02040.21430.55100.2143HEL50.23470.31630.15310.16330.1327HUAT240.15310.34690.19390.3061 HUAT270.03060.07140.15310.28570.33670.1224IDGVA270.14290.40820.36730.0816IDUGA460.84690.01020.05100.08160.0102ILSTS0050.97960.0204TGLA440.12240.44900.4286TGLA530.09180.29590.34690.24490.0204ETH30.91840.07140.0102

表7 4個(gè)群體中微衛(wèi)星標(biāo)記PCR擴(kuò)增條件、片段大小、等位基因數(shù)及PIC值

Table 7PICvalue，length and the number of alleles of all SSRs loci in 4 populations

位點(diǎn)Loci產(chǎn)物長(zhǎng)度/bpLength等位基因數(shù)Numberofallele多態(tài)信息含量PIC晉南牛Jinnan郟縣紅牛JiaxianRed魯西黃牛Luxi秦川牛Qinchuan總體TotalILSTS005172～17820.55290.52690.59900.56010.5834BM2113123～14740.41110.54500.56330.48430.5126IDVGA2795～11840.60680.57270.57500.46290.5654ETH225141～16850.37490.42100.62380.30140.5741BM1824176～19850.58880.70140.41310.60890.6915HEL5143～16950.26230.64710.57960.54380.6136HUAT24148～17050.71230.69620.71800.74210.7275TGLA44185～20850.46900.53390.56770.67440.6613TGLA53108～14250.67830.70290.63900.72340.7053ETH3116～13650.52070.32190.50930.60980.5877HUAT27143～16660.58930.48820.46740.25960.5225BM1818226～24660.10640.10640.07520.03910.0830IDVGA46149～18270.67150.58170.53820.51410.6668BM1225205～23980.70350.61300.67100.67250.6839ETH10190～22480.58700.71170.50150.14250.6482

由表8～12所示，本試驗(yàn)所選15個(gè)微衛(wèi)星多態(tài)位點(diǎn)在4個(gè)群體中平均有效等位基因數(shù)為3.067 6，晉南牛為2.682 5，郟縣紅牛為2.810 7，魯西黃牛為2.665 2，秦川牛為2.668 5。4個(gè)群體中平均純合度觀測(cè)值為0.614 8，晉南牛為0.622 7，郟縣紅牛為0.562 7，魯西黃牛為0.635 4，秦川牛為0.639 5。4個(gè)群體的平均雜合度觀測(cè)值為0.385 2，晉南牛為0.377 3，郟縣紅牛為0.562 7，魯西黃牛為0.364 6，秦川牛為0.360 5。4個(gè)群體中平均純合度期望值為0.359 7，晉南牛為0.418 9，郟縣紅牛為0.394 2，魯西黃牛為0.402 9，秦川牛為0.448 6。4個(gè)群體中平均雜合度期望值為0.640 3，晉南牛為0.581 1，郟縣紅牛為0.605 8，魯西黃牛為0.597 1，秦川牛為0.551 4。4個(gè)群體中平均Nei氏期望雜合度值為0.638 7，晉南牛為0.575 3，郟縣紅牛為0.599 7，魯西黃牛為0.591 0，秦川牛為0.545 8。4個(gè)群體中總平均雜合度值為0.578 0。

2.3 群體遺傳分化

H.Takahashi等[3]和M.Nei[4]通過(guò)計(jì)算機(jī)模擬對(duì)各種遺傳距離進(jìn)行研究，證明在分析物種內(nèi)群體間的遺傳變異時(shí)，運(yùn)用DA遺傳距離是獲得準(zhǔn)確系統(tǒng)發(fā)生樹(shù)的最有效的方法，且用UPGMA法優(yōu)于NJ法。4個(gè)牛群之間的DA遺傳距離見(jiàn)表13，晉南牛與魯西黃牛、郟縣紅牛及秦川牛的遺傳距離分別為：0.809 3、0.759 8、0.807 1?；?個(gè)牛群間DA遺傳距離構(gòu)建的NJ遺傳關(guān)系樹(shù)，明顯地聚為3大支：第一支為晉南牛，第二支為秦川牛，第三支為郟縣紅牛和魯西牛(圖4)。

圖4 基于NJ法構(gòu)建的4個(gè)牛群之間的遺傳關(guān)系樹(shù)Fig.4 Neighbour-joining (NJ) tree of 4 populations based on genetic distance

3 討 論

3.1 各等位基因與頻率

微衛(wèi)星座位上等位基因數(shù)目和頻率的差異可以反映出家畜品種內(nèi)和品種間的差異，近年來(lái)，使用微衛(wèi)星DNA標(biāo)記已經(jīng)開(kāi)展了一些地方牛群體遺傳特征和群體遺傳變異的工作。孫維斌等[5]利用TGLA227、ETH225、HEL9、CSSM66、TGLA1216等5個(gè)微衛(wèi)星檢測(cè)晉南牛遺傳多態(tài)性，結(jié)果發(fā)現(xiàn)，位點(diǎn)均處于不平衡狀態(tài)(P<0.01)，平均有效等位基因數(shù)為10.163 6。畢偉偉[6]利用8個(gè)微衛(wèi)星研究了魯西牛群體的遺傳多態(tài)性，共檢測(cè)到34個(gè)等位基因，每個(gè)微衛(wèi)星座位的等位基因數(shù)為3～6個(gè)，平均等位基因數(shù)為4.25個(gè)。王洪程[7]利用微衛(wèi)星研究秦川牛群體的遺傳多態(tài)性，在144頭秦川牛的7個(gè)微衛(wèi)星位點(diǎn)檢測(cè)到164個(gè)等位基因，平均每個(gè)位點(diǎn)23.43個(gè)等位基因，在IDVGA2位點(diǎn)上檢測(cè)到等位基因最多，為28個(gè)，在IDVGA27位點(diǎn)上檢測(cè)到等位基因最少，為16個(gè)。李榮嶺等[8]利用微衛(wèi)星標(biāo)記對(duì)12個(gè)中外牛品種群體遺傳結(jié)構(gòu)的研究表明，郟縣紅牛的平均等位基因數(shù)最多(10.58)，其他地方品種牛都在8.4以上，而外國(guó)引進(jìn)品種的等位基因數(shù)較少(7～7.8)。本研究中，15個(gè)微衛(wèi)星在4個(gè)群體中總共檢測(cè)到80個(gè)等位基因，其中晉南牛為63個(gè)，郟縣紅牛為65個(gè)，魯西牛65個(gè)，秦川牛68個(gè)，等位基因多態(tài)性比較豐富。研究中檢測(cè)發(fā)現(xiàn)在15個(gè)微衛(wèi)星中，IDVGA46位點(diǎn)僅有晉南牛有B等位基因，為特有等位基因，而在TGLA44位點(diǎn)僅有晉南牛沒(méi)有B等位基因，為共有等位基因。郟縣紅牛在ETH10位點(diǎn)缺少E等位基因，同時(shí)在BM1818有F等位基因，在HUAT24存在E等位基因，在ETH位點(diǎn)檢測(cè)到C等位基因。這些特有等位基因，若在更多的品種中檢測(cè)驗(yàn)證，則可作為代表品種的分子標(biāo)記。

表8 4個(gè)群體微衛(wèi)星位點(diǎn)遺傳參數(shù)統(tǒng)計(jì)

Table 8 Genetic parameter statistics of all SSRs loci in 4 populations

位點(diǎn)Loci樣品數(shù)Number有效等位基因數(shù)Effectivenumberofalleles純合度觀測(cè)值Obs.Hom雜合度觀測(cè)值Obs.Het純合度期望值*Exp.Hom雜合度期望值*Exp.HetNei氏期望雜合度**Nei’sExp.Het**平均雜合度Ave.HetBM12254002.70760.48480.51520.36770.63230.63070.6149ETH104002.45050.46460.53540.40660.59340.59190.5799ETH2254002.65930.52020.47980.37450.62550.62400.6146BM18184002.77460.53030.46970.35880.64120.63960.5169BM18244003.76110.80300.19700.26400.73600.73410.6382BM21134002.97600.59090.40910.33430.66570.66400.5587HEL54004.28760.70200.29800.23130.76870.76680.7579HUAT244003.47990.60100.39900.28560.71440.71260.6231HUAT274003.92260.41920.58080.25300.74700.74510.7274IDVGA274002.85870.95960.04040.34820.65180.65020.5610IDVGA464002.41200.74240.25760.41310.58690.58540.5070ILSTS0054001.09500.91920.08080.91300.08700.08680.0860TGLA444003.50400.63640.36360.28360.71640.71460.6382TGLA534003.70410.39390.60610.26810.73190.73000.7115ETH34003.38130.45450.54550.29400.70600.70430.5343Mean1003.06760.61480.38520.35970.64030.63870.5780St.Dev0.78710.17730.17730.16330.16330.16290.1554

*.期望雜合度和純合度由Levene計(jì)算而來(lái)；**.Nei’s期望雜合度。表9～12同

*.Expected homozygosty and heterozygosity were computed using Levene (1949)；**.Nei’s (1973) expected heterozygosity.The same as Table 9-12

表9 晉南牛微衛(wèi)星位點(diǎn)遺傳參數(shù)統(tǒng)計(jì)

Table 9 Genetic parameter statistics of all SSRs loci in Jinnan cattle

位點(diǎn)Loci樣品數(shù)Number有效等位基因數(shù)Effectivenumberofalleles純合度觀測(cè)值Obs.Hom.雜合度觀測(cè)值Obs.Het純合度期望值*Exp.Hom雜合度期望值*Exp.HetNei氏期望雜合度**Nei’sExp.Het平均雜合度Ave.HetETH101001.78640.62000.38000.38770.61230.60620.6149BM12251002.53940.54000.46000.55540.44460.44020.5799ETH2251003.00840.52000.48000.32570.67430.66760.6146BM18181001.99920.58000.42000.49520.50480.49980.5169BM18241002.91550.80000.20000.33640.66360.65700.6382BM21131001.40960.66000.34000.70650.29350.29060.5587HEL51004.08500.66000.34000.23720.76280.75520.7579HUAT241002.10440.58000.42000.46990.53010.52480.6231HUAT271003.64960.54000.46000.26670.73330.72600.7274IDGVA271002.45581.00000.00000.40120.59880.59280.5610IDUGA461002.79640.70000.30000.35110.64890.64240.5070ILSTS0051001.12710.88000.12000.88610.11390.11280.0860TGLA441003.57910.50000.50000.27210.72790.72060.6382TGLA531003.95260.40000.60000.24550.75450.74700.7115ETH31002.82970.36000.64000.34690.65310.64660.5343Mean1002.68250.62270.37730.41890.58110.57530.5780St.Dev0.89560.17120.17120.18170.18170.17990.1554

表10 郟縣紅牛微衛(wèi)星位點(diǎn)遺傳參數(shù)統(tǒng)計(jì)

Table 10 Genetic parameter statistics of all SSRs loci in Jiaxian Red cattle

位點(diǎn)Loci樣品數(shù)Number有效等位基因數(shù)Effectivenumberofalleles純合度觀測(cè)值Obs.Hom雜合度觀測(cè)值Obs.Het純合度期望值*Exp.Hom雜合度期望值*Exp.HetNe氏期望雜合度*Nei’sExp.Het平均雜合度Ave_HetETH101002.53160.46000.54000.38890.61110.60500.6149BM12251002.50130.40000.60000.39370.60630.60020.5799ETH2251002.66100.38000.62000.36950.63050.62420.6146BM18181002.08940.38000.62000.47330.52670.52140.5169BM18241003.85800.76000.24000.25170.74830.74080.6382BM21131003.39440.72000.28000.28750.71250.70540.5587HEL51003.84320.78000.22000.25270.74730.73980.7579HUAT241002.55490.76000.24000.38530.61470.60860.6231HUAT271003.91850.36000.64000.24770.75230.74480.7274IDGVA271001.67560.92000.08000.59270.40730.40320.5610IDUGA461002.23510.68000.32000.44180.55820.55260.5070ILSTS0051001.12710.88000.12000.88610.11390.11280.0860TGLA441002.65530.44000.56000.37030.62970.62340.6382TGLA531002.96560.34000.66000.33050.66950.66280.7115ETH31004.01930.18000.82000.24120.75880.75120.5343Mean1002.81070.56270.43730.39420.60580.59970.5780St.Dev0.86240.23110.23110.16730.16730.16560.1554

表11 魯西黃牛微衛(wèi)星位點(diǎn)遺傳參數(shù)

Table 11 Genetic parameter statistics of all SSRs loci in Luxi cattle

位點(diǎn)Loci樣品數(shù)Number有效等位基因數(shù)Effectivenumberofalleles純合度觀測(cè)值Obs.Hom雜合度觀測(cè)值Obs.Het純合度期望值*Exp.Hom雜合度期望值*Exp.HetNei氏期望雜合度**Nei’sExp.Het平均雜合度Ave.HetETH101002.86340.46940.53060.34250.65750.65080.6149BM12251002.47020.53060.46940.39870.60130.59520.5799ETH2251002.69930.59180.40820.36400.63600.62950.6146BM18181003.09210.44900.55100.31640.68360.67660.5169BM18241001.93080.91840.08160.51290.48710.48210.6382BM21131002.73620.46940.53060.35890.64110.63450.5587HEL51004.15760.85710.14290.23270.76730.75950.7579HUAT241002.73310.59180.40820.35940.64060.63410.6231HUAT271003.10810.30610.69390.31470.68530.67830.7274IDGVA271002.36091.00000.00000.41760.58240.57640.5610IDUGA461002.27260.81630.18370.43430.56570.56000.5070ILSTS0051001.08500.91840.08160.92090.07910.07830.0860TGLA441002.55700.77550.22450.38480.61520.60890.6382TGLA531003.48480.51020.48980.27960.72040.71300.7115ETH31002.42650.32650.67350.40610.59390.58790.5343Mean1002.66520.63540.36460.40290.59710.59100.5780St.Dev0.69270.22660.22660.15820.15820.15660.1554

表12 秦川牛微衛(wèi)星位點(diǎn)遺傳參數(shù)統(tǒng)計(jì)

Table 12 Genetic parameter statistics of all SSRs loci in Qincuan cattle

位點(diǎn)Loci樣品數(shù)Number有效等位基因數(shù)Effectivenumberofalleles純合度觀測(cè)值Obs.Hom雜合度觀測(cè)值Obs.Het純合度期望值*Exp.Hom雜合度期望值*Exp.HetNei氏期望雜合度**Nei’sExp.Het平均雜合度Ave.HetETH101002.48550.38780.61220.39620.60380.59770.6149BM12251003.16340.38780.61220.30910.69090.68390.5799ETH2251002.16010.59180.40820.45740.54260.53710.6146BM18181001.58690.71430.28570.62630.37370.36980.5169BM18241003.05670.73470.26530.32020.67980.67280.6382BM21131002.52600.51020.48980.38960.61040.60410.5587HEL51004.48790.51020.48980.21480.78520.77720.7579HUAT241003.63510.46940.53060.26760.73240.72490.6231HUAT271004.17570.46940.53060.23160.76840.76050.7274IDGVA271003.04310.91840.08160.32170.67830.67140.5610IDUGA461001.37590.77550.22450.72400.27600.27320.5070ILSTS0051001.04161.00000.00000.95960.04040.04000.0860TGLA441002.49840.83670.16330.39410.60590.59980.6382TGLA531003.61320.32650.67350.26930.73070.72320.7115ETH31001.17840.95920.04080.84700.15300.15140.5343Mean1002.66850.63950.36050.44860.55140.54580.5780St.Dev1.06880.22360.22360.23230.23230.22990.1554

表13 4個(gè)牛品種間Nei’s 遺傳距離(DA，對(duì)角線上方)和Nei's 標(biāo)準(zhǔn)遺傳距離(DS，對(duì)角線下方)

Table 13 Nei’s genetic distance (above the diagonal) and Nei's standard genetic distance (below the diagonal) among 4 indigenous cattle breeds

牛群Population晉南牛Jinnancattle郟縣紅牛JiaxianRedcattle魯西黃牛Luxicattle秦川牛Qinchuancattle晉南牛Jinnan****0.75980.80930.8071郟縣紅牛JiaxianRed0.2747****0.83820.8384魯西黃牛Luxi0.21150.1765****0.8027秦川牛Qinchuan0.21430.17630.2197****

本研究所檢測(cè)的微衛(wèi)星位點(diǎn)中，等位基因數(shù)的范圍是2～7個(gè)，由于群體的規(guī)模有限，所測(cè)的等位基因數(shù)目與文獻(xiàn)報(bào)道有些差異，其中HEL9座位沒(méi)有檢測(cè)到多態(tài)，這與羅永生等的結(jié)果不同[9]，ILSTS005位點(diǎn)等位基因數(shù)最少，僅檢測(cè)到2個(gè)，ETH10和BM1225位點(diǎn)等位基因數(shù)最多，共檢測(cè)到8個(gè)。也反映了微衛(wèi)星位點(diǎn)在種間、群體間的差異，體現(xiàn)了其保守性。

從各微衛(wèi)星的基因頻率來(lái)看，等位基因分布并不均勻，每個(gè)位點(diǎn)中都有一個(gè)或幾個(gè)優(yōu)勢(shì)等位基因存在，且群體中頻率最高的等位基因是該物種中最為保守的，最為原始的，其他的等位基因則是由于該基因位點(diǎn)的突變引起的[10]。

3.2 位點(diǎn)遺傳特性分析

多態(tài)信息含量(PIC)是衡量標(biāo)記多態(tài)性較好的指標(biāo)，D.Bostein等[11]提出了衡量基因變異程度高低的多態(tài)信息指標(biāo)：當(dāng)PIC>0.5時(shí)，為高度多態(tài)性；當(dāng)0.25

孫維斌等[5]檢測(cè)晉南牛TGLA1126位點(diǎn)雜合度、遺傳距離及PIC值，分別為0.976 5、8.940 7、0.885 8。5個(gè)微衛(wèi)星平均雜合度為0.980 9，平均多態(tài)信息含量為0.904 9。王洪程等[7]利用微衛(wèi)星研究秦川牛群體的遺傳多態(tài)性，7個(gè)微衛(wèi)星位點(diǎn)在秦川牛群中表觀雜合度最低值與最高值分別為0.563 9和0.985 5，平均值為0.763 4，期望雜合度最低值與最高值分別為0.886 5和0.951 2，其平均值為0.923 2。7個(gè)位點(diǎn)PIC>0.5，其平均值為0.914 1，均呈高度多態(tài)性。本研究中，4個(gè)群體的平均雜合度期望值為0.640 3，晉南牛為0.581 1，郟縣紅牛為0.605 8，魯西黃牛為0.597 1，秦川牛為0.551 4。4個(gè)群體的平均Nei氏期望雜合度值為0.638 7，晉南牛為0.575 3，郟縣紅牛為0.599 7，魯西黃牛為0.591 0，秦川牛為0.545 8。4個(gè)群體的總平均雜合度值為0.578 0。所以期望的多樣性要高于觀測(cè)的多樣性，因此這4個(gè)種群可能存在選擇與近交[12]。

多態(tài)信息含量、有效等位基因數(shù)等都是衡量群體內(nèi)遺傳變異大小的指標(biāo)，這些數(shù)值在大小上均有一致性，多態(tài)信息含量大，有效等位基因數(shù)相對(duì)大[13-14]。這幾個(gè)參數(shù)較大時(shí)說(shuō)明群體在該位點(diǎn)的變異性高，有較大的選擇潛力，即可利用該位點(diǎn)進(jìn)行與生產(chǎn)性狀相關(guān)的標(biāo)記輔助選擇[15]。

3.3 群體遺傳分化

晉南牛與郟縣紅牛、魯西黃牛、秦川牛的遺傳距離DA分別為0.759 8、0.809 3和0.807 1，距離值不太遠(yuǎn)，可能是因?yàn)檫@4個(gè)品種在發(fā)展進(jìn)化的過(guò)程中由于在地域上的封閉性及差異造成。晉南牛相對(duì)與其他3種牛比較獨(dú)立，可能由于晉南牛在選育過(guò)程中較為封閉，與其他品種的牛血緣上還沒(méi)有過(guò)多的交叉，具有典型的地域性，同時(shí)，由于郟縣紅牛與魯西黃牛在近年來(lái)突破了地域的局限性，基因相互有些滲透，所以同屬于共同的進(jìn)化分支。M.A.Cronin 等利用微衛(wèi)星標(biāo)記研究美國(guó)平原野牛與森林野牛，結(jié)果表明，它們?cè)谌后w遺傳結(jié)構(gòu)上存在有更多的同源性[16]。T.Keros等利用微衛(wèi)星對(duì)克羅地亞本地灰牛群分類后在遺傳結(jié)構(gòu)上與美洲牛群比較，證明其更傾向于歐洲的牛群[17]。

本研究對(duì)晉南牛的遺傳結(jié)構(gòu)及遺傳分化做了分子水平的研究，晉南牛在分子遺傳關(guān)系樹(shù)上獨(dú)成一支，這與雷初朝等利用mtDNA研究牛的分類所得出結(jié)論相一致[18]。這種群體遺傳特性上的獨(dú)立性，從分子水平上證明了晉南牛為我國(guó)寶貴的遺傳資源，因此對(duì)其的保種顯得尤為重要。利用微衛(wèi)星檢測(cè)遺傳結(jié)構(gòu)，可指導(dǎo)牛群的分類及保種工作。L.D.Pham等利用微衛(wèi)星標(biāo)記研究越南地方牛的遺傳多樣性，對(duì)6個(gè)類群從遺傳結(jié)構(gòu)上進(jìn)行分類分群，從而指導(dǎo)保種，大大節(jié)約了保種的費(fèi)用，提高了效率[19]。Y.T.Utsunomiya 等利用AFLP遺傳標(biāo)記研究西亞與南亞的瘤牛、水牛及已經(jīng)滅絕的歐洲野牛牛群的遺傳結(jié)構(gòu)，對(duì)牛群的保種起到積極的指導(dǎo)作用[20]。C.Acosta等對(duì)古巴5個(gè)牛群的遺傳結(jié)構(gòu)利用微衛(wèi)星進(jìn)行了研究，結(jié)果表明，5個(gè)牛群可以接近于分成兩個(gè)牛群，對(duì)于牛群的保種有重要意義[21]。E.Armstrong等利用微衛(wèi)星將烏拉圭牛與美洲牛群作對(duì)比，結(jié)果表明，遺傳漂變等可能會(huì)引起群體的遺傳變異，最終使得烏拉圭牛有別于美洲牛的遺傳群體，因此對(duì)其的保種有著非常重要的意義[22]。

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(編輯 郭云雁)

Analyses of Genetic Diversity among Jinnan Cattle and Three other Chinese Indigenous Cattle Breeds

WANG Xi1，ZHANG Yuan-qing1*，HE Dong-chang1，ZHANG Xi-zhong1，LI Bo1，WANG Dong-cai1，JIN Guang1，LI Fu-xing2，YANG Xiao-min1，XU Fang1

(1.InstituteofAnimalScienceandVeterinaryMedicine，ShanxiAcademyofAgriculturalScience，Taiyuan030032China；2.YunchengYellowCattleFarm，Yuncheng044500，China)

The genetic diversity and genetic structure of Jinnan cattle，Jiaxian Red cattle，Luxi cattle and Qinchuan cattle were detected by microsatellite technology.Allele frequencies and distribution of 16 microsatellite markers were detected in 4 cattle populations.Number of effective alleles of the other 15 loci was from 2 to 8 except HEL9 as a monomorphic loci in all individuals，and the average number of effective alleles was 3.067 6.BM1818 was low genetic polymorphisms (PIC=0.083 0，PIC<0.25)，and the others were high genetic polymorphisms (PIC>0.5)，with the highestPICvalue at HUAT24 locus (PIC=0.727 5).80 alleles were identified in 4 populations (63 alleles in Jinnan cattle，65 in Jiaxian Red cattle，65 in Luxi cattle and 68 in Qinchuan cattle，respectively).The allele B at IDVGA46 was detected only in Jinnan cattle，and the allele B at TGLA44 was only lack in Jinnan cattle.Mean observed heterozygosity，mean expected heterozygosity and mean heterozygosity were 0.385 2，0.640 3 and 0.578 0，respectively in 4 populations.Jinnan cattle was independently clustered in the NJ tree and the genetic distance (DA) with Luxi cattle，Jiaxian Red cattle and Qinchuan cattle were 0.809 3，0.759 8 and 0.807 1.There were special genetic characterics and sufficient genetic diversity in Jinnan cattle，and it was a relatively closed population in its evolutionary process.

microsatellites；genetic diversity；Jinnan cattle

10.11843/j.issn.0366-6964.2015.06.006

2014-07-28

“十二五”國(guó)家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃)子課題(2011AA100307-05)；山西省科技攻關(guān)項(xiàng)目(20130311024-1；20120311021-1；20130311024-2；20140311018-1)；山西省農(nóng)業(yè)科學(xué)院育種工程項(xiàng)目(11yzgc017)；山西省農(nóng)業(yè)科學(xué)院博士基金(YBSJJ1402)

王 曦(1975-)，男，山西嵐縣人，副研究員，博士，主要從事牛遺傳育種與功能基因組研究，E-mail：wxphilip@aliyun.com

*通信作者：張?jiān)獞c，副研究員，E-mail：yuanqing_zhang@163.com

S813.1；S823

A

0366-6964(2015)06-0911-13