DNA條形碼技術(shù)鑒定中國(guó)地方雞品種的重新評(píng)估

黃勛和，陳潔波，何丹林，張細(xì)權(quán)，鐘福生

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DNA條形碼技術(shù)鑒定中國(guó)地方雞品種的重新評(píng)估

黃勛和1，陳潔波1，何丹林2，張細(xì)權(quán)2，鐘福生1

（1嘉應(yīng)學(xué)院生命科學(xué)學(xué)院，廣東梅州 514015；2華南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)學(xué)院，廣州 510642）

【目的】探討COI基因作為標(biāo)準(zhǔn)DNA條形碼技術(shù)鑒定外形差異較小的地方雞品種的可行性?！痉椒ā恳匀A南地區(qū)9種優(yōu)質(zhì)地方雞（懷鄉(xiāng)雞、清遠(yuǎn)麻雞、惠陽(yáng)胡須雞、中山沙欄雞、陽(yáng)山雞、杏花雞、五華三黃雞、文昌雞和廣西三黃雞）和國(guó)外引進(jìn)品種隱性白羽雞為試驗(yàn)材料，測(cè)定標(biāo)準(zhǔn)的DNA條形碼技術(shù)的線粒體細(xì)胞色素C 氧化酶亞基I （cytochromeoxidase subunit I，COI），同時(shí)下載已發(fā)表的31條家雞和原雞及綠頭鴨的COI基因序列，分析品種遺傳多樣性與遺傳距離，構(gòu)建單倍型中介網(wǎng)絡(luò)圖和系統(tǒng)發(fā)生鄰接樹，界定區(qū)分品種特異的單倍型。【結(jié)果】除去PCR引物序列，獲得了695 bp COI基因片段。根據(jù)標(biāo)準(zhǔn)的DNA條形碼序列，截取648 bp 線粒體COI基因序列進(jìn)行分析。10個(gè)雞品種203個(gè)個(gè)體共檢測(cè)到110個(gè)變異位點(diǎn)，占分析位點(diǎn)的16.98%，其中90個(gè)單一位點(diǎn)突變，20個(gè)簡(jiǎn)約信息位點(diǎn)。平均核苷酸多樣性為0.00394（0.00349—0.00560），平均單倍型多樣性為0.832（0.763—0.905），其中五華三黃雞最高，中山沙欄雞次之，文昌雞最低。定義了84種單倍型，單倍型1為9個(gè)地方雞種所共享，出現(xiàn)頻率為64次；單倍型9和5為家雞和隱性白羽雞共享，出現(xiàn)頻率分別為29次和19次；每個(gè)雞品種均有品種特異的單倍型。廣西三黃雞、五華三黃雞與中山沙欄雞的單倍型數(shù)最多，為13個(gè)，隱性白羽雞與清遠(yuǎn)麻雞的最少，為8個(gè)。不同品種的單倍型分布差異較大，如杏花雞的單倍型主要分布在1，清遠(yuǎn)麻雞主要分布在1和9，惠陽(yáng)胡須雞主要分布在1、5和9，隱性白羽雞主要分布在9和79。10個(gè)雞種品種間遺傳距離范圍為0.003—0.006，凈遺傳距離為0—0.003；雞品種間的遺傳距離一般大于雞品種內(nèi)的遺傳距離；綠頭鴨與雞品種間的遺傳距離大于0.2。中介網(wǎng)絡(luò)圖將84個(gè)單倍型分為3條進(jìn)化枝，呈現(xiàn)出一定的品種特異性，如以單倍型9為起點(diǎn)的進(jìn)化枝沒有廣西三黃雞和文昌雞分布，但另外兩枝未表現(xiàn)出此特征；1為祖先單倍型，由此逐漸衍生出其他單倍型。鄰接樹顯示中國(guó)家雞與紅原雞聚為一簇，與黑尾原雞、灰原雞和綠原雞分開；中國(guó)地方雞聚為同一簇，且存在明顯的交叉現(xiàn)象，無(wú)顯著的品種特異性?！窘Y(jié)論】COI基因可作為研究雞品種遺傳多樣性的候選分子標(biāo)記。僅依靠標(biāo)準(zhǔn)的DNA條形碼技術(shù)無(wú)法有效區(qū)分差異外形較小的地方雞種，需要聯(lián)合多種分子標(biāo)記如COI基因、細(xì)胞色素b、 AFLP指紋技術(shù)、微衛(wèi)星位點(diǎn)LEI0258、基因組SNP和品種特異的外貌特征。

線粒體COI基因；DNA條形碼；地方雞種；品種鑒定；遺傳多樣性

0 引言

【研究意義】作為分布最廣泛的家禽，家雞（）在人類生產(chǎn)生活中扮演著重要角色。中國(guó)家雞資源豐富，僅列入《中國(guó)畜禽遺傳資源志·家禽志》的地方雞品種就有107個(gè)[1]。隨著外來(lái)引進(jìn)品種和大量商品雞的飼養(yǎng)，地方雞的種質(zhì)特性受到極大的影響。尋求快速簡(jiǎn)便的區(qū)分外形多樣、遺傳復(fù)雜的地方雞品種的技術(shù)方法是科學(xué)家和育種專家急需解決的問(wèn)題。由于線粒體細(xì)胞色素C 氧化酶亞基I（cytochromeoxidase I，COI）具有相對(duì)保守與穩(wěn)定的變異性的特性，已作為DNA條形碼（DNA barcoding）研究的標(biāo)準(zhǔn)基因廣泛應(yīng)用于脊椎動(dòng)物和無(wú)脊椎動(dòng)物的系統(tǒng)分類、種類鑒別、群體遺傳多樣性和分子進(jìn)化學(xué)研究[2-7]?！厩叭搜芯窟M(jìn)展】自加拿大生物學(xué)家Hebert等[8]首先倡導(dǎo)將648 bp COI基因作為DNA條形碼編碼技術(shù)的標(biāo)準(zhǔn)基因應(yīng)用于生物物種鑒定以來(lái)，獲得了蓬勃的發(fā)展。目前BOLD系統(tǒng)（The Barcode of Life Data System）記錄的標(biāo)本量達(dá)5 259 477件，其中條形碼研究3 881 860，物種數(shù)為235 994[9]。Kerr等[10]通過(guò)研究643種北美鳥類COI基因，發(fā)現(xiàn)94%的種類擁有明顯的條形碼分支，剩余的6%大部分是有規(guī)律雜交的物種。Ward[11]總結(jié)了FISH-BOL（The Fish Barcode of Life Initiative）的研究成果表明，DNA條形碼技術(shù)可以有效區(qū)分98%的海洋魚類和93%的淡水魚類。WILSON等[12]建立了馬來(lái)西亞半島鱗翅目蝴蝶的DNA條形碼參考數(shù)據(jù)庫(kù)并定義了亞種的參考閾值。孟瑋等[13-16]分別驗(yàn)證了COI基因作為DNA條形碼在魚類物種鑒定、鳥類分類、銀鯧群體遺傳多樣性、動(dòng)物藥材鑒定等方面的可行性。動(dòng)物品種鑒定方面，高玉時(shí)等[2,17-18]采用DNA條形碼技術(shù)驗(yàn)證了差異較大的地方雞品種鑒定的可行性和有效性?！颈狙芯壳腥朦c(diǎn)】之前的地方雞DNA條形碼鑒定研究主要針對(duì)于外觀差異較大、地緣分布較遠(yuǎn)的品種，而對(duì)于外觀相近，地緣集中的品種則尚未開展研究?！緮M解決的關(guān)鍵問(wèn)題】本研究以華南9個(gè)優(yōu)質(zhì)地方雞和國(guó)外引進(jìn)品種隱性白羽雞作為試驗(yàn)材料，采用標(biāo)準(zhǔn)的DNA條形碼技術(shù)對(duì)10個(gè)雞品種進(jìn)行分子鑒定，評(píng)估COI基因作為DNA條形碼鑒定外形差異較小的雞品種的可行性。

1 材料與方法

1.1 試驗(yàn)材料

以華南9種優(yōu)質(zhì)地方雞和國(guó)外引進(jìn)品種隱性白羽雞共203個(gè)個(gè)體為試驗(yàn)材料（表1）。除五華三黃雞之外，其他8種地方雞種均入選《中國(guó)畜禽遺傳資源志·家禽志》[1]，而五華三黃雞入選《中國(guó)禽類遺傳資源》[19]。五華三黃雞、陽(yáng)山雞采自其原產(chǎn)地，文昌雞、清遠(yuǎn)麻雞采自商品群，其余6種采自保種場(chǎng)，五華三黃雞的樣品類型為羽毛，其他品種均為血液；從基因庫(kù)下載已發(fā)表的雞品種如黃郎雞、桃源雞、仙居雞等和原雞如紅原雞、灰尾原雞和綠原雞等的COI基因全序列，同時(shí)下載綠頭鴨COI基因作為計(jì)算遺傳距離的參考外群（http://www.ncbi.nlm.nih.gov/nuccore/）。使用試劑盒HiPure Blood/Tissue DNA Mini Kit（美基生物，廣州）提取樣品基因組DNA，-20℃保存?zhèn)溆谩?/p>

表1 樣品信息

1.2 PCR擴(kuò)增與序列測(cè)定

為提高PCR擴(kuò)增的特異性和效率，參照鳥類DNA條形碼通用引物和已發(fā)表的黃郎雞線粒體全基因組序列（GU261701）微調(diào)COI基因的擴(kuò)增引物（BirdF1：5′-TTCTCCAACCACAAAGACATTGGCAC-3′[20]，COIR：5′- CGTGGGAAATTATTCCGAAACCTG -3′）。PCR反應(yīng)體系為30 μL，含10×PCR buffer 3μL，dNTP mixture（2.5 mmol·L-1）2.4 μL，引物各0.3 μL（20 μmol·L-1），rTaq DNA聚合酶（寶生物, 大連）1.5 U，DNA模板100 ng。擴(kuò)增條件為94℃預(yù)變性4 min，然后35個(gè)循環(huán)（94℃變性30 s，58℃退火1 min，72℃延伸50 s），最后72℃延伸7 min。PCR產(chǎn)物于1.5%瓊脂糖凝膠電泳檢測(cè)后送上海立菲生物技術(shù)有限公司（廣州）雙向測(cè)序。

1.3 序列分析

Bioedit 軟件讀取測(cè)序序列，對(duì)每個(gè)序列進(jìn)行人工逐個(gè)堿基檢查校對(duì)，并通過(guò)每個(gè)樣品雙向測(cè)序和每個(gè)單倍型重新測(cè)序進(jìn)行雙重校正。采用標(biāo)準(zhǔn)的648 bp DNA條形碼序列（對(duì)應(yīng)小鼠COI基因第58—705位堿基）作為分析數(shù)據(jù)。所獲序列用Clustal X軟件[21]進(jìn)行對(duì)位排列后，用DnaSP5.0軟件[22]定義單倍型，計(jì)算單倍型多樣性（Haplotype diversity，）、核苷酸多樣性（nucleotide diversity，π）和核苷酸差異均數(shù)（）。通過(guò)NETWORK 4.6.1.4 軟件[23]構(gòu)建單倍型中介連接網(wǎng)絡(luò)圖（Median-joining networks）。以綠頭鴨為外群（，KJ883269），用MEGA6.0 軟件[24]中的Kimura 2-parameter（K2P）模型計(jì)算遺傳距離；應(yīng)用鄰接法（neighbor-joining，NJ）構(gòu)建系統(tǒng)進(jìn)化樹。

2 結(jié)果

2.1 COI基因序列的核苷酸位點(diǎn)變異、核苷酸多樣性和單倍型多樣性

除去PCR引物序列，獲得了695 bp COI基因片段。根據(jù)標(biāo)準(zhǔn)的DNA條形碼序列，截取648 bp 線粒體COI基因序列進(jìn)行分析。10個(gè)雞種203個(gè)個(gè)體線粒體COI基因共檢測(cè)到110個(gè)變異位點(diǎn)，占分析位點(diǎn)的16.98%，其中單一位點(diǎn)突變90個(gè)（2型20個(gè)，3型2個(gè)），簡(jiǎn)約信息位點(diǎn)20個(gè)。五華三黃雞的平均核苷酸差異數(shù)最大，為3.629，文昌雞的最小，為1.455；相應(yīng)地，五華三黃雞的核苷酸多樣性最高，文昌雞的最?。ū?）。在110個(gè)變異位點(diǎn)中，定義了84種單倍型，其中單倍型1出現(xiàn)頻率最高，為64次（9個(gè)地方雞種所共有，隱性白羽雞除外），其次是單倍型9，為29次（8個(gè)雞種共有，杏花雞、文昌雞除外），第三是單倍型5，為19次（9個(gè)雞種共有，清遠(yuǎn)麻雞除外）（表3）。廣西三黃雞、五華三黃雞與中山沙欄雞的單倍型數(shù)最多，為13個(gè)，隱性白羽雞與清遠(yuǎn)麻雞的最少，為8個(gè)。不同品種的單倍型分布差異較大，如隱性白羽雞單倍型主要分布在9和79，清遠(yuǎn)麻雞主要分布在1和9，惠陽(yáng)胡須雞主要分布在1、5和9，杏花雞主要分布在1（表3）。

表2 10個(gè)雞種COⅠ基因序列變異位點(diǎn)數(shù)、平均核苷酸差異、核苷酸多樣性和單倍型多樣性

表3 10個(gè)雞品種線粒體COI基因的單倍型及其在不同品種的分布

續(xù)表3 Continued table 3

單倍型Haplotype變異位點(diǎn)Variable sites (in bp)單倍型在品種的分布（頻率）Breeds (frequency) 1111111111111111122222222222222222223333333333333333334444444444444444444455555555555555666122344555566677899011123455666789990111122344556888999011123466788888999002233344555667777890000113555689901490121492378247473091035379155674046850368058051710461261048752697035680457868467041271825781702792611251104294 Hap27.............................G................................................................................SL(1) Hap28.........C........C................G.......................G..................................................SL(1) Hap29.........C........C................G...........................................C..................A...........SL(1) Hap30.........C........C................G....................A.....................................................SL(1),WH(1) Hap31...................G..........................................................................................SL(1),YS(1) Hap32...............................................................................................T........G.....SL(1) Hap33......................................................................C.......................................SL(1) Hap34.........C........C................G........C.................................................................SL(1) Hap35.........C......T.C................G..........................................................................YS(1) Hap36...............................................................................A..............................YS(1) Hap37.................G.................................C..........................................................YS(1) Hap38.........C........C................G..............C............................................T...G..........YS(1) Hap39......................G.......................................................................................YS(1) Hap40.........C........C.............G..G..........................................................................YS(1) Hap41......................................................................................................G.....A.XH(1) Hap42....................................................G.........................................................XH(1) Hap43.........CT.......C................G..........................................................................XH(1) Hap44.........................................................................C......................A.............XH(1) Hap45.........C........C....G...........G...................................C..................T...................XH(1) Hap46.....................................................C........................................................XH(1) Hap47.........C........C................G......................G....G....................................C.........XH(1) Hap48......................................................................................................G.......XH(1) Hap49...........................C...................................................................T..............XH(1) Hap50..........................T....................................................................T..............WH(1) Hap51.............................................C................................................................WH(1) Hap52.........C........C................G....................A........................................C............WH(1) Hap53............................T.................................................................................WH(1) Hap54.........C..AA....C...........G....G..................TCA.....G...AC.........G.........................A..G...WH(1) Hap55.........C........C......C.........G..........................................................................WH(1)

續(xù)表3 Continued table 3

單倍型Haplotype變異位點(diǎn)Variable sites (in bp)單倍型在品種的分布（頻率）Breeds (frequency) 1111111111111111122222222222222222223333333333333333334444444444444444444455555555555555666122344555566677899011123455666789990111122344556888999011123466788888999002233344555667777890000113555689901490121492378247473091035379155674046850368058051710461261048752697035680457868467041271825781702792611251104294 Hap56..................C............................................................................T..............WH(1) Hap57.........C........C................G............................G.............................................WH(1) Hap58.....C.........................................G..............................................................WC(1) Hap59........................................G.....................................................................WC(2) Hap60...........................C........G.........................................................................WC(1) Hap61..........................................................................C...................................WC(1) Hap62................................................................................C.............................WC(1) Hap63........................................................................C.....................................WC(1) Hap64................................................T..............................................T..............WC(1) Hap65......A.......................................................................................................WC(1) Hap66....A.A.......................................................................................................WC(1) Hap67...........T..................................................................................................WC(1) Hap68......................................................................................T.......................WC(1) Hap69..............................................C..................................C............................GX(1) Hap70.................................................T..................C.........................................GX(1) Hap71C.............................................................................................................GX(1) Hap72.......T......................................................................................................GX(1) Hap73...A..........................................................................................................GX(1) Hap74......................................G.......................................................................GX(1) Hap75.....................A........................................................................................GX(1) Hap76............................................................T...................C.............................GX(1) Hap77..A....................................G......................................................................GX(1) Hap78..............G..........................................................................G.....T..............GX(1) Hap79.........C........C................G.......................................G..................................RW(6) Hap80.........C........C................G.........................................................................CRW(1) Hap81.........C........C.....C..........G.......................................G..................................RW(1) Hap82.........C........C................G.........................G................................................RW(1) Hap83...............A.......................................................C.......................T..............RW(1) Hap84..................C........................G...................................................T..............RW(1)

圓點(diǎn)表示與單倍型Hap1具有相同的堿基 Dots within the nucleotide position indicate the same nucleotides as given in Hap1

2.2 10個(gè)雞品種群體內(nèi)與群體間遺傳距離

對(duì)10個(gè)雞品種COI基因序列K2P距離和凈遺傳距離（Da）研究顯示，綠頭鴨與雞品種的遺傳距離明顯大于雞品種之間的遺傳距離（表4）。雞品種間遺傳距離范圍為0.003—0.006，隱性白羽雞與9種地方雞種的遺傳距離較遠(yuǎn)；五華三黃雞品種內(nèi)的遺傳距離最大，為0.006，文昌雞品種內(nèi)遺傳距離最小，為0.002。10個(gè)雞種品種凈遺傳距離為0—0.003（表4）。

表4 10個(gè)雞品種間Kiumura 雙參數(shù)距離和凈遺傳距離（Da）

上三角為凈遺傳距離Da，下三角為Kiumura 雙參數(shù)距離

Above dialogue was net distance (Da), down diagonal was K2P distance of COIgene among 10 chicken breeds

2.3 COI基因中介網(wǎng)絡(luò)圖和系統(tǒng)發(fā)育樹

利用表3的單倍型數(shù)據(jù)構(gòu)建中介網(wǎng)絡(luò)圖。中介網(wǎng)絡(luò)圖主要分為3個(gè)進(jìn)化枝，第1枝是以Hap1為中心節(jié)點(diǎn)，另外2枝是從Hap1衍生出來(lái)的以Hap5、Hap9為中心節(jié)點(diǎn)（圖1）。Hap1最有可能是祖先單倍型，逐漸衍生出其他單倍型。以Hap9為中心的進(jìn)化枝表現(xiàn)出一定的品種特異性，如文昌雞和廣西三黃雞不在此枝；另外兩枝則未表現(xiàn)出此特征。

紅色方塊是軟件NETWORK生成的媒介載體，代表在實(shí)際樣本未觀察到的推測(cè)的中間單倍型。圓的大小對(duì)應(yīng)于單倍型頻率。不同雞品種用不同顏色標(biāo)注

采用K2P模型構(gòu)建本研究定義的單倍型（每個(gè)品種所有的單倍型，共121條序列）和下載已發(fā)表的雞COI基因序列（648 bp，29條序列）的系統(tǒng)發(fā)育樹（鄰接樹）。發(fā)育樹分為三枝，第一枝是黑尾原雞和灰原雞，第二枝是綠原雞，第三枝是包括五個(gè)紅原雞亞種在內(nèi)的地方雞（圖2）。第三枝又細(xì)分為兩枝，存在亞種/品種的個(gè)體交叉現(xiàn)象，沒有發(fā)現(xiàn)亞種/品種特異性。

品種名稱縮寫見表1，數(shù)字表示樣品編號(hào) The breed Abbr. please see Table 1 in details, digit represent the sample number

3 討論

3.1 10個(gè)雞品種COI基因序列的遺傳多樣性

本研究獲得了9個(gè)地方雞種和1個(gè)引進(jìn)雞品種共203個(gè)個(gè)體的648 bp線粒體COI基因序列，共檢測(cè)到110個(gè)變異位點(diǎn)，定義了84種單倍型。其中五華三黃雞的遺傳多樣性最高，中山沙欄雞次之，文昌雞最低。由于分布在偏遠(yuǎn)山村，基因交流較少，未進(jìn)行系統(tǒng)選育且樣品為隨機(jī)采集如五華三黃雞[25]，或采自祖代群如中山沙欄雞，或采樣時(shí)間較早如陽(yáng)山雞，這些品種保留著較高的遺傳多樣性。而來(lái)自商品群的地方雞遺傳多樣性則較低，如文昌雞（品種內(nèi)遺傳距離0.002，表4）。84種單倍型在不同的品種的分布有所差異，如單倍型1出現(xiàn)64次，除了隱性白羽雞，其他9個(gè)地方雞都有分布；其次是單倍型9和5；隱性白羽雞主要分布在單倍型9和79，清遠(yuǎn)麻雞主要分布在單倍型1和9，惠陽(yáng)胡須雞主要分布在單倍型1、5和9，杏花雞分布在單倍型1，而其他7個(gè)品種單倍型分布較為分散（表3）。本文研究的雞品種的COI基因序列變異位點(diǎn)數(shù)、平均核苷酸差異、核苷酸多樣性和單倍型多樣性比同等類型研究相近或更高[2,17-18]，說(shuō)明本文選取的雞品種保護(hù)潛力較大，證明標(biāo)準(zhǔn)DNA條形碼用于研究雞品種遺傳多樣性的有效性。另外，以綠頭鴨為外群計(jì)算遺傳距離時(shí)，綠頭鴨與雞品種間的遺傳距離明顯大于雞品種間的遺傳距離，表明COI基因可作為計(jì)算雞品種間的遺傳距離的分子標(biāo)記。

3.2 中國(guó)地方雞品種COI基因系統(tǒng)發(fā)生關(guān)系

鄰接樹顯示中國(guó)地方雞與紅原雞分為兩枝，雖然有些品種分布較為集中（如惠陽(yáng)胡須雞），但均存在亞種/品種的個(gè)體交叉現(xiàn)象（圖2）。當(dāng)提高建樹可信度時(shí)（1000步bootstrap、50% cut-off），所有中國(guó)地方雞與紅原雞聚為一枝（數(shù)據(jù)未顯示），說(shuō)明應(yīng)用標(biāo)準(zhǔn)的DNA條形碼技術(shù)無(wú)法將中國(guó)地方雞品種有效區(qū)分開來(lái)。而中國(guó)地方雞和紅原雞與黑尾原雞、灰原雞、綠原雞分開，間接支持了中國(guó)地方雞與紅原雞親緣關(guān)系較近的觀點(diǎn)。主要原因可能有兩點(diǎn)：一是本研究的9個(gè)地方雞種在廣東飼養(yǎng)量較大，因地域鄰近和經(jīng)貿(mào)頻繁，不同的雞種在品種形成與進(jìn)化過(guò)程中不可避免存在不同程度的雜交，出現(xiàn)了個(gè)別品種間的交叉現(xiàn)象（圖2）；二是COI基因的相對(duì)保守性，品種間的遺傳差異較小（表4）。在本研究中，雖然部分單倍型為特定品種所有，如單倍型12—19為清遠(yuǎn)麻雞特有，但這些單倍型與其他雞品種部分單倍型聚在同一枝（圖2）；并且該結(jié)果是基于有限的樣品（10個(gè)品種，每個(gè)品種18—23只，共203只）得出的，在增加樣品數(shù)量或品種后是否還是清遠(yuǎn)麻雞特有還有待研究。因此，雜交使得亞種/品種鑒定變得困難，分析時(shí)需要在足夠的樣品數(shù)量和聯(lián)合多種方法后才能得出比較可靠的結(jié)論[2,12]。

3.3 應(yīng)用COI基因鑒定地方雞品種

研究表明DNA條形碼對(duì)鑒定差異較大的雞品種鑒定具有可行性和有效性[2,26]。在相近的COI基因片段長(zhǎng)度下，本研究在10個(gè)雞種203只個(gè)體共檢測(cè)到110個(gè)變異位點(diǎn)，多于15個(gè)差異較大的雞種227個(gè)個(gè)體的38個(gè)變異位點(diǎn)[2]，說(shuō)明本研究采用的COI基因片段信息含量較高。雖然不同的品種有特異的單倍型，但分布不集中，且大多只有1個(gè)個(gè)體（隱性白羽雞除外，單倍型79有6個(gè)個(gè)體），因此，無(wú)法確定是否能夠作為品種鑒定的診斷依據(jù)，需加大樣本量進(jìn)行驗(yàn)證。在本研究的9種華南家雞中，外形特征較為顯著，同時(shí)按照品種標(biāo)準(zhǔn)采集樣品，因同品種異名而導(dǎo)致COI無(wú)法區(qū)分開這9種地方雞品種的可能性極低。此外，有趣的是，單倍型1為9個(gè)地方雞品種所共有，隱性白羽雞除外，可作為中國(guó)地方雞品種的鑒定依據(jù)。由于地方雞品種復(fù)雜的形成與進(jìn)化歷史和COI基因自身的屬性，運(yùn)用標(biāo)準(zhǔn)的DNA條形碼技術(shù)無(wú)法鑒定本研究的9個(gè)親緣關(guān)系較近、地域鄰近的華南地方雞種。因此，有效的品種鑒定還需聯(lián)合多種分子標(biāo)記如COI基因[26]、AFLP指紋技術(shù)[27]、細(xì)胞色素b[28]、LEI0258[29]、基因組SNP[30]等，以及與品種特定的外形特征如清遠(yuǎn)麻雞的“一楔、二細(xì)、三麻身”、杏花雞的“兩細(xì)（頭細(xì)、腳細(xì)）、三黃（羽黃、腳黃、喙黃）、三短（頸短、體軀短、腿短）”。

4 結(jié)論

COI基因可用于地方雞的遺傳多樣性研究，但標(biāo)準(zhǔn)的DAN條形碼技術(shù)無(wú)法有效區(qū)分差異較小的雞品種，需要聯(lián)合其他分子標(biāo)記以及外形特征進(jìn)行鑒定。

致謝：感謝鄒志冠、劉少豐提供部分試驗(yàn)樣品，感謝杜炳旺教授、古文良、陳浩、劉怡然、溫金星、黃豐勛、徐迪宗、陳勇杰、曾德鑫在收集樣品時(shí)提供的幫助。

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（責(zé)任編輯 林鑒非）

DNA Barcoding of Indigenous Chickens in China: a Reevaluation

HUANG Xun-he1, CHEN Jie-bo1, HE Dan-lin2, ZHANG Xi-quan2, ZHONG Fu-sheng1

(1School of Life Sciences, Jiaying University, Meizhou 514015, Guangdong;2College of Animal Science, South China Agricultural University, Guangzhou 510642)

【Objective】 The aim of this study is to determine the feasibility of utility of mitochondrial cytochromeoxidase subunit I (COI) gene as DNA barcoding to identify indigenous chicken breeds with nearer appearances.【Method】 COI gene of 648 bp in length was obtained from nine indigenous chicken breeds of South China (Huaixiang, Qingyuan spotted, Huiyang bearded, Zhongshan shalan, Yangshan, Xinghua, Wuhua three-yellow, Wenchang and Guangxi yellow) and one commercial breed, Recessive White, with the method of direct sequencing of PCR products; while other COI sequences were downloaded from GenBank, including Chinese indigenous chickens and wild jungles and mallard (). These sequences were then used to analyze genetic diversity and genetic distance, construct median-joining network and phylogenetic tree based on haplotypes, as well as define breed specific haplotypes. 【Result】 COI gene of 695 bp in length was obtained after deletion of the primers sequences. And then 648 bp of standard barcoding was used for analysis. A total of 110 mutation sites were detected from 203 individuals of 10 breeds with 16.98% in all sites, of which 90 were singleton variable sites and the remaining 20 were parsimony informative sites. The average nucleotide diversity and haplotype diversity were 0.00394 (0.00349-0.00560) and 0.832 (0.763-0.905), respectively. Wuhua three-yellow chicken had the highest levels of genetic diversity, Zhongshan Shalan chicken had the second higher one, but Wenchang chicken had the lowest ones. A total of 84 haplotypes were defined, haplotype 1 had the highest frequency in nine indigenous chicken breeds. Haplotyes 9 and 5 were sharing both in indigenous and Recessive White chicken breeds, with the frequencies of 29 and 19, respectively. Each breed had its own haplotypes. Guangxi yellow, Wuhua three-yellow and Zhongshan shalan chickens had most 13 haplotypes, while Qingyuan spotted and Recessive White had least 8 haplotypes. The distribution of haplotypes of different breeds had a little of difference. For example, the haplotypes of Xinghua chicken was mostly distributed at 1, where Qingyuan spotted, Huiyang bearded and Recessive White was mostly distributed at 1 and 9, 1, 5 and 9, and 9 and 79, respectively. The genetic distance and net genetic distance between 10 breeds were ranged from 0.003 to 0.006 and from 0 to 0.003, respectively. The genetic distance among breeds was higher than those of within a breed; those of betweenand chickens were higher than 0.2. The 84 haplotyes of median-joining networks of were classified into three clusters with the characteristic of breed specific. For example, the cluster originated from in haplotype 9 had no Guangxi yellow and Wenchang chicken breeds. Other haplotypes were the descendent of 1. The Neighbor-joining tree showed that indigenous chickens in China andwere clustered into one branch, separating from,and. No branch with breed specificity was found. 【Conclusion】The results presented herein indicated that COI gene can be used as a candidate molecular marker for elucidate genetic diversity of indigenous chickens. It’s less effectivity of utility of standard COI gene as DNA barcoding to identify indigenous chicken with nearer appearance, there is highly need to incorporate multiple molecular markers such as COI gene, Cytochrome b gene, AFLP (Amplified Fragment Length Polymorphism), SNP (Single Nucleotide Polymorphism) and breed specific appearances.

mitochondrial cytochromeoxidase subunit I gene; DNA barcode; indigenous chicken; breed identification; genetic diversity

2015-06-23；接受日期：2016-05-14

廣東省自然科學(xué)基金（2014A030307018）、嘉應(yīng)學(xué)院“創(chuàng)新強(qiáng)校工程”項(xiàng)目（CQX019）、廣東省公益研究與能力建設(shè)項(xiàng)目（2015A020208020）

黃勛和，E-mail：hxh826@126.com。通信作者鐘福生，E-mail：zfs@jyu.edu.cn