基于線粒體CO Ⅰ基因和控制區(qū)序列的遼寧沿海彎棘斜棘群體遺傳多樣性和遺傳結(jié)構(gòu)分析

李玉龍 劉修澤 于旭光 李軼平 付 杰 董 婧

(遼寧省海洋水產(chǎn)科學(xué)研究院, 遼寧省海洋生物資源與生態(tài)學(xué)重點實驗室, 大連 116023)

基于線粒體CO Ⅰ基因和控制區(qū)序列的遼寧沿海彎棘斜棘群體遺傳多樣性和遺傳結(jié)構(gòu)分析

李玉龍 劉修澤 于旭光 李軼平 付 杰 董 婧

(遼寧省海洋水產(chǎn)科學(xué)研究院, 遼寧省海洋生物資源與生態(tài)學(xué)重點實驗室, 大連 116023)

為研究遼寧沿海彎棘斜棘(Repomucenus curvicornis)自然群體的遺傳多樣性及遺傳結(jié)構(gòu), 采用PCR擴增獲得遼寧沿海彎棘斜棘遼東灣群體(n=22)及黃海北部群體(n=18)線粒體的COⅠ及控制區(qū)(CR)部分DNA序列片段, 進行序列比較及遺傳多樣性分析。獲得彎棘斜棘COⅠ基因片段624 bp, 其A、T、C、G平均含量分別為24.09%、31.04%、25.28%和19.59%; CR片段460 bp, 其A、T、C、G平均含量分別為32.96%、32.80%、14.86%和19.38%。基于COⅠ基因和CR序列得到的兩群體變異位點數(shù)、平均核苷酸差異數(shù)、單倍型多樣性指數(shù)以及核苷酸多樣性指數(shù)分別為: 38、4.67、0.96±0.02和0.0075±0.0042; 26、3.35、0.97±0.02和0.0073±0.0043。序列分析結(jié)果均顯示, 遼東灣群體的遺傳多樣性低于黃海北部群體。分子方差(AMOVA)分析結(jié)果顯示, 基于COⅠ基因片段遼東灣與黃海北部群體間無明顯遺傳分化(Fst=0.0091, P=0.25) 而基于CR序列兩群體間具有較小但接近顯著的遺傳分化(Fst=0.0264, P=0.09)。研究表明, 線粒體CR序列與COⅠ基因均可作為檢測彎棘斜棘群體遺傳多樣性的有效分子標(biāo)記, 但CR序列遺傳分化的敏感度要高于COⅠ基因, 更適合作為彎棘斜棘群體遺傳研究的分子標(biāo)記。

彎棘斜棘; 線粒體DNA; COⅠ基因; 控制區(qū)序列; 遺傳多樣性; 遺傳分化

1 材料與方法

1.1 樣品采集及種類鑒定

1.2 DNA提取、擴增和測序

采用CTAB法提取基因組DNA后, 分別利用引物COⅠ a: 5′-CCTGCAGGAGGAGGAGAYCC-3′和COⅠb: 5′-ATGCATATCTATCTGCCATTTTAG-3′[21]和DL-F: 5′-CCCACCACTAACTCCCAAAGC-3′; DL-R: 5′-CTGGAAAGAACGCCCGGCATG-3′[22]對40個樣品進行擴增, 反應(yīng)體系25 μL, 包括: 0.2 mmol/L每種dNTPs, 0.2 μmol/L每種引物, 1 μL DNA模板, 1U Taq, 2.0 mmol/L MgCl2, 2.5 μL 10×緩沖液, 滅菌超純水補足剩余體系。PCR擴增在Bio-Rad C1000型PCR儀上進行, 反應(yīng)程序: 95℃預(yù)變性3min后, 95℃變性30s, 55—60℃退火35s, 72℃延伸50s, 運行35個循環(huán), 最后72℃下延伸5min。擴增后進行雙向測序[生工生物工程(上海)股份有限公司]。

1.3 數(shù)據(jù)分析

測序所得線粒體COⅠ基因和CR序列片段利用Bioedit軟件[23]進行拼接并輔以人工校對, 通過BLAST (http://www.ncbi.nlm.gov/BLAST/)檢索確定為目的片段。應(yīng)用CLUSTAL X 1.83軟件對序列進行比對分析。根據(jù)樣品的地理來源, 將渤海遼東灣海域采集的22個樣品作為遼東灣群體(LD), 黃海北部附近海域采集的18個樣品歸為黃海北部群體(HB)。DnaSP v5軟件確定單倍型, Mega5.0軟件[24]統(tǒng)計堿基含量、變異位點, 采用Kimura雙參數(shù)模型計算單倍型間遺傳距離。Arlequin3.5[25]軟件計算單倍型數(shù)、多態(tài)位點數(shù)、單倍型多態(tài)度 (H)、核苷酸多態(tài)度(π)等分子多樣性指數(shù)以及兩兩群體之間的Fst統(tǒng)計值, 其顯著性通過參數(shù)重抽樣法進行檢驗。使用Arlequin3.5軟件中的分子變異分析(AMOVA)來評估群體間遺傳變異, 其顯著性通過1000次重抽樣來檢驗, 群體間的遺傳距離采用Kimura 2 parameter模型計算。此外, 為探討彎棘斜棘單倍型的譜系結(jié)構(gòu), 采用中介網(wǎng)絡(luò)法[26]構(gòu)建單倍型網(wǎng)絡(luò)關(guān)系圖。

圖 1 研究區(qū)域彎棘斜棘?樣品取樣圖Fig. 1 The sampling sites of Repomucenus curvicornis

2 結(jié)果

2.1 遺傳多樣性

PCR擴增共得到39條COⅠ基因同源序列, 經(jīng)比對后得到624 bp序列用于分析, 定義了28種單倍型(Hap1—28)。在28個單倍型中, Hap8、Hap12、Hap 20為兩群體共享單倍型, 其中Hap12、Hap 20為主體單倍型, 其所占頻率分別為12.8%和17.9%,剩余25個單倍型(89%)為群體特有單倍型且只在一個個體中檢測到 (表 1)。在研究的COⅠ序列中, A、T、C、G堿基的平均含量分別為24.09%、31.04%、25.28%和19.59%, A+T (55.13%)含量高于G+C (44.87%)含量(表 2)。共檢測到38個多態(tài)位點,包括16個簡約信息位點和22個單態(tài)核苷酸變異位點, 其中3個突變發(fā)生在第一密碼子, 其余都發(fā)生在第三密碼子, 僅1個突變位點導(dǎo)致編碼氨基酸的改變(M-V)。種內(nèi)個體間COⅠ序列遺傳距離為0—2.1%, 平均遺傳距離0.8%。

表 1 彎棘斜棘不同單倍型COⅠ序列的變異位點分布Tab. 1 Variable sites of COⅠfragments of different haplotypes of Repomucenus curvicornis

單倍型Haplotype 00000001111112223333333333444445555556 群體Population 11345670036882690022344459246790135770 29027925892092770617628970615421675060 LD HB Hap1 CACGGCTGCTCCTAAATACTTGTCAATAGACCCCTTAG 1 Hap2 .G.................................... 1 Hap3 .G......................T .....T....C.. 1 Hap4 .G ......T..TC ........AC .....A ..TGA .... 1 Hap5 .G..A ..A...TCG ..............A ..TGA .... 1 Hap6 .G.A .......TC......C........A ..T.A .... 1 Hap7 .G .A ........................A ..TGA .... 1 Hap8 .G....C .....................A ..TGA .... 1 1 Hap9 .G....CA....................A ..TGA .... 1 Hap10 TG ........................C .A ..TGA .... 1 Hap11 TG ....C .........C .A .........A ..TGA .... 1 Hap12 .G .........................GA ..TGA .C .. 4 1 Hap13 .G.........................GAG.TGA .C .. 1 Hap14 .G ......T ..................GA ..TGA .C .. 1 Hap15 TG .........................GA ..TGA .C .. 1 Hap16 TG .............G ............A ..TGA .C .. 1 Hap17 .G..........................A ..TGA .C .. 1 Hap18 .G ..........................A ..TGAG ... 1 Hap19 .G..........................A ..TGAA... 1 Hap20 .G ..........................A ..TGA .... 5 2 Hap21 .G ..........................A ..TGA ..G . 1 Hap22 .G...........G ........T........TG ..... 1 Hap23 .G.......C..........C ....T.....TG ..... 1 Hap24 .G ...............G ...A .........T ...... 1 Hap25 T ............G .......A .........T ...... 1 Hap26 ........T......................T ...... 1 Hap27 ...............................T .....A 1 Hap28 .T ...G....T...G ................T ....G . 1合計Total 2217

表 2 彎棘斜棘線粒體COⅠ與CR片段的序列組成Tab. 2 Nucleotide compositions of COⅠmitochondrial fragment and CR gene in Repomucenus curvicornis (%)

表 2 彎棘斜棘線粒體COⅠ與CR片段的序列組成Tab. 2 Nucleotide compositions of COⅠmitochondrial fragment and CR gene in Repomucenus curvicornis (%)

群體Population A T C G COⅠ CR COⅠCR COⅠCR COⅠCR遼東灣Liaodong Bay 24.1232.9831.0332.82 25.2614.8419.5919.36黃海北部North Yellow Sea 24.0532.93 31.0532.78 25.3014.88 19.6019.41平均Mean24.0932.9631.0432.8025.2814.8619.5919.38

經(jīng)測序得到36條CR同源序列, 比對后得到460 bp序列用于分析, 定義了27種單倍型(Hap1—27)。在27個單倍型中, Hap3、Hap6、Hap13、Hap 20為兩群體共享單倍型, 其中Hap20為主體單倍型,其所占頻率為14%, 21個單倍型(81%)為群體特有單倍型且只在一個個體中檢測到, 余下的2個單倍型不止在一個個體中被發(fā)現(xiàn), 且這些個體僅分布于一個群體中(表 4)。在所分析CR序列中, A、T、C、G的平均含量分別為32.96%、32.80%、14.86%和19.38%, A+T (65.76%)含量明顯高于G+C (34.24%)含量(表 2), 符合海水魚類線粒體控制區(qū)序列特征。共發(fā)現(xiàn)多態(tài)位點26個, 包括13個簡約信息位點和13個單態(tài)核苷酸變異位點。種內(nèi)個體間CR序列遺傳距離為0—2.4%, 平均遺傳距離0.7%。

2.2 群體遺傳分化

而基于線粒體CR序列計算的分子方差分析(AMOVA)的結(jié)果則顯示, 97.36%的差異屬于群體內(nèi)變異, 群體間差異為2.64%, Fst值為0.0264但差異接近顯著(P=0.09), 表明本研究中遼寧沿海兩個彎棘斜棘群體可能存在較小的遺傳分化(表 6)。

3 討論

表 3 基于線粒體COⅠ基因和CR序列的彎棘斜棘遺傳多樣性參數(shù)Tab. 3 The genetic diversity of Repomucenus curvicornis based on COⅠ and CR sequence

表 3 基于線粒體COⅠ基因和CR序列的彎棘斜棘遺傳多樣性參數(shù)Tab. 3 The genetic diversity of Repomucenus curvicornis based on COⅠ and CR sequence

群體Sample單倍型數(shù)No. of haplotypes樣本數(shù)n 變異位點Variable sites平均核苷酸差異數(shù)k單倍型多樣性h核苷酸多樣性π COⅠ CR COⅠ CR COⅠ CR COⅠ CR COⅠ CR COⅠ CR遼東灣Liaodong Bay 22 22 22 20 15 18 4.06 2.95 0.93±0.040.97±0.02 0.0065±0.00380.0064±0.0039黃海北部North Yellow Sea 17 14 29 16 16 12 5.50 3.95 0.99±0.020.98±0.03 0.0088±0.00500.0086±0.0051合計Total 39 36 38 26 28 27 4.67 3.35 0.96±0.020.97±0.02 0.0075±0.00420.0073±0.0043

表 4 彎棘斜棘不同單倍型控制區(qū)序列的變異位點分布Tab. 4 Variable sites of CR fragment of different haplotypes of Repomucenus curvicornis

表 4 彎棘斜棘不同單倍型控制區(qū)序列的變異位點分布Tab. 4 Variable sites of CR fragment of different haplotypes of Repomucenus curvicornis

單倍型Haplotype 00001112222222222233333333 群體Population 77790370011133558901137778 18948648912524397013662890 LD HB Hap1 CAAGGAGAAGGTGGAATTTGAAAGTC 1 Hap2 T...............C......... 1 Hap3 T......................A.. 1 1 Hap4 T ..........C .T .........A .. 1 Hap5 T ............T .C .......A .. 1 Hap6 T.G....G ...............AC. 1 1 Hap7 T .......G ..............AC . 1 Hap8 T .G ...........G ........A .. 1 Hap9 T .G ...........G .......GA .. 1 Hap10 T .G ...................GA .. 3 Hap11 T.G...................G... 1 Hap12 T .G ....G ..............GA .. 1 Hap13 T.G ......A ...........G .A.. 1 1 Hap14 T.G..................G .A.. 1 Hap15 T .G ................A .GGA .. 1 Hap16 T.G........C..........GA.. 2 Hap17 T .G ........C ........G ..A .. 1 Hap18 T .G .........A .......G ..A .. 1 Hap19 T .G .........A ..........A .. 1 Hap20 T.G ....................A.. 3 2 Hap21 T .G ...............A ....A .. 1 Hap22 T .G ..............C .....A .. 1 Hap23 T.G.......A......C.....A.. 1 Hap24 T .GACGT ................A .. 1 Hap25 T .T ....................A .. 1 Hap26 TC.....G.T................ 1 Hap27 T.......TA..........TG ...A 1合計Total 2214

表 5 基于COⅠ序列的遼寧沿海兩個彎棘斜棘群體的AMOVA分析Tab. 5 COⅠsequence-based analysis of molecular variation for populations of Repomucenus curvicornis

表 5 基于COⅠ序列的遼寧沿海兩個彎棘斜棘群體的AMOVA分析Tab. 5 COⅠsequence-based analysis of molecular variation for populations of Repomucenus curvicornis

變異來源Source of variation自由度df方差總和Sum of squares變異組分Variance components變異貢獻率Percentage of variation FstP群體間Among population 1 2.756 0.02155 0.91 0.0091 0.25群體內(nèi)Within population 37 86.665 2.34230 99.09總數(shù)Total 38 89.421 2.36385 100.00

表 6 基于CR序列的遼寧沿海兩個彎棘斜棘群體的AMOVA分析Tab. 6 CR sequence-based analysis of molecular variation for populations of Repomucenus curvicornis

表 6 基于CR序列的遼寧沿海兩個彎棘斜棘群體的AMOVA分析Tab. 6 CR sequence-based analysis of molecular variation for populations of Repomucenus curvicornis

變異來源Source of variation自由度df方差總和Sum of squares變異組分variance components變異貢獻率Percentage of variation FstP群體間Among population 1 2.439 0.04518 2.64 0.0264 0.09群體內(nèi)Within population 34 56.645 1.66603 97.36總數(shù)Total 35 59.084 1.71121 100.00

海洋魚類在整個生活史中會受到多種因素的影響, 一般而言, 底棲生活海洋魚類由于移動能力較差阻礙了不同地理群體間的基因交流易于產(chǎn)生遺傳分化; 而開放的海洋環(huán)境中產(chǎn)浮性卵魚類由于其生活史早期易受海域內(nèi)洋流的作用而具有較強的擴散潛力導(dǎo)致遺傳分化不明顯。本研究基于兩個分子標(biāo)記計算的分子方差分析(AMOVA)的結(jié)果并不一致, 基于COⅠ基因序列的結(jié)果顯示彎棘斜棘遼東灣群體和黃海北部群體幾乎無遺傳分化(Fst=0.0091, P=0.25); 而基于CR序列的結(jié)果則表明兩者間存在較小但接近顯著的遺傳分化(Fst=0.0264, P=0.09), 提示彎棘斜棘不同群體間可能存在復(fù)雜的遺傳結(jié)構(gòu)。彎棘斜棘復(fù)雜遺傳結(jié)構(gòu)的形成可能與晚更新世末-全新世初黃渤海海域發(fā)生的多次大范圍的海侵和洋面的回升以及彎棘斜棘自身的生態(tài)習(xí)性有關(guān)。彎棘斜棘為暖水性底棲小型魚類, 其游泳能力較差, 但產(chǎn)浮性卵, 在其整個生活史階段可以營短暫的浮游生活。由于多次大范圍的海侵, 在不同海域進化的彎棘斜棘群體可能在黃海發(fā)生過重新混合的現(xiàn)象, 后來隨著海侵過程或者沿岸流一部分個體由黃海遷入渤海, 但由于分化的時間較短兩個群體間尚未形成明顯的遺傳分化。由于受采樣范圍及數(shù)量限制, 有關(guān)中國近海彎棘斜棘不同地理種群間遺傳結(jié)構(gòu)及遺傳多樣性的研究仍有待繼續(xù)深入。

3.3 兩種分子標(biāo)記在海水魚類群體遺傳研究中的適用性

魚類線粒體控制區(qū)序列受選擇壓力較小、進化速率快, 而COⅠ基因作為線粒體編碼基因, 其進化速度相對較慢。不同硬骨魚類的線粒體CR序列進化速率并不一致, 在大多物種中, 其進化速率快于蛋白編碼基因, 而在另一些種類中兩者的進化速率差異不大[19,20]。在本研究中, 彎棘斜棘線粒體控制區(qū)序列的遺傳變異水平與COⅠ基因相當(dāng)(表 3),這種現(xiàn)象在銀鯧[10]、澳洲彩虹魚[11]以及東非洲慈鯛屬(Haplochromis)[12]等魚類中都曾被發(fā)現(xiàn)。而在一些無脊椎動物如蕁麻蛺蝶(Aglais urticae), 其CR序列的遺傳變異甚至要低于COⅠ基因[36]。

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POPULATION GENETIC STRUCTURE AND DIVERSITY ANALYSIS OF REPOMUCENUS CURVICORNIS IN THE LIAONING COAST BASED ON DNA SEQUENCES OF THE MITOCHONDRIAL COⅠ GENE AND CONTROL REGION

LI Yu-Long, LIU Xiu-Ze, YU Xu-Guang, LI Yi-Ping, FU Jie and DONG Jing
(Liaoning Key Laboratory of Marine Biological Resources and Ecology, Liaoning Ocean and Fishery Science Research Institute, Dalian 116023, China)

To analyze the genetic diversity and genetic structure of Repomucenus curvicornis from the Liaodong Bay (n=22) and the North Yellow Sea (n=18), the mitochondrial DNA cytochrome oxidase I (COⅠ) gene and control region fragments were obtained by PCR amplification. The average contents of A, T, C and G of 624 bp COⅠgene fragments were 24.09%, 31.04%, 25.28%, and 19.59%, respectively. The average contents of A, T, C and G of CR fragments (460 bp) were 32.96%, 32.80%, 14.86% and 19.38%, respectively. The total variable sites, mean pairwise nucleotide differences (k), haplotype diversity (H) and nucleotide diversity (π) based on COⅠgene fragments were 38, 4.67, 0.96±0.02 and 0.0075±0.0042, respectively. The same parameters based on CR fragments were 26, 3.35, 0.97±0.02 and 0.0073±0.0043, respectively. Based on mitochondrial COⅠgene and control region, the genetic diversity of Liaodong Bay population was lower than that of North Yellow Sea population. The AMOVA analysis based on CR fragments revealed almost significant genetic divergence between the Liaodong Bay and North Yellow Sea populations, while there was no significant genetic divergence based on COⅠgene. The results showed that CR and COⅠgene were effective molecular markers for detecting the genetic diversity of Repomucenus curvicornis population, while CR was more reliable than COⅠgene in detecting the genetic structure. In conclusion, CR is a appropriate marker for marine fish population genetic analysis.

Repomucenus curvicornis; mtDNA; COⅠgene; Control region; Genetic diversity; Genetic differentiation

Q346+.5

A

1000-3207(2017)03-0581-08

10.7541/2017.75

2016-06-06;

2016-09-10

海洋公益性行業(yè)科研專項(201405010); 遼寧省海洋與漁業(yè)科研項目(201401)資助 [Supported by the Public Science and Technology Research Funds Project of Ocean (201405010); the Research Project of Marine and Fishery of Liaoning Province (201401)]

李玉龍(1981—), 男, 山東臨沂人; 副研究員; 主要從事漁業(yè)資源增殖放流及海洋生物分子生物學(xué)研究。E-mail: liyudragon@ 126.com

董婧(1966—), 女, 遼寧沈陽人; 研究員; 主要從事漁業(yè)資源調(diào)查及大型水母生物學(xué)研究。E-mail: 1024470248@qq.com