渔业科学进展  2018, Vol. 39 Issue (5): 27-35  DOI: 10.19663/j.issn2095-9869.20170814001
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引用本文 

孙超, 刘洪波, 姜涛, 轩中亚, 杨健. 不同鲚属鱼类Cyt b和D-loop序列的比较及其判别早期生活史个体的潜力分析[J]. 渔业科学进展, 2018, 39(5): 27-35. DOI: 10.19663/j.issn2095-9869.20170814001.
SUN Chao, LIU Hongbo, JIANG Tao, XUAN Zhongya, YANG Jian. Comparison of D-loop and Cytochrome b Sequences Among Different Coilia Species and Their Potential Use in Analysis for the Identification of Species at Early Life Stages[J]. Progress in Fishery Sciences, 2018, 39(5): 27-35. DOI: 10.19663/j.issn2095-9869.20170814001.

基金项目

中央级公益性科研院所基本科研业务费专项资金项目(2016PT01)和国家自然科学基金项目(31372533; 31602159)共同资助

作者简介

孙超, E-mail: sunchao159@126.com

通讯作者

杨健, 研究员, E-mail: jiany@ffrc.cn

文章历史

收稿日期:2017-08-14
收修改稿日期:2017-10-16
不同鲚属鱼类Cyt b和D-loop序列的比较及其判别早期生活史个体的潜力分析
孙超1, 刘洪波2, 姜涛2, 轩中亚1, 杨健1,2     
1. 南京农业大学无锡渔业学院 无锡 214081;
2. 中国水产科学研究院淡水渔业研究中心 中国水产科学研究院内陆渔业生态环境与资源重点开放实验室 无锡 214081
摘要:使用mtDNA作为分子标记,基于1022 bp和1322 bp左右的部分序列分析刀鲚(Coilia nasus)、湖鲚(C. nasus taihuensis)、七丝鲚(C. grayii)及凤鲚(C. mystus)成鱼之间的遗传关系。结果显示,刀鲚和湖鲚间的遗传距离分别为0.0036±0.0008和0.0038±0.0008,凤鲚与刀鲚、湖鲚的遗传距离分别为0.1215±0.0111、0.1228±0.0111(Cyt b)和0.1075±0.0108、0.1067±0.0107 (D-loop),七丝鲚与刀鲚、湖鲚的遗传距离分别为0.0342±0.0056、0.0351±0.0057 (Cyt b)和0.0527±0.0069、0.0529±0.0070 (D-loop),七丝鲚和凤鲚间的遗传距离分别为0.1158±0.0111和0.1123±0.0111。用Kimura双参数模型构建的2种序列的NJ分子系统树均显示,湖鲚和刀鲚不能形成独立的分支,而是混合聚在一起形成1个分支;七丝鲚和凤鲚则形成另外2个分支。首先根据采样点不同可初步断定未知仔幼鱼为刀鲚,稚鱼为湖鲚,而后对未知种仔幼鱼、稚鱼和刀、凤鲚鱼卵的2种序列的分析发现,不同采集点内部的这些早期生活个体间的遗传距离分别为0.0024~0.0032和0.0025~0.0082。在Kimura双参数模型构建的NJ分子系统树中,未知种仔幼鱼、稚鱼、刀鲚鱼卵与刀鲚、湖鲚聚为一类,凤鲚鱼卵与凤鲚聚为一支。由此可见,Cyt b基因序列和D-loop序列作为分子标记,虽然可以区分刀鲚、凤鲚以及七丝鲚的仔幼、稚鱼及鱼卵,但不能有效区分湖鲚和刀鲚的早期生活个体。
关键词鲚属    早期生活史    Cyt b基因    D-loop序列    
Comparison of D-loop and Cytochrome b Sequences Among Different Coilia Species and Their Potential Use in Analysis for the Identification of Species at Early Life Stages
SUN Chao1, LIU Hongbo2, JIANG Tao2, XUAN Zhongya1, YANG Jian1,2     
1. Fisheries College, Nanjing Agricultural University, Wuxi 214081;
2. Key Laboratory of Fishery Ecological Environment Assessment and Resource Conservation in Middle and Lower Reaches of the Yangtze River, Chinese Academy of Fishery Sciences; Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081
Corresponding author: YANG Jian, E-mail: jiany@ffrc.cn
Fund: This work was supported by Special Scientific Research Funds for Central Non-Profit Institutes(2016PT01), and National Natural Science Foundation of China (31372533, 31602159)
Abstract: Based on 1022 bp and 1322 bp sequences, partial nucleotide sequences of Cyt b and D-loop segments of mtDNA were analyzed for Coilia nasus, C. nasus taihuensis, C. grayii, and C. mystus to develop a reference dataset for species identification in the genus Coilia. The genetic distances were 0.00360±0.00083 and 0.00378±0.00082 between C. nasus and C. nasus taihuensis, 0.12146±0.01106 and 0.10753±0.01076 between C. mystus and C. nasus, 0.12207±0.01111 and 0.10674±0.01074 between C. mystus and C. nasus taihuensis, 0.03418±0.00561 and 0.05267±0.00686 between C. nasus and C. grayii, 0.03506±0.00568 and 0.05287±0.00698 between C. nasus taihuensis and C. grayii, as well as 0.11585± 0.01109 and 0.11233±0.01114 between C. grayii and C. mystus, respectively. Based on the sequences of the Cyt b gene and D-loop and the neighbor-joining (NJ) molecular dendrogram using Kimura 2-parameters, individuals of C. nasus and C. nasus taihuensis were combined in a cluster while C mystus and C. grayii formed another two clusters. First, based on sampling sites, the identities of the unknown larvae from the estuary of the Yangtze River and unknown juveniles from the Taihu Lake could be deduced preliminarily as C. nasus and C. nasus taihuensis, respectively. Then, nucleotide sequences of Cyt b and D-loop of larvae and eggs of genus Coilia were analyzed and compared with the reference established above. Genetic distance ranged from 0.0024 to 0.0032 and 0.0025 to 0.0082, respectively, within the larva and egg individuals collected from different places. The NJ molecular dendrogram showed that all the unknown larvae, the eggs of C. nasus, adult C. nasus, and C. nasus taihuensis clustered together. In contrast, individual eggs of C. mystus and adult C. mystus formed a separate cluster. Thus, Cyt b and D-loop sequence analysis could be used to identify unknown Coilia nasus, C. grayii, and C. mystus at early life stages, but could not effectively be used to identify unknown individuals at early life stages between C. nasus and C. nasus taihuensis.
Key words: Genus Coilia    Early life stage    Cyt b gene sequence    D-loop sequence    

对处于早期生活史的鱼类的种类进行准确的鉴定是进行渔业资源量评估的重要方面,也是进行鱼类整个生活史研究的基础工作。传统的形态学鉴定方法一般基于鱼类的形态学和解剖学特征(何文平等, 2011),操作简单,样品可重复使用。在鱼类早期生活史阶段,可通过光学显微镜观察鱼卵的形状、颜色、纹理、卵直径和胚胎色素沉着等特征以及胚胎发育过程中鱼卵形态的连续变化来鉴别鱼卵所属物种(Mork et al, 2011; Hofmann et al, 2017);也可通过在显微镜下细数仔稚鱼、幼鱼的一些形态学参数判别其所属物种(Webb et al, 2006; 何文平等, 2011)。但这种传统方法操作过程费时费力、依赖经验、主观性较大。不仅如此,形态特征在不同的物种间(特别是早期生活史个体)还会产生重叠(张冬良等, 2009; Mork et al, 2011),容易产生误判;有时采样只采到形态不完整的个体(Webb et al, 2006),这时利用传统的物种鉴定方法很难准确判别,而分子生物学方法能克服形态学鉴定中主观性强、要求研究者具备较高的形态学研究素养、要求样本个体完整性高等局限,且具有重复性好、灵敏度高等优点(孙超等, 2011)。

线粒体DNA(Mitochondrial DNA, mtDNA)是一种闭合的环状分子,分子结构简单,遵循严格的母系遗传,几乎不发生重组,进化速度相对较快,且不同的区域进化速度存在差异,因而,可以选择不同区域进行不同时间尺度的进化分析,是鱼类分子群体遗传学和分子系统学研究中的重要标记(Xiao et al, 2000)。其中,细胞色素b基因(Cyt b)进化速率适中,能很好地反映系统演化的较晚期历史(Xiao et al, 2000; 孙超等, 2013);线粒体控制区序列(D-loop)无编码选择压力,进化速率较其他线粒体基因快,适合于研究种内种群遗传结构和遗传分化(Liu et al, 2004)。线粒体控制区序列(D-loop)较mtDNA的其他序列,其进化速率较快(Liu et al, 2004; Chan et al, 2016)。因此,结合线粒体Cyt b和D-loop两种分子序列标记研究鱼类种内的遗传变异和不同的科、属、种的系统进化关系更可靠(胡玉婷等, 2015)。这2种线粒体基因已应用于不同物种、群体间的遗传多样性和系统发育物种鉴定研究等方面(阎雪岚等, 2009; 何文平等, 2011; Chan et al, 2016; Mansourkiaei et al, 2016),如针对水生生物的物种鉴定(李渊等, 2013; Cheng et al, 2013; 张静等, 2016)、鱼卵仔稚鱼鉴定(Perez et al, 2005; Pegg et al, 2006; 周美玉等, 2015)、水产品检测(Shen et al, 2016; 安丽艳等, 2016)、隐存种发掘(Zemlak et al, 2009; Chen et al, 2015)新纪录种的发现(Guo et al, 2010; Gao et al, 2011)等。然而,从鲚属鱼类的研究来看,仅有基于Cyt b基因序列的刀鲚和凤鲚仔稚鱼判别尝试(何文平等, 2011),但尚未有涉及鱼卵的相关研究报道。本研究采用Cyt b基因和D-loop序列作为分子标记,将凤鲚、刀鲚鱼卵、未知鲚属仔稚鱼和幼鱼所得序列与刀鲚[Coilia nasus,含其陆封生态型湖鲚(C. nasus taihuensis)]、凤鲚(C. mystus)、七丝鲚(C. grayii)成鱼的相应序列进行比对,通过计算遗传距离、构建分子系统树等方法对处于早期生活史的鲚属鱼类个体所属的种进行判别,为分子生物学技术应用于鲚属鱼卵、仔稚鱼鉴别提供理论参考和技术支撑。

1 材料与方法 1.1 样本采集

于2007年5月~2012年2月间采集样本。湖鲚采自太湖平台山,七丝鲚采自广东十五涌,凤鲚鱼卵采自瓯江七都岛永加蒲东村段所获亲鱼,刀鲚鱼卵采自鄱阳湖星子县所获亲鱼,未知鲚属稚鱼采自太湖仙岛,未知鲚属仔幼鱼分别采自长江口崇明岛区域和长江口东滩区域。其中,凤鲚鱼卵(4~5期卵)与刀鲚鱼卵(3~4期卵)(Li et al, 2007)分别取自雌性凤鲚和刀鲚体内。研究中刀鲚、湖鲚、七丝鲚和凤鲚成鱼均按照生态习性和形态特征加以鉴别(袁传宓等, 1980)。样本信息见表 1。其中,未知鲚属仔鱼、稚鱼和幼鱼(袁传宓等, 1978; 张冬良等, 2009)的体长分别为16.4~18.3 mm,43.5~53.25 mm和100.6~122.5 mm;但部分未知鲚属样本形态不完整,鱼体残缺无法进行测量。

表 1 本研究中鲚属鱼类样本的背景 Table 1 Background information of Coilia species in the present study
1.2 基因组DNA提取、PCR扩增及目的片段的纯化

总DNA提取采用传统的“酚–氯仿”法(Sambrook et al, 1989)。使用0.5%的琼脂糖凝胶电泳检测DNA质量。Cyt b基因扩增使用通用引物L14724(5'-GAC TTG AAA AAC CAC CGT TC-3')和H15915(5'-CTC CGA TCT CCG GAT TAC AAG AC-3')(Xiao et al, 2001),PCR扩增体系和条件参照何文平等(2011)。D-loop序列扩增使用引物序列为DF1(5'-CTA ACT CCC AAA GCT AGA ATT CT-3')和DR2(5'-ATC TTA GCA TCT TCA GTG-3'),PCR扩增体系和条件参照唐文乔等(2007)。PCR扩增完成后,用1%琼脂糖凝胶电泳检测PCR扩增产物。扩增产物用生工生物工程(上海)股份有限公司的UNIQ-10柱式PCR产物纯化试剂盒回收。

1.3 目的片段的克隆和测序

用购自TaKaRa公司的连接试剂盒(pMD®18-T Vector)将经纯化回收的目的基因片段与载体进行连接(16℃)。将重组子转化至DH5α感受态细胞(由上海生工高效感受态Cell制备试剂盒制备)中,涂布于LB氨苄抗生素(Amp)抗性平板,经37℃过夜培养后,挑取明显的白斑进行菌液PCR验证(菌液量为5 μl)。对确认含有目的基因片段的菌液进行扩大培养,之后送上海生工进行双向测序。部分样本直接采用PCR扩增产物纯化后产物进行测序,测序不理想样本经上述步骤后进行菌液测序。

1.4 数据分析

使用MEGA 5.05软件(Tamura et al, 2011)中的ClustalW程序对比排列所得序列并辅以手工校正。同时,利用该软件中的Kimura双参数模型计算序列之间的遗传距离(K2P距离),并根据K2P距离采用邻接法(Neighbour-joining method, NJ)构建种间分化的系统树。通过自展分支检验(Bootstrapping),获得系统树分支的置信度,重复次数为1000次。

2 结果

鲚属成鱼、未知鲚属仔稚鱼、幼鱼及鱼卵样本的Cyt b基因和D-loop序列均在1300 bp左右。经过序列比对和手工校正后,分别选择其中的1022 bp和1322 bp进行进一步分析。其中,本研究所得鲚属成鱼序列已上传至GenBank(登录号分别为JX030385~ JX030394、JX030382~JX030383和JX442546)。部分成鱼序列载自GenBank,即Cyt b: CE01~10 (GU371509~ 18), CET01~02 (EU694405~06); D-loop: CE01~10 (GU371625, 27, 29, 31, 33, 34, 37, 39, 41, 43), CM01~04 (EF419828~31), CET01~08 (EF419804, 06, 08, 22, 23, 25~27), CG02~05 (EF419800~03)。部分序列孙超等(2013),即Cyt b: CM01~05 (JX030395~ 96), D-loop: CM05 (JX030384)。

Kimura双参数模型估算刀鲚、湖鲚、七丝鲚、凤鲚成鱼、未知鲚属仔幼鱼、稚鱼及刀鲚、凤鲚鱼卵间Cyt b基因片段和D-loop序列的遗传距离见表 2。结果显示,刀鲚和湖鲚间的遗传距离分别为0.0036±0.0008和0.0038±0.0008,凤鲚与刀鲚、湖鲚的遗传距离分别为0.1215±0.0111、0.1228±0.0111 (Cyt b)和0.1075±0.0108、0.1067±0.0107 (D-loop),七丝鲚与刀鲚、湖鲚的遗传距离分别为0.0342±0.0056、0.0351±0.0057 (Cyt b)和0.0527±0.0069、0.0529±0.0070 (D-loop),七丝鲚和凤鲚间的遗传距离分别为0.1158±0.0111 (Cyt b)和0.1123±0.0111 (D-loop)。Kimura双参数模型估算,未知仔幼鱼、稚鱼、刀鲚和凤鲚鱼卵4类各类别内个体间的平均遗传距离分别为0.0032±0.0009、0.0029±0.0010、0.0024±0.0009和0.0029±0.0008 (Cyt b);0.0076±0.0015、0.0062±0.0015、0.0082±0.0020和0.0025±0.0007(D-loop)。数据显示,未知仔幼鱼与刀鲚、湖鲚间的遗传距离分别为0.0037±0.0008、0.0028±0.0007 (Cyt b)和0.0060±0.0012、0.0060±0.0011 (D-loop);而未知仔幼鱼与七丝鲚与凤鲚间的遗传距离较大,分别为0.0349±0.0057、0.1222±0.0111 (Cyt b)和0.0534±0.0069、0.1089±0.1078 (D-loop);未知稚鱼与刀鲚、湖鲚间的遗传距离分别为0.0037±0.0008、0.0026±0.0008 (Cyt b)和0.0056±0.0012、0.0054±0.0012 (D-loop),而未知稚鱼与七丝鲚、凤鲚间的遗传距离较大,分别为0.0344±0.0056、0.1219±0.0111 (Cyt b)和0.0528±0.0068、0.1085±0.0107(D-loop);凤鲚鱼卵与凤鲚间的遗传距离最小为0.0016±0.0005 (Cyt b) 0.0037±0.0009 (D-loop),与刀鲚、湖鲚、七丝鲚的遗传距离分别为0.1208±0.0109、0.1215±0.0110、0.1152±0.0110 (Cyt b)和0.1071±0.0108、0.1063±0.0107、0.1121±0.0112 (D-loop);刀鲚鱼卵与刀鲚、湖鲚的遗传距离都较小,分别为0.0033±0.0008、0.0025±0.0007 (Cyt b)和0.0068±0.0015、0.0070±0.0015 (D-loop),而与七丝鲚、湖鲚的遗传距离较大,分别为0.0344±0.0056、0.1220±0.0112 (Cyt b)和0.0522±0.0067、0.1083±0.0107 (D-loop)。

表 2 不同鲚属鱼类及其早期生活史个体的平均Kimura双参数遗传距离 Table 2 Mean value of the Kimura's two-parameter distance among different Coilia species and corresponding eggs, larvae and juveniles

用Kimura双参数模型构建的包括刀鲚、湖鲚、七丝鲚和凤鲚成鱼Cyt b基因片段序列和D-loop序列的NJ树明显分为三支:刀鲚和湖鲚不能形成彼此独立的分支,混合聚在一起形成一支;七丝鲚和凤鲚各自形成一单支(图略)。将未知鲚属仔幼鱼、稚鱼和刀鲚、凤鲚鱼卵一并加入分析后,未知鲚属仔幼鱼、稚鱼、刀鲚鱼卵、刀鲚成鱼和湖鲚成鱼混合聚在一起形成一支(支持率分别为99%和100%),不能各自形成独立的分支;七丝鲚成鱼形成一单支(支持率分别为98%和100%);凤鲚成鱼和凤鲚鱼卵混合在一起形成一支(支持率分别为99%和100%),没有各自形成独立的分支(图 1)。

图 1 不同鲚属鱼类基于Cyt b基因(A)和D-loop序列(B)构建的NJ分子系统发生树 Figure 1 Neighbour-joining molecular dendrogram of different Coilia species based on the sequences of Cyt b gene (A) and D-loop (B) *:载自GenBank;**孙超等(2013);节点处数字为大于50%的支持率的自展分支检验值 *: Loaded from GenBank; **: Sun et al(2013); Numbers at nodes represented bootstrap values, and only bootstrap values greater than 50% were shown
3 讨论 3.1 不同鲚属鱼类Cyt b和D-loop序列的比较

由于湖鲚在生活习性和形态上与洄游型刀鲚有一定的差异,袁传宓等(19761980)认为,湖鲚属于刀鲚的一个亚种。然而,由于刀鲚和湖鲚的可数和可量性状上存在较大重叠(唐文乔等, 2007),因此,湖鲚的亚种地位一直存在着争议,部分学者(Cheng et al, 2004; 刘文斌等, 1995; 唐文乔等, 2007; 许志强等, 2009; 周晓犊等, 2010)通过形态学、生物化学和分子生物学方法对湖鲚亚种的有效性秉持否定的观点,认为湖鲚是刀鲚的一个不同生态型种群,而非一个亚种。本研究用线粒体Cyt b和D-loop序列构建的系统树显示刀鲚和湖鲚不具单系性,两种序列构成的系统树中都有湖鲚构成小的分支后与刀鲚相聚或湖鲚直接与刀鲚构成一个分支的情况,这与许志强等(2009)的研究结果相同;刀鲚与湖鲚间的平均遗传距离分别为0.0036±0.0008和0.0038±0.0008,介于二者各自内部个体间的遗传距离(Cyt b:0.0023~0.004;D-loop: 0.0034~0.0042),且明显小于湖鲚与凤鲚和七丝鲚间的平均遗传距离。因此,从线粒体Cyt b和D-loop序列分析看,湖鲚与刀鲚是同一物种的不同生态型,并未达到亚种水平。因此,应用线粒体Cyt b和D-loop序列虽然可以区分刀鲚、凤鲚以及七丝鲚的仔幼、稚鱼及鱼卵,但很难有效区分湖鲚和刀鲚的早期生活个体。

3.2 基于Cyt b和D-loop序列判别鲚属鱼类早期生活史个体的潜力

本研究中未知鲚属仔幼鱼、稚鱼和刀鲚、凤鲚鱼卵4类早期生活个体各类别内个体间基于线粒体Cyt b和D-loop序列的平均遗传距离最大分别为0.0032和0.0082,远小于相应序列得出的刀鲚(湖鲚)、凤鲚和七丝鲚等3种不同种的成鱼之间的遗传距离,表明该4类早期生活个体各自属于同一物种。用Kimura双参数模型分别计算4类早期生活个体与3种不同成鱼的遗传距离(表 2)并构建NJ系统树(图 1),可以看出,未知鲚属仔幼鱼及稚鱼与刀鲚和湖鲚的遗传距离明显低于不同种之间的遗传距离,且系统树中与刀鲚、湖鲚、刀鲚鱼卵混在一起,因此,这些未知鲚属仔幼鱼及稚鱼可能是刀鲚或湖鲚,另外,由于刀鲚的另一种淡水定居生态型-短颌鲚与刀鲚、湖鲚也不能有效区分(许志强等, 2009; 周晓犊等, 2010),所以也有可能是短颌鲚,但长江口和太湖区域一般没有短颌鲚分布(袁传宓等, 1980; 袁传宓等, 1984)。因此未知鲚属仔幼鱼及稚鱼应属于刀鲚或湖鲚。由于湖鲚一般分布于太湖,而在长江口出现的可能性不大(袁传宓等, 1980),所以采自太湖的稚鱼应为湖鲚,采自长江口的未知鲚属仔鱼和幼鱼应判别为刀鲚,而稚鱼采自太湖,应均为湖鲚(李秀启等, 2015)。同时,刀鲚、凤鲚鱼卵在遗传距离和NJ树中也能准确定种。

凤鲚和刀鲚在长江口的繁殖时间有重叠(袁传宓等, 1980),因此,有理由怀疑凤鲚和刀鲚的早期生活个体有一定的混杂,但目前关于长江口仔稚鱼分布的大量调查中(蒋玫等, 2006; 蒋日进等, 2008; 葛珂珂等, 2009),尚未有在同一次调查中的同一站点发现刀鲚和凤鲚并存的报道。何文平等(2011)认为,长江口刀鲚和凤鲚在仔鱼阶段的空间生态位存在明显的分离,在本研究中未知鲚属幼鱼于2月采自长江口东滩样点,根据序列分析结果及生态分布分析表明该批幼

鱼都为刀鲚。该结果说明了长江口刀鲚和凤鲚在幼鱼阶段的空间生态位也存在明显的分离。与此同时,本研究采用Cyt b基因和D-loop序列对采自刀鲚和凤鲚体内的鱼卵成功进行了区分,由于mtDNA呈母系遗传,雌鱼体内鱼卵与未来的受精卵应该有着同一套mtDNA,而七丝鲚一般分布于闽江、九龙江和珠江口区域(袁传宓等, 1980; 姜涛等, 2015),不会在长江口与刀鲚、凤鲚产生混淆;因此,本研究的分子生物学方法应该具有判别野外采集到的鲚属鱼类受精卵的潜力。其将有利于今后进一步研究长江口刀鲚和凤鲚早期生活个体间的空间分布和环境因子的关系,闽江、九龙江和珠江口凤鲚和七丝鲚早期生活个体的空间分布特征,从对总体揭示鲚属鱼类的相互作用关系及资源(鱼卵和仔稚鱼)补充过程差异提供重要的理论支撑。

致谢: 本研究相关基因序列分析得到了中国水产科学研究院淡水渔业研究中心傅洪拓研究员、乔慧博士的支持;论文的撰写得到了南京农业大学无锡渔业学院LY Sokta女士、Khumbanyiwa Davison Daniel先生的协助,谨此感谢。
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