Species status of Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata using ITS and AFLP markers
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摘要:
我国合浦珠母贝Pinctada fucata的命名较混乱,与日本的P. fucata martensii和澳大利亚的P. imbricata的分类关系存在争议。用ITSs序列和AFLP标记对这3个种的遗传关系进行了分析。结果表明这3个地理种的遗传分化很低,种内和种间的遗传距离相互重叠,AFLP数据的聚类分析表明澳大利亚种群大部分个体单独聚合成一支,主成分分析结果相似。种间的遗传距离与分化程度与其地理距离呈正相关。分子方差结果也表明种间的差异较小,低于6%。这些结果表明这3个种实际上是同一物种。由于大西洋的P. mbricata与澳大利亚的不同,因此根据分类命名的优先原则,正确的种名应为P. fucata。
Abstract:Species status for Chinese Pinctada fucata, Japanese P. fucata martensii and Australian P. imbricata was investigated using ITSs and AFLP. The results indicated that genetic differentiation among populations of the three nominal species is low, and intraspecific and interspecific genetic distances are overlapping, as revealed by ITSs and AFLP markers. Based on AFLP data, phylogenetic analysis showed that most individuals from P. imbricata population are clustered together, consistent with the result from principal component analysis. It was found that the three nominal species are genetically isolated by geographic distance. AMOVA indicated that no more than 6% of the total variation is attributed to interspecific differences. These observations support the hypothesis that the three species are conspecific. According to the priority rule of nomination, the correct name of the species should be P. fucata because Atlantic P. imbricata is reported to be genetically different from Australian P. imbricata.
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金焰笛鲷(Lutjanus fulviflamma)又称火斑笛鲷,属硬骨鱼纲(Osteichthyes)、鲈形目(Perciformes)、笛鲷科(Lutjanidae),主要分布于印度洋和太平洋西部海域,我国见于南海和东海南部,是一种颇受人们欢迎的经济鱼类。近年来受市场需求的驱动,我国南海的捕捞强度明显增加,加之环境的恶化,使金焰笛鲷的种群数量和产量都明显降低,种质资源受到威胁。因此开展其遗传多样性的研究对其种质资源的保护和恢复都有重要的意义。
目前针对金焰笛鲷遗传多样性的研究主要有刘丽等[1]利用RAPD技术对金焰笛鲷进行了分析,发现金焰笛鲷个体间的平均遗传相似系数为0.6879,平均遗传距离0.3121,平均遗传多样性指数为0.1353,同时利用SSR技术计算出金焰笛鲷群体的杂合度为0.593,多态信息含量为0.554,并认为目前南海的金焰笛鲷的遗传多样性比较丰富;王中铎等[2]利用RFLP技术对其mt DNA进行了分析,结果同样表明目前金焰笛鲷的遗传多样性比较丰富,并利用cyt-b部分序列构建了5种笛鲷的分子系统树。
真核生物的核糖体DNA(rDNA)中的ITS-1和ITS-2分别位于18S和5.8S以及5.8S和28S rRNA之间,属于非编码区。由于ITS最终不加入成熟核糖体,所以受到的选择压力较小,进化速度较快,可以从中获得较多的信息[3]。并且变异的速度在不同的物种间也存在着较大的差异,因此ITS非常适合用于各种分子操作。目前在微生物、植物和无脊椎动物中利用ITS探讨不同生物之间的系统关系、种质鉴定和进化方式的研究已经比较成熟[4-5]。但是利用ITS对鱼类进行分析的研究还非常少,目前在这一方面研究比较深入的是华盛顿大学的PHILLIPS教授,在国内还没有见到在鱼类中利用ITS区进行研究的报道。本研究以南海常见的经济鱼类金焰笛鲷为研究对象,对其rDNA的ITS-1进行序列分析,作为一种新的DNA标记,以期能进一步对南海笛鲷的种群遗传多样性、进化关系和系统发生的研究奠定基础。
1. 材料与方法
1.1 实验材料
金焰笛鲷于2002年6月采集于广东省湛江海区的野生群体,依照《南海鱼类志》[6]进行形态学鉴定,从尾静脉抽取血液,加ACD(acid citrate dextrose)抗凝,-70℃保存备用。
1.2 实验方法
1.2.1 基因组DNA的提取
每尾鱼取50 μL血液,采用常规的苯酚/氯仿方法[7]提取基因组总DNA,溶解于TE中,-70°保存备用。
1.2.2 PCR扩增
引物ITS-1F/R(由华盛顿大学的PHILLIPS教授设计[8])由上海生工公司合成,序列为:ITS-1 F:5-AAA AAG CTT CCG TAG GTG AAC CTG CG-3;ITS-1 R:5-AGC TTG CTG CGT TCT TCA TCG A-3,PCR扩增仪为Hema 8000型,反应总体积为25 μL,其中含有1×PCR Buffer 2.5 μL,0.2 mM dNTPs,0.2 μM双向引物,1.3 mM MgCl2,1.5U Taq酶(以上试剂均为上海生工公司产品),DNA模板1 μL,用灭菌超纯水补足至25 μL。PCR扩增条件优化为:94℃预变性5 min;然后94℃变性50 s,56℃退火1 min,72℃延伸1 min,共35个循环;最后在72℃延伸5 min,4℃保存。
1.2.3 PCR产物克隆
扩增产物以1.5%的琼脂糖凝胶电泳检测确定为单一条带后,将PCR产物利用UNIQ-5柱式胶回收试剂盒(SK1132,Sangon)纯化回收后,将纯化产物与pUCm-T载体(PCR产物克隆试剂盒BS425,BBI公司)连接,连接反应体系为10 μL(含1 μL载体,1 μL PEG,1 μL Ligase buffer,1 μL T4连接酶,5 μL纯化产物,1 μL ddH2O),16℃连接3 h后,42℃转化到DH5a感受态细胞,然后通过蓝白斑进行阳性克隆筛选,挑取白色菌落用LB培养基继续扩大培养,用M13+/-引物对克隆进行检测[7]。
1.2.4 测序
选择含有目的插入片段的克隆由上海生工公司进行测序,测序引物用M13+/-通用引物,进行双向测序。
1.2.5 数据处理
测序结果经过人工校正核对,除去测得的引物序列和扩增的部分18S和5.8S rRNA序列,得到完整的ITS-1序列,利用MEGA3.1软件计算碱基组成含量。
2. 结果与讨论
选用的引物在金焰笛鲷扩增出了约640 bp的DNA片段(图 1),该片段克隆后用M13+/-通用引物进行检测大约850 bp。引物是根据rDNA的18S和5.8S rRNA基因的保守性设计的,最初是用来扩增大西洋鲑的ITS-1,由于18S、5.8S和28S rRNA的高度保守性,本试验尝试利用此引物来扩增笛鲷属的鱼类,得到目的片段,说明此引物有很好的通用性,适用于笛鲷属鱼类的研究,目前已经对笛鲷属的红鳍笛鲷L. erythropterus、勒氏笛鲷L. russelli、紫红笛鲷L. argentimaculatus和画眉笛鲷L. vitta的ITS-1进行了序列测定和分析,以期能利用ITS序列对南海笛鲷属鱼类的系统发生关系进行可靠的分析。
由于ITS在不同种内变异比较大,因此适合用作种以上分类阶元或不同地理种群的系统学分析[4-5],但在同种内一般变异不大,本试验随机选取2个样本进行克隆测序,测得的序列长度均为566 bp,随机选择一个递交GenBank(序列如图 2),登录号为:DQ354988。该序列的碱基组成分别为:14.1%(A)、16.1%(T)、30.2%(G)、39.6%(C),G+C(69.8%)含量明显高于A+T含量(30.2%),这和在GenBank中检索到的其他鲈形目鱼类的ITS的碱基组成相似。
用BLAST在GenBank中进行搜索比对,发现此序列与许多物种的ITS-1有着较高的同源性,其中与目前在GenBank中登录的鳜鱼的同源性最高,而它们都同属于鲈形目,并且符合传统的分类地位,与GenBank中登录的其他属的鱼比较后发现有较大的变异,并且由于ITS-1片段的长度适合测序分析,可以提供较多的位点信息,因此它是一个很好的可用于分析种、属及以上分类单元研究的序列。
本研究还对笛鲷属其他几种鱼的ITS-1进行分析,发现ITS-1在不同种间存在着比较大的差异,这些差异主要体现在序列长度上,如同为笛鲷科的勒氏笛鲷ITS-1片段长度为769 bp,两者相差200多个碱基,但是它们的碱基组成相差并不大;同时几种鱼的比对结果显示ITS-1在靠近5.8S rRMA端同源性非常高,而在靠近18S端却差异非常大。这些数据同样可以说明ITS区完全可以作为一个很好的分子标记用来分析物种的分子系统发生,种类和种群的分类鉴定以及遗传多样性的分析。
目前,对ITS区的研究主要集中在微生物、植物和无脊椎动物中,在水产经济动物中目前开展的研究还主要集中在软体动物上。喻达辉和朱嘉濠[9-10]以及喻达辉等[11-12]对珍珠贝的ITS-2序列特征进行了分析,并利用ITS-1和ITS-2对珠母贝属主要种类进行了系统发育研究;何毛贤[13]对长耳珠母贝的ITS-2序列进行了分析;陈琳林等[14]对魁蚶的ITS-1和ITS-2进行了克隆测序,同时对5种双壳贝类进行了序列比较;魏晓华[15]将测得的4种扇贝的ITS-1序列与其他4种扇贝构建了系统发育树,发现与其他方式构建的有非常强的一致性;KONG[16]对太平洋牡蛎的ITS区进行了分析,分析了其碱基组成。YU[17]分析了栉孔扇贝的ITS-1和ITS-2,认为这2个变异性较大的序列在扇贝种群中应用潜力很大,可广泛用于种内群体间遗传变异研究、种质鉴别及系统学研究。在鱼类的ITS研究中,目前国内尚未见报道,在国外主要集中在鲑科,DOMANICO[18]等通过对rDNA的ITS-1和ITS-2区基因的测序结果,综合分析了大西洋鲑的系统发育关系,分析认为细鳞大麻哈鱼(pink salmon)和大麻哈鱼(chum salmon)以及银大麻哈鱼(coho salmon)和大鳞大麻哈鱼(chinook salmon)之间有最近的亲缘关系。LINN[19]等通过对白鲑种类rDNA-ITS-1的序列分析,构建了较大范围的系统发育树,分别与蛋白质和mtDNA所构建的系统发育树进行了比较,得到了较为一致的结果。
本研究在国内水产经济鱼类中首先进行了ITS-1的序列分析,对引物的可用性、实验的可行性及其应用和前景进行了探讨。相信随着分子生物学技术的发展以及人们对ITS研究规律认识的深入,序列数据库的不断充实,这一方面的研究将在水产动物的系统发生、遗传变异和种质鉴定方面起着重要的作用。
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表 1 ITS1的等位基因及其在各种群中的分布
Table 1 Alleles of ITS1 and their distributions in populations of each species
种群
populationh01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 CN 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 JP 3 3 1 AUS 1 种群
populationh16 h17 h18 h19 h20 h21 h22 h23 h24 h25 h26 h27 h28 h29 sum CN 1 18 JP 1 1 1 1 12 AUS 1 3 1 1 1 3 1 1 1 14 注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
Note: h01, h02, …: allele;CN. Chinese population;JP. Japanese population;AUS. Australian population表 2 ITS2的等位基因及其在种群中的分布
Table 2 Alleles of ITS2 and their distributions in populations of each species
种群
populationh01 h02 h03 h04 h05 h06 h07 h08 h09 h10 h11 h12 h13 h14 h15 sum CN 2 2 1 2 1 1 3 1 1 1 1 16 JP 1 3 1 2 1 8 AUS 3 1 3 1 2 10 注:h01, h02, …: 等位基因;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
Note:h01, h02, …:allele;CN. Chinese population;JP. Japanese population;AUS. Australian population表 3 ITS1和ITS2的种内和种间的平均K2P遗传距离及AFLP的Nei & Li平均遗传距离
Table 3 Average intra- and interspecific K2P genetic distances from ITS1 & 2, and Nei and Li′s (1979) interspecific genetic distances from AFLP data
种群
population种内平均遗传距离/%
intraspecific genetic distance种间遗传距离/%(下三角: ITS 1,上三角: ITS2)
interspecific genetic distance (below diagonal: ITS1, above diagonal: ITS2)AFLP的Nei&Li′s
遗传距离
Nei&Li′s distance for AFLPITS 1 ITS 2 CN JP AUS CN JP CN 0.87
(0~2.00)0.77
(0~1.80)- 1.25
(0~3.10)0.87
(0~2.20)JP 0.69
(0~1.50)1.20
(0~1.80)0.90
(0~2.00)- 1.11
(0~2.60)0.74 AUS 0.54
(0~1.20)0.79
(0~1.80)0.84
(0~2.00)0.67
(0~1.30)- 2.39 2.49 注:CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
Note: CN. Chinese population;JP. Japanese population;AUS. Australian population表 4 基于DNA序列信息或等位基因频率的遗传分化显著性检验
Table 4 Significant test on genetic differentiation among species based on sequence information (K*ST) or allelic frequencies (FST)
KST* FST ITS1 ITS2 AFLP 种群
populationCN AUS CN AUS CN AUS AUS 0.057*** 0.004ns 0.084* JP 0.034* 0.034* 0.095*** 0.113* 0.021* 0.092* 注:ns. 不显著;*. 0.01 < P < 0.05;***. P < 0.001;CN. 中国种群;JP. 日本种群;AUS. 澳大利亚种群
Note:ns. not significant;*. 0.01 < P < 0.05;***.P < 0.001;CN. Chinesse P. fucata;JP. Japanese P. fucata martensii;AUS. Australian P. imbricata -
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