10种帘蛤科贝类COI基因序列分析及系统发育研究

应用通用引物扩增了凸加夫蛤(Gafrarium tumidum)、锯齿巴非蛤(Paphiagallus)、细纹卵蛤(Pitar striatum)、钝缀锦蛤(Tapes dorsatus)、裂纹格特蛤(Marcia hiantina)5种帘蛤科贝类COI基因片段,并与GenBank数据库收录的加夫蛤(Gafrarium pectinatum)、沟纹巴非蛤(Paphia exarata)、日本卵蛤(Pitar japonicum)、日本格特蛤(Marcia japonica)、四射缀锦蛤(Tapes belcheri)5种帘蛤科贝类的同源序列进行比对分析.结果表明:所有物种扩增片段长度均为616 bp,序列A+T平均含量(62.9％)明显高于G+C含量.在616个位点中,保守位点数为282个,变异位点数为334个,其中简约信息位点数为283个.以COI基因片段序列为标记,以海螂科砂海螂(Mya arenaria)作外群,构建了帘蛤科贝类的系统进化树,其拓扑结构显示:细纹卵蛤和日本卵蛤聚为一枝,凸加夫蛤和加夫蛤聚为一枝,锯齿巴非蛤和沟纹巴非蛤聚为一枝,四射缀锦蛤单独聚为一枝,钝缀锦蛤、裂纹格特蛤和日本格特蛤聚为一枝,最后所有帘蛤科物种聚为一枝,与外群相区别,其结果与传统形态分类基本一致.研究表明,线粒体COI基因作为帘蛤科贝类DNA条形码在物种鉴定方面具有可靠性,可以作为物种分类的重要辅助手段.     

基于线粒体COI基因序列的文蛤属（软体动物门：帘蛤科）系统发育关系

测定了4种文蛤属贝类的15个个体的COI基因序列,并从GenBank下载了短文蛤(M petechialis)的相应序列.比对后的序列长度为574bp,包括93个简约信息位点,A、T、C和G的平均含量分别为21.15%、44.71%、14.05%和20.09%.通过对序列的分析,共定义了12个单倍型:文蛤(M.meretrix)4个,斧文蛤(M.lamarckii)2个,丽文蛤(M.lusoria)3个,琴文蛤(M.lyrata)1个,短文蛤2个.以青蛤(cylina sinensis)为外群,用MP法和贝叶斯法构建系统树.结果显示,短文蛤、丽文蛤和文蛤为亲缘关系较近的物种,支持短文蛤和丽文蛤为文蛤的同物异名的观点.     

基于线粒体COI基因序列的文蛤属(软体动物门:帘蛤科)系统发育关系(英文)

测定了4种文蛤属贝类的15个个体的COI基因序列,并从GenBank下载了短文蛤 (M. petechialis)的相应序列。比对后的序列长度为574 bp,包括93个简约信息位点,A、T、C和G的平均含量分别为21.15%、44.71%、14.05%和20.09%。通过对序列的分析,共定义了12个单倍型：文蛤 (M. meretrix) 4个,斧文蛤 (M. lamarckii) 2个,丽文蛤 (M. lusoria) 3个,琴文蛤 (M. lyrata) 1个,短文蛤2个。以青蛤(Cylina sinensis)为外群,用MP法和贝叶斯法构建系统树。结果显示,短文蛤、丽文蛤和文蛤为亲缘关系较近的物种, 支持短文蛤和丽文蛤为文蛤的同物异名的观点。     

10种帘蛤科贝类COI基因序列分析及系统发育研究

应用通用引物扩增了凸加夫蛤（Gafrarium tumidum）、锯齿巴非蛤（Paphia gallus）、细纹卵蛤（Pitar striatum）、钝缀锦蛤（Tapes dorsatus）、裂纹格特蛤（Marcia hiantina） 5种帘蛤科贝类COI基因片段，并与GenBank数据库收录的加夫蛤（Gafrarium pectinatum）、沟纹巴非蛤（Paphia exarata）、日本卵蛤（Pitar japonicum）、日本格特蛤（Marcia japonica）、四射缀锦蛤（Tapes belcheri ）5种帘蛤科贝类的同源序列进行比对分析。结果表明：所有物种扩增片段长度均为616 bp，序列A+T平均含量（62.9%）明显高于G+C含量。在 616个位点中，保守位点数为282个，变异位点数为334个，其中简约信息位点数为283个。以COI基因片段序列为标记，以海螂科砂海螂（Mya arenaria）作外群，构建了帘蛤科贝类的系统进化树，其拓扑结构显示：细纹卵蛤和日本卵蛤聚为一枝，凸加夫蛤和加夫蛤聚为一枝，锯齿巴非蛤和沟纹巴非蛤聚为一枝，四射缀锦蛤单独聚为一枝，钝缀锦蛤、裂纹格特蛤和日本格特蛤聚为一枝，最后所有帘蛤科物种聚为一枝，与外群相区别，其结果与传统形态分类基本一致。研究表明，线粒体COI基因作为帘蛤科贝类DNA条形码在物种鉴定方面具有可靠性，可以作为物种分类的重要辅助手段。     

双壳纲贝类18S rRNA基因序列变异及系统发生

双壳纲贝类栖息于环境多变的海域,是一个形态学和生态学都具有多样性的类群,清晰而可靠的进化关系对于养殖与相关种类的管理具重要意义。然而,目前对双壳类宏观分子系统学研究的报道较少。研究用18S rRNA基因(18S)分析了双壳类3个亚纲贝类的系统发育关系。从GenBank下载帘蛤目、海螂目、贻贝目、胡桃蛤目、蚶目、珍珠贝目6个目94个种类的18S全/部分序列107个,通过ClustalX软件进行序列比对, 用MEGA4.1软件和PHyML软件计算遗传距离, 构建系统发育树, 研究了双壳类18S变异规律及其在系统发生研究中的应用。结果显示18S有插入/缺失序列, 存在长度多态性。序列比对显示有5段约30 70bp的保守区, 4段约130 550bp的高变区。碱基组成平均为T:24.4%, C:23.6%, A:24.5%, G:27.5%。G+C含量为51.1%。在1796个比对位点中, 变异位点占31.7%, 简约信息位点占24.0%。目内科间遗传距离为0.003 0.043, 目间遗传距离为0.026 0.093。NJ树和ML树显示贻贝目、珍珠贝目、胡桃蛤目、蚶目和海螂目的缝栖蛤科先分别聚为支持率很高(BPN=94 100)的单系支, 后聚为一大支(BPN=100)。蛤蜊科与帘蛤目的其他科分离形成一置信度很高的单系支(BPN=93)。帘蛤科种类聚为置信度较低(BPN=60)的一支。海螂目、帘蛤目的种类没能完全聚到所属支系, 彼此嵌套,缝栖蛤科的种类从海螂目中分离出来。18S资料揭示帘蛤目的蛤蜊科、海螂目的缝栖蛤科已经进化为独立的支系。     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

基于COI序列的DNA条形码在中国沿海缀锦蛤亚科贝类中的应用分析

该研究探讨了将COI序列应用于中国沿海缀锦蛤亚科贝类物种鉴定的可行性,获得了该亚科5属11种贝类51个个体的43个单倍型序列.碱幕替换饱和性分析表明,颠换未出现饱和现象,而转换在序列分化达到10%至15%时即到达饱和.单倍犁Hap33可能是由杂交引起的,排除此瞥倍型,种内个体间遗传距离在0%到2.02%之间,平均为0.46%,属内不同种个体间遗传距离在17.21%～32.24%之间,平均为24.96%,存在条形码问隙:11种缀锦蛤哑科贝类在邻接树和贝叶斯树上都独立的单系群.该研究表明,基于COI的DNA条形码技术能够将研究所涉及的约98%的缀锦蛤亚科贝类鉴定剑种的水平,因此,利用DNA条形码技术可以对缀锦蛤亚科贝类进行有效地分类鉴定.     

线蛱蝶亚科蝶类部分类群线粒体COI基因的分子系统发生分析

以线粒体细胞色素氧化酶I（COI）基因作分子标记,对线蛱蝶亚科蝴蝶进行序列测定．序列分析的结果表明．经比对和处理后的序列总长度是645bp,其中有199个变异位点,147个简约信息位点;所编码的氨基酸序列中有18个变异位点,7个信息位点．A＋T平均含量为69．6％,G＋C平均含量为30．4％,碱基组成出现AT偏斜．以蛱蝶亚科及秀蛱蝶亚科物种为外类群,用NJ、MP及贝叶斯法重建了该亚科的系统发生树,探讨了它们主要类群间的系统发生关系．分子系统树显示,线蛱蝶亚科由以下3大支系：环蛱蝶族＋翠蛱蝶族、线蛱蝶族、丽蛱蝶族构成;其中,环蛱蝶族为单系群（NJ树也支持线蛱蝶族的单系性）;翠蛱蝶族与环蛱蝶族亲缘关系较近：丽蛱蝶族可能是该亚科较早分化出的一支．     

基于线粒体COI基因全序列的直翅目部分类群系统发育关系分析

该研究基于直翅目56种昆虫的COI基因全序列构建了该目部分类群间的系统发育关系,同时也分析了COI基因编码的氨基酸序列构建直翅目系统发育关系的可靠性。将COI序列按照密码子一、二、三位点划分,分别计算PBS(partioned Bremer support)值,评估蛋白质编码基因密码子不同位点的系统发生信号强度。分析结果支持螽亚目和蝗亚目的单系性;剑角蝗科、斑腿蝗科、斑翅蝗科、网翅蝗科和槌角蝗科5科均不是单系群,科间的遗传距离在0.107~0.153之间变化,与其他科相比遗传距离较小,符合将这5科合并为一科(即蝗科)的分类系统,瘤锥蝗科和锥头蝗科归为锥头蝗总科,癞蝗科单独成为一科,这也与Otte(1997)系统的划分一致。根据PBS值的大小推断密码子第三、第一位点对系统树分支的贡献比第二位点大,并且较长的序列含有较多的信息位点。研究也证实将各物种COI基因之间的遗传距离作为直翅目划分科级阶元的工具是可行的。     

扬子鳄MHCⅡ类B基因第二外元的克隆及序列分析

3头扬子鳄血样取自宣城安徽省扬子鳄繁殖研究中心。利用一对简并引物对MHCⅡ类B基因第二外元的部分片段进行扩增;通过克隆、单链构象多态性分析、测序,并将测得序列与下载的8个物种MHC序列比对,确定序列差异和变异位点;利用MEGA软件构建NJ树,PAUP4．0构建MP树。结果得到10种不同的序列,片段长166bp。核苷酸序列中有38个变异位点,氨基酸序列中有23个变异位点;推定的抗原结合位点非同义替换(dN)明显高于同义替换(dS)。10种序列的NJ树和MP树极为相似,均为A、B两个分支,两个分支明显的特异性位点核苷酸序列中有9个。氨基酸序列中有7个。表明扬子鳄MHCⅡ类B基因第二外元有较高的多态性,有利于扬子鳄饲养种群的遗传保护。     

The complete mitochondrial genome of the hard clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

Veneridae is a diverse, commercially important, and cosmopolitan family. Here we present the complete mitochondrial genome of the hard clam Meretrix meretrix (Bivalvia: Veneridae). The entire mitochondrial genome (mitogenome) sequence of M. meretrix is 19,826 bp in length, and contains 37 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 23 tRNAs. All genes are encoded on the heavy strand. In contrast to the typical animal mitochondrial genome, it lacks the protein-coding gene ATP8, and has only one copy of the tRNA^Ser gene, but three duplications of the tRNA^Gln, which is the first report among the present molluscan mtDNAs. We observed that the gene arrangement between M. meretrix and M. petechialis is same except one more tRNAGln gene in M. meretrix., and the sequence similarity is as high as 99%, indicating that M. petechialis and M. meretrix could be treated as a junior synonym of M. meretrix. Maximum Likelihood and Bayeslan analysis of 12 concatenated protein-coding amino acid sequences place the Unionidae as a sister group to other bivalves, which reflects the general opinion that the Unionidae deverged very early in Bivalvia evolution.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Isolation and characterization of a virulent Vibrio sp. bacterium from clams (Meretrix meretrix) with mass mortality

MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (Yue et al., 2010). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84 h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P < 0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix.     

Three EST-SSR Markers Associated with QTL for the Growth of the Clam Meretrix meretrix Revealed by Selective Genotyping

The clam Meretrix meretrix is a member of widely cultured, commercially important clams. A marker–trait association analysis was performed using expressed sequence tag (EST) simple sequence repeat (SSR) markers for marker-assisted selection in M. meretrix. Three markers, MM1272, MM2034, and MM7721, were found to be significantly associated with quantitative trait loci (QTLs) controlling shell length (P?<?0.0001) in clams of a fast-growing population (JSF) and a control population (JSC). The 144-bp allele of MM1272, the 154-bp allele of MM2034, and the 152- and 165-bp alleles of MM7721 showed a significantly higher frequency in the JSF population (17.65, 36.41, 28.67, and 29.33 %) than in the JSC population (4.65, 8.33, 3.47, and 5.56 %). The three markers showed lower values for the number of alleles and observed heterozygosity as well as a higher proportion of homozygotes in JSF than in JSC population. The three markers have been further confirmed in the high and low tails of another population (09G₃SPSB); similarly, lower values for the number of alleles and observed heterozygosity as well as a higher proportion of homozygotes were found in 09G₃SPSB_H. The putative functions of the three gene fragments containing MM1272, MM2034, and MM7721 also suggested that the three SSR-containing genes might be involved in growth of M. meretrix. All the results suggest that the three EST-SSR markers associated with growth QTLs would be useful for marker-assisted selection in M. meretrix breeding.     

Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality

A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of ∼6 × 10⁶ CFU ml⁻¹. MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding.

Methodology/Principal Findings

Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique.

Conclusions/Significance

A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.     

The Complete Female- and Male-Transmitted Mitochondrial Genome of Meretrix lamarckii

Bivalve mitochondrial genomes show many uncommon features, like additional genes, high rates of gene rearrangement, high A-T content. Moreover, Doubly Uniparental Inheritance (DUI) is a distinctive inheritance mechanism allowing some bivalves to maintain and transmit two separate sex-linked mitochondrial genomes. Many bivalve mitochondrial features, such as gene extensions or additional ORFs, have been proposed to be related to DUI but, up to now, this topic is far from being understood. Several species are known to show this unusual organelle inheritance but, being widespread only among Unionidae and Mytilidae, DUI distribution is unclear. We sequenced and characterized the complete female- (F) and male-transmitted (M) mitochondrial genomes of Meretrix lamarckii, which, in fact, is the second species of the family Veneridae where DUI has been demonstrated so far. The two mitochondrial genomes are comparable in length and show roughly the same gene content and order, except for three additional tRNAs found in the M one. The two sex-linked genomes show an average nucleotide divergence of 16%. A 100-aminoacid insertion in M. lamarckii M-cox2 gene was found; moreover, additional ORFs have been found in both F and M Long Unassigned Regions of M. lamarckii. Even if no direct involvement in DUI process has been demonstrated so far, the finding of cox2 insertions and supernumerary ORFs in M. lamarckii both strengthens this hypothesis and widens the taxonomical distribution of such unusual features. Finally, the analysis of inter-sex genetic variability shows that DUI species form two separate clusters, namely Unionidae and Mytilidae+Veneridae; this dichotomy is probably due to different DUI regimes acting on separate taxa.