Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.     

Identification of bloodmeals in haematophagous Diptera by cytochrome B heteroduplex analysis

We developed a DNA assay for bloodmeal identification in haematophagous insects. Specific host cytochrome B gene sequences were amplified by PCR and classified on the basis of their mobility in a heteroduplex assay. In the blackfly Simulium damnosum s.l. (Diptera: Simuliidae), human cytochrome B DNA sequences were identifiable up to 3 days following ingestion of the bloodmeal. In the tsetse Glossina palpalis (Diptera: Glossinidae) collected from tsetse traps in Ivory Coast, bloodmeals were identified as taken from domestic pigs on the basis of their heteroduplex pattern and DNA sequence. Evidently the cytochrome B sequence shows sufficient interspecific variation to distinguish between mammalian host samples, while exhibiting minimal intraspecific variation. The stability of DNA in bloodmeals, for several days post-ingestion by haematophagous insects, allows PCR-HDA assays to be used reliably for host identification.     

DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.     

12S rRNA和Cyt b基因序列测定在獐乳制品鉴定中的应用

采用改良的蛋白酶K消化和酚/氯仿抽提的方法从脂肪含量很高的动物乳制品及胃组织中提取出基因组总DNA,利用特异引物扩增了线粒体12S rRNA基因和Cyt b基因的部分片段,测定了12S rRNA和Cyt b基因的PCR扩增产物序列,使用BLAST搜索软件将其序列与GenBank中的同源序列进行比对,并利用DNAMAN分析软件分析序列同源性。结果表明3件检材均来源于獐Hydropotes inermis。本研究所用方法在野生动物乳制品鉴定中具有较高的应用价值。     

通用引物双重PCR在节肢动物物种鉴定中的应用

【目的】本研究旨在使用基于线粒体基因通用引物的双重PCR技术同时扩增单一样本中两条标记基因,从而达到简化节肢动物物种鉴定流程的目的。【方法】在一次PCR实验中同时加入可扩增线粒体COI基因和16S rDNA两个不同分子标记的引物,对3纲8目14科的14种节肢动物物种标本的基因组DNA进行扩增;扩增产物经电泳和胶回收后测序,并BLAST在线搜索相似序列,验证基于通用引物的双重PCR在不同的动物类群中用于物种鉴定的有效性。【结果】应用基于COI和16S rDNA的引物从分属于3纲8目14科的14种节肢动物基因组DNA中均可成功扩增目的基因;扩增产物测序结果进一步证实了扩增的准确性。【结论】通过本方法进行物种的分子鉴定,不仅可以保证物种鉴定的高准确率,还可以明显减少时间与DNA样本量的消耗,这对需要快速准确鉴定物种或珍稀的材料样本十分重要。     

mtDNA标记在几种海关进出口动物产品鉴定中的应用

从海关查获的动物及其制品中提取基因组DNA,通过PCR技术扩增mtDNA细胞色素b(cytochrome b,cyt b)和12S rRNA基因并测定其序列。利用互联网上丰富的基因数据库进行BIAST。搜索,准确地鉴定了动物的种类,从而为海关查获的走私珍稀野生动物的鉴定提供了一条灵敏和可靠的途径。     

不同品种金鱼和鲫鱼的分子系统发育关系研究

为探讨不同品种金鱼的系统进化关系,利用PCR技术扩增了金鱼的7个代表品种红龙睛(Red dragongoldfish)、红帽子(Red cap goldfish)、虎头(Tiger head goldfish)、琉金(Gold plating goldfish)、墨龙睛(Black dragongoldfish)、水泡眼(Water vesicle goldfish)、珍珠(Genuine pearl goldfish)的线粒体DNA上细胞色素b的部分核苷酸序列,长度为597 bp.结合GenBank中红鲫、野鲫、日本白鲫、银鲫、鲤鱼的序列进行比较分析,结果显示,这7个金鱼品种之间的同源性都很高,在99.5%～100%之间;7种金鱼和红鲫的同源性也很高,为99.5%～99.8%,与野鲫的同源性在96.8%～97.2%,与日本白鲫、银鲫的同源性为93.1%～94.3%,与鲤鱼的同源性相对较低,为88.3%～88.6%.利用DNAstar软件构建了不同品种金鱼和鲫鱼的分子系统树,从分子水平进一步证实了金鱼起源于野鲫.     

DNA barcoding of Oryx leucoryx using the mitochondrial cytochrome C oxidase gene

The massive destruction and deterioration of the habitat of Oryx leucoryx and illegal hunting have decimated Oryx populations significantly, and now these animals are almost extinct in the wild. Molecular analyses can significantly contribute to captive breeding and reintroduction strategies for the conservation of this endangered animal. A representative 32 identical sequences used for species identification through BOLD and GenBank/NCBI showed maximum homology 96.06% with O. dammah, which is a species of Oryx from Northern Africa, the next closest species 94.33% was O. gazella, the African antelope. DNA barcode sequences of the mitochondrial cytochrome C oxidase (COI) gene were determined for O. leucoryx; identification through BOLD could only recognize the genus correctly, whereas the species could not be identified. This was due to a lack of sequence data for O. leucoryx on BOLD. Similarly, BLAST analysis of the NCBI data base also revealed no COI sequence data for the genus Oryx.     

Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys

DNA barcoding is a powerful tool for species detection, identification and discovery. Metazoan DNA barcoding is primarily based upon a specific region of the cytochrome c oxidase subunit I gene that is PCR amplified by primers HCO2198 and LCO1490 (‘Folmer primers’) designed by Folmer et al. (Molecular Marine Biology and Biotechnology, 3 , 1994, 294). Analysis of sequences published since 1994 has revealed mismatches in the Folmer primers to many metazoans. These sequences also show that an extremely high level of degeneracy would be necessary in updated Folmer primers to maintain broad taxonomic utility. In primers jgHCO2198 and jgLCO1490, we replaced most fully degenerated sites with inosine nucleotides that complement all four natural nucleotides and modified other sites to better match major marine invertebrate groups. The modified primers were used to amplify and sequence cytochrome c oxidase subunit I from 9105 specimens from Moorea, French Polynesia and San Francisco Bay, California, USA representing 23 phyla, 42 classes and 121 orders. The new primers, jgHCO2198 and jgLCO1490, are well suited for routine DNA barcoding, all‐taxon surveys and metazoan metagenomics.     

斗鱼属鱼类亲缘关系的Cyt b基因序列和RAPD分析

采用mtDNA Cyt b基因序列分析和RAPD两种分子标记技术,研究了中国分布的叉尾斗鱼(Macropodus opercularis)、圆尾斗鱼(M.chinensis)和香港斗鱼(M.hongkongensis)以及越南的红鳍斗鱼(M.erythropterus)4种鱼类之间的亲缘关系.获得4种斗鱼14条Cyt b基因全序列(1 155 bp),结合GenBank中搜索到的近缘物种同源序列进行分析.从133条随机引物中筛选到36条引物,在优化的反应条件下,10个群体96个个体共扩增出清晰稳定的条带749条,构建矩阵进行分析和聚类.基于Cyt b全序列以邻接法和最小进化法构建的系统树及RAPD数据UPGMA聚类分析的结果都显示,香港斗鱼和红鳍斗鱼先聚为一分支,再与叉尾斗鱼聚类,圆尾斗鱼处于外缘.本研究结果反映了圆尾斗鱼与其他斗鱼的亲缘关系较远,种间遗传距离为0.184 5～0.225 3(Cyt b)和0.653 6～0.746 5(RAPD),两者为同一单系群中两个独立演化的自然类群;香港斗鱼与叉尾斗鱼间遗传分化明显,Cyt b碱基差异为11.00%,RAFD遗传距离达0.577 7,支持其为独立物种的观点,且香港斗鱼群体间遗传差异较大,Cyt b碱基差异为3.12%,RAPD遗传距离0.060 1;叉尾斗鱼群体间Nei's基因多样度和Shannon信息指数分别为0.058 2和0.086 9,而各群体内的数值分别为0.016 1～0.031 7和0.023 5～0.046 7,表明遗传差异主要来自群体间,并按分布流域分别聚类.     

EST derived PCR-based markers for functional gene homologues in cotton.

We investigated the utility of the Gossypium arboreum EST sequences in the GenBank database for developing PCR-based markers targeting known-function genes in cultivated tetraploid cottons, G. hirsutum and G. barbadense. Four hundred sixty-five randomly selected ESTs from this library were subjected to BLASTn search against all GenBank databases, of which putative function was assigned to 93 ESTs based on high nucleotide homology to previously studied genes. PCR primers were synthesized for 89 of the known-function ESTs. A total of 57 primer pairs amplified G. arboreum genomic DNA, but only 39 amplified in G. hirsutum and G. barbadense, suggesting that sequence divergence may be a factor causing non-amplification for some sites. DNA sequence analysis showed that most primer pairs were targeting the expected homologous loci. While the amplified products that were of larger size than the corresponding EST sequences contain introns, the primer pairs with a smaller amplicon than predicted from the flanking EST sequences did not amplify the expected orthologous gene sequences. Among the 39 primer pairs that amplified tetraploid cotton DNA, 3 detected amplicon size polymorphisms and 10 detected polymorphisms after digestion with one of six restriction enzymes. Ten of the polymorphic loci were subsequently mapped to an anchor RFLP map. Digestion of PCR-amplified sequences offers one means by which cotton genes can be mapped to their chromosomal locations more quickly and economically than by RFLP analysis.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

基于线粒体Cyt b基因的全长序列探讨闭壳龟类的系统进化

采用PCR技术对淡水龟科具闭壳结构的黄缘盒龟、黄额盒龟、金头闭壳龟、潘氏闭壳龟、锯缘龟和白腹摄龟的线粒体Cytb基因的全长序列进行了PCR扩增和序列测定,并结合GenBank中16种淡水龟科物种的同源序列,进行了序列变异和系统发生分析。经C lustalX1.8软件对位排列后共有1154个位点,其中可变位点413个,简约信息位点301个;A＋T的平均含量（56.5%）高于G＋C（43.5%）。在氨基酸密码子中,第一位富含A,第二位富含T,第三位富含C;碱基转换/颠换率为5.97,碱基替换多发生在密码子第三位。以中华鳖和马来鳖为外群,通过最大简约法,最大似然法和贝叶斯法重建了分子系统树,均具有一致的拓扑结构,结果表明：金头闭壳龟和潘氏闭壳龟最先聚成一支,再和三线闭壳龟聚成一组,说明形态上相似的三种闭壳龟亲缘关系最近;闭壳龟属、盒龟属和单种锯缘龟属聚成一个单系的闭壳龟群,建议合并为闭壳龟属;齿缘龟属和果龟属聚为一支,它们与新的闭壳龟属关系较远,揭示闭壳结构的形成不是由一个共同祖先分化而来;乌龟属、花龟属和拟水龟属三属为并系起源,建议三属可以合并为一属。     

Reconstructing mammalian phylogenies: a detailed comparison of the cytochrome B and cytochrome oxidase subunit I mitochondrial genes

The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cyt b more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cyt b correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cyt b also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5-2.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.     

一枚恐龙蛋内细胞色素b基因片段的序列分析

用一对根据人线粒体细胞色素ｂ基因保守区序列设计的引物,以一枚恐龙蛋内ＤＮＡ为模板,在ＰＣＲ上扩增出大约１７０ｂｐ的基因片段。序列测定后经ＩＮＴＥＲＮＥＴ联网的ＢＬＡＳＴ数据库类似性检索,结果表明,该序列与人类线粒体细胞色素ｂ基因的序列完全相同（９６－１００％相似）。因此,可以肯定实验材料本身有严重污染。     

Identifying the last supper: utility of the DNA barcode library for bloodmeal identification in ticks

Ticks are among the most important vectors of disease in the Northern Hemisphere, and a better understanding of their feeding behaviour and life cycle is critical to the management and control of tick-borne zoonoses. DNA-based tools for the identification of residual bloodmeals in hematophagous arthropods have proven useful in the investigation of patterns of host use in nature. Using a blind test approach, we challenged the utility of the DNA barcode library for the identification of vertebrate bloodmeals in engorged, field-collected Ixodes scapularis. Universal vertebrate primers for the COI barcode region successfully amplified DNA from the host bloodmeal and only rarely amplified tick DNA. Of the 61 field-collected ticks, conclusive genus- and species-level identification was possible for 72% of the specimens. In all but two cases, barcode-based identification of the bloodmeal was consistent with the morphological identification of the vertebrate host the ticks were collected from. Possible explanations for mismatches or ambiguities are presented. This study validates the utility of the DNA barcode library as a valuable and reliable resource for the identification of unknown bloodmeals in arthropod vectors of disease. Future directions aimed at the refinement of these techniques to gain additional information and to improve the amplification success of digested vertebrate DNA in tick bloodmeals are discussed.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

High-resolution melting (HRM) of the cytochrome B gene: a powerful approach to identify blood-meal sources in Chagas disease Vectors

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate.