Complete sequence of the Tibetan Mastiff mitochondrial genome and its phylogenetic relationship with other Canids ( Canis, Canidae)

In this study, the complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined, and the phylogenetic relationships between the Tibetan Mastiff and other species of Canidae were analyzed using the coyote (Canis latrans) as an outgroup. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16 710 bp, and included 22 tRNA genes, 2S rRNA gene, 13 protein-coding genes and one non-coding region (D-loop region), which is similar to other mammalian mitochondrial genomes. The characteristics of the protein-coding genes, non-coding region, tRNA and rRNA genes among Canidae were analyzed in detail. Neighbor-joining and maximum-parsimony trees of Canids constructed using 12 mitochondrial protein-coding genes showed that as the coyotes and Tibetan wolves clustered together, so too did the gray wolves and domestic dogs, suggesting that the Tibetan Mastiff originated from the gray wolf as did other domestic dogs. Domestic dogs clustered into four clades, implying at least four maternal origins (A to D). The Tibetan Mastiff, which belongs to clade A, appears to be closely related to the Saint Bernard and the Old English Sheepdog.     

Origin and phylogenetic analysis of Tibetan Mastiff based on the mitochondrial DNA sequence

At present, the Tibetan Mastiff is the oldest and most ferocious dog in the world. However, the origin of the Tibetan Mastiff and its Phylogenetic relationship with other large breed dogs such as Saint Bernard are unclear. In this study, the primers were designed according to the mitochondrial genome sequence of the domestic dog, and the 2,525 bp mitochondrial sequence, containing the whole sequence of Cytochrome b, tRNA-Thr, tRNA-Pro, and control region of the Tibetan Mastiff, was obtained. Using grey wolves and coyotes as outgroups, the Tibetan Mastiff and 12 breeds of domestic dogs were analyzed in phylogenesis. Tibetan Mastiff, domestic dog breeds, and grey wolves were clustered into a group and coyotes were clustered in a group separately. This indicated that the Tibetan Mastiff and the other domestic dogs originated from the grey wolf, and the Tibetan Mastiff belonged to Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris on the animal taxonomy. In domestic dogs, the middle and small breed dogs were clustered at first; German Sheepdog, Swedish Elkhound, and Black Russian Terrier were clustered into one group, and the Tibetan Mastiff, Old English Sheepdog, Leonberger, and Saint Bernard were clustered in another group. This confirmed the viewpoint that many of the famous large breed dogs worldwide Such as Saint Bernard possibly had the blood lineage of the Tibetan Mastiff, based on the molecular data. According to the substitution rate, we concluded that the approximate divergence time between Tibetan Mastiff and grey wolf was 58,000 years before the present (YBP), and the approximate divergence time between other domestic dogs and grey wolf was 42,000 YBP, demonstrating that the time of origin of the Tibetan Mastiff was earlier than that of the other domestic dogs.     

Comparative genomic analysis of high-altitude adaptation for Mongolia Mastiff,Tibetan Mastiff,and Canis Lupus

Tibetan Mastiff has adapted to the extreme environment of the Qinghai-Tibetan Plateau. Yet, the underlying mechanisms of its high-altitude-adaptation and origin remains elusive. Here, we generated the draft genomes of Mongolia Mastiff, Tibetan Mastiff, and Canis Lupus. The phylogenetic tree uncovered that Tibetan Mastiff and Mongolia Mastiff were derived from Canis Lupus species. The comparative genomic analyses identified that the expansion of gene families related to DNA repair and damage response, and contraction related to ATPase activity revealed the genetic adaptations of Tibetan Mastiff and Canis Lupus to high altitude. In addition, the Tibetan Mastiff and Canis Lupus had signals of positive selection for genes involved in fatty-acid α/β- oxidation for highland adaptation. Notably, the positively selected TERT of Tibetan Mastiff should be an adaptive trait for correcting DNA damage. These findings suggested that the Tibetan Mastiff and Canis Lupus evolves basic strategies for adaptation to high altitude.     

Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)

DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms (ca. 20,000 species) has not been established. We succeeded in generating 167 partial COI sequences (~450 bp) representing ~100 morphospecies from ~650 collections of Agaricomycotina using several sets of new primers. Large introns (~1500 bp) at variable locations were detected in ~5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (~30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.     

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648‐bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.     

基于mtDNA COI基因的离腹寡毛实蝇属常见种DNA条形码识别和系统发育分析

【目的】离腹寡毛实蝇属Bactrocera昆虫是最具经济重要性的实蝇类害虫,本研究依据mtDNA COI基因碱基序列对离腹寡毛实蝇属常见实蝇种类进行识别鉴定与系统发育分析。【方法】以口岸经常截获的离腹寡毛实蝇属8个亚属21种实蝇为对象,采用DNA条形码技术,通过对mtDNA COI基因片段 (约650 bp)的测序和比对,以MEGA软件的K2-P双参数模型计算种内及种间遗传距离,以邻接法(NJ) 构建系统发育树。【结果】聚类分析与形态学鉴定结果一致,除11种单一序列实蝇外,其他10种实蝇均各自形成一个单系,节点支持率为99%以上。种内（10种）遗传距离为0.0003~0.0068,平均为0.0043;种间（21种）遗传距离为0.0154~0.2395,平均为0.1540;种间遗传距离为种内遗传距离的35.8倍,而且种内、种间遗传距离没有重叠区域。【结论】基于mtDNA COI基因的DNA条形码技术可以用于离腹寡毛实蝇属昆虫的快速鉴定识别,该技术体系的建立对实蝇类害虫的检测监测具有重要意义。     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

DNA barcoding of Caenogastropoda along coast of China based on the COI gene

DNA barcoding provides an efficient method for species-level identifications. In this study, we have amplified partial sequences of mitochondrial cytochrome c oxidase I (COI) gene from 110 specimens of 45 species of Caenogastropoda collected from the coast along China to evaluate whether DNA barcodes can distinguish these species accurately. The average Kimura 2-parameter (K2P) distances within species, genera and families were 0.44%, 13.96% and 22.27%, respectively. Both the neighbour-joining tree and the Bayesian tree showed a clear discrimination of all the species in our study with highly supported clades. These results proved that the species of Caenogastropoda can be efficiently and accurately identified by DNA barcoding based on the COI gene.     

COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae)

The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.     

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.     

Universal COI primers for DNA barcoding amphibians

DNA barcoding is a proven tool for the rapid and unambiguous identification of species, which is essential for many activities including the vouchering tissue samples in the genome 10K initiative, genealogical reconstructions, forensics and biodiversity surveys, among many other applications. A large‐scale effort is underway to barcode all amphibian species using the universally sequenced DNA region, a partial fragment of mitochondrial cytochrome oxidase subunit I COI. This fragment is desirable because it appears to be superior to 16S for barcoding, at least for some groups of salamanders. The barcoding of amphibians is essential in part because many species are now endangered. Unfortunately, existing primers for COI often fail to achieve this goal. Herein, we report two new pairs of primers (?, ?) that in combination serve to universally amplify and sequence all three orders of Chinese amphibians as represented by 36 genera. This taxonomic diversity, which includes caecilians, salamanders and frogs, suggests that the new primer pairs will universally amplify COI for the vast majority species of amphibians.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.     

藻类DNA条形码研究进展

DNA barcode,又称为DNA条形码,是指利用短的标准DNA序列的核苷酸多样性进行物种的鉴定和快速识别.目前该方法在动物分类研究中应用广泛,其中线粒体的细胞色素c氧化酶亚基1(cytochrome c oxidase subunit 1,COI或cox 1)基因中的约700bp长度的一段被用来作为标准DNA片段.在陆地植物条形码研究中,生命-植物条形码联盟会(Consortium for the Barcode of Life-Plant Working Group,CBOL-Plant Working Group)近期推荐将植物叶绿体中的两个基因片段rbcL+ matK作为初步的陆生植物条形码,此组合能在70％的程度上进行植物物种的鉴别.在海藻的分类研究中,DNA条形码的应用较少,已有的研究主要集中在硅藻、红藻和褐藻,尚没有学者明确提出适合藻类的DNA条形码.总结了能够作为藻类DNA条形码的序列特点、应用流程及分析方法,综述了DNA条形码在藻类中的研究现状和存在的问题,展望了藻类DNA条形码的应用前景.     

Phylogenetic Analysis of Brine Shrimp (Artemia) in China Using DNA Barcoding

DNA barcoding is a powerful approach for characterizing species of organisms,especially those with almost identical morphological features, thereby helping to to establish phylogenetic relationships and reveal evolutionary histories. In this study, we chose a 648-bp segment of the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), as a standard barcode region to establish phylogenetic relationships among brine shrimp (Artemia) species from major habitats around the world and further focused on the biodiversity of Artemia species in China, especially in the Tibetan Plateau. Samples from five major salt lakes of the Tibetan Plateau located at altitudes over 4,000 m showed clear differences from other Artemia populations in China. We also observed two consistent amino acid changes, 153A/V and 183L/F, in the COI gene between the high and low altitude species in China.Moreover, indels in the COI sequence were identified in cyst and adult samples unique to the Co Qen population from the Tibetan Plateau, demonstrating the need for additional investigations of the mitochondrial genome among Tibetan Artemia populations.     

DNA barcoding and phylogenetic analysis of Pectinidae (Mollusca: Bivalvia) based on mitochondrial COI and 16S rRNA genes

DNA sequence data enable not only the inference of phylogenetic relationships but also provide an efficient method for species-level identifications under the terms DNA barcoding or DNA taxonomy. In this study, we have sequenced partial sequences of mitochondrial COI and 16S rRNA genes from 63 specimens of 8 species of Pectinidae to assess whether DNA barcodes can efficiently distinguish these species. Sequences from homologous regions of four other species of this family were gathered from GenBank. Comparisons of within and between species levels of sequence divergence showed that genetic variation between species exceeds variation within species. When using neighbour-joining clustering based on COI and 16S genes, all species fell into reciprocally monophyletic clades with high bootstrap values. These evidenced that these scallop species can be efficiently identified by DNA barcoding. Evolutionary relationships of Pectinidae were also examined using the two mitochondrial genes. The results are almost consistent with Waller’s classification, which was proposed on the basis of shell microstructure and the morphological characteristics of juveniles.     

Single nucleotide polymorphism barcoding of cytochrome c oxidase I sequences for discriminating 17 species of Columbidae by decision tree algorithm

DNA barcodes are widely used in taxonomy, systematics, species identification, food safety, and forensic science. Most of the conventional DNA barcode sequences contain the whole information of a given barcoding gene. Most of the sequence information does not vary and is uninformative for a given group of taxa within a monophylum. We suggest here a method that reduces the amount of noninformative nucleotides in a given barcoding sequence of a major taxon, like the prokaryotes, or eukaryotic animals, plants, or fungi. The actual differences in genetic sequences, called single nucleotide polymorphism (SNP) genotyping, provide a tool for developing a rapid, reliable, and high‐throughput assay for the discrimination between known species. Here, we investigated SNPs as robust markers of genetic variation for identifying different pigeon species based on available cytochrome c oxidase I (COI) data. We propose here a decision tree‐based SNP barcoding (DTSB) algorithm where SNP patterns are selected from the DNA barcoding sequence of several evolutionarily related species in order to identify a single species with pigeons as an example. This approach can make use of any established barcoding system. We here firstly used as an example the mitochondrial gene COI information of 17 pigeon species (Columbidae, Aves) using DTSB after sequence trimming and alignment. SNPs were chosen which followed the rule of decision tree and species‐specific SNP barcodes. The shortest barcode of about 11 bp was then generated for discriminating 17 pigeon species using the DTSB method. This method provides a sequence alignment and tree decision approach to parsimoniously assign a unique and shortest SNP barcode for any known species of a chosen monophyletic taxon where a barcoding sequence is available.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.     

DNA条形码在鞘翅目昆虫分子系统学研究中的应用

近年来,DNA条形码(DNA Barcoding)技术已经成为生物分类学研究中备受关注的新型技术,并在鞘翅目昆虫系统发育研究中得到广泛应用。本文总结了鞘翅目昆虫DNA条形码研究所用COⅠ基因序列,概述了DNA条形码在鞘翅目昆虫的物种分类鉴定、发现新种和隐存种、系统发育关系研究等方面的应用,并对DNA条形码研究技术新进展和标准序列筛选需要注意的问题进行了讨论。     

First large-scale DNA barcoding assessment of reptiles in the biodiversity hotspot of Madagascar, based on newly designed COI primers

Background

DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar.

Methodology/Principal Findings

Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family.

Conclusions/Significance

The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge.