Markers For Phylogenetic Analysis Research Articles

New Insights on Tools for Detecting β-Tubulin Polymorphisms in Trichuris trichiura Using rhAmpTM SNP Genotyping.

Soil-transmitted helminth (STH) infections, commonly treated with benzimidazoles, are linked to resistance through single nucleotide polymorphisms (SNPs) at position 167, 198, or 200 in the β-tubulin isotype 1 gene. The aim of this study was to establish a novel genotyping assay characterized by its rapidity and specificity. This assay was designed to detect the presence of SNPs within the partial β-tubulin gene of Trichuris trichiura. This was achieved through the biallelic discrimination at codons 167, 198, and 200 by employing the competitive binding of two allele-specific forward primers. The specificity and reliability of this assay were subsequently confirmed using Trichuris samples isolated from captive primates. Furthermore, a molecular study was conducted to substantiate the utility of the β-tubulin gene as a molecular marker. The assays showed high sensitivity and specificity when applied to field samples. Nevertheless, none of the SNPs within the β-tubulin gene were detected in any of the adult worms or eggs from the analyzed populations. All specimens consistently displayed an SS genotype. The examination of the β-tubulin gene further validated the established close relationships between the T. trichiura clade and Trichuris suis clade. This reaffirms its utility as a marker for phylogenetic analysis.

Analysis of Selected Marker Studies on Minor Poaceae Forages

Pastures are very important for agriculture since the main feeding source of the enormous cattle herd in the world is forage. Pastures comprise plants from several genera of grasses and legumes. In terms of the physical variety, ecology, and economic significance, the Poaceae is among the most significant families of Angiosperms. Regarding stress tolerance, species within this family exhibit a very wide range of variation. In recent years, the importance of using molecular markers in phylogenetic analyses of numerous organisms has increased. The development of genomic technologies and infrastructure has progressed sufficiently for their use in marker- aided selection (MAS) to be studied in several important perennial fodder species. Differences can be directly attributed to minor variations in the genetic code such as phenotype, single sequence repeats (SSRs), and single nucleotide polymorphisms (SNPs). Therefore, breeders can benefit significantly from developing and characterizing new genetic markers. This paper gives a brief analysis of some international studies on some minor Poacea forages.

Cytomolecular analysis of mutants, breeding lines, and varieties of camelina (Camelina sativa L. Crantz).

Camelina sativa L. Crantz (Brassicaceae family), known as camelina, has gained new attention as a re-emerging oil seed crop. With a unique seed oil profile, with the majority of the fatty acids consisting of linolenic (C18:3), oleic (C18:1), linoleic (C18:2), and eicosenoic (C20:1), camelina oil is reported to be useful as a food oil and biofuel. However, there are still many unknown factors about the structure and genetic variability of this crop. Chromosomal localization of ribosomal DNA was performed using fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as molecular probes on mitotic chromosomes of enzymatically digested root-tip meristematic cells. Here, we present for the first time a comparative analysis of selected genotypes (cultivars, breeding lines and mutants) of C. sativa with the use of cytogenetic techniques. The main aim of the study was to determine the intraspecific and interspecific polymorphisms in the structure of chromosomes of selected accessions using conserved 5S and 25S rDNA repetitive sequences as molecular probes. The results were compared with C. microcarpa (closely related to C. sativa) rDNA gene loci distribution. The presence of minor rDNA sites was discussed and compared with other Brassicaceae species. In addition, demonstration karyograms of C. sativa and C. microcarpa mapped with rDNA probes were prepared based on the cv. "Przybrodzka" and GE2011-02 genotype, respectively. The use of 5S and 25S rDNA probes provided an insight on the genome structure of C. sativa at the cytogenetic level and can help to understand the genome organization of this crop. The putative role of cytogenetic markers in phylogenetic analyses of camelina was discussed, as well.

Low-Grade Appendiceal Mucinous Neoplasm (LAMN)-Case Report

The evolutionary origin and significance of spliceosomal introns have been the subject of many investigations. Two theories, "introns-early" theory and "introns-late" theory, have been proposed to explain the evolution of introns in eukaryotic genes. Intron position is generally conserved in paralogue and orthologue genes. Some introns occur at similar but not necessarily identical positions in homologous genes, which were separated by great evolutionary distances. This event can be explained by insertion, loss or movement of the intron over short distances. Intron loss and gain events are unique in evolution and can be useful as markers for phylogenetic analyses. The insertion of introns at an identical position suggests a common ancestor gene. Here we analyzed, using PCR and RT-PCR, the structure of the 1,3-beta-glucan synthase gene (FKS) in several clinical isolates of Paracoccidioides brasiliensis (Pb): isolates Pb 01, Pb 4940, Pb 8515, Pb 8311, Pb 8334, Pb 4268, Pb 1668, and Pb E. Our results showed that seven of the isolates examined showed identical structures concerning the position of introns in PbFKS1. PbFKS4940 showed the intron described at the 3' end and had lost that one at the 5' end. The presence of the PbFKS4940 transcript suggests that it could be a functional gene. These data suggest a divergent evolution for introns with regard to the 1,3-beta-glucan synthase gene in P. brasiliensis isolates.

Mayahuelin, a Type I Ribosome Inactivating Protein: Characterization, Evolution, and Utilization in Phylogenetic Analyses of Agave.

Agaves resist extreme heat and drought. In A. tequilana var. azul, the central spike of the rosette -containing the shoot apical meristem and folded leaves in early stages of development- is remarkably heat tolerant. We found that the most abundant protein in this organ is a 27 kDa protein. This protein was named mayahuelin to honor Mayáhuel, the agave goddess in the Aztec pantheon. LC-MS/MS analyses identified mayahuelin as a type I RIP (Ribosome Inactivating Protein). In addition to the spike, mayahuelin was expressed in the peduncle and in seeds, whereas in mature leaves, anthers, filaments, pistils, and tepals was absent. Anti-mayahuelin antibody raised against the A. tequilana var. azul protein revealed strong signals in spike leaves of A. angustifolia, A. bracteosa, A. rhodacantha, and A. vilmoriniana, and moderate signals in A. isthmensis, A. kerchovei, A. striata ssp. falcata, and A. titanota, indicating conservation at the protein level throughout the Agave genus. As in charybdin, a type I RIP characterized in Drimia maritima, mayahuelin from A. tequilana var. azul contains a natural aa substitution (Y76D) in one out of four aa comprising the active site. The RIP gene family in A. tequilana var. azul consists of at least 12 genes and Mayahuelin is the only member encoding active site substitutions. Unlike canonical plant RIPs, expression of Mayahuelin gene in S. cerevisiae did not compromise growth. The inhibitory activity of the purified protein on a wheat germ in vitro translation system was moderate. Mayahuelin orthologs from other Agave species displayed one of six alleles at Y76: (Y/Y, D/D, S/S, Y/D, Y/S, D/S) and proved to be useful markers for phylogenetic analysis. Homozygous alleles were more frequent in wild accessions whereas heterozygous alleles were more frequent in cultivars. Mayahuelin sequences from different wild populations of A. angustifolia and A. rhodacantha allowed the identification of accessions closely related to azul, manso, sigüín, mano larga, and bermejo varieties of A. tequilana and var. espadín of A. angustifolia. Four A. rhodacantha accessions and A. angustifolia var. espadín were closer relatives of A. tequilana var. azul than A. angustifolia wild accessions or other A. tequilana varieties.

Molecular phylogeny of mulberries reconstructed from ITS and two cpDNA sequences.

BackgroundSpecies in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information.MethodsWe sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry.ResultsWe found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades.ConclusionsTwo species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

Botryosphaerialean fungi causing canker and dieback of tree hosts from Mount Yudu in China

The order Botryosphaeriales includes many latent fungal pathogens with a wide range of woody hosts. The taxonomy of these pathogens has been difficult due to the use of poorly informing markers in phylogenetic analyses and the lack of good morphological characters. Many genera and families in this order have not yet been systematically studied in different hosts and from different regions. In this study, a total of 29 fungal strains from the Aplosporellaceae and Botryosphaeriaceae were isolated from branches or twigs with symptoms of canker and dieback disease in Mount Yudu of China. Morphology and multigene analyses (ITS, LSU and TEF1-α) indicated five distinct lineages, including Aplosporella javeedii, Botryosphaeria dothidea, Diplodia quercicola sp. nov., Phaeobotryon aplospora sp. nov. and Phaeobotryon rhois. Diplodia quercicola is characterized by multiloculate conidiomata, producing oblong to cylindrical, thick-walled, hyaline, aseptate conidia. Phaeobotryon aplospora is characterized by pulvinate, multiloculate conidiomata, producing ellipsoid to oblong, brown, aseptate conidia. The new species differ from related species phylogenetically and ecologically and in morphological features.

Evolution of organellar genes of chlorophyte algae: Relevance to phylogenetic inference

Protein-coding genes in organellar genomes have been widely used to resolve relationships of chlorophyte algae. The mode of evolution of these protein-coding genes affects relationship estimations, yet selection effects on genes commonly used as markers in phylogenetic analyses are insufficiently well understood. To gain more understanding about the effects of green algal organelle protein-coding genes on phylogenies, more information is needed about the mode of gene evolution. We used phylogenetic frameworks to examine evolutionary relationships of 58 protein-coding genes present in the organellar genomes of chlorophyte and streptophyte algae at multiple levels: organelle, biological function, and individual gene, and calculated pairwise dN/dS ratios of algal organellar protein-coding genes to investigate mode of evolution. Results indicate that mitochondrial genes have evolved at a higher rate than have chloroplast genes. Low dN/dS ratios indicating relatively high level of conservation indicate that nad2, nad5, atpA, atpE, psbC, and psbD might be particularly good candidates for use as markers in chlorophyte phylogenies. Chlorophycean atp6, nad2, atpF, clpP, rps2, rps3, rps4, and rps7 protein-coding sequences exhibited selective mutations, suggesting that changes in proteins encoded by these genes might have increased fitness in Chlorophyceae.

Testing the higher-level phylogenetic classification of Digenea (Platyhelminthes, Trematoda) based on nuclear rDNA sequences before entering the age of the 'next-generation' Tree of Life.

Digenea Carus, 1863 represent a highly diverse group of parasitic platyhelminths that infect all major vertebrate groups as definitive hosts. Morphology is the cornerstone of digenean systematics, but molecular markers have been instrumental in searching for a stable classification system of the subclass and in establishing more accurate species limits. The first comprehensive molecular phylogenetic tree of Digenea published in 2003 used two nuclear rRNA genes (ssrDNA = 18S rDNA and lsrDNA = 28S rDNA) and was based on 163 taxa representing 77 nominal families, resulting in a widely accepted phylogenetic classification. The genetic library for the 28S rRNA gene has increased steadily over the last 15 years because this marker possesses a strong phylogenetic signal to resolve sister-group relationships among species and to infer phylogenetic relationships at higher levels of the taxonomic hierarchy. Here, we have updated the database of 18S and 28S rRNA genes until December 2017, we have added newly generated 28S rDNA sequences and we have reassessed phylogenetic relationships to test the current higher-level classification of digeneans (at the subordinal and subfamilial levels). The new dataset consisted of 1077 digenean taxa allocated to 106 nominal families for 28S and 419 taxa in 98 families for 18S. Overall, the results were consistent with previous higher-level classification schemes, and most superfamilies and suborders were recovered as monophyletic assemblages. With the advancement of next-generation sequencing (NGS) technologies, new phylogenetic hypotheses from complete mitochondrial genomes have been proposed, although their power to resolve deep levels of trees remains controversial. Since data from NGS methods are replacing other widely used markers for phylogenetic analyses, it is timely to reassess the phylogenetic relationships of digeneans with conventional nuclear rRNA genes, and to use the new analysis to test the performance of genomic information gathered from NGS, e.g. mitogenomes, to infer higher-level relationships of this group of parasitic platyhelminths.

Plastid genome and composition analysis of two medical ferns: Dryopteris crassirhizoma Nakai and Osmunda japonica Thunb.

BackgroundDryopteris crassirhizoma Nakai and Osmunda japonica Thunb. are ferns that are popularly used for medicine, as recorded by the Chinese pharmacopoeia, and are distributed in different regions of China. However, O. japonica is not record in the Standards of Chinese Herbal Medicines in Hong Kong. Research on identification methods of D. crassirhizoma and O. japonica is necessary and the phylogenetic position of the two species should be identified. The plastid genome is structurally highly conserved, providing valuable sources of genetic markers for phylogenetic analyses and development of molecule makers for identification.MethodsThe plastid genome DNA was extracted from both fern species and then sequenced on the Illumina Hiseq 4000. Sequences were assembled into contigs by SOAPdenovo2.04, aligned to the reference genome using BLAST, and then manually corrected. Genome annotation was performed by the online DOGMA tool. General characteristics of the plastid genomes of the two species were analyzed and compared with closely related species. Additionally, phylogenetical trees were reconstructed by maximum likelihood methods. The content of dryocrassin of the two species were determined according to the Standards of Chinese Herbal Medicines in Hong Kong.ResultsThe genome structures of D. crassirhizoma and O. japonica have different characteristics including the genome size, the size of each area, gene location, and types. Moreover, the (simple sequence repeats) SSRs of the plastid genomes were more similar to other species in the same genera. Compared with D. fragrans, D. crassirhizoma shows an inversion (approximately 1.6 kb), and O. japonica shows two inversions (1.9 kb and 216 bp). The nucleotide diversity (polymorphism information, Pi) analysis showed that the psbK gene and rpl14-rpl16 region have the highest Pi value in Dryopteris, and the ycf2-CDS3 and rpl14-rpl16 regions show the highest Pi vale in O. japonica. Phylogenetic analyses showed that the two species were grouped in two separate clades from each other, with both individually located with other members of their genus. The marker content of dryocrassin is not found in O. japonica.ConclusionsThe study is the first to identify plastid genome features of D. crassirhizoma and O. japonica. The results may provide a theoretical basis for the identification and the application of the two medically important fern species.

Analysis of genetic distance and phylogenetic relationships of “natural/admixed” and “introgressive” hybridization of Fasciola spp. in Vietnam

Fasciola hepatica, F. gigantica and “intermediate” forms of Fasciola species, are responsible for fascioliasis in ruminant animals and humans in many countries. There are two forms: “natural/admixed” and “introgressive” hybridization. Vietnam is a "hot spot" country, where the “hybrid” Fasciola forms have been found nationwide. Determination of phylogenetic relationships and genetic distance of these hybrid forms with those in family Fasciolidae and class Trematoda (Phyllum Platyhelminthes) is necessary to confirm their taxonomic classification. In this study, we sequenced the ITS1 and ITS2 for discrimination of two kinds of hybrid Fasciola mentioned above. The deduced amino acid sequences of the entire mitochondrial genes, cytochrome b (cob), nicotinamide dehydrogenase 1 (nad1) and cytochrome oxidase 1 (cox1), from two hybrid Fasciola spp. including Fsp-DL11-VN (from buffalo, admixed hybrid), Fsp-FH1-VN (from human, introgressive hybrid) and “pure” F. gigantica (Fgig-T4V-VN, from cattle) were obtained and combined (cob+nad1+cox1). These sequences were used as markers for phylogenetic analysis together with 28 representative isolates/species of Fasciolidae and Trematoda. Genetic distances between 13 isolates/species in the family Fasciolidae have also been determined to accurately account for their species-relationships. As results, pairwise genetic distance calculation showed that the distance rate was only 0.4% – 0.7% between Fasciola spp. hybrids of Vietnam and China, slightly higher, 1.3 – 2.0% with “pure” F. gigantica, whilist this rate was quite high compared with F. hepatica (5.7% – 5.9%), with Fasciolopsis buski (20.6% – 21.0%), and two other fasciolids, Fasciola jacksoni and Fascioloides magna (11.0% – 12.6%). The phylogenetic tree of 31 strains/species showed 5 distinct groups, corresponding to 5 families, ie., Fasciolidae, Echinostomatidae, Echinochasmidae, Heterophyidae, Opisthorchiidae and an outgroup, Schistosomatidae. Two "hybrid" Fasciola species of Vietnam (Fsp-FH1-VN and Fsp-DL11-VN) grouped with the reference "hybrid" Fsp-GHL-CN strain of China; and “pure” Fgig-T4V-VN with the two “pure” F. gigantica, Fgig-Bali-ID (Indonesia) and Fgig-GX-CN (China). These results confirmed that “hybrid” Fasciola spp. were of the maternally inherited mitochondrial lineage from F. gigantica.

Complete mitochondrial genome sequence of Macromia daimoji Okumura, 1949 (Odonata: Macromiidae)

The dragonfly Macromia daimoji Okumura, 1949 (Odonata: Macromiidae) has been listed as an Endangered insect in South Korea. We sequenced the complete 15,198 bp mitochondrial genome (mitogenome) of this organism, which is the first mitogenome sequence reported from the family Macromiidae. The genome includes a typical set of genes [13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes) and one non-coding region with an arrangement identical to that observed in most insect genomes. Phylogenetic analyses using concatenated sequences of the 13 PCGs and 2 rRNA genes using the Bayesian inference (BI) method placed Macromiidae, represented by M. daimoji, as a sister group to Libellulidae with the highest nodal support [Bayesian posterior probabilities (BPP) = 1]. Unlike conventional phylogenetic analysis, the suborders Anisozygoptera and Zygoptera formed a strong sister group (BPP =1), justifying the use of different molecular markers for phylogenetic analysis.

Two mitogenomes in Gruiformes (Amaurornis akool/A. phoenicurus) and the phylogenetic placement of Rallidae

Rallidae, with 34 genera including 142 species, is the largest family in the Gruiformes, the phylogenetic placement of this family was still in debate. The complete mitochondrial genomes (mitogenomes), with many advantageous characters, have become popular markers in phylogenetic analyses. We sequenced the mitogenomes of brown crake (Amaurornis akool) and white-breasted waterhen (Amaurornis phoenicurus), analyzed the genomic characters of mitogenomes in Rallidae, and explored the phylogenetic relationships between Rallidae and other four families in Gruiformes based on mitogenome sequences of 32 species with Bayesian method. The mitogenome of A. akool/A. phoenicurus was 16,950/17,213 bp in length, and contained 37 genes typical to avian mitogenomes and one control region, respectively. The genomic characters of mitogenomes in Rallidae were similar. The phylogenetic results indicated that, among five families, Rallidae had closest relationship with Heliornithidae, which formed a sister taxa to Gruidae, while Rhynochetidae located in the basal lineage. Within Rallidae, Rallina was ancestral clade. Gallirallus & Rallus and Aramides were closely related, Gallicrex & Amaurornis and Fulica & Gallinula had close relationships, and these two taxa formed a sister clade to Porphyrio & Coturnicops. Our phylogenetic analyses provided solid evidence for the phylogenetic placement of Rallidae and the evolutionary relationships among different genus within this family. In addition, the mitogenome data presented here provide useful information for further molecular systematic investigations on Gruiformes as well as conservation biology research of these species.

Testing the contribution of individual genes in mitochondrial genomes for assessing phylogenetic relationships in Vetigastropoda

Phylogenetic analyses for molluscs frequently make use of the mitochondrial (mt) markers COX1, 12S and 16S, in part because of the availability of universal primers (e.g. Plazzi & Passamonti, 2010; Williams et al., 2010; Aktipis & Giribet, 2011; Plazzi et al., 2011; Wakabayashi et al., 2012). Recent phylogenetic studies have expanded on gene sampling, sequenced entire mt genomes and undertaken phylogenetic analyses using all protein-coding and rRNA genes for determining relationships within Mollusca, generally resulting in well-resolved trees with highly supported nodes (e.g. White et al., 2011; Williams, Foster & Littlewood, 2014; Uribe et al., 2015). However, several papers have pointed out that trees based purely on mtDNA can produce erroneous deep relationships between different molluscan taxa (Stoger & Schrodl, 2013; Bernt et al., 2013a). This is perhaps unsurprising, given that mtDNA generally evolves more quickly than the nuclear genome (Burton & Barreto, 2012), and deeper relationships may therefore be beyond the saturation point of mtDNA. However, with the recent development of next-generation sequencing, entire mt genomes can be sequenced rapidly and relatively cheaply. It is therefore important to determine the utility and reliability of the mt genome as a marker for phylogenetic analysis.

Insufficient resolving power of mitogenome data in deciphering deep phylogeny of Holometabola

AbstractThe mitochondrial genome (mitogenome) is one of the most widely used markers for phylogenetic analysis. Compared with whole‐genome data, mitogenome data are less expensive to obtain and easier to manipulate. However, compositional bias and accelerated evolutionary rate reduce the effectiveness of the mitogenome in determining insect phylogeny. This study shows that mitogenome data are not suitable to reconstruct deep holometabolan evolution, even with a most comprehensive data coding scheme and the more realistic CAT model. For the deep levels of divergence within Holometabola, protein‐coding genes only retain weak phylogenetic signals, leading to peculiar interordinal relationships. Consensus relationships in the Holometabola phylogeny, such as the monophyly of Holometabola, the most basal position of Hymenoptera, and the sister group relationship between the Strepsiptera and Coleoptera were rarely resolved in our analyses. The relationships of the holometabolan groups as inferred by mitogenomes are highly vulnerable to gene types, data coding regimes, model choice, and optimality criteria, and no consistent alternative hypothesis of Holometabola's relationships is supported. Thus, we suggest that the slowly evolving nuclear genes or genome‐scale approaches may be better options for resolving deep‐level phylogeny of Holometabola.

Molecular phylogenetic relationship among three Acanthodactylus species (Reptilia: Lacertidae) in Egypt based on 18S rRNA Sequences

Acanthodactylus is a genus of lacertid lizard, commonly referred to as fringe-fingered lizards or fringe-toed lizards. The systematics of the genus Acanthodactylus was classically based on external morphological traits, osteological characters and morphology of the hemipenes. In this study, partial sequences of 18S rRNA gene were used to estimate the relationships among three lacertide species from Egypt: Acanthodactylus boskianus, Acanthodactylus scutellatus and Acanthodactylus longipes. These data were used together with morphological information, to evaluate the relationships among these three species. DNA was extracted from the collected specimens, 988 bp fragment of 18S rRNA was amplified using universal primers and sequenced. The obtained sequences were analyzed and a phylogenetic tree was constructed. Results of analysis revealed that A. scutellatus and A. longipes form a very well supported clade with 86% bootstrap. A. boskianus was found to constitute a separate clade. The resultant tree based on DNA sequence data was similar to that produced from a differentially weighted data set of morphological characters. In conclusion, the 18S rRNA gene sequence seems to be a good marker for phylogenetic analysis of Acanthodactylus species.

An integrated phylogenomic approach toward pinpointing the origin of mitochondria.

Overwhelming evidence supports the endosymbiosis theory that mitochondria originated once from the Alphaproteobacteria. However, its exact position in the tree of life remains highly debated. This is because systematic errors, including biased taxonomic sampling, high evolutionary rates and sequence composition bias have long plagued the mitochondrial phylogenetics. In this study, we address this issue by 1) increasing the taxonomic representation of alphaproteobacterial genomes by sequencing 18 phylogenetically novel species. They include 5 Rickettsiales and 4 Rhodospirillales, two orders that have shown close affiliations with mitochondria previously, 2) using a set of 29 slowly evolving mitochondria-derived nuclear genes that are less biased than mitochondria-encoded genes as the alternative “well behaved” markers for phylogenetic analysis, 3) applying site heterogeneous mixture models that account for the sequence composition bias. With the integrated phylogenomic approach, we are able to for the first time place mitochondria unequivocally within the Rickettsiales order, as a sister clade to the Rickettsiaceae and Anaplasmataceae families, all subtended by the Holosporaceae family. Our results suggest that mitochondria most likely originated from a Rickettsiales endosymbiont already residing in the host, but not from the distantly related free-living Pelagibacter and Rhodospirillales.

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Corbicula regularis is a freshwater mussel found in the Indian sub-continent. In the present study, phylogenetic characterization of this important bivalve was attempted using 18S ribosomal RNA gene markers. Genomic DNA was extracted and 18S rRNA gene was amplified by universal primers. The amplification product was sequenced and compared with the nucleotide databases available online to evaluate phylogenetic relationship of the animal under study. Results indicated that 18S rRNA gene sequences of C. regularis showed high degree of similarity to another freshwater mussel, C. fluminea. This work constitutes the first ever sequence deposition of the C. regularis in the nucleotide databases highlighting the usefulness of 18S ribosomal gene markers for phylogenetic analysis.

Study of alternative oxidase as possible molecular marker for phylogenetic analysis of the Botrytis cinerea

Botrytis cinerea (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is able to attack several economically important plants causing gray rot. Botrytis cinerea species complex includes two cryptic species (B. cinerea and B. pseudocinerea) that tolerate fungicides differently. On the basis of classical taxonomic markers, the two related species are very difficult to be distinguished; therefore, their separation is usually performed using molecular methods based on the time-consuming molecular analysis of several markers. Our goal was to find markers, which are suitable for the differentiation. Testing the nucleotide sequences of the alternative oxidase encoding gene, B. cinerea and B. pseudocinerea strains were clearly differentiated. Moreover, the analysis of the protein sequences of the enzyme with the maximum likelihood method reflected well the taxonomic relationships of the different fungi.

Sequence variation and protein structure of pipo gene in Potato virus Y

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.