Phylogeny and molecular evolution of the DMC1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy nuclear disrupted meiotic cDNA (DMC1) gene within the StH genome species, two DMC1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from seven diploid taxa representing the St and H genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) there is a close relationship among North American StH genome species; (2) the DMC1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) the StH genome polyploids have higher levels of sequence diversity in the St genome homoeolog than the H genome homoeolog; (4) the DMC1 sequence may evolve faster in the polyploid species than in the diploids; (5) high dN and dN/dS values in the St genome within polyploid species could be caused by low selective constraints or AT-biased mutation pressure. Our result provides some insight on evolutionary dynamics of duplicate DMC1 gene, the polyploidization events and phylogeny of the StH genome species.     

Comparison of Acetyl-CoA carboxylase 1 (Acc-1) gene diversity among different Triticeae genomes

It has widely been documented that life form and mating system have significant influences on genetic diversity. In the tribe Triticeae, several genera contain both annual and perennial species, whereas other genera comprise strictly annual or perennial species. It was suggested that Triticeae annuals have originated from Triticeae perennials. The present study aims to analyze nucleotide diversity of Acc-1 gene among different Triticeae genomes, and attempts to link effects of life history (annuals and perennials) and mating systems. The nucleotide diversity of 364 Acc-1 sequences in Triticeae species was characterized. The highest estimates of nucleotide diversity values (π = 0.01919, θ = 0.03515) were found for the Ns genome among the genomes analyzed. Nucleotide diversities in the D genome and Ns genome of polyploids are higher than those in respective genomes of diploids, while in the St genome of polyploids, it is lower than that in the St genome of diploids. The averaged π value (0.013705) in the genomes of perennials is more than twice of the value (0.00508) in the genomes of annuals. The averaged π value (0.01323) in the genomes of outcrossing species is two-fold of the value (0.005664) in the genomes of selfer. Our results suggested that the evolutionary history and mating system may play an important role in determining nucleotide diversity of Acc-1 gene in each genome.     

Genetic diversity and phylogeny in Hystrix (Poaceae, Triticeae) and related genera inferred from Giemsa C-banded karyotypes

The phylogenetic relationships of 15 taxa from Hystrix and the related genera Leymus (NsXm), Elymus (StH), Pseudoroegneria (St), Hordeum (H), Psathyrostachys (Ns), and Thinopyrum (E) were examined by using the Giemsa C-banded karyotype. The Hy. patula C-banding pattern was similar to those of Elymus species, whereas C-banding patterns of the other Hystrix species were similar to those of Leymus species. The results suggest high genetic diversity within Hystrix, and support treating Hy. patula as E. hystrix L., and transferring Hy. coreana, Hy. duthiei ssp. duthiei and Hy. duthiei ssp. longearistata to the genus Leymus. On comparing C-banding patterns of Elymus species with their diploid ancestors (Pseudoroegneria and Hordeum), there are indications that certain chromosomal re-arrangements had previously occurred in the St and H genomes. Furthermore, a comparison of the C-banding patterns of the Hystrix and Leymus species with the potential diploid progenitors (Psathyrostachys and Thinopyrum) suggests that Hy. coreana and some Leymus species are closely related to the Ns genome of Psathyrostachys, whereas Hy. duthiei ssp. duthiei, Hy. duthiei ssp. longearistata and some of the Leymus species have a close relationship with the E genome. The results suggest a multiple origin of the polyploid genera Hystrix and Leymus.     

Molecular phylogeny of RPB2 gene reveals multiple origin, geographic differentiation of H genome, and the relationship of the Y genome to other genomes in Elymus species

It has been hypothesized from isozymic and cytological studies of Elymus species that the Old and New World taxa may be of separate origin of the H genome in the StH genome species. To test this hypothesis, and estimate the phylogenetic relationships of polyploid Elymus species within the Triticeae, the second largest subunit of RNA polymerase II (RPB2) sequence of 36 Elymus accessions containing StH or StY genomes was analyzed with those of Pseudoroegneria (St), Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum(Ee), Thinopyrum(Eb) and Dasypyrum (V). Our data indicated that the H genome in Elymus species is differentiated in accordance with geographical origin, and that the Eurasian and American StH genome species have independent alloploid origins with different H-genome donors. Phylogenetic analysis of Y genome sequences with other genome donors (St, H, P, W) of Elymus revealed that W and P genomes are sister to Y genome with a 87% bootstrap support, and that StY and StH species group might have acquired their RPB2 St sequences from distinct Pseudoroegneria gene pools. Our data did not support the suggestion that the St and Y genomes have the same origin as put forward in a previous study using ITS data. Our result provides some insight on the origin of Y genome and its relationship to other genomes in Elymus.     

Phylogeny of the StH genome species in Triticeae (Poaceae): Evidence from chloroplast trnL-F and mitochondria COXII intron sequences

The StH genome species in Triticeae exhibit different morphological variations and extensive geographic distribution. To estimate the phylogenetic relationship of the StH genome species in Triticeae, mitochondria COXII intron and chloroplast trnL-F sequences of 16 StH genome species were analyzed with those of four Pseudoroegneria species (St) and four Hordeum species (H). Sequence diversity and genealogical analysis suggested that (1) the trnL-F and COXII sequence may evolve faster in the polyploid species than in the diploids; (2) the COXII intron has a high evolutionary rate compared to trnL-F sequence and would provide potentially useful phylogenetic analysis in the StH genome species; (3) different Pseudoroegneria species might serve as the maternal donor during the polyploid speciation of the StH genome species; (4) phylogenetic relationships of the StH genome species may be not linked with the inter-continental disjunction between Eurasian and North American.     

Chloroplast Genome Evolution in Early Diverged Leptosporangiate Ferns

In this study, the chloroplast (cp) genome sequences from three early diverged leptosporangiate ferns were completed and analyzed in order to understand the evolution of the genome of the fern lineages. The complete cp genome sequence of Osmunda cinnamomea (Osmundales) was 142,812 base pairs (bp). The cp genome structure was similar to that of eusporangiate ferns. The gene/intron losses that frequently occurred in the cp genome of leptosporangiate ferns were not found in the cp genome of O. cinnamomea. In addition, putative RNA editing sites in the cp genome were rare in O. cinnamomea, even though the sites were frequently predicted to be present in leptosporangiate ferns. The complete cp genome sequence of Diplopterygium glaucum (Gleicheniales) was 151,007 bp and has a 9.7 kb inversion between the trnL-CAA and trnV-GCA genes when compared to O. cinnamomea. Several repeated sequences were detected around the inversion break points. The complete cp genome sequence of Lygodium japonicum (Schizaeales) was 157,142 bp and a deletion of the rpoC1 intron was detected. This intron loss was shared by all of the studied species of the genus Lygodium. The GC contents and the effective numbers of co-dons (ENCs) in ferns varied significantly when compared to seed plants. The ENC values of the early diverged leptosporangiate ferns showed intermediate levels between eusporangiate and core leptosporangiate ferns. However, our phylogenetic tree based on all of the cp gene sequences clearly indicated that the cp genome similarity between O. cinnamomea (Osmundales) and eusporangiate ferns are symplesiomorphies, rather than synapomorphies. Therefore, our data is in agreement with the view that Osmundales is a distinct early diverged lineage in the leptosporangiate ferns.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

Molecular detection and phylogenetic analysis of the alkane 1-monooxygenase gene from Gordonia spp.

The alkB gene encodes for alkane 1-monooxygenase, which is a key enzyme responsible for the initial oxidation of inactivated alkanes. This functional gene can be used as a marker to assess the catabolic potential of bacteria in bioremediation. In the present study, a pair of primers was designed based on the conserved regions of the AlkB amino acid sequences of Actinobacteria, for amplifying the alkB gene from the genus Gordonia (20 Gordonia strains representing 13 species). The amplified alkB genes were then sequenced and analyzed. In the phylogenetic tree based on the translated AlkB amino acid sequences, all the Gordonia segregated clearly from other closely related genera. The sequence identity of the alkB gene in Gordonia ranged from 58.8% to 99.1%, which showed higher sequence variation at the inter-species level compared with other molecular markers, such as the 16S rRNA gene (93.1–99.8%), gyrB gene (77.5–97.3%) or catA gene (72.4–99.5%). The genetic diversity of four selected loci also showed that the alkB gene might have evolved faster than rrn operons, as well as the gyrB or catA genes, in Gordonia. All the available actinobacterial alkB gene sequences derived from the whole genome shotgun sequencing projects are phylogenetically characterized here for the first time, and they exclude the possibility of horizontal gene transfer of the alkB gene in these bacterial groups.     

Molecular Characterization of Taenia multiceps Isolates from Gansu Province,China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.     

Nucleotide diversity and minisatellite in chloroplast Asp(GUC)–Thr(GGU) region in Elymus trachycaulus complex, Elymus alaskanus and Elymus caninus

Nucleotide variation in chloroplast Asp(GUC)–Thr(GGU) intergenic region and genetic relationships among this group were examined among Elymus trachycaulus complex, Elymus alaskanus and Elymus caninus. The estimates of nucleotide diversity (π) ranged from 0.00111 for Elymus virescens to 0.03086 for E. caninus. Highest nucleotide diversity was found for E. caninus among the taxa analyzed here and followed by Elymus hyperarcticus. E. virescens accessions are genetically very uniform. Phylogenetic analysis suggested that E. caninus is paraphyletic. Elymus violaceus is genetically distinct from both E. alaskanus and E. trachycaulus. Our result indicates that Asp (GUC)–Thr (GGU) intergenic region has a high rate of evolutionary in Elymus species. Large indels detected in this region appear to have a highly rate of evolution and are thus more prone to homoplasy. We also first reported a minisatellite discovered in Asp (GUC)–Thr (GGU) region in Elymus species. The minisatellite identified here is an excellent candidate marker for studying population structures of Elymus species.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

Molecular Identification of Diphyllobothrium nihonkaiense from 3 Human Cases in Heilongjiang Province with a Brief Literature Review in China

Human diphyllobothriasis is a widespread fish-borne zoonosis caused by the infection with broad tapeworms belonging to the genus Diphyllobothrium. In mainland China, so far 20 human cases of Diphyllobothrium infections have been reported, and the etiologic species were identified as D. latum and D. nihonkaiense based on morphological characteristics or molecular analysis. In the present study, proglottids of diphyllobothriid tapeworms from 3 human cases that occurred in Heilongjiang Province, China were identified as D. nihonkaiense by sequencing mitochondrial cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 5 (nad5) genes. Two different cox1 gene sequences were obtained. One sequence showed 100% homology with those from humans in Japan. The remaining cox1 gene sequence and 2 different nad5 gene sequences obtained were not described previously, and might reflect endemic genetic characterizations. D. nihonkaiense might also be a major causative species of human diphyllobothriasis in China. Meanwhile, the finding of the first pediatric case of D. nihonkaiense infection in China suggests that infants infected with D. nihonkaiense should not be ignored.     

A unique tRNA gene family and a novel,highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae)

Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS^− AGY and trnQ genes, a trnM isomerism, but it lacks trnS^− CUN. We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP → trnS^− AGY) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, “Oxidored_q1_N”, which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available bivalve mt tRNA and atp8 genes.     

Molecular Variation in the Paragonimus heterotremus Complex in Thailand and Myanmar

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. Of the 7 members of the genus known in Thailand until recently, only P. heterotremus has been confirmed as causing human disease. An 8th species, P. pseudoheterotremus, has recently been proposed from Thailand, and has been found in humans. Molecular data place this species as a sister species to P. heterotremus, and it is likely that P. pseudoheterotremus is not specifically distinct from P. heterotremus. In this study, we collected metacercariae of both nominal species (identification based on metacercarial morphology) from freshwater crabs from Phetchabun Province in northern Thailand, Saraburi Province in central Thailand, and Surat Thani Province in southern Thailand. In addition, we purchased freshwater crabs imported from Myanmar at Myawaddy Province, western Thailand, close to the Myanmar-Thailand border. The DNAs extracted from excysted metacercariae were PCR-amplified and sequenced for ITS2 and cox1 genes. The ITS2 sequences were nearly identical among all samples (99-100%). Phylogenies inferred from all available partial cox1 sequences contained several clusters. Sequences from Indian P. heterotremus formed a sister group to sequences from P. pseudoheterotremus-type metacercariae. Sequences of P. heterotremus from Thailand, Vietnam, and China formed a separate distinct clade. One metacercaria from Phitsanulok Province was distinct from all others. There is clearly considerable genetic variation in the P. heterotremus complex in Thailand and the form referred to as P. pseudoheterotremus is widely distributed in Thailand and the Thai-Myanmar border region.     

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA^His differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.