Phylogenetic and Population Analyses on P1, HC-Pro, and P3 of Potato Virus Y Isolates Infecting Potato in the Central Region of Türkiye

The soft-shell turtles Apalone spinifera (AS) and Apalone ferox (AF) are two important economic species. AF is found in the Yellow River of China, and is a confirmed member of the Trionychidae family. However, the classification of AS was in dispute. Mitochondrial genomes (mitogenomes) have been widely used for species identification, as well as population and phylogenetic analysis. In order to understand the phylogenetic and mitogenomic features of AS and AF, the complete mitogenomes were sequenced, annotated and analyzed in this study. The complete mitogenomes of AS and AF are 16,817bp and 16,756bp in length, respectively. Both mitogenomes contain 37 genes, seven short intergenic spacers and two long intergenic spacers. Comparative analysis showed that there are 1,137 variation sites (6.79%) between the two mitogenomes. AS and AF mitogenomes both show a usage preference in terms of nucleotides, codons and amino acids. In addition, the non-synonymous substitution rate/synonymous substitution rate indicates that all protein-coding genes (PCGs) have undergone a strong purifying selection. Phylogenetic trees constructed by 13 PCGs show a clear phylogenetic relationship of the soft-shell turtles and suggest that AS is a sister species to AF of the genus Apalone. The data could be useful for further research of species identification, population analysis and the mitogenomic features of soft-shell turtles.

Despite four decades of control of Chagas disease in Venezuela, domestic infestations still persist and transmission of Trypanosoma cruzi may be increasing. This is in contrast to the Southern cone region where control has successfully eliminated domestic populations of the main vector Triatoma infestans over large areas. However unlike T. infestans, the main vector in Venezuela, Rhodnius prolixus, has a widespread silvatic distribution occurring primarily in palm trees, which are a ubiquitous feature of the Venezuelan landscape. The palm tree is an important part of campensino life and is maintained for fruit, shade and for use in house construction. Control failures may be due to reinvasion of houses by these prevalent silvatic populations. However, debate exists as to whether the silvatic populations are in fact Rhodnius robustus, a related species of minor epidemiological importance, and therefore no threat to control. With an estimated 800,000 people infected with T. cruzi in Venezuela and a further 3 million at risk of infection, an effective control programme is required, which necessitates that this relationship between silvatic and domestic populations is resolved. This study was undertaken in order to (1) confirm the identity of silvatic populations of Rhodnius in Venezuela and (2) determine if domestic and silvatic populations are isolated, thus to clarify the role of silvatic populations in maintaining house infestations. To achieve these aims field collected silvatic and domestic populations of Rhodnius from 5 States were analysed by genetic methods, direct sequencing (cytochrome b, D2) and microsatellites, and by geometric morphometric analysis. A total of 551 specimens from 31 localities in six Venezuelan states were analysed by direct sequencing of cytochrome b (cytb). Results confirmed the presence of R. prolixus in both silvatic and domestic ecotopes, dispelling the belief that all silvatic populations are R.robustus. Rhodnius robustus does however occur and was found in this study in the Andean State Trujillo. Here it was limited to the silvatic environment. This project found that silvatic and domestic populations of R. prolixus are not isolated, sharing 6 haplotypes, including four silvatic haplotypes also detected in domestic nymphs, indicating that silvatic specimens are capable of domestic colonisation. This was also confirmed from the analysis of adjacent domestic and silvatic populations in Portuguesa and Barinas State where population homogeneity was detected. Additionally cytb analysis identified an introgression event between Amazonian R. robustus and R prolixus, confirmed by incongruence of cytb and D2 nuclear characterisation. Phylogenetic analysis of specimens was also undertaken. To investigate further fine scale population heterogeneity a panel of microsatellite markers, hitherto unavailable, was developed for R prolixus, using an enrichment technique. A panel of 10 loci was available for analysis following PCR screening and linkage analysis. A total of 555 specimens were analysed from 33 populations . Microsatellite analysis also detected population homogeneity between ecotopes, including adjacent populations, indicating that silvatic populations are not isolated. Population heterogeneity was greater among localities in Portuguesa than Barinas, may be due to landscape variation. Differences between control programmes may also play a role. Geometric morphometries identified shape similarity between populations across all States. However, shape convergence by ecotope was detected and results indicated that morphometries might be of limited use for the analysis of populations of R prolixus, with the exception of post-control reinvasion\recrudescent studies. The three methods did not always concur precisely. However comparison was difficult due to detected introgression and shape convergence distorting, respectively, mitochondrial and morphometric analysis. Results indicated that a combined use of microsatellites and morphometries would be beneficial in the analysis of adjacent domestic and silvatic populations. A similar pattern of a lack of isolation between silvatic and domestic ecotopes was detected by both genetic methods, with limited morphometries overlap detected; broadly all three methods showed that populations from differing ecotopes are not isolated. This is also supported by a parallel project on risk-factor analysis. From this study it is clear that silvatic populations of R prolixus present an unquestionable threat to the successful control of Chagas disease in some endemic regions of Venezuela and, unlike the Southern cone, elimination of domestic infestations may not be possible in areas where silvatic R prolixus occur. A restructuring of the control programme in Venezuela is required to deal with this silvatic threat. This may include increased vigilance and methodical respraying and the incorporation of novel approaches to deal with silvatic invasion such as use of insecticide treated curtains; ultimately more investment in the improvement of the rural rancho is required.

Beet black scorch virus (BBSV) was surveyed in major sugar beet cultivation areas in Iran in 2008–2013 growing seasons. A total of 148 out of 308 samples (48%) collected in seven Iranian provinces were BBSV-infected, as shown by RT-PCR. To investigate the genetic diversity of Iranian BBSV isolates, sequences of the coat protein (CP) gene and of the 3′ untranslated region (3′UTR) were compared to corresponding sequences in GenBank. The CP nucleotide sequence identity among Iranian isolates was 87.9–99.9%. They shared nucleotide sequence identities of 88–89.8% with those of other BBSV isolates available at GenBank. Phylogenetic analyses of CP sequences demonstrated that Iranian isolates were distant from Chinese and US isolates at both nucleotide and amino acid levels. Three isolates from western provinces of the country showed independent divergent lineage compared with other Iranian isolates. Comparison of 3′UTR region of BBSV indicated that all Iranian isolates, except four isolates from the west of the country, were distinct from European, Chinese, and US isolates. Population genetic analyses revealed that BBSV populations from west of Iran had distinct evolutionary divergence and differentiated from eastern BBSV isolates. Our results indicated that purifying selection might have contributed to evolution of the isolates belonging to the four identified BBSV subgroups with infrequent genetic exchanges occurring between them. Phylogenetic analyses and estimation of genetic distance indicated that Iranian isolates in both CP and 3′UTR genomic regions had wider genetic diversity than isolates from other parts of the world. Based on the result of phylogenetic and population analyses, we suggest discriminating two different groups, namely BBSV western and BBSV eastern strains.

The genus Vibrio includes bacteria with different morphological and metabolic characteristics responsible for different human and animal diseases. An accurate identification is essential to assess the risks in regard to aquatic organisms and consequently to public health. The Multilocus Sequence Analysis (MLSA) scheme developed on the basis of 4 housekeeping genes (gyrB, pyrH, recA and atpA) was applied to identify 92 Vibrio strains isolated from crustaceans in 2011. Concatenated sequences were used for the phylogenetic and population analyses and the results were compared with those from biochemical identification tests. From the phylogenetic analysis, 10 clusters and 4 singletons emerged, whereas the population analysis highlighted 12 subpopulations that were well supported by phylogeny with few exceptions. The retrospective analysis allowed correct re-attribution of isolated species, indicating how, for some pathogens, there may be an overestimation of phenotypic identification (e.g. V. parahaemolyticus). Use of the PubMLST Vibrio database highlighted a possible genetic link between Sequence Type (ST) 529 and ST195 (V. alginolyticus) isolated from a human case in Norway during 2018. In addition to the identification of major risk groups of V. cholerae, V. vulnificus and V. parahaemolyticus, MLSA could be a valid support for species considered a minor risk, such as V. alginolyticus, V. mimicus and V. fluvialis. Due to the increased incidence of vibriosis in Europe, the application of different tools will also have to be considered to investigate the possible epidemiological links of the various species in the perspective of Open Science to protect the consumer.

The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%-2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration.

A phylogenetic analysis of heterotrophic bacterial populations inhabiting streams and groundwater contaminated by acid mine drainage (AMD) was conducted. The samples were collected from sites around an inactive underground Dae-Sung coal mine at Keumsan, Korea. The investigation showed pigment-forming bacteria to be the major strains inhabiting the pollutant, accounting for up to approximately 50% of all isolates. Twenty-six pigment-forming bacteria were isolated and their taxonomic characteristics determined by phenotypic, chemotaxonomic and phylogenetic analyses. Based on a phylogenetic analysis using 16S ribosomal RNA gene nucleotide sequences, these isolates were found to fall within four major phylogenetic groups: α, β, and γ subdivisions of Proteobacteria; and low-G+C gram-positive bacteria. The α-Proteobacteria were further separated into α-1, α-2 and α-4 subclasses. Many isolates from the polluted stream (site P5) and groundwater (site G) were identified as Sphingomonas of the Proteobacteria α-4 subclass. Because strains P5-21 and P5-11 appeared to be novel species within the genus Sphingomonas, the discussion was focused on their taxonomy as well as abundance in the polluted regions.

Blackbuck (Antilope cervicapra) is a threatened species endemic to the Indian subcontinent. Many populations of blackbuck are found in southern India. Populations of blackbuck are negatively affected in many places for various reasons, such as habitat destruction and poaching. Their range decreased sharply during the 20th century. There is very limited information available on the population dynamics of blackbuck in southern India. For the phylogenetic and genetic diversity analyses of blackbuck populations among different distribution ranges in southern India, we sequenced mt DNA of cytochrome b (Cyt b) for 120, cytochrome c oxidase subunit-1 (COI) for 137 and the control region (CR) for 137 fecal pellets from eleven different locations in southern India. We analyzed the genetic structure of three mitochondrial markers, the CR, Cyt b and the COI region, separately and in a combined dataset. The haplotype diversity and nucleotide diversity of CR were 0.969 and 0.047, respectively, and were higher than those of Cyt b and COI. A Bayesian phylogeny and an MJ network based on the CR and combined dataset (105 sequences) signified several distinct haplotype clusters within blackbuck, whereas no clusters were identified with the Cyt b and COI phylogenetic analyses. The analysis of molecular variance of the combined data set revealed 52.46% genetic variation within the population. Mismatch distribution analysis revealed that blackbuck populations underwent complex changes with analysis of the combined dataset in each population and analysis of each marker separately in the overall population. The results provide evidence that blackbuck in different geographic locations has a distinct population structure due to habitat fragmentation after the formation of the Western and Eastern Ghats.

Genetic variation and population structure in the threatened ghost bat, Macroderma gigas

In 2020, 264 samples were collected from potato fields in the Turkish provinces of Bolu, Afyon, Kayseri and Niğde. RT-PCR tests, with primers which amplified its coat protein (CP), detected potato virus S (PVS) in 35 samples. Complete CP sequences were obtained from 14 samples. Phylogenetic analysis using non-recombinant sequences of (i) the 14 CP’s, another 8 from Tokat province and 73 others from GenBank; and (ii) 130 complete ORF, RdRp and TGB sequences from GenBank, found that they fitted within phylogroups, PVSI, PVSII or PVSIII. All Turkish CP sequences were in PVSI, clustering within five subclades. Subclades 1 and 4 were in three to four provinces, whereas 2, 3 and 5 were in one province each. All four genome regions were under strong negative selection constraints (ω = 0.0603–0.1825). Considerable genetic variation existed amongst PVSI and PVSII isolates. Three neutrality test methods showed PVSIII remained balanced whilst PVSI and PVSII underwent population expansion. The high fixation index values assigned to all PVSI, PVSII and PVSIII comparisons supported subdivision into three phylogroups. As it spreads more readily by aphid and contact transmission, and may elicit more severe symptoms in potato, PVSII spread constitutes a biosecurity threat for countries still free from it.

Genetic population analyses for Nile fishes are very scarce. Nile pufferfish Tetraodon lineatus (Linnaeus, 1758), is a widely-distributed freshwater fish, with no known major widespread threats. It has been classed as 'Endangered'. Little is known concerning its biology and genetics in Egypt. Hence, this work was designed to study the genetic diversity and conservation status of T. lineatus for the first time in Egypt and Africa. DNA barcoding was carried out through PCR-amplification and sequencing of the cytochrome c oxidase gene barcode 5´ region of forty-five samples obtained from three different localities in Upper Egypt. Only three haplotypes could be characterized in all samples. The other population analyses showed clear population loss of extension and potential bottleneck, what may explain the severe drop of species records in the Northern areas of the Nile. Phylogenetic analysis exhibited the monophyletic origin T. lineatus and other African freshwater pufferfishes, more probably as descendants from an Indo-West Pacific ancestor. We highly recommend the fulfilling of more studies concerning the biology, ecology, and genetics of the species as major steps towards its proper conservation and understanding of its adaptation to different natural and man-made constraints in the River Nile system.

Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning.

The characterization of the different taxa of the highly diverse genus Monodelphis is poorly understood, as is the case of their distribution. Historically, taxonomic studies of Monodelphis have been restricted to a few or single species, whereas molecular approaches have been used for estimating phylogenetic relationships between species. We carried out phylogenetic analyses of 14 Monodelphis species, including Monodelphis domestica, based on cytochrome b mitochondrial DNA sequences. Forty-five complete (1149 bp) sequences of this gene were obtained from 39 specimens of M. domestica collected in 23 localities of the Brazilian Cerrado, Pantanal, and Caatinga morphoclimatic domains; one of Monodelphis umbristriata, two of Monodelphis americana, and two of Monodelphis dimidiata. A total of 72 haplotypes were analyzed, 48 only in M. domestica. Analyses were carried out in conjunction with 46 other sequences retrieved from GenBank, including M. domestica, Monodelphis brevicaudata, Monodelphis glirina, Monodelphis emiliae, Monodelphis peruviana, Monodelphis osgoodi, Monodelphis handleyi, Monodelphis kunsi, Monodelphis americana, Monodelphis dimidiata, Monodelphis iheringi, Monodelphis reigi, and Monodelphis adusta, with six other different didelphid species used as outgroups. Maximum likelihood and Bayesian inference were similar in depicting phylogenetic relationships of different Monodelphis taxa. Two clades of M. domestica were defined on the basis of these results. Genetic distance estimates ranged from 3.2% to 6.2% between these clades of M. domestica. Population analyses were carried out to infer the likely demographic scenarios and the relationship between M. domestica haplotypes. Median-joining and spatial analyses showed two populations related to different morphoclimatic domains (Cerrado/Pantanal and Caatinga). These results indicate a population structure in M. domestica and the possibility that this taxon might represent a species complex.

BackgroundAdvances in next-generation sequencing technologies have provided an opportunity to perform population-level comparative genomic analysis to discover unique genomic characteristics of domesticated animals. Duck is one of the most popular domesticated waterfowls, which is economically important as a source of meat, eggs, and feathers. The objective of this study is to perform population and functional analyses of Korean native duck, which has a distinct meat flavor and texture phenotype, using whole-genome sequencing data. To study the distinct genomic features of Korean native duck, we conducted population-level genomic analysis of 20 Korean native ducks together with 15 other duck breeds.ResultsA total of 15.56 million single nucleotide polymorphisms were detected in Korean native duck. Based on the unique existence of non-synonymous single nucleotide polymorphisms in Korean native duck, a total of 103 genes related to the unique genomic characteristics of Korean native duck were identified in comparison with 15 other duck breeds, and their functions were investigated. The nucleotide diversity and population structures among the used duck breeds were then compared, and their phylogenetic relationship was analyzed. Finally, highly differentiated genomic regions among Korean native duck and other duck breeds were identified, and functions of genes in those regions were examined.ConclusionsThis is the first study to compare the population of Korean native duck with those of other duck breeds by using whole-genome sequencing data. Our findings can be used to expand our knowledge of genomic characteristics of Korean native duck, and broaden our understanding of duck breeds.

DNA sequences of an 847 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) gene and a 514 bp fragment of 16s rRNA gene were determined to examine the phylogenetic relationships of 12 Penaeoidea shrimp species (Penaeus chinensis, Penaeus japonicus, Penaeus penicillatus, Penaeus vannamei, Penaeus canaliculatus, Trachypenaeus curvirostris, Metapenaeus affinis, Metapenaeus ensis, Metapenaeopsis barbata, Parapenaeus fissuroides, Parapenaeopsis hardiwickii, Solenocera crassicomis). Both fragments of the swimming crab Portunus trituberculaus chosen as the outgroup were also sequenced. Intraspecific sequence divergence of 0.24-1.2% in the COI gene was found in 5 species, while no intraspecific variation was observed in the 16s rRNA gene. Three phylogenetic trees based on the 1361 bp combined sequences of COI and 16s rRNA were concordant in indicating the following suggestions: (1) phylogenetic relationship of the 11 Penaeidae species based on our result support the opinion of Burkenroad (Burkenroad, M.D. (1983). Crustacean Issues 3:279-290) on the basis of morphological features; (2) it seems more reasonable to class Solenocera crassicorni in the family Penaeidae; (3) the fragment of the COI gene chosen here appears to be a good marker for speciation studies and population analysis in Crustaceans, while the 16s rRNA gene fragment here seems suitable for examining phylogenetic relationships at the species or genus levels in Crustaceans. Our time estimates suggest that Penaeus and Metapenaeus might have separated about 6.38 x 10(6)-7.98 x 10(6) years BP in the post-Miocene, and the species separation within Metapenaeus and Penaeus might occur 0.08 x 10(6)-0.4 x 10(6) years BP in the late Pleistocene.

The Chilean Terrier is a known breed in Chile that has not been genetically assessed despite its distinctive color patterns, agility, and hardiness across the diversity of climates encountered within the Chilean landscape. The population structure and its relatedness with other breeds, as well as the actual origin of the breed, remain unknown. We estimated several population parameters using samples from individuals representing the distribution of the Chilean Terrier across the country. By utilizing the Illumina HD canine genotyping array, we computed the effective population size (Ne ), individual inbreeding, and relatedness to evaluate the genetic diversity of the breed. The results show that linkage disequilibrium was relatively low and decayed rapidly; in fact, Ne was very high when compared to other breeds, and similar to other American indigenous breeds (such as the Chihuahua with values of Ne near 500). These results are in line with the low estimates of genomic inbreeding and relatedness and the relatively large number of effective chromosome segments (Me = 2467) obtained using the properties of the genomic relationship matrix. Between population analysis (cross-population extended haplotype homozygosity, di ) with other breeds such as the Jack Russell Terrier, the Peruvian-Inca Orchid, and the Chihuahua suggested that candidate regions harboring FGF5, PAX3, and ASIP, probably explained some morphological traits, such as the distinctive color pattern characteristic of the breed. When considering Admixture estimates and phylogenetic analysis, together with other breeds of American and European origin, the Chilean Terrier does not have a recent European ancestry. Overall, the results suggest that the breed has evolved independently in Chile from other terrier breeds, from an unknown European terrier ancestor.