Nucleotide Sites Research Articles

Molecular Characterization of the Coding Region and 5’ UTR of HSP70 Gene in Indian Riverine Buffalo Breeds

Background: HSP70 (Heat Shock Protein 70), plays a crucial role in nascent protein folding; the added challenges due to physiological factors demand stringent role-playing of such chaperones for tropical livestock such as water buffalo (Bubalus bubalis). Therefore to evaluate the variations at nucleotide level in HSP70 that could potentially unravel the molecular basis of thermal adaptation in the riverine buffalo breeds of India, the current study was targeted to sequence the CDS (Coding Sequence) and UTR (Untranslated Region) of the gene in a panel of 16 Indian riverine buffalo breeds. Methods: Blood samples were collected and genomic DNA was isolated followed by PCR standardized for the amplification of different fragments of the HSP70 gene using different sets of primer pairs covering the entire coding region and 5’UTR. Multiple amplicons generated to cover the entire gene were sequenced. Sequences were further analyzed manually for the identification of heterozygous animals to detect the polymorphic nucleotide sites and variation between breeds documented. Result: The HSP70 results suggest, the highly conserved nature of gene in buffalo. The only non-synonymous polymorphic site was found in the Toda buffalo breed (g.SNPC greater than T at position 14), resulting in amino acid change 5M greater than T. A total of 7 polymorphic sites were found in the 5’UTR flanking region. Additionally, two insertion/deletions (INDEL) of 30 and 1 nucleotide length were found in the 5’UTR.

Pseudomonas spp. Mediate defense response in sugarcane through differential exudation of root phenolics

Pseudomonas spp., a ubiquitous biocontrol agent, protects the plants from phytopathogens by suppressing them directly by reinforcing the plant’s intrinsic defense mechanism. Root exudated phenolics play an important role in establishing the rhizobacteria population and cross the host boundaries in beneficial plant–microbe interaction. In this study, Pseudomonas spp. HU-8 & HU-9 antagonized the sugarcane red rot pathogen (C. falcatum) and showed a positive chemotactic response against different concentrations (10–30 µM) of synthetic phenolic acids like p-coumaric, vanillic, and 3,4 di-hydroxybenzoic acid. In a pot experiment, they effectively colonized the sugarcane rhizosphere and mediated defense response in sugarcane plants challenged with red rot pathogen C. falcatum by regulating the exudation of root phenolics under hydroponic conditions. They significantly induced the activity of the antioxidant enzymes CAT (1.24–1.64 fold), PO (0.78–1.61 fold), PAL (0.77–0.97 fold), and PPO (3.67–3.73 fold) over untreated plants in sugarcane. They also induced the total phenolic contents (TPC) in sugarcane in the presence (6.56–10.29 mg/g GAE) and absence (2.89–4.16 mg/g GAE) of the pathogen quantified through the Folin-Ciocalteu (FC) method. However, their effect was lower than that of the pathogen (4.34–8 mg/g GAE). The Pseudomonas spp. significantly colonized the sugarcane rhizosphere by maintaining a cell population of (1.0E + 07–1.3E + 08 CFU/mL). A significant positive Pearson’s correlation was observed between the root exudated total phenolic contents, antioxidant enzymatic activities, and rhizospheric population of inoculated bacteria. The 16S rRNA and rpoD gene analysis showed sequence conservation (C: 0.707), average number of nucleotide differences (k: 199.816), nucleotide diversity, (Pi): 0.09819), average number of informative nucleotide sites per site (Psi: 0.01275), GC content (0.57), and polymorphic sites (n = 656). These diverse Pseudomonas spp. could be an ideal bio-inoculants for a broad range of hosts especially graminaceous crops.

Revisiting the evolution of Ostrinia moths with phylogenomics (Pyraloidea: Crambidae: Pyraustinae)

AbstractReconstructing a robust phylogenetic framework is key to understanding the ecology and evolution of many economically important taxa. The crambid moth genus Ostrinia contains multiple agricultural pests, and its classification and phylogeny has remained controversial because of the paucity of characters and the lack of clear morphological boundaries for its species. To address these issues, we inferred a molecular phylogeny of Ostrinia using a phylogenomic dataset containing 498 loci and 115 197 nucleotide sites and examined whether traditional morphological characters corroborate our molecular results. Our results strongly support the monophyly of one of the Ostrinia species groups but surprisingly do not support the monophyly of the other two. Based on the extensive morphological examination and broadly representative taxon sampling of the phylogenomic analyses, we propose a revised classification of the genus, defined by three species groups (Ostrinia nubilalis species group, Ostrinia obumbratalis species group, and Ostrinia penitalis species group), which differs from the traditional classification of Mutuura & Munroe (1970). Morphological and molecular evidence reveal the presence of a new North American species, Ostrinia multispinosa Yang sp.n., closely related to O. obumbratalis. Our analyses indicate that the Ostrinia ancestral larval host preference was for dicots, and that O. nubilalis (European corn borer) and Ostrinia furnacalis (Asian corn borer) independently evolved a preference for feeding on monocots (i.e., maize). Males of a few Ostrinia species have enlarged, grooved midtibiae with brush organs that are known to attract females to increase mating success during courtship, which may represent a derived condition. Our study provides a strong evolutionary framework for this agriculturally important insect lineage.

Zoledronic Acid as a Novel Dual Blocker of KIR6.1/2-SUR2 Subunits of ATP-Sensitive K+ Channels: Role in the Adverse Drug Reactions

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10−6 and 2.1 ± 3.7 × 10−10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10−10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10−10 M and 7.1 ± 3.1 × 10−6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

The first outbreak of dengue fever and molecular tracing in Puyang, 2019

Objective: This study retrospectively analyzed an outbreak of dengue fever in Puyang of Henan province in 2019, in order to find the sources of infection. Methods: Dengue virus IgM/IgG and NS1 antigen were tested by colloidal gold method. E gene was amplified by PCR. MegaX was used for sequences alignment to construct evolutionary distance trees. Results: After clinical and laboratory confirmation, there were 81 cases of dengue fever, 17 of which were imported case who were local farmers and worked in Combadia and Thailand, and 64 of which were indigenous cases. The E gene alignment results showed that the pathogen of this epidemic was Vietnamese 1 and highly homologous with the Vietnamese strain. After the local outbreak, dengue virus E gene developed a nucleotide site mutation which can be steadily transmission. Conclusion: The dengue fever outbreak in Puyang was a local outbreak caused by dengue virus type 1, which was associated with imported cases. Gene sequencing showed that the imported pathogen had a relatively stable and transmissible nucleotide mutation after the local epidemic.

Molecular identification and phylogenetics of local pearl millet cultivars using internal-transcribed spacers of nuclear ribosomal DNA

AbstractLocal cultivars of pearl millet in Saudi Arabia are known to tolerate extreme heat and drought stress. In the current study, the sequences of internal-transcribed spacers (ITSs) of six pearl millet cultivars were sequenced and analysed to investigate the genetic diversity among the local cultivars. The nucleotide polymorphism, secondary structures and phylogenetics were analysed for ITS sequences of the six local cultivars. The obtained sequences were 772–774 base pairs (bp) in length, including complete sequences of the ITS1–5.8S–ITS2 region and partial sequences of 18S and 26S rRNA. The nucleotide diversity among cultivars was higher in ITS2 sequences than ITS1 sequences. The ITS2 had four variable nucleotide sites in three native cultivars, whereas the ITS1 contained one base insertion. The secondary structures of the ITS1 and 5.8S region were highly conserved among the six cultivars and contained some motifs that are conserved across Viridiplantae. However, the ITS2 secondary structure for the two native cultivars, Sayah and Jazan, was distinct from the other cultivars, which confirms the applicability of the ITS2 sequence in distinguishing between genetically close taxa. Additionally, the phylogenetic analysis of the six investigated cultivars and 31 pearl millet accessions from the NCBI database showed close relationships between the local accessions and NCBI accessions from India and France. However, the local cultivar Sayah appeared to be distinct from the other cultivars in the phylogenetic trees. This study provides insights into the polymorphism within local pearl millet cultivars which is important for the identification and conservation of these cultivars.

Evidence for Persistent Heteroplasmy and Ancient Recombination in the Mitochondrial Genomes of the Edible Yellow Chanterelles From Southwestern China and Europe.

Mitochondrial genes and genomes have patterns of inheritance that are distinctly different from those of nuclear genes and genomes. In nature, the mitochondrial genomes in eukaryotes are generally considered non-recombining and homoplasmic. If heteroplasmy and recombination exist, they are typically very limited in both space and time. Here we show that mitochondrial heteroplasmy and recombination may not be limited to a specific population nor exit only transiently in the basidiomycete Cantharellus cibarius and related species. These edible yellow chanterelles are an ecologically very important group of fungi and among the most prominent wild edible mushrooms in the Northern Hemisphere. At present, very little is known about the genetics and population biology of these fungia cross large geographical distances. Our study here analyzed a total of 363 specimens of edible yellow chanterelles from 24 geographic locations in Yunnan in southwestern China and six geographic locations in five countries in Europe. For each mushroom sample, we obtained the DNA sequences at two genes, one in the nuclear genome and one in the mitochondrial genome. Our analyses of the nuclear gene, translation elongation factor 1-alpha (tef-1) and the DNA barcode of C. cibarius and related species, suggested these samples belong to four known species and five potential new species. Interestingly, analyses of the mitochondrial ATP synthase subunit 6 (atp6) gene fragment revealed evidence of heteroplasmy in two geographic samples in Yunnan and recombination within the two new putative species in Yunnan. Specifically, all four possible haplotypes at two polymorphic nucleotide sites within the mitochondrial atp6 gene were found distributed across several geographic locations in Yunnan. Furthermore, these four haplotypes were broadly distributed across multiple phylogenetic clades constructed based on nuclear tef-1 sequences. Our results suggest that heteroplasmy and mitochondrial recombination might have happened repeatedly during the evolution of the yellow chanterelles. Together, our results suggest that the edible yellow chanterelles represent an excellent system from which to study the evolution of mitochondrial-nuclear genome relationships.

Evidence for a force favoring GC over AT at short intronic sites in Drosophila simulans and Drosophila melanogaster.

Population genetics studies often make use of a class of nucleotide site free from selective pressures, in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. Here, we investigate evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and Drosophila simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila, we find a bias in the spectrum of mutations toward AT basepairs. In addition, we find that this bias is stronger in the D. melanogaster lineage than in the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. Here, we provide evidence for a weak force favoring GC in both species, which is correlated with the GC content of introns and is stronger in D. simulans than in D. melanogaster.

Unbiased population heterozygosity estimates from genome‐wide sequence data

Abstract Heterozygosity is a metric of genetic variability frequently used to inform the management of threatened taxa. Estimating observed and expected heterozygosities from genome‐wide sequence data has become increasingly common, and these estimates are often derived directly from genotypes at single nucleotide polymorphism (SNP) markers. While many SNP markers can provide precise estimates of genetic processes, the results of ‘downstream’ analysis with these markers may depend heavily on ‘upstream’ filtering decisions. Here we explore the downstream consequences of sample size, rare allele filtering, missing data thresholds and known population structure on estimates of observed and expected heterozygosity using two reduced‐representation sequencing datasets, one from the mosquito Aedes aegypti (ddRADseq) and the other from a threatened grasshopper, Keyacris scurra (DArTseq). We show that estimates based on polymorphic markers only (i.e. SNP heterozygosity) are always biased by global sample size (N), with smaller N producing larger estimates. By contrast, results are unbiased by sample size when calculations consider monomorphic as well as polymorphic sequence information (i.e. genome‐wide or autosomal heterozygosity). SNP heterozygosity is also biased when differentiated populations are analysed together while autosomal heterozygosity remains unbiased. We also show that when nucleotide sites with missing genotypes are included, observed and expected heterozygosity estimates diverge in proportion to the amount of missing data permitted at each site. We make three recommendations for estimating genome‐wide heterozygosity: (a) autosomal heterozygosity should be reported instead of (or in addition to) SNP heterozygosity; (b) sites with any missing data should be omitted and (c) populations should be analysed in independent runs. This should facilitate comparisons within and across studies and between observed and expected measures of heterozygosity.

Unexpected Discovery of Hypermutator Phenotype Sounds the Alarm for Quality Control Strains.

Microbial strains with high genomic stability are particularly sought after for testing the quality of commercial microbiological products, such as biological media and antibiotics. Yet, using mutation–accumulation experiments and de novo assembled complete genomes based on Nanopore long-read sequencing, we find that the widely used quality-control strain Shewanella putrefaciens ATCC-8071, also a facultative pathogen, is a hypermutator, with a base-pair substitution mutation rate of 2.42 × 10−8 per nucleotide site per cell division, ∼146-fold greater than that of the wild-type strain CGMCC-1.6515. Using complementation experiments, we confirm that mutL dysfunction, which was a recent evolutionary event, is the cause for the high mutation rate of ATCC-8071. Further analyses also give insight into possible relationships between mutation and genome evolution in this important bacterium. This discovery of a well-known strain being a hypermutator necessitates screening the mutation rate of bacterial strains before any quality control or experiments.

Hunting for Beneficial Mutations: Conditioning on SIFT Scores When Estimating the Distribution of Fitness Effect of New Mutations.

The distribution of fitness effects (DFE) of new mutations is a key parameter of molecular evolution. The DFE can in principle be estimated by comparing the site frequency spectra (SFS) of putatively neutral and functional polymorphisms. Unfortunately, the DFE is intrinsically hard to estimate, especially for beneficial mutations because these tend to be exceedingly rare. There is therefore a strong incentive to find out whether conditioning on properties of mutations that are independent of the SFS could provide additional information. In the present study, we developed a new measure based on SIFT scores. SIFT scores are assigned to nucleotide sites based on their level of conservation across a multispecies alignment: the more conserved a site, the more likely mutations occurring at this site are deleterious, and the lower the SIFT score. If one knows the ancestral state at a given site, one can assign a value to new mutations occurring at the site based on the change of SIFT score associated with the mutation. We called this new measure δ. We show that properties of the DFE as well as the flux of beneficial mutations across classes covary with δ and, hence, that SIFT scores are informative when estimating the fitness effect of new mutations. In particular, conditioning on SIFT scores can help to characterize beneficial mutations.

Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP.

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

Protein Labeling and Crosslinking by Covalent Aptamers.

We developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated through introduction of a proximity-driven electrophile at specific nucleotide sites. Using thrombin as a proof-of-concept, we demonstrate that covalent aptamers can selectively transfer a variety of functional handles and/or irreversibly crosslink to the target protein. This approach offers broad programmability and high target specificity. Furthermore, it addresses issues common to aptamers such as instability towards endogenous nucleases and residence times during target engagement. Covalent aptamers are new tools that enable specific protein modification and sensitive protein detection. Moreover, they provide prolonged, nuclease-resistant enzyme inhibition.

Protein Labeling and Crosslinking by Covalent Aptamers

AbstractWe developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated through introduction of a proximity‐driven electrophile at specific nucleotide sites. Using thrombin as a proof‐of‐concept, we demonstrate that covalent aptamers can selectively transfer a variety of functional handles and/or irreversibly crosslink to the target protein. This approach offers broad programmability and high target specificity. Furthermore, it addresses issues common to aptamers such as instability towards endogenous nucleases and residence times during target engagement. Covalent aptamers are new tools that enable specific protein modification and sensitive protein detection. Moreover, they provide prolonged, nuclease‐resistant enzyme inhibition.

Transport unplugged: KCCs are regulated through an N-terminal plug of the ion pathway.

The ability to regulate transmembrane ion transport in response to various cues is vital to any living cell. In neurons, one key example of critical ion control relates to the extrusion of chloride mediated by the potassium-chloride-cotransporters (KCC1-4). In a recent hallmark study, Chi et␣al (2021) report cryo-EM structures of human KCC1 and KCC3b, delineating in detail how regulation by phosphorylation inhibits the transport activity. The authors also identify a stabilizing binding site for nucleotides and speculate on its functional role.

Population differentiation of Rhodobacteraceae along with coral compartments.

Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.

Morphology and molecular phylogeny of a new brackish pleurostomatid ciliate, Loxophyllum paludosum sp. n. (Ciliophora, Litostomatea, Haptoria), from a mangrove wetland in China

A new ciliate species of the genus Loxophyllum Dujardin, 1841, Loxophyllum paludosum sp. n., is described from a mangrove wetland near Daya Bay in Guangdong Province, southern China, based on morphological and molecular analyses. The new species can be distinguished from its congeners by a combination of the following characters: (1) 12–14 right kineties and 4–6 left kineties; (2) two macronuclear nodules and one micronucleus; (3) a single contractile vacuole located terminally; (4) extrusomes bar-shaped, evenly spaced along entire ventral margin, and clustered to form 5–7 warts along dorsal margin; and (5) presence of three ridges on the left side of cell. The new species is divergent from its congeners from 0.4% to 6.7% (5–104 nucleotide sites) based on the small subunit (SSU) rRNA gene sequence data. The validity of the new species is also supported by molecular phylogenetic trees inferred from SSU rRNA gene sequences.

The genetic variability, phylogeny and functional significance of E6, E7 and LCR in human papillomavirus type 52 isolates in Sichuan, China

BackgroundVariations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China.MethodsLCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database.Results50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387–7391 (TTATG) and 7698–7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287–7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY.ConclusionThis study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.

Distinguishing among Pisum accessions using a hypervariable intron within Mendel’s green/yellow cotyledon gene

Distinguishing among commercial varieties or accessions in a germplasm repository presents considerable challenges when dealing with common crops or large collections. In the genus Pisum L. the third intron of the gene STAYGREEN (SGR), responsible for the green/yellow cotyledon variation, contains a complex of repeat sequences that display considerable indel and single nucleotide (SNP) polymorphism. In a sample of 137 Pisum L. accessions obtained from various germplasm collections 76 alleles were identified, ranging from about 600 to 1500 nucleotides in length. In most cases alleles differed by insertion/deletion (indel) polymorphisms in addition to single base changes. The amplicon was particularly useful for distinguishing wild accessions (those with dehiscent pods and dormant seeds). Among the 51 P. sativum L. accessions examined with a definite wild phenotype there were 44 SGR alleles. When this allelic variation was combined with location data for the wild accessions, each allele could be assigned a unique geographical location. Similarly, of the 10 P. fulvum Sibth. & Sm. accessions analyzed, only a group of three had the same allele, two of which are known to be replicates. We were able to confirm the identities of several P. sativum accessions from different germplasm collections, as well as identifying two cases where supposedly synonymous accessions gave different intron sequences. In addition, a group of wild pea accessions from Greece, Sicily, and Portugal exhibited a unique repeat sequence not found in any other wild accession, suggesting that these accessions constitute a distinct lineage. The rate of nucleotide site variation in this fragment of the SGR locus was found to be about 2.22 × 10–8 substitutions per site per year. This value is similar to those found for many other sequences in herbaceous papilionoid tribes, although it is at the faster end of the distribution of herbaceous legume substitution rates. We propose that both a fast substitution rate as well as the formation and rearrangement of indels have contributed to the high level of variation we detected at SGR. This PCR product should prove valuable in the identification of Pisum accessions, for quality control operations in germplasm repositories, and for exploring the evolutionary relationships within the genus.

The genome of the lowland anoa (Bubalus depressicornis) illuminates the origin of river and swamp buffalo

Two types of domestic water buffalo are currently recognized: the river buffalo from the Indian subcontinent and Mediterranean countries and the swamp buffalo from China and Southeast Asia. To test the hypothesis of two separate species of water buffalo, we sequenced the genome of the lowland anoa, Bubalus depressicornis, which is a dwarf wild buffalo endemic to Sulawesi, and two genomes of swamp buffalo, and made comparisons with 12 additional genomes. Three genomic data sets were constructed to infer phylogenetic relationships: the mitochondrial genome (15,468 bp; maternal transmission), two concatenated Y-chromosomal genes, AMELY and DDX3Y (20,036 bp; paternal transmission), and a selection of 30 nuclear genes representing all cattle chromosomes (364,887 bp; biparental transmission). The comparisons between our 30 nuclear gene sequences obtained by read mapping and those directly extracted from Bos taurus and Bubalus bubalis genome assemblies show that the mapping approach revealed higher levels of heterozygosity at both nucleotide sites and indels (insertions and deletions) (0.09–0.15%), as well as several sequence errors (0.07%). Our phylogenetic and molecular dating analyses provide strong evidence that the lowland anoa, river buffalo, and swamp buffalo are three distinct taxa which separated rapidly from each other during the Pleistocene epoch. We therefore conclude that two species of domestic water buffalo should be distinguished: Bubalus bubalis for the river buffalo and Bubalus kerabau for the swamp buffalo. The new classification can have deep implications for understanding the evolution and selection of domesticated forms and for the conservation and management of wild buffalo populations in South and Southeast Asia.