Molecular Evolutionary Analysis Research Articles

Random Amplified Polymorphic DNA (RAPD) Markers Protocol of Bacterial Isolates from Two selected General Hospitals Wastewater (HWW)

In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 103Cfu/ml to 24.0 x 103Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 103 and 18.0 x103 Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways

Gene Rearrangements in the Mitochondrial Genome of Gonatopsis borealis and Onychoteuthis compacta Reveal Their Phylogenetic Implications for Oegopsida.

The complete mitochondrial genome provides crucial information for comprehending gene rearrangement, molecular evolution, and phylogenetic analysis. Here, we have determined the complete mitogenome sequence of Gonatopsis borealis and Onychoteuthis compacta for the first time. Their genome sizes were 20,148 bp and 20,491 bp, respectively, including 18 protein-coding genes, COI-COIII, ATP6, and ATP8 are duplicated, 23 transfer RNA genes, and 2 ribosomal RNA (rRNA) genes (12S and 16S rRNA). Specifically, the overall A+T content is 70.69% and 72.67%. It shows a significant AT bias. The whole mitogenomes indicate positive AT skew (0.070 and 0.062). Furthermore, the gene order has been rearranged within Oegopsida. The tandem duplication random loss model was determined as most likely to explain the observed gene rearrangements. Phylogenetic analysis was performed, and the result tree was found to be consistent with the morphological identification classification. Estimation of divergence time for 35 species showed that the main differentiation of Oegopsida occurred in 140.70Mya. These results will help to better understand the gene rearrangements and evolution of G. borealis and O. compacta and lay a foundation for further phylogeny genetic studies of Oegopsida.

Genomic insights into Yak (Bos grunniens) adaptations for nutrient assimilation in high-altitudes

High-altitude environments present formidable challenges for survival and reproduction, with organisms facing limited oxygen availability and scarce nutrient resources. The yak (Bos grunniens), indigenous to the Tibetan Plateau, has notably adapted to these extreme conditions. This study delves into the genomic basis of the yak’s adaptation, focusing on the positive selection acting on genes involved in nutrient assimilation pathways. Employing techniques in comparative genomics and molecular evolutionary analyses, we selected genes in the yak that show signs of positive selection associated with nutrient metabolism, absorption, and transport. Our findings reveal specific genetic adaptations related to nutrient metabolism in harsh climatic conditions. Notably, genes involved in energy metabolism, oxygen transport, and thermoregulation exhibited signs of positive selection, suggesting their crucial role in the yak’s successful colonization of high-altitude regions. The study also sheds light on the yak's immune system adaptations, emphasizing genes involved in response to various stresses prevalent at elevated altitudes. Insights into the yak’s genomic makeup provide valuable information for understanding the broader implications of high-altitude adaptations in mammalian evolution. They may contribute to efforts in enhancing livestock resilience to environmental challenges.

Comparative evolutionary analyses of peste des petits ruminants virus genetic lineages.

Peste des petits ruminants virus (PPRV) causes a highly infectious disease affecting mainly goats and sheep in large parts of Africa, Asia, and the Middle East and has an important impact on the global economy and food security. Full genome sequencing of PPRV strains has proved to be critical to increasing our understanding of PPR epidemiology and to inform the ongoing global efforts for its eradication. However, the number of full PPRV genomes published is still limited and with a heavy bias towards recent samples and genetic Lineage IV (LIV), which is only one of the four existing PPRV lineages. Here, we generated genome sequences for twenty-five recent (2010-6) and seven historical (1972-99) PPRV samples, focusing mainly on Lineage II (LII) in West Africa. This provided the first opportunity to compare the evolutionary pressures and history between the globally dominant PPRV genetic LIV and LII, which is endemic in West Africa. Phylogenomic analysis showed that the relationship between PPRV LII strains was complex and supported the extensive transboundary circulation of the virus within West Africa. In contrast, LIV sequences were clearly separated per region, with strains from West and Central Africa branched as a sister clade to all other LIV sequences, suggesting that this lineage also has an African origin. Estimates of the time to the most recent common ancestor place the divergence of modern LII and LIV strains in the 1960s-80s, suggesting that this period was particularly important for the diversification and spread of PPRV globally. Phylogenetic relationships among historical samples from LI, LII, and LIII and with more recent samples point towards a high genetic diversity for all these lineages in Africa until the 1970s-80s and possible bottleneck events shaping PPRV's evolution during this period. Molecular evolution analyses show that strains belonging to LII and LIV have evolved under different selection pressures. Differences in codon usage and adaptative selection pressures were observed in all viral genes between the two lineages. Our results confirm that comparative genomic analyses can provide new insights into PPRV's evolutionary history and molecular epidemiology. However, PPRV genome sequencing efforts must be ramped up to increase the resolution of such studies for their use in the development of efficient PPR control and surveillance strategies.

Disappearance of Imported Cases of Omicron Lineage BA.2.40 in West Kalimantan, Indonesia.

The World Health Organization has declared Omicron as the fifth variant of concern with more than 50 mutations, particularly in the spike protein. Given increased viral infectivity due to mutations, worldwide genomic surveillance and detection of severe acute respiratory syndrome 2 (SARS-CoV-2) is essential. The present study aimed to track Omicron lineage BA.2.40 in West Kalimantan, Indonesia. In May-August 2022, nasopharyngeal swab samples (n=3,642) were collected from international travelers to West Kalimantan (active surveillance), and patients hospitalized due to SARS-CoV-2 infection (baseline surveillance). The samples were tested for Omicron lineages based on ORF1ab, N, and HV69-70del genes, followed by whole-genome sequencing. The sequences were then identified using two genomic databases, aligned against the reference genome (Wuhan/Hu-1/2019), and then compared with BA.2.40 lineage detected across the world. Phylogenetic analysis between the samples and other SARS-CoV-2 isolates was performed using molecular evolutionary genetics analysis software. Based on the genomic databases, 10 isolates were identified as BA.2.40. All samples tested positive for the ORF1ab and N genes, but negative for the HV69-70del gene, which is a marker to detect the Omicron variant. Phylogenetic analysis showed the isolates were closely related to an isolate from Malaysia, an area dominated by BA.2.40. Omicron lineage BA.2.40 has no HV69-70 deletion in the spike protein, a marker used to screen for the Omicron variant. BA.2.40 showed a high similarity to an isolate from Malaysia and was detected only during certain periods, indicating the effect of internationally imported cases.

Evaluating the Genetic Diversity of the Plasmodium vivax siap2 Locus: A Promising Candidate for an Effective Malaria Vaccine?

Malaria is the deadliest parasitic disease in the world. Traditional control measures have become less effective; hence, there is a need to explore alternative strategies, such as antimalarial vaccines. However, designing an anti-Plasmodium vivax vaccine is considered a challenge due to the complex parasite biology and the antigens´ high genetic diversity. Recently, the sporozoite invasion-associated protein 2 (SIAP2) has been suggested as a potential antigen to be considered in vaccine design due to its significance during hepatocyte invasion. However, its use may be limited by the incomplete understanding of gene/protein diversity. Here, the genetic diversity of pvsiap2 using P. vivax DNA samples from Colombia was assessed. Through PCR amplification and sequencing, we compared the Colombian sequences with available worldwide sequences, revealing that pvsiap2 displays low genetic diversity. Molecular evolutionary analyses showed that pvsiap2 appears to be influenced by directional selection. Moreover, the haplotypes found differ by a few mutational steps and several of them were shared between different geographical areas. On the other hand, several conserved regions within PvSIAP2 were predicted as potential B-cell or T-cell epitopes. Considering these characteristics and its role in hepatocyte invasion, the PvSIAP2 protein emerges as a promising antigen to be considered in a multi-antigen-multi-stage (multivalent) fully effective vaccine against P. vivax malaria.

Diversity of astroviruses in wild animals in Yunnan province, China

BackgroundAstroviruses (AstVs) are single-stranded RNA viruses that have been detected in a wide range of mammals and birds. They are associated with numerous interspecies transmissions and viral recombination events, posing a threat to human and animal health.MethodsWe collected 1,333 samples from wild animals, including bats, rodents, wild boars, and birds, from various states and cities in the Yunnan Province, China, between 2020 and 2023 to investigate the presence of AstVs. AstVs were detected using a polymerase chain reaction targeting the RdRp gene. Finally, the Molecular Evolutionary Genetics Analysis software was used to construct the phylogenetic tree.ResultsThe overall positivity rate for AstVs was 7.12% in four species, indicating their widespread occurrence in the region. High genetic diversity among AstVs was observed in different animal species, suggesting the potential for interspecies transmission, particularly among rodents and birds. Additionally, we identified a novel AstV strain and, for the first time, provided information on the presence of bastroviruses in Yunnan, China.ConclusionsThe widespread distribution and high genetic diversity of AstVs, along with the observed potential for interspecies transmission, highlight the importance of further investigation and surveillance in the region. The findings emphasize the need for increased attention to AstVs and their potential impact on human and animal health in Yunnan and other regions.

Nuclear phylogenomics of Asteraceae with increased sampling provides new insights into convergent morphological and molecular evolution

Convergent morphological evolution is widespread in flowering plants, and understanding this phenomenon relies on well-resolved phylogenies. Nuclear phylogenetic reconstruction using transcriptome datasets has been successful in various angiosperm groups, but it is limited to taxa with available fresh materials. Asteraceae, which are one of the two largest angiosperm families and are important for both ecosystems and human livelihood, show multiple examples of convergent evolution. Nuclear Asteraceae phylogenies have resolved relationships among most subfamilies and many tribes, but many phylogenetic and evolutionary questions regarding subtribes and genera remain, owing to limited sampling. Here, we increased the sampling for Asteraceae phylogenetic reconstruction using transcriptomes and genome-skimming datasets and produced nuclear phylogenetic trees with 706 species representing two-thirds of recognized subtribes. Ancestral character reconstruction supports multiple convergent evolutionary events in Asteraceae, with gains and losses of bilateral floral symmetry correlated with diversification of some subfamilies and smaller groups, respectively. Presence of the calyx-related pappus may have been especially important for the success of some subtribes and genera. Molecular evolutionary analyses support the likely contribution of duplications of MADS-box and TCP floral regulatory genes to innovations in floral morphology, including capitulum inflorescences and bilaterally symmetric flowers, potentially promoting the diversification of Asteraceae. Subsequent divergences and reductions in CYC2 gene expression are related to the gain and loss of zygomorphic flowers. This phylogenomic work with greater taxon sampling through inclusion of genome-skimming datasets reveals the feasibility of expanded evolutionary analyses using DNA samples for understanding convergent evolution.

Molecular evolution and phylogeographic analysis of wheat dwarf virus.

Wheat dwarf virus (WDV) has caused considerable economic loss in the global production of grain crops. Knowledge of the evolutionary biology and population history of the pathogen remain poorly understood. We performed molecular evolution and worldwide phylodynamic analyses of the virus based on the genes in the protein-coding region of the entire viral genome. Our results showed that host-driven and geography-driven adaptation are major factors that affects the evolution of WDV. Bayesian phylogenetic analysis estimates that the average WDV substitution rate was 4.240 × 10-4 substitutions/site/year (95% credibility interval, 2.828 × 10-4-5.723 × 10-4), and the evolutionary rates of genes encoding proteins with virion-sense transcripts and genes encoding proteins with complementary-sense transcripts were different. The positively selected sites were detected in only two genes encoding proteins with complementary-sense, and WDV-barley are subject to stronger purifying selection than WDV-wheat. The time since the most recent common WDV ancestor was 1746 (95% credibility interval, 1517-1893) CE. Further analyses identified that the WDV-barley population and WDV-wheat population experienced dramatic expansion-decline episodes, and the expansion time of the WDV-barley population was earlier than that of the WDV-wheat population. Our phylogeographic analysis showed that the WDV population originating in Iran was subsequently introduced to Europe, and then spread from Eastern Europe to China.

Adaptive evolution and loss of a putative magnetoreceptor in passerines.

Migratory birds possess remarkable accuracy in orientation and navigation, which involves various compass systems including the magnetic compass. Identifying the primary magnetosensor remains a fundamental open question. Cryptochromes (Cry) have been shown to be magnetically sensitive, and Cry4a from a migratory songbird seems to show enhanced magnetic sensitivity in vitro compared to Cry4a from resident species. We investigate Cry and their potential involvement in magnetoreception in a phylogenetic framework, integrating molecular evolutionary analyses with protein dynamics modelling. Our analysis is based on 363 bird genomes and identifies different selection regimes in passerines. We show that Cry4a is characterized by strong positive selection and high variability, typical characteristics of sensor proteins. We identify key sites that are likely to have facilitated the evolution of an optimized sensory protein for night-time orientation in songbirds. Additionally, we show that Cry4 was lost in hummingbirds, parrots and Tyranni (Suboscines), and thus identified a gene deletion, which might facilitate testing the function of Cry4a in birds. In contrast, the other avian Cry (Cry1 and Cry2) were highly conserved across all species, indicating basal, non-sensory functions. Our results support a specialization or functional differentiation of Cry4 in songbirds which could be magnetosensation.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Avian infectious bronchitis virus (AIBV) review by continent.

Infectious bronchitis virus (IBV) is a positive-sense, single-stranded, enveloped RNA virus responsible for substantial economic losses to the poultry industry worldwide by causing a highly contagious respiratory disease. The virus can spread quickly through contact, contaminated equipment, aerosols, and personal-to-person contact. We highlight the prevalence and geographic distribution of all nine genotypes, as well as the relevant symptoms and economic impact, by extensively analyzing the current literature. Moreover, phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA-6), which provided insights into the global molecular diversity and evolution of IBV strains. This review highlights that IBV genotype I (GI) is prevalent worldwide because sporadic cases have been found on many continents. Conversely, GII was identified as a European strain that subsequently dispersed throughout Europe and South America. GIII and GV are predominant in Australia, with very few reports from Asia. GIV, GVIII, and GIX originate from North America. GIV was found to circulate in Asia, and GVII was identified in Europe and China. Geographically, the GVI-1 lineage is thought to be restricted to Asia. This review highlights that IBV still often arises in commercial chicken flocks despite immunization and biosecurity measures because of the ongoing introduction of novel IBV variants and inadequate cross-protection provided by the presently available vaccines. Consequently, IB consistently jeopardizes the ability of the poultry industry to grow and prosper. Identifying these domains will aid in discerning the pathogenicity and prevalence of IBV genotypes, potentially enhancing disease prevention and management tactics.

Mycotoxic effects of entomopathogenic fungi of fall armyworm (Spodoptera frugiperda J.E. Smith) on poultry feed safety

This study was carried out to analyze mycotoxins of entomopathogenic fungi of fall armyworm (Spodoptera frugiperda J.E. Smith) and poultry feed safety. An experiment was set up to assess the types of mycotoxins produced by entomopathogenic fungal parasite of fall armyworm larvae and their subsequent effect on the safety of the larvae as a feed ingredient. Molecular characterization was done to estimate the diversity of entomopathogenic fungi on fall armyworm larvae specimens from the treatment plots. Sequenced data was analyzed and processed using Molecular Evolutionary Genetic Analysis 6.0 software. The results showed relative diversity of fall armyworm larvae with 11 species isolated belonging to Aspergillus, Penicillium, Fusarium, Trichoderma, Bipolaris and Irpex genus. Some of these are potential mycotoxin producing fungi. Although isolated fungi potentially produce Ochratoxin, Fumonisin, Zearalenone and Trichothecene mycotoxins, only aflatoxins were analyzed in this study. About 3.98 μg /kg of aflatoxin was observed using the ELISA total assay which is within the threshold toxicity levels set in Kenya for feed of about 20 μg /kg. While this is under the threshold set by Kenya, it is still enough to cause concern as the cumulative exposure of even low doses can have impacts. This study therefore concludes that, Spodoptera frugiperda can be potentially contaminated with aflatoxins and when formulating poultry diets, there is need to monitor production so that the quality is not compromised and feed safety is ensured. Further studies are recommended to determine how much produced aflatoxins are then transferred into the poultry products such as eggs and meat.

Demographic dynamics and molecular evolution of the rare and endangered subsect. Gerardianae of Pinus: insights from chloroplast genomes and mitochondrial DNA markers.

The divergence of subsect. Gerardianae was likely triggered by the uplift of the Qinghai-Tibetan Plateau and adjacent mountains. Pinus bungeana might have probably experienced expansion since Last Interglacial period. Historical geological and climatic oscillations have profoundly affected patterns of nucleotide variability, evolutionary history, and species divergence in numerous plants of the Northern Hemisphere. However, how long-lived conifers responded to geological and climatic fluctuations in East Asia remain poorly understood. Here, based on paternally inherited chloroplast genomes and maternally inherited mitochondrial DNA markers, we investigated the population demographic history and molecular evolution of subsect. Gerardianae (only including three species, Pinus bungeana, P. gerardiana, and P. squamata) of Pinus. A low level of nucleotide diversity was found in P. bungeana (π was 0.00016 in chloroplast DNA sequences, and 0.00304 in mitochondrial DNAs). The haplotype-based phylogenetic topology and unimodal distributions of demographic analysis suggested that P. bungeana probably originated in the southern Qinling Mountains and experienced rapid population expansion since Last Interglacial period. Phylogenetic analysis revealed that P. gerardiana and P. squamata had closer genetic relationship. The species divergence of subsect. Gerardianae occurred about 27.18 million years ago (Mya) during the middle to late Oligocene, which was significantly associated with the uplift of the Qinghai-Tibetan Plateau and adjacent mountains from the Eocene to the mid-Pliocene. The molecular evolutionary analysis showed that two chloroplast genes (psaI and ycf1) were under positive selection, the genetic lineages of P. bungeana exhibited higher transition and nonsynonymous mutations, which were involved with the strongly environmental adaptation. These findings shed light on the population evolutionary history of white pine species and provide striking insights for comprehension of their species divergence and molecular evolution.

Genome-wide molecular evolution analysis of the GRF and GIF gene families in Plantae (Archaeplastida)

BackgroundPlant growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) interact with each other and collectively have important regulatory roles in plant growth, development, and stress responses. Therefore, it is of great significance to explore the systematic evolution of GRF and GIF gene families. However, our knowledge and understanding of the role of GRF and GIF genes during plant evolution has been fragmentary.ResultsIn this study, a large number of genomic and transcriptomic datasets of algae, mosses, ferns, gymnosperms and angiosperms were used to systematically analyze the evolution of GRF and GIF genes during the evolution of plants. The results showed that GRF gene first appeared in the charophyte Klebsormidium nitens, whereas the GIF genes originated relatively early, and these two gene families were mainly expanded by segmental duplication events after plant terrestrialization. During the process of evolution, the protein sequences and functions of GRF and GIF family genes are relatively conservative. As cooperative partner, GRF and GIF genes contain the similar types of cis-acting elements in their promoter regions, which enables them to have similar transcriptional response patterns, and both show higher levels of expression in reproductive organs and tissues and organs with strong capacity for cell division. Based on protein–protein interaction analysis and verification, we found that the GRF–GIF protein partnership began to be established in pteridophytes and is highly conserved across different terrestrial plants.ConclusionsThese results provide a foundation for further exploration of the molecular evolution and biological functions of GRF and GIF genes.

Genome-Wide Identification of Vitellogenin Gene Family and Comparative Analysis of Their Involvement in Ovarian Maturation in Exopalaemon carinicauda.

Vitellogenin (Vtg) is a precursor of yolk proteins in egg-laying vertebrates and invertebrates and plays an important role in vitellogenesis and embryonic development. However, the Vtg family remains poorly characterized in Exopalaemon carinicauda, a major commercial mariculture species found along the coasts of the Yellow and Bohai Seas. In this study, 10 Vtg genes from the genomes of E. carinicauda were identified and characterized. Phylogenetic analyses showed that the Vtg genes in crustaceans could be classified into four groups: Astacidea, Brachyra, Penaeidae, and Palaemonidae. EcVtg genes were unevenly distributed on the chromosomes of E. carinicauda, and a molecular evolutionary analysis showed that the EcVtg genes were primarily constrained by purifying selection during evolution. All putative EcVtg proteins were characterized by the presence of three conserved functional domains: a lipoprotein N-terminal domain (LPD_N), a domain of unknown function (DUF1943), and a von Willebrand factor type D domain (vWD). All EcVtg genes exhibited higher expression in the female hepatopancreas than in other tissues, and EcVtg gene expression during ovarian development suggested that the hepatopancreas is the main synthesis site in E. carinicauda. EcVtg1a, EcVtg2, and EcVtg3 play major roles in exogenous vitellogenesis, and EcVtg3 also plays a major role in endogenous vitellogenesis. Bilateral ablation of the eyestalk significantly upregulates EcVtg mRNA expression in the female hepatopancreas, indicating that the X-organ/sinus gland complex plays an important role in ovarian development, mostly by inducing Vtg synthesis. These results could improve our understanding of the function of multiple Vtg genes in crustaceans and aid future studies on the function of EcVtg genes during ovarian development in E. carinicauda.

Molecular evolution analysis of MYB5 in Brassicaceae with specific focus on seed coat color of Brassica napus

BackgroundMYB transcription factors are splay a vital role in plant biology, with previous research highlighting the significant impact of the R2R3-MYB-like transcription factor MYB5 on seed mucilage biosynthesis, trichome branching, and seed coat development. However, there is a dearth of studies investigating its role in the regulation of proanthocyanidin (PA) biosynthesis.ResultsIn this study, a total of 51 MYB5 homologous genes were identified across 31 species belonging to the Brassicaceae family, with particular emphasis on Brassica napus for subsequent investigation. Through phylogenetic analysis, these genes were categorized into four distinct subclasses. Protein sequence similarity and identity analysis demonstrated a high degree of conservation of MYB5 among species within the Brassicaceae family. Additionally, the examination of selection pressure revealed that MYB5 predominantly underwent purifying selection during its evolutionary history, as indicated by the Ka/Ks values of all MYB5 homologous gene pairs being less than one. Notably, we observed a higher rate of non-synonymous mutations in orthologous genes compared to paralogous genes, and the Ka/Ks value displayed a stronger correlation with Ka. In B. napus, an examination of expression patterns in five tissues revealed that MYB5 exhibited particularly high expression in the black seed coat. The findings from the WGCNA demonstrated a robust correlation between MYB5 and BAN(ANR) associated with PA biosynthesis in the black seed coat, providing further evidence of their close association and co-expression. Furthermore, the results obtained from of the analysis of protein interaction networks offer supplementary support for the proposition that MYB5 possesses the capability to interact with transcriptional regulatory proteins, specifically TT8 and TT2, alongside catalytic enzymes implicated in the synthesis of PAs, thereby making a contribution to the biosynthesis of PAs. These findings imply a plausible and significant correlation between the nuique expression pattern of MYB5 and the pigmentation of rapeseed coats. Nevertheless, additional research endeavors are imperative to authenticate and substantiate these findings.ConclusionsThis study offers valuable insights into the genetic evolution of Brassicaceae plants, thereby serving as a significant reference for the genetic enhancement of Brassicaceae seed coat color.

Positive selection and functional diversification of transcription factor Cmr1 homologs in Alternaria

Transcription factor Cmr1 (Colletotrichum melanin regulation 1) and its homologs in several plant fungal pathogens are the regulators of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis pathway and have evolved functional diversification in morphology and pathogenicity. The fungal genus Alternaria comprises the group of “black fungi” that are rich in DHN-melanin in the primary cell wall and septa of the conidia. Some Alternaria species cause many economically important plant diseases worldwide. However, the evolution and function of Cmr1 homologs in Alternaria remain poorly understood. Here, we identified a total of forty-two Cmr1 homologs from forty-two Alternaria spp. and all contained one additional diverse fungal specific transcription factor motif. Phylogenetic analysis indicated the division of these homologs into five major clades and three branches. Dated phylogeny showed the A and D clades diverged latest and earliest, respectively. Molecular evolutionary analyses revealed that three amino acid sites of Cmr1 homologs in Alternaria were the targets of positive selection. Asmr1, the homolog of Cmr1 in the potato early blight pathogen, Alternaria solani was amplified and displayed the sequence conservation at the amino acid level in different A. solani isolates. Asmr1 was further confirmed to have the transcriptional activation activity and was upregulated during the early stage of potato infection. Deletion of asmr1 led to the decreased melanin content and pathogenicity, deformed conidial morphology, and responses to cell wall and fungicide stresses in A. solani. These results suggest positive selection and functional divergence have played a role in the evolution of Cmr1 homologs in Alternaria.Key points• Cmr1 homologs were under positive selection in Alternaria species• Asmr1 is a functional transcription factor, involved in spore development, melanin biosynthesis, pathogenicity, and responses to cell wall and fungicide stresses in A. solani• Cmr1 might be used as a potential taxonomic marker of the genus Alternaria

PHYLOGENETIC RELATIONSHIP BETWEEN RABIES VIRUS (RABIES LYSSAVIRUS) VARIANTS FROM TWO DIFFERENT HOST SPECIES

Rabies is a fatal zoonotic disease that affects several mammals. Bats are recognized hosts of the rabies virus, and their main food source is the blood of other mammals, particularly cattle. During feeding, bats transmit the virus to cattle, which are victims of the disease, contributing to economic losses and increasing the risk of infection for humans. Based on this affinity in the rabies cycle between bats and cattle, the objective of this study was to analyze the phylogenetic relationships of rabies virus samples in cattle and bats. The G gene of the rabies virus was chosen for this study because it is directly related to the infection process. Nucleotide sequences of the viral G gene were selected from GenBank for samples obtained from infected cattle and bats. Maximum parsimony analyses were conducted using the Molecular Evolutionary Genetics Analysis software. The Maxima Parsimony tree indicated a phylogenetic relationship between the G genes of both hosts, indicating that the virus evolved from bats to cattle. Analysis of parsimoniously informative sites revealed that the viral G gene carried specific mutations in each host. Knowledge of the evolutionary relationships between the rabies virus and its hosts is critical for identifying potential new hosts and the possible routes of infection for humans.

A novel in-situ-process technique constructs whole circular cpDNA library

BackgroundThe chloroplast genome (cp genome) is directly related to the study and analysis of molecular phylogeny and evolution of plants in the phylogenomics era. The cp genome, whereas, is highly plastic and exists as a heterogeneous mixture of sizes and physical conformations. It is advantageous to purify/enrich the circular chloroplast DNA (cpDNA) to reduce sequence complexity in cp genome research. Large-insert, ordered DNA libraries are more practical for genomics research than conventional, unordered ones. From this, a technique of constructing the ordered BAC library with the goal-insert cpDNA fragment is developed in this paper.ResultsThis novel in-situ-process technique will efficiently extract circular cpDNA from crops and construct a high-quality cpDNA library. The protocol combines the in-situ chloroplast lysis for the high-purity circular cpDNA with the in-situ substitute/ligation for the high-quality cpDNA library. Individually, a series of original buffers/solutions and optimized procedures for chloroplast lysis in-situ is different than bacterial lysis in-situ; the in-situ substitute/ligation that reacts on the MCE membrane is suitable for constructing the goal-insert, ordered cpDNA library while preventing the large-insert cpDNA fragment breakage. The goal-insert, ordered cpDNA library is arrayed on the microtiter plate by three colonies with the definite cpDNA fragment that are homologous-corresponds to the whole circular cpDNA of the chloroplast.ConclusionThe novel in-situ-process technique amply furtherance of research in genome-wide functional analysis and characterization of chloroplasts, such as genome sequencing, bioinformatics analysis, cloning, physical mapping, molecular phylogeny and evolution.