The Diverse Evolutionary Histories of Domesticated Metaviral Capsid Genes in Mammals.

Selfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae, giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals.

Host-specific co-evolution likely driven by diet in Buchnera aphidicola.

BACKGROUND: Russian wheat aphid (Diuraphis noxia Kurd.) is a severe pest to wheat, and even though resistance varieties are available to curb this pest, they are becoming obsolete with the development of new virulent aphid populations. Unlike many other aphids, D noxia only harbours a single endosymbiont, Buchnera aphidicola. Considering the importance of Buchnera, this study aimed to elucidate commonalities and dissimilarities between various hosts, to better understand its distinctiveness within its symbiotic relationship with D. noxia. To do so, the genome of the D. noxia's Buchnera was assembled and compared to those of other aphid species that feed on diverse host species. RESULTS: The overall importance of several features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. Buchnera protein coding genes were found to have percentage GC contents that tended towards a mean of ~ 26% which had strong correlation to their identity to their E. coli homologs. Several SNPs were identified between different aphid populations and multiple isolates of Buchnera were confirmed in single aphids. CONCLUSIONS: Establishing the strong correlation of percentage GC content of protein coding genes and gene identity will allow for identifying which genes will be lost in the continually shrinking Buchnera genome. This is also the first report of a parthenogenically reproducing aphid that hosts multiple Buchnera strains in a single aphid, raising questions regarding the benefits of maintaining multiple strains. We also found preliminary evidence for post-transcriptional regulation of Buchnera genes in the form of polyadenylation.

Histone H3K27 Methylation Perturbs Transcriptional Robustness and Underpins Dispensability of Highly Conserved Genes in Fungi.

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 underpins the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation, we used a framework of 23 species across the Fusarium genus including three species covered with histone methylation data. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. In contrast, genes marked by H3K27me3 do not show such associations. Furthermore, highly conserved genes marked by H3K27me3 encode smaller proteins, exhibit weaker codon usage bias, higher levels of hydrophobicity, show lower intrinsically disordered regions, and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival during host infection. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related Fusarium fungi are associated with transcriptional perturbation at the microevolutionary scale. In conclusion, we show how repressive histone marks are entangled in the evolutionary fate of highly conserved genes across evolutionary timescales.

Female Germ Cell Development in Chickens and Humans: The Chicken Oocyte Enriched Genes Convergent and Divergent with the Human Oocyte.

The development of germ cells and other physiological events in the differentiated ovary of humans are highly conserved with several mammalian species, except for the differences in timing. However, comparative knowledge on this topic is very scarce with respect to humans and lower vertebrates, such as chickens. In chickens, female germ cells enter into meiosis around embryonic day (E) 15.5 and are arrested in meiotic prophase I as primary oocytes. The oocytes arrested in meiosis I are accumulated in germ-cell cysts; shortly after hatching, they are enclosed by flattened granulosa cells in order to form primordial follicles. In humans, the process of meiotic recombination in female germ cells begins in the 10-11th week of gestation, and primordial follicles are formed at around week 20. In this review, we comprehensively elucidate both the conservation and the species-specific differences between chickens and humans with respect to germ cell, oocyte, and follicle development. Importantly, we provide functional insights into a set of chicken oocyte enriched genes (from E16 to 1 week post-hatch) that show convergent and divergent expression patterns with respect to the human oocyte (from week 11 to 26).

Mycoplasma species in the male reproductive organs and the fresh and frozen semen of the Hungarian native goose.

The objective of this study was to clarify whether the most common species of Mycoplasma can be detected in the reproductive organs and the cloaca, as well as in the semen of asymptomatic native Hungarian male geese. As it is necessary for the semen of that breed to be preserved pathogen-free in an in vitro gene-conservation programme, the presence of and sources of infection, as well as prevention of the survival of pathogens following semen cryopreservation, are key issues. Ten asymptomatic, 2-year-old ganders were tested. For the detection of mycoplasmas, samples were taken from both fresh and frozen/thawed semen, cloaca, phallus lymph, testes and vas deferens; that is five samples from each of the 10 ganders. The semen was statically frozen using dimethyl-formamide as a cryoprotectant and stored in liquid nitrogen at -196°C. Species-specific PCR systems targeting M. anserisalpingitidis, M. anseris and M. cloacale were used for screening and identification. Results of this study have shown, for the first time, that (1) among the three Mycoplasma species examined, all were detectable in the indigenous Hungarian ganders, with no clinical signs; (2) the pathogens could be detected in the cloaca, in both fresh and cryopreserved semen samples, but remained undetected within the inner reproductive organs; and (3) as pathogens were able to survive the freezing/storing/thawing procedures, the possibility of vertical transmission of the pathogens during artificial inseminations does exist, which causes problems in the in vitro gene-conservation programmes for this breed.

Fine-scale spatial genetic structure, mating, and gene dispersal patterns in Parkia biglobosa populations with different levels of habitat fragmentation.

PREMISE: A good understanding of genetic variation and gene dispersal in tree populations is crucial for their sustainable management, particularly in a context of rapid environmental changes. West African Sudanian savannahs are being fragmented and degraded, partly due to expansion of crop cultivation and monocultures that reduce tree density and may impact pollinators. The population dynamics of important indigenous trees could also be affected. We investigated the influence of habitat fragmentation on patterns of genetic diversity and gene dispersal of a key Sudanian agroforestry tree species, Parkia biglobosa. METHODS: Using 10 highly polymorphic nuclear microsatellites, we genotyped 2475 samples from reproductive trees, seedlings, and embryos in four tree populations presenting different levels of habitat fragmentation. RESULTS: Parkia biglobosa presented similar high genetic diversity across the four populations studied. Genetic diversity and inbreeding were similar between adults and embryo cohorts. In all four populations, the selfing rate was less than 1%. The effective number of pollen donors per tree was high (NEP ~ 18-22), as was the pollen immigration rate (from 34 to 74%). Pollen dispersal was characterized by a fat-tailed distribution with mean estimates exceeding 200 m. In three populations, stem diameter had a pronounced effect on male reproductive success. Here, the highest male reproductive success was observed in trees with a diameter at breast height between 60 and 75 cm. CONCLUSIONS: At the scale analyzed, fragmentation does not seem to pose limitations to gene flow in any of the sites investigated, regardless of the landscape configuration associated with the different tree stands. The study provides useful insights on the reproductive biology of an important tree species in the West African savannahs.

Hypothesis: protein and RNA attributes are continuously optimized over time.

BACKGROUND: Little is known why proteins and RNAs exhibit half-lives varying over several magnitudes. Despite many efforts, a conclusive link between half-lives and gene function could not be established suggesting that other determinants may influence these molecular attributes. RESULTS: Here, I find that with increasing gene age there is a gradual and significant increase of protein and RNA half-lives, protein structure, and other molecular attributes that tend to affect protein abundance. These observations are accommodated in a hypothesis which posits that new genes at 'birth' are not optimized and thus their products exhibit low half-lives and less structure but continuous mutagenesis eventually improves these attributes. Thus, the protein and RNA products of the oldest genes obtained their high degrees of stability and structure only after billions of years while the products of younger genes had less time to be optimized and are therefore less stable and structured. Because more stable proteins with lower turnover require less transcription to maintain the same level of abundance, reduced transcription-associated mutagenesis (TAM) would fixate the changes by increasing gene conservation. CONCLUSIONS: Consequently, the currently observed diversity of molecular attributes is a snapshot of gene products being at different stages along their temporal path of optimization.

Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew.

BACKGROUND: Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. RESULTS: We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620-6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116-175 genes encoding CSEPs with limited gene amplification. CONCLUSIONS: Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.

A vertebrate model for the study of lipid binding/transfer protein function: conservation of OSBP-related proteins between zebrafish and human.

Oxysterol-binding protein (OSBP) and OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a family of lipid-binding/transfer proteins (LTPs) present in eukaryotes from yeast to man. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and act as either lipid transporters or sensors that control lipid metabolism, cell signaling, and vesicle transport. Zebrafish, Danio rerio, has gained increasing popularity as a model organism in developmental biology, human disease, toxicology, and drug discovery. However, LTPs in the fish are thus far unexplored. In this article we report a series of bioinformatic analyses showing that the OSBPL gene family is highly conserved between the fish and human. The OSBPL subfamily structure is markedly similar between the two organisms, and all 12 human genes have orthologs, designated osbpl and located on 11 chromosomes in D. rerio. Interestingly, osbpl2 and osbpl3 are present as two closely related homologs (a and b), due to gene duplication events in the teleost lineage. Moreover, the domain structures of the distinct ORP proteins are almost identical between zebrafish and man, and molecular modeling in the present study suggests that ORD liganding by phosphatidylinositol-4-phosphate (PI4P) is a feature conserved between yeast Osh3p, human ORP3, and zebrafish Osbpl3. The present analysis identifies D. rerio as an attractive model to study the functions of ORPs in vertebrate development and metabolism.

Exploring the evolution of CHS gene family in plants.

Chalcone synthase (CHS) is a key enzyme that catalyzes the first committed step of flavonoid biosynthetic pathway. It plays a vital role not only in maintaining plant growth and development, but also in regulating plant response to environmental hazards. However, the systematic phylogenomic analysis of CHS gene family in a wide range of plant species has not been reported yet. To fill this knowledge gap, a large-scale investigation of CHS genes was performed in 178 plant species covering green algae to dicotyledons. A total of 2,011 CHS and 293 CHS-like genes were identified and phylogenetically divided into four groups, respectively. Gene distribution patterns across the plant kingdom revealed the origin of CHS can be traced back to before the rise of algae. The gene length varied largely in different species, while the exon structure was relatively conserved. Selection pressure analysis also indicated the conserved features of CHS genes on evolutionary time scales. Moreover, our synteny analysis pinpointed that, besides genome-wide duplication and tandem duplication, lineage specific transposition events also occurred in the evolutionary trajectory of CHS gene family. This work provides novel insights into the evolution of CHS gene family and may facilitate further research to better understand the regulatory mechanism of traits relating to flavonoid biosynthesis in diverse plants.

Strict Conservation yet Non-Essential Nature of Plasmid Gene bba40 in the Lyme Disease Spirochete Borrelia burgdorferi.

The highly segmented genome of Borrelia burgdorferi, the tick-borne bacterium that causes Lyme disease, is composed of a linear chromosome and more than 20 co-existing endogenous plasmids. Many plasmid-borne genes are unique to B. burgdorferi and some have been shown to provide essential functions at discrete points of the infectious cycle between a tick vector and rodent host. In this study, we investigated the role of bba40, a highly conserved and differentially expressed gene on a ubiquitous linear plasmid of B. burgdorferi. In a prior genome-wide analysis, inactivation of bba40 by transposon insertion was linked with a noninfectious phenotype in mice, suggesting that conservation of the gene in the Lyme disease spirochete reflected a critical function of the encoded protein. To address this hypothesis, we moved the bba40::Tn allele into a similar wild-type background and compared the phenotypes of isogenic wild-type, mutant and complemented strains in vitro and throughout the in vivo mouse/tick infectious cycle. In contrast to the previous study, we identified no defect in the ability of the bba40 mutant to colonize the tick vector or murine host, or to be efficiently transmitted between them. We conclude that bba40 joins a growing list of unique, highly conserved, yet fully dispensable plasmid-borne genes of the Lyme disease spirochete. We infer that the experimental infectious cycle, while including the tick vector and murine host, lacks key selective forces imposed during the natural enzootic cycle. IMPORTANCE The key finding of this study contradicts our premise that the ubiquitous presence and strict sequence conservation of a unique gene in the Lyme disease spirochete, Borrelia burgdorferi, reflect a critical role in either the murine host or tick vector in which these bacteria are maintained in nature. Instead, the outcome of this investigation illustrates the inadequate nature of the experimental infectious cycle currently employed in the laboratory to fully model the enzootic cycle of the Lyme disease spirochete. This study also highlights the importance of complementation for accurate interpretation of mutant phenotypes in genetic studies of Borrelia burgdorferi.

The diverse evolutionary histories of domesticated metaviral capsid genes in mammals.

Selfish genetic elements and their remnants comprise at least half of the human genome. Active transposons duplicate by inserting copies at new sites in a host genome. Following insertion, transposons can acquire mutations that render them inactive; the accrual of additional mutations can render them unrecognizable over time. However, in rare instances, segments of transposons become useful for the host, in a process called gene domestication. Using the first complete human genome assembly and 25 additional vertebrate genomes, we analyzed the evolutionary trajectories and functional potential of genes domesticated from the capsid genes of Metaviridae, a retroviral-like retrotransposon family. Our analysis reveals four families of domesticated capsid genes in placental mammals with varied evolutionary outcomes, ranging from universal retention to lineage-specific duplications or losses and from purifying selection to lineage-specific rapid evolution. The four families of domesticated capsid genes have divergent amino-terminal domains, inherited from four distinct ancestral metaviruses. Structural predictions reveal that many domesticated genes encode a previously unrecognized RNA-binding domain retained in multiple paralogs in mammalian genomes both adjacent to and independent from the capsid domain. Collectively, our study reveals diverse outcomes of domestication of diverse metaviruses, which led to structurally and evolutionarily diverse genes that encode important, but still largely-unknown functions in placental mammals. (207).

Epidemiology and Phylogeny of Anaplasma ovis with a Note on Hematological and Biochemical Changes in Asymptomatic Goats Enrolled from Four Districts in Punjab, Pakistan.

Background: Anaplasma ovis is an intra-erythrocytic gram negative rickettsial bacterium that infects small ruminants, resulting in huge economic losses worldwide. Materials and Methods: The present investigation aims at reporting the molecular prevalence of A. ovis in 1200 asymptomatic goats that were enrolled from 4 districts (Layyah, Lohdran, Dera Ghazi Khan, and Rajanpur) in Punjab, Pakistan by targeting the msp4 gene of bacterium. Risk factors associated with the prevalence of A. ovis and phylogeny of bacterium were also documented. Results: 184 out of 1200 (15%) goat blood samples were infected with A. ovis. The prevalence of the pathogen varied with the sampling sites (p = 0.005), and the highest prevalence was detected in goats from Layyah (19%) followed by Rajanpur (17%), Dera Ghazi Khan (15%), and Lohdran district (9%). The represented partial msp4 gene amplicon was confirmed by Sanger sequencing and deposited to GenBank (OP225957-59). Phylogenetic analysis revealed that the amplified isolates resembled the msp4 sequences reported from Iran, Mangolia, Sudan, and the United States. Sex and age of goats, herd composition and size, and the presence of ticks on goats and dogs associated with herds were the rick factors associated with the prevalence of A. ovis. Red blood cells, lymphocytes (%), neutrophils (%), hemoglobin, and hematocrit levels in blood and Aspartate amino transferase, urea, and creatinine levels in serum were disturbed in A. ovis infected goats when compared with uninfected animals. Conclusion: We are reporting the prevalence of A. ovis in Pakistani goats from four districts of Punjab and these data will help in developing the integrated control policies against this tick-borne pathogen that is infecting our goat breeds.

A framework for identifying fertility gene targets for mammalian pest control.

Fertility-targeted gene drives have been proposed as an ethical genetic approach for managing wild populations of vertebrate pests for public health and conservation benefit.This manuscript introduces a framework to identify and evaluate target gene suitability based on biological gene function, gene expression, and results from mouse knockout models.This framework identified 16 genes essential for male fertility and 12 genes important for female fertility that may be feasible targets for mammalian gene drives and other non-drive genetic pest control technology. Further, a comparative genomics analysis demonstrates the conservation of the identified genes across several globally significant invasive mammals.In addition to providing important considerations for identifying candidate genes, our framework and the genes identified in this study may have utility in developing additional pest control tools such as wildlife contraceptives.

Genomics and adaptation in forest ecosystems.

Rapid human-induced environmental changes like climate warming represent a challenge for forest ecosystems. Due to their biological complexity and the long generation time of their keystone tree species, genetic adaptation in these ecosystems might not be fast enough to keep track with conditions changing at such a fast pace. The study of adaptation to environmental change and its genetic mechanisms is therefore key for ensuring a sustainable support and management of forests. The 4-day conference of the European Research Group EvolTree (https://www.evoltree.eu) on the topic of "Genomics and Adaptation in Forest Ecosystems" brought together over 130 scientists to present and discuss the latest developments and findings in forest evolutionary research. Genomic studies in forest trees have long been hampered by the lack of high-quality genomics resources and affordable genotyping methods. This has dramatically changed in the last few years; the conference impressively showed how such tools are now being applied to study past demography, adaptation and interactions with associated organisms. Moreover, genomic studies are now finally also entering the world of conservation and forest management, for example by measuring the value or cost of interspecific hybridization and introgression, assessing the vulnerability of species and populations to future change, or accurately delineating evolutionary significant units. The newly launched conference series of EvolTree will hopefully play a key role in the exchange and synthesis of such important investigations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11295-022-01542-1.

Evolutionary selection identifies critical immune-relevant genes in lung cancer subtypes.

In an evolving population, proliferation is dependent on fitness so that a numerically dominant population typically possesses the most well adapted phenotype. In contrast, the evolutionary "losers" typically disappear from the population so that their genetic record is lost. Historically, cancer research has focused on observed genetic mutations in the dominant tumor cell populations which presumably increase fitness. Negative selection, i.e., removal of deleterious mutations from a population, is not observable but can provide critical information regarding genes involved in essential cellular processes. Similar to immunoediting, "evolutionary triage" eliminates mutations in tumor cells that increase susceptibility to the host immune response while mutations that shield them from immune attack increase proliferation and are readily observable (e.g., B2M mutations). These dynamics permit an "inverse problem" analysis linking the fitness consequences of a mutation to its prevalence in a tumor cohort. This is evident in "driver mutations" but, equally important, can identify essential genes in which mutations are seen significantly less than expected by chance. Here we utilized this new approach to investigate evolutionary triage in immune-related genes from TCGA lung adenocarcinoma cohorts. Negative selection differs between the two cohorts and is observed in endoplasmic reticulum aminopeptidase genes, ERAP1 and ERAP2 genes, and DNAM-1/TIGIT ligands. Targeting genes or molecular pathways under positive or negative evolutionary selection may permit new treatment options and increase the efficacy of current immunotherapy.

Comparative Genomics of Six Lytic Bacillus subtilis Phages from the Southwest United States.

Background: Despite their importance to microbial dynamics involving Bacillus subtilis, we have a limited understanding of the diversity of phages that can lyse this model organism. Materials and Methods: Phages were isolated from soil samples collected from various sites in the southwest U.S. deserts on a wild B. subtilis strain. Their genomes were assembled, characterized, and bioinformatically compared. Results: Six Siphoviruses with high nucleotide and amino acid similarity to each other (>80%) but very limited similarity to phages currently in GenBank were isolated. These phages have double-stranded DNA genomes (55,312 to 56,127 bp) with 86-91 putative protein coding genes, and a low GC content. Comparative genomics reveal differences in loci encoding proteins that are putatively involved in bacterial adsorption with evidence for genomic mosaicism and a possible role for small genes. Conclusions: A comparative approach provides insights into phage evolution, including the role of indels in protein folding.

Forty Years without Family: Three Novel Bacteriophages with High Similarity to SPP1 Reveal Decades of Evolutionary Stasis since the Isolation of Their Famous Relative.

SPP1, an extensively studied bacteriophage of the Gram-positive Bacillus subtilis, is a model system for the study of phage-host interactions. Despite progress in the isolation and characterization of Bacillus phages, no previously fully sequenced phages have shared more than passing genetic similarity to SPP1. Here, we describe three virulent phages very similar to SPP1; SPP1 has greater than 80% nucleotide sequence identity and shares more that 85% of its protein coding genes with these phages. This is remarkable, given more than 40 years between the isolation of SPP1 and these phages. All three phages have somewhat larger genomes and more genes than SPP1. We identified a new putative gene in SPP1 based on a conserved sequence found in all phages. Gene conservation connotes purifying selection and is observed in structural genes and genes involved in DNA metabolism, but also in genes of unknown function, suggesting an important role in phage survival independent of the environment. Patterns of divergence point to genes or gene domains likely involved in adaptation to diverse hosts or different environments. Ultimately, comparative genomics of related phages provides insight into the long-term selective pressures that affect phage-bacteria interactions and alter phage genome content.

Seasonal Investigation of Anaplasma marginale Infection in Pakistani Cattle Reveals Hematological and Biochemical Changes, Multiple Associated Risk Factors and msp5 Gene Conservation.

Bovine anaplasmosis is a tick-borne disease caused by an obligate intercellular Gram-negative bacterium named Anaplasma (A.) marginale. In this study, we report the seasonal prevalence, potentially associated risk factors and phylogeny of A. marginale in cattle of three different breeds from Multan District, Southern Punjab, Pakistan. A total of 1020 blood samples (crossbred, n = 340; Holstein Friesian, n = 340; and Sahiwal breed, n = 340) from apparently healthy cattle were collected on a seasonal basis from March 2020 to April 2021. Based on PCR amplification of the msp5 partial sequence, overall, the A. marginale prevalence rate was estimated at 11.1% (113/1020) of the analyzed cattle samples. According to seasons, the highest prevalence rate was observed in autumn (16.5%), followed by winter (10.6%) and summer (9.8%), and the lowest was recorded in the spring (7.5%). The crossbred and Sahiwal cattle were the most susceptible to A. marginale infection, followed by Holstein Friesian cattle (7.9%). Analysis of epidemiological factors revealed that cattle reared on farms where dairy animals have tick loads, dogs coinhabit with cattle and dogs have tick loads have a higher risk of being infected with A. marginale. In addition, it was observed that white blood cell, lymphocyte (%), monocyte (%), hematocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentrations were significantly disturbed in A. marginale-positive cattle compared with non-infested cattle. Genetic analysis of nucleotide sequences and a phylogenetic study based on msp5 partial sequencing demonstrated that this gene appears to be highly conserved among our isolates and those infecting apparently healthy cattle from geographically diverse worldwide regions. The presented data are crucial for estimating the risk of bovine anaplasmosis in order to develop integrated control policies against bovine anaplasmosis and other tick-borne diseases infecting cattle in the country.

Genetic diversity and genetic structure of Acer monspessulanum L. across Zagros forests of Iran using molecular markers.

Acer monspessulanum L. is an important tree species found in the temperate Zagros forests of Iran. Despite its importance, the long-term persistence of its small and fragmented populations is jeopardised by genetic erosion and hence, monitoring its genetic resource and variability is practically required for providing conservation measures of the species germplasm in Zagros woodland ecosystem. The present study aimed to provide the first data on genetic diversity and genetic differentiation pattern of 19 natural populations comprising 240 individuals of A. monspessulanum across its growing area in Zagros forests using three molecular tools including inter-simple sequence repeats (ISSR), start codon targeted (SCoT), and simple sequence repeat (SSR). In total, ISSR and SCoT primers generated a total of 141 and 121 clear and scorable bands for analysis with the polymorphism rate of 90.50 and 90.02% and a mean of 10.85 and 11 fragments per marker, respectively. In addition, 73 alleles were achieved using 10 polymorphic SSR loci from the studied accessions with 100% polymorphism, ranging between 5 and 10 alleles per locus. Average percentage of polymorphic alleles per population for ISSR, SCoT, and SSR data was 84.02%, 83%, and 100%, respectively, and generally, Nei's gene diversity (H) and Shannon's index of diversity (I) values for all populations demonstrated moderate to high levels of genetic diversity (H = 0.267-0.707; I = 0.38-1.38). The AMOVA results of the three marker systems attributed higher genetic variation to individuals within in each population than among populations. Furthermore, overall GST value for all populations detected the moderate to high levels of genetic differentiation, indicating a limited gene flow occurrence among the populations. STRUCTURE analysis (K = 5) clustered the populations into four to five distinct groups, in accordance with geographical distances. These results could represent an important contribution for effective germplasm characterization and could be eventually used in in situ or ex situ conservation of A. monspessulanum genetic resources.