SNP analysis infers that recombination is involved in the evolution of Amitraz resistance in Rhipicephalus microplus

Rhipicephalus microplus, better known as the Asiatic cattle tick, is a largely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale, particularly cattle. Tick-borne diseases (babesiosis and anaplasmosis) transmitted by R. microplus are alarming as they decrease the quality of livestock health and production. In sub-Saharan Africa, cattle represent a major source of meat and milk, but this region of the world is severely affected by the Rhipicephalus microplus tick. The principal method for tick control is the use of chemical acaricides, notably amitraz, which was implemented in the 1990’s after resistance to other acaricides surfaced. However, the efficiency of chemical control is hindered by an increase in the frequency of mutant resistance alleles to amitraz in tick populations. Presently, the only way to assess amitraz resistance is by means of larval packet tests, but this technique is time-consuming and not particularly cost effective. The main aims of this study were three-fold. First, we attempted to correlate two known SNPs in the octopamine/tyramine (OCT/Tyr) receptor with amitraz resistance in South African field samples of R. microplus. Second, we calculated gametic disequilibrium for these SNPs to determine whether they are randomly associated. Lastly, we conducted a study to assess the evolutionary effects of recombination within the OCT/ Tyr receptor. Our results confirmed that the two SNPs are associated with amitraz resistance in the South African tick strain, and that they are in gametic disequilibrium. Additionally, recombination was detected in the OCT/Tyr receptor generating two recombinant haplotypes. These results are of concern to farmers in sub-Saharan Africa, and the emergence of amitraz resistance should be closely monitored in future. Therefore, we present a quick and affordable RFLP based diagnostic technique to assess amitraz resistance in field samples of R. microplus.S1 Fig. Subpopulation structure of ticks across South Africa. Ticks from each farm were placed into subpopulations (1–15) depending on the region from which they were collected. Grid blocks were constructed 300 x 300 km over the country for accurate overall segregation of populations. The farms from which tick samples were analyzed are indicated in the table, along with their grid block number and province. Farm numbers correspond with sample number, e.g. sample 44.1MF is sample 1 of female R. microplus from farm 44.S1 Table. GenBank accession numbers for all R. microplus OCT/Tyr receptor sequences.S2 Table. Genotypes of field samples of R. microplus ticks at the two published SNP positions.S3 Table. Rhipicephalus microplus larval packet test results.Funding was provided by (a) Gauteng Department of Agriculture and Rural Development, C Maritz-Olivier. (b) Zoetis South Africa (Pty) Ltd., C Maritz-Olivier (c) National Research Foundation, THRIP grant nr: 83890.http://www.plosone.orgam201

Generic concepts in Nectriaceae

The ascomycete family Nectriaceae (Hypocreales) includes numerous important plant and human pathogens, as well as several species used extensively in industrial and commercial applications as biodegraders and biocontrol agents. Members of the family are unified by phenotypic characters such as uniloculate ascomata that are yellow, orange-red to purple, and with phialidic asexual morphs. The generic concepts in Nectriaceae are poorly defined, since DNA sequence data have not been available for many of these genera. To address this issue we performed a multi-gene phylogenetic analysis using partial sequences for the 28S large subunit (LSU) nrDNA, the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), the large subunit of the ATP citrate lyase (acl1), the RNA polymerase II largest subunit (rpb1), RNA polymerase II second largest subunit (rpb2), α-actin (act), β-tubulin (tub2), calmodulin (cmdA), histone H3 (his3), and translation elongation factor 1- alpha (tef1) gene regions for available type and authentic strains representing known genera in Nectriaceae, including several genera for which no sequence data were previously available. Supported by morphological observations, the data resolved 47 genera in the Nectriaceae. We re-evaluated the status of several genera, which resulted in the introduction of six new genera to accommodate species that were initially classified based solely on morphological characters. Several generic names are proposed for synonymy based on the abolishment of dual nomenclature. Additionally, a new family is introduced for two genera that were previously accommodated in the Nectriaceae.This study was financially supported by the NWO Joint Scientific Thematic Research Programme – Joint Research Projects 2012 ALW file number 833.13.2005 titled “Building the fungal quarantine & quality barcode of life database to ensure plant health”.http://www.studiesinmycology.org/am2016Forestry and Agricultural Biotechnology Institute (FABI)Genetic

Diversity and movement of indoor Alternaria alternata across the mainland USA

Alternaria spp. from sect. Alternaria are frequently associated with hypersensitivity pneumonitis, asthma and allergic fungal rhinitis and sinusitis. Since Alternaria is omnipresent in the outdoor environment, it is thought that the indoor spore concentration is mainly influenced by the outdoor spore concentration. However, few studies have investigated indoor Alternaria isolates, or attempted a phylogeographic or population genetic approach to investigate their movement. Therefore, the aim of the current study was to investigate the molecular diversity of indoor Alternaria isolates in the USA, and to test for recombination, using these approaches. Alternaria isolates collected throughout the USA were identified using ITS, gapdh and endoPG gene sequencing. This was followed by genotyping and population genetic inference of isolates belonging to Alternaria sect. Alternaria together with 37 reference isolates, using five microsatellite markers. Phylogenetic analyses revealed that species of Alternaria sect. Alternaria represented 98% (153 isolates) of the indoor isolates collected throughout the USA, of which 137 isolates could be assigned to A. alternata, 15 to the A. arborescens species complex and a single isolate to A. burnsii. The remaining 2% (3 isolates) represented sect. Infectoriae (single isolate) and sect. Pseudoulocladium (2 isolates). Population assignment analyses of the 137 A. alternata isolates suggested that subpopulations did not exist within the sample. The A. alternata isolates were thus divided into four artificial subpopulations to represent four quadrants of the USA. Forty-four isolates representing the south-western quadrant displayed the highest level of uniqueness based on private alleles, while the highest level of gene flow was detected between the south-eastern (32 isolates) and south-western quadrants. Genotypic diversity was high for all quadrants, and a test for linkage disequilibrium suggested that A. alternata has a cryptic sexual cycle. These statistics could be correlated with environmental factors, suggesting that indoor A. alternata isolates, although extremely diverse, have a continental distribution and high levels of gene flow over the continent.Dutch Ministry of Education, Culture and Science through an endowment of the FES programme ‘‘Making the tree of life work’’.http://www.elsevier.com/locate/yfgbihb201

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the host’s haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein family’s evolution within R. microplus adds to current knowledge of this economically important tick.This project was partially funded by the Wellcome Trust, United Kingdom, under the ‘Animal Health in the Developing World’ initiative through Project 0757990 entitled ‘Adapting recombinant anti-tick vaccines to livestock in Africa’.http://www.elsevier.com/locate/ijpar

Intron derived size polymorphism in the mitochondrial genomes of closely related chrysoporthe species

In this study, the complete mitochondrial (mt) genomes of Chrysoporthe austroafricana (190,834 bp), C. cubensis (89,084 bp) and C. deuterocubensis (124,412 bp) were determined. Additionally, the mitochondrial genome of another member of the Cryphonectriaceae, namely Cryphonectria parasitica (158,902 bp), was retrieved and annotated for comparative purposes. These genomes showed high levels of synteny, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as intron positions. Comparative analyses revealed signatures of duplication events, intron number and length variation, and varying intronic ORFs which highlighted the genetic diversity of mt genomes among the Cryphonectriaceae. These mt genomes showed remarkable size polymorphism. The size polymorphism in the mt genomes of these closely related Chrysoporthe species was attributed to the varying number and length of introns, coding sequences and to a lesser extent, intergenic sequences. Compared to publicly available fungal mt genomes, the C. austroafricana mt genome is the second largest in the Ascomycetes thus far.S1 Fig. Midrooted phylogenetic tree of rnpb genes. Rnpb genes were mannualy retrieved from annotated fungal mt genomes publicly available in GenBank. Phylogenetic analysis was performed using maximum likelihood method implemented in RAxML with HKY model of selection. Branch support was calculated using 1000 bootstrap replicates. Green boxes depict atp6 gene, red; small sub-unit of ribosomal RNA (rns) and dark red; small sub-unit of ribosomal RNA (rnl). (PDF)S2 Fig. Maximum likelihood phylogeny of Rps3. Phylogenetic analysis of orf540, orf551 and orf551 from C. austroafricana, C. cubensis and C. deuterocubensis which show sequence similarity to C. parasitica S5 ribosomal protein/maturase fusion protein. Sequences used in this phylogeny were retrieved from GenBank using BLAST. The LG+G model of substitution was used. Branch support for was calculated using the bootstrap method with 1000 replicates. (PDF)S3 Fig. RPKM values for 14 OXPHOS genes. The graph shows the average expression for genes that are involved in oxidative phosphorylation and electron transport and the rnpb gene of Chrysoporthe austroafricana grown in complete and minimal media. (PDF)S4 Fig. Physical maps of the mt genomes of C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica showing locations of all intronic and intergenic ORFs. Intronic ORFs are depicted by yellow arrowed boxes on introns of genes where found and are labelled according to the gene and intron position. Intergenic ORFs are also depicted by yellow arrowed boxes and are labelled with “IN” prefix. (PDF)S1 Table. Accession number and species names for sequences used for rps3 gene phylogeny. (PDF)S2 Table. Codon usage analysis. Comparison of codon usage and tRNAs for the 14 genes involved in oxidative phosphorylation and electron transport in the mitochondrial genomes of Chrysoporthe austroafricana, C. cubensis, C. deuterocubensis and Cryphonectria parasitica. (PDF)S3 Table. BLAST analysis of all identified introns against mt genome sequences deposited in NCBI GenBank. The best hits for each query (intron) is shown. Default BLAST parameters were used. The prefix CA, CC, CD and CP is added to the intron names for clarity. (PDF)S4 Table. BLAST analysis of identified intron encoded and free standing HEG protein sequences. Results from all possible comparisons were calculated. Only Blast hits with a threshold of over 50% alignment coverage, 50% sequence identity and E-value of 0.00005 are shown. (PDF)S5 Table. BLAST analysis of intronic and free standing HEG protein sequences against mt genomes deposited in NCBI GenBank database. HEG type were annotated using the NCBI Conserved Domain Database (CDD). (PDF)The Genomics Research Institute (GRI) University of Pretoria, the University of Pretoria Research Development Programme, the DST/NRF Centre of Excellence in Tree Health Biotechnology (FABI, University of Pretoria), and the National Research Foundation (NRF) (grant number 87332).http://www.plosone.orgam2016Forestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Intron derived size polymorphism in the mitochondrial genomes of closely related chrysoporthe species

In this study, the complete mitochondrial (mt) genomes of Chrysoporthe austroafricana (190,834 bp), C. cubensis (89,084 bp) and C. deuterocubensis (124,412 bp) were determined. Additionally, the mitochondrial genome of another member of the Cryphonectriaceae, namely Cryphonectria parasitica (158,902 bp), was retrieved and annotated for comparative purposes. These genomes showed high levels of synteny, especially in regions including genes involved in oxidative phosphorylation and electron transfer, unique open reading frames (uORFs), ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as intron positions. Comparative analyses revealed signatures of duplication events, intron number and length variation, and varying intronic ORFs which highlighted the genetic diversity of mt genomes among the Cryphonectriaceae. These mt genomes showed remarkable size polymorphism. The size polymorphism in the mt genomes of these closely related Chrysoporthe species was attributed to the varying number and length of introns, coding sequences and to a lesser extent, intergenic sequences. Compared to publicly available fungal mt genomes, the C. austroafricana mt genome is the second largest in the Ascomycetes thus far.S1 Fig. Midrooted phylogenetic tree of rnpb genes. Rnpb genes were mannualy retrieved from annotated fungal mt genomes publicly available in GenBank. Phylogenetic analysis was performed using maximum likelihood method implemented in RAxML with HKY model of selection. Branch support was calculated using 1000 bootstrap replicates. Green boxes depict atp6 gene, red; small sub-unit of ribosomal RNA (rns) and dark red; small sub-unit of ribosomal RNA (rnl). (PDF)S2 Fig. Maximum likelihood phylogeny of Rps3. Phylogenetic analysis of orf540, orf551 and orf551 from C. austroafricana, C. cubensis and C. deuterocubensis which show sequence similarity to C. parasitica S5 ribosomal protein/maturase fusion protein. Sequences used in this phylogeny were retrieved from GenBank using BLAST. The LG+G model of substitution was used. Branch support for was calculated using the bootstrap method with 1000 replicates. (PDF)S3 Fig. RPKM values for 14 OXPHOS genes. The graph shows the average expression for genes that are involved in oxidative phosphorylation and electron transport and the rnpb gene of Chrysoporthe austroafricana grown in complete and minimal media. (PDF)S4 Fig. Physical maps of the mt genomes of C. austroafricana, C. cubensis, C. deuterocubensis and C. parasitica showing locations of all intronic and intergenic ORFs. Intronic ORFs are depicted by yellow arrowed boxes on introns of genes where found and are labelled according to the gene and intron position. Intergenic ORFs are also depicted by yellow arrowed boxes and are labelled with “IN” prefix. (PDF)S1 Table. Accession number and species names for sequences used for rps3 gene phylogeny. (PDF)S2 Table. Codon usage analysis. Comparison of codon usage and tRNAs for the 14 genes involved in oxidative phosphorylation and electron transport in the mitochondrial genomes of Chrysoporthe austroafricana, C. cubensis, C. deuterocubensis and Cryphonectria parasitica. (PDF)S3 Table. BLAST analysis of all identified introns against mt genome sequences deposited in NCBI GenBank. The best hits for each query (intron) is shown. Default BLAST parameters were used. The prefix CA, CC, CD and CP is added to the intron names for clarity. (PDF)S4 Table. BLAST analysis of identified intron encoded and free standing HEG protein sequences. Results from all possible comparisons were calculated. Only Blast hits with a threshold of over 50% alignment coverage, 50% sequence identity and E-value of 0.00005 are shown. (PDF)S5 Table. BLAST analysis of intronic and free standing HEG protein sequences against mt genomes deposited in NCBI GenBank database. HEG type were annotated using the NCBI Conserved Domain Database (CDD). (PDF)The Genomics Research Institute (GRI) University of Pretoria, the University of Pretoria Research Development Programme, the DST/NRF Centre of Excellence in Tree Health Biotechnology (FABI, University of Pretoria), and the National Research Foundation (NRF) (grant number 87332).http://www.plosone.orgam2016Forestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Host switching between native and non-native trees in a population of the canker pathogen Chrysoporthe cubensis from Colombia

The purpose of this study was to test the hypothesis that Chrysoporthe cubensis on native trees in South America could be the source of the pathogen that causes severe stem cankers and often mortality in commercially propagated Eucalyptus trees. This was done by investigating populations originating from two adjacent Eucalyptus (Myrtaceae) plantations in Colombia, and wild Miconia rubiginosa trees (Melastomataceae) growing alongside these stands. Polymorphic microsatellite markers were used to quantify allele sizes in 20 and 39 isolates from the two Eucalyptus stands and 32 isolates from adjacent M. rubiginosa trees. Gene and genotypic diversities were calculated from these data, and population differentiation and assignment tests were performed to ascertain whether the populations were genetically different. Results showed that there were no differences between any of the populations using these techniques, and that they can be treated as a single population. Therefore, the results support the hypothesis that host switching has occurred in C. cubensis in Colombia.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3059am201

Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.Zoetis (Pty) Ltd., South Africa, the National Research Foundation Technology and Human Resources for Industry Programme (Grant number TP12082911252) and the Belgium Development Cooperation (DGD) FA3 project.http://www.elsevier.com/locate/ttbdis2017-06-30hb2016GeneticsVeterinary Tropical Disease

Transmission ratio distortion in an interspecific cross between Fusarium circinatum and Fusarium subglutinans

Previously, an interspecific cross between Fusarium circinatum and Fusarium subglutinans was used to generate a genetic linkage map. A ca. 55 % of genotyped markers displayed transmission ratio distortion (TRD) that demonstrated a genome-wide distribution. The working hypothesis for this study was that TRD would be non-randomly distributed throughout the genetic linkage map. This would indicate the presence of distorting loci. Using a genome-wide threshold of α = 0.01, 79 markers displaying TRD were distributed on all 12 linkage groups (LGs). Eleven putative transmission ratio distortion loci (TRDLs), spanning eight LGs, were identified in regions containing three or more adjacent markers displaying distortion. No epistatic interactions were observed between these TRDLs. Thus, it is uncertain whether the genome-wide TRD was due to linkage between markers and genomic regions causing distortion. The parental origins of markers followed a non-random distribution throughout the linkage map, with LGs containing stretches of markers originating from only one parent. Thus, due to the nature of the interspecific cross, the current hypothesis to explain these observations is that the observed genome-wide segregation was caused by the high level of genomic divergence between the parental isolates. Therefore, homologous chromosomes do not align properly during meiosis, resulting in aberrant transmission of markers. This also explains previous observations of the preferential transmission of F. subglutinans alleles to the F1 progenyThe National Research Foundation (NRF), University of Pretoria, Forestry and Agricultural Biotechnology Institute (FABI), the DST/NRF Center of Excellence in Tree Health Biotechnology (CTHB), members of the Tree Protection Co-operative Programme (TPCP), and the Andrew Mellon Foundation.http://link.springer.com/journal/13258hb201

Mitochondrial introgression and interspecies recombination in the Fusarium fujikuroi species complex

The Fusarium fujikuroi species complex (FFSC) is an economically important monophyletic lineage in the genus Fusarium. Incongruence observed among mitochondrial gene trees, as well as the multiple non-orthologous copies of the internal transcribed spacer region of the ribosomal RNA genes, suggests that the origin and history of this complex likely involved interspecies gene flow. Based on this hypothesis, the mitochondrial genomes of non-conspecific species should harbour signatures of introgression or introgressive hybridization. The aim of this study was therefore to search for recombination between the mitochondrial genomes of different species in the FFSC. Using methods based on mt genome sequence similarity, five significant recombinant regions in both gene and intergenic regions were detected. Using coalescent-based methods and the sequences for individual mt genes, various ancestral recombination events between different lineages of the FFSC were also detected. These findings suggest that interspecies gene flow and introgression are likely to have played key roles in the evolution of the FFSC at both ancient and more recent time scales.The South African National Department of Science and Technology (DST), National Research Foundation (NRF), the Technology and Human Resources of Industry Programme (THRIP) (includes Grant specific unique reference number (UID) 83924), the Tree Protection Cooperative Programme (TPCP), L’Oréal/UNESCO for Women in Science in Sub-Saharan Africa, the Claude Leon Foundation, and the University of Pretoria.http://www.imafungus.orgam2019BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog