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Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species.

Generic delimitation in Piptadenia and allies (mimosoid legumes) has been in a state of flux, particularly caused by over-reliance on fruit and seed morphology to segregate species out of Piptadenia into the genera Parapiptadenia , Pityrocarpa and Pseudopiptadenia . Although supporting their segregation from Piptadenia , previous phylogenetic analyses suggested that some of these segregated genera are not monophyletic. Here, we test the monophyly of Parapiptadenia , Pityrocarpa and Pseudopiptadenia with dense taxon sampling across these genera, including the type species of each genus. Our analysis recovers Parapitadenia as monophyletic, but places Pseudopiptadenia species in two distinct lineages, one of which includes all three species of Pityrocarpa . Given that the type species of both Pseudopiptadenia and Pityrocarpa are nested in the same clade, we subsume Pseudopiptadenia under the older name Pityrocarpa . The remaining Pseudopiptadenia species are assigned to the new genus Marlimorimia . Alongside high molecular phylogenetic support, recognition of Parapiptadenia , Pityrocarpa and Marlimorimia as distinct genera is also supported by combinations of morphological traits, several of which were previously overlooked.

• Background and Aims The genus Arachis contains 80 described species. Section Arachis is of particular interest because it includes cultivated peanut, an allotetraploid, and closely related wild species, most of which are diploids. This study aimed to analyse the genetic relationships of multiple accessions of section Arachis species using two complementary methods. Microsatellites allowed the analysis of inter- and intraspecific variability. Intron sequences from single-copy genes allowed phylogenetic analysis including the separation of the allotetraploid genome components. • Methods Intron sequences and microsatellite markers were used to reconstruct phylogenetic relationships in section Arachis through maximum parsimony and genetic distance analyses. • Key Results Although high intraspecific variability was evident, there was good support for most species.However, some problems were revealed, notably a probable polyphyletic origin for A. kuhlmannii. The validity of the genome groups was well supported. The F, K and D genomes grouped close to the A genome group. The 2n = 18 species grouped closer to the B genome group. The phylogenetic tree based on the intron data strongly indicated that A. duranensis and A. ipaënsis are the ancestors of A. hypogaea and A. montícola. Intron nucleotide substitutions allowed the ages of divergences of the main genome groups to be estimated at a relatively recent 2.3-2.9 million years ago. This age and the number of species described indicate a much higher speciation rate for section Arachis than for legumes in general. • Conclusions The analyses revealed relationships between the species and genome groups and showed a generally high level of intraspecific genetic diversity. The improved knowledge of species relationships should facilitate the utilization of wild species for peanut improvement. The estimates of speciation rates in section Arachis are high, but not unprecedented. We suggest these high rates may be linked to the peculiar reproductive biology of Arachis.

Crenea Aubl. (Lythraceae) is a ditypic genus of subshrubs occurring in mangrove vegetation on the coasts of northern South America. Phylogenetic analyses based on morphology have offered unresolved and conflicting phylogenetic positions for the genus in the family. This study presents the first molecular sequences for Crenea, from nrITS, rbcL, trnL, trnL-F, and matK regions. Molecular phylogenetic analyses find full support for Crenea within Ammannia L., a relationship not previously recognized. Ammannia is a globally distributed genus of terrestrial to amphibious herbs mostly occurring in freshwater marshes and wetlands. It was recently reconfigured based on phylogenetic evidence to include the genera Nesaea Comm. ex Kunth and Hionanthera A. Fern. & Diniz. The transfer of Crenea to Ammannia further extends the morphological, ecological, and biogeographical diversity of Ammannia and provides the final evidence defining Ammannia as a monophyletic lineage of the Lythraceae. A revised circumscription of Ammannia s.l. adds several new morphological character states and the first species in the genus restricted to mangrove vegetation. Two changes in taxonomic status are made: Ammannia maritima (Aubl.) S. A. Graham, P. W. Inglis, & T. B. Cavalc., comb. nov., and Ammannia patentinervius (Koehne) S. A. Graham, P. W. Inglis, & T. B. Cavalc., comb. nov. The new combinations are described, a list of exsiccatae examined is provided, and the effects of the reconfiguration to the morphology and biogeography of the genus are detailed.

• The genus Manihot, with around 120 known species, is native to a wide range of habitats and regions in the tropical and subtropical Americas. Its high species richness and recent diversification only c. 6 million years ago have significantly complicated previous phylogenetic analyses. Several basic elements of Manihot evolutionary history therefore remain unresolved. • Here, we conduct a comprehensive phylogenomic analysis of Manihot, focusing on exhaustive sampling of South American taxa. • We find that two recently described species from northeast Brazil’s Atlantic Forest were the earliest to diverge, strongly suggesting a South American common ancestor of Manihot. Ancestral state reconstruction indicates early Manihot diversification in dry forests, with numerous independent episodes of new habitat colonization, including into savannas and rainforests within South America. We identify the closest wild relatives to Manihot esculenta, including the crop cassava, and we quantify extensive wild introgression into the cassava gene pool from at least five wild species, including Manihot glaziovii, a species used widely in breeding programs. Finally, we show that this wild-to-crop introgression substantially shapes the mutation load in cassava. • Our findings provide a detailed case study for neotropical evolutionary history in a diverse and widespread group, and a robust phylogenomic framework for future Manihot and cassava research.

In a comparative analysis of genome sequences from isolates of the baculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from Brazil and Guatemala, we identified a subset of isolates possessing chimeric genomes. We identified six distinct phylogenetically incongruous regions (PIRs) dispersed in the genomes, of between 279 and 3345 bp in length. The individual PIRs possessed high sequence similarity among the affected ChinNPV isolates but varied in coverage in some instances. The donor for four of the PIRs implicated in horizontal gene transfer (HGT) was identified as Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), an alphabaculovirus closely related to ChinNPV, or another unknown but closely related virus. BLAST searches of the other two PIRs returned only ChinNPV sequences, but HGT from an unknown donor baculovirus cannot be excluded. Although Chrysodeixis includens and Trichoplusia ni are frequently co-collected from soybean fields in Brazil, pathogenicity data suggest that natural coinfection of C. includens larvae with ChinNPV and TnSNPV is probably uncommon. Additionally, since the chimeric ChinNPV genomes with tracts of TnSNPV sequence were restricted to a single monophyletic lineage of closely related isolates, a model of progressive restoration of the native DNA sequence by recombination with ChinNPV possessing a fully or partially non-chimeric genome is reasonable. However, multiple independent HGT from TnSNPV to ChinNPV during the evolution of these isolates cannot be excluded. Mortality data suggest that the ChinNPV isolates with chimeric genomes are not significantly different in pathogenicity towards C. includens when compared to most other ChinNPV isolates. Exclusion of the PIRs prior to phylogenetic analysis had a large impact on the topology of part of the maximum-likelihood tree, revealing a homogenous clade of three isolates (IB, IC and ID) from Paraná state in Brazil collected in 2006, together with an isolate from Guatemala collected in 1972 (IA), comprising the lineage uniquely affected by HGT from TnSNPV. The other 10 Brazilian ChinNPV isolates from Paraná, Mato Grosso, and Minas Gerais states showed higher variability, where only three isolates from Paraná state formed a monophyletic group correlating with geographical origin.

Arachis batizocoi is a wild relative of cultivated peanut (A. hypogaea), an allotetraploid with an AABB genome. Arachis batizocoi was once considered the ancestral donor of the peanut B genome, but cytogenetics and DNA phylogenies have indicated a new genome classification, 'K'. These observations seem inconsistent with genetic studies and breeding that have shown that A. batizocoi can behave as a B genome. The genetic behaviour, genome composition and phylogenetic position of A. batizocoi were studied using controlled hybridizations, induced tetraploidy, whole-genome in situ fluorescent hybridization (GISH) and molecular phylogenetics. Sterile diploid hybrids containing AK genomes were obtained using A. batizocoi and the A genome species A. duranensis, A. stenosperma, A. correntina or A. villosa. From these, three types of AAKK allotetraploids were obtained, each in multiple independent polyploidy events. Induced allotetraploids were vigorous and fertile, and were hybridized to A. hypogaea to produce F1 hybrids. Even with the same parental combination, fertility of these F1 hybrids varied greatly, suggesting the influence of stochastic genetic or epigenetic events. Interestingly, hybrids with A. hypogaea ssp. hypogaea were significantly more fertile than those with the subspecies fastigiata. GISH in cultivated × induced allotetraploids hybrids (harbouring AABK genomes) and a molecular phylogeny using 16 intron sequences showed that the K genome is distinct, but more closely related to the B than to the A genome. The K genome of A. batizocoi is more related to B than to the A genome, but is distinct. As such, when incorporated in an induced allotetraploid (AAKK) it can behave as a B genome in crosses with peanut. However, the fertility of hybrids and their progeny depends upon the compatibility of the A genome interactions. The genetic distinctness of A. batizocoi makes it an important source of allelic diversity in itself, especially in crosses involving A. hypogaea ssp. hypogaea.

Background In Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression. Results Chromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure. Conclusion Our data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression.

 Generic delimitation in Piptadenia and allies (mimosoid legumes) has been in a state of flux, particularly caused by over-reliance on fruit and seed morphology to segregate species out of Piptadenia into the genera Parapiptadenia , Pityrocarpa and Pseudopiptadenia . Although supporting their segregation from Piptadenia , previous phylogenetic analyses suggested that some of these segregated genera are not monophyletic. Here, we test the monophyly of Parapiptadenia , Pityrocarpa and Pseudopiptadenia with dense taxon sampling across these genera, including the type species of each genus. Our analysis recovers Parapitadenia as monophyletic, but places Pseudopiptadenia species in two distinct lineages, one of which includes all three species of Pityrocarpa . Given that the type species of both Pseudopiptadenia and Pityrocarpa are nested in the same clade, we subsume Pseudopiptadenia under the older name Pityrocarpa . The remaining Pseudopiptadenia species are assigned to the new genus Marlimorimia . Alongside high molecular phylogenetic support, recognition of Parapiptadenia , Pityrocarpa and Marlimorimia as distinct genera is also supported by combinations of morphological traits, several of which were previously overlooked. Keywords: Caesalpinioideae , Fabaceae , Leguminosae , Parapiptadenia , Stryphnodendron clade, tropical America