The genome assembly of Island Oak (Quercus tomentella), a relictual island tree species.

Island oak (Quercus tomentella) is a rare relictual island tree species that exists only on six islands off the coast of California and Mexico, but was once widespread throughout mainland California. Currently, this species is endangered by threats such as non-native plants, grazing animals, and human removal. Efforts for conservation and restoration of island oak currently underway could benefit from information about its range-wide genetic structure and evolutionary history. Here we present a high-quality genome assembly for Q. tomentella, assembled using PacBio HiFi and Omni-C sequencing, developed as part of the California Conservation Genomics Project (CCGP). The resulting assembly has a length of 781 Mb, with a contig N50 of 22.0 Mb and a scaffold N50 of 63.4 Mb. This genome assembly will provide a resource for genomics-informed conservation of this rare oak species. Additionally, this reference genome will be the first one available for a species in Quercus section Protobalanus, a unique oak clade present only in western North America.

Genomic signatures of ecological divergence between savanna and forest populations of a Neotropical tree.

BACKGROUND AND AIMS: In eastern Neotropical South America, the Cerrado, a large savanna vegetation, and the Atlantic Forest harbour high biodiversity levels, and their habitats are rather different from each other. The biomes have intrinsic evolutionary relationships, with high lineage exchange that can be attributed, in part, to a large contact zone between them. The genomic study of ecotypes, i.e. populations adapted to divergent habitats, can be a model to study the genomic signatures of ecological divergence. Here, we investigated two ecotypes of the tree Plathymenia reticulata, one from the Cerrado and the other from the Atlantic Forest, which have a hybrid zone in the ecotonal zone of Atlantic Forest-Cerrado. METHODS: The ecotypes were sampled in the two biomes and their ecotone. The evolutionary history of the divergence of the species was analysed with double-digest restriction site-associated DNA sequencing. The genetic structure and the genotypic composition of the hybrid zone were determined. Genotype-association analyses were performed, and the loci under putative selection and their functions were investigated. KEY RESULTS: High divergence between the two ecotypes was found, and only early-generation hybrids were found in the hybrid zone, suggesting a partial reproductive barrier. Ancient introgression between the Cerrado and Atlantic Forest was not detected. The soil and climate were associated with genetic divergence in Plathymenia ecotypes and outlier loci were found to be associated with the stress response, with stomatal and root development and with reproduction. CONCLUSIONS: The high genomic, ecological and morphophysiological divergence between ecotypes, coupled with partial reproductive isolation, indicate that the ecotypes represent two species and should be managed as different evolutionary lineages. We advise that the forest species should be re-evaluated and restated as vulnerable. Our results provide insights into the genomic mechanisms underlying the diversification of species across savanna and forest habitats and the evolutionary forces acting in the species diversification in the Neotropics.

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas.

Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex - ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes. Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios. We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae. Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

Genetic diversity, asexual reproduction and conservation of the edible fruit tree Spondias purpurea L. (Anacardiaceae) in the Costa Rican tropical dry forest.

The term circa situm has been used to describe different conservation strategies within agricultural landscapes. Circa situm conserves planted or remnant species in farmlands, where natural vegetation has been modified through anthropogenic intervention. It has been proposed that trees planted or retained under circa situm conditions may contribute to maintaining genetic diversity, however information on the role of this strategy in preserving genetic diversity is scarce. The aim of this study was to determine the levels of genetic diversity and structure, and mating patterns in planted and unmanaged stands of the tropical fruit tree Spondias purpurea L. in north western Costa Rica. In three localities, we used seven polymorphic microsatellite loci and genotyped 201 adults and 648 seeds from planted and wild stands. We found no differences in genetic diversity among planted and wild stands. Genetic structure analysis revealed that gene flow occurs among planted and wild stands within localities. Clones were present and their diversity and evenness were both high and similar between planted and wild stands. The number of pollen donors per progeny array was low (Nep = 1.01) which resulted in high levels of correlated paternity (rp = 0.9). Asexual seeds were found in 4.6% of the progeny arrays, which had multilocus genotypes that were identical to the maternal trees. Our results show that although planted stands under circa situm conditions can maintain similar levels of genetic diversity than wild stands, the low number of sires and asexual seed formation could threaten the long term persistence of populations.

Comparative Seeds Storage Transcriptome Analysis of Astronium fraxinifolium Schott, a Threatened Tree Species from Brazil.

Astronium fraxinifolium Schott (Anacardiaceae), also known as a 'gonçalo-alves', is a tree of the American tropics, with distribution in Mexico, part of Central America, Argentina, Bolivia, Brazil and Paraguay. In Brazil it is an endangered species that occurs in the Cerrado, Caatinga and in the Amazon biomes. In support of ex situ conservation, this work aimed to study two accessions with different longevity (p50) of A. fraxinifolium collected from two different geographic regions, and to evaluate the transcriptome during aging of the seeds in order to identify genes related to seed longevity. Artificial ageing was performed at a constant temperature of 45 °C and 60% relative humidity. RNA was extracted from 100 embryonic axes exposed to control and aging conditions for 21 days. The transcriptome analysis revealed differentially expressed genes such as Late Embryogenesis Abundant (LEA) genes, genes involved in the photosystem, glycine rich protein (GRP) genes, and several transcription factors associated with embryo development and ubiquitin-conjugating enzymes. Thus, these results contribute to understanding which genes play a role in seed ageing, and may serve as a basis for future functional characterization of the seed aging process in A. fraxinifolium.

Rhodnius (Stål, 1859) (Hemiptera, Triatominae) genus in Bolivian Amazonia: a risk for human populations?

BACKGROUND: Chagas disease, one of the most important neglected tropical diseases in the countries of Latin America, is considered to be a particularly important public health concern in the Amazon region due to increases in the number of outbreaks of acute Chagas disease and increased local transmission in the last 20 years. However, relative to other countries, in Bolivia there is little information available on its transmission in the Amazon region. The aim of this study was to investigate the infestation of palm trees, the main habitat of Triatominae in the region, in several localities, to evaluate the danger they represent to inhabitants. METHODS: Triatominae were collected using live bait traps left overnight in six localities in Pando and Beni Departments, Bolivia. DNA extraction and sequencing were used to establish the Triatominae species (Cytb, 16S and 28S-D2 gene fragments), and the blood meal sources (Cytb fragment). Trypanosoma sp. infection was analyzed by sequencing gene fragments (GPX, GPI, HMCOAR, LAP, PDH and COII) or by mini-exon multiplex PCR. RESULTS: A total of 325 Rhodnius were captured (97.3% of nymphs) from the 1200 traps placed in 238 palm trees and 32 burrows/ground holes. Sequence analyses on DNA extracted from 114 insects and phylogeny analysis identified two triatomine species: Rhodnius stali (17%) and Rhodnius montenegrensis (equated to Rhodnius robustus II, 83%). These were found in palm trees of the genera Attalea (69%), Astrocaryum (13%), Copernicia (12%), Euterpe (2%) and Acrocomia (1%). The infection rate was around 30% (165 analyzed insects), with 90% of analyzed insects infected by Trypanosoma cruzi (only the TcI discrete typing unit was detected), 3% infected by Trypanosoma rangeli (first time found in Bolivian Triatominae) and 7% infected by mixed T. cruzi (TcI)-T. rangeli. Rhodnius specimens fed on Didelphidae, rodents, gecko and humans. CONCLUSIONS: The results of this study highlight the epidemiological importance of Rhodnius in the Bolivian Amazon region. The huge geographical distribution of Rhodnius and their proximity to the human dwellings, high infection rate and frequent meals on the human population highlight a risk of transmission of Chagas disease in the region.

Correlation of genome methylation of fig tree accessions with natural nematode and rust incidence.

Commercial fig tree cultivation in Brazil involves a single cultivar, 'Roxo-de-Valinhos'. The use of a single cultivar results in serious diseases and related problems. The aim of this study was to characterize fig accessions by analyzing the natural root-knot nematode and leaf rust incidence in relation to the epigenomic profile of the plant, since epigenetic variations affect plant-pathogen interactions. All plants were attacked by nematodes, indicating susceptibility; Meloidogyne incognita was the root-knot nematode species involved. Joint analysis of data showed that methylation and leaf rust incidence were correlated when observed in the same phenological phase, presenting initial evidence of the same factorial pressure loads in genotypes, suggesting similar behavior within these genotypes.

Higher frequency of legitimate pollinators and fruit set of autotetraploid trees of Libidibia ferrea (Leguminosae) compared to diploids in a mixed tropical urban population.

In mixed-ploidy populations, newly formed polyploids initially occur at low frequencies when compared to diploids. However, polyploidy may lead to morphological and phenological changes, which promote reproductive isolation and favor polyploid establishment and reproductive success. Additionally, previous studies have shown that polyploidy can confer some adaptive advantages to organisms in stressful environments. Here, we investigate variation in reproductive phenology, floral traits and reproductive success between diploid and autotetraploid trees of Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz (Leguminosae) in a mixed tropical urban population, a stressful environment. We assessed ploidy levels, flowering and fruiting phenology, flowering synchrony, floral and reproductive biology, pollination and fruit and seed set. We tested the hypothesis that autotetraploid individuals have a higher frequency of pollinators and higher fruit and seed set per inflorescence (as a proxy of reproductive success) than diploids in an urban green space. Libidibia ferrea is a good model to test our hypothesis because it is self-incompatible (i.e. relies on pollinators to set fruits). In the urban ecosystem studied, we found that diploids flowered for 6-7 months/year and autotetraploids for 3-5 months/year. Flowering synchrony was low between and within cytotypes and even though autotetraploids and diploids exhibited some overlap in flowering period, diploids flowered alone for 2-3 months. Autotetraploids had significantly more flowers per inflorescences, larger flowers and larger pollen grains (as expected for polyploids), but also a higher frequency of visits by legitimate pollinators including two exclusive ones, and higher fruit and seed set per inflorescence when compared to diploids, despite having a shorter flowering period. Our findings reveal some advantages for polyploids over their related diploids in a tropical urban green space. Also, our results highlight the need for more studies that seek to understand abiotic mechanisms affecting reproductive output of polyploids in urban ecosystems.

Comparative population genomics in Tabebuia alliance shows evidence of adaptation in Neotropical tree species.

The role of natural selection in shaping spatial patterns of genetic diversity in the Neotropics is still poorly understood. Here, we perform a genome scan with 24,751 probes targeting 11,026 loci in two Neotropical Bignoniaceae tree species: Handroanthus serratifolius from the seasonally dry tropical forest (SDTF) and Tabebuia aurea from savannas, and compared with the population genomics of H. impetiginosus from SDTF. OutFLANK detected 29 loci in 20 genes with selection signal in H. serratifolius and no loci in T. aurea. Using BayPass, we found evidence of selection in 335 loci in 312 genes in H. serratifolius, 101 loci in 92 genes in T. aurea, and 448 loci in 416 genes in H. impetiginosus. All approaches evidenced several genes affecting plant response to environmental stress and primary metabolic processes. The three species shared no SNPs with selection signal, but we found SNPs affecting the same gene in pair of species. Handroanthus serratifolius showed differences in allele frequencies at SNPs with selection signal among ecosystems, mainly between Caatinga/Cerrado and Atlantic Forest, while H. impetiginosus had one allele fixed across all populations, and T. aurea had similar allele frequency distribution among ecosystems and polymorphism across populations. Taken together, our results indicate that natural selection related to environmental stress shaped the spatial pattern of genetic diversity in the three species. However, the three species have different geographical distribution and niches, which may affect tolerances and adaption, and natural selection may lead to different signatures due to the differences in adaptive landscapes in different niches.

Does clustering of DNA barcodes agree with botanical classification directly at high taxonomic levels? Trees in French Guiana as a case study.

Characterizing biodiversity is one of the main challenges for the coming decades. Most diversity has not been morphologically described, and barcoding is now complementing morphological-based taxonomy to further develop inventories. Both approaches have been cross-validated at the level of species and OTUs. However, many known species are not listed in reference databases. One path to speed up inventories using barcoding is to directly identify individuals at coarser taxonomic levels. We therefore studied in barcoding of plants whether morphological-based and molecular-based approaches are in agreement at genus, family and order levels. We used Agglomerative Hierarchical Clustering (with Ward, Complete and Single Linkage) and Stochastic Block Models (SBM), with two dissimilarity measures (Smith-Waterman scores, kmers). The agreement between morphological-based and molecular-based classifications ranges in most of the cases from good to very good at taxonomic levels above species, even though it decreases when taxonomic levels increase, or when using the tetramer-based distance. Agreement is correlated with the entropy of morphological-based classification and with the ratio of the mean within- and mean between-groups dissimilarities. The Ward method globally leads to the best agreement, whereas Single Linkage can show poor behaviours. SBM provides a useful tool to test whether or not the dissimilarities are structured by the botanical groups. These results suggest that automatic clustering and group identification at taxonomic levels above species are possible in barcoding.

High genetic diversity and low structure in an endemic long-lived tree, Yucca capensis (Asparagaceae).

Endemic species distributed in fragmented habitats are highly vulnerable to extinction because they may have low genetic diversity. However, some life-history traits can mitigate the effect of genetic drift on populations. We analysed the level and distribution of genetic variation and ancestral population size of Yucca capensis, a long-lived endemic plant of the Baja California Peninsula, Mexico. Its populations are scattered across a habitat that is suffering accelerated transformation. We used six nuclear microsatellites to genotype 224 individuals from 17 locations across the entire species' geographic range. We estimated polymorphisms, heterozygosity and genetic structure. We also evaluated the ancestral and recent effective size and time since the population started to change. We found high heterozygosity, high polymorphism and low differentiation among locations, suggesting a panmictic population across the range. We also detected a large ancestral effective population size, which suffered a strong reduction in the Mid-Holocene. Despite changes in environmental conditions caused by habitat modification, the high diversity and low differentiation in Y. capensis may result from its large ancestral effective size and life-history traits, such as plant longevity, clonal growth and mating system, which reduce the rate of loss of genetic variation. However, the dependence on a specialist pollinator that displays short flight range can reduce gene flow among the plant populations and could, shortly, lead them into an extinction vortex.

Fast and novel botanical exploration of a 320-km transect in eastern Amazonia using DNA barcoding / Exploração botânica rápida e inovadora de um transecto de 320 km no leste da Amazônia usando código de barras de DNA

We explored a 320-km transect in the Tumucumaque mountain range along the border between southern French Guiana and Brazil, sampling all trees and lianas with DBH ≥ 10 cm in seven 25 x 25-m plots installed near seven boundary milestones. We isolated DNA from cambium tissue and sequenced two DNA barcodes (rbcLa and matK) to aid in species identification. We also collected fertile herbarium specimens from other species (trees/shrubs/herbs) inside and outside the plots. The selected DNA barcodes were useful at the family level but failed to identify specimens at the species level. Based on DNA barcoding identification, the most abundant families in the plots were Burseraceae, Fabaceae, Meliaceae, Moraceae, Myristicaceae and Sapotaceae. One third of the images of sampled plants posted on the iNaturalist website were identified by the community to species level. New approaches, including the sequencing of the ITS region and fast evolving DNA plastid regions, remain to be tested for their utility in the identification of specimens at lower taxonomic levels in floristic inventories in the Amazon region.(AU)

Um transecto de 320 km foi explorado na Serra do Tumucumaque, ao longo da fronteira entre o sul da Guiana Francesa e o Brasil por meio da amostragem de todas as árvores e lianas com DAP ≥ 10 cm em sete parcelas de 25 x 25 m instaladas perto de sete marcos fronteiriços. Isolamos DNA de tecido cambial e sequenciamos dois códigos de barra de DNA (rbcLa e matK) para auxiliar na identificação das espécies. Também coletamos espécimes de herbário férteis de outras espécies (árvores/arbustos/ervas) dentro e fora das parcelas. Os códigos de barra de DNA selecionados foram úteis em nível de família, mas não conseguiram identificar espécimes em nível de espécie. Com base na identificação de DNA barcoding, as famílias mais abundantes nas parcelas foram Burseraceae, Fabaceae, Meliaceae, Moraceae, Myristicaceae e Sapotaceae. Um terço das imagens de plantas amostradas postadas no website iNaturalist foram identificadas em nível de espécie. Novas abordagens, incluindo o sequenciamento da região ITS e regiões de DNA plastidial de rápida evolução, ainda precisam ser testadas quanto à sua utilidade na identificação de espécimes até níveis taxonômicos mais baixos em inventários florísticos na região amazônica.(AU)

Genetic diversity of Peltophorum dubium (Spreng.) Taub. progenies from the states of Minas Gerais and Mato Grosso do Sul, Brazil / Diversidade genética de progênies de Peltophorum dubium (Spreng.) Taub. procedentes de Minas Gerais e Mato Grosso do Sul, Brasil

There is little information regarding the genetic diversity of native species aimed at identifying the best viable progenies for in situ and ex situ conservation. Furthermore, there is a lack of future forest improvement programs. We aimed to know the genetic diversity of 64 Peltophorum dubium (Spreng.) Taub. (Fabaceae) progenies. We determined this species' dendrometric characteristics, and when using multivariate techniques and cluster analysis, we verified the differences between the progenies and groups with less heterogeneity. The progeny and provenance test was installed in Dourados (Mato Grosso do Sul - MS), with seeds collected in three MS regions (Vale do Ivinhema, Serra de Maracaju, and Serra da Bodoquena) and in the micro-region of Lavras (Minas Gerais - MG). The experiment was conducted in an alpha lattice 8 x 8 with four repetitions. We found genetic variability among and within P. dubium populations for all height, diameter, circumference at breast height, volume, and basal area characters. We suggest that P. dubium populations have high genetic variability, which indicates possible genetic improvement through best progeny selection. The UPGMA and Tocher methods grouped the progenies into three and nine groups, respectively, in which the most divergent individuals come from MG and the Bonito region in MS. Based on morphological characters, P. dubium progenies identified as 45, 47, 49, 50, 55, and 59 from MG are the most promising, while progenies 6 and 9 were the least promising.

Informações quanto à diversidade genética de espécies nativas visando identificar melhores progênies viáveis para conservação in situ e ex situ, além de futuros programas de melhoramento florestal são incipientes. Objetivamos conhecer a diversidade genética de 64 progênies de Peltophorum dubium (Spreng.) Taub. (Fabaceae). Determinamos os caracteres dendrométricos dessa espécie, e ao utilizarmos técnicas multivariadas e análises de agrupamento verificamos as diferenças entre as progênies e grupos de menor heterogeneidade. O teste de progênies e procedências foi instalado em Dourados (MS), cujas sementes foram coletadas em três regiões de MS (Vale do Ivinhema, Serra de Maracaju e Serra da Bodoquena) e na microrregião de Lavras-MG. O experimento foi conduzido em alfa-látice 8 x 8, com quatro repetições. Constatamos variabilidade genética entre e dentro das populações de P. dubium para todos os caracteres altura, diâmetro, circunferência à altura do peito, volume e área basal. Sugerimos que as populações de P. dubium tem alta variabilidade genética, o que indicou possibilidade de melhoramento genético pela seleção das melhores progênies. Os métodos UPGMA e Tocher agruparam as progênies em três e nove grupos, respectivamente, em que os indivíduos mais divergentes são procedentes de MG e da região de Bonito em MS. As progênies de P. dubium de identificação 45, 47, 49, 50, 55 e 59, todas de MG, são as mais promissoras, enquanto que as progênies 6 e 9, foram as menos promissoras, baseando-se nos caracteres morfológicos.

Patterns of genetic diversity, spatial genetic structure and gene flow in Campomanesia xanthocarpa: insights from SSR markers of different genomic origins.

Campomanesia xanthocarpa (Mart.) O. Berg is a South American fruit tree species with important ecological and medicinal properties, which remnants are currently found mainly in isolated forest fragments. In this study, SSR markers from three different genomic origins (gene-linked, nuclear neutral, and organellar) were used to evaluate the patterns of genetic diversity, fine-scale spatial genetic structure and historical gene flow in fragmented forest formations of C. xanthocarpa from the Atlantic Forest in southern Brazil. Our results show that the forest fragments present moderate to high levels of genetic diversity in comparison to species presenting similar life traits, although a trend opposite to expected was observed concerning gene-linked and neutral SSR markers. The fine-scale spatial genetic structure revealed different patterns in short and large distance classes, with a distinct influence of gene-linked and neutral markers in driving the genetic structure in each distance class. The presence of an isolation-by-adaptation pattern implies the need for maintenance of the current remnants to assure the conservation of the private alleles. Finally, as the genetic diversity is found predominantly within forest fragments, programs of seed collection and/or genetic rescue should prioritize a larger number of individuals within each fragment, to increase the sampled diversity.

Achievements and Challenges of Genomics-Assisted Breeding in Forest Trees: From Marker-Assisted Selection to Genome Editing.

Forest tree breeding efforts have focused mainly on improving traits of economic importance, selecting trees suited to new environments or generating trees that are more resilient to biotic and abiotic stressors. This review describes various methods of forest tree selection assisted by genomics and the main technological challenges and achievements in research at the genomic level. Due to the long rotation time of a forest plantation and the resulting long generation times necessary to complete a breeding cycle, the use of advanced techniques with traditional breeding have been necessary, allowing the use of more precise methods for determining the genetic architecture of traits of interest, such as genome-wide association studies (GWASs) and genomic selection (GS). In this sense, main factors that determine the accuracy of genomic prediction models are also addressed. In turn, the introduction of genome editing opens the door to new possibilities in forest trees and especially clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). It is a highly efficient and effective genome editing technique that has been used to effectively implement targetable changes at specific places in the genome of a forest tree. In this sense, forest trees still lack a transformation method and an inefficient number of genotypes for CRISPR/Cas9. This challenge could be addressed with the use of the newly developing technique GRF-GIF with speed breeding.

Detection and application of novel SSR markers from transcriptome data for Astronium fraxinifolium Schott, a threatened Brazilian tree species.

Astronium fraxinifolium is an endangered tree species from Brazil. Due to its significance in environmental reforestation, as well as the continued exploitation of its wood, it is necessary to develop management programs that support the conservation of the species. Simple sequence repeats (SSR) or microsatellite markers are widely used in population genetic studies across a range of diverse organisms. In this study, we present the first SSR markers developed for A. fraxinifolium as well as their frequency and distribution based on transcriptome data. From transcriptome data, we identified more than 100 thousand sequences presenting microsatellites, with a predominant distribution of trinucleotide repeats. From the initial screening, we selected 20 microsatellite loci which were validated and evaluated for genetic indices in two natural populations. All loci were polymorphic, ranging from four to 11 alleles per locus. The observed and expected heterozygosities ranged from 0 to 1.0 and from 0.533 to 1.0, respectively, while the genetic differentiation (GST = 0.363) was greater within than between populations. The developed SSR loci from RNA-Seq data provides a foundation for future studies on genetic diversity and population structure, mating system, and gene flow for A. fraxinifolium populations and related species, aiming at conservation and management.

Water requirement and growth indicators of forest tree species seedlings produced with automated irrigation management.

The lack of information regarding the water requirement of tree species results in water waste in the seedlings production in nurseries. Water requirement, the growth plant factors and water efficiencies for height and diameter were determined for Schizolobium parahyba (Vell.) Blake, Cytharexylum myrianthum Cham. and Ceiba speciosa Ravenna seedlings, under automated irrigation management and greenhouse conditions, located at 22o45'53" S and 43o41'50" W. We used sewage sludge biosolids as substrate in the seedling phase (280 cm-3 tube), and sandy soil material in the initial pot growth phase (18 dm-3 pot). In the seedlings phase, four water replacement levels were applied to the substrate, by drip irrigation, corresponding to average replacement ranging from 40 (V1) to 100% (V4) of the species water requirement. Seedlings developed properly and 80 days after emergence, S. parahyba, C. myrianthum and C. speciosa seedlings received, respectively, 2.40, 1.08 and 0.85 L per plant, for V4. After growth phase (230 DAE), the total water volumes were, respectively, 70.0, 50.3 and 52.7 L per plant. Under adequate water supply, there were rapid recovery and growth of the species, even for the seedlings which showed different height and diameter in the tube phase. The growth plant factors values found were below 0.5 for all species indicating low sensitivity to growth, both in height and in diameter, in response to water deficit. Water efficiency indicators point to distinct trends between the two phases, and C. speciosa has higher values of water efficiencies for height (80.7 and 17.0 cm L-1) and diameter (2.1 and 0.5 mm L-1) in both phases.

A 3K Axiom SNP array from a transcriptome-wide SNP resource sheds new light on the genetic diversity and structure of the iconic subtropical conifer tree Araucaria angustifolia (Bert.) Kuntze.

High-throughput SNP genotyping has become a precondition to move to higher precision and wider genome coverage genetic analysis of natural and breeding populations of non-model species. We developed a 44,318 annotated SNP catalog for Araucaria angustifolia, a grandiose subtropical conifer tree, one of the only two native Brazilian gymnosperms, critically endangered due to its valuable wood and seeds. Following transcriptome assembly and annotation, SNPs were discovered from RNA-seq and pooled RAD-seq data. From the SNP catalog, an Axiom® SNP array with 3,038 validated SNPs was developed and used to provide a comprehensive look at the genetic diversity and structure of 15 populations across the natural range of the species. RNA-seq was a far superior source of SNPs when compared to RAD-seq in terms of conversion rate to polymorphic markers on the array, likely due to the more efficient complexity reduction of the huge conifer genome. By matching microsatellite and SNP data on the same set of A. angustifolia individuals, we show that SNPs reflect more precisely the actual genome-wide patterns of genetic diversity and structure, challenging previous microsatellite-based assessments. Moreover, SNPs corroborated the known major north-south genetic cline, but allowed a more accurate attribution to regional versus among-population differentiation, indicating the potential to select ancestry-informative markers. The availability of a public, user-friendly 3K SNP array for A. angustifolia and a catalog of 44,318 SNPs predicted to provide ~29,000 informative SNPs across ~20,000 loci across the genome, will allow tackling still unsettled questions on its evolutionary history, toward a more comprehensive picture of the origin, past dynamics and future trend of the species' genetic resources. Additionally, but not less importantly, the SNP array described, unlocks the potential to adopt genomic prediction methods to accelerate the still very timid efforts of systematic tree breeding of A. angustifolia.

A cost-effective approach to DNA methylation detection by Methyl Sensitive DArT sequencing.

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).

SNP Markers as a Successful Molecular Tool for Assessing Species Identity and Geographic Origin of Trees in the Economically Important South American Legume Genus Dipteryx.

Dipteryx timber has been heavily exploited in South America since 2000s due to the increasing international demand for hardwood. Developing tools for the genetic identification of Dipteryx species and their geographical origin can help to promote legal trading of timber. A collection of 800 individual trees, belonging to 6 different Dipteryx species, was genotyped based on 171 molecular markers. After the exclusion of markers out of Hardy-Weinberg equilibrium or with no polymorphism or low amplification, 83 nuclear, 29 chloroplast, 13 mitochondrial single nucleotide polymorphisms (SNPs), and 2 chloroplast and 5 mitochondrial INDELS remained. Six genetic groups were identified using Bayesian Structure analyses of the nuclear SNPs, which corresponded to the different Dipteryx species collected in the field. Seventeen highly informative markers were identified as suitable for species identification and obtained self-assignment success rates to species level of 78-96%. An additional set of 15 molecular markers was selected to determine the different genetic clusters found in Dipteryx odorata and Dipteryx ferrea, obtaining self-assignment success rates of 91-100%. The success to assign samples to the correct country of origin using all or only the informative markers improved when using the nearest neighbor approach (69-92%) compared to the Bayesian approach (33-80%). While nuclear and chloroplast SNPs were more suitable for differentiating the different Dipteryx species, mitochondrial SNPs were ideal for determining the genetic clusters of D. odorata and D. ferrea. These 32 selected SNPs will be invaluable genetic tools for the accurate identification of species and country of origin of Dipteryx timber.