DNA sequence variation of drought-response candidate genes in Austrocedrus chilensis

Background: Austrocedrus chilensis (D. Don) Pic. Ser. et Bizzarri commonly known as Patagonian cypress is a member of the Cupressaceae family, characterized by a high adaptive potential for growing in marginal areas and good timber quality. The species grows over a wide area and under a wide range of rainfall. This study assessed adaptive genetic variation at SNP level in candidate genes involved in response to drought stress. Results: A total of 18 single nucleotide polymorphisms (SNPs) were found among 1,428 bp. Average nucleotide diversity value (π = 0.00312) was similar to those previously reported in other Cupressaceae. The Fst average among genes and populations was 0.163 and the lowest differentiation was observed in continuous and humid populations. A number of neutrality tests were applied to find evidence of positive selection in our candidate gene set, but only AcAQP2 gene in Pedregoso and San Ramón populations revealed significant departures from neutrality with positive values suggesting balancing selection. Conclusions: In this study we report the levels of nucleotide diversity searched in some drought stress candidate genes in Austrocedrus chilensis and the selective factors that may be acting on this species.Fil: Pomponio, Maria Florencia. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias. Centro de Invest.de Recursos Naturales. Instituto de Recursos Biologicos; ArgentinaFil: Torales, Susana. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias. Centro de Invest.de Recursos Naturales. Instituto de Recursos Biologicos; ArgentinaFil: Gallo, Leonardo. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.patagonia Norte. Estacion Exptal.agrop.s.c.de Bariloche. Grupo de Genetica Forestal; ArgentinaFil: Pastorino, Mario Juan. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.patagonia Norte. Estacion Exptal.agrop.s.c.de Bariloche. Grupo de Genetica Forestal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marchelli, Paula. Instituto Nacional de Tecnologia Agropecuaria. Centro Reg.patagonia Norte. Estacion Exptal.agrop.s.c.de Bariloche. Grupo de Genetica Forestal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cervera, María Teresa. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Centro de Investigación Forestal; EspañaFil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias. Centro de Invest.de Cs.veterinarias y Agronomicas. Instituto de Biotecnologia; Argentin

De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba

Background: Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus. Results: Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads. Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic. Conclusions: This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data. The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera.Fil: Torales, Susana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pomponio, María Florencia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: González, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández, Paula del Carmen. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: López Lauenstein, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Verga, Aníbal Ramón. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Geneticos Vegetales; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

Análise da diversidade genética em dois plantios de Pinus caribaea var. hondurensis

Forest tree breeding activities in plantations with exotic species implies several instances of material selection where genetic variation can be affected. The objective of the work was to verify the genetic variability present in two plantations of Pinus caribaea var. hondurensis by microsatellite markers, initially designed for Pinus taeda fluorescent-labeled. Thus, 299 individuals were analyzed using eight polymorphic microsatellite markers. The results indicate that both plantations have adequate levels of genetic diversity that are representative of the Pinus genus. Through the Bayesian method, it was possible to detect two different population genetic structures (K = 2) between both plantations analyzed. In conclusion, this study suggests that microsatellites markers are useful tools to monitor genetic variation in genetic breeding programs. The genetic diversity estimated in both plantations is similar and typical for Pinus plantations, and as expected, there was a slight decrease in genetic variability in the commercial plantation in comparation with the base plantation.As atividades de melhoramento genético florestal em plantios com espécies exóticas envolvem várias instâncias de seleção de material, onde a variação genética pode ser afetada. O objetivo do trabalho foi verificar a variabilidade genética presente em duas plantações de Pinus caribaea var. hondurensis através de marcadores microssatélites, inicialmente desenhados para Pinus taeda. Assim, 299 indivíduos foram analisados utilizando oito marcadores microssatélites polimórficos. Os resultados indicam que ambas plantações têm níveis adequados de diversidade genética que são representativos do gênero Pinus. Através do método Bayesiano, foi possível detectar duas populações genéticas estruturadas (K = 2) entre as duas plantações analisadas. Em conclusão, este estudo sugere que os marcadores microssatélites são ferramentas úteis para monitorar a variação genética nos programas de melhoramento genético. A diversidade genética estimada em ambas plantações é semelhante e típica das plantações de Pinus, e como se esperava verificou-se uma ligeira diminuição da variabilidade genética na plantação comercial, em comparação com a plantação base

De novo transcriptome sequencing and SSR markers development for Cedrela balansae C. DC., a native tree species of northwest Argentina

The endangered Cedrela balansae C.DC. (Meliaceae) is a high-value timber species with great potential for forest plantations that inhabits the tropical forests in Northwestern Argentina. Research on this species is scarce because of the limited genetic and genomic information available. Here, we explored the transcriptome of C. balansae using 454 GS FLX Titanium next-generation sequencing (NGS) technology. Following de novo assembling, we identified 27,111 non-redundant unigenes longer than 200 bp, and considered these transcripts for further downstream analysis. The functional annotation was performed searching the 27,111 unigenes against the NR-Protein and the Interproscan databases. This analysis revealed 26,977 genes with homology in at least one of the Database analyzed. Furthermore, 7,774 unigenes in 142 different active biological pathways in C. balansae were identified with the KEGG database. Moreover, after in silico analyses, we detected 2,663 simple sequence repeats (SSRs) markers. A subset of 70 SSRs related to important “stress tolerance” traits based on functional annotation evidence, were selected for wet PCR-validation in C. balansae and other Cedrela species inhabiting in northwest and northeast of Argentina (C. fissilis, C. saltensis and C. angustifolia). Successful transferability was between 77% and 93% and thanks to this study, 32 polymorphic functional SSRs for all analyzed Cedrela species are now available. The gene catalog and molecular markers obtained here represent a starting point for further research, which will assist genetic breeding programs in the Cedrela genus and will contribute to identifying key populations for its preservation.Fil: Torales, Susana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Rivarola, Maximo Lisandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Gonzalez, Sergio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Inza, María Virginia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Pomponio, María Florencia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Fernández, Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zelener, Noga. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Fornes, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Tucuman-Santiago del Estero; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Belgrano. Facultad de Ciencias Exactas y Naturales; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

DNA sequence variation of drought-response candidate genes in Austrocedrus chilensis

Background: Austrocedrus chilensis (D. Don) Pic. Ser. et Bizzarri commonly known as Patagonian cypress is a member of the Cupressaceae family, characterized by a high adaptive potential for growing in marginal areas and good timber quality. The species grows over a wide area and under a wide range of rainfall. This study assessed adaptive genetic variation at SNP level in candidate genes involved in response to drought stress. Results: A total of 18 single nucleotide polymorphisms (SNPs) were found among 1,428 bp. Average nucleotide diversity value (\u3c0 = 0.00312) was similar to those previously reported in other Cupressaceae. The Fst average among genes and populations was 0.163 and the lowest differentiation was observed in continuous and humid populations. A number of neutrality tests were applied to find evidence of positive selection in our candidate gene set, but only AcAQP2 gene in Pedregoso and San Ram\uf3n populations revealed significant departures from neutrality with positive values suggesting balancing selection. Conclusions: In this study we report the levels of nucleotide diversity searched in some drought stress candidate genes in Austrocedrus chilensis and the selective factors that may be acting on this species

Transcriptome dynamics of rooting zone and leaves during in vitro adventitious root formation in eucalyptus nitens

Wood properties and agronomic traits associated with fast growth and frost tolerance make Eucalyptus nitens a valuable forest alternative. However, the rapid age-related decline in the adventitious root (AR) formation (herein, meaning induction, initiation, and expression stages) limits its propagation. We analyzed transcriptomic profile variation in leaves and stem bases during AR induction of microcuttings to elucidate the molecular mechanisms involved in AR formation. In addition, we quantified expressions of candidate genes associated with recalcitrance. We delimited the ontogenic phases of root formation using histological techniques and Scarecrow and Short-Root expression quantification for RNA sequencing sample collection. We quantified the gene expressions associated with root meristem formation, auxin biosynthesis, perception, signaling, conjugation, and cytokinin signaling in shoots harvested from 2- to 36-month-old plants. After IBA treatment, 702 transcripts changed their expressions. Several were involved in hormone homeostasis and the signaling pathways that determine cell dedifferentiation, leading to root meristem formation. In part, the age-related decline in the rooting capacity is attributable to the increase in the ARR1 gene expression, which negatively affects auxin homeostasis. The analysis of the transcriptomic variation in the leaves and rooting zones provided profuse information: (1) To elucidate the auxin metabolism; (2) to understand the hormonal and signaling processes involved; (3) to collect data associated with their recalcitrance.Instituto de BiotecnologíaFil: Ayala, Paula Gabriela. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste. Laboratorio de Biotecnología Aplicada y Genómica Funcional; ArgentinaFil: Ayala, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ayala, Paula Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Concordia; ArgentinaFil: Acevedo, Raúl M. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste. Laboratorio de Biotecnología Aplicada y Genómica Funcional; ArgentinaFil: Acevedo, Raúl M. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Luna, Claudia Verónica. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste. Laboratorio de Biotecnología Aplicada y Genómica Funcional; ArgentinaFil: Luna, Claudia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Marcucci Poltri, Susana Noemi. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: González, Ana M. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste. Laboratorio de Biotecnología Aplicada y Genómica Funcional; ArgentinaFil: González, Ana M. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sansberro, Pedro Alfonso. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste. Laboratorio de Biotecnología Aplicada y Genómica Funcional; ArgentinaFil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in Eucalyptus globulus

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus . SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus : cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus. This set of markers is then appropriate for characterizing genetic variation, with potential usefulness for quantitative trait loci (QTL) mapping in different eucalypts genetic pedigrees and other applications such as fingerprinting and marker assisted diversity management

Transcriptome survey of Patagonian southern beech Nothofagus nervosa (= N. Alpina): assembly, annotation and molecular marker discovery

Nothofagus nervosa is one of the most emblematic native tree species of Patagonian temperate forests. Here, the shotgun RNA-sequencing (RNA-Seq) of the transcriptome of N. nervosa, including de novo assembly, functional annotation, and in silico discovery of potential molecular markers to support population and associations genetic studies, are described.Fil: Torales, Susana Leonor. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; ArgentinaFil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pomponio, Maria Florencia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; ArgentinaFil: Fernandez, Paula Del Carmen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Marchelli, Paula Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. ArgentinaFil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Azpilicueta, María M. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gallo, Leonardo A. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. ArgentinaFil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marcucci Poltri, Susana Noemi. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin