Carolina Vianna Morgante

Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection

BackgroundCultivated peanut (Arachis hypogaea) is one of the most widely grown grain legumes in the world, being valued for its high protein and unsaturated oil contents. Worldwide, the major constraints to peanut production are drought and fungal diseases. Wild Arachis species, which are exclusively South American in origin, have high genetic diversity and have been selected during evolution in a range of environments and biotic stresses, constituting a rich source of allele diversity. Arachis stenosperma harbors resistances to a number of pests, including fungal diseases, whilst A. duranensis has shown improved tolerance to water limited stress. In this study, these species were used for the creation of an extensive databank of wild Arachis transcripts under stress which will constitute a rich source for gene discovery and molecular markers development.ResultsTranscriptome analysis of cDNA collections from A. stenosperma challenged with Cercosporidium personatum (Berk. and M.A. Curtis) Deighton, and A. duranensis submitted to gradual water limited stress was conducted using 454 GS FLX Titanium generating a total of 7.4 x 105 raw sequence reads covering 211 Mbp of both genomes. High quality reads were assembled to 7,723 contigs for A. stenosperma and 12,792 for A. duranensis and functional annotation indicated that 95% of the contigs in both species could be appointed to GO annotation categories. A number of transcription factors families and defense related genes were identified in both species. Additionally, the expression of five A. stenosperma Resistance Gene Analogs (RGAs) and four retrotransposon (FIDEL-related) sequences were analyzed by qRT-PCR. This data set was used to design a total of 2,325 EST-SSRs, of which a subset of 584 amplified in both species and 214 were shown to be polymorphic using ePCR.ConclusionsThis study comprises one of the largest unigene dataset for wild Arachis species and will help to elucidate genes involved in responses to biological processes such as fungal diseases and water limited stress. Moreover, it will also facilitate basic and applied research on the genetics of peanut through the development of new molecular markers and the study of adaptive variation across the genus.

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

BackgroundWild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut.ResultsA set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied.ConclusionsThis first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.

A Study of Gene Expression in the Nematode Resistant Wild Peanut Relative, Arachis stenosperma, in Response to Challenge with Meloidogyne arenaria

Peanut (Arachis hypogaea) is amongst the most important legume crops in the world. One of its main yield constraints is the root-knot nematode Meloidogyne arenaria. A number of wild Arachis species, including A. stenosperma, are resistant to nematodes, and are a potential source of new resistance alleles for cultivated peanut. Using in silico subtraction of ESTs and macroarray analysis, we identified genes differentially expressed in A. stenosperma roots during its resistance response to M. arenaria. The three most differentially expressed genes [Auxin Repressed Protein (AsARP), Cytokinin Oxidase (AsCKX) and Metallothionein Type 2 (AsMET2)] were further analyzed using northern-blot and showed distinct expression profiles in the resistant A. stenosperma and susceptible A. hypogaea, both after, and sometimes even before, challenge with nematodes. Of the three most differentially expressed genes, AsARP and AsCKX are potentially involved in plant hormonal balance, and AsMET2 may be related to the reactive oxygen reaction triggered by the hypersensitive response (HR).

A survey of genes involved in Arachis stenosperma resistance to Meloidogyne arenaria race 1

Root-knot nematodes constitute a constraint for important crops, including peanut (Arachis hypogaea L.). Resistance to Meloidogyne arenaria has been identified in the peanut wild relative Arachis stenosperma Krapov. & W. C. Greg., in which the induction of feeding sites by the nematode was inhibited by an early hypersensitive response (HR). Here, the transcription expression profiles of 19 genes selected from Arachis species were analysed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), during the early phases of an A. stenosperma-M. arenaria interaction. Sixteen genes were significantly differentially expressed in infected and non-infected roots, in at least one of the time points analysed: 3, 6, and 9 days after inoculation. These genes are involved in the HR and production of secondary metabolites related to pathogen defence. Seven genes encoding a resistance protein MG13, a helix-loop helix protein, an ubiquitin protein ligase, a patatin-like protein, a catalase, a DUF538 protein, and a resveratrol synthase, were differentially expressed in all time points analysed. Transcripts of two genes had their spatial and temporal distributions analysed by in situ hybridisation that validated qRT-PCR data. The identification of candidate resistance genes involved in wild peanut resistance to Meloidogyne can provide additional resources for peanut breeding and transgenic approaches.

Transcriptome Profiling of Wild Arachis from Water-Limited Environments Uncovers Drought Tolerance Candidate Genes

Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.

Root Transcriptome Analysis of Wild Peanut Reveals Candidate Genes for Nematode Resistance.

Wild peanut relatives (Arachis spp.) are genetically diverse and were adapted to a range of environments during the evolution course, constituting an important source of allele diversity for resistance to biotic and abiotic stresses. The wild diploid A. stenosperma harbors high levels of resistance to a variety of pathogens, including the root-knot nematode (RKN) Meloidogyne arenaria, through the onset of the Hypersensitive Response (HR). In order to identify genes and regulators triggering this defense response, a comprehensive root transcriptome analysis during the first stages of this incompatible interaction was conducted using Illumina Hi-Seq. Overall, eight cDNA libraries were produced generating 28.2 GB, which were de novo assembled into 44,132 contigs and 37,882 loci. Differentially expressed genes (DEGs) were identified and clustered according to their expression profile, with the majority being downregulated at 6 DAI, which coincides with the onset of the HR. Amongst these DEGs, 27 were selected for further qRT-PCR validation allowing the identification of nematode-responsive candidate genes that are putatively related to the resistance response. Those candidates are engaged in the salycilic (NBS-LRR, lipocalins, resveratrol synthase) and jasmonic (patatin, allene oxidase cyclase) acids pathways, and also related to hormonal balance (auxin responsive protein, GH3) and cellular plasticity and signaling (tetraspanin, integrin, expansin), with some of them showing contrasting expression behavior between Arachis RKN-resistant and susceptible genotypes. As these candidate genes activate different defensive signaling systems, the genetic (HR) and the induced resistance (IR), their pyramidding in one genotype via molecular breeding or transgenic strategy might contribute to a more durable resistance, thus improving the long-term control of RKN in peanut.

Vegetative desiccation tolerance of Tripogon spicatus (Poaceae) from the tropical semiarid region of northeastern Brazil

The vegetative desiccation tolerance of Tripogon spicatus (Nees) Ekman was confirmed by its ability to recover the physiological functionality of intact plants previously subjected to extreme dehydration. Photosynthesis became undetectable when leaf relative water content (RWCleaf) achieved ~60%, whereas photochemical variables showed a partial decrease. Until the minimum RWCleaf of 6.41%, total chl decreased by 9%, and total carotenoids increased by 29%. Superoxide dismutase (SOD) activity decreased by 57%, on average, during dehydration, but catalase (CAT) and peroxidase (APX) activities showed no significant differences throughout the experiment. Malondialdehyde (MDA) content increased by 151%, total leaf and root amino acids decreased by 62% and 77%, respectively, whereas leaf and root proline decreased by 40% and 61%, respectively, until complete desiccation. After rehydration, leaves completely recovered turgidity and total chl contents. Carotenoids and MDA remained high, whereas SOD was 60% lower than the measured average measured before dehydration. With the exception of root amino acid contents, total amino acids and proline concentrations recovered completely. Gas exchange and photochemical variables remained substantially higher 4 days after rehydration, compared with the control. Besides increasing MDA, the overall physiological results showed that membrane functionality was preserved, leading to the vegetative desiccation tolerance of T. spicatus during the dehydration-rehydration cycle.

Evaluation of cassava germplasm for drought tolerance under field conditions

The development of cassava (Manihot esculenta Crantz) with a high yield under water-deficit conditions is one of the goal of the breeding programs. The objective of this study was to evaluate the performance and to select cassava accessions based on drought tolerance indices and productive potential under water stress. Forty-nine accessions were evaluated for five agronomic traits (plant height—PH, root yield—RoY, shoot yield—ShY, harvest index—HI; and dry matter content of roots—DMC) under full irrigation conditions and drought stress (DS). The accessions were selected based on: (i) high yield under drought conditions (HY-DS) and (ii) high drought tolerance (Dr-To) based on six different indices. Overall, water stress dramatically reduced the traits’ means (RoY—72.98%, ShY—54.95%, DMC—26.15%, HI—31.05%, and PH—32.95%). Low coincidence among the top ten accessions was identified based on HY-DS and Dr-To criteria. Therefore, considering only the most important traits (RoY and ShY), five accessions (BGM0815, BGM0598, 9624-09, BGM0818, and BRS Formosa) presented high HY-DS. In contrast, to Dr-To criterion, eight and nine accessions were selected for high yield of the aerial part (ShY and PH) and roots (RoY and DMC), respectively. The mean productivity, geometric mean productivity, and drought tolerance indices were the most promising to identify genotypes with high agronomic attributes, while drought susceptibility index, susceptibility, and yield stability index were suitable to identify the most drought tolerant accessions. This set of selected accessions can be used in breeding programs aimed at high yield and drought tolerance.

Características fisiológicas, produção total de raízes e de parte aérea em acessos de Manihot esculenta em condições de déficit hídrico (Physiological characteristics, total root and shoot production in accessions of Manihot esculenta under water ...)

Na regiao Nordeste a cultura da mandioca destaca-se como alternativa de uso agronomico e de subsistencia para a maioria dos agricultores rurais familiares. No entanto pesquisas relacionadas ao desenvolvimento de cultivares recomendaveis para sistemas produtivos dependentes de chuva sao escassas. Genotipos mais produtivos em termos de raizes e parte aerea sob condicao de seca podem ser indicados para programas de melhoramento voltados para obtencao de materiais para alimentacao humana e animal. Neste estudo, objetivou-se a identificacao, em campo, de acessos com maior capacidade de producao total de raizes e de parte aerea nas condicoes irrigada e de deficit hidrico em 49 acessos de mandioca, e identificar quais variaveis fisiologicas estao relacionadas a maior producao. A produtividade total de raizes (PTR, t/ha), foi calculada pela soma de raizes comerciais e nao comerciais, e a produtividade de parte aerea (PPA, t/ha) pela massa total de ramos e folhas, da parcela como um todo. Foram determinadas as variaveis progressao do diâmetro de caule, de diâmetro da copa, de altura da planta, da taxa relativa de expansao foliar, do indice de area foliar, do conteudo relativo de clorofila total e da eficiencia fotoquimica dos fotossistemas II. O acesso Formosa foi o mais produtivo em termos PTR tanto na condicao de irrigacao quanto de deficiencia hidrica, enquanto BGM541 foi superior quanto a PPA em ambas condicoes. Considerando a soma de PTR e PPA, o acesso Formosa se manteve como o mais produtivo. As variaveis fisiologicas avaliadas apresentaram baixa correlacao com PTR e PPA. ABSTRACT In the Northeastern region of Brazil, cassava plants stand out as an alternative to agronomical use and livelihood for most rural family farmers. Research related to the development of recommended cultivars for rain-dependent production systems are scarce. This study aimed to identify accessions with a higher total root and shoot production capacity in irrigated and water stress field conditions between 49 cassava accessions, and identify which physiological variables are related to higher production. Total Root Production (TRP, t/ha) was calculated as the sum of fresh commercial and non-commercial roots of each plot, and Shoot Production (SP, t/ha) as the sum of fresh stems and leaves. They were determined the progression of the variables crown and stem diameters, plant height, relative rate of leaf expansion, relative content of total chlorophyll, potential quantum efficiency of photosystem II, and leaf area index. The Formosa accession was the most productive in terms of TRP in the two conditions, while BGM541 accession was most productive in SP. Considering the sum of TRP and SP, the accession Formosa remained as the most productive. The physiological parameters showed low correlation with TRP and SP. Key-words: Cassava, water deficit, productivity