Peanut (Arachis hypogaea) Expressed Sequence Tag Project: Progress and Application

Many plant ESTs have been sequenced as an alternative to whole genome sequences, including peanut because of the genome size and complexity. The US peanut research community had the historic 2004 Atlanta Genomics Workshop and named the EST project as a main priority. As of August 2011, the peanut research community had deposited 252,832 ESTs in the public NCBI EST database, and this resource has been providing the community valuable tools and core foundations for various genome-scale experiments before the whole genome sequencing project. These EST resources have been used for marker development, gene cloning, microarray gene expression and genetic map construction. Certainly, the peanut EST sequence resources have been shown to have a wide range of applications and accomplished its essential role at the time of need. Then the EST project contributes to the second historic event, the Peanut Genome Project 2010 Inaugural Meeting also held in Atlanta where it was decided to sequence the entire peanut genome. After the completion of peanut whole genome sequencing, ESTs or transcriptome will continue to play an important role to fill in knowledge gaps, to identify particular genes and to explore gene function

Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection

<p>Abstract</p> <p>Background</p> <p>Peanut (<it>Arachis hypogaea </it>L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of <it>Aspergillus parasiticus </it>and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against <it>Aspergillus </it>infection and subsequent aflatoxin contamination.</p> <p>Results</p> <p>We constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to <it>Aspergillus </it>infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to <it>Aspergillus </it>with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by <it>A. parasiticus </it>and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (<it>R</it>) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of <it>R </it>> 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to <it>Arabidopsis thaliana</it>, maize (<it>Zea mays</it>), <it>Medicago truncatula</it>, rapeseed (<it>Brassica napus</it>), rice (<it>Oryza sativa</it>), soybean (<it>Glycine max</it>) and wheat (<it>Triticum aestivum</it>) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.</p> <p>Conclusion</p> <p>The developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers <ext-link ext-link-type="gen" ext-link-id="ES702769">ES702769</ext-link> to <ext-link ext-link-type="gen" ext-link-id="ES724546">ES724546</ext-link>.</p

Resistance to Thrips in Peanut and Implications for Management of Thrips and Thrips-Transmitted Orthotospoviruses in Peanut

Thrips are major pests of peanut (Arachis hypogaea L.) worldwide, and they serve as vectors of devastating orthotospoviruses such as Tomato spotted wilt virus (TSWV) and Groundnut bud necrosis virus (GBNV). A tremendous effort has been devoted to developing peanut cultivars with resistance to orthotospoviruses. Consequently, cultivars with moderate field resistance to viruses exist, but not much is known about host resistance to thrips. Integrating host plant resistance to thrips in peanut could suppress thrips feeding damage and reduce virus transmission, will decrease insecticide usage, and enhance sustainability in the production system. This review focuses on details of thrips resistance in peanut and identifies future directions for incorporating thrips resistance in peanut cultivars. Research on thrips–host interactions in peanut is predominantly limited to field evaluations of feeding damage, though, laboratory studies have revealed that peanut cultivars could differentially affect thrips feeding and thrips biology. Many runner type cultivars, field resistant to TSWV, representing diverse pedigrees evaluated against thrips in the greenhouse revealed that thrips preferred some cultivars over others, suggesting that antixenosis “non-preference” could contribute to thrips resistance in peanut. In other crops, morphological traits such as leaf architecture and waxiness and spectral reflectance have been associated with thrips non-preference. It is not clear if foliar morphological traits in peanut are associated with reduced preference or non-preference of thrips and need to be evaluated. Besides thrips non-preference, thrips larval survival to adulthood and median developmental time were negatively affected in some peanut cultivars and in a diploid peanut species Arachis diogoi (Hoehne) and its hybrids with a Virginia type cultivar, indicating that antibiosis (negative effects on biology) could also be a factor influencing thrips resistance in peanut. Available field resistance to orthotospoviruses in peanut is not complete, and cultivars can suffer substantial yield loss under high thrips and virus pressure. Integrating thrips resistance with available virus resistance would be ideal to limit losses. A discussion of modern technologies such as transgenic resistance, marker assisted selection and RNA interference, and future directions that could be undertaken to integrate resistance to thrips and to orthotospoviruses in peanut cultivars is included in this article

Induction of Plant Resistance in Tobacco (Nicotiana tabacum) against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco (Nicotiana tabacum) plants against TSWV. Double-stranded RNA (dsRNA) molecules for the NSs (silencing suppressor) and N (nucleoprotein) genes were produced by a two-step PCR approach followed by in vitro transcription. When topically applied to tobacco leaves, both molecules elicited a resistance response. Host response to the treatments was measured by determining the time to symptom expression, and the level of resistance by absolute quantification of the virus. We also show the systemic movement of dsRNA_N from the inoculated leaves to younger, non-inoculated leaves. Post-application, viral siRNAs were detected for up to nine days in inoculated leaves and up to six days in non-inoculated leaves. The topical application of dsRNAs to induce RNAi represents an environmentally safe and efficient way to manage TSWV in tobacco crops and could be applicable to other TSWV-susceptible crops

Comparative Study of Phosphorous-Acid-Containing Products for Managing Phytophthora Blight of Bell Pepper

Phytophthora blight of pepper caused by Phytophthora capsici is a major constraint to bell pepper (Capsicum annuum) production. The long-term effectiveness of chemicals currently in use against P. capsici is uncertain due to the development of fungicide resistance by this pathogen. Hence, the efficacy of alternative chemicals such as phosphorous-acid-containing products was evaluated in this study. In in vitro tests, ProPhyt, K-Phite, Lexx-A-Phos, Agri-Fos, and Nutri-Phite were less effective in inhibiting mycelial growth (EC50 = 50.5 to 324.4 µg mL−1) and sporangium formation (EC50 = 6.1 to 225.7 µg mL−1) of two P. capsici isolates, but more effective against zoospore germination compared with mefenoxam. Among phosphorous-acid-containing products tested, Nutri-Phite was most effective in inhibiting mycelial growth of both P. capsici isolates. In greenhouse studies, Nutri-Phite was effective against Phytophthora blight used as drench. The use of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could induce systemic resistance against foliar blight when applied to the root and potting mix. The results indicated that some phosphorous-acid-containing products have the potential to lower disease occurrence and delay Phytophthora blight of bell pepper without phytotoxic effects. The utility of the systemic protection induced by these products is promising in Phytophthora blight management

Comparative Study of Phosphorous-Acid-Containing Products for Managing Phytophthora Blight of Bell Pepper

Phytophthora blight of pepper caused by Phytophthora capsici is a major constraint to bell pepper (Capsicum annuum) production. The long-term effectiveness of chemicals currently in use against P. capsici is uncertain due to the development of fungicide resistance by this pathogen. Hence, the efficacy of alternative chemicals such as phosphorous-acid-containing products was evaluated in this study. In in vitro tests, ProPhyt, K-Phite, Lexx-A-Phos, Agri-Fos, and Nutri-Phite were less effective in inhibiting mycelial growth (EC50 = 50.5 to 324.4 &micro;g mL&minus;1) and sporangium formation (EC50 = 6.1 to 225.7 &micro;g mL&minus;1) of two P. capsici isolates, but more effective against zoospore germination compared with mefenoxam. Among phosphorous-acid-containing products tested, Nutri-Phite was most effective in inhibiting mycelial growth of both P. capsici isolates. In greenhouse studies, Nutri-Phite was effective against Phytophthora blight used as drench. The use of Nutri-Phite, Agri-Fos, ProPhyt, and K-Phite could induce systemic resistance against foliar blight when applied to the root and potting mix. The results indicated that some phosphorous-acid-containing products have the potential to lower disease occurrence and delay Phytophthora blight of bell pepper without phytotoxic effects. The utility of the systemic protection induced by these products is promising in Phytophthora blight management

Host plant resistance against tomato spotted wilt virus in peanut (Arachis hypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behavior and survival

Tomato spotted wilt virus (TSWV) severely affects peanut production in the southeastern United States. Breeding efforts over the last three decades resulted in the release of numerous peanut genotypes with field resistance to TSWV. The degree of field resistance in these genotypes has steadily increased over time, with recently released genotypes exhibiting a higher degree of field resistance than older genotypes. However, most new genotypes have never been evaluated in the greenhouse or laboratory against TSWV or thrips, and the mechanism of resistance is unknown. In this study, TSWV-resistant and -susceptible genotypes were subjected to TSWV mechanical inoculation. The incidence of TSWV infection was 71.7 to 87.2%. Estimation of TSWV nucleocapsid (N) gene copies did not reveal significant differences between resistant and susceptible genotypes. Parsimony and principal component analyses of N gene nucleotide sequences revealed inconsistent differences between virus isolates collected from resistant and susceptible genotypes and between old (collected in 1998) and new (2010) isolates. Amino acid sequence analyses indicated consistent differences between old and new isolates. In addition, we found evidence for overabundance of nonsynonymous substitutions. However, there was no evidence for positive selection. Purifying selection, population expansion, and differentiation seem to have influenced the TSWV populations temporally rather than positive selection induced by host resistance. Choice and no-choice tests indicated that resistant and susceptible genotypes differentially affected thrips feeding and survival. Thrips feeding and survival were suppressed on some resistant genotypes compared with susceptible genotypes. These findings reveal how TSWV resistance in peanut could influence evolution, epidemiology, and management of TSWV