Association of differentially expressed R-gene candidates with leaf spot resistance in peanut (Arachis hypogaea L.).

Early leaf spot (ELS) and late leaf spot (LLS) are major fungal diseases of peanut that can severely reduce yield and quality. Development of acceptable genetic resistance has been difficult due to a strong environmental component and many major and minor QTLs. Resistance genes (R-genes) are an important component of plant immune system and have been identified in peanut. Association of specific R-genes to leaf spot resistance will provide molecular targets for marker-assisted breeding strategies. In this study, advanced breeding lines from different pedigrees were evaluated for leaf spot resistance and 76 candidate R-genes expression study was applied to susceptible and resistant lines. Thirty-six R-genes were differentially expressed and significantly correlated with resistant lines, of which a majority are receptor like kinases (RLKs) and receptor like proteins (RLPs) that sense the presence of pathogen at the cell surface and initiate protection response. The largest group was receptor-like cytoplasmic kinases (RLCKs) VII that are involved in pattern-triggered kinase signaling resulting in the production reactive oxygen species (ROS). Four R-genes were homologous to TMV resistant protein N which has shown to confer resistance against tobacco mosaic virus (TMV). When mapped to peanut genomes, 36 R-genes were represented in most chromosomes except for A09 and B09. Low levels of gene-expression in resistant lines suggest expression is tightly controlled to balance the cost of R-gene expression to plant productively. Identification and association of R-genes involved in leaf spot resistance will facilitate genetic selection of leaf spot resistant lines with good agronomic traits.

Identification of expressed R-genes associated with leaf spot diseases in cultivated peanut.

Peanut (Arachis hypogaea L.) is an important food and oilseed crop worldwide. Yield and quality can be significantly reduced by foliar fungal diseases, such as early and late leaf spot diseases. Acceptable levels of leaf spot resistance in cultivated peanut have been elusive due to environmental interactions and the proper combination of QTLs in any particular peanut genotype. Resistance gene analogs, as potential resistance (R)-genes, have unique roles in the recognition and activation of disease resistance responses. Novel R-genes can be identified by searches for conserved domains such as nucleotide binding site, leucine rich repeat, receptor like kinase, and receptor like protein from expressed genes or through genomic sequences. Expressed R-genes represent necessary plant signals in a disease response. The goals of this research are to identify expressed R-genes from cultivated peanuts that are naturally infected by early and late spot pathogens, compare these to the closest diploid progenitors, and evaluate specific gene expression in cultivated peanuts. Putative peanut R-genes (381) were available from a public database (NCBI). Primers were designed and PCR products were sequenced. A total of 214 sequences were produced which matched to proteins with the corresponding R-gene motifs. These R-genes were mapped to the genome sequences of Arachis duranensis and Arachis ipaensis, which are the closest diploid progenitors for tetraploid cultivated peanut, A. hypogaea. Identification and association of specific gene-expression will elucidate potential disease resistance mechanism in peanut and may facilitate the selection of breeding lines with high levels of leaf spot resistance.

Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

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.

Expressed sequence tags from the red imported fire ant, Solenopsis invicta: annotation and utilization for discovery of viruses.

An expression library was created and 2304 clones sequenced from a monogyne colony of Solenopsis invicta. The primary intention of the project was to utilize homologous gene identification to facilitate discovery of viruses infecting this ant pest that could potentially be used in pest management. Additional genes were identified from the ant host and associated pathogens that serve as an important resource for studying these organisms. After assembly and removal of mitochondrial and poor quality sequences, 1054 unique sequences were yielded and deposited into the GenBank database under Accession Nos. EH412746 through EH413799. At least nine expressed sequence tags (ESTs) were identified as possessing microsatellite motifs and 15 ESTs exhibited significant homology with microsporidian genes. These sequences most likely originated from Thelohania solenopsae, a well-characterized microsporidian that infects S. invicta. Six ESTs exhibited significant homology with single-stranded RNA viruses (3B4, 3F6, 11F1, 12G12, 14D5, and 24C10). Subsequent analysis of these putative viral ESTs revealed that 3B4 was most likely a ribosomal gene of S. invicta, 11F1 was a single-stranded RNA (ssRNA) virus contaminant introduced into the colony from the cricket food source, 12G12 appeared to be a plant-infecting tenuivirus also introduced into the colony as a field contaminant, and 3F6, 14D5, and 24C10 were all from a unique ssRNA virus found to infect S. invicta. The sequencing project illustrates the utility of this method for discovery of viruses and pathogens that may otherwise go undiscovered.

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. RESULTS: Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. CONCLUSION: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut.

Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins.

Development and Utilization of InDel Markers to Identify Peanut (Arachis hypogaea) Disease Resistance.

Peanut diseases, such as leaf spot and spotted wilt caused by Tomato Spotted Wilt Virus, can significantly reduce yield and quality. Application of marker assisted plant breeding requires the development and validation of different types of DNA molecular markers. Nearly 10,000 SSR-based molecular markers have been identified by various research groups around the world, but less than 14.5% showed polymorphism in peanut and only 6.4% have been mapped. Low levels of polymorphism limit the application of marker assisted selection (MAS) in peanut breeding programs. Insertion/deletion (InDel) markers have been reported to be more polymorphic than SSRs in some crops. The goals of this study were to identify novel InDel markers and to evaluate the potential use in peanut breeding. Forty-eight InDel markers were developed from conserved sequences of functional genes and tested in a diverse panel of 118 accessions covering six botanical types of cultivated peanut, of which 104 were from the U.S. mini-core. Results showed that 16 InDel markers were polymorphic with polymorphic information content (PIC) among InDels ranged from 0.017 to 0.660. With respect to botanical types, PICs varied from 0.176 for fastigiata var., 0.181 for hypogaea var., 0.306 for vulgaris var., 0.534 for aequatoriana var., 0.556 for peruviana var., to 0.660 for hirsuta var., implying that aequatoriana var., peruviana var., and hirsuta var. have higher genetic diversity than the other types and provide a basis for gene functional studies. Single marker analysis was conducted to associate specific marker to disease resistant traits. Five InDels from functional genes were identified to be significantly correlated to tomato spotted wilt virus (TSWV) infection and leaf spot, and these novel markers will be utilized to identify disease resistant genotype in breeding populations.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p ≤ 0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng · g(-1) of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng · g(-1). This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

Putative protease inhibitor gene discovery and transcript profiling during fruit development and leaf damage in grapefruit (Citrus paradisi Macf.).

Seven putative protease inhibitor (PPI) cDNAs, representing four protein families, were isolated from a grapefruit (Citrus paradisi Macf. Cv. Marsh) immature fruit flavedo cDNA library. Cloned open reading frames encoded proteins with similarity to, and protein signatures for: legume Kuntiz inhibitors (lkiL-1, lkiL-2, lkiL-3), potato trypsin inhibitor I (ptiIL-1), serpins (serpL-1), cystatins (cystL-1), and gamma thionins (gthL-1). Response of transcript abundance to fruit development and leaf wounding was determined for all but lkiL-1 using real-time RT-PCR. Immature leaves had the highest transcript levels for all PPIs. The gthL-1 transcript in immature leaves was the most abundant transcript but was absent from healthy mature leaves. In fruit flavedo, transcripts for all PPIs were most abundant in youngest fruit (<15 mm dia. fruit), and declined during development, but displayed different patterns of developmental change. Mechanical or Diaprepes root weevil (DRW) feeding damage to leaves caused a <10-fold reduction or had no effect on transcript level with the exception of gthL-1 which, as a result of damage, increased >50-fold in mature leaves and decreased >1400-fold in immature leaves. This developmental control of transcript response to wounding in a woody perennial is opposite of what has been observed for defensive proteinase inhibitors (PIs) in other plants (typically herbaceous and/or annual plants), where younger leaves typically invoke a higher defensive proteinase inhibitor transcript accumulation than older tissues. Except for gthL-1, the PPI transcripts were minimally responsive or unresponsive to wounding. Changes in PPI transcript levels suggest diverse roles for the products of these genes in citrus, with only gthL-1 responding in a defense-like manner.

Evaluation of five peanut (Arachis hypogaea) genotypes to identify drought responsive mechanisms utilising candidate-gene approach.

Drought can significantly limit yield and quality in peanut (Arachis hypogaea L.), depending on its timing, duration and severity. The objective of this study was to identify potential molecular mechanism(s) utilising a candidate-gene approach in five peanut genotypes with contrasting drought responses. An early season drought stress treatment was applied under environmentally controlled rain-out shelters. When water was completely withheld for 3 weeks, no physical differences were observed for treated plants compared with their fully irrigated counterparts as indicated by relative water content; however, yield, grades (total sound mature kernel, TSMK), specific leaf area, and leaf dry matter content showed significant differences. Comparing expression levels of candidate genes, 'C76-16' exhibited significantly higher levels for CuZnSOD, NsLTP and drought protein 1 week earlier compared to the other genotypes, followed by significantly lower levels for the same genes. This suggested an early recognition of drought in C76-16 followed by an acclimation response. Cultivar 'Georgia Green' showed different patterns of gene-expression than C76-16. AP-3, a susceptible genotype, showed generally lower levels of gene-expression than C76-16 and Georgia Green. Myo-inositol phosphate synthase gene-expression showed high levels in irrigated treatment, ranging from 4-fold for 08T-12 to 12-fold for Georgia Green, but were significantly inhibited in drought treatment after 2 weeks of drought and after recovery.

Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization.

We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5' proximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3' proximal ORF2 (nucleotides 4390-7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st-2nd) and late (3rd-4th) instars, worker pupae, workers, sexual pupae, alates ( male symbol and female symbol ), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavated from the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.