Abstract

Media summary

Key Words

Arachis hypogaea, cultivated peanut, resistance gene, drought tolerance, EST/functional genomics.

Introduction

Expressed sequence tag (EST) libraries and databases have proven to be powerful tools for gene discovery, gene mapping and for the analysis of quantitative traits. ESTs are generated by large-scale sequencing of randomly picked clones from cDNA libraries constructed from mRNA isolated at a particular development stage and/or tissue. Peanut (Arachis hypogaea L.) is an important crop internationally for both direct human food and oil production. Industry and consumers have emphasized the food quality of peanut, however, peanuts are susceptible to Aspergillus infection, which can result in aflatoxin contamination (Holbrook et al. 1994). Peanuts also have proteins that result in allergic reactions (Li et al. 2000). Improvement of insect resistance, drought tolerance, oil quality and flavor are also great challenges for breeding programs (Holbrook and Stalker 2003).

Although an abundance of morphological variation within A. hypogaea is known, many agronomical traits are difficult to select using conventional selection techniques. This is in large part because most agronomically important traits in peanut are quantitatively inherited (Wynne and Coffelt 1982), and significant genotype and environmental interactions exist. Molecular genetic research with peanut has mainly focused on molecular marker-assisted selection (Stalker and Mozingo 2001) and genetic transformation (Ozias-Akins and Gill 2001). Molecular markers are useful for crop improvement and studies of crop evolution in many species (Mohan et al. 1997). Unfortunately, little variation has been observed using molecular markers in A. hypogaea (Stalker and Mozingo 2001), resulting in limited application. A reliable regeneration and transformation system in peanut has been established, and several genes for insect and virus resistance have been successfully introduced into peanut (Ozias-Akins and Gill 2001). Although the application of transgenic technologies has enormous potential for enhancing trait improvement, the critical component needed for peanut cultivar development is identification of agronomically useful genes. Functional genomics research may aid in the development of polymorphic molecular markers (He et al. 2003), and provide useable genes for transgenic research.

The National Center for Biotechnology Information’s (GenBank) dbEST database contains (June 25, 2004) 22,165,266 ESTs (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). Human ESTs are 5,643,076, with 4,191,008 ESTs for mouse and rat. Among plants, Triticum aestivum (wheat) has the most ESTs deposited with NCBI, at 552,245, and Zea mays (maize) second at 397,515. Glycine max (soybean) has 334,668, Oryza sativa (rice) has 284,006, Arabidopsis thaliana has 258,825, and Medicago truncatula has 187,763. Peanut (Arachis hypogaea) is pathetically low on the list for such an important crop, with only 1,366 ESTs listed, in which we released 1,345 in May 2003. There is great interest in the ESTs from peanut research community, U.S. Industries and USDA. At the Peanut Genomics Research Workshop held in Atlanta, Georgia, March 23, 2004, a Strategic Plan for Peanut Genomics was developed and EST Project was on the top list. The target is to have at least 50,000 ESTs available publicly from in 2006 and 150,000 ESTs in 2008. In the present research, two peanut genotypes were used to construct cDNA libraries and generate ESTs. One genotype (C34-24) was selected for its resistance to leaf spots and TSWV, and the other genotype (A13) was selected for its drought tolerance and reduced preharvest aflatoxin contamination. Simple sequence repeat (SSR) markers have been characterized and microarray chips with 400 unigenes have been produced for gene expression analysis.

Methods

cDNA libraries construction and transformation

The peanut lines used in this research were C34-24 (leaves) and A13 (immature pots). A total of 24 genotypes were evaluated for SSR. Total RNA was isolated from leaf and pods using TRIZOL reagent and mRNA was purified from total RNA using PolyATtract mRNA isolation kit. Two cDNA libraries were constructed using mRNA from leaves or immature pods.

Plasmid DNA purification and DNA sequencing

Plasmid DNA isolated from randomly selected colonies by using 96 TurboFilter Miniprep kits and a Qiagen BioRobot 9600. Ninety-six  individual plasmids from random selected clones were used as template for PCR amplification of the cloned cDNA by using T3 and T7 universal primers. The resulting amplicons were analyzed by agarose gel electrophoresis to identify the size of inserts. The PCR products were concentrated by ethanol precipitation. Pellets were re-suspended in 15 µl of formamide and amplicons were sequenced using BigDye Terminator Cycle Sequencing Kit. Sequencing was performed on ABI Prism 3700 Sequencer.

EST processing and sequence analysis

Sequences were edited by the software SEQUENCHER^® V4.1.4 and by manual trimming to remove the vector sequence. Sequences <150 nucleotides were removed. The remaining ESTs were compared with the GenBank non-redundant protein database using BLASTX algorithm (Altschul et al. 1990). A match was considered significant when the score was higher than 100 (optimized similarity score) with E-value scores ≤ 10^-10. Novel ESTs were identified by comparison with sequences in non-redundant nucleotide and EST databases of GenBank using BLASTn algorithm. In addition, the sequences of each contig were aligned by using the fragment assembly program of SEQUENCHER^®, and consensus sequences were generated with 90% identity over a minimum of 50 nucleotides.

ESTs functional analysis, simple sequence repeat (SSR) characterization, and real-time PCR

EST putative functions were classified according to the Munich Information Center for Protein Sequences (MIPS) functional classification system applied to Arabidopsis (Mewes et al. 2002; Schoof et al. 2002). A total of 1345 ESTs from these two cDNA libraries have been submitted to GenBank database under accession numbers: CD037499 to CD038843. These sequences were searched for SSR using BLASTn software to identify di- and tri- nucleotide and some tetranucleotide SSR motifs. SSR motifs, which repeat more than seven repeats in dinucleotide, five in trinucleotide, and four in tetranucleotide were counted. Primer pairs were designed for 44 SSRs based on the number of repeats and the sequences of the flanking region using the Primer3 software (Rozen and Skaletsky 2000) (code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html). Twenty four cultivated peanut genotypes were screened for polymorphisms. Four hundred unigenes have been arrayed on glass slides for gene expression analysis. The microarray analysis data are validated by RT-PCR and real-time PCR.

Results

Quality of cDNA libraries and ESTs

Two cDNA libraries were constructed, one from bulked leaf samples of C34-24 and one from bulked immature pod samples of A13. In the leaf cDNA library, the insert size distribution ranged from 200 to 1,500 bp, with an average of 550 bp. In the immature pod cDNA library, the insert size distribution ranged from 400 to 1,500 bp, with an average of 650 bp. Sequence analysis showed 769 high quality ESTs from the leaf library and 1056 from the immature-pod library. Three hundred and ninety-one of the 769 leaf ESTs and 378 of the 1056 immature-pod ESTs were assembled to contigs, resulting 536 and 800 unique ESTs in the two libraries, respectively.

Putative function analysis of ESTs

To identify the putative function of these ESTs, the EST sequences were compared with the sequences in the UniGene (Schuler 1997; Wheeler et al. 2003) database of GenBank using a BLASTx algorithm. A nucleotide homology search indicated that 52.8% of the clones from the leaf library and 78.6% of the clones from the immature pod library matched known function genes. A further search in dbEST database of GenBank based on BLASTn algorithm indicated that 27.3% of 363 clones from the leaf library and 22.1% of 226 clones from the immature pod library with un-known function matched sequences in dbEST database.

Based on the MIPS Functional Catalogue criteria (Mewes et al. 2002; Schoof et al. 2002) and the putative functions, the EST sequences in the two cDNA libraries were further characterized by functional category sorting into 15 categories (Fig. 1). Each putative transcript was assigned to a category in the MIPS Functional Catalogue (Mewes et al. 2002). The metabolism-related genes accounted for 6.6% of the cDNA clones in the leaf library and 6.8% in the immature-pod library. In the protein synthesis category, cDNA clones encoding various ribosomal proteins and other formations from simpler components of a protein, rather than of proteins in general, were common in both libraries (Fig. 1). The cDNA clones related to cellular transport and transport mechanisms were low in both libraries (0.1%).

Figure 1. Distribution of ESTs from leaf and immature cDNA libraries among functional categories. MIPS functional categories: 1-Metabolism; 2-Energy; 3-Cell growth, division and DNA synthesis; 4-Transcription; 5-Protein synthesis; 6-Protein destination; 7-Transport facilitation; 8-Cellular transport and transport mechanisms; 9-Cellular biogenesis; 10-Cellular communication/ signal transduction mechanism; 11-Cell rescue, defense and virulence; 12-Ionic homeostasis; 13-Cellular organization; 14-Development; 15-Unclassified protein.

Genes of higher expression

Redundancy of EST sequences is a reflection of the importance of certain genes in different development stages and environment conditions. Because of the small numbers of ESTs generated from these two libraries, ESTs with redundancy of 4 and above were selected in this analysis. In the leaf library, genes related with photosynthesis were ubiquitous among the redundant genes, and 13.9% of total ESTs were coding for Rubisco (ribulose bisphosphate carboxylase). Redundant ESTs for drought-induced genes were observed in both cDNA libraries. The types of drought-induced genes were different in leaf and immature-pod cDNA libraries. The functions of several redundant ESTs from these two cDNA libraries could not be identified from the public database. Most of these ESTs with unknown function do not have homologous sequences in the GenBank and may represent genes unique to peanut.

Putative adversity tolerance genes

To identify genes that control tolerance to drought and disease resistance traits, the ESTs from these two cDNA libraries were characterized according to their putative functions related to previously reported adversity resistance genes. These genes were sorted into different catalogues and distributed among many defense-related pathways.

EST-derived SSR markers and gene discovery

In the expressed sequences there were only three types of dimeric repeat motifs [(GA)_n, (CT)_n, and (AT)_n]. Trimeric repeat motifs are more frequent than dimeric repeats. Tetrameric and hexameric repeat motifs were also found. EST-derived SSR motifs were identified, and 44 SSRs from the released 1350 ESTs were designed. Nine of the 44 primer pairs resulted in polymorphism among 24 genotypes. The number of alleles at each locus ranged from two to five. The rate of detecting polymorphism among peanut lines is higher using EST-derived SSR markers (over 20%) than SSR derived from genomic sequence of peanut (He, personal communication).

To identify novel genes, 400 unigenes were selected from these two libraries and arrayed on glass slides for gene expression analyses. Some data were validated by RT-PCR and real-time PCR (Fig. 2). The information in genomic research could help geneticists and breeders identify genes expressed in a particular tissue or under a specific condition and could aid in understanding the complexity of gene expression and regulation.

Figure 2. Quantitative expression analysis of genes with putative function of disease resistance and drought tolerance by RT-PCR (A, B, C, D). Total RNA was extracted from peanut C34-24 leaf samples of normal plants (A), drought stressed plants (B), or plants challenged by fungal leaf spots (C), and from peanut A13 immature seeds challenged by fungus Aspergillus flavus (D). Genes examined: 1-Histone H3.3 (positive internal control), 2-Allergen Ara h3/4, 3-10kDa protein, 4-Leucine-rich protein, 5-Bax inhibitor, 6-Heat shock cognate protein, 7-Non-specific lipid transfer protein, 8-drought inducible 22 kDa protein, 9-drought inducible RPR 10, 10-Defensin protein.

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