ESAP plus: a web-based server for EST-SSR marker development.

BACKGROUND: Simple sequence repeats (SSRs) have become widely used as molecular markers in plant genetic studies due to their abundance, high allelic variation at each locus and simplicity to analyze using conventional PCR amplification. To study plants with unknown genome sequence, SSR markers from Expressed Sequence Tags (ESTs), which can be obtained from the plant mRNA (converted to cDNA), must be utilized. With the advent of high-throughput sequencing technology, huge EST sequence data have been generated and are now accessible from many public databases. However, SSR marker identification from a large in-house or public EST collection requires a computational pipeline that makes use of several standard bioinformatic tools to design high quality EST-SSR primers. Some of these computational tools are not users friendly and must be tightly integrated with reference genomic databases. RESULTS: A web-based bioinformatic pipeline, called EST Analysis Pipeline Plus (ESAP Plus), was constructed for assisting researchers to develop SSR markers from a large EST collection. ESAP Plus incorporates several bioinformatic scripts and some useful standard software tools necessary for the four main procedures of EST-SSR marker development, namely 1) pre-processing, 2) clustering and assembly, 3) SSR mining and 4) SSR primer design. The proposed pipeline also provides two alternative steps for reducing EST redundancy and identifying SSR loci. Using public sugarcane ESTs, ESAP Plus automatically executed the aforementioned computational pipeline via a simple web user interface, which was implemented using standard PHP, HTML, CSS and Java scripts. With ESAP Plus, users can upload raw EST data and choose various filtering options and parameters to analyze each of the four main procedures through this web interface. All input EST data and their predicted SSR results will be stored in the ESAP Plus MySQL database. Users will be notified via e-mail when the automatic process is completed and they can download all the results through the web interface. CONCLUSIONS: ESAP Plus is a comprehensive and convenient web-based bioinformatic tool for SSR marker development. ESAP Plus offers all necessary EST-SSR development processes with various adjustable options that users can easily use to identify SSR markers from a large EST collection. With familiar web interface, users can upload the raw EST using the data submission page and visualize/download the corresponding EST-SSR information from within ESAP Plus. ESAP Plus can handle considerably large EST datasets. This EST-SSR discovery tool can be accessed directly from: http://gbp.kku.ac.th/esap_plus/ .

Changes in Protein Expression in Peanut Leaves in the Response to Progressive Water Stress.

This investigation was carried out at the Department of Plant Science and Agricultural Resources, Khon Kaen University in the rainy season of 2011. The objective of this study was to identify proteins in leaves of drought-susceptible peanut plants when regulated by progressive water stresses. The drought-susceptible peanut plants of Khon Kaen 4 cultivar were grown in pots under controlled environment. At day 30 after seed emergence, the plants were subjected to stress conditions for 5 and 6 days. The results showed that withheld water supply for 5 and 6 days gave moderately and severely water stresses, respectively. Under moderate water stress conditions, two up-regulated and eight down-regulated proteins were attained. The up-regulated proteins were striated fibre assembling and flap endonuclease 1. The down-regulated proteins were peptidyl-prolyl cis-trans isomerase FKBP4, tRNA(Ile)-lysidine synthase, chloroplastic, chloroplastic thioredoxin F-type, cytidinedeaminase 7, ALF domain class transcription factor, nudix hydrolase 8, pentatricopeptide repeat super-family protein, putative and ribulose-1,5-bisphosphate carboxylase/oxygenase, a large sub-unit. Under severe water stress conditions, two proteins, i.e., tRNA(Ile) lysidine synthase, chloroplastic and ALF domain class transcription factor did not change their relative abundance significantly where it indicated drought acclimation. The remaining proteins displayed significant changes and the changes were the same as those found in the peanut leaves when deprived water for 5 days. Up-regulated proteins are responsible for alleviating oxidative damages to plant genome and mediating plants responsive to the environmental factors in providing mechanical support, barriers and a rapid transport route. Down-regulated proteins were associated with drought susceptibility of the Khon Kaen 4 peanut plants.

Isolation and identification of peanut leaf proteins regulated by water stress.

Water deficits trigger signaling cascades leading to modulation of protein expression in plant tissues. Identification of peanut leaf proteins regulated by water stress provides some insights of cellular and molecular response of peanut plants to drought stress. Peanut variety Khon Kaen 4, a water-stress sensitive variety, was grown in a growth chamber under controlled environment. Water stress was imposed on day 30 after seedling emergence by withholding watering peanut plants for 6 days as compared to plants adequately supplied with water. Total protein were prepared from a leaflet of fully expanded leaf on the main stem. Proteins were separated in duplicated gels using two-dimensional gel electrophoresis and visualized by silver nitrate staining. Image analysis was performed using ImageMaster 2D Platinum 5.0 to determine proteins regulated by water stress. Molecular mass and isoelectric point of each regulated protein were used in database queries for protein identification. One protein was induced under water stress and the homologous protein was identified as Serine/threonine-protein phosphatase PP 1. Five proteins were down-regulated by water deficit. The homologous proteins were chaperone protein DNAJ, auxin-responsive protein IAA29, peroxidase 43, caffeoyl-CoA O-methyltransferase and SNF1-related protein kinase regulatory subunit beta-2. Down-regulated proteins may be associated with sensitivity of the peanut variety to water stress.