2008 Annual Report 1a.Objectives (from AD-416) Identify biomarkers of high quality cotton grown under critical temperature and drought conditions; describe fundamental functional genomic mechanisms of cellulose synthesis of cotton grown under stress; and define genetic pathways that can be targeted for reniform nematode control. 1b.Approach (from AD-416) Identify differentially-expressed mRNA and proteins in mature cotton leaves, fibers in elongation phase, fibers in deposition phase, and pollen and ovules in plants grown under critical temperature and drought conditions using nucleotide microarray and high-throughput expression proteomics technologies; identify key regulatory nodes in genetic networks, under temperature and drought stress conditions by combining microarray and expression proteomics data with computational systems-biology network modeling; and physically map and sequence the reniform nematode genome using ultra-high-throughput genome sequencing. 3.Progress Report This project has 3 aims:. 1)identify high-quality-cotton biomarkers;. 2)identify genetic mechanisms of cotton fiber quality under climate stress; and. 3)define genetic pathways that can be selectively targeted for reniform nematode control. Aims 1 and 2 are being accomplished together. Cotton, cultivar TM-1, plants were grown from seed in the Soil-Plant-Atmosphere Research facility at optimum conditions for vegetative and reproductive growth. A combination of drought and heat stress was then imposed at mid-fruiting. Fiber samples were collected for different age class bolls of 10, 15, 20, 25 and 35 d after anthesis from the plants to be compared by microarray and expression proteomics with plants that were not stressed. Due to the high content of cellulose and phenolic compounds, protein isolation from cotton fiber, particularly in the late developmental stage, is highly challenging, but we have established a novel, highly efficient phenol extraction method for protein isolation from cotton fibers at different developmental stages. We have developed a novel nano spray-LC-MS/MS and label-free quantification for cotton proteomics and have completed expression proteomics of cotton fiber proteome at different developmental stages, including elongation, transition, and deposition stage. To effectively reveal the gene expression profile in the cotton fiber of different developmental stages, we have established a method to isolate the RNA samples from cotton fiber efficiently. Our RNA samples are ready for Solexa or 454 sequencing. For aim 3, reniform nematode eggs were harvested and utilized to construct a DNA bacterial artificial chromosome (BAC) library of 21,504 individually-archived clones, affording 32-fold coverage of the nematode’s genome. Inserts from 6912 BACs (10.4-fold genome coverage) were isolated and each was sheared into 1.5 kb fragments; 3456 oligonucleotide adaptors (each possessing a 454 Technologies bead attachment sequence and a novel 6 bp “barcode”) were designed. Adaptors are currently being synthesized by IDT Technologies. For each fragmented BAC, one barcode adaptor will be ligated to the 3’ end of each of its fragments. Two pools, each of 3456 uniquely barcoded clones, will be sequenced using a 454 FLX DNA Analyzer (unidirectional sequencing, 1 Gb per run) to give 4X coverage/BAC. All sequences from a given run sharing a barcode (i.e., pieces of the same BAC) will be grouped and assembled. ADODR used site visit and telephone conferences to monitor activities of the project.