Patricia Klein - US grants

The long term goal of this research is to elucidate the molecular mechanisms that regulate fruit abscission in higher plants. It has been documented that cellulase (beta, 1:4 glucan glucanhydrolase) increases in the abscission zone of Phaseolus vulgaris during leaf fracture. It is also known that this increase in cellulase activity can be accelerated by the addition of exogenous ethylene. A cDNA clone, complementary to bean cellulase mRNA has been recently isolated and messenger RNA complementary to it has been shown to increase dramatically in the abscission zone several hours after ethylene treatment of bean. The kinetics suggest that other cellular events precede cellulase appearance in signalling abscission. One approach to identifying the mechanism through which ethylene promotes fruit abscission is to examine those genes which respond rapidly to externally applied ethylene. Dr. Klein proposes here to isolate and characterize ethylene responsive genes that are rapidly induced in the abscission zone and examine their expression during fruit abscission in tomato.

The long term goal of this project is to identify, characterize, and understand the function of genes and naturally occurring allelic variants present in the sorghum germplasm collection that contribute to plant adaptation to the environment. The research team will map and sequence cDNA tags (short DNA sequences copied from mRNA) encoded by several gene dense regions of the sorghum genome using 'direct selection' technology and identify sorghum abiotic response genes by sequence similarity and cross species 'direct selection'. This research will further develop direct selection technology for gene mapping, physical map construction, and cross species analysis.

The research team is constructing an integrated genetic, physical, and gene content map of the sorghum genome using a novel approach based on multi-dimensional pooling of Bacteria Artificial Chromosome (BAC) DNA, Amplified Fragment Length Polymorphism (AFLP) technology, and DNA fingerprinting. A high throughput 'direct selection' gene mapping technology has been developed to map genes across the gene-dense portions of the sorghum genome. This activity will provide valuable information about gene order in grasses, identify candidate genes in trait loci, help construct integrated genetic and physical maps, and create a deep collection of gene tags for microarrays.

Initial NSF PGRP funding has permitted the development of a public sorghum unigene resource that will approach 14,000 members during the coming year. This renewal proposal is designed to augment the size, and thus value, of this resource while simultaneously using it to explore the genomic/genetic/biochemical basis of sorghum's special adaptation to adverse environments. The results of this research effort will reveal potential strategies for improvement of the productivity of not only this, but other, cereals that do not have sorghum's ability to withstand environmental extremes such as drought. Fusion of the differing strengths of two research groups will lead to a synergistic outcome. The University of Georgia (UGA) group has developed substantial expertise in construction of a comprehensive unigene resource and in development and mining of a secure and versatile Oracle-based relational database management system (RDBMS). The Texas A & M University and USDA/ARS group has extensive experience with the experimental system (sorghum), its physiology, genetics, and genome characteristics, and has recently developed a powerful new tool for identifying on a combined physical/genetic sorghum map the location of many of the ESTs that already have been, and will continue to be, developed at UGA.
We propose to increase the sorghum unigene set to ~20,000 members by selective sequencing of 50,000 cDNAs from ~20 new libraries. Libraries will be enriched in full-length cDNAs through a no-cost collaboration with Dr. Sumio Sugano. The UGA RDBMS will be enhanced so that we can mine 5' and 3' UTRs to explore their participation in regulation of genome expression in this monocot. The RDBMS has already been modified to incorporate all returned BLAST data in readily mined tables. We will use this information to investigate as well the potential role of alternative splicing in regulation of genome expression. The unigene set will be maintained and distributed as a public resource, and the UGA web site will be enhanced to make this information available to the scientific community. Data will also be made available to the developing Gramene database resource.
Sorghum genome function will be explored by coordinated microarray experiments designed to permit the construction of a compendium of gene expression profiles. While conducting assays that will answer specific biological questions, we will simultaneously design experimental protocols with this objective in mind. Specific immediate objectives include examination of well-characterized genotypes to explore relationships between genome expression, light perception and flowering time and to characterize pre- and post-flowering responses (drought tolerance) in contrasting genotypes. The UGA RDBMS will be expanded to include all relevant information, not only array data, but also experimental parameters, phenotype, and so on. Microarray experiments will be designed around a common core set of parameters such that variations in abiotic factors within a single genotype or between different genotypes and a standard reference genotype can inasmuch as possible be related always to this core. Such experimental design will permit the development of a true compendium that will be available for extensive mining beginning during this project period, but also extending well beyond it.

Deliverables

1. sorghum ESTs

ESTs are available from GenBank (dbEST) and from http://fungen.botany.uga.edu/Projects/Sorghum/Sorghum.htm

ESTs have been mapped to the rice genome at http://www.gramene.org

2. sorghum unigene set

information about sorghum unigenes is available at http://fungen.botany.uga.edu/Projects/Sorghum/Sorghum.htm

unigenes have been mapped to the rice genome at http://www.gramene.org

3. sorghum cDNA clones

clones are available by directing email to Dr. Marie-Michele Cordonnier-Pratt at mmpratt@uga.edu (full instructions are at http://fungen.botany.uga.edu/Projects/Sorghum/Sorghum.htm

Cereals provide seventy percent of the world's calories and water is the most limiting natural resource for grain production worldwide. Sorghum, a grass with its origin in Africa, is the fifth most important cereal worldwide. Sorghum has evolved characteristics that permit grain production in hot dry environments (i.e., thick leaf wax, deep root system) and that facilitate continued growth when water availability is limited. Sorghum is also similar in gene sequence to other important cereals such as rice, corn, and wheat and has a relatively small genome (only twice the size of rice and 20-fold smaller than wheat). Some sorghum genotypes show distinct drought-tolerance. Of particular interest are plants that continue to grow when experiencing water limitation, thus showing a 'stay-green' phenotype. A genomics approach will allow scientists to understand the network of genes that form the basis to stay green traits. To accomplish this, genomic resources will be further developed in Sorghum. The TAMU-ARS sorghum genetic and physical map, already established, will be further developed and will be aligned to the rice genome sequence. Sorghum chromosomes will be characterized and a virus-induced gene silencing system (VIGS/RNAi) will be established. Breeders are developing the genetic populations that permit precise chromosomal localization of drought tolerance genes, which is essential for candidate gene identification. All these genetic tools will be used to fine map, isolate, and characterize this network of genes that control expression of the sorghum stay-green trait that is central to grain production in water limited environments.

Information will be made publicly available through scientific and popular publications, presentations at meetings, and through collaboration with the groups developing Gramene, a national relational database for grasses (http://www.gramene.org) and on a local project web site (http://SorghumGenome.tamu.edu). The complete sequence of several sorghum BACs, BAC sequence and other sequence data will be submitted to GenBank and DNA sequence data will be provided to the curators of Gramene. The information and technology generated will help public and private cereal breeders to improve US crop drought tolerance and productivity and will facilitate the transfer of the 'stay green' trait to other cereal species that lack sorghum's tolerance to drought.

During the past 10 years, there has been a major revolution in the biological/life sciences as a result of the remarkable development and subsequent generation of a vast quantity of data from genome sequencing efforts. Genome sequencing is a process that determines the identity and order of an organism's DNA; thus providing a blueprint for the genetic code underlying all of the processes of life. This has been highlighted by the recently completed draft sequence of the human genome, a notable technical achievement made possible by the development of powerful computational hardware and software. The availability of complete genome sequences is providing the foundation for understanding how organisms (from microbes to humans) function, develop, evolve and interact with pathogens and assorted environmental cues. This has led to a transformational change in the field of biology stemming from the development of next-generation ultra high-throughput, ultra low-cost DNA sequencing technology ( e.g. Roche 454), which serves as the basis for this proposal. What used to take several months, can now be done in several hours. The Roche 454 FLX will be used by more than 60 faculty members and their research staff, including post-doctoral fellows, graduate and undergraduate students at Texas A&M University. Applications and research programs include: dissection of sorghum drought tolerance, functional and metagenomic analysis of biological communities for cattle and rumen bacteria, disease resistance and an analysis of microflora in ocean drilling programs, among others. Dissemination of data and project outcomes will be available on the Institute for Plant Genomics and Biotechnology website (http://ipgb.tamu.edu), research publications, relevant scientific meetings and during our scheduled campus-wide annual meeting.