The Structure and Function of the Expressed Portion of the Wheat Genomes

1. The Structure and Function of the Expressed Portion of the Wheat Genomes

2. The Structure and Function of the Expressed Portion of the Wheat Genomes The long term goal of this project is to decipher the chromosomal location and biological function of all genes in the wheat genomes.

3. Worldwide, wheat (bread and durum) is the most widely grown crop. With its high protein content, wheat is the single most important source of plant protein in the human diet. In the USA, wheat is the fourth most economically significant crop and the leading export crop. Wheat products: seed flourbeverageglutenstarch Value and uses of wheat The Structure and Function of the Expressed Portion of the Wheat Genomes

5. Cal Qualset, U Calif DANR, Davis James Anderson, U Minnesota Olin Anderson, U Calif DANR, Albany Timothy Close, U Calif Riverside Jorge Dubcovsky, U Calif Davis Jan Dvorak, U Calif Davis Bikram Gill, Kansas State U Kulvinder Gill, U Nebraska J Perry Gustafson, U Missouri Shahryar Kianian, N Dakota State U Nora Lapitan, Colorado State U Henry Nguyen, Texas Tech U Mark Sorrells, Cornell U M Kay Walker-Simmons, Washington State U

6. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy.

7. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy. 2. Determine the base-pair sequence of these cDNAs, yielding ESTs. Carry out 5’end sequencing of cDNA libraries, with 3’ sequencing of putative singletons.

8. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy. 2. Determine the base-pair sequence of these cDNAs, yielding ESTs. Carry out 5’end sequencing of cDNA libraries, with 3’ sequencing of putative singletons. 3. Map into wheat deletion stocks a set of 10,000 unique ESTs. Map ESTs into bins defined by wheat deletion stocks; target is 10,000 mapped ESTs.

9. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy. 2. Determine the base-pair sequence of these cDNAs, yielding ESTs. Carry out 5’end sequencing of cDNA libraries, with 3’ sequencing of putative singletons. 3. Map into wheat deletion stocks a set of 10,000 unique ESTs. Map ESTs into bins defined by wheat deletion stocks; target is 10,000 mapped ESTs. 4. Determine the expression of the mapped ESTs relative to reproductive biology of wheat. Using arrays, analyze the expression of the mapped ESTs, focusing on five aspects of wheat reproduction.

10. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy. 2. Determine the base-pair sequence of these cDNAs, yielding ESTs. Carry out 5’end sequencing of cDNA libraries, with 3’ sequencing of putative singletons. 3. Map into wheat deletion stocks a set of 10,000 unique ESTs. Map ESTs into bins defined by wheat deletion stocks; target is 10,000 mapped ESTs. 4. Determine the expression of the mapped ESTs relative to reproductive biology of wheat. Using arrays, analyze the expression of the mapped ESTs, focusing on five aspects of wheat reproduction. 5. Process, analyze, and display data accumulated in this project (bioinformatics). Develop and enhance means to analyze, interpret, and visualize project data (data processing, database modifications, and web page maintenance).

11. Objectives and Experimental Approach 1. Produce cDNA libraries from as many tissue and condition combinations as possible. Produce multiple cDNA libraries with a target of 30 total after quality testing, normalizing and subtraction to reduce redundancy. 2. Determine the base-pair sequence of these cDNAs, yielding ESTs. Carry out 5’end sequencing of cDNA libraries, with 3’ sequencing of putative singletons. 3. Map into wheat deletion stocks a set of 10,000 unique ESTs. Map ESTs into bins defined by wheat deletion stocks; target is 10,000 mapped ESTs. 4. Determine the expression of the mapped ESTs relative to reproductive biology of wheat. Using arrays, analyze the expression of the mapped ESTs, focusing on five aspects of wheat reproduction. 5. Process, analyze, and display data accumulated in this project (bioinformatics). Develop and enhance means to analyze, interpret, and visualize project data (data processing, database modifications, and web page maintenance). 6. Analyze gene density and distribution of mapped ESTs and thus genes in the wheat genomes (genome structure and evolution). Analyze densities and distribution of related genes determined from deletion map locations combined with functionality.

12. EST Arrays DeletionMapping SAGE ComparativeMapping SequenceMatching Obj. 5. Bioinformatics Obj. 4.FunctionalGenomics Obj. 3.Mapping Objs. 1 & 2.EST Production cDNA librariesScreening/normalizationsSequencingData analysisDNA storage/distribution Obj. 6.GenomeStructure &Evolution The Structure and Function of the Expressed Portion of the Wheat Genomes

13. EST Arrays DeletionMapping SAGE ComparativeMapping SequenceMatching Project Coordinator: Calvin QualsetProject Manager: Patrick McGuire Objectives 1 and 2.EST ProductionCoordinator:Olin Anderson Objective 5.BioinformaticsCoordinator:Olin Anderson Obj. 5. Bioinformatics Obj. 4.FunctionalGenomics Obj. 3.Mapping Objs. 1 & 2.EST Production cDNA librariesScreening/normalizationsSequencingData analysisDNA storage/distribution Objective 3.MappingCoordinator:Bikram Gill Objective 4.Functional GenomicsCoordinator:Mark Sorrells Obj. 6.GenomeStructure &Evolution Objective 6.Genome Structure & EvolutionCoordinator:Jan Dvorák The Structure and Function of the Expressed Portion of the Wheat Genomes

14. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available.

15. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B If probe X produces a signal in this stock, ... In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available.

16. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B If probe X produces a signal in this stock, ... … and in this stock, ... In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available.

17. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B If probe X produces a signal in this stock, ... … and in this stock, ... … but not in this stock, then ... In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available.

18. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B If probe X produces a signal in this stock, ... … one concludes that the DNA sequence in this chromosome corresponding to the EST represented by probe X is physically located in this region or ‘bin’. … and in this stock, ... … but not in this stock, then ... In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available.

19. 6B #1 #2 #3 Use of chromosome deletion stocksExample: chromosome 6B If probe X produces a signal in this stock, ... … one concludes that the DNA sequence in this chromosome corresponding to the EST represented by probe X is physically located in this region or ‘bin’. … and in this stock, ... … but not in this stock, then ... In this physical model of the chromosome, the dark areas are the heterochromatic bands which serve as landmark patterns identifying this chromosome uniquely. The arrows indicate breakpoints of the deletion stocks available. Cumulatively, these deletion stocks define ‘bins’ of various sizes in the physical chromosomes. The full 10,000 ESTs developed in this project will be mapped into these bins.

20. Meiosis Flowering Signals Seed Development Pollen Development Dormancy & Germination Functional Genomics: five aspects of wheat reproduction The Structure and Function of the Expressed Portion of the Wheat Genomes

21. 30 cDNA libraries, normalized and enhanced for abundance of different sequences Sequence database for 10,000 singleton ESTs for bread wheat Physical bin map of the singleton ESTs in the wheat genomes Arrays of the mapped singleton ESTs for gene discovery efforts Expression profiles for wheat genes during five stages of reproduction: flowering initiation; meiosis; pollen development and nucleo-cytoplasmic interactions; seed development; and seed dormancy/germination Informatics sites and software for sequence analyses, comparisons, and display and for differential display analyses of probed arrays A minimum of 13 young professionals with training and experience with tools and technology of functional genomics A nucleus of 13 public labs equipped, experienced, and networked for gene discovery and deployment and training with wheat or other species Deliverables

22. Triticum aestivum L., tribe Triticeae, family Poaceae Allohexaploid: six component genomes, AABBDD, 2n=6x=42 Bread wheat originated from two hybridization events, the first between two diploid species producing an AABB genome tetraploid, the second between the tetraploid species and another diploid species Closest relatives to bread wheat in the tribe:the crops durum wheat (tetraploid, AABB), einkorn wheat (diploid, AA), barley (HH), rye (RR), and triticale (AABBRR), numerous weedy annual species of Aegilops and Triticum, and numerous forage species Diploid species are the sources of the wheat genomes: A from Triticum urartu B from a primitive form of Aegilops speltoides or an extinct close relative D from Aegilops tauschii The molecular genetic map has some 2000 markers. Each chromosome pair has a unique pattern of heterochromatic bands allowing unequivocal cytological identification Wheat tolerates aneuploidy and a huge library of aneuploid stocks exists in many different genotypes. Monosomics Deletions Ditelosomics Translocations Nullisomics-tetrasomics Alien introgression stocks Aneuploids have been critical in gene discovery, gene mapping, chromosome manipulation, and gene transfer in classical cytogenetics and breeding. Coupled with molecular technology, the aneuploids offer tools for greater progress in all these areas plus gene cloning, transformation, and physical mapping. Aneuploids have facilitated the understanding of syntenic relationships among thee three genomes: seven homoeologous groups have been defined, each with a chromosome from each genome. The Structure and Function of the Expressed Portion of the Wheat Genomes Biology of bread wheat