Academic journal article Genetics

Development of an Expressed Sequence Tag (EST) Resource for Wheat (Triticum Aestivum L.): EST Generation, Unigene Analysis, Probe Selection and Bioinformatics for a 16,000-Locus Bin-Delineated Map

Academic journal article Genetics

Development of an Expressed Sequence Tag (EST) Resource for Wheat (Triticum Aestivum L.): EST Generation, Unigene Analysis, Probe Selection and Bioinformatics for a 16,000-Locus Bin-Delineated Map

Article excerpt

ABSTRACT

This report describes the rationale, approaches, organization, and resource development leading to a large-scale deletion bin map of the hexaploid (2n = 6x = 42) wheat genome (Triticum aestivum L.). Accompanying reports in this issue detail results from chromosome bin-mapping of expressed sequence tags (ESTs) representing genes onto the seven homoeologous chromosome groups and a global analysis of the entire mapped wheat EST data set. Among the resources developed were the first extensive public wheat EST collection (113,220 ESTs). Described are protocols for sequencing, sequence processing, EST nomenclature, and the assembly of ESTs into contigs. These contigs plus singletons (unassembled ESTs) were used for selection of distinct sequence motif unigenes. Selected ESTs were rearrayed, validated by 5' and 3' sequencing, and amplified for probing a series of wheat aneuploid and deletion stocks. Images and data for all Southern hybridizations were deposited in databases and were used by the coordinators for each of the seven homoeologous chromosome groups to validate the mapping results. Results from this project have established the foundation for future developments in wheat genomics.

HEXAPLOID wheat (2n = 6x = 42, Triticum aestivum L.) is one of the world's cornerstone crops, feeds more people than any other crop (~600 million tons is produced annually), and is the most widely adapted of the major crops, thus offering potential for increased food production. Hexaploid wheat is composed of three genomes (A, B, and D), each of which contains seven pairs of chromosomes, which have been identified and characterized by SEARS (1966), who established that there is a strong homoeologous relationship among chromosomes belonging to the three genomes. The wheat genome, while complex, offers a unique opportunity for enhancing our understanding of variation in gene density and evolution between and within plant chromosomes.

Technical complexities in studying the wheat genome include that it is an allohexaploid composed of ~16,000 Mb of DNA (ARUMUGANATHAN and EARLE 1991), ~40 times the size of the rice (Oryza sativa L.) genome. However, even with the large size of this hexaploid genome, the genes within the three component genomes remain largely colinear (VAN DEYNZE et al. 1995). Extensive aneuploid stocks have been developed, including nullisomic-tetrasomic and ditelosomic lines (SEARS 1954, 1966; SEARS and SEARS 1978). The ability of the homoeologous chromosomes of polyploid wheat to buffer losses of chromosome fragments has been shown and developed (ENDO 1988, 1990) and a collection of overlapping deletion lines involving all chromosome arms has been accumulated and characterized (ENDO and GILL 1996; QI et al. 2003). The breakpoints of the sequential deletions available for a chromosome arm define physical segments (bins) for that arm. This deletion series offers a unique opportunity to perform bin mapping of all single-dose restriction fragments by their presence or absence in DNA from members of the deletion population.

Expressed sequence tags (ESTs) are short cDNA sequences that serve to "tag" the gene from which the messenger RNA (mRNA) originated and that can serve multiple important uses. Typically, anonymous ESTs are single-pass sequenced to yield a 200-700 bp sequence that can be used to search DNA and protein databases for similar genes (ADAMS et al. 1991). Information from the search can be used to determine if a specific gene (or sequence motif) has been found in the same or other organisms and if its function has been determined. Until recently the lack of ESTs from species of the Triticeae tribe [wheat, barley (Hordeum vulgare L.), rye (Secale cereale L.)] had been a serious limitation to gene-sequence-based research for wheat. In May 2000, GenBank contained only 9 ESTs for wheat, 86 for barley, and none for rye. A large EST data set was a high priority for large-scale efforts to characterize the wheat genome more fully. …

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