"Frontmatter". In: Plant Genomics and Proteomics
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Christopher A. Cullis - Plant Genomics and Proteomics-J. Wiley & Sons (2004)
CHAPTER
3 S E Q U E N C I N G S T R AT E G I E S O VERVIEW The central goal in any genome sequencing project is the discovery of all the genes in that organism, which in essence can only be known for certain when every nucleotide in the genome is known and placed in its appropriate context. On December 14, 2000 the first complete genome sequence of a plant was reported in which the analysis of sequenced regions covered 115.4 Mb of a total genome size of 125 Mb (The Arabidopsis Genome Initiative, 2000). How was this achieved? Essentially, the Arabidopsis genome was sequenced with the traditional approach to genome sequencing. This involved cutting the genome into a large number of subfragments, cloning the pieces, and then grouping the pieces together as large regions of overlapping clones. The chromosomal locations of all of these large groups were determined with the high-density genetic map. Groups of researchers then took on the task of sequencing a specific chromosome or chromosome segment. This sequencing strategy is slow and expensive but provides the most precise and complete sequence across the entire genome. The resulting sequence only represented the inbred progeny of a single isolate of Arabidopsis, so any vari- ation between ecotypes was not immediately available. Complete genome sequences, even those that are made up of a very large number of relatively short segments, of a variety of plants will provide pow- erful tools for biologists. The sequences will aid in understanding how gene families have been created, amplified, and diverged, resulting in the creation of new biological activities and specificities. The gene content of related species can be investigated and compared to identify which pathways are shared among many species and which are restricted to some parts of the plant kingdom. The advantage of having a full set of genes for a compre- Plant Genomics and Proteomics, by Christopher A. Cullis ISBN 0-471-37314-1 Copyright © 2004 John Wiley & Sons, Inc. 4 7 hensive characterization of gene expression especially, for example, when plants are growing under stressed conditions, will lead to the understand- ing and subsequent manipulation of plant growth for improved agronomic performance. The efficient use of high-throughput approaches like micro- array hybridization and analysis will be much more efficient if the complete suite of genes for the plant is available for experimentation. Not only will geneticists have access to the genes underlying quantitative trait loci (QTLs), but they will also be able to generate essentially an unlimited number of DNA markers. One of the major uses of a genome sequence would be to perform map-based cloning of genes and to associate candidate genes with important traits. A well-integrated physical and genetic map is essential for map-based cloning, rendering a pure shotgun sequence less valuable than an anchored sequence. Therefore, the genes identified by sequencing projects will provide the basis for determining why and how some characteristics are shared among particular lineages of organisms while others have a more limited representation in the branches of the tree of life. A complete genome sequence is a first step toward the understand- ing of biological processes, but it still must be followed by detailed studies of gene function. The costs and benefits of following a complete genome strategy as opposed to just trying to identify the genes themselves need to be consid- ered. Relatively few plant species have a comprehensive sequence analysis available. Across-species comparisons are very valuable, but how many species are needed to be able to use comparative and syntenic relationships to draw valid conclusions? Therefore, how many genomes need to be sequenced? What are the alternative approaches to getting sufficient infor- mation that do not necessarily aim to generate the complete genome sequence but will serve to add enormous value to the existing genome resources? The traditional approach to genome sequencing (the stepwise sequenc- ing of overlapping clones) is slow and expensive. A faster and less expen- sive method is the shotgun sequence analysis of small-insert clones (Venter et al., 1996). This latter approach was used to generate the rice genome sequence by Syngenta’s Torrey Mesa Research Institute and the Beijing Genomics Institute (Goff et al., 2002). This method, which produced draft sequences, only cost approximately 10% of the International Rice Genome Sequencing Project (ISGRP), which followed the traditional sequencing method. Shotgun sequencing alone does not provide the locations of the sequence segments on the genetic or physical map. In the traditional sequencing approach, however, the positions of the sequenced regions are already known. An alternative to generating a whole genome sequence is to devise a gene enrichment strategy that will result in the sequences of all the genes but without the need to generate the sequences of all the repetitive regions. This gene enrichment strategy will provide a paradigm for a cost- 4 8 3. S E Q U E N C I N G S T R AT E G I E S effective sequencing of large complex genomes of plants that would other- wise be cost prohibitive to produce by whole genome sequence. In this chapter we consider the processes for generating both whole genome sequences and partial genome sequences enriched for genes. The organization of a whole genome sequence by the traditional and shotgun methods is considered. Various forms of gene enrichment strategies including expressed sequence tags (ESTs), kinetic and methylation- dependent fractionations of the genome, and the use of transposons are explained. The reliance on bioinformatics to assemble such sequences is also considered. A compilation of the “best approach” to obtaining useful genome sequences is described. Finally, the potential plant targets for genome sequencing in the near future and the effect of technology on any predictions are discussed. Download 1.13 Mb. Do'stlaringiz bilan baham: |
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