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American Society of Plant Biologists
Mapping and Sequencing the Rice GenomeDepartment of Biology Brookhaven National Laboratory Upton, NY 11973 burr{at}bnl.gov
In 1997, a group of scientists met in Singapore and agreed to collaborate on sequencing the rice genome. It was agreed at the outset to work on a single cultivar, to share materials, to use a clone-by-clone approach, and to accept the policy of immediate sequence release (Sasaki and Burr, 2000
Having a robust physical map is critical to the success of the project. From the outset, this project had the advantage of having an extremely well-mapped plant genome. About 1450 genetically mapped expressed sequence tags (ESTs) were used previously to anchor a yeast artificial chromosome (YAC)–based physical map that covered 63% of the genome (Saji et al., 2001 In this issue of The Plant Cell, Wu et al. (pages 525–535) and Chen et al. (pages 537–545) present two complementary studies that greatly increase the number of mapped ESTs and refine the physical maps to provide 90% coverage of the rice genome. In addition, they provide new estimates of the size of the rice genome and indicate the distribution of genes on the rice chromosomes.
In the first article, Wu and colleagues obtained 3' end sequences from >20,000 clones of their rice cDNA libraries. 3' end sequences tend to be the least redundant and thus the most likely to give gene-specific markers. From these, they selected 8440 unique sequences as templates for polymerase chain reaction primers. After screening, they retained 6713 sequences that amplified a single band of the predicted size from both rice genomic DNA and the pooled YAC library. Subsequently, they screened pools from the YAC library to identify the YAC clones containing the EST markers. Most of these markers identified YACs that were part of the physical map, permitting immediate mapping of these markers. Approximately 1500 ESTs identified YACs not placed on the physical map previously. A subset of 431 ESTs were mapped genetically, which allowed the placement of more clones on the YAC-based physical map, increasing coverage from 63 to 80%. Finally, a centromere-specific primer was used to identify YACs covering 11 of the 12 rice centromeres. In the end, an additional 6591 EST markers were placed on the physical map. This high marker density will be important in identifying BACs that fill the remaining gaps in the tiling path and in anchoring unplaced BAC contigs (a contig is a contiguous set of overlapping clones or sequences).
Previous work ties the physical map to the genetic map so that the two can be aligned. Because the genetic distance is not uniform with respect to the physical distance, these two maps show different spacing between the markers. Typically, recombination is reduced around centromeres so that genetic distances tend to be condensed, whereas they are more spread out on the chromosome arms. The work of Wu et al. (2002)
BACs and PACs are the primary templates for the clone-by-clone sequencing approach. The article by Chen et al. (2002)  represents the culmination of several years of work by Rod Wing and his colleagues at the Clemson University Genetics Institute (CUGI) to create a BAC-based physical map of rice that covers 100% of the euchromatin and >90% of the genome. Two BAC libraries were created from HindIII and BamHI restriction enzyme partial digests that together represent 25-fold coverage of the genome. The ends of each clone were sequenced, and  110,000 end sequences called sequence tag connectors (STCs) were obtained. STCs are used to pick clones flanking sequenced BACs with minimum overlap. The standard approach in building a physical map is to use DNA fingerprinting. In this method, individual BACs are digested to completion and displayed by high-resolution gel electrophoresis with molecular markers so that the fragments can be sized accurately. A collection of 25 or more sized fragments becomes the fingerprint for each BAC clone. FPC (FingerPrinted Contigs) software (Soderlund et al., 2000) is used to assemble the fingerprints to find overlapping BACs. These assemblies then must be examined manually to edit the contigs. Additionally, primer probes were developed from end sequences of terminal clones in many of the assemblies to identify overlapping clones or contigs. The contigs were placed on the genetic map by probing with mapped markers from the IRGSP. Carol Soderlund, the author of FPC, has written software that generates DNA fingerprints from sequenced BACs as they appear in GenBank and brings them into the assemblies, further anchoring the assemblies to the physical map.
The Monsanto Company conducted an independent genome sequence project for rice (Barry, 2001
Estimates of the genome size of rice and the physical length of rice chromosomes are important issues for rice genome sequencers, who need to know how much must be sequenced. Arumuganathan and Earle (1991) reported that the 2C (twice the gametic) value for Oryza sativa ssp. japonica ranged from 0.86 to 0.91 pg and that the haploid genome size was 430 Mb. Arumuganathan (personal communication) later measured O. japonica cv Nipponbare and found a 2C value of 0.90 ± 0.02 pg. Assuming a mass of 650 D per base pair, this value places the haploid genome of cv Nipponbare at 417 Mb.
Chen et al. (2002) The current estimate of the length of each chromosome was calculated assuming a genome size of 430 Mb and dividing this figure by each chromosome's fraction of the total genetic distance measured in the Nipponbare x Kasalanth mapping population. Now that sequencing for chromosomes 1, 4, and 10 is nearly complete, we can see that, except for chromosome 1, these are reasonable estimates of the sizes of most of the chromosomes. The size of chromosome 1 is not known accurately because there are still a few gaps, including in the centromere region. The sequencing groups working on chromosomes 1, 4, and 10 estimate sizes of 47, 36, and 24.5 Mb, respectively. The sizes estimated from the integrated physical map are 44.3, 36.6, and 25.7 Mb.
The work of Wu et al. (2002)
Arumuganathan, K., and Earle, E.D. (1991). Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 3, 208–218.
Barry, G. (2001). The use of the Monsanto draft rice genome sequence in research. Plant Physiol. 125, 1164–1165.
Chen, M., et al. (2002). An integrated physical and genetic map of the rice genome. Plant Cell 14, 537–545.
Eckardt, N.A. (2000). Sequencing the rice genome. Plant Cell 12, 2011–2017. Saji, S., Umehara, Y., Antonio, B.A., Yamane, H., Tanoue, H., Baba, T., Aoki, H., Ishige, N., Wu, J., Koike, K., Matsumoto, T., and Sasaki, T. (2001). A physical map with yeast artifical chromosome (YAC) clones covering 63% of the 12 rice chromosomes. Genome 44, 32–37.[Medline] Sasaki, T., and Burr, B. (2000). International Rice Genome Sequencing Project: The effort to completely sequence the rice genome. Curr. Opin. Plant Biol. 3, 138–141.[CrossRef][Web of Science][Medline]
Soderlund, C., Humphray, S., Dunham, A., and French, L. (2000). Contigs built with fingerprints, markers and FPC V4.7. Genome Res. 10, 1772–1787.
Wu, J., et al. (2002). A comprehensive rice transcript map containing 6591 ex-pressed sequence tag sites. Plant Cell 14, 525–535. Related articles in Plant Cell:
This article has been cited by other articles:
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