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Over the past two years, selected regions of the rice genome have been sequenced and shown to be colinear at the sequence level with limited regions of other cereal genomes. A large number of expressed gene sequences and molecular markers have accumulated in the public databases. Large insert clone libraries of the rice genome have been
constructed, and rice has become an increasingly attractive candidate for whole genome sequencing.
Addresses
Novartis Agricultural Discovery Institute, 3050 Science Park Road, San Diego, CA 92121, USA; e-mail: [email protected]
Current Opinion in Plant Biology1999, 2:86–89 http://biomednet.com/elecref/1369526600200086 © Elsevier Science Ltd ISSN 1369-5266
Abbreviations
BAC bacterial artificial chromosome
cM centi Morgan
EST expressed sequence tag
Kbp kilobase pairs
Mbp megabase pairs
QTL quantitative trait loci
RFLP restriction fragment length polymorphism
RGP rice genome project
YAC yeast artificial chromosome
Introduction
Rice is one of three cereals produced annually at worldwide levels of approximately half a billion tons. Unlike the other major cereals, more than 90% of rice is consumed by humans. Approximately half of the world’s population derives a significant proportion of their caloric intake from rice consumption. Given the predicted rise in the world’s human population, it is likely that rice consumption and, therefore, demand will increase over the next several decades. As little new acreage is available to increase rice cultivation, larger yields will be needed to meet the antici-pated higher demand. Application of molecular techniques to rice improvement will help to achieve better yields.
In addition to rice being an important cereal crop for human consumption, it is also becoming clear that it could play a major role as a model for cereal genomics. Rice has a genome size considerably smaller than the other major cereals. The size of the rice genome is estimated at 420 to 450 megabase pairs (Mbp or millions of base pairs). Sorghum, maize, barley, and wheat have significantly larg-er genomes (1000, 3000, 5000, and 16000 Mbp, respectively). The smaller genome size of rice results in a higher gene density relative to the other cereals. Assuming a total of 30,000 genes in each of the cereal genomes, rice will have on average one gene approxi-mately every 15 kilobase pairs (Kbp). Maize and wheat will have one gene approximately every 100 and 500 Kbp,
respectively. This higher gene density in rice makes it an attractive target for cereal gene discovery efforts and genomic sequence analysis.
Although the genes in rice are present at a higher relative density than in other cereals, they are predicted to be arranged in a similar general order within the genome. Comparisons of the physical and genetic maps of cereal genomes have lead to reports that a significant amount of colinearity of gene order exists among the various cereal genomes studied [1]. For this reason, the use of rice as a model for cereal genome analysis has been proposed and recently reviewed [2].
In addition to the general conservation of gene order among the cereals, studies of a number of individual genes demon-strate that there is also considerable homology among various cereal gene families. This conservation of gene (and protein) sequence suggests that studies on the functions of genes or proteins from one cereal could lead to elucidation of the functions of orthologous genes/proteins in other cere-als. Non-coding regulatory regions of the genome may also retain similar function between the various cereals. For example strong constitutive or tissue-specific promoters from one cereal are likely to retain function when intro-duced as a portion of a transgene in another species.
Yeast and bacterial artificial chromosomes (YACs and BACs respectively) carrying large continuous regions of the rice genome have been constructed by various groups. Expressed sequence tags (ESTs) representing genes expressed in a particular organ or tissue type are being gen-erated for several cereals. Taken together, these developments and approaches make rice an attractive model for cereal genome research. In this paper, I will review a selection of recent publications on rice genomics, sequenc-ing, transformation, and comparative cereal genomics.
Progress in rice DNA sequencing
Expressed genes from a variety of rice tissues have been cloned and sequenced. The number of rice ESTs in the dbEST public database now exceeds 35,000 (as of November 1998). This is more than an order of magni-tude greater than the number of ESTs for any other cereal and the second largest number of plant ESTs avail-able to the public [3]. The rice genome project (RGP) in Japan has generated over 36,000 ESTs [4], and the Korean rice EST database has approximately 7000 entries [4]. Approximately 25% of the ESTs generated by the RGP in Japan are reported to have significant homology to known genes [5]. An estimated 60% or more of these ESTs are redundant. Additional large-scale rice EST sequencing projects currently in progress will increase the number of available rice gene sequences.
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Interest in sequencing the rice genome has also attracted much attention and planning. Workshops held in February and in April of 1998 described the formation of the International Rice Genome Sequencing Project [6,7], and outlined the research plans as well as the anticipated fund-ing for this genome sequencfund-ing project. Although government funding agencies have recognized the possibil-ity of advancing cereal genomics through specific rice genomics efforts [8], funding has suffered unexpected delays [9]. The delay in the initiation of rice genome sequencing within the US is probably due to the focus of federal funding on maize research. The perception of rice as an excellent model for cereal genomics may require fur-ther evidence before a large publicly funded genome project is fully supported.
Rice physical mapping progress
Large insert BAC and YAC libraries covering the majority of the rice genome have been constructed [10–14], and are being fingerprinted and aligned in preparation for a future genome sequencing effort [14]. These large-insert clones cover the majority of the genome, and their positions rela-tive to the genetic map are being determined. The large-insert clones are also being mapped relative to one another by clone fingerprinting and cross-hybridization. This process results in clone coverage of continuous genomic regions (contigs of clones). As a number of physi-cal markers are mapped to more than a single cereal genetic map, these so-called ‘anchor’ markers [15] are also being mapped to the rice large insert contigs. The result is that the physical and genetic maps of several cereal genomes of commercial interest will be linked. These large insert libraries and physical markers are invaluable for synteny studies, positional cloning, and sequencing of the genome.
Over 2200 markers from ESTs, randomly selected genomic clones, YAC-end clones, barley, maize, and wheat clones have recently been mapped to the rice genome under the RGP in Japan [16•]. This effort has increased the number of available markers to more than 3,000 [4,17]. On average, these markers should be located at approximately 150 Kbp intervals (0.5 cM) assuming a random distribution. 39 gaps larger than 5 cM still exist, however, due to the non-random distribution of markers [16•]. More than 600 of these markers show significant similarity to genes from other species, and they include single copy genes as well as members of gene families.
Progress of cereal colinearity studies
Studies on the structural similarities of cereal genomes have led to the proposal that cereal genomes arose from a common ancestor [18–23], and can be viewed as a sin-gle genetic system [24]. These studies of large numbers of molecular or genetic markers provide support that the position and order of genes in the cereal genomes has been conserved. Evidence providing further support for the fine scale colinearity (microcolinearity or microsyn-teny) of the rice genome with the genomes of other
cereals has recently been reported. Sequence level analysis of the genomic regions carrying the well studied
sh2and a1genes and a predicted transcription factor in rice, maize, and sorghum have been reported [25••,26••]. The orientation of these genes in each of the cereals is conserved. The sh2 gene sequence in rice is 82% identi-cal to the maize gene and 83% identiidenti-cal to the sorghum gene within the exons, whereas the non-coding introns are less than 60% conserved on a nucleotide sequence level. A similar conservation of exon sequence was found for the rice a1and X genes relative to the maize a1gene and sorghum a1 and X genes. The physical distances between the sh2 and a1genes of rice, sorghum and maize are reported at 20 Kb, 22 Kb, and 140 Kb respectively. The sequences representing these intergenic regions display no detectable conservation, and the distance and sequence differences are likely due to transposable ele-ment activity [25••,26••,27].
DNA regions known as matrix attachment regions (MARs) interact with the structural components of the nucleus and are also conserved between rice and sorghum [28••]. MARs position active regions of the genome to allow the transcriptional machinery to properly regulate gene expression. Studies once again focused on the region of the genome containing the sh2/a1 genes. The positions of the MARs within the rice and sorghum regions were determined by binding to isolated nuclear matrices. The relative positions of the rice and sorghum MARs flanking the sh2, X, and a1 genes were reported to be conserved. Genes within this genomic region cross hybridize between rice and sorghum, whereas most repetitive DNA flanking the genes does not.
In contrast to these studies, genes conferring resistance to plant pathogens (R gene homologues) are not in con-served locations within the rice, barley, and foxtail millet cereal genomes [28••]. These genes encode proteins with conserved nucleotide binding sites (NBSs) and leucine rich repeats (LRRs), and were isolated from these species via a degenerate PCR amplification approach. Fourteen classes of predicted R gene homologues were isolated from rice, and mapped by restriction fragment length polymorphism (RFLP) analysis. Although a number of these gene fragments were found to map close to resis-tance loci, only 5 of the 17 gene fragments mapped to syntenic positions in the cereal genomes tested. These results suggest that the NBS-LRR genes are rapidly reor-ganized in the cereal genomes studied. Such rapidly reorganized regions of the genome may not be amenable to comparative genomics studies.
Application of markers and colinearity in rice genomic research
to isolate polymorphic markers in barley, and shown to map to syntenic regions in rice and barley. The rice genome should be generally useful in the positional cloning of syn-tenic genes of interest from other cereal species.
Rice marker technology has recently been used to iden-tify trait improving quantitative trait loci (QTL) alleles from a wild relative of cultivated rice [31••]. Molecular markers were used to introduce an average of approxi-mately 5% of the genome of a wild relative into a modern productive variety, and several phenotypes such as plant height, panicle length and 1000-grain weight were scored. A total of 68 QTL were identified and 35 (51%) of these had trait-improving alleles. Trait-improving alle-les were found for all phenotypes except plant height where any change is considered negative. Of the 35 trait-improving alleles, 19 had no effect on other phenotypes whereas 16 had deleterious effects on other traits. This study demonstrates that valuable traits can be intro-gressed from wild relatives, and underscores the value of molecular marker technology for yield improvement.
Synteny between rice and barley has recently been reported in the the genomic region carrying malting quality QTLs [32], and use of synteny between cereals for positional cloning efforts is likely to add considerable value to rice genome analysis. Likewise, mapping of the liguless region of sorghum, a region containing a devel-opmental control gene, was facilitated using molecular markers from a syntenic region of the rice genome [33].
Progress in rice transformation
Efficient rice transformation and regeneration is critical to the development of new rice lines through biotechnology approaches. Rice transformation has been accomplished by microprojectile bombardment [34], electroporation of pro-toplasts, and Agrobacterium-mediated transformation. Improved methods of rice transformation have been devel-oped, and the expression patterns and inheritance of multiple transgenes in rice has been reported [35]. Rice transformation is sufficiently advanced to be routinely used as a rice genomics research tool.
Bioinformatics progress
The increase in available sequence information for rice and other cereals as well as the cross-referencing of the var-ious cereal genomes will result in large interconnected data sets. These large data sets will need sufficient data storage systems, sequence analysis software, and visualization tool development. Database query software is commonly avail-able over the Internet at a variety of sites. Display software for rice and other cereal genomic map data has been reported [36], or is under development [37], and mapping software to visualize synteny between genomes has also been developed [38]. Despite these software develop-ments, insufficient funding has been directed toward the development of database and visualization tools that will be required for genomics in the near future.
Perspectives in genome sequencing progress
DNA sequencing technology has advanced rapidly in the past several years. The introduction of automated capillary DNA sequencing instruments will decrease the labor, length of time and cost genome sequencing efforts will require. In addition, a random fragment whole genome approach, such as that proposed for the human genome, may provide an inexpensive route to completion of the first cereal genome. Application of this enhanced DNA sequencing technology to rice appears to be the most rea-sonable route to obtain an entire set of cereal genes, and to advance the use of rice as a model cereal.
Acknowledgements
The author thanks Jane Glazebrook for comments on the manuscript.
References and recommended reading
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