Flor (1) proposed a model based upon the genetic studies using flax and the flax rust pathogen. The ¡°gene-for-gene¡± model predicts that plant resistance will occur only when a plant possesses a dominant resistance gene (R) and the pathogen expresses the complementary dominant avirulence gene (Avr). An alteration or loss of the plant resistance gene or the pathogen Avr determinant leads to disease on the host as the outcome. The R gene products are hypothesized to act as receptors for the products of the avrulence locus. The model holds true for many host-pathogen interactions.
Cloning and characterization of several disease resistance genes in different plant species has revealed common structural features in their predicted protein products, including NBS and LRR domains. This suggests that common mechanisms for perception and transduction of pathogen signals exist in diverse plant species (2). In rice, the most serious fungal disease of rice is blast caused by the fungus Magnaporthe grisea and the most serious bacterial diseases of rice in Africa and Asia is bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo). Of the four rice disease resistance genes cloned from rice so far, three, Xa1 (3), Pib (4) and Pita (5), possess sequences encoding the NBS-LRR domains, whereas one, Xa21, codes for an LRR receptor kinase-like protein (6).
The R gene-mediated resistance is known to be an economical method to control losses in the field. The combined presence of R loci ensures a mechanism for conferring long-term and durable resistance (7). Using molecular markers of isolated genes or tightly linked to resistance loci, simultaneous selection of multiple resistance loci, called marker assisted selection, has been facilitated for pyramiding R genes in a certain cultivar.
Positional cloning, also called map-based cloning, is the process of identifying the genetic basis of a phenotype, i.e. resistance, by looking for linkage to markers whose physical location in the genome is known. The amount of effort required for map-based cloning of genes in rice has dropped dramatically in recent years. Saturation mapping, a new approach to produce markers in a very small interval of several hundred kb pairs, is indispensable for positional cloning.
Over the last few years major advances in rice genomics have made positional cloning in rice much more efficient. A high-density genetic linkage map and a YAC-, PAC-, and BAC-based contig map have been constructed for the rice cultivar Nipponbare (8). Over 110,000 sequence-tagged connectors (STCs) have been generated by sequencing both ends of every BAC clone (9). A fingerprint-based contig (FPC) of BAC clones has been anchored with RFLP markers onto the genetic map (10). In addition, Nipponbare is the rice genotype being sequenced by the International Rice Genome Sequencing Project Consortium (11).
Nipponbare appeared to be susceptible to many M. grisea strains. Recent studies, however, demonstrated that the genetic resource of Nipponbare lacking an R gene can be efficiently utilized to develop markers required for saturation mapping of the R locus (12-14). Here we describe in detail an efficient method of the construction of physical map for positional cloning in rice using the Nipponbare genetic resources. First, an initial markers linked to an R gene are mapped on the high-density genetic map of Nipponbare/Kasalath. Secondly, Nipponbare BAC clones physically spanning the region are identified using the AGI database (http://www.genome.arizona.edu/fpc/rice/). Third, additional genetic markers for saturation mapping are produced using subclones of the identified BACs. Fourth, a small interval of Nipponbare corresponding to the R gene region is delimited by determining recombination breakpoints. Fifth, a physical map of the R locus is constructed using flanking markers and a BIBAC library generated from the R gene-containing line.
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