32. Location of the rice blast resistance locus Pi5 (t) in Moroberekan by AFLP bulk segregant analysis

D.CHEN1, G.L. WANG2 and P.C. RONALD1

1) Department of Plant Pathology, University of California, Davis, CA 95616
2) Institute of Molecular Agrobiology, The National University of Singapore, 59A The Fleming, 1 Sci.
Park Dr.,Singapore. 118240@

Moroberekan, a West African upland rice variety, is considered to confer durable resistance to rice blast disease. At least six major blast resistance loci have been identified in Moroberekan. Four major blast resistance loci [Pi5 (t), Pi7 (t) (Wang et al. 1994). Pi12 (t) (Inukai et al. 1996), and Pi45 (t) (Chen et al. in preparation)] have been identified in Moroberekan using the Philippine isolates of Pyricularia grisea. Using Indian isolates of P. grisea, two major resistance loci [Pi10 (t), Pil57 (t)] were identified (Naqvi and Chattoo 1996). To understand the durability of blast resistance and evolutionary relatedness among the resistance genes in Moroberekan, we are initiating the cloning of blast resistance genes Pi5 (t) and Pi7 (t). We are here reporting the chromosomal location of Pi5 (t) by amplified fragment length polymorphism (AFLP) analysis.

Pi5 (t), identified using blast isolate PO6-6, was located on chromosome 4, flanked by RFLP markers RG498 and RG864 (Wang et al. 1994). To facilitate map-based cloning of Pi5 (t), a fine map of Pi5 (t) is required. Three recombinant inbred lines (RIL206, RIL249 and RIL260), derived from a cross of CO39 with Moroberekan, were selected to be the candidates for fine mapping Pi5 (t), based on molecular and phenotypic dataset from the previous study (Wang et al. 1994).

RIL206, RIL249 and RIL260 were crossed with CO36. Three F2 populations each consisting of 50 progeny, were obtained from the crosses. The F2 progeny and their F3 families were inoculated with isolate PO6-6 21 days after sowing (17 to 20 seedings per family). Disease was evaluated 7 days after inoculation. DNA from 17 to 20 seedlings of each F3 families was extracted to represent the F2 individual. Bulked DNA segregant analysis by AFLP was applied to identify DNA markers associated with the resistance to isolate PO6-6. The AFLP analysis was essentially following the protocol developed by Zabeau and Vos (1993).

All three F2 populations were segregating against blast isolate PO6-6 in a 3:1 ratio, indicating a single locus conferring resistance to isolate PO6-6. The F2 inoculation results were confirmed in two replicated inoculations of F3 families with the same isolate.

EcoRI and MseI AFLP primers were surveyed for DNA markers associated with the resistance locus in two resistant and two susceptible bulks for each of the three F2 populations. DNA markers associated with resistance were confirmed in the F2 individuals. Out of 350 primer pairs surveyed, 10 and II AFLP markers were identified to be associated with the resistance in the RIL206-derived and RIL249-derived F2 populations, respectively. Out of 750 primers surveyed, 12 linked markers were identified to be associated with the resistance in the RIL260-derived F2 population. One marker showed polymorphism in both the RIL206-derived and RIL249-derived populations, and three markers were associated with resistance in both the RIL249- and RIL260-derived populations. These results suggest that the resistance locus in these three lines was likely to be the same or tightly linked. Though six markers were co-segregating with the resistance in one of the three small populations, the exact genetic distance need to be determined in a larger mapping population.

Primer pairs generating the linked marker were used to analyze the parents of a mapping population Black Gora/Labell (provided by D.J. Mackill: Redona and Mackill 1996: Mackill et al. 1996). Nine primer pairs (B14/M03, S04/G03, S10/F07, S08/G24, S10/ M04A, S10/M04B, B11/G19, R01/G23A, R01/G23B) gave rise to polymorphism between Black Gora and Labell at the loci associated with the resistance to PO6-6. The segregation of the 80 F2 progeny of Black Gora/Labell at these loci fits a 3:1 ratio. Linkage analysis with Mapmaker and Joinmap showed that B14/M03 from the RIL206-derived population was mapped to chromosome 11, while other eight markers from the RIL249-derived and RIL260-derived populations were placed on chromosome 9 (Fig. 1). These results suggest that the loci conferring resistance PO6-6 in RIL249 and RIL260 were most likely the same or tightly linked. Since B14/M03 was the only one among ten linked markers from the RIL206-derived population showing polymorphism in Black Gora/Labell population, and the map location of this marker was conflicted with other markers, the location of the resistance locus in RIL206 needs to be determined using more markers in different mapping populations.

The results presented here differ from the previous study, in which Pi5 (t) was mapped to chromosome 4. The location of Pi5 (t) on chromosome 9 corroborates the finding of Inukai who mapped the resistance locus in a segregating population generated from RIL249 (Inukai, personal communication). Whether Pi5 (t) in our three populations is the same as that located on chromosome 4 designated Pi5 (t) by Wang et al. (1994) needs to be confirmed. For fine mapping of Pi5 (t) in Moroberekan, RIL260 was retained for further marker analysis in a larger mapping population.

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