Rapid identification of resistance gene analogs linked to two blast resistance genes in rice

Rapid identification of resistance gene analogs linked to two blast resistance

J.-Y. ZHUANG�, J.-L. Wu�, R.-Y. CHAI2, Y.-Y. FAN�, M.-Z. JIN2, H. LEUNG3 and K.-L. ZHENG�

Biotechnology Department, China National Rice Research Institute, Hangzhou, 310006, China

Institute of Plant Protection, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Entomology and Plant Pathology Division, International Rice Research Institute, 1099 Manila, Philip

In recent years, a number of disease resistance genes have been cloned from several crop species. Remarkably, most resistance genes against diverse pathogens, such as viruses, bacteria and fungi, share structural similarity. By utilizing the conserved motif of disease resistance genes, a PCR-based approach to isolate new resistance genes and to develop markers tightly linked to resistance genes has been widely used in various crop species (Kanazin et a!. 1996; Leister et a!. 1996; Yu et a!. 1996). A large proportion of resistance gene analogs (RGAs) amplified were clustered in the chromosomal regions of respective crop species where known resistance genes are located.

An F8 recombinant inbred population was produced from a cross of indica varieties Zhong 156/Gumei 2 using single seed descent. Genetic control of the resistance to the blast fungus race ZC,5 was studied using 146 recombinant inbred lines (RILs), and RAPD markers were tagged to two resistance genes (Zhuang et a!. 1997). In this study, the population was used to identify RGAs linked to the blast resistance genes.

Eight pairs of RGA primers were used (Table 1), all of which are being maintained at the Genetics Laboratory of the Entomology and Plant Pathology Division, International Rice Research Institute. The primers were designed based on the conserved motif of various disease resistance genes. PCR amplification, polyacrylamide electrophoresis and silver staining were performed following the procedure described by Chen et a!. (1998). All the eight primers detected polymorphisms between the parents and each generated 3-13 polymorphic bands. Altogether, 57 polymorphic RGA bands were scored. Most of them were inherited as dominant markers, but two pairs of bands appeared to be allelic and were suggested to be co-dominant markers by their segregation characteristics. Of the 55 RGA markers, 43 (76.4%) segregated in the expected 1:1 ratio, indicating that the majority of the RGA markers were inherited as a single locus. In comparison with parental survey using 190 RFLP probes and 280 RAPD primers, polymorphism was detected with only 35 probes (18%) and 69 RAPD primers (25%), and 63.2% of the RFLP markers and 84.6% of the RAPD markers segregated in the 1:1 ratio among the RILs.

Linkage analysis was then conducted using the computer software MAPMAKER/ EXP version 3.Ob (Lincoln et a!. 1992). One resistance gene was mapped onto the interval of Pi-2 (t) and Pi-9 (t) on chromosome 6 (Fig. 1). Its resistance allele was derived from Gumei 2. Two RGA markers were found to be linked to this gene. Another gene was mapped onto the interval of Pi-4(t) and Pi-6(t) on chromosome 12 (Fig. 1). Its resistance allele was derived from Thong 156. Three RGA markers were found to be linked to this gene.

Table 1. A list of RGA primers used in this study

Table 1. A list of RGA primers used in this study

GGIGGIGTIGOIAAIACIAC IAGIGCIAGIGGIAGICC

TGCTACGTFCTCCGGG TCAGGCCGTGAAAAATAT

TAGGGCCTCTFGCATCGT TATAAAAAGTGCCGGACT

CCGTFGGACAGGAAGGAG CCCATAGACCGGACTGTF

TFGTCAGGCCAGATACCC GAGGAAGGACAGGTTGCC

GCATFGGAACAAGGTGAA AGGGGGACCACCACGTAG

TAGTFCGGACG1TFACAT AGTGTC�ITGTAGGGTATC

ARIGCTARIGGIARICC GGIGGIGTIGGIAAIACIAC

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