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29. Identification of overlapping BAC clones carrying the xa13 locus in rice A.C. SANCHEZ1, L. ILAG2, D. YANG1, D.S. BRAR1, F. AUSUBEL2, G.S. KHUSH1, M. YANO3,T. SASAKI3,N. HUANG1,4 and Z. LI1 1) International Rice Research Institute, P.O. Box 933,
1099 Manila, Philippines We report the genetic and physical
mapping of the recessive gene xa13, which confers specific
resistance to Philippine race 6 (PXO99) of Xoo. Resistance conferred
by recessive genes like xa13 may represent very different and
largely unknown biochemical pathways in the host defense system. Nine
selected DNA markers and two F2 populations (NIL cross n
= 131 and NPT cross n = 230) were used to construct a genetic map of
the xa13 region of rice chromosome 8. Four DNA markers, RG136,
R2027, S14003 and G1149, closely flanking xa13 were used to
identify bacterial artificial chromosome (BAC) clones potentially harboring
the xa13 locus from a rice BAC library constructed using IR64
(Yang et al. 1997). All candidate BAC clones identified by
colony hybridization were subjected to Southern hybridization analysis
to confirm their overlaps. Clones were built into contigs based on HindIII
fingerprint analysis using digested clones as probes. Additional probes
for chromosome walking and contig orientation were generated by TAIL-PCR
of the positive BAC clones detected from colony hybridization (Liu and
Whittier 1995). The construction of a contig map with overlapping BAC clones encompassing the xa13 locus represented a significant step toward our final goal to isolate this gene. Our results indicated that in the xa13 region of chromosome 8, each cM of genetic distance is roughly equivalent to 96 kb, a nearly 3-fold reduction in the ratio of physical/genetic map as compared to the average ratio of 260-280 kb/cM estimated by Wu and Tanksley (1993) and by Harushima et al. (1998). This reduction was apparently due to enhanced recombination in the region. The following steps to isolate xa13 including construction of the xa13 cDNA library, identification of the positive cDNA clones for the resistant allele of xal3, sub-cloning of clone 21H14, genetic complementation, etc., are underway.
References Harushima, Y., M. Yano, A. Shomura, M. Sato, T. Shimano, Y. Kuboki,T. Yamamoto, S.Y. Lin, B.A. Antonio, A. Parco, H. Kajiya, N. Huang, K. Yamamoto, Y. Nagamura, N. Kurata, G.S. Khush and T. Sasaki, 1998. A high density rice genetic linkage map with 2275 markers using a single F2 population. Genetics 148: 479-494. Liu, Y.G. and R.F. Whittier, 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674-681. Wu, K.S. and S.D. Tanksley, 1993. PFGE analysis of the rice genome: estimation of the fragment sizes, organization of the repetitive sequences and relationships between genetic and physical distances. Plant Mol. Biol. Rep. 23: 243-254. Yang, D., A. Parco, S. Nandi, S. Subudhi, Y. Zhu, G. Wang and N. Huang, 1997. Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4- specific RFLP markers in rice. Theor. Appl. Genet. 95: 1147-1154. |
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