Soybean gene golfing: Applications on cloning Rhg1 and Rhg4 disease
resistance genes
Khalid Meksem1, Kimberley Zobrizt1, Aziz Jamai1, Jeffery Schultz1, David A. Lightfoot1
Chengcang Wu2, Hongbin Zhang2
1
Department of Plant Soil and General Agriculture, Southern Illinois University at
Carbondale, Carbondale, IL 62901-4415
2
Department of Soil and Crop Sciences, Crop Biotechnology Center, Texas A & M University,
College Station, TX 77843-2123
DNA marker analysis of genomes has revolutionized genetic studies of organisms in the past
decade; genetic maps have been developed for all major crops. The development of
technologies; for large DNA fragment cloning such as bacterial artificial chromosomes (BAC),
for fingerprinting and for contig assembly, provided powerful tools to rapidly generate genome
wide physical maps. A physical map integrated with a DNA marker genetic map provides
improved efficiency in isolating genes known only by their phenotypes and the study of
genome structure and organization, this improvement has been termed gene golfing (Zhang and
Wing 1997). A partial physical map integrated with the existing SSR genetic map
(http://www.siu.edu/~pbgc), for the entire soybean genome is available and will provide a new
strategy to clone genes known only by their phenotypes by gene golfing.
Inheritance of field resistance to soybean cyst nematode (SCN) race 3 (Heterodera glycines I.)
in soybean [Glycine max (L.) Merr.] in ‘Forrest’ cultivar (Peking source) is conditioned by two
loci: Rhg1 on linkage group G and Rhg4 on linkage group A2 (Meksem et al., 2001). Using
BSA (Bulked Segregant Analysis), AFLP and microsatellite markers, we have constructed, a
high density genetic map for the intervals carrying Rhg1 and Rhg4, and a large insert DNA
library from Forest in the binary pCLD04541 vector. The library is providing clones for
physical mapping of the soybean genome and for chromosome walking and landing. Candidate
clones containing target genes can be directly used to transform plants for genetic
complementation tests via Agrobacterium-mediated methods. Using the fingerprinting
technology for contig assembly (Tao and Zhang 1998); we constructed a 350 Kb contig in the
2.5 cM interval spanning Rhg1 and 250 Kb contig in the region around Rhg4. Two overlapping
clones (73P6 and 21d9) containing the two markers flanking Rhg1 in Forrest were subcloned.
Sequencing of subclones targeted resistance genes sequences has identified candidate
resistance genes with high homology to the rice Xa21 and tomato Cf genes, homologues to
Rhg1 are found in Rhg4 interval suggesting gene duplication and genome rearrangements have
occurred. Transformation with candidate gene clones is in progress.