Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-n t soybean cDNA encoding a portion of the bifunctional enzyme aspartukinase- homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locu s, conferring a pigmented seed coat, on linkage group A, hence, it is close ly linked to the Rhg(4) locus conferring resistance to race 3 of the soybea n cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select p rogeny containing the Rhg(4) allele. The polymorphic bands were sequenced t o determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg(4)) yielded thre e DNA fragments, la (1160 bp), Ib (1146 bp) and 3 (996 bp). Amplification o f DNA from the cultivar Kent (rhg(4)) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments la, Ib, 2 and 4 were also polymorphic be tween the soybean lines PI 290136 and BARC-2(Rj(4)). A segregating populati on of 80 F-2 and F-3 plants derived from the cross PI 290136 x BARC-2 (Rj(4 )) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg(4) locus on linkage group A. The nucleotide seque nces of fragments Ib, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the diffe rent fragments, whereas the exon was well conserved.