Advances in the positional cloning of nodulation genes in soybean

The positional cloning of genes involved in plant control of infection and nodule formation in legumes requires the development of improved tools for the analysis of large genomes and cloning of high molecular weight DNA. We have used bulked segregant analysis (BSA) and DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers to detect polymorphisms linked so genes important to nodulation in soybean (Glycine max L. Merrill). Three loci controlling legume nodulation (nts-1, rjI and rj6) and one involved in early nodule development (enod2) were studied. Wild-type Bragg and EMS-induced mutants defective in autoregulation (nts382) or nodulation (nod49 and nod 139) were crossed with G. soja (Sieb. and Zucc.), for the analysis of segregants in F₂ and F₃ populations. DNA pools from wild-type and mutant individuals, or DNA pools based on RFLP pattern, were screened with sets of structured (mini-hairpin) and unstructured primers, identifying polymorphic DNA. DAF with mini-hairpin primers, template endonuclease cleaved DAF (tecMAAP), and arbitrary signatures from amplification profiles (ASAP) were especially successful in detecting linked polymorphisms. Putative markers were confirmed by individual analysis of segregants, and some of them converted into sequence-characterized amplified regions (SCAR). These markers will be used for high density mapping of the relevant genomic regions and as landmarks for linking cloned soybean DNA from yeast and bacterial artificial chromosome (YAC and BAC) libraries. As present, she partial YAC library (avg. insert size=200 kb) represents about 20% of the soybean genome. YAC endclones were isolated using vectorette PCR, and consisted of unique or repeated DNA. Together with YAC-specific signatures generated with mini-hairpin primers, they will be used in the construction of contigs for positional cloning.