The focus of this investigation was towards the map-based cloning of nodulation genes required for nodule formation or regulation of nodule number in soybean (Glycine max (L.) Merr.). Major emphasis was given towards cloning the supemodulation (nts-1) gene, which also confers a nitrate-tolerant symbiosis (nts) phenotype.
Using bulked segregant analysis (BSA), approximately 2600 polymorphic amplified fragment length polymorphism (AFLP) markers were mapped relative to the supernodulation locus. Analysis of F2 individuals confirmed eleven AFLP markers to be closely linked to the locus, nine from G. max and two from G. soja. The closest AFLP marker (UQC-IS1) was converted to a codominant PCR marker and mapped on 155 F2 supernodulating plants. UQC-IS1 was shown to be located 1.9 cM from nts-1, on the other side of the locus from the previously described RFLP marker pUTG132a (Kolchinsky et al., 1997). In the same mapping population, pUTG132a was shown to be 1.3 cM from nts-1. Using DNA markers UQC-IS1 and pUTG132a BAC contigs on both sides of the soybean supernodulation (nts-1) locus were initiated. BAC contig ends were isolated, sequenced, codominant PCR markers designed and mapped on a F2 mapping population to order the contig ends relative to nts-1. On the UQC-ISl side, BAC-end UQC-IS2 was demonstrated to be closer than UQC-IS1 to nts-1, 0.9 cM away from the locus. On the other side, BAC-end 17107 was demonstrated to be closer than pUTG132a and 0.9 cM from nts-1. Thus, the nts-1 locus was shown to reside on a 1.8 cM chromosomal fragment between UQC-IS2 and 17107. DNA markers from both contigs in the vicinity of the nts-1 locus were used to map a chromosomal deletion in a fast neutron supernodulating mutant, FN37. This mutant retained UQC-IS1 and UQC-IS2 but lacked both pUTG132a and 171O7.
Three revertants, one with wild-type nodulation and two with intermediate supernodulation, were identified after ethyl methanesulfonate (EMS) mutagenesis of the extreme supernodulating line PS55. Preliminary complementation tests indicated the new mutations in the revertants were alleles of the supernodulation locus (L. Zhao, pers. comm.) and the three revertants were confirmed to be derived from PS55 by DNA fingerprinting. A chromosomal rearrangment was identified near the nts-1 locus at DNA marker UQC-IS1 in the wild-type revertant R1695. Experiments are being conducted to determine if this DNA rearrangement is associated with the reversion from supernodulation to wild-type nodulation. Along with existing independent mutants at the nts-1 locus (Carroll et al., 1985a), these revertants may be extremely valuable in confirming the cloning of the locus.
Simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers were mapped relative to three nodulation loci, nod49, nod139-1 and nod139-2 in soybean. Soybean is an ancient tetraploid, and nod139-1 and nod139-2 were demonstrated to represent a duplicated gene at unlinked loci. 1300 dominant AFLP markers were mapped relative to each loci, but none were confirmed to be less than 10 cM from the loci. Three different BSA strategies were implemented to successfully identify codominant SSR markers linked to each locus. Analysis of F2 individuals confirmed the respective SSR markers were closely linked and within 5 cM of each locus. SSR marker Satt071 was less than 3 cM from nod139-1, Satt426 was located 5 cM from nod139-2 and Satt459 was 3 cM from nod49. Integration of the closely linked SSR markers to nod49, nod139-1 and nod139-2 into the soybean physical map will accelerate the cloning of these genes.