Background: Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU). Results: We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template. Conclusion: The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

Summary: The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode–plant interactions and assist in breeding nematode‐resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co‐localized with a resistance‐gene‐like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1‐4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1‐4 encodes a protein of 1136 amino acids that contains Toll‐interleukin 1 receptor (TIR), nucleotide‐binding (NB), leucine‐rich repeat (LRR) homology domains and a C‐terminal domain with unknown function. The deduced Gro1‐4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon‐like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT‐PCR products showed that Gro1‐4 is expressed, among other members of the family including putative pseudogenes, in non‐infected roots of nematode‐resistant plants. RT‐PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.

Two types of resistant soybean (Glycine max(L.) Merr.) sources are widely used against soybean cyst nematode (SCN,Heterodera glycinesIchinohe). These include Peking-type soybean, whose resistance requires both therhg1-aandRhg4alleles, and PI 88788-type soybean, whose resistance requires only therhg1-ballele. Multiple copy number of PI 88788-typeGmSNAP18,GmAAT, andGmWI12in one genomic segment simultaneously contribute torhg1-bresistance. Using an integrated set of genetic and genomic approaches, we demonstrate that therhg1-aPeking-typeGmSNAP18is sufficient for resistance to SCN in combination withRhg4. The two SNAPs (soluble NSF attachment proteins) differ by only five amino acids. Our findings suggest that Peking-typeGmSNAP18is performing a different role in SCN resistance than PI 88788-typeGmSNAP18. As such, this is an example of a pathogen resistance gene that has evolved to underlie two types of resistance, yet ensure the same function within a single plant species.