Functional Genomics of Legumes

Abstract

Advances in the field of gene discovery related to genes involved in nodulation, nitrogen fixation and root development are described. Focus is given to two legumes, namely the crop plant soybean, and the model legume Lotus japonicus. Two approaches for gene isolation are described. The first involves map-based cloning, in which an inherited mutant phenotype is first mapped close to molecular markers, then isolated through the detection of high molecular weight DNA fragments cloned into bacterial artificial chromosomes (BACs). We illustrate advances in screening a BAC library for BACs anchored at the pUTG-132a marker, which was placed less than 1 cM (about 450 kb) from the supernodulation locus nts-1. The second approach utilizes insertional mutagenesis using T-DNA derived from Agrobacterium. The T-DNA is modified so that close to the left border one finds a promoter-less ß-glucuronidase (gus) gene, which will only be expressed, if inserted in an active transcriptional unit. This form of promoter trapping produces not only the valuable entry point for flanking DNA isolation and gene discovery, but also may yield homozygous insertions in the progeny, allowing a coupling of gene structure, gene expression pattern, and symbiotic phenotype. The high transformation ability of L. japonicus via the A. rhizogenes and A. tumefaciens methods has permitted the isolation of nearly 100 tagged lines (10% of total), which show expression of gus in either root, vasculature, epidermis, nodule or meristem tissue. Inverse PCR was used to isolate the first flanking DNA sequences.