Construction and characterization of high molecular weight DNA clones of legumes for positional cloning

The purpose of this study was to construct and characterize high molecular weight DNA (HMW DNA) clones for further use in the positional cloning of legume nodulation genes. Bacterial and Yeast Artificial Chromosomes (BACs and YACs), two of the most powerful and popular HMW DNA tools were constructed and characterized. Later work mostly concentrated on BACs. This work forms a part of a larger project aimed at studying genes that control nodulation, a natural process by which leguminous plants, in association with specific soil-inhabiting bacteria form specialized root structures called nodules. For this purpose, this thesis is focussed on two plant species, one, the crop plant soybean (Glycine maxL. Merr) and the other, a model legume Lotus japonicus(Regal) Larsen. The specific phenotype that was followed up for positional cloning in soybean was nitrate tolerant symbiosis (nts), a mutant that exhibits supernodulation -a condition in which the autoregulational control of nodulation is diminished.

Conditions were standardized for the construction of appropriate sized HMW DNA clones of soybean and Lotus. A HMW DNA library of Lotus was also constructed.Clones were characterized for insert size and stability. Pulsed Field Gradient Electrophoresis (PFGE) was used to prepare HMW DNA for cloning. Difficulties encountered in the construction of clones are described along with solutions. A comparison of BACs and YACs as the HMW DNA clones of choice was done. Screening of BAC clones identified candidates for an RFLP marker pUTG-132a that is tightly linked to the nts-1 gene. End clones were isolated from these candidates to facilitate construction of contigs and chromosome walking. The general utilization of HMW DNA clones in genome analysis is described.