Organelle DNA isolation and RFLP analysis

Abstract

The analysis of restriction fragment length polymorphisms (RFLPs) in organelle DNA is radically different from that in nuclear DNA in both purpose and execution. The fundamental reason for these differences is genome size: in land plants, chloroplast DNA (cpDNA) is 1-2 x 102 kb in size [38, 39] and mitochondrial DNA (mtDNA) 2-20 x 102 kb [14, 26, 37, 39], whereas most nuclear genomes are 1-10 x 106 kb [2]. The larger size of nuclear genomes is reflected in our relatively poor understanding of their structure and the fact that only a small fraction of nuclear genes have been isolated and analyzed. Physical mapping of RFLPs for an entire, average-sized nuclear genome is a forbidding task whose completion is at least several years away, and the physical mapping of even a 1 % region of the genome presents a significant challenge. As a consequence, nuclear RFLPs are presently used primarily as genetic markers in linkage studies aimed at identifying and selecting traits of agronomic importance [27, 44] and at examining modes of chromosomal evolution [15, 54]. Nuclear RFLPs are just beginning to be used as physical markers in chromosome walking efforts to clone mapped genes of interest [58].