A chloroplast DNA hypervariable region in eucalypts

Eucalypt chloroplast DNA (cpDNA) provides useful markers for phylogenetic and population research including gene flow and maternity studies. All cpDNA studies in Eucalyptus to date have been based on the RFLP technique, which requires relatively large amounts of clean DNA. The objective of this study was to develop PCR-based cpDNA markers for Eucalyptus. The chloroplast genome of Eucalyptus, like that of most angiosperms, possesses inverted repeats (IR). The two junctions between the IRs and the large single copy (LSC) regions are defined as J(LA) and J(LB). The region surrounding the J(LA) junction was sequenced from 26 Eucalyptus DNA samples (21 of E. globulus, plus 5 other species), and the J(LB) region was sequenced using 5 of these samples. The samples were chosen to represent all major haplotypes identified in previous cpDNA RFLP studies. The J(LA) products were 150-210 bp in size, while those from J(LB) were approximately 500 bp in size. Eighteen mutations were scored in total. Extensive variation was found in the IR within the intergenic spacer between rpl2 and the IR/LSC junctions. Many of these characters were complex indels. One sample of E. globulus possessed a relatively large (38 bp) insertion which caused displacement of the IR/LSC junctions. This is the first report of intraspecific variation in the position of IR/LSC junctions; interspecific variation was also found. The LSC region near J(LB) had a low rate of mutation and none were informative. The LSC region near J(LA) possessed 2 informative mutations. The variation revealed from these sequences reflects the differentiation previously uncovered in an extensive RLFP analysis on the same samples. These results suggest that the J(LA) region will provide very useful cpDNA polymorphisms for future studies in Eucalyptus.