POLYMORPHIC SIMPLE SEQUENCE REPEAT REGIONS IN CHLOROPLAST GENOMES - APPLICATIONS TO THE POPULATION-GENETICS OF PINES

Simple sequence repeats (SSRs), consisting of tandemly repeated multip le copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquito us in eukaryotic genomes and are frequently used as genetic markers, t aking advantage of their length polymorphism, We have examined the pol ymorphism of such sequences in the chloroplast genomes of plants, by u sing a PCR-based assay, GenBank searches identified the presence of se veral (dA)(n) .(dT)(n) mononucleotide stretches in chloroplast genomes , A chloroplast (cp) SSR was identified in three pine species (Pinus c ontorta, Pinus sylvestris, and Finns thunbergii) 312 bp upstream of th e psbA gene, DNA amplification of this repeated region from 11 pine sp ecies identified nine length variants, The polymorphic amplified fragm ents were isolated and the DNA sequence was determined, confirming tha t the length polymorphism was caused by variation in the length of the repeated region, In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinu s leucodermis Ant. revealed the presence of four variants with intrapo pulational diversities ranging from 0.000 to 0.629 and an average of 0 .320. Restriction fragment length polymorphism analysis of cpDNA on th e same populations previously failed to detect any variation, Populati on subdivision based on cpSSR was higher (G(st) = 0.22, where G(st) is coefficient of gene differentiation) than that revealed in a previous isozyme study (G(st) = 0.05) We anticipate that SSR loci within the c hloroplast genome should provide a highly informative assay for the an alysis of the genetic structure of plant populations.