A set of primers for the amplification of 20 chloroplast microsatellites in Pinaceae

genetic diversity (Müller-Starck et al. 1992) and therefore an accurate analysis of the entity and distribution of their genetic resources requires the screening of many populations and many individuals within populations. Hence the choice of appropriate genetic markers assumes great importance in order to reduce costs and to save time. In particular genetic markers should be highly polymorphic and inherited in a Mendelian codominant manner. Microsatellites, or simple sequence repeats (SSR), that are tandem repeats of six base pairs or less (Litt & Luty 1989; Weber & May 1989), have these characteristics and therefore seem to be suitable genetic markers for population genetic studies also in forest tree species. The length variation of the tandem repeats can easily be scored following amplification of the SSR-containing fragments by the polymerase chain reaction using unique flanking primers. In plants the possibility to screen highly polymorphic microsatellite regions also in the chloroplast genome, which shows lower mutation rates than the nuclear genome and is usually uniparentally inherited, might increase the knowledge about the biology of these organisms. In particular chloroplast microsatellites may be very useful for studying cytoplasmic diversity, cytonuclear interactions and monitoring gene flow. Powell et al. (1995a,b) recently demonstrated the utility of the analysis of the length variation of chloroplast microsatellites. In particular they analysed (Powell et al. 1995b) a mononucleotide microsatellite located in the intergenic region between the trnK and psbA genes for assaying genetic variation in pine species. It was shown that the primers worked in all the species tested and that the amplified fragment showed high levels of polymorphisms due to size variations between and within species. Sequencing of the fragments confirmed that the size differences were due to variation in the repeat units forming the microsatellite region. The level of within and between population diversity detected in Pinus leucodermis Ant. , a tertiary relic and highly endangered species, were much higher than those previously reported for the same populations with isozyme markers (Boscherini et al. 1994). PCR–RFLP analysis (Boscherini et al. 1994) had previously failed to detect any polymorphism in the cpDNA molecule in this species. These observations suggest that the identification of other tandem repeats localized in the chloroplast genome may be very useful. The search for cp-microsatellites is greatly facilitated by the availability of the full sequence of the cpDNA for an increasing number of species. Due to the high degree of sequence conservation of the chloroplast genome (Clegg et al. 1992), especially in its coding sequences, it should be possible to design primers that could work across a wide range of species. The persistence of homologous A/T stretches in the chloroplast DNA of distantly related species such as Oenothera and Nicotiana has been observed beyond theoretical expectations (Tachida & Iizuka 1992) and provides further support to the use of primers targeting such regions across different species. Recently Wakasugi et al. (1994) sequenced the complete chloroplast genome of the black pine (Pinus thunbergii Parl.). As reported by Powell et al. (1995a,b) 19 A/T and one G/C mononucleotide SSRs (flanking another A/T stretch) of 10 or more repeat units are present in it and no dinucleotide SSRs of similar length are found. 20 primer pairs flanking the mononucleotide stretches in the P. thunbergii sequence were designed (Table 1) using the computer program P R I M E R version 0.5 provided by S. Lincoln, M. Daly and E. Lander (Whitehead Institute, Cambridge, MA, USA). One region with a complex stretch of Ts and Cs (Pt15169) was added to the 19 above mentioned ones, even though neither of the repeated regions in it was 10 repeats long. Primers were designed in order to allow multiplexing by size and thus save considerable time in amplification and/or running gels. The primers were then used to amplify DNA samples extracted from P. leucodermis. DNA was extracted from germinating embryos according to the CTAB procedure outlined by Doyle & Doyle (1990). PCR PRIMER NOTE