Dna extraction

Pelser, P. B., Nickrent, D. L. & Barcelona, J. F., 2018, A conservation genetic study of Rafflesia speciosa (Rafflesiaceae): patterns of genetic diversity and differentiation within and between islands, Blumea 63 (2), pp. 93-101 : 96

DNA extraction and microsatellite genotyping

Using the DNeasy plant mini kit (Qiagen, Germantown, Mary- land), total genomic DNA was extracted from tissue of a total of 105 R. speciosa flowers or flower buds. The results of a pilot study in which the amplification of 17 microsatellite markers de- veloped for R. lagascae ( Pelser et al. 2017) was tested (results not reported here) showed that 15 of these loci amplified well for R. speciosa (Man78, Man80, Man109, Man111, Man120, Man142, Man144, Man166, Man171, Man273, Man553, Man714, Man866, Man1134, Man1169), but that the loci Man788 and Man1193 frequently failed to yield unambigu- ous genotyping profiles. These 15 loci (see Pelser et al. 2017 for primer and microsatellite information) were subsequently genotyped for all available R. speciosa samples. Multiplex PCR analyses using the Qiagen Type-it microsatellite PCR kit and up to four primer combinations per PCR sample followed an M13 protocol as described in Pelser et al. (2017). The PCR products were run on an ABI 3130xL Genetic Analyzer at the University of Canterbury. Geneious 6.1.7 (Biomatters Ltd, Auckland, New Zealand) was used to determine fragment lengths.