Dna extraction

Beyer, J., Esser, H. - J., Eurlings, M. C. M. & Welzen, P. C. van, 2023, A revision of the genus Croton (Euphorbiaceae) in Sumatra (Indonesia), Blumea 68 (1), pp. 1-25 : 2

DNA extraction , amplification and sequencing

Most of the herbarium specimens of Croton from Sumatra are of very old age, from the late 19th century until 1980, and in a degraded state due to outdated collection and drying methods. Therefore, the extraction method and primers used were chosen for the highest chance of yield instead of the best quality. Initial DNA extraction of the Sumatran material was done using small (c. 1 cm 2) pieces of leaves from herbarium specimens, using the CTAB DNA extraction method ( Doyle & Doyle 1987). Due to the age and state of the material, ITS was sequenced in two parts. First ITS1 with primers M13F-17SE and M13R-5.8I1 and secondly ITS2 with primers M13F-5.8I2 and M13R-26SE ( Sun et al. 1994). Amplifications for ITS were conducted in a total volume of 25 µL containing 1 µL template 20 × diluted DNA , 13.4 µL mQ water (Ultrapure, Uithoorn, The Netherlands), 5 µL 5 × PCR buffer (Thermo Fisher Scientific, Waltham, USA), 1 µL 25mM MgCL 2 (Qaigen, Valencia, USA), 1 µL 10 mg /ml BSA (Promega, Madison, USA), 1.3 µL of each primer with a concentration of 10 pMol/uL, 0.5 µL 2.5 mM dNTP and 0.5 µL Phire Green Hot Start II DNA Polymerase (Thermo Fisher Scientific, Waltham USA). Amplification conditions included an initial denaturation of 60 s at 98 °C, followed by 40 cycles of 10 s denaturation at 98 °C, 10 s annealing at 52 °C, and 20 s extension at 72 °C, followed by a final 5 min extension at 72 °C. A disadvantage of this method is that the 5.8S region between the two ITS regions is not sequenced. For the trn L-F region, the primers used were M13F- trn L-g and M13R- trn L-h ( Taberlet et al. 2007) which are renowned for amplifying old and degraded material due to the small size of the target fragment (~100 bp). These proved to be too small and did not contain any phylogenetic information at the species level and are thus ignored in the Bayesian analysis. High throughput Sequencing took place at Baseclear (Leiden, the Netherlands).