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- Rapid, simple and potentially universal method for DNA extraction from Opuntia spp. fresh cladode tissues suitable for PCR amplificationPublication . Raimundo, Joana; Reis, C.M.G.; Ribeiro, M.M.A.In Opuntia spp., the cladode tissues contain many polysaccharides and secondary metabolites that interfere with obtaining high-quality deoxyribonucleic acid (DNA), using currently available methods. To circumvent this problem, three commercial kits, three modified versions of the conventional cetyltrimethylammonium bromide method (CTAB) method and one combined method were tested in Opuntia ficus-indica, O. robusta, O. dillenii and O. elata species. We obtained a rapid and simple protocol that allows the extraction of DNA from all the tested species with good DNA yield and purity, namely, the combined method. With this method (DNeasy® Plant Mini Kit combined with the CTAB method), DNA yields from 13.2 ± 7.8 to 15.9 ± 11.3 µg.g-1 of fresh tissue were obtained in the four Opuntia species. The purity, evaluated by the ratio A260/A280 ratio, ranged from 1.67 ± 0.12 to 2.01 ± 0.25, revealing low levels of problematic metabolites. The extracted DNA quality was confirmed by amplifying a set of nuclear microsatellites obtained for the genus. Reliable reproducible bands and electropherogram profiles were obtained. The combined method has potential to be universal for good-quality DNA extraction in cacti, particularly in the Opuntia genus and other difficult-to-extract species.
- Assessment of Genetic Diversity in Opuntia spp. Portuguese Populations Using SSR Molecular MarkersPublication . Reis, C.M.G.; Raimundo, Joana; Ribeiro, M.M.A.The Opuntia spp., most likely few individuals, were introduced in the Iberian Peninsula in the beginning of the 16th century, after the discovery of America, spreading afterwards throughout the Mediterranean basin. We analysed, for the first time, the genetic diversity in a set of 19 Portuguese Opuntia spp. populations from the species O. ficus-indica, O. elata, O. dillenii and O. robusta using nuclear microsatellite (nuSSR) markers. The Italian cultivars ‘Bianca’, ‘Gialla’ and ‘Rossa’ were included in the study for comparison purposes. The nuSSR amplifications produced from five to 16 alleles, with an average of 9.2 alleles per primer pair, and average polymorphism information content of 0.71. The estimated Dice coefficient among populations varied from 0.26 to 1.0, indicating high interspecific genetic diversity but low genetic diversity at the intraspecific level. The hierarchical clustering analysis revealed four major groups that clearly separated the four Opuntia species. Among the O. ficus-indica populations, two sub-clusters were found, one including the white pulp fruits (with cv. Bianca) and the other with the orange pulp ones and including the cv. Gialla, the cv. Rossa, and one pale yellow pulp population. No genetic differences were found between the inermis form, O. ficus-indica f. ficus-indica, and the rewilded spiny one, O. ficus-indica f. amyclaea. The dendrogram indicated that the clustering pattern was unrelated to geographical origin. The results revealed a low level of genetic diversity among the Portuguese populations of O. ficus-indica.