A DNA-based diagnostic for differentiating among New Zealand endemic Podocarpus

Abstract

Species of the genus Podocarpus are primarily found in the Southern Hemisphere. In New Zealand, there are four endemic species—Podocarpus acutifolius, Podocarpus nivalis, Podocarpus totara and Podocarpus cunninghamii. The last mentioned two species, tōtara and Hall’s tōtara, are the most economically and culturally important and have been used extensively for carving, timber and medicinal purposes. However, these species are often difficult to distinguish morphologically as seedlings and adults. Useable Po. totara and Po. cunninghamii timber resources are now scarce, and replanting of tōtara is very costly; therefore, cheap diagnostics for ensuring species identity would be useful for replanting. Using expressed sequence tag (EST)-aligned genomic DNA sequences from putative Po. totara × Po. cunninghamii hybrids, we designed 120 primers for high-resolution melting (HRM) assays. These were evaluated in a multi-stage screening process to identify markers that discriminate among New Zealand endemic Podocarpus species. Ten markers reproducibly differentiated at least one species from the other three, and six differentiated two or more species. One marker differentiated all four species. Moreover, two markers were able to identify ‘artifical’ F1 hybrids of Po. totara and Po. cunninghamii that had been created from mixing equal amounts of DNA from one genotype of each species. Markers also differentiated a non-New Zealand endemic, Podocarpus lawrencei. Phylogenetic analyses indicated that Po. acutifolius accessions were genetically most similar to those of Po. totara, while Po. nivalis was the most genetically distinct species. Our results show that HRM markers can be easily developed from small amounts of next-generation sequence data and used to identify species and determine their phylogenetic relationships.