Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)

Abstract

Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii. Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations. Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations. Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.