Genetic diversity of Eurycoma longifolia inferred from single nucleotide polymorphisms

Abstract

Eurycoma longifolia Jack. is a treelet that grows in the forests of Southeast Asia and is widely used throughout the region because of its reported medicinal properties. Widespread harvesting of wild-grown trees has led to rapid thinning of natural populations, causing a potential decrease in genetic diversity among E. longifolia. Suitable genetic markers would be very useful for propagation and breeding programs to support conservation of this species, although no such markers currently exist. To meet this need, we have applied a genome complexity reduction strategy to identify a series of single nucleotide polymorphisms (SNPs) within the genomes of several E. longifolia accessions. We have found that the occurrence of these SNPs reflects the geographic origins of individual plants and can distinguish different natural populations. This work demonstrates the rapid development of molecular genetic markers in species for which little or no genomic sequence information is available. The SNP markers that we have developed in this study will also be useful for identifying genetic fingerprints that correlate with other properties of E. longifolia, such as high regenerability or the appearance of bioactive metabolites.

Figure 1

A, Peninsular Malaysia depicting geographic distribution of populations from which E. longifolia accessions were collected. The geographic origin of materials propagated in tissue culture was not known. Although domesticated populations were similarly maintained on a plantation in southwest Malaysia, the geographic origins of the founders of this population were not known. B, Summary of SNP data for individual plants from six collections. Homozygosity and heterozygosity are indicated by color: blue, homozygous for the major allele; red, homozygous for the minor allele, green, heterozygous; white, no/inconclusive data, except in the final (summary) column for each population, where white indicates the presence of polymorphic loci within that population. C, Dendrogram from unweighted pair group method (UPGMA) cluster analysis based on the unbiased genetic distance in E. longifolia populations of Nei (1978). Color coding corresponds to populations as shown in A. D, Dendrogram from UPGMA cluster analysis based on the unbiased genetic distance between individuals of Nei (1978). Annotations indicate populations from which individuals originated: J, Johor, M, plantation grown; P, Pahang, TC, tissue culture collection; T, Terengganu; and L, Langkawi. E, Dendrogram from Ward's analysis of genetic distance between individuals.

Figure 2

Examples of SNP genotyping of 47 E. longifolia samples. In each case, identical blots were prepared carrying LS-PCR products from each of the different accessions; then one blot was hybridized with the ASO for the first allele, and the second blot was hybridized with the ASO for the second allele. The circle indicates an individual that is homozygous for the second allele, whereas the box adjacent to it demonstrates that another accession was heterozygous for these two alleles. The lack of heterozygosity detected with SNP98 illustrates how some SNPs reported starkly different results.