New microsatellite markers for population studies of Phytophthora cinnamomi, an important global pathogen

Abstract

Phytophthora cinnamomi is the causal agent of root rot, canker and dieback of thousands of plant species around the globe. This oomycete not only causes severe economic losses but also threatens natural ecosystems. In South Africa, P. cinnamomi affects eucalyptus, avocado, macadamia and indigenous fynbos. Despite being one of the most important plant pathogens with a global distribution, little information is available regarding origin, invasion history and population biology. This is partly due to the limited number of molecular markers available for studying P. cinnamomi. Using available genome sequences for three isolates of P. cinnamomi, sixteen polymorphic microsatellite markers were developed as a set of multiplexable markers for both PCR and Gene Scan assays. The application of these markers on P. cinnamomi populations from avocado production areas in South Africa revealed that they were all polymorphic in these populations. The markers developed in this study represent a valuable resource for studying the population biology and movement of P. cinnamomi and will aid in the understanding of the origin and invasion history of this important species.

Figure 1

Schematic illustration of the SSR marker development pipeline used in this study.

Figure 2

Agarose gel electrophoresis of PCR products obtained from (a) 16 individual SSR markers (b) multiplex-1 (c) multiplex-2 (d) multiplex of all 16 markers. This figure has been cropped and edited for illustration purposes – the original images are presented in the Supplementary Fig. S1. Additionally, electropherograms obtained from genescan analysis of all 16 markers multiplexed together are presented in the Supplementary Fig. S2.

Figure 3

Population structure of Phytophthora cinnamomi isolates from South Africa (a) PCA analysis using Bruvo’s distances of P. cinnamomi isolates. The sources of isolates are indicated in red circles for Limpopo and blue circles for Mpumalanga (b) Minimum spanning network using Bruvo’s distances. The size of the nodes is proportional to the number of represented clones and the thickness of the lines represent the Bruvo genetic distance between two nodes (thicker lines denote smaller genetic distance) (c) Bar chart displaying the membership coefficients (based on Structure) of P. cinnamomi isolates used in this study for K = 2 and K = 3. Sources of isolates are indicated below the chart.