Identification and development of new polymorphic microsatellite markers using genome assembly for Ganoderma boninense, causal agent of oil palm basal stem rot disease

Abstract

Ganoderma boninense is a telluric lignicolous basidiomycete and the causal agent of basal stem rot, one of the most devastating diseases of the oil palm (Elaeis guineensis). While the fight against G. boninense is of major concern in Southeast Asia, little information is available regarding the genetic diversity and evolutionary history of the fungus. In this context, the development of an informative molecular marker set to characterize the diversity of G. boninense is a key step towards understanding the biology of this pathogen. A G. boninense draft genome sequence of 63 Mbp, assembled using 454 and Illumina sequencing technology, was used to identify and develop a set of microsatellite markers (simple sequence repeats, SSRs). A total of 2487 SSRs were identified, for which 145 SSR primer pairs were designed. These SSRs are characterized by di-to hexanucleotide motifs with 5 to 34 repetitions. Ninety-seven SSR loci were successfully amplified on an initial small set of G. boninense isolates from Indonesia. A collection of 107 isolates from several regions in Southeast Asia were screened to characterize each locus for allele number, polymorphism information criterion and the presence or absence of null alleles at each locus. These results allowed us to propose an effective set of 17 SSRs for studying genetic diversity within G. boninense.

About Ganoderma boninense in oil palm plantations of Sumatra and peninsular Malaysia: Ancient population expansion, extensive gene flow and large scale dispersion ability

“…Nucleic acid extraction and genotyping were performed on all 357 samples according to the protocol for the 17 microsatellites described in Merciere et al (2015). The microsatellite marker characteristics are summarized in Supplementary Table 2.…”

“…The microsatellite marker characteristics are summarized in Supplementary Table 2. Microsatellites were amplified in 384 well plates with a volume of 10 ml per PCR reaction following the composition described by Merciere et al (2015). The PCR program was developed following a classical touchdown protocol: first a Taq activation step 4 min at 94 C, 25 cycles consisting of 45 s at 94 C, 60 Ce55 C (with a decrease of 0.5 C each cycle for ten cycles), and 1 min 15 s at 72 C. To finalize elongation, a last step of 30 min at 72 C was performed.…”

“…As SSR loci with null alleles may also cause bias in population genetics analysis (Paetkau & Strobeck 1995;Chapuis & Estoup 2007), we used ML-NullFreq (Kalinowski & Taper 2006) and Microchecker (Van Oosterhout et al 2004) software to identify them, as recommended by Dabrowski et al (2014), and we also applied the decision rule described by Merciere et al (2015). SSRs exhibiting evidence of null alleles were removed from the population genetics analysis.…”

“…These 11 SSRs included the nine SSRs previously identified by Merciere et al (2015) as null-allele-free and two additional SSRs in which null alleles had formerly been detected. This difference may be due to the fact that our sampling was larger and unstructured.…”

“…We used previously developed microsatellite (also called Simple Sequence Repeats or SSR) markers (Merciere et al 2015) to genotype fruiting bodies (basidiomata) collected from several oil palm plantations spread over peninsular Malaysia and Sumatra (Indonesia). Microsatellite markers were chosen because they are easy to score, codominant, reproducible, and highly polymorphic (Jarne & Lagoda 1996) and also that they can be easily used to infer evolutionary history and demographic parameters (Michalakis & Excoffier 1996;Cornuet et al 1999;Luikart et al 2003).…”