Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality

Abstract

Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobroma cacao L., the source of chocolate. FPR occurs in most of the cacao producing countries in the Western Hemisphere, causing yield losses up to 80%. Genetic diversity within the FPR pathogen population may allow the population to adapt to changing environmental conditions and adapt to enhanced resistance in the host plant. The present study developed single nucleotide polymorphism (SNP) markers from RNASeq results for 13 M. roreri isolates and validated the markers for their ability to reveal genetic diversity in an international M. roreri collection. The SNP resources reported herein represent the first study of RNA sequencing (RNASeq)-derived SNP validation in M. roreri and demonstrates the utility of RNASeq as an approach for de novo SNP identification in M. roreri. A total of 88 polymorphic SNPs were used to evaluate the genetic diversity of 172 M. roreri cacao isolates resulting in 37 distinct genotypes (including 14 synonymous groups). Absence of heterozygosity for the 88 SNP markers indicates reproduction in M. roreri is clonal and likely due to a homothallic life style. The upper Magdalena Valley of Colombia showed the highest levels of genetic diversity with 20 distinct genotypes of which 13 were limited to this region, and indicates this region as the possible center of origin for M. roreri.

Keywords: RNAseq; SNP; biodiversity; genotyping; homothallism; monilia pod rot.

FIGURE 1

The overview of the single nucleotide polymorphism (SNP) mining strategy from Moniliophthora roreri RNASequencing (RNASeq) libraries. Each library from M. roreri isolates Co17, Co7, E18, Co15, E16, E43, C13, Co12, Co8, Co11, E32, P1, and MCA2977 was aligned against the M. roreri genome (Meinhardt et al., 2014) and variant calling was performed. Reference for bioinformatics tools: ¹Langmead et al., 2009;²Li et al., 2009; ³Larkin et al., 2007, and ⁴DNASTAR, 1999. Abbreviation: QUAL (quality): Phred-scaled quality score for the assertion made in alternate base; DP: Combined depth across samples; GQ: Conditional genotype quality.

FIGURE 2

Sanger sequencing of partial genomic DNA flanking the putative heterozygous SNP marker 485_1_1286 as called by SAMtools mpileup and bcftools with default parameters from M. roreri isolates C13. PCR-amplicons were cloned into pCR4-TOPO vector followed by transformation into DH5-alpha Escherichia coli and 9–11 clones from each amplicon were randomly selected to be sequenced from both ends using M13F and M13R primers. ClustalW2 (Larkin et al., 2007) was used to compare the DNA sequences of each clone and a distance tree of 100 bootstrapped data sets was generated by using the Phylogeny.fr program (http://phylogeny.lirmm.fr/) and the neighbor-joining method.

FIGURE 3

Genetic relationships based on 88 SNP markers among 172 M. roreri isolates collected from frosty pod rot affected areas of South and Central America. (A) Principal Coordinates Analysis based on the pairwise distance matrix. The plane of the first three main PCO axes accounted for 75.1% of total variation. First axis = 59.48% of total information, the second = 9.15%, and the third = 6.47%. (B) Cluster analysis and a consensus dendrogram were generated from the resulting distance matrix using the neighbor-joining algorithm (Saitou and Nei, 1987) and visualized using Figtree ver. 1.3.1 (Rambaut, 2009). Nei et al.’s (1983) genetic distance was calculated using microsatellite analyzer (Dieringer and Schlötterer, 2003) and 100 bootstrap replications were applied.

FIGURE 4

Geographical distributions of M. roreri isolates collected from frosty pod rot affected areas of South and Central America and their phylogenetic relation. Isolates were collected from infected cacao pods from 1999 to 2013. Approximate geographical locations were indicated using Google map application software. See Figure 3 for phylogenetic relationships based on the color code and Table 2 for synonymous group codes and isolates with unique genotypes.