Inferring outcrossing in the homothallic fungus Sclerotinia sclerotiorum using linkage disequilibrium decay

Abstract

The occurrence and frequency of outcrossing in homothallic fungal species in nature is an unresolved question. Here we report detection of frequent outcrossing in the homothallic fungus Sclerotinia sclerotiorum. In using multilocus linkage disequilibrium (LD) to infer recombination among microsatellite alleles, high mutation rates confound the estimates of recombination. To distinguish high mutation rates from recombination to infer outcrossing, 8 population samples comprising 268 S. sclerotiorum isolates from widely distributed agricultural fields were genotyped for 12 microsatellite markers, resulting in multiple polymorphic markers on three chromosomes. Each isolate was homokaryotic for the 12 loci. Pairwise LD was estimated using three methods: Fisher's exact test, index of association (IA) and Hedrick's D'. For most of the populations, pairwise LD decayed with increasing physical distance between loci in two of the three chromosomes. Therefore, the observed recombination of alleles cannot be simply attributed to mutation alone. Different recombination rates in various DNA regions (recombination hot/cold spots) and different evolutionary histories of the populations could explain the observed differences in rates of LD decay among the chromosomes and among populations. The majority of the isolates exhibited mycelial incompatibility, minimizing the possibility of heterokaryon formation and mitotic recombination. Thus, the observed high intrachromosomal recombination is due to meiotic recombination, suggesting frequent outcrossing in these populations, supporting the view that homothallism favors universal compatibility of gametes instead of traditionally believed haploid selfing in S. sclerotiorum. Frequent outcrossing facilitates emergence and spread of new traits such as fungicide resistance, increasing difficulties in managing Sclerotinia diseases.

Figure 1

(a) Locations where population samples (triangles labeled 1 to 8) were taken from. Numbers correspond to sample numbers in Table 1. (b) Relative locations on supercontigs (corresponding chromosomes in parenthesis) of microsatellite loci tested in this study. Locus with an asterisk (*) is polymorphic and used final LD analyses and locus without an asterisk was either monomorphic or hypervariable (9D) and was not suitable for population analyses.

Figure 2

Scatter plots and regressions showing the relationships of the three LD estimates for three supercontigs. (a) I_A vs P-values of Fisher's exact test; (b) Hedrick's D′ vs I_A; and (c) Hedrick's D′ vs P-values of Fisher's exact test.

Figure 3

Relationship between pairwise LD and the physical distance between markers on three supercontigs. Trend lines are shown only for r²>0.5. (a) Supercontig 3 (chromosome 4); (b) supercontig 9 (chromosome 6); and (c) supercontig 19 (chromosome 5).