3.4 | Relationship between multilocus genotype and
traits related to quantitative pathogenicity
Analysis of multilocus genotypes was conducted on the 63 isolates
individually sequenced across the 28 regions in which haplotypes could
be defined. No significant multilocus disequilibrium was detected in the
two populations considering the two haplotypes previously defined across
the 28 regions (0.008 > \(\overline{r}\)D> 0.009), the 12 regions only associated with DLA-R (0
> \(\overline{r}\)D > 0.007),
or the eight regions only with DLA-S (0 >\(\overline{r}\)D > 0.009). Furthermore,
based on the same haplotypes, no significant pairwise linkage
disequilibrium (LD) was detected between the 28 regions in the two
populations (CU1S2 and CU1R2). However, the number of advantageous
haplotypes in the resistant cultivar accumulated per isolate ranged from
2 to 10 (out of 12 possible advantageous haplotypes) and the correlation
of this number with the level of aggressiveness in the resistant
cultivar (measured with DLA-R) was positive (Spearman’s correlation
coefficient ρ=0.39, p=2.21e-16). On the other hand,
the number of advantageous haplotypes in the susceptible cultivar
accumulated per isolate ranged from 0 to 7 (out of 8 possible
advantageous haplotypes) and the correlation of this number with the
level of aggressiveness in the susceptible cultivar (measured with
DLA-S) was also positive (Spearman’s correlation coefficient ρ=0.38 and
p=4.093 e-9). These results suggested that the most
aggressive isolates in both cultivars tend to accumulate more
advantageous haplotypes in the corresponding cultivars across the
candidate regions but in different combinations.
The frequencies of haplotype 1 or haplotype 2 defined from individual
sequencing in the 17 genomic regions associated with DLA-R and/or DLA-S
(Table 3) were estimated in the 14 study pools with the software harp
(71, Table S4).
This program uses an
expectation-maximization (EM) algorithm to infer the maximum-likelihood
estimated frequencies of a known haplotype in a pool of individuals. To
validate this approach, we first compared the frequencies of the two
haplotypes between estimates resulting from individual and pool
sequencing in samples from location 1 for which both kinds of data were
available (Chi2 test). Haplotype frequencies differed significantly in
pool and individual sequencing in 4/17 regions (S1R3-Cu, S2R3-Cu,
S4R2-Cu, S4R4-Cu). Visual examination of the alignments from the
sequencing of individuals revealed a large number of recombinant
haplotypes, missing data, and/or point mutations in these regions that
could have biased our estimation of haplotype frequencies in pools.
These four regions were consequently not used for further analysis. A
significant difference in haplotype frequencies (Fisher’s exact test)
was detected between population pairs in some of the 13 remaining
regions and in some of the locations, indicating high heterogeneity
between populations but a tendency was nevertheless observed (Table 4).
For the seven regions correlated with DLA-R, haplotype 1, which was
advantageous in the resistant cultivars, was always significantly more
frequent in the populations of this cultivar sampled, with one exception
(region S8R1-Cu). In the four regions correlated with DLA-S, the
advantageous haplotype 2 in the susceptible cultivar was always more
frequently found in the populations of this cultivar sampled. In the
S2R1-Cu region correlated with both DLA-R and DLA-S, haplotype 1 was
more aggressive considering both traits but was found to be more
frequent only in the population of the resistant variety sampled in
location 3 in 2013. Fot the region S9R3-Cu haplotypes 1 and 2 were
considered to be advantageous in the resistant and susceptible cultivar,
respectively. In this region in location 1 in 2011 and 2013, haplotypes
1 and 2 were significantly more frequent in populations of resistant
cultivars and susceptible cultivars, respectively, suggesting
diversifying selection. Overall, our results concerning multilocus
haplotypes suggested a low degree of convergence between the different
locations, and the regions involved in the two traits are not
necessarily the same from one location to another. However, some
populations accumulated several haplotypes across regions that are
advantageous in their cultivar of origin.