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# Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in northern Tunisia

Mekki Ben Khalifa, Vincent Simon, M. Marrakchi, Hatem Fakhfakh, Benoît Moury

► To cite this version:

Mekki Ben Khalifa, Vincent Simon, M. Marrakchi, Hatem Fakhfakh, Benoît Moury. Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in northern Tunisia. Plant Pathology, 2009, 58 (4), pp.763-772. 10.1111/j.1365-3059.2009.02064.x. hal-02667583

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PVY epidemiology in pepper in Tunisia

1 **Contribution of host plant resistance and geographic distance**

2 to the structure of *Potato virus Y* (PVY) populations in

3 **pepper in Northern Tunisia.**

4

5 M. Ben Khalifaª, V. Simonᵇ, M. Marrakchiª, H. Fakhfakhª and B.

6 Mouryᵇ*

7 ª Laboratoire de Génétique Moléculaire Immunologie et Biotechnologie,

8 Faculté des Sciences de Tunis, Campus Universitaire, 2092, El-Manar,

9 Tunisia.

10 ᵇ INRA, UR407 Pathologie Végétale, F-84143 Montfavet cedex, France.

11

12 *Corresponding author: Benoît Moury ; INRA, Unité de Pathologie

13 Végétale, Domaine St Maurice, BP94, F-84143 Montfavet Cedex, France ;

14 phone: 04 32 72 28 43 ; fax: 04 32 72 28 42 ; e-mail:

15 **moury@avignon.inra.fr**

16
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PVY epidemiology in pepper in Tunisia

17 Abstract

18 The impact of plant resistances on the structure of targeted viral populations
19 has rarely been studied, even though it is of importance to optimize the
20 management of genetic control methods. We investigated the genetic
21 structure of *Potato virus Y* (PVY) populations in naturally-infected pepper
22 fields, collected at eight different localities of Northern Tunisia, where 23%
23 of the sampled plants were homozygous for the *pvr2*<sup>1</sup> recessive resistance
24 allele, while the other plants carried the dominant susceptibility allele *pvr2*<sup>+</sup>.

25 Restriction fragment length polymorphism analysis at three PVY genome
26 segments allowed detecting a high level of viral diversity, a majority of
27 cases of co-infections of individual plants by several PVY haplotypes and a
28 strong genetic differentiation of viral populations collected in the different
29 localities. We detected a strong effect of geographic distances on the
30 differentiation of PVY populations and isolation by distance among these
31 populations was significant. On the opposite, the occurrence of the *pvr2*<sup>1</sup>
32 resistance allele did not contribute to the structure of viral populations,
33 suggesting that the virulence properties of the virus did not affect
34 significantly its fitness, that the larger deployment of the *pvr2*<sup>1</sup> gene would
35 probably not be a suitable strategy to control PVY and that other resistance
36 genes should be preferred.

37
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PVY epidemiology in pepper in Tunisia

38 **Keywords:** eukaryotic initiation factor 4E (eIF4E), *Potyvirus*, restriction

39 fragment length polymorphism (RFLP), isolation by distance.

40
PVY epidemiology in pepper in Tunisia

41 **Introduction**

42 *Potato virus Y* (PVY) is the type member of the genus *Potyvirus*, the largest
43 among plant viruses, which belongs to the family *Potyviridae*, a group of
44 single-stranded RNA viruses. PVY is transmitted in a nonpersistent manner
45 by more than 70 aphid species (Powell, 1991; Lopez-Abella et al., 1988)
46 and is the causal agent of major diseases in cultivated solanaceous crops
47 including potato, pepper, tobacco and tomato. Based on their host range and
48 the symptoms they induce in potato and tobacco as well as on serological
49 characterization, PVY isolates have been divided into three main groups (O,
50 N and C), which correspond to three monophyletic groups. Several
51 recombinant isolates have also been characterized, mainly between the N
52 and O groups (Revers et al., 1996; Glais et al., 2002; Ogawa et al., 2008).
53 Almost all pepper isolates of PVY belong to the C group (Blanco-Urgoiti et
54 al., 1998).

55 Genetic resistance in plants is an efficient way to control virus diseases but
56 the more or less rapid selection of virus isolates adapted to resistance genes
57 (i.e. virulent isolates) has been documented in several cases (e.g. Pelham et
58 al., 1970; Fletcher 1992). The impact of plant resistance genes on the
59 structure of the viral populations has however never been explored, though
60 the analysis of that structure could provide essential information regarding
61 the selective impact of plant resistance and the occurrence of fitness costs
62 associated to virus virulence. In these regards, it is essential to disentangle
PVY epidemiology in pepper in Tunisia

63 the effects of geographical location and host genotype on the structure of
64 virus populations.

65 In this study, we analysed the genetic diversity of PVY infecting pepper in
66 the regions of Bizerte and Cap Bon, the most important open-field pepper
67 producing areas in Tunisia, with the aims to understand the structure of
68 PVY populations and to estimate the respective impacts of pepper genotype,
69 notably the presence of a recessive resistance allele, and geographic location
70 on that structure.

71

72 **Materials and methods**

73 **Field PVY isolates**

74 PVY isolates were collected according to a hierarchical (nested) sampling
75 design. Collections were made in 21 fields in six localities in the regions of
76 Bizerte, northern Tunisia, and two localities in the region of Cap Bon,
77 North-eastern Tunisia (Fig. 1) from October to December 2006. A total of
78 192 young pepper (Capsicum annuum L.) fruits (one fruit per plant) were
79 randomly collected from symptomatic plants of the local cultivars
80 ‘Baklouti’ and ‘Bar Abid’. The variety Baklouti produces hot, large fruits
81 which become red at maturity, whereas the variety Bar Abid produces long,
82 thin, red-fleshed hot fruits. Samples were first analysed by double antibody
83 sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with
PVY epidemiology in pepper in Tunisia

84 antibodies raised against PVY and CMV, the two most prevalent pepper viruses in Tunisia (Mnari-Hattab et al., 1999).

86

87 cDNA synthesis and RFLP analysis of virus isolates

88 The viral population from PVY-infected samples was characterized by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) analysis of the cDNAs obtained from three genomic regions encoding respectively the P3 protein, the viral protein genome-linked (VPg) and the coat protein (CP). These regions were chosen because they affect PVY virulence properties (VPg; Moury et al., 2004) or because they are variable but flanked by relatively conserved regions, allowing the design of PVY-polyvalent primers (P3 and CP). Total RNAs were purified from a 0.5 g piece of flesh of PVY-infected pepper fruits with the Tri-Reagent kit (Molecular Research Center Inc., Cincinnati, USA), and were used as template for RT-PCR (Moury et al., 2004). Parts of the P3 and VPg cistrons, and the entire CP cistron were amplified using primers listed in Table 1. These primers were designed to be polyvalent for all PVY groups and amplified 1145 nucleotides of the P3 cistron, 547 nucleotides of the VPg cistron and a 1159 nucleotide fragment overlapping the CP cistron. Restriction endonucleases used for RFLP analyses of RT-PCR products were chosen based on genomic sequences available for PVY group C, which includes almost all pepper isolates, in order to reveal polymorphism

100

101

102

103

104

105
PVY epidemiology in pepper in Tunisia

106 within this group. For RFLP analysis, 2 µl of RT-PCR products were digested with endonucleases *Rsa*I, *Cla*I and *Bgl*III for the P3 cistron, *Rsa*I, *Hind*III and *Hinf*I for the VPg cistron and *Mse*I and *Hae*III for the CP cistron. A fourth endonuclease, *Sac*I, was used for the VPg cistron in order to reveal the presence of PVY isolates belonging to the N or O groups.
107
108
109
110
111 Indeed, a *Sac*I site is present in 56 sequences of the VPg cistron of 58 available sequences from the N and O groups (or N×O recombinants) but absent among the 22 available sequences of the VPg cistron of the C group.

114 Genotyping of the *pvr2* locus of sampled pepper plants

115 The *pvr2* gene from eleven randomly-sampled pepper plants were amplified by RT-PCR using specific primers (Table 1) and the obtained PCR products were directly sequenced by Genome Express (Meylan, France). Occurrence of the two nucleotide substitutions that distinguish alleles *pvr2*<sup>+</sup> and *pvr2*<sup>1</sup> (Ruffel 2004; Ayme et al., 2007) was then checked for all sampled plants using dCAPS analysis (Neff et al., 2002) of RT-PCR products obtained from the RNA extracts previously used to analyse PVY diversity. The dCAPS analysis used mismatches in one of the two PCR primers flanking the mutations to create a specific *Nru*I recognition site in the *pvr2*<sup>+</sup> or *pvr2*<sup>1</sup> sequence (Table 1).

125 Statistical analyses

126 For each genomic region, haplotypes were defined as the different combinations of RFLP patterns observed with the different endonucleases
PVY epidemiology in pepper in Tunisia

128 among the PVY isolates. A number of PVY samples showed mixtures of
129 two different RFLP patterns for some of the genome segments and some of
130 the endonucleases (Fig 2). These observations were considered to be the
131 result of mixed infections of the plants by several PVY isolates belonging to
132 different haplotypes. In these cases, when only one endonuclease revealed a
133 mixture of two different RFLP patterns, we considered that the samples
134 were composed of two different PVY isolates belonging to two haplotypes.

135 When two or more endonucleases showed mixtures of two different RFLP
136 patterns, neither the number of isolates composing these PVY samples nor
137 the haplotypes of these isolates could be known. For instance, for a given
138 genome segment and a given PVY sample let A and B be the two
139 haplotypes observed for a first endonuclease and C and D be the two
140 haplotypes observed for a second endonuclease. This PVY sample can be
141 composed of two to four isolates whose haplotypes could be, non-
142 exhaustively, A-C and B-D; A-D and B-D; A-C, A-D and B-C or A-C, A-D,
143 B-C and B-D. Such PVY samples were excluded from further analyses.

144 Further analyses were conducted separately for each genome region and/or
145 for the combination of the three genome regions. In the latter case,
146 haplotypes were defined in the same way as described above for the
147 combination of results obtained with different endonucleases.

148 Completeness of sampling was evaluated by the use of “rarefaction curves”,
149 where the cumulative number of observed haplotypes was plotted versus
PVY epidemiology in pepper in Tunisia

150 sample size, and of the richness of the PVY populations (i.e. the actual number of haplotypes) estimated with the SPADE software (Chao and Shen, 2003).

153 The observed haplotype distribution and that expected in a neutral evolution model were compared using the Ewens-Watterson test (Watterson, 1978).

155 Pairwise genetic differentiation between PVY populations in different localities (hereafter subpopulations) was estimated with the Wright's fixation index ($F_{ST}$; Wright, 1951). Two populations with similar distributions of haplotype frequencies will give an $F_{ST}$ value not statistically different from zero. Following Rousset's recommendations (1997), the hypothesis of isolation by distance (IBD) was explored by examining the correlation between the matrices representing $F_{ST}/(1-F_{ST})$ and the natural logarithm of geographic distance (ln d) for pairs of subpopulations.

163 Significance of the correlation was assessed by a Mantel test using 10,000 permutations using the R software ( Ihaka and Gentleman, 1996).

165 The effects of the sampling locality, pepper variety or *pvr2* allele composition of sampled plants on the variation of haplotype distribution were estimated by hierarchically partitioning the sampled population among these factors, and determining the contribution of each separate factor to the observed genetic variation. For this purpose, comparison by analysis of molecular variance (AMOVA; Excoffier et al., 1992) was performed by
PVY epidemiology in pepper in Tunisia

171 considering the type and frequency of haplotypes in each group of samples
172 and by estimating the probability that their distribution was random.

173 The Ewens-Watterson test, *F* statistic and AMOVAs were performed using
174 the software Arlequin version 3.11 (Excoffier et al., 2005). *F* statistics and
175 AMOVAs were computed both using haplotypes distributions and distance
176 matrices.

177

178 **Results**

179 **High diversity level and frequency of mixed infections in PVY samples**
180 **from Tunisian pepper plants**

181 DAS-ELISAs revealed the infection of 114 pepper samples with PVY, 104
182 of which were simultaneously infected with CMV. Genetic diversity among
183 these 114 samples was examined by RT-PCR-RFLP analysis of three
184 genomic regions in the P3, VPg and CP cistrons. For each genomic region
185 and each endonuclease, RT-PCR-RFLP analyses revealed from 2 to 4 DNA
186 profiles (Fig. 2), except SacI for the VPg cistron which was monomorphic
187 and uncut. Seven different haplotypes were observed for the P3 and VPg
188 cistrons and eight different haplotypes were observed for the CP cistron
189 (Table 2). Evidence of mixed infections with isolates belonging to different
190 haplotypes was found for 51.6% of the PVY samples, based on observation
191 of two RFLP profiles for at least one endonuclease with one genome
192 segment. In addition, for some of the mixed-infected samples, the haplotype
PVY epidemiology in pepper in Tunisia

193 composition could not be known (see the Materials and Methods section).

194 Consequently, only 98 of the 114 PVY positive samples were analysed for

195 at least one genome segment and 75 were analysed for the three genome

196 segments. Note that the number of analysed isolates can be larger than 98

197 (Table 2) due to the presence of two isolates belonging to different

198 haplotypes in a single initial PVY sample.

199 The haplotype distribution and frequency for each genomic region was used

200 to analyse the representativeness of sampling. Rarefaction curves presented

201 quite similar shapes for each genome region and reached asymptotes as the

202 number of samples increased, suggesting that PVY populations were

203 exhaustively sampled (Fig. 3). This was supported by the nonparametric

204 Chao1 richness estimates of the actual number of haplotypes (Table 3),

205 which were close to the observed numbers of haplotypes and presented

206 narrow confidence intervals. Thirty-six different haplotypes were observed

207 when taking into account the RFLP analyses of the three genomic regions

208 and an even larger number of haplotypes could have been expected, had the

209 sampling effort been more intense (Table 3). This number of haplotypes is

210 however somewhat smaller than that expected if the haplotypes defined

211 separately in the three genome regions were randomly combined (95%

212 confidence interval: 45.0-60.0; Table 3).

213

214 High genetic differentiation of PVY subpopulations
PVY epidemiology in pepper in Tunisia

215 Overall, the highest diversity of haplotypes was found in Aousja and Touiba (from three to six haplotypes for each genomic region) and the lowest diversity was found in El-Marja (which contained only three samples) and Sounine (one or two haplotypes, depending on the genomic region).

219 Whatever the genome segment examined, the distribution of haplotypes was quite dissimilar between subpopulations. For each segment, a predominant haplotype was observed which accounted for 45-63% of the total, but this major haplotype was minor or even absent in several subpopulations. There was no specific association between particular haplotypes and the sampled varieties of pepper. In contrast, several haplotypes were observed in one locality only (for Aousja, Utique, Touiba, Boucharraya and El-Marja), usually at low frequencies.

227 Comparing the observed and expected homozygosity values with the Ewens-Watterson test did not reveal any significant departure from neutrality for the P3 or CP cistrons (Table 4). This was the same for the VPg cistron, except for the subpopulation from Aousja, where the observed homozygosity was significantly lower than the expected one (P<0.01). In that locality and for that genome segment, six different haplotypes were observed, four of which at frequencies 15-27%. The Ewens-Watterson test was not significant when combining the three genome regions (Table 4). Consequently, further analyses of the structure of the PVY populations
PVY epidemiology in pepper in Tunisia

236 based on these viral genomic segments will globally not be obscured by
237 strong selection or demography effects.

238 Genetic differentiation between PVY subpopulations was shown by
239 pairwise F<sub>ST</sub>. From 16 to 21 of 28 pairwise F<sub>ST</sub> comparisons were
240 significant (P<0.05), depending on the genome segment and, remarkably,
241 almost all pairwise F<sub>ST</sub> were significant for at least one genome segment
242 (data not shown). Similarly, for the combination of the three genome
243 regions, 26 of 28 pairwise F<sub>ST</sub> comparisons were significant (Table 5).

244 Results were similar using distance matrices instead of raw haplotypes
245 distributions to compute F<sub>ST</sub> values. A hypothesis for this high genetic
246 differentiation could be a lack of PVY migration between sites. Confirming
247 that hypothesis, we noticed that five of seven haplotypes for the P3 cistron,
248 two of seven haplotypes for the VPg cistron and six of eight haplotypes for
249 the CP cistron were observed in two or less localities, suggesting lack of
250 virus dissemination.

251 Combining the three genome regions and using the estimated F<sub>ST</sub> values
252 between subpopulations, a small but significant correlation was observed
253 between geographic distances and PVY subpopulation differentiation
254 (Pearson's coefficient of correlation r=0.36; P-value=0.013, Mantel test;
255 Fig. 4). This correlation was higher and significance increased at smaller
256 distances, i.e. using the samples from the region of Bizerte only, which were
PVY epidemiology in pepper in Tunisia

collected in a 16×6 km area (Pearson's coefficient of correlation r=0.66; P-value=0.006, Mantel test; Fig. 4).

**Relative contributions of geographical location and host genotype to the structure of the PVY population**

Since the alleles present at the *pvr2* locus determine resistance or susceptibility of pepper plants to PVY isolates and could be important to understand the differentiation of PVY populations, each RNA sample was checked for occurrence of two nucleotide substitutions differentiating *pvr2*<sup>+</sup> from *pvr2*<sup>1</sup> (Ruffel 2004; Ayme et al., 2007), and both *pvr2*<sup>+</sup> and *pvr2*<sup>1</sup> from other *pvr2* alleles, by dCAPS analysis. Three kinds of plants were observed: 66% of *pvr2*<sup>+</sup>/*pvr2*<sup>+</sup> and 11% of *pvr2*<sup>+</sup>/*pvr2*<sup>1</sup> plants (both kinds of plants called “susceptible” in the following, given that resistance is recessive) and 23% of *pvr2*<sup>1</sup>/*pvr2*<sup>1</sup> plants (hereafter called “resistant”). The *pvr2*<sup>2</sup> and *pvr2*<sup>3</sup> alleles were absent from the analysed plants since none of the samples showed uncut RT-PCR products simultaneously for both nucleotide substitutions. Resistant plants were present in all localities at frequencies between 12 and 28%, except in Sounine and El-Marja (0%) and Saaden (75%). The estimated haplotype richness (Chao1 estimators) was around four times higher in susceptible than in resistant plants (Table 3), although 95% confidence intervals were large and did slightly overlap. This could indicate a selective effect of the *pvr2*<sup>1</sup>/*pvr2*<sup>1</sup> resistant plants and the lack of
PVY epidemiology in pepper in Tunisia

279 fitness cost associated with virulence toward *pvr2*¹, since the richness in
280 susceptible plants was similar to that in the whole population (Table 3).

281 AMOVAs were conducted to evaluate the contribution of various factors to
282 the differentiation of PVY subpopulations. The total variation observed for
283 the combination of the three genomic regions was shared between (i)
284 variations among the different groups considered (localities, pepper
285 varieties, susceptibility or resistance of sampled plant), (ii) between fields or
286 plants within the same group and (iii) within the field or plant population.

287 The locality contributed significantly to the total variation (10.2% of total
288 variation) (Table 6). 8.1% of the variation was contributed by the field level
289 within localities while the largest part of the variance (81.7%) was at the
290 within-field level. On the contrary, there was no effect of the pepper variety
291 or of the *pvr2* allele in the host plants on the differentiation of PVY
292 populations (Table 6). Similar results were obtained using distance matrices
293 to perform AMOVAs and by the separate analysis of the three genome
294 regions (data not shown).

295

296 Discussion

297 Genetic variation in PVY populations was studied in pepper crops in
298 Northern Tunisia by analysing RFLP haplotypes of three genomic regions.

299 Analysis of the VPg cistron with the *SacI* endonuclease suggested that all
300 collected isolates belonged to the C group, since almost all available
PVY epidemiology in pepper in Tunisia

301 sequences of PVY O and N groups possess at least one *SacI* restriction site
302 in the VPg cistron. This was confirmed later by sequencing the VPg cistron
303 of 20 randomly chosen samples (data not shown). Potato crops are common
304 in the vicinity of the sampled pepper fields around Bizerte and Cap Bon.
305 These crops are mainly infected with N and O group PVY recombinants
306 (Boukhris Bouhachem et al., 2007), which were not found in pepper crops,
307 confirming that PVY does not spread from potato to pepper (Gebre Selassie
308 et al., 1985).

309

310 Overall, a high PVY diversity was observed, since a limited number of
311 restriction enzymes permitted to estimate that the actual richness in the PVY
312 population examined varied between 45.5 and 131.0 haplotypes (Table 3).
313 Also, a high percentage (51.6%) of plants was infected simultaneously by
314 several PVY isolates belonging to different haplotypes. This percentage is
315 certainly an underestimation because of the small number of endonucleases
316 used and of the lack of power to detect PVY isolates present at low
317 concentrations by RT-PCR-RFLP. Indeed, artificial mixtures of PVY
318 isolates with different RFLP profiles showed that this method does not
319 allow detecting isolates representing less than 10 to 20% of the entire virus
320 population (data not shown). A potential consequence of this detection limit
321 could have been to decrease the number of observed PVY isolates and,
322 consequently, to decrease the ability to differentiate genetically the PVY
PVY epidemiology in pepper in Tunisia

subpopulations. This was however not the case (Table 5). Co-infection of
plants with different closely-related virus variants is usually rare (<10%;
Bodaghi et al., 2004; Rubio et al., 2001; McNeil et al., 1996) although
higher frequencies of mixed infections have occasionally been observed,
especially in perennial plants (Vigne et al., 2004). However, detection of
viral co-infections in plants depends largely on the level of polymorphism of
the virus population and on the use of appropriate molecular tools, such as
RFLP or sequencing of a number of DNA clones representing the viral
population. Therefore, we still lack an exhaustive view of the occurrence of
viral co-infections (García-Arenal et al., 2001).

333

There was no evidence of differences in the selective constraints exerted on
the different genome regions analysed. Indeed, similar results were obtained
for the three regions by analyses of homozygosity (Table 4), F statistics or
AMOVAs (data not shown). Except one genome segment for one PVY
subpopulation, the haplotype distributions did not deviate significantly from
the neutral model, thus making our data suitable for analyses of migration
patterns and population differentiation independently of demography and
selection processes. The only exception concerns the VPg segment in the
Aousja subpopulation, where there is an excess of haplotypes at
intermediate frequencies compared to the neutral model. This could be due
to repeated selection of VPg variants by the host plant, since the VPg was
PVY epidemiology in pepper in Tunisia

345 shown to be involved in adaptation to resistance alleles at the *pvr2* locus
346 (Moury et al., 2004; Ayme et al., 2006; 2007).

347 When analysing the haplotypes observed for the combination of the three
348 genome regions, we noticed a near-random association of haplotypes
349 between these regions (Table 3). This may be caused by high recombination
350 and/or mutation frequencies in these populations. No estimations of
351 recombination frequency are available for PVY. However, genome analyses
352 have revealed the widespread occurrence of recombination at the intra-
353 specific level (Revers et al., 1996; Glais et al., 2002; Ogawa et al., 2007)
354 which, together with the high frequency of mixed-infected plants, makes
355 recombination a plausible mechanism for the generation of the haplotype
356 diversity observed.

357 A strong genetic differentiation was observed between the sampled
358 subpopulations as assessed by the *F* statistic (Table 5). In theory, that
359 structure could be attributed either to ecological substructuring of the virus
360 population into ecotypes associated with distinct habitats, such as different
361 plant genotypes, or to the geographical distance separating the sampled
362 localities, which could be here considered as the null hypothesis for
363 population differentiation. A significant IBD was indeed observed when
364 comparing the geographic and genetic distances separating the
365 subpopulations (Fig. 4). The correlation between geographic and genetic
366 distances increased when analysing the samples from the region of Bizerte
PVY epidemiology in pepper in Tunisia

367 only. It is known that sampling at large distances can reduce the capacity to
368 detect IBD because the variance of FST estimators is probably higher at
369 large distances (Rousset, 1997). Confirming this, significant correlations
370 were observed between the logarithm of the distance (ln d) and the residuals
371 of the linear regression between FST/(1-FST) and (ln d), either for the whole
372 dataset or the region of Bizerte only (P-values=0.014 and 0.002,
373 respectively; Mantel tests).

374 AMOVAs showed that the strong effect of the locality in PVY genetic
375 differentiation was due to the location of the site itself but not to the pepper
376 variety cultivated in these sites (Table 6). The varieties Bar Abid and
377 Baklouti are local pepper populations which are propagated from seeds
378 saved from one season to the next by the growers. Relatively high rates of
379 allogamy have been observed in pepper (Tanksley, 1984) and can be
380 responsible for maintaining polymorphism at the *pvr2* locus in these plant
381 populations. In spite of the strong resistance level conferred by the *pvr2*<sup>1</sup>
382 gene against avirulent PVY isolates (Moury et al., 2004), the polymorphism
383 at the *pvr2* locus did not contribute significantly to the genetic
384 differentiation of PVY, as assessed by AMOVA (Table 6). Eight of the ten
385 haplotypes (defined from the combination of the three genome regions)
386 observed in *pvr2*<sup>1</sup>/*pvr2*<sup>1</sup> resistant plants were also observed frequently in
387 susceptible plants carrying the *pvr2*<sup>+</sup> allele, suggesting limited, if any,
388 fitness cost associated to the virulence toward *pvr2*<sup>1</sup>. The remaining two
PVY epidemiology in pepper in Tunisia

389 haplotypes were a singleton and a doubleton (haplotype observed twice). It
390 should be noted that co-infections with CMV do not modify the level of
391 PVY resistance conferred by the *pvr2*<sup>l</sup> allele in pepper (M. Ben Khalifa and
392 B. Moury, data not shown). These observations indicate that the host plant is
393 only weakly involved in the genetic structure of PVY populations and the
394 observed PVY diversity is mostly explained by the geographic distances
395 separating the subpopulations. The strong genetic differentiation of PVY
396 populations observed at relatively short distances could be due to the fact
397 that nonpersistent viruses such as PVY bind only transiently to their aphid
398 vectors (a few hours at maximum and frequently less when aphids feed on
399 healthy plants), which is certainly responsible for a lack of long-distance
400 virus dispersal and for a strong differentiation of PVY populations. In
401 addition, genetic drift caused by narrow population bottlenecks both during
402 plant infection (French and Stenger, 2003; Sacristán et al., 2003) and during
403 PVY transmission by aphids between plants (Moury et al., 2007) may also
404 contribute to strong population differentiation.

405 Measuring fitness costs imposed by virulence properties is important to
406 analyse the emergence of pathogen variants and to optimize the use of plant
407 resistance genes (Leach et al., 2001). In plant viruses, fitness comparisons
408 between virulent and avirulent variants have been essentially performed in
409 controlled conditions with a very limited number of virus genotypes and of
410 environmental conditions (Ayme et al. 2006; Desbiez et al. 2003; Jenner et al.
PVY epidemiology in pepper in Tunisia

411 2002). The representativeness of these experiments regarding the problems
412 of plant resistance management and durability can therefore be questioned
413 and measures of fitness costs in more realistic conditions would be highly
414 desirable. This study provides a first analysis of the impact of a plant
415 resistance gene on the genetic structure of targeted virus populations in
416 epidemiological conditions. Our results suggest the lack of strong fitness
417 costs associated to the virulence towards the *pvr2*¹ resistance gene. This
418 could be because the mutations responsible for the virulence towards *pvr2*¹
419 are not costly for the virus or that an initial fitness cost conferred by these
420 mutations was compensated for by additional mutations. Consequently, the
421 larger deployment of pepper cultivars carrying the *pvr2*¹ resistance would
422 probably not help control PVY in pepper in Northern Tunisia. Instead, other
423 resistance genes such as *pvr2*² (Ayme et al. 2007) or *Pvr4*, which proved to
424 be durable, would be more appropriate.

425

426 Acknowledgments

427 This study was supported by a Franco-Tunisian CORUS program from the
428 French Ministère des Affaires Etrangères, the Tunisian Ministère de
429 l'Éducation Supérieure, de la Recherche Scientifique et de la Technologie, a
430 program "Ecologie pour la gestion des Ecosystèmes et de leurs Ressources"
431 and a program "Agriculture et Développement Durable" from the Agence
432 Nationale de la Recherche. We gratefully acknowledge Pierre Franck for
PVY epidemiology in pepper in Tunisia

433 advice concerning the analyses and Carole Caranta and Carine Charron for

434 sharing oligonucleotides for the *pvr2* gene. We also acknowledge the

435 Tunisian Ministry of Agriculture for help with the surveys.

436
PVY epidemiology in pepper in Tunisia

437 References

438 Ayme V, Souche S, Caranta C, Jacquemond M, Chadœuf J, Palloix A, Moury B, 2006. Different mutations in the VPg of Potato virus Y confer virulence on the *pvr2³* resistance in pepper. *Molecular Plant-Microbe Interactions* **19**, 557-563.

442 Ayme V, Petit-Pierre J, Souche S, Palloix A, Moury B, 2007. Molecular dissection of the potato virus Y VPg virulence factor reveals complex adaptations to the *pvr2* resistance allelic series in pepper. *Journal of General Virology* **88**, 1594-1601.

446 Blanco-Urgoiti B, Sanchez F, Perez de san Roman C, Dopazo J, Ponz F, 1998. PVY<sup>C</sup> isolates are a homogeneous pathotype but two different genetic strains. *Journal of General Virology* **79**, 2037-2042.

449 Bodaghi S, Mathews DM, Dodds JA, 2004. Natural incidence of mixed infections and experimental cross protection between two genotypes of *Tobacco mild green mosaic virus*. *Phytopathology* **94**, 1337-1341.

452 Boukhris-Bouhachem S, Khamassy N, Glais L, Kerlan C, 2007. Occurrence in Tunisia of potato tuber necrotic ringspot disease (PTNRD) caused by variant PVY<sup>NTN</sup> of *Potato virus Y*. *New Disease Reports* **15**.

455 Chao A, Shen T-J, 2003. Program SPADE (Species Prediction And Diversity Estimation). Program and User's Guide published at http://chao.stat.nthu.edu.tw.
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458 Desbiez C, Gal-On A, Girard M, Wipf-Scheibel C, Lecoq H, 2003. Increase in Zucchini yellow mosaic virus symptom severity in tolerant zucchini
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462 with a loss of relative fitness on susceptible plants. *Phytopathology* **93**, 1478-1484.

463 Excoffier L, Laval G, Schneider S, 2005. Arlequin ver. 3.0: An integrated
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478 naturelle des souches du virus Y de la pomme de terre dans les cultures
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481 Glais L, Tribodet M, Kerlan C, 2002. Genetic variability in *Potato potyvirus* *Y* (PVY): Evidence that PVY<sup>NW</sup> and PVY<sup>NTN</sup> variants are single to multiple recombinants between PVY<sup>O</sup> and PVY<sup>N</sup> isolates. *Archives of Virology* **147**, 363-378.

485 Ihaka R, Gentleman R, 1996. R: A language for data analysis and graphics.
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489 Leach JE, Vera Cruz CM, Bai J, Leung H, 2001. Pathogen fitness penalty as a predictor of durability of disease resistance genes. *Annual Review of Phytopathology* **39**, 187-224.

492 Lopez-Abella D, Bradley RHE, Harris KF, 1988. Correlation between stylet paths made during superficial probing and the ability of aphids to transmit nonpersistent viruses. *Advances in Disease and Vector Research* **5**, 251-285.

496 McNeil JE, French R, Hein GL, Baezinger PS, Eskridge KM, 1996.
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499 Mnari-Hattab M, Ezzaier K, Gebre-Selassie K, Marchoux G, Gognalons P,
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501 Tunisia. In: Proceedings of the 7th International plant virus
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504 Moury B, Morel C, Johansen E, Guilbaud L, Souche S, Ayme V, Caranta C,
505 Palloix A, Jacquemond M, 2004. Mutations in *Potato virus Y* genome-
506 linked protein determine virulence toward recessive resistances in
507 *Capsicum annum* and *Lycopersicon hirsutum*. *Molecular Plant-Microbe*
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509 Moury B, Fabre F, Senoussi R, 2007. Estimation of the number of virus
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512 Neff MM, Turk E, Kalishman M, 2002. Web-based primer design for single
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514 Ogawa T, Tomitaka Y, Nakagawa A, Ohshima K, 2008. Genetic structure
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518 Pelham J, Fletcher JT, Hawkins JH, 1970. The establishment of a new strain
519 of tobacco mosaic virus resulting from the use of resistant varieties of
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521 Powell G, 1991. Cell membrane punctures during epidermal penetrations by aphids: consequences for the transmission of two potyviruses. *Annals of Applied Biology* **119**, 313-321.

524 Revers F, Le Gall O, Candresse T, Le Romancer M, Dunez J, 1996. Frequent occurrence of recombinant potyvirus isolates. *Journal of General Virology* **77**, 1953-1965.

527 Rousset F, 1997. Genetic differentiation and estimation of gene flow from *F*-statistics under isolation by distance. *Genetics* **145**, 1219-1228.

529 Rubio L, Ayllón MA, Kong P, Fernández A, Polek M, Guerri J, Moreno P, Falk BW, 2001. Genetic variation of *Citrus tristeza virus* isolates from California and Spain: evidence for mixed infections and recombination. *Journal of Virology* **75**, 8054-8062.

533 Ruffel S, 2004. Résistances récessives aux potyvirus chez les solanacées et facteurs du complexe d'initiation de la traduction. PhD thesis, University of Aix Marseille II.

536 Sacristán S, Malpica JM, Fraile A, García-Arenal F, 2003. Estimation of population bottlenecks during systemic movement of *Tobacco mosaic virus* in tobacco plants. *Journal of Virology* **77**, 9906-9911.

539 Tanksley SD, 1984. High rates of cross-pollination in chile pepper. *Hortscience* **19**, 580-582.

541 Vigne E, Bergdoll M, Guyader S, Fuchs M, 2004. Population structure and genetic variability within isolates of *Grapevine fanleaf virus* from a
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543 naturally infected vineyard in France: evidence for mixed infection and

544 recombination *Journal of General Virology* **85**, 2435–2445.

545 Watterson G, 1978. The homozygosity test of neutrality. *Genetics* **88**, 405-

546 417.

547 Wright S, 1951. The genetical structure of populations. *Annals of Eugenics*

548 **15**, 323-354.

549
PVY epidemiology in pepper in Tunisia

550 **Figure 1** Sampling sites in the regions of Bizerte (Northern Tunisia) and
551 Cap-Bon (North-eastern Tunisia).

552 **Figure 2** Restriction profiles obtained for the P3, VPg and CP cistrons of
553 PVY by digestion of corresponding RT-PCR products with endonucleases
554 *Rsa*I (lines: 1 to 3), *Cla*I (lines: 4 and 5) and *Bgl*II (lines 6 to 9) for P3, *Rsa*I
555 (lines 1 and 2), *Hind*III (lines 3 to 5) and *Hinf*I (lines 6 and 7) for VPg, and
556 *Mse*I (lines 1 to 4) and *Hae*III (lines 5 to 7) for CP. An example of mixed
557 infection is shown for the CP cistron and *Mse*I endonuclease (lines 1, 2, 3,
558 5: profiles corresponding to single haplotypes; lines 4, 6, 7: profiles
559 corresponding to a mixture of the same haplotypes). M: size marker. ND:
560 non digested RT-PCR products.

561 **Figure 3** Observed haplotype richness of *Potato virus Y* samples collected
562 in Tunisian pepper fields versus sample size (rarefaction curves). The
563 number of haplotypes observed for a given sample size was averaged over
564 100 simulations. Diversity was revealed from RFLP of three genomic
565 regions.

566 **Figure 4** Isolation by distance pattern between genetic differentiation,
567 measured as *F*<sub>ST</sub> / (1 - *F*<sub>ST</sub>), for haplotypes distributions combining the three
568 genome regions, and geographic distances (natural logarithm of the
569 distances in km) for pairwise PVY subpopulations. The correlation was
570 significant as assessed by Mantel test, both for samples from the region of
PVY epidemiology in pepper in Tunisia

571 Bizerte only (boxed), or for the whole dataset (P=0.006 and P=0.013,

572 respectively).

573
PVY epidemiology in pepper in Tunisia

<caption>Table 1 Primers used for reverse transcription or PCR amplifications</caption>

574
575

<table><thead><tr><th>Name</th><th>Polarity</th><th>Primer sequence (5' to 3')<sup>a</sup></th><th>Binding site</th></tr></thead><tbody><tr><td>P3-F</td><td>+</td><td>TCACCNTTYAGAGARGGNGG</td><td>2445-2464<sup>b</sup></td></tr><tr><td>P3-R</td><td>-</td><td>CARTCRCTCCTTTCAGCATC</td><td>3570-3589<sup>b</sup></td></tr><tr><td>VPg-F</td><td>+</td><td>GAATYCAAGCHYTRAAGTTTCG</td><td>5734-5755<sup>b</sup></td></tr><tr><td>VPg-R</td><td>-</td><td>GCTTCATGYTCYACHTCCTG</td><td>6261-6280<sup>b</sup></td></tr><tr><td>CP-F</td><td>+</td><td>GCTGAACACAGGCTCGAAG</td><td>8289-8307<sup>b</sup></td></tr><tr><td>CP-R</td><td>-</td><td>TAAAAGTAGTACAGGAAAAGCCA</td><td>9425-9447<sup>b</sup></td></tr><tr><td>Pvr2-F</td><td>+</td><td>AAAAGCACACAGCACCAACA</td><td>9-28<sup>c</sup></td></tr><tr><td>Pvr2-R</td><td>-</td><td>TATTCCGACATTGCATCAAGAA</td><td>716-737<sup>c</sup></td></tr><tr><td>dC67<sup>d</sup></td><td>-</td><td>GAGCTACCCCAAGCAGCTTGTTTCGATTTCGCG</td><td>229-261<sup>c</sup></td></tr><tr><td>dC79<sup>d</sup></td><td>-</td><td>CTTCAACAGTGGAGAAAGTGTAGACGTTGCGT</td><td>265-296<sup>c</sup></td></tr></tbody></table>

576 <sup>a</sup> N: A, C, G or T; Y: C or T; R: A or G; H: A, C or T.

577 <sup>b</sup> referring to PVY strain SON41p (accession number AJ439544).

578 <sup>c</sup> referring to the cDNA of the *pvr2*<sup>+</sup> allele from *Capsicum annuum* cv. Yolo

579 Wonder (accession number AY122052).

580 <sup>d</sup> The primers were designed from sequences AY122052 (*pvr2*<sup>+</sup>) and

581 AF521964 (*pvr2*<sup>1</sup>) with the help of dCAPS Finder 2.0 (Neff et al., 2002).

582 The mismatches creating the specific *NruI* restriction sites are underlined.
PVY epidemiology in pepper in Tunisia

<table><caption>**Table 2** Distribution of RFLP haplotypes of three genome regions of *Potato virus Y* among pepper samples collected in different localities of Northern Tunisia</caption><thead><tr><th rowspan="2">Locality</th><th rowspan="2">Variety</th><th rowspan="2">No. fields</th><th rowspan="2">No. samples</th><th colspan="7">P3 cistron</th><th colspan="7">VPg cistron</th><th colspan="8">CP cistron</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>a</th><th>b</th><th>c</th><th>d</th><th>e</th><th>f</th><th>g</th><th>α</th><th>β</th><th>χ</th><th>δ</th><th>ε</th><th>φ</th><th>γ</th><th>η</th></tr></thead><tbody><tr><td rowspan="2">Aousja</td><td>Baklouti</td><td>2</td><td>16</td><td>6</td><td>10</td><td>1</td><td>2</td><td>1</td><td>-</td><td>-</td><td>5</td><td>6</td><td>8</td><td>5</td><td>2</td><td>1</td><td>-</td><td>17</td><td>11</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Bar Abid</td><td>1</td><td>7</td><td>1</td><td>1</td><td>5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>1</td><td>1</td><td>-</td><td>-</td><td>3</td><td>-</td><td>6</td><td>-</td><td>-</td><td>5</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Saaden</td><td>Baklouti</td><td>1</td><td>14</td><td>12</td><td>6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>15</td><td>-</td><td>-</td><td>4</td><td>1</td><td>-</td><td>-</td><td>10</td><td>6</td><td>6</td><td>-</td><td>1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Sounine</td><td>Baklouti</td><td>1</td><td>7</td><td>7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Ghar el</td><td>Baklouti</td><td>2</td><td>6</td><td>5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>9</td><td>4</td><td>2</td><td>4</td><td>-</td><td>-</td><td>-</td><td>6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Melh</td><td>Bar Abid</td><td>1</td><td>9</td><td>9</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1</td><td>2</td><td>-</td><td>-</td><td>1</td><td>-</td><td>-</td><td>4</td><td>-</td><td>-</td><td>-</td><td>5</td><td>-</td><td>-</td></tr><tr><td>Utique</td><td>Baklouti</td><td>2</td><td>12</td><td>-</td><td>9</td><td>-</td><td>2</td><td>-</td><td>1</td><td>-</td><td>9</td><td>-</td><td>3</td><td>-</td><td>2</td><td>-</td><td>-</td><td>2</td><td>-</td><td>8</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td></tr><tr><td>Touiba</td><td>Baklouti</td><td>5</td><td>6</td><td>1</td><td>2</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>2</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>2</td><td>1</td><td>-</td><td>1</td><td>-</td><td>2</td><td>-</td><td>1</td></tr><tr><td>Boucharraya</td><td>Bar Abid</td><td>5</td><td>18</td><td>4</td><td>9</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>13</td><td>-</td><td>-</td><td>2</td><td>1</td><td>-</td><td>1</td><td>6</td><td>8</td><td>-</td><td>-</td><td>-</td><td>4</td><td>-</td><td>-</td></tr><tr><td>El-Marja</td><td>Bar Abid</td><td>1</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody><tfoot><tr><td>Total</td><td></td><td>21</td><td>98</td><td>45</td><td>37</td><td>6</td><td>4</td><td>4</td><td>3</td><td>1</td><td>55</td><td>20</td><td>19</td><td>15</td><td>7</td><td>4</td><td>1</td><td>63</td><td>26</td><td>14</td><td>7</td><td>6</td><td>6</td><td>2</td><td>1</td></tr></tfoot></table>

583

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585

586
<table><caption>**Table 3** Estimations of plant virus population richness (actual number of haplotypes)</caption><thead><tr><th>Genome region and dataset</th><th>N<sup>a</sup></th><th>n<sup>b</sup></th><th>S<sub>Chao1</sub><sup>c</sup></th><th>CI<sub>95%</sub></th></tr></thead><tbody><tr><td>P3</td><td>100</td><td>7</td><td>7.0</td><td>7.0-7.0<sup>c</sup></td></tr><tr><td>VPg</td><td>120</td><td>7</td><td>7.0</td><td>7.0-7.0<sup>c</sup></td></tr><tr><td>CP</td><td>125</td><td>8</td><td>8.5</td><td>8.0-16.4<sup>c</sup></td></tr><tr><td>Combination of the three regions</td><td>107</td><td>36</td><td>66.1</td><td>45.5-131.0<sup>c</sup></td></tr><tr><td>Random sampling of the three regions</td><td>107</td><td>52.2</td><td>-</td><td>45.0-60.0<sup>d</sup></td></tr><tr><td>Resistant (pvr2<sup>l</sup>/pvr2<sup>l</sup>) plants (3 regions)</td><td>25</td><td>10</td><td>14.5</td><td>10.5-50.9<sup>c</sup></td></tr><tr><td>Susceptible (pvr2<sup>+</sup>/-) plants (3 regions)</td><td>82</td><td>34</td><td>64.1</td><td>43.5-129.0<sup>c</sup></td></tr></tbody></table>

<sup>a</sup> Number of virus samples.

<sup>b</sup> Observed number of haplotypes.

<sup>c</sup> nonparametric estimator of actual richness of the population and 95% confidence intervals (CI<sub>95%</sub>) obtained with the SPADE software (Chao and Shen, 2003).

<sup>d</sup> CI<sub>95%</sub> obtained with 1,000 Monte Carlo simulations with the R software (Ihaka and Gentleman, 1996) for random sampling of the three genomic regions.

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<table><caption>**Table 4** Observed and expected homozygosity according to the neutral model with infinite alleles and probability of the Ewens-Watterson test of neutrality</caption><thead><tr><th rowspan="2">Localities</th><th colspan="3">P3 cistron</th><th colspan="3">VPg cistron</th><th colspan="3">CP cistron</th><th colspan="3">Three genome regions</th></tr><tr><th>Obs.</th><th>Exp.</th><th>P</th><th>Obs.</th><th>Exp.</th><th>P</th><th>Obs.</th><th>Exp.</th><th>P</th><th>Obs.</th><th>Exp.</th><th>P</th></tr></thead><tbody><tr><td>Aousja</td><td>0.29</td><td>0.39</td><td>0.20</td><td>0.19</td><td>0.34</td><td>0.007*</td><td>0.43</td><td>0.61</td><td>0.13</td><td>0.09</td><td>0.09</td><td>0.52</td></tr><tr><td>Saaden</td><td>0.56</td><td>0.72</td><td>0.24</td><td>0.60</td><td>0.56</td><td>0.69</td><td>0.33</td><td>0.46</td><td>0.14</td><td>0.20</td><td>0.20</td><td>0.62</td></tr><tr><td>Sounine</td><td>1.00</td><td>-</td><td>-</td><td>0.76</td><td>0.65</td><td>1.00</td><td>1.00</td><td>-</td><td>-</td><td>0.68</td><td>0.61</td><td>1.00</td></tr><tr><td>Ghar el Melh</td><td>1.00</td><td>-</td><td>-</td><td>0.30</td><td>0.37</td><td>0.28</td><td>0.56</td><td>0.71</td><td>0.25</td><td>0.18</td><td>0.25</td><td>0.10</td></tr><tr><td>Utique</td><td>0.60</td><td>0.51</td><td>0.84</td><td>0.48</td><td>0.53</td><td>0.47</td><td>0.50</td><td>0.51</td><td>0.53</td><td>0.34</td><td>0.23</td><td>1.00</td></tr><tr><td>Touiba</td><td>0.39</td><td>0.43</td><td>0.60</td><td>0.38</td><td>0.38</td><td>1.00</td><td>0.30</td><td>0.32</td><td>0.72</td><td>0.38</td><td>0.38</td><td>1.00</td></tr><tr><td>Boucharraya</td><td>0.57</td><td>0.70</td><td>0.32</td><td>0.60</td><td>0.42</td><td>0.95</td><td>0.36</td><td>0.55</td><td>0.06</td><td>0.21</td><td>0.26</td><td>0.23</td></tr><tr><td>El-Marja</td><td>1.00</td><td>-</td><td>-</td><td>1.00</td><td>-</td><td>-</td><td>1.00</td><td>-</td><td>-</td><td>1.00</td><td>-</td><td>-</td></tr></tbody></table>

Obs.: observed haplotype distributions; Exp.: haplotype distributions expected under the neutral model. *Significant difference (p<0.05).

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<table><caption>**Table 5** *F*<sub>ST</sub> values between pairs of PVY subpopulations for the combination of three coding regions using haplotypes distributions</caption><thead><tr><th></th><th>Aousja</th><th>Saaden</th><th>Sounine</th><th>Ghar el Melh</th><th>Utique</th><th>Touiba</th><th>Boucharraya</th></tr></thead><tbody><tr><td>Saaden</td><td>0.103*</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sounine</td><td>0.163*</td><td>0.312*</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ghar el Melh</td><td>0.067*</td><td>0.074*</td><td>0.190*</td><td></td><td></td><td></td><td></td></tr><tr><td>Utique</td><td>0.148*</td><td>0.080*</td><td>0.397*</td><td>0.201*</td><td></td><td></td><td></td></tr><tr><td>Touiba</td><td>0.019</td><td>0.161*</td><td>0.403*</td><td>0.151*</td><td>0.233*</td><td></td><td></td></tr><tr><td>Boucharraya</td><td>0.070*</td><td>0.098*</td><td>0.319*</td><td>0.118*</td><td>0.211*</td><td>0.094</td><td></td></tr><tr><td>El-Marja</td><td>0.301*</td><td>0.375*</td><td>0.742*</td><td>0.363*</td><td>0.472*</td><td>0.520*</td><td>0.385*</td></tr></tbody></table>

<footnote>*Significant difference (p<0.05).</footnote>
<table><caption>**Table 6** Analysis of molecular variance for haplotypes distributions when the subpopulations are grouped by locality, pepper variety or resistance status of sampled plants</caption><thead><tr><th>Source of variation</th><th>d.f.</th><th>Sum of squares</th><th>Components of variance</th><th>Percent variation</th><th>F-statistics</th></tr></thead><tbody><tr><td>Between localities</td><td>7</td><td>9.3</td><td>0.05</td><td>10.2*</td><td>F<sub>CT</sub>= 0.10*</td></tr><tr><td>Between fields in each locality</td><td>10</td><td>5.8</td><td>0.04</td><td>8.1*</td><td>F<sub>SC</sub>= 0.09*</td></tr><tr><td>Within fields</td><td>91</td><td>36.1</td><td>0.40</td><td>81.7*</td><td>F<sub>ST</sub>= 0.18*</td></tr><tr><td>Total</td><td>108</td><td>51.1</td><td>0.49</td><td></td><td></td></tr><tr><td>Between varieties</td><td>1</td><td>1.1</td><td>0.003</td><td>0.5</td><td>F<sub>CT</sub>= 0.01</td></tr><tr><td>Between fields with the same variety</td><td>16</td><td>14.0</td><td>0.08</td><td>17.2*</td><td>F<sub>SC</sub>= 0.17*</td></tr><tr><td>Within fields</td><td>91</td><td>36.1</td><td>0.40</td><td>82.3*</td><td>F<sub>ST</sub>= 0.18*</td></tr><tr><td>Total</td><td>108</td><td>51.2</td><td>0.48</td><td></td><td></td></tr><tr><td>Between resistant and susceptible plants</td><td>1</td><td>0.5</td><td>-0.01</td><td>-2.6</td><td>F<sub>CT</sub>=-0.03</td></tr><tr><td>Between fields with the same category of plants</td><td>24</td><td>18.0</td><td>0.09</td><td>18.9*</td><td>F<sub>SC</sub>=0.18*</td></tr><tr><td>Within fields</td><td>83</td><td>32.7</td><td>0.39</td><td>83.7*</td><td>F<sub>ST</sub>=0.16*</td></tr><tr><td>Total</td><td>108</td><td>51.2</td><td>0.47</td><td></td><td></td></tr></tbody></table>

607
608
609

<footnote>* Significant at P<0.01.</footnote>