Institut National de la Recherche Agronomique, UMR Biologie et Gestion des Adventices, Dijon Cédex, France
Correspondence: E-mail: delye{at}dijon.inra.fr.
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Abstract |
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Key Words: acetyl coenzyme A carboxylase Alopecurus myosuroides herbicide local selection population structure
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Introduction |
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We considered the selection pressure exerted by herbicides in the grass weed Alopecurus myosuroides Huds. (black grass). Black grass is a very widespread, annual, allogamous (Chauvel and Gasquez 1994) weed of cereal crops in northwestern Europe. To control this weed, herbicides targeting the chloroplastic acetyl coenzyme A carboxylase (ACCase, EC 6.1.4.2) have been broadly used since the end of the 1980s. Chloroplastic ACCase is a vital enzyme that catalyzes the first committed step in fatty acid biosynthesis (Harwood 1988). In Poaceae (grasses), the chloroplastic ACCase is a multidomain enzyme (Konishi et al. 1996) encoded by a nuclear gene (Gornicki et al. 1994; Podkowinski et al. 1996) of about 12,500 bp, with a complex intron-exon structure (Podkowinski et al. 1996). It encodes a protein of about 250 kDa, containing three distinct functional domains, biotin-carboxylase, biotin-carboxyl carrier protein, and carboxyl-transferase (CT) (Gornicki et al. 1994). Herbicides targeting ACCase are known to specifically bind to the CT domain of grass chloroplastic ACCase, thus blocking fatty acid biosynthesis and causing plant death (Gronwald 1991). Recently, mutant ACCase alleles with reduced sensitivity to ACCase-inhibiting herbicides were identified in black-grass populations (Délye, Calmès, and Matéjicek 2002; Délye et al. 2003), suggesting that, in agricultural fields, strong selection pressure can drive fast adaptation of weed populations. Hence, the ACCase gene in black grass is an excellent candidate for use in the exploration of the selective effects of herbicides in a weed.
In this work, we examined nucleotide variation in ACCase CT domain within and between 18 black-grass populations to find out whether a signature of the selective pressure exerted by herbicides specifically targeting ACCase could be detected. We found that ACCase CT domain was highly conserved both between and within grass species. The distribution of DNA polymorphisms found within ACCase sequences revealed a differentiation of black-grass populations much higher than expected for an allogamous plant. We demonstrated that within-population and between-population distribution of polymorphisms in ACCase sequences are a consequence of local, recent, positive, herbicide-driven selection.
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Materials and Methods |
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Data Analysis
Eighteen black-grass populations were investigated. A total of 86 black-grass seedlings were used for sequencing experiments. Forty-seven seedlings contained two distinct ACCase alleles. The 39 remaining seedlings contained two identical ACCase alleles. For each seedling, the two ACCase sequences obtained were used for analysis. A total of 172 sequences were thus used for analysis. To achieve adequate sample sizes for statistical analyses, only sequences obtained from the eight populations collected in 2000 in which at least four individual plants have been studied were considered for detailed between-population and within-population analyses (tables 1 and 2).
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Differentiation between populations was measured by FST as implemented in the MEGA version 2.1 software (Kumar S., K. Tamura, I.B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software Bioinformatics 17:12441245). Pairwise genetic distances between populations were computed using MEGA 2.1 as the mean number of sites differing between sequences (Nei and Kumar 2000). Genetic distances were used in the nearest neighbor statistic (Snn) permutation test (Hudson 2000) to assess whether there was a significant pairwise and global differentiation between the populations. Correlation between pairwise genetic distances and pairwise geographical distances between populations was assessed using a Mantel test with 1,000 random permutations.
Recombination detection within the alignment of ACCase sequences was performed using the "phylogenetic profiles" method (Weiller 1998). This method computes the correlation coefficient of pairwise distances between a given sequence and all other sequences upstream and downstream of each sequence position using a sliding-window procedure. Phylogenetic profiles were computed by the software PhylPro (Weiller 1998). The width of the sliding window for comparisons was set to 100 nucleotides. Haplotypes that gave the smallest phylogenetic correlations, and thus the strongest recombination signals, were discarded from further phylogenetic analysis as described (Weiller 1998). The program AMOVA in the software Arlequin version 2.00 (Schneider S., D. Roessli, and L. Excoffier. 2000. Arlequin ver. 2.00: a software for population genetics data anlaysis. Genetics and Biometry laboratory, University of Geneva, Geneva, Switzerland) was used with 5,000 repetitions to construct a minimum-spanning network connecting all nonrecombinant haplotypes.
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Results |
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The genetic differentiation between the eight populations was very high, with an FST value of 0.416. The Snn test for differentiation was highly significant when applied to the whole set of sequences obtained from the eight populations (Snn = 0.6415, P < 103). Application of the Snn test to all possible pairs of populations revealed significant differentiation for 27 pairs out of 28. Mantel test detected no significant association between pairwise genetic and geographical distances.
The reference population LF85 had a level of nucleotide diversity similar to those of the other populations (table 1). However, population LF85 contained a low number of private alleles, all being singletons (table 1). Population LF85 shared three, one, and one alleles with one, two, and five other populations, respectively. Pairwise Snn tests computed between population LF85 and each of the eight populations used for detailed analysis were significant for only three pairs out of eight.
Linkage Disequilibrium and Selection
To obtain a high power of detection, recombination and linkage disequilibrium were analyzed using the whole set of 172 sequences. All four gametes were found in 12.7% of all pairs of sites analyzed. The minimum number of recombination events needed to explain these data was Rm = 20. Despite this indication for frequent occurrence of recombination, significant linkage disequilibrium was detected by Fisher's exact test with Bonferroni correction in 284 pairs of sites out of 2,145 tested.
Tajima's D statistic was either close to 0 or slightly negative, as expected for an excess of low-frequency polymorphisms. However, D values were never significantly different from 0 (table 2). Fay and Wu's H test uses the frequency distribution of derived polymorphisms to test for an excess of high-frequency variants compared with equilibrium neutral expectations. It was applied to the coding region in the sequences we studied. Among the 49 positions where substitutions were observed in the coding sequence in the region we investigated in the gene encoding black-grass ACCase, 32 contained the same nucleotide in the sequences from wheat, maize, and foxtail millet. This nucleotide was, therefore, deemed "ancestral." The "ancestral state" of the 17 remaining positions was assumed to be that found in the sequence from wheat, which is most closely related to that from black grass. In contrast with Tajima's D statistic, H test values were always negative. They were significantly different from 0 in five out of the eight populations, as well as for the overall set of 17 populations. The H test was not significant for the reference population LF85 (table 2).
Phylogenetic Relationships Between Haplotypes
Phylogenetic profile analysis revealed strong recombination signals in 34 out of the 72 haplotypes. Removing any of those haplotypes from the analysis improved the phylogenetic correlation values for at least 85% of the remaining sequences. The minimum-spanning haplotype network connecting the 38 remaining haplotypes revealed three main clusters that centered on haplotypes Am22, Am37, and Am64 (fig. 4). When considering the distribution of haplotypes containing nonsynonymous substitutions using black-grass, herbicide-sensitive ACCase sequence (EMBL accession number AJ310767) as a reference, we found that haplotypes containing a Lys-to-Arg substitution at position 2264 all clustered around haplotype Am64. In contrast, haplotypes containing Ile-to-Leu and Ile-to-Asn substitutions at positions 1781 and 2041, respectively, were found in several clusters (fig. 4).
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Discussion |
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High Level of Population Differentiation in an Allogamous, Annual Plant
In the literature, few studies of gene nucleotide diversity have considered both within-population and between-population variation in allogamous plants (table 3). Most studies only considered a very small number of populations; hence, there may be high variation in parameter estimation. However, most computed FST values ranged between 0 and 0.3 (table 3). From these data, the FST value we computed for eight black-grass populations (0.416) is exceptionally high for an allogamous, annual plant. Furthermore, this very high FST value is conflicting with a previous survey of population diversity in black grass (Chauvel and Gasquez 1994). In this study, genetic differentiation between 19 black-grass populations from various countries assessed at seven isoenzyme loci was only 0.023. This value is in agreement with the biology of black grass (allogamous mating system, wind pollination, and large populations), in the absence of selective pressure. In contrast, we found high FST values for ACCase, which were confirmed by significant pairwise and overall Snn tests. These values are in contradiction with neutral expectations, suggesting diversifying and/or local selection exists between populations. Two possible explanations can be proposed: (1) diversifying and/or local selection may act only indirectly upon ACCase (i.e., the ACCase gene is in strong disequilibrium with another gene undergoing diversifying selection), or (2) ACCase itself may be the target for diversifying and/or local selection. The second hypothesis is not conflicting with the occurrence of purifying selection at ACCase proposed in the previous section. It only implies that diversifying and/or local selection targets the few nonsynonymous mutations, most of which are involved in resistance to herbicides.
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Reference population LF85 was collected before the use of ACCase-inhibiting herbicides. The H test was not significant for population LF85. Both the presence of several shared alleles and the nonsignificant pairwise Snn tests involving population LF85 indicated a low level of differentiation between population LF85 and the populations collected in 2000. Population LF85 may, thus, be considered as representing preherbicide ACCase allelic diversity. Several distinct substitutions associated with herbicide resistance are found in haplotypes occurring in most of the populations sampled in 2000 but not in population LF85. The most likely scenario for selection consistent with this data is that positive selection targeting haplotypes resistant to ACCase-inhibiting herbicides occurred after population LF85 was collected (i. e., after 1985). This time lag represents at most 15 black-grass generations. Among the eight populations studied in detail, all except population Lux contained at least one of the five mutations associated with resistance to ACCase-inhibiting herbicides (see Purifying Selection at ACCase above). These mutations were found in intermediate to high frequencies in the seven remaining population (0.10 and 0.20 in populations 010 and 006, and from 0.60 to 0.80 in the five remaining populations). Population Lux contained one haplotype with an Ala-to-Thr substitution at position 2241 and one with a Cys-to-Ser substitution at position 1610. The effects of both substitutions upon sensitivity to ACCase-inhibiting herbicides remain to be characterized. The origin of resistant ACCase haplotypes may be caused by de novo mutation or by the introduction of resistant alleles from migration. Assuming an initial frequency of 106, complete dominance and no cost for resistance in a deterministic model (Wright 1977), a net selection coefficient (s) value of at least 0.85 would be sufficient to attain an observed resistant ACCase haplotype frequency as high as 0.80 within 10 generations (not shown). Such an s value would correspond to a herbicide killing rate (mortality of herbicide-sensitive individuals) of 85%. This is largely consistent with literature. The average killing rate for herbicides ranges from 90% to 99% (Jasieniuk, Brûlé-Babel, and Morrison 1996), with ACCase-inhibiting herbicides displaying a killing rate of 95% to 97% (Foster, Ward, and Hewson 1993).
On the minimum-spanning network connecting nonrecombinant haplotypes, herbicide-resistant haplotypes containing Leu and Asn at positions 1781 and 2041 were found in two and three distinct clusters, respectively (fig. 4). This implies several independent appearances of each of those nonsynonymous changes. One given resistant ACCase haplotype most often occurs within a single population. The multiple, independent appearance of resistant haplotypes would, thus, be a consequence of population structure, suggesting that herbicide selection is a local, population-level process. However, resistant haplotypes with clearly distinct origins were sometimes recorded in a single population (e.g., haplotypes Am07 and Am36 carrying an Asn residue at position 2041 in population 099) (fig. 4). This would imply either that some populations have large enough effective sizes to allow several independent appearances of identical amino acid substitutions or that some resistant haplotypes may have been introduced from migration. Clearly, appearance and spread of resistant ACCase alleles is a complex process strongly dependent on the population structure of black grass.
Our study is the first gene-level analysis carrying evidence for an ongoing selection process resulting from anthropic action upon natural plant populations. Studies considering the effects of pesticide-mediated selection pressure at the gene level are scarce and most often considered insects or human parasites, which are prone to rapid, active, or passive (i.e., human-mediated) long-range dissemination. In most cases, one or a few distinct resistance gene(s) appeared once and spread throughout all populations of the insect or parasite undergoing drug or pesticide-based selection pressure (e.g., Raymond et al. [1998], Daborn et al. [2002], and Wootton et al. [2002]). Here, we demonstrated that, because of the very strong associated selection pressure, the use of ACCase-inhibiting herbicides, although very recent, had clearly visible effect upon the genetic variation at the ACCase gene. However, in contrast with the studies mentioned above, our black-grass population sampling showed that herbicide selection pressure is a local selection process leading to the selection of several independently arisen, resistant ACCase alleles. This is consistent with herbicide-spraying programs being designed at the field level and with the absence of known, efficient, long-range black-grass dissemination. Local adaptation at the molecular level has already been documented in short-lived plants, especially in A. thaliana, with respect to flowering time (e.g., Le Corre, Roux, and Reboud [2002]) and resistance to herbivores or pathogens (e.g., Bergelson et al. [2001] and Berger, Mitchell-Olds, and Stotz [2002]), but never for human-mediated selection pressure. Direct studies of nucleotide sequence variability of a major adaptive gene, thus, provided insight into the ecological genetics of short-lived plant populations and showed that adaptive response to a drastic selection pressure can arise within very few generations.
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Footnotes |
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Spencer V. Muse, Associate Editor
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