Zentrum für angewandte Genetik, Universität für Bodenkultur, Wien, Vienna, Austria;
Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Vienna, Austria
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Abstract |
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Introduction |
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Recently, it has been suggested that for the identification of genes that contribute to variation in complex traits, association-based methods could be more powerful than linkage studies (Risch and Merikangas 1996
). The principle underlying association tests is that the joint distribution of genotypes and phenotypes in population samples are studied (Long and Langley 1999
). At its most extreme, an association study is based on every polymorphic site in the genome, including the polymorphism on which the quantitative trait is based. Until further technological advances are made, a realistic association study relies on the detection of polymorphic DNA markers that are in linkage disequilibrium with the trait-causing site. Alternatively, association studies could take advantage of the recent progress made in genomics and developmental genetics and use the available set of candidate genes (Long et al. 1998
; Long and Langley 1999
).
Arabidopsis trichomes are single epidermal cells on leaves, stems, petioles, and sepals. Among Arabidopsis thaliana accessions, the density of trichomes has been shown to vary in a quantitative manner. Genetic analyses have identified several candidate loci which have been demonstrated to affect trichome number in A. thaliana. Two genes, GLABROUS1 (GL1) and TRANSPARENT TESTA GLABRA (TTG), are essential for trichome initiation, and null mutants produce completely glabrous plants (Koornneef, Dellaert, and van der Veen 1982
; Oppenheimer et al. 1991
). Mutations in GL3 cause a reduced number of trichomes and also affect the branching pattern of the trichomes (Koornneef, Dellaert, and van der Veen 1982
; Payne, Zhang, and Lloyd 2000
). CAPRICE (CPC) and TRIPTYCHON (TRY) have also been shown to influence trichome number (Hülskamp, Miséra, and Jürgens 1994
; Wada et al. 1997
). Furthermore, in a QTL study, one locus, called REDUCED TRICHOME NUMBER (RTN), could be associated with a smaller number of trichomes in the accession Landsberg erecta when compared with Columbia plants (Larkin et al. 1996
).
Most of these candidate genes have already been cloned. This provides the opportunity to test naturally occurring molecular variation at the candidate genes for an association with the trichome phenotype (e.g., trichome density). Thus, the analysis of natural populations may complement classical mutational analyses to understand gene function and provide insight into the molecular changes required for adaptation.
Here, we studied the GL1 gene, which belongs to the group of R2R3-MYB transcription factors. This class of proteins is characterized by two repeat motifs, which are most similar to the second and third repeats of the three animal MYB DNA-binding domains. We analyzed the molecular variation of 3-kb genomic sequence containing the GL1 locus. Phenotypic analysis of trichome number and distribution in the same set of A. thaliana accessions was used to search for an association with the molecular variation at the GL1 locus.
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Materials and Methods |
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DNA Sequencing
Genomic DNA was isolated from leaves by a modified CTAB method as described in Hauser et al. (1998)
. The GL1 sequence was determined by direct sequencing of PCR products. GL1 was amplified in two pieces using the PCR primers Gl1AF (5'-TTA TAG CCA TGA TTA CAC AAA G 3-') and Gl1R (5'-CCA TGA TCC GAA GAG ACT AT-3'), as well as the primers Gl1F (5'-ATA TTG AGT ACT GCC TTT AG 3-') and Gl1HR (5'-ATG TAT GTT TAC ATT TCG AGT GC 3-'). A BLAST search was performed with all PCR primers to verify that only the GL1 locus was amplified. All PCR primers had homology to published GL1 sequences only. PCR conditions were as follows: a 3-min denaturing step at 94°C was followed by 35 cycles each consisting of 55°C for 15 s, 72°C for 1.5 min, and 94°C for 15 s. The two sequences overlapped by 519 bp. The orthologous GL1 sequence from A. lyrata was amplified using the PCR primers Gl1AF and Gl1R and the primers Gl1BF (5'-CCA CAA GCT CCT CGG CAA TAG-3') and Gl1IR (5'-CTA CGC GGA AGA TAT CAA CAC AAC-3'). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Sequencing primers were located approximately 550 bp apart. Each accession was sequenced in both directions on an ABI automated sequencer using the BigDye terminator sequencing chemistry. The DNA sequences are available from GenBank (accession numbers AF263690AF263721).
Data Analysis
Sequence contigs were generated with the AutoAssember software (Perkin Elmer), and all mutations were individually verified using the original electropherograms. Sequences were aligned using CLUSTAL W (Thompson, Higgins, and Gibson 1994
) and manually adjusted. Because of the large indels between A. thaliana and A. lyrata sequences, homologous regions were identified with a dot matrix. A table of polymorphic sites was generated using the SITES software (Hey and Wakeley 1997
). Tests of neutrality, recombination, and linkage disequilibrium were performed using DNA SP 3.14 (Rozas and Rozas 1999
). Indel polymorphisms were excluded for tests of neutrality, recombination analysis, and estimates of nucleotide diversity (
). Complex mutations consisting of an insertion and deletion were treated as single events. Similarly, microsatellite polymorphisms were treated as a single indel at each microsatellite stretch. For all tests requiring an outgroup, the hairy A. lyrata individual was used.
Phylogenetic reconstruction was carried out with the PUZZLE 4.0 software (Strimmer and von Haeseler 1996
) using the Tamura-Nei model of sequence evolution (Tamura and Nei 1993
) and eight categories of rate heterogeneity. Treeview (Page 1996
) was employed for the graphical representation of the tree. DNA slider software was used (10,000 replicates) to test for heterogeneity in the distribution of base substitutions (McDonald 1996, 1998
). In particular, we used the three most powerful test statistics: the runs test (runs), the mean sliding G statistic (average G), and the Kolmogorov-Smirnov statistic (K-S) (McDonald 1998
).
ANOVA analysis was carried out with the SPSS software package. Trichome densities were ln-transformed to achieve normality. To evaluate the statistical significance of differences in trichome densities among A. thaliana accessions, we used trichome density measurements of one to seven plants per accession for a one-way ANOVA. The measurements of trichome densities were averaged for each accession to determine the influence of molecular variation on trichome density. Since A. thaliana has a low rate of effective recombination, sites in the 3-kb fragment of the GL1 locus are not independent of each other. To account for the correlation of molecular variation, we used a cladistic grouping of the A. thaliana accessions (see table 2
and fig. 3
). Three different grouping levels were considered in addition to the ANOVA based on individual accessions. It should be noted that our cladistic analysis differs from the one proposed by Templeton, Boerwinkle, and Sing (1987)
in the way we defined cladistic groups.
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Results |
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To test whether the presence of two diverged clades could explain the observed deviation from neutrality, we repeated the sliding-window analysis for the two GL1 clades independently. The pairwise nucleotide differences among the members of a clade were found to be very similar over the entire sequence (fig. 1
). Similarly, the test statistics implemented in DNA Slider were not significant for either of the two clades (table 4
). These results would be consistent with the hypothesis of balancing selection acting on the 3' region of the GL1 locus. To further test to what extent the observed sequence variability at the GL1 locus is influenced by natural selection, we used several neutrality tests. While Tajima's D for all 26 A. thaliana GL1 sequences was positive, as expected under balancing selection (Tajima 1989
), it was very small and not significant (D = 0.39, P > 0.1). Similarly, Fu and Li's (1993)
test statistics were also not significant (D = 0.41, P > 0.1; F = 0.43, P > 0.1). A McDonald-Kreitman test (McDonald and Kreitman 1991
) based on the A. lyrata accession with trichomes and 26 wild-type A. thaliana accessions did not reveal a significant deviation from neutral expectations (G = 0.03, P = 0.87). In summary, the evidence for a deviation from neutral evolution of the GL1 locus is not very strong and is based only on an observed heterogeneity in the ratio of fixed to polymorphic sites.
Linkage Disequilibrium at the GL1 Locus
Significant linkage disequilibrium was detected at the GL1 locus, with most significant pairwise comparisons in the 3' region (positions 16013998; fig. 4
). Out of 86 base substitutions and 21 indels in the 3' region, 44 substitutions and 17 indels were fixed between the assigned clades. These 61 polymorphisms were distributed over the entire 3' region. Any recombination event between the clades would result in shared mutations between the two clades. Because the large number of fixed differences between the two clades are distributed over the entire 3' region, very little or no recombination has occurred between them for a long time. The distribution of polymorphism in the 5' region shows the opposite trend. While in the 3' region about 51% of the base substitutions were fixed between the clades, none of the 21 polymorphic sites in the 5' region are fixed between the two clades. Only a single indel polymorphism (positions 327330) was consistent with the assignment to the two clades for 23 of 26 accessions, resulting in a significant pairwise linkage disequilibrium with the 44 substitutions fixed between clades A and B (D = 0.723, P < 0.001; fig. 4
).
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Analysis of 26 Worldwide Wild-Type Accessions
We counted the numbers of trichomes on different parts of the plant in most of the accessions sequenced. Substantial variation was detected in the distribution and density of trichomes between these accessions (table 1
). An ANOVA indicated that much of the observed phenotypic variation could be attributed to differences among accessions (P < 0.001). The highest trichome density was observed in the accession Can, with an average of 3.84 trichomes/mm2, which is more than twice as high as the trichome density observed in Cond, the accession with the second-highest trichome density. The lowest trichome density was observed in Gr-1, with 0.3 trichomes/mm2.
While it would be highly desirable to test all polymorphic sites at the GL1 locus for an association between phenotype and molecular variation, the low effective recombination rate of A. thaliana significantly complicates the interpretation of such an analysis, as the polymorphic sites are highly correlated. Thus, we used a cladistic approach to group the A. thaliana accessions in our study according to their shared history. Three different cladistic levels were analyzed by ANOVA to test for a correlation with trichome density, but none of the analyses were statistically significant (table 2 ). Interestingly, the extreme sequence divergence in the 3' region of the GL1 locus, dividing the GL1 sequences into two clades, could not be associated with trichome density. In addition, no pattern emerged for the other plant organs (table 1 ).
Analysis of Glabrous Plants
We used four natural accessions which were described as glabrous in the Nottingham stock center seed catalog. In two of these accessions (Wil-2 and Est), PCR amplification of GL1 failed completely despite the use of several primer combinations. Hence, we conclude that the glabrous phenotype may be caused by a large deletion encompassing the entire GL1 locus. The remaining two natural GL1 mutants (Br-0, Mir-0) contained a single-base-pair deletion in the second exon at the beginning of the R3 motif (table 5
). The resulting frameshift caused a premature stop after amino acid 93. Phenotypically, these accessions were identical to gl1-1, Wil-2, and Est, which have lost the GL1 gene. Hence, no trichomes could be detected on those plants.
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Analysis of A. lyrata
For comparison, we also sequenced GL1 in two A. lyrata individuals, one showing a glabrous phenotype. Interestingly, both individuals carried a 7-bp insertion in the third exon causing a frameshift at amino acid 215 and a stop codon 11 codons downstream. Because this mutation occurred independent of the trichome phenotype, it probably has no implications for the functionality of GL1. The glabrous plant carried an additional insertion of 4 bp at the beginning of the third exon in the R3 domain. This mutation leads to a frameshift of 10 codons and a truncation of 81 amino acids of the predicted GL1 gene product. We regard this mutation as the possible cause of the glabrous phenotype. An important conclusion from this naturally occurring glabrous phenotype is that, as in A. thaliana, GL1 is also required for trichome initiation in A. lyrata.
Cross-species comparisons indicated seven amino acid replacements between A. thaliana and A. lyrata. No amino acid replacement occurred in the conserved myb R2 (amino acids 1466, nucleotide positions 206491 in our alignment) and R3 (amino acids 67117, nucleotide positions 4921507) domains. We detected most amino acid replacements (86%) in the 110 terminal amino acids, suggesting only a limited requirement for the C-terminus.
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Discussion |
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Whether or not a simple historical explanation, such as a secondary contact of diverged A. thaliana populations (Innan et al. 1996
), fits the pattern of nucleotide polymorphism better requires more data but should be borne in mind as an alternative explanation.
Functional Analysis of Molecular Variation at GL1
One of the major goals of this study was to link phenotypic and genotypic variation to learn more about the function of the GL1 locus. Our analysis revealed substantial phenotypic and molecular variation. Most of the molecular variation could be attributed to the presence of two diverged sequence clades. Despite this extensive molecular divergence between the two clades, our ANOVA did not detect a significant effect of the molecularly defined clades on the measured trichome densities. Further ANOVA analyses also relying on a cladistic grouping of GL1 sequences did not show a statistically significant effect.
In principle, several different explanations for the lack of a statistically significant result could be invoked. The first explanation would be that an association between molecular variation and trichome density exists, but the statistical power to detect the association was too low. This hypothesis is supported by the large within-accession variance in trichome density observed in this study as well as in a previous report (Larkin et al. 1996
). A second explanation assumes an influence of additional genes. For example, the co-evolution of GL1 and genes interacting with GL1 could result in a nonsignificant ANOVA result. Assuming that each sequence variant is associated with a corresponding, co-evolved allele of one or several interacting genes, we would not expect to see an effect of the two sequence clades on trichome distribution. Recently, a similar scenario was tested for two genes (APETALA3 and PISTILLATA) involved in flower development which are known to interact with each other. The authors of the study detected significant linkage disequilibrium within genes (largely due to two allelic classes) but no significant intergenic association between polymorphic sites (Purugganan and Suddith 1999
). Hence, their results are not consistent with a co-evolution of the two classes of allelic variants. One further possibility is that the observed sequence variation at GL1 has an effect on trichome density, but a large effect of variation at another locus obscures the association of the phenotype with GL1 variation. Finally, GL1 may be an important gene for initiation of trichome formation, but it has no influence on trichome densities. We used a one-way ANOVA for three different cladistic groupings to test whether the partitioning of molecular variation at GL1 could explain the variation in trichome number among accessions (table 1
). While we did not observe a significant ANOVA, this could be simply due to the lack of statistical power. Nevertheless, a closer inspection of the three different cladistic levels indicates that the proportion of variance that could be explained by the grouping of accessions into different clades increases with the number of groups. This observation further corroborates the conclusion that shared polymorphic sites, as indicated by the cladistic grouping, fail to explain the observed variance in trichome density among accessions. Further evidence against the involvement of GL1 in trichome density comes from accessions with the identical sequence at GL1. Although these accessions (Es-0, As-0, Mt-0, RLD, and Tsu-0) lack sequence variation at GL1, they differ significantly in trichome density (ANOVA, P = 0.04). This observation is also consistent with a recent QTL analysis which identified one gene, not mapping to the GL1 locus, with a significant effect on trichome density (Larkin et al. 1996
).
The combined analysis of molecular variation in glabrous plants and the closely related species A. lyrata provides some insight into the functionally important regions of the GL1 gene. One important observation was that those mutations which resulted in a predicted truncation of the GL1 protein differed in the extent to which various plant organs were affected. The most extreme mutation, which did not affect trichome density, was a C-terminal insertion of 7 bp in A. lyrata. The resulting frameshift caused a replacement of 10 amino acids and a deletion of six terminal amino acids. A stop codon at amino acid position 202 in A. thaliana resulted in a weak phenotype in which mainly late rosette leaves had a reduced trichome formation (Esch, Oppenheimer, and Marks 1994
). The stop codon at position 181 resulted in a lack of trichomes on the rosette leaves and the stem. Cauline leaves of this mutant had a reduced number of trichomes. A frameshift mutation located in the highly conserved R3 domain (exon 3) which resulted in a stop codon at position 104, however, was completely glabrous.
Hence, the emerging picture is that the effect on trichome density becomes weaker the closer the truncation is located to the C-terminal end of the protein. Furthermore, late rosette leaves are generally more affected than the stem and cauline leaves. In particular, the latter organ showed some robustness toward truncations of the GL1 protein.
Interestingly, a single amino acid replacement (G100R) observed in the EMS-induced mutant gl1-65 in the R3 domain (nucleotide position 1454), which is conserved among many members of the myb proteins, was sufficient to produce a phenotype comparable to a deletion of 48 amino acids in the C-terminal region of the protein.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Keywords: trichome
Arabidopsis thaliana
Arabidopsis lyrata
balancing selection
GLABROUS1.
2 Address for correspondence and reprints: Christian Schlötterer, Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Josef-Baumann Gasse 1, A-1210 Vienna, Austria. christian.schloetterer{at}vu-wien.ac.at
.
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