Nucleotide polymorphism at the Atmyb2 locus of the wild plant Arabidopsis thaliana

DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (pi) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (pi = 0.0036) had a larger variation than the non-MYB region (pi = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.     

DNA variation in the basic chitinase locus (ChiB) region of the wild plant Arabidopsis thaliana.

Nucleotide variation in a 2.2-kbp region of basic chitinase (ChiB) locus in 17 ecotypes of Arabidopsis thaliana was compared with previously investigated regions to investigate genetic mechanisms acting on DNA polymorphism. In the ChiB region, dimorphic DNA variation was detected, as in the Adh and ChiA regions. Nucleotide diversity (pi) of the entire region was 0.0091, which was similar to those of the two other regions. About half of polymorphic sites (37/87) in the ChiB region were observed in only two ecotypes. Tajima's D was negative but not significantly, while Fu and Li's D* was positive. Neither McDonald-Kreitman nor Hudson, Kreitman, Aguadé tests showed a significant result, indicating that these loci were under similar evolutionary mechanisms before and after speciation. Linkage disequilibria were observed within the three regions because of dimorphic polymorphisms. Interlocus linkage disequilibrium was not detected between the Adh and the two chitinase regions, but was observed between the ChiA and ChiB regions. This could be due to epistatic interaction between the two chitinase loci, which are located on different chromosomes.     

DNA variation in the acidic chitinase locus (ChiA) region in Arabis gemmifera and its related species

We analyzed DNA variation at the acidic chitinase (ChiA) locus of Arabis gemmifera and among its eight related species. Nucleotide diversity (pi) of the entire locus in A. gemmifera was 0.0032, which was one third that of A. thaliana. In A. gemmifera, an excess of unique polymorphisms yielded significantly negative results with the tests of Tajima and Fu and Li. The McDonald and Kreitman test revealed that the ratio of nucleotide replacement to synonymous changes in A. gemmifera was significantly greater than those between A. gemmifera and A. glabra, A. gemmifera and A. griffithiana, A. gemmifera and A. korshinskyi, A. gemmifera and A. wallichii, and A. gemmifera and A. himalaica. These results indicated that the neutrality assumption, the equilibrium population assumption, or both, could not be applied to the ChiA locus of A. gemmifera. The small size and relative isolation of local subpopulations of A. gemmifera could explain the excess of unique polymorphisms and the high proportion of replacement changes. The specific sampling scheme of this study, where one strain each was sampled from each local subpopulation might also have led to an excess of singletons. Interspecific comparison among Arabidopsis, Arabis and Cardaminopsis species showed that Ka was always lower than Ks, providing evidence against the adaptive evolution of ChiA. However, Ka/Ks was greater between closely related species than between more distant related species. ChiA had a higher level of replacement divergence and a lower level of synonymous divergence compared than did Adh. We suggest that both the mutation rate at the nucleotide level and the selective constraints at the protein level are lower in ChiA than in Adh.     

DNA polymorphism at the cytosolic phosphoglucose isomerase (PgiC) locus of the wild plant Arabidopsis thaliana

DNA variation in a 4.7-kb region of the cytosolic phosphoglucose isomerase (PgiC) locus was investigated for 21 ecotypes of Arabidopsis thaliana. The estimated nucleotide diversity was 0.0038, which was one-third of those in previously investigated loci. Since most of the nucleotide variations (93%) were singleton and doubleton, Tajima's test statistic was significantly negative. About 50% of nucleotide polymorphisms in exons were replacement, which caused significance in McDonald and Kreitman's test when compared with Arabis gemmifera and Cardaminopsis petraea. These results indicated that DNA polymorphism at the PgiC locus was not under neutrality. There were two divergent sequence types in the PgiC region, which were associated with allozyme variation. The Fast allozyme was shown to have originated from the Slow allozyme, since two outgroup species had the Slow form. A phylogenetic tree of ecotypes with the Fast allozyme had the shape of a star phylogeny. Mismatch distribution of the Fast allozyme ecotypes resembled that expected under an expanding population model. These results suggest positive selection for the Fast allozyme of the PGIC in A. thaliana.     

DNA polymorphism at the ACAULIS5 locus of the wild plant Arabidopsis thaliana

Nucleotide variation in the ACL5 gene region, which encodes spermine synthase, was analyzed for 21 Arabidopsis thaliana ecotypes and one accession of Arabis gemmifera. In A. thaliana, dimorphism was also detected in the ACL5 region, as in other nuclear genes of this plant. The nucleotide diversity (pi) of the entire region, exon and intron was 0.0163, 0.0042 and 0.0293, respectively. The level of nucleotide variation in this region was among the highest of those reported for genes in this plant species. The neutrality tests of Tajima, and Fu and Li did not detect significant deviation from test assumptions for the polymorphism data. However, the HKA test indicated that the level of polymorphism in the intron was significantly high, compared with A. gemmifera. The high nucleotide variation in the intron is responsible for the high level of nucleotide variation in the entire region. These results can be explained by elevated mutation rate in the ACL5 region in the A. thaliana lineage after the two species were split.     

Nucleotide variation at the CHALCONE ISOMERASE locus in Arabidopsis thaliana

An approximately 1.9-kb region encompassing the CHI gene, which encodes chalcone isomerase, was sequenced in 24 worldwide ecotypes of Arabidopsis thaliana (L.) Heynh. and in 1 ecotype of A. lyrata ssp. petraea. There was no evidence for dimorphism at the CHI region. A minimum of three recombination events was inferred in the history of the sampled ecotypes of the highly selfing A. thaliana. The estimated nucleotide diversity theta(TOTAL) = 0.004, theta(SIL) = 0. 005 was on the lower part of the range of the corresponding estimates for other gene regions. The skewness of the frequency spectrum toward an excess of low-frequency polymorphisms, together with the bell-shaped distribution of pairwise nucleotide differences at CHI, suggests that A. thaliana has recently experienced a rapid population growth. Although this pattern could also be explained by a recent selective sweep at the studied region, results from the other studied loci and from an AFLP survey seem to support the expansion hypothesis. Comparison of silent polymorphism and divergence at the CHI region and at the Adh1 and ChiA revealed in some cases a significant deviation of the direct relationship predicted by the neutral theory, which would be compatible with balancing selection acting at the latter regions.     

DNA variation in the wild plant Arabidopsis thaliana revealed by amplified fragment length polymorphism analysis.

To investigate the level and pattern of DNA variation of Arabidopsis thaliana at the entire genome level, AFLP analysis was conducted for 38 ecotypes distributed throughout the world. Ten pairs of selective primers were used to detect a total of 472 bands, of which 374 (79. 2%) were polymorphic. The frequency distribution of polymorphic bands was skewed toward an excess of singleton variation. On the basis of AFLP variation, nucleotide diversity for the entire genome was estimated to be 0.0106, which was within the range reported previously for specific nuclear genes. The frequency distribution of pairwise distance was bimodal because of an ecotype (Fl-3) with a large number of unique bands. Linkage disequilibrium between polymorphic AFLPs was tested. The proportion of significant linkage disequilibria was close to random expectation after neglecting the ecotype Fl-3. This result indicates that the effect of recombination could not be ignored in this selfing species. A neighbor-joining tree was constructed on the basis of the AFLP variation. This tree has a star-like topology and shows no clear association between ecotype and geographic origin, suggesting a recent spread of this plant species and limited migration between its habitats.     

Nucleotide polymorphism in the Adh2 region of the wild rice Oryza rufipogon

DNA variation in the alcohol dehydrogenase (Adh2) region of the wild rice Oryza rufipogon and its related species was analyzed to clarify maintenance mechanisms of the DNA variation in these species. A dimorphic pattern was detected in the Adh2 region of O. rufipogon. The silent nucleotide diversity (π) in the Adh2 region in O. rufipogon was 0.011, which was higher than that of the Adh1 region in O. rufipogon. Especially, a high nucleotide diversity was detected at synonymous sites of the catalytic domain 1. Average nucleotide diversity at silent sites within each of the dimorphic sequence types of the Adh2 region was similar to that in the Adh1 region, indicating that the high level of silent polymorphism in the Adh2 region was caused by the difference between the dimorphic sequence types. On the other hand, the level of replacement polymorphism in the Adh2 region was as low as that in the Adh1 region. The neutrality test of Fu and Li indicated significantly negative deviation from the neutral mutation model for the replacement sites of the Adh2 region. This result suggests purifying selection on the replacement sites of the Adh2 region, as detected for the Adh1 region. Significant linkage disequilibria (16.4% of the tests) were detected between the Adh1 and Adh2 regions. Even when nonrandom association was tested for the strains belonging to one of the divergent sequence types of the Adh2 region, significant interlocus linkage disequilibria were detected. The close physical distance and/or epistasis between the two Adh regions could be invoked to explain these nonrandom associations.     

Contrasting patterns of nucleotide polymorphism at the alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsis thaliana

Nucleotide variation at the alcohol dehydrogenase locus (Adh) was studied in the outcrossing Arabidopsis lyrata, a close relative of the selfing Arabidopsis thaliana. Overall, estimated nucleotide diversity in the North American ssp. lyrata and two European ssp. petraea populations was 0.0038, lower than the corresponding specieswide estimate for A. thaliana at the same set of nucleotide sites. The distribution of segregating sites across the gene differed between the two species. Estimated sequence diversity within an A. lyrata population with a large sample size (0.0023) was much higher than has previously been observed for A. thaliana. This North American population has an excess of sites at intermediate frequencies compared with neutral expectation (Tajima's D = 2.3, P < 0.005), suggestive of linked balancing selection or a recent population bottleneck. In contrast, an excess of rare polymorphisms has been found in A. thaliana. Polymorphism within A. lyrata and divergence from A. thaliana appear to be correlated across the Adh gene sequence. The geographic distribution of polymorphism was quite different from that of A. thaliana, for which earlier studies of several genes found low within-population nucleotide site polymorphism and no overall continental differentiation of variation despite large differences in site frequencies between local populations. Differences between the outcrossing A. lyrata and the selfing A. thaliana reflect the impact of differences in mating system and the influence of bottlenecks in A. thaliana during rapid colonization on DNA sequence polymorphism. The influence of additional variability-reducing mechanisms, such as background selection or hitchhiking, may not be discernible.     

Linkage disequilibrium between incompatibility locus region genes in the plant Arabidopsis lyrata

We have studied diversity in Arabidopsis lyrata of sequences orthologous to the ARK3 gene of A. thaliana. Our main goal was to test for recombination in the S-locus region. In A. thaliana, the single-copy ARK3 gene is closely linked to the non-functional copies of the self-incompatibility loci, and the ortholog in A. lyrata (a self-incompatible species) is in the homologous genome region and is known as Aly8. It is thus of interest to test whether Aly8 sequence diversity is elevated due to close linkage to the highly polymorphic incompatibility locus, as is theoretically predicted. However, Aly8 is not a single-copy gene, and the presence of paralogs could also lead to the appearance of elevated diversity. We established a typing approach based on different lengths of Aly8 PCR products and show that most A. lyrata haplotypes have a single copy, but some have two gene copies, both closely linked to the incompatibility locus, one being a pseudogene. We determined the phase of multiple haplotypes in families of plants from Icelandic and other populations. Different Aly8 sequence types are associated with different SRK alleles, while haplotypes with the same SRK sequences tend to have the same Aly8 sequence. There is evidence of some exchange of sequences between different Aly8 sequences, making it difficult to determine which ones are allelic or to estimate the diversity. However, the homogeneity of the Aly8 sequences of each S-haplotype suggests that recombination between the loci has been very infrequent over the evolutionary history of these populations. Overall, the results suggest that recombination rarely occurs in the interval between the S-loci and Aly8 and that linkage to the S-loci can probably account for the observed high Aly8 diversity.     

Translational polymorphism as a potential source of plant proteins variety in Arabidopsis thaliana

MOTIVATION: According to scanning model, 40S ribosomal subunits can either initiate translation at start AUG codon in suboptimal context or miss it and initiate translation at downstream AUG(s), thereby producing several proteins. Functional significance of such a protein translational polymorphism is still unknown. RESULTS: We compared predicted subcellular localizations of annotated Arabidopsis thaliana proteins and their potential N-terminally truncated forms started from the nearest downstream in-frame AUG codons. It was found that localizations of full and N-truncated proteins differ in many cases: 12.2% of N-truncated proteins acquired sorting signals de novo and 5.7% changed their predicted subcellular locations (mitochodria, chloroplast or secretory pathway). It is likely that the in-frame downstream AUGs may be frequently utilized to synthesize proteins possessing new functional properties and such a translational polymorphism may serve as an important source of cellular and organelle proteomes.     

The pattern of polymorphism in Arabidopsis thaliana

We resequenced 876 short fragments in a sample of 96 individuals of Arabidopsis thaliana that included stock center accessions as well as a hierarchical sample from natural populations. Although A. thaliana is a selfing weed, the pattern of polymorphism in general agrees with what is expected for a widely distributed, sexually reproducing species. Linkage disequilibrium decays rapidly, within 50 kb. Variation is shared worldwide, although population structure and isolation by distance are evident. The data fail to fit standard neutral models in several ways. There is a genome-wide excess of rare alleles, at least partially due to selection. There is too much variation between genomic regions in the level of polymorphism. The local level of polymorphism is negatively correlated with gene density and positively correlated with segmental duplications. Because the data do not fit theoretical null distributions, attempts to infer natural selection from polymorphism data will require genome-wide surveys of polymorphism in order to identify anomalous regions. Despite this, our data support the utility of A. thaliana as a model for evolutionary functional genomics.     

A new locus (NIA 1) in Arabidopsis thaliana encoding nitrate reductase.

We have isolated two nitrate reductase genes and their corresponding cDNAs from Arabidopsis thaliana. Sequences of the two cDNAs, when compared to a sequence of a barley cDNA clone, confirm their identity as nitrate reductase clones and show that they are closely related. The two genes have been mapped using restriction fragment length polymorphisms; gNR2 is close to the previously identified chl-3 locus and is probably identical to it, while gNR1 maps to a new locus (NIA1) on chromosome 1, near gl-2.     

Trimorphic DNA variation in the receptor-like protein kinase gene in the F18L15-130 region of the wild plant Arabidopsis thaliana

DNA variation in the F18L15-130 region, which contains a receptor-like protein kinase gene, was analyzed for the wild plants Arabidopsis thaliana and Arabis gemmifera. In A. thaliana, at least three divergent sequence types were observed (trimorphism), instead of two distinct sequence types (dimorphism) detected in most of other nuclear gene regions studied for this plant species. Intragenic recombinations among the divergent sequence types generated four recombinant sequence types, although pattern of intragenic recombinations was complex. The estimated nucleotide variation in A. thaliana was the highest (pi = 0.0226 and theta = 0.0228) among genes investigated in this plant so far. The high level of nucleotide variation is due to divergence among the three distinct sequence types, each of which has a low level of nucleotide variation. Tests of Tajima, and Fu and Li did not detected deviation from neutrality, except for replacement sites, for which significantly negative Fu and Li's test value was detected. These results suggest that the receptor-like protein kinase gene in this region is functional. Possible causes for the trimorphic pattern of DNA polymorphism was discussed.     

Correlative controls of senescence and plant death in Arabidopsis thaliana (Brassicaceae)

Like most monocarpic plants, longevity of Arabidopsis thaliana plants is controlled by the reproductive structures; however, they appear to work differently from most dicots studied. Neither male- and female-sterility mutations (ms1-1 and bell1, respectively) nor surgical removal of the stems with inflorescences (bolts) at various stages significantly increased the longevity of individual rosette leaves, yet the mutants and treated plants lived 20-50 d longer, measured by the death of the last rosette and/or the last cauline leaf. A series of growth mutations (clv2-4, clv3-2, det3, vam1 enh, and dark green) also increased plant longevity by 20-30 d but did not delay the overall development of the plants. The mutations prolonged plant life through the production of new leaves and stems with inflorescences (bolts) rather than by extending leaf longevity. In growing stems, the newly-formed leaves may induce senescence in the older leaves; however, removal of the younger leaves did not significantly increase the life of the older leaves on the compressed stems of Arabidopsis. Since plants that produce more bolts also live longer, the reproductive load (dry weight) of the bolts did not seem to drive leaf or whole plant senescence here. The developing reproductive structures caused the death of the plant by preventing regeneration of leaves and bolts, which are green and presumably photosynthetic. They also exerted a correlative control (repression) on the development of additional reproductive structures.     

Targeted disruption of the TGA3 locus in Arabidopsis thaliana

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non-selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A β-glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Km^r calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient, transformation methodologies, gene replacement studies in plants could become a routine technique.