Nucleotide polymorphism in the Adh1 locus region of the wild rice Oryza rufipogon.

Nucleotide variation in the alcohol dehydrogenase (Adh1) locus region of the wild rice Oryza rufipogon and its related species was analysed to clarify the maintenance mechanism of DNA variation in Oryza species. The estimated nucleotide diversity in the Adh1 locus region of O. rufipogon was 0.002, which was one of the lowest values detected in nuclear loci of plant species investigated so far. Tests of neutrality detected significantly negative deviation from the neutral mutation model for the coding region, especially for replacement sites. When each of the ADH1 domains was considered, significance was detected only for the catalytic domain 1. These results suggest purifying selection in the Adh1 coding region. In the phylogenetic tree of Oryza species based on Adh1 variation, cultivated rice O. sativa subspp. japonica and indica were included in the cluster of O. rufipogon. The genetic distance of the Adh1 region between O. rufipogon and O. sativa was as low as the nucleotide diversity of O. rufipogon. These results imply that O. rufipogon and O. sativa cannot be classified based on the nucleotide variation of Adh1. No replacement divergence between O. rufipogon and the other three A-genome species (O. glumaepatula, O. barthii and O. meridionalis) were detected, indicating that ADH1 is conserved in the A-genome species. On the other hand, between O. rufipogon and the E-genome species O. australiensis, replacement changes were detected only in the catalytic domain 1. The difference in replacement substitutions between the A- and E-genome species may be related to adaptive changes in the ADH1 domains, reflecting environmental differences where the species encounter anaerobic stress.

DNA variation in the 5' upstream region of the Adh locus of the wild plants Arabidopsis thaliana and Arabis gemmifera.

To investigate the level and pattern of DNA polymorphism in the noncoding regulatory region in the plant nuclear genome, 2.4 kb of nucleotide sequence of the 5' upstream region of ADH: was determined for 14 ecotypes of Arabidopsis thaliana and five accessions of Arabis gemmifera. Using this data set and previously determined ADH: sequence data, DNA variation was analyzed in a 4.4-kb region of the locus. Two divergent sequence types detected in the transcriptional unit of ADH: were not present in the 5' region of the ADH: gene in A. thaliana. Nucleotide diversity of the entire 5' region was estimated to be 0.0040, which is lower than that in the transcriptional unit. The level of variation was not uniform. There were peaks of variations in a approximately 400-bp region where cis-regulatory elements for ADH: expression were clustered and in exon 4. In interspecific comparison with A. gemmifera, lower divergence was observed in the 5' flanking region than in the exons. High peaks of divergence in the 400-bp regulatory region and exon 4 were also detected, although there were many other peaks. These results indicate that regions of functional importance have a high level of polymorphism and divergence in the ADH: locus of these genera. The possibility of balancing selection in the ADH: gene of these plants is discussed.

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 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 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.

Intra- and interspecific DNA variation and codon bias of the alcohol dehydrogenase (Adh) locus in Arabis and Arabidopsis species.

Sequence variation at the alcohol dehydrogenase (Adh) locus was analyzed for six species each of the genera Arabis and Arabidopsis. Phylogenetic analysis showed that investigated species were grouped into three clusters, and the generic classification did not correspond to the clusterings. The results indicated that the genera could not be distinguished on the basis of the Adh variation. A significant difference in the ratio of silent to replacement sites was detected by MK test in two comparisons, with Arabidopsis thaliana polymorphism due to excess silent divergence. Silent changes were predominant in the evolution of the Adh locus in Arabis and Arabidopsis. To infer evolutionary significance of silent substitutions, codon bias was studied. The degree of codon bias of the Adh region was relatively constant over Arabis and Arabidopsis species. "Preferred" codons of A. thaliana were determined. No evidence of natural selection on codon change was detected in the Adh regions of A. thaliana and Arabis gemmifera.

Subfamily divergence in the multigene family of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) in Triticeae and its relatives.

To investigate genetic mechanisms acting on multigene family in plants, we analyzed sequence variation in the rbcS gene of 13 species of Triticeae and one species each of related tribes (Bromeae and Aveneae). A total of 36 rbcS genes were analyzed. Based on dimorphism in the length of intron, the rbcSs of investigated species were classified into two subfamilies A and B. The difference in intron length was caused by an indel of about 200 bp in the middle of the intron. The two subfamilies of rbcS were present in the three tribes, indicating that the divergence of rbcS subfamilies occurred before the split of these tribes. Generally, variation between the two subfamilies of rbcS was larger than that within subfamily, but these two measures were about the same at the tribe level. This result suggested that divergence of the subfamilies of rbcS occurred at about the same time of tribe diversification. The level of nucleotide variation in the exon region between subfamilies was reduced in the Triticeae, but clear change was not detected in the intron sequence. This result suggested that the exon sequences between subfamilies of rbcS were homogenized without affecting the intron sequence in the Triticeae lineage.

Nucleotide polymorphism in the acidic chitinase locus (ChiA) region of the wild plant Arabidopsis thaliana.

To investigate DNA variation in natural plant populations, a 1.8-kb region of the acidic chitinase locus (ChiA)was analyzed for 17 ecotypes of Arabidopsis thaliana sampled worldwide and 3 Arabis species in Japan. As in the Adh region, dimorphism was detected throughout the investigated ChiA region, suggesting the possibility that dimorphic DNA variation exists in the entire nuclear genome of A. thaliana. The ChiA region was divided into two blocks by an intragenic recombination between two parental sequence types, which diverged 7.4 MYA under the assumption that nucleotide mutation rate per site per year is mu = 10(-9). Nucleotide diversity in the entire ChiA region was 0.0104. Tajima's test was significantly negative for both nucleotide and indel variations, which was manifested as an excess of unique polymorphisms. However, the level and pattern of polymorphism in the ChiA region were inconsistent with simple theoretical explanations. The HKA test detected no difference in the levels of intra- and interspecific variations between the ChiA and Adh regions. In the ChiA coding region, no difference in the patterns of synonymous and replacement variation was found in intra- and interspecific comparisons by the MK test. Although it was difficult to determine the exact genetic mechanism acting on the ChA locus, these results suggested that the ChA locus region was under the same genetic mechanism before and after the establishment of A. thaliana as a species.

DNA polymorphism at the Pgi locus of a wild yam, Dioscorea tokoro.

To study the origin and maintenance mechanisms of the PGI allozyme polymorphism of a wild plant, Dioscorea tokoro, DNA sequences of the entire coding region (1701 bp) and two intronic regions (total 2049 bp) of the Pgi gene as well as a part of the Adh gene (590 bp) were analyzed. Two replacement substitutions were revealed to be responsible for the differentiation of three allozymes alleles (Pgi-a, Pgi-b and Pgi-c) that occur in natural population in intermediate frequencies. Interspecific comparison of DNA sequences identified Pgi-b as the oldest allele, from which two other alleles were derived probably within the last 150,000 years. The level of DNA polymorphism at D. tokoro Pgi locus was low. No elevated level of DNA polymorphism was detected in the close vicinity of the two replacement sites differentiating the three allozymes. Departures from the neutral mutation hypothesis were detected by Fu and Li's and MK tests. The observed patterns of DNA polymorphism are explainable by both (1) the neutral mutation hypothesis with an assumption of small effective size of D. tokoro population, and (2) the positive selection hypothesis that the allele frequencies of Pgi-a and Pgi-c have increased in a short time by their selective advantages.

Microsatellite polymorphism in natural populations of the wild plant Arabidopsis thaliana.

Variation in repeat number at 20 microsatellite loci of Arabidopsis thaliana was studied in a worldwide sample of 42 ecotypes to investigate the pattern and level of polymorphism in repetitive sequences in natural plant populations. There is a substantial amount of variation at microsatellite loci despite the selfing nature of this plant species. The average gene diversity was 0.794 and the average number of alleles per locus was 10.6. The distribution of alleles was centered around the mean of repeat number at most loci, but could not be regarded as normal. There was a significantly positive correlation between the number of repeats and the amount of variation. For most loci, the observed number of alleles was between the expected values of the infinite allele and stepwise mutation models. The two models were rejected by the sign test. Linkage disequilibrium was detected in 12.1% of the pairwise comparisons between loci. In phylogenetic tree, there was no association between ecotype and geographic origin. This result is consistent with the recent expansion of A. thaliana throughout the world.

Variation in coxII intron in the wild ancestral species of wheat.

To study the maternal lineage and evolution of polyploid species of wheat, variation in mitochondrial DNA was investigated in Triticum and Aegilops by PCR-aided RFLP analysis. A 1.3 kb region containing the intron of coxII was studied using 20 accessions from five species of Sitopsis section of Aegilops, one species of Einkorn wheat, four species of tetraploid wheat, and one species of common wheat. Only three restriction site changes and a single deletion/insertion were found among 884 restriction fragments surveyed. This fact suggests the highly conserved nature of this region within Triticum and Aegilops. Four haplo-types were recognized in coxII intron. A parsimonious relationship indicated that three haplo-types were independently derived from one prototype which was found in wild Einkorn and Aegilops species except for Ae. speltoides. All but one accession of Ae. speltoides possessed a derivative haplo-type, common in Timopheevi wheat. The result supported the hypothesis that Ae. speltoides donated the G genome to Timopheevi wheat; however did not agree with that Ae. speltoides was the B genome donor to the Emmer and common wheat.

Plasmon analyses of Triticum (wheat) and Aegilops: PCR-single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs.

To investigate phylogenetic relationships among plasmons in Triticum and Aegilops, PCR-single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13. 7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28. 9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn, Triticum, and Aegilops). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one Aegilops species, Ae. speltoides, was included in the Triticum group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.

Polymerase chain reaction-single strand conformational polymorphism analysis of intra- and interspecific variations in organellar DNA regions of Aegilops mutica and related species.

In order to study the phylogeny of Aegilops mutica in the genera of Triticum and Aegilops, variations in chloroplast and mitochondrial DNA regions were investigated by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. Nine lines, each of Ae. mutica and Ae. speltoides, were studied together with nine other Triticum and Aegilops species, including T. aestivum. By analyzing 9.7-kb chloroplast and 13.1-kb mitochondrial DNA regions, a total of 268 bands were detected, of which 176 (65.7%) showed variation within and/or between species. The level of intraspecific variation of Ae. mutica was lower than that of Ae. speltoides. The low level of the intraspecific variation of Ae. mutica was contrary to the expectation from previous studies on morphological and cytolo-gical characters. In the phylogenetic trees based on SSCP, Ae. mutica, Ae. speltoides and the other four species of the section Sitopsis (the subsection Emarginata) were separated into three different clusters. In addition, T. aestivum was included in the cluster of Ae. speltoides in the phylogenetic trees. This result suggests that Ae. speltoides is the cytoplasmic donor of common wheat.

Intragenic recombination in the Adh locus of the wild plant Arabidopsis thaliana.

Nucleotide variation in the Adh region of the wild plant Arabidopsis thaliana was analyzed in 17 ecotypes sampled worldwide to investigate DNA polymorphism in natural plant populations. The investigated 2.4-kb Adh region was divided into four blocks by intragenic recombinations between two parental sequence types that diverged 6.3 million years (Myr) ago, if the nucleotide mutation rate mu = 10(-9) is assumed. Within each block, dimorphism of segregating variations was observed with intermediate frequencies, which caused a substantial amount of nucleotide variation in A. thaliana at the species level. The first recombination introduced the divergent variation that resulted in dimorphism in this plant species approximately 3.3 Myr ago, and three subsequent intragenic combinations have occurred sporadically in approximately 1.1-Myr intervals. It was shown that there was only a limited number (six) of sequence types in this species and that no clear association was observed between sequence type and geographic origin. Taken together, these results suggest that A. thaliana has spread over the world only recently. It can be concluded that recombination played an important role in the evolutionary history of A. thaliana, especially through the generation of DNA polymorphism in the natural populations of this plant species.

Wheat phylogeny determined by RFLP analysis of nuclear DNA. 3. Intra- and interspecific variations of five Aegilops Sitopsis species.

The level of intra- and interspecific variations on nuclear DNA in five Aegilops species of the Sitopsis section were investigated using restriction fragment length polymorphism (RFLP) analysis. A total of 18 accessions, i.e. 7 of Ae. speltoides, 3 of Ae. longissima, 2 of Ae. searsii, 3 of Ae. sharonensis and 3 of Ae. bicornis, were used. One accession each of Triticum aestivum, T. durum, T. urartu and Ae. squarrosa was included as reference material. Five enzymes and 20 probes were used. Among the five Sitopsis species studied, Ae. speltoides had the largest intraspecific variation (π=0.061), which was as high as the interspecific variation observed among the other four species. The section Sitopsis was divided into two distinct groups: one containing only Ae. speltoides and the other, Ae. longissima, Ae. searsii, Ae. sharonensis and Ae. bicornis. This grouping by RFLP analysis is in agreement with the taxonomical classification of the subsections.

DNA fingerprinting study on the intraspecific variation and the origin of Prunus yedoensis (Someiyoshino).

In order to investigate the intraspecific variation of Prunus yedoensis (Someiyoshino) and interspecific relationship among P. yedoensis, P. lannesiana (Oshimazakura) and P. pendula (Edohigan), DNA fingerprinting study was conducted by using two different kinds of probes, M13 repeat sequence and (GACA)4 synthetic oligonucleotide. In this study, 68 plants of P. yedoensis grown in 46 prefectures in Japan were investigated. All the P. yedoensis individuals investigated showed the completely same banding pattern, indicating their clonal origin from a single plant. On the other hand, each of P. lannesiana and P. pendula individuals investigated showed a unique banding pattern, suggesting a considerable amount of genetic variation in these two species. About 90% of bands in DNA fingerprints of P. yedoensis were detected in either P. lannesiana or P. pendula. This result supports the hypothesis that P. yedoensis is an interspecific hybrid between P. lannesiana and P. pendula. From those results, it is concluded that P. yedoensis was produced only once through hybridization between P. lannesiana and P. pendula, and that this particular hybrid plant has been spread vegetatively all over Japan.

Molecular variation in chloroplast DNA regions in ancestral species of wheat.

Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.

Restriction map polymorphism in the forked and vermilion regions of Drosophila melanogaster.

Restriction map polymorphism at two X linked foci, forked and vermilion of Drosophila melanogaster was studied in three natural populations. The estimates of nucleotide variation were theta = 0.003 and pi = 0.002 for the forked region and theta = 0.004 and pi = 0.002 for the vermilion region. Three insertions (> 500 bp) were observed at each locus. Typical of other regions of this species each of these large insertions was unique in the sample. Non-random association among polymorphisms was common at the vermilion locus, while the forked locus was not polymorphic enough to test linkage disequilibrium. The amounts of restriction site and size variation in the vermilion and forked were within the range observed for other loci of D. melanogaster.

Statistical methods for estimating the effective number of alleles, expected heterozygosity and genetic distance in self-incompatibility locus.

In order to understand the evolutionary process of self-incompatibility, we must know the genetic variability of the self-incompatibility genes within and between populations. Statistical methods for estimating the effective number of alleles, expected heterozygosity and genetic distance from pollination experiments were developed, which can be applied to both gametophytic and sporophytic self-incompatibility systems. In these methods, bud-pollination, which is necessary for obtaining homozygotes, is not required. Since bud-pollination, which is time-consuming, is not required in the present methods, they might be useful.