Rhizospheric bacterial community structure of Triticum and Aegilops revealed by pyrosequencing analysis of the 16S rRNA gene: dominance of the A genome over the B and D genomes.

This study examined the relationship between host plant and rhizospheric bacterial community structure, including composition and diversity, in Triticum and Aegilops species (12 and two accessions, respectively) as well as three closely related species, barley, rye and oat (four accessions), to explore the possibility that wheat root and rhizosphere interaction can be utilized for wheat breeding and biotechnology in the future. For this purpose, DNA was isolated from rhizospheric soil samples and one control non-rhizospheric soil sample, and the 16S rRNA gene region was amplified and subjected to DNA pyrosequencing. A total of 132,888 amplicons were analyzed. Bacterial composition at the phylum level was similar among the 18 rhizospheric samples; however, the proportion of Acidobacteria was much lower in these samples than in the control non-rhizospheric soil sample, indicating that rhizospheres influenced the bacterial composition even at the higher taxonomic level. Across host plant genome types (three levels of ploidy and three major genomes, A, B and D), there was no detectable difference in phylum composition or species diversity. Estimated bacterial species diversity was higher in the control soil sample than in plant rhizospheric soils, implying that bacterial species diversity was reduced in rhizospheres. A PCoA plot and UPGMA dendrogram based on the bacterial species composition showed that control soil was distantly located from the plant rhizospheric samples and that Triticum, Aegilops and related species were well separated. PERMANOVA analysis detected statistically significant differentiation among these four groups. Clustering of Triticum species suggested that the A genome was dominant over the B and D genomes, with respect to the influence on rhizospheric bacterial species composition. Although the cause was not investigated in this study, these results clearly indicated that the genetic constitution of the plant host exerted a strong influence on rhizospheric bacterial community structure.

Geographical variation in soil bacterial community structure in tropical forests in Southeast Asia and temperate forests in Japan based on pyrosequencing analysis of 16S rRNA.

Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (ß-diversity) and direct distance between forests. Both α- and ß-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.

Contrasting soil bacterial community structure between the phyla Acidobacteria and Proteobacteria in tropical Southeast Asian and temperate Japanese forests.

Soil bacterial community structures of six dominant phyla (Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes, Bacteroidetes and Actinobacteria) and unclassified bacteria detected in tropical Sarawakian and temperate Japanese forests were compared based on 16S rRNA gene sequence variation. The class composition in each phylum was similar among the studied forests; however, significant heterogeneities of class frequencies were detected. Acidobacteria and Proteobacteria were the most dominant phyla in all six forests, but differed in the level of bacterial species diversity, pattern of species occurrence and association pattern of species composition with physicochemical properties in soil. Species diversity among Acidobacteria was approximately half that among Proteobacteria, based on the number of clusters and the Chao1 index, even though a similar number of sequence reads were obtained for these two phyla. In contrast, species diversity within Planctomycetes and Bacteroidetes was nearly as high as within Acidobacteria, despite many fewer sequence reads. The density of species (the number of sequence reads per cluster) correlated negatively with species diversity, and species density within Acidobacteria was approximately twice that within Proteobacteria. Although the percentage of forest-specific species was high for all bacterial groups, sampling site-specific species varied among bacterial groups, indicating limited inter-forest migration and differential movement of bacteria in forest soil. For five of the seven bacterial groups, including Acidobacteria, soil pH appeared to strongly influence species composition, but this association was not observed for Proteobacterial species. Topology of UPGMA trees and pattern of NMDS plots among the forests differed among the bacterial groups, suggesting that each bacterial group has adapted and evolved independently in each forest.

Soil bacterial community structure in five tropical forests in Malaysia and one temperate forest in Japan revealed by pyrosequencing analyses of 16S rRNA gene sequence variation.

Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40-50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40-60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro- and macro-environments, but macro-environmental diversity made a larger contribution to total bacterial diversity in forest soil.

DNA polymorphism in the blast disease resistance gene Pita of the wild rice Oryza rufipogon and its related species.

Intra-and interspecific DNA variations in the blast resistance gene Pita in wild rice (Oryza rufipogon), cultivated rice (O. sativa), and two other related wild rice species (O. meridionalis and O. officinalis) were analyzed to elucidate the nucleotide polymorphism maintenance mechanisms and evolution of Pita in these species. Nucleotide diversity at silent sites of O. rufipogon Pita was 0.0101, an intermediate value relative to other O. rufipogon nuclear genes. A dimorphic pattern of nucleotide polymorphism was detected in the O. rufipogon Pita region. Inoculation of the blast fungus Magnaporthe oryzae verified that the O. rufipogon Pita gene resides in a dimorphic sequence type. The resistance Pita allele had lower levels of variation than the susceptibility pita allele. A hypothesis of evolutionary relationships indicated that the amino acid mutation in the O. rufipogon Pita protein responsible for the difference between resistance and susceptibility occurred relatively recently. These results suggested that the resistance Pita originated from the susceptibility pita. Nucleotide diversity at replacement sites of the leucine-rich domain (LRD) of both the resistance and susceptibility O. rufipogon pita was low. In tests of neutrality, significantly negative values were detected for the LRD of O. rufipogon susceptibility pita. The low nucleotide diversity at replacement sites of the LRD of the susceptibility pita could be explained by purifying selection. Comparison of Pita between O. rufipogon and O. officinalis revealed an excess of nonconservative amino acid substitutions in the LRD, which could be related to the host-pathogen interaction.

DNA polymorphism in the SUPERWOMAN1 (SPW1) locus of the wild rice Oryza rufipogon and its related species.

The level and pattern of nucleotide variation in the SUPERWOMAN1(SPW1) locus of the wild rice Oriza rufipogon and its related species were analyzed. SPW1 is orthologous to APETELA3(AP3) in Arabidopsis thaliana. The estimated level of nucleotide variation for the entire region (pi = 0.0046) was intermediate among those for other genes of O. rufipogon, although the estimates varied considerably among the SPW1 domains. Complicated haplotype structure was detected, resulting in a high proportion of significant linkage disequilibrium. Deviation from the neutrality was not detected. However, purifying selection was suggested by lack of replacement variation in the MADS-box and I-domain regions, which function as DNA-binding domain. On the other hand, an excess of drastic amino acid changes was detected in the C-domain, as in the AP3 region of A. thaliana. Taken together, these results imply that different types of natural selection are acting among different domains of a single protein. In the phylogenetic tree, O. sativa strains were included in the same cluster of O. rufipogon, supporting the hypothesis that O. rufipogon is the wild ancestor of O. sativa.

Large-scale DNA polymorphism study of Oryza sativa and O. rufipogon reveals the origin and divergence of Asian rice.

Polymorphism over approximately 26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (pi = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (pi = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to approximately 50 kb in O. sativa ssp. indica.

DNA variation in the metallothionein genes in wild rice Oryza rufipogon: relationship between DNA sequence polymorphism, codon bias and gene expression.

This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism.

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 (pi) 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.

Patterns of codon usage bias in three dicot and four monocot plant species.

Codon usage in nuclear genes of four monocot and three dicot species was analyzed to find general patterns in codon choice of plant species. Codon bias was correlated with GC content at the third codon position. GC contents were higher in monocot species than in dicot species at all codon positions. The high GC contents of monocot species might be the result of relatively strong mutational bias that occurred in the lineage of the Poaceae species. In both dicot and monocot species, the effective number of codons (ENCs) for most genes was similar to that for the expected ENCs based on the GC content at the third codon positions. G and C ending codons were detected as the "preferred" codons in monocot species, as in Drosophila. Also, many "preferred" codons are the same in dicot species. Pyrimidine (C and T) is used more frequently than purine (G and A) in four-fold degenerate codon groups.

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.

DNA polymorphism in active gene and pseudogene of the cytosolic phosphoglucose isomerase (PgiC) loci in Arabidopsis halleri ssp. gemmifera.

DNA variations in two PgiC loci were investigated in 15 strains of Arabidopsis halleri ssp. gemmifera. In a 5.5-kb region of the PgiC1 locus, 127 nucleotide substitutions and 33 length variations were observed. In a 6.0-kb region of the PgiC2 locus, 138 nucleotide substitutions and 33 length variations were observed. Frame shift, novel stop codons, and large length variations were observed in the PgiC2 coding region. These findings suggested that PgiC2 may be a pseudogene. The nucleotide diversities (pi) for the entire regions of both PgiC loci were approximately 0.0033. Tajima's test of both PgiC loci yielded significantly negative results. In the coding regions, the high proportions of replacement substitutions caused significant deviations from neutrality in McDonald and Kreitman's test. An excess of singletons and a high proportion of replacement polymorphic sites have been observed in the Adh and ChiA regions of A. halleri ssp. gemmifera. Thus, the A. halleri ssp. gemmifera population may not have reached equilibrium, and thus nonneutral patterns of DNA polymorphism were observed.

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.

Characterization of duplicated two cytosolic phosphoglucose isomerase (PgiC) loci in Arabidopsis halleri ssp. gemmifera.

Arabidopsis halleri ssp. gemmifera has two cytosolic phosphoglucose isomerase (PgiC) loci. A 48-bp deletion was observed in the junction of exon 17 and intron 17 for a locus (PgiC2). PCR-RFLP analysis using cDNA template did not detect the PgiC2 locus. Another locus (PgiC1) has common structure with A. thaliana and expressed normally. A phylogenetic tree of PgiC sequences revealed that duplication of the two loci in A. gemmifera occurred after species splitting of A. thaliana and A. gemmifera. More than 12 kb region encompassing PgiC was sequenced for both loci. In both PgiC1 and PgiC2, sequence homologous to A. thaliana PgiC 5' upstream region was not detected. A gene located on chromosome 4 of A. thaliana was detected in the 5' upstream of PgiC2. This result suggested that the microsyntheny around the PgiC region between A. thaliana and A. gemmifera is not established.

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.

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.