Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

Genetic architecture of leaf photosynthesis in rice revealed by different types of reciprocal mapping populations.

The improvement of leaf net photosynthetic rate (An) is a major challenge in enhancing crop productivity. However, the genetic control of An among natural genetic accessions is still poorly understood. The high-yielding indica cultivar Takanari has the highest An of all rice cultivars, 20-30% higher than that of the high-quality japonica cultivar Koshihikari. By using reciprocal backcross inbred lines and chromosome segment substitution lines derived from a cross between Takanari and Koshihikari, we identified three quantitative trait loci (QTLs) where the Takanari alleles enhanced An in plants with a Koshihikari genetic background and five QTLs where the Koshihikari alleles enhanced An in plants with a Takanari genetic background. Two QTLs were expressed in plants with both backgrounds (type I QTL). The expression of other QTLs depended strongly on genetic background (type II QTL). These beneficial alleles increased stomatal conductance, the initial slope of An versus intercellular CO2 concentration, or An at CO2 saturation. Pyramiding of these alleles consistently increased An. Some alleles positively affected biomass production and grain yield. These alleles associated with photosynthesis and yield can be a valuable tool in rice breeding programs via DNA marker-assisted selection.

A novel QTL associated with rice canopy temperature difference affects stomatal conductance and leaf photosynthesis.

Canopy temperature can be a good indicator of stomatal conductance. To understand the genetic basis of phenotypic differences in stomatal conductance between average and high-yielding rice (Oryza sativa L.) cultivars, we conducted a quantitative trait locus (QTL) analysis of canopy temperature. We developed reciprocal series of backcross inbred lines (BC1F6) derived from a cross between the average-yielding japonica cultivar 'Koshihikari' and the high-yielding indica cultivar 'Takanari'. A stable QTL, qCTd11 (QTL for canopy temperature difference on chromosome 11) on the short arm of chromosome 11, accounted for 10.4 and 19.8% of the total phenotypic variance in the two lines; the 'Takanari' allele decreased the canopy temperature difference value. A chromosome segment substitution line carrying the Takanari qCTd11 showed a greater reduction in canopy temperature than 'Koshihikari', and had higher stomatal conductance and photosynthetic rate. These results suggest that qCTd11 is not only involved in canopy temperature, but is also involved in both stomatal conductance and photosynthetic rate.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Higher sterol content regulated by CYP51 with concomitant lower phospholipid content in membranes is a common strategy for aluminium tolerance in several plant species.

Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species.

Genetic mechanisms underlying yield potential in the rice high-yielding cultivar Takanari, based on reciprocal chromosome segment substitution lines.

BACKGROUND: Increasing rice yield potential is a major objective in rice breeding programs, given the need for meeting the demands of population growth, especially in Asia. Genetic analysis using genomic information and high-yielding cultivars can facilitate understanding of the genetic mechanisms underlying rice yield potential. Chromosome segment substitution lines (CSSLs) are a powerful tool for the detection and precise mapping of quantitative trait loci (QTLs) that have both large and small effects. In addition, reciprocal CSSLs developed in both parental cultivar backgrounds may be appropriate for evaluating gene activity, as a single factor or in epistatic interactions. RESULTS: We developed reciprocal CSSLs derived from a cross between Takanari (one of the most productive indica cultivars) and a leading japonica cultivar, Koshihikari; both the cultivars were developed in Japan. Forty-one CSSLs covered most of the Takanari genome in the Koshihikari background and 39 CSSLs covered the Koshihikari genome in the Takanari background. Using the reciprocal CSSLs, we conducted yield trials under canopy conditions in paddy fields. While no CSSLs significantly exceeded the recurrent parent cultivar in yield, genetic analysis detected 48 and 47 QTLs for yield and its components in the Koshihikari and Takanari backgrounds, respectively. A number of QTLs showed a trade-off, in which the allele with increased sink-size traits (spikelet number per panicle or per square meter) was associated with decreased ripening percentage or 1000-grain weight. These results indicate that increased sink size is not sufficient to increase rice yield in both backgrounds. In addition, most QTLs were detected in either one of the two genetic backgrounds, suggesting that these loci may be under epistatic control with other gene(s). CONCLUSIONS: We demonstrated that the reciprocal CSSLs are a useful tool for understanding the genetic mechanisms underlying yield potential in the high-yielding rice cultivar Takanari. Our results suggest that sink-size QTLs in combination with QTLs for source strength or translocation capacity, as well as careful attention to epistatic interactions, are necessary for increasing rice yield. Thus, our findings provide a foundation for developing rice cultivars with higher yield potential in future breeding programs.

Characterization of AtSTOP1 orthologous genes in tobacco and other plant species.

Aluminum (Al) and proton (H⁺) tolerances are essential traits for plants to adapt to acid soil environments. In Arabidopsis (Arabidopsis thaliana), these tolerances are mediated by a zinc-finger transcription factor, SENSITIVE TO PROTON RHIZOTOXICITY1 (AtSTOP1), which regulates the transcription of multiple genes critical for tolerance to both stressors. Here, the functions of orthologous proteins (STOP1-like proteins) in other plant species were characterized by reverse genetics analyses and in planta complementation assays. RNA interference of a gene for NtSTOP1 repressed Al and H⁺ tolerances of tobacco (Nicotiana tabacum) roots. Tobacco roots released citrate in response to Al, concomitant with the up-regulated transcription of an ortholog of an Al tolerance gene encoding a citrate-transporting multidrug and toxic compound extrusion protein. The RNA interference repression of NtSTOP1 blocked this process and also repressed the transcription of another orthologous gene for Al tolerance, ALUMINUM SENSITIVE3, which encodes a prokaryote-type transporter. These results demonstrated that NtSTOP1 regulates Al tolerance in tobacco through the transcriptional regulation of these genes. The in planta complementation assays revealed that other plant species, including woody plants, a legume, and a moss (Physcomitrella patens), possess functional STOP1-like proteins that can activate several H⁺ and Al-tolerance genes in Arabidopsis. Knocking out the gene encoding the STOP1-like protein decreased the Al tolerance of P. patens. Together, our results strongly suggest that transcriptional regulation by STOP1-like proteins is evolutionarily conserved among land plants and that it confers the ability to survive in acid soils through the transcriptional regulation of Al- and H⁺-tolerance genes.

The source of "fairy rings": 2-azahypoxanthine and its metabolite found in a novel purine metabolic pathway in plants.

Rings or arcs of fungus-stimulated plant growth occur worldwide; these are commonly referred to as "fairy rings". In 2010, we discovered 2-azahypoxanthine (AHX), a compound responsible for the fairy-ring phenomenon caused by fungus; AHX stimulated the growth of all the plants tested. Herein, we reveal the isolation and structure determination of a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH). AHX is chemically synthesized from 5-aminoimidazole-4-carboxamide (AICA), and AHX can be converted into AOH by xanthine oxidase. AICA is one of the members of the purine metabolic pathway in animals, plants, and microorganisms. However, further metabolism of AICA remains elusive. Based on these results and facts, we hypothesized that plants themselves produce AHX and AOH through a pathway similar to the chemical synthesis. Herein, we demonstrate the existence of endogenous AHX and AOH and a novel purine pathway to produce them in plants.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene.

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1-1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

Potential of spectroscopic analyses for non-destructive estimation of tea quality-related metabolites in fresh new leaves.

Spectroscopic sensing provides physical and chemical information in a non-destructive and rapid manner. To develop non-destructive estimation methods of tea quality-related metabolites in fresh leaves, we estimated the contents of free amino acids, catechins, and caffeine in fresh tea leaves using visible to short-wave infrared hyperspectral reflectance data and machine learning algorithms. We acquired these data from approximately 200 new leaves with various status and then constructed the regression model in the combination of six spectral patterns with pre-processing and five algorithms. In most phenotypes, the combination of de-trending pre-processing and Cubist algorithms was robustly selected as the best combination in each round over 100 repetitions that were evaluated based on the ratio of performance to deviation (RPD) values. The mean RPD values were ranged from 1.1 to 2.7 and most of them were above the acceptable or accurate threshold (RPD = 1.4 or 2.0, respectively). Data-based sensitivity analysis identified the important hyperspectral regions around 1500 and 2000 nm. Present spectroscopic approaches indicate that most tea quality-related metabolites can be estimated non-destructively, and pre-processing techniques help to improve its accuracy.

Tissue-Dependent Variation Profiles of Tea Quality-Related Metabolites in New Shoots of Tea Accessions.

Several metabolites define tea quality in new tea shoots composed of leaf and stem. To improve tea quality for breeding, it is important to understand the tissue-dependent genetic mechanisms and metabolic network responsible for the profile of tea quality-related metabolites. We analyzed the volatiles and specialized metabolites as the tea quality-related metabolites in leaves and stems of new shoots in 30 tea accessions to understand the tissue variation and network between tea quality-related metabolites. Our results provided the tissue-dependent variation network in the tea quality-related metabolites, including volatiles in new leaves and stems in tea accessions. Each volatile content in tea accessions showed the coefficient of variation ranging from 58.7 to 221.9% and 54.2 to 318.3% in new leaves and new stems, respectively. The accumulation pattern of tea quality-related metabolites in new leaves and stems varied depending on the accession. When comparing tea genetic populations, the profile of tea quality-related metabolites of new leaves, but not new stems, was the key to distinguishing tea genetic populations by chemical indicators. We described the network between tea quality-related metabolites, especially the dense network in new leaves. These results also will provide the key information for metabolic engineering and the selection of breeding materials in tea plants based on the tea quality-related metabolites and aid in understanding their molecular mechanisms and network of metabolic variation.

Cesium uptake and translocation from tea cutting roots (Camellia sinensis L.).

To estimate the uptake of radiocesium (137Cs) by tea plant roots, 1-year-old rooted tea cuttings (Camellia sinensis L. cv. Yabukita) at the time of bud opening were cultivated hydroponically for 27 days in pots containing nutrient solutions with or without 137CsCl (600 Bq mL-1). Total 137Cs radioactivity of whole tea plants were 6.1 kBq g-1 dry weight. The plant/solution 137Cs transfer factors of different tissues were in the range of 2.6 (in mature leaves) to 28.2 mL g-1 dry weight (in roots), which were lower than those reported in wheat and spinach. In total, 69% of 137Cs remained in roots and 31% was transported from roots to shoots. The results indicated that 137Cs was preferentially translocated to new shoots, which are used for manufacturing tea, over mature leaves.

Dissection of hyperspectral reflectance to estimate nitrogen and chlorophyll contents in tea leaves based on machine learning algorithms.

Nondestructive techniques for estimating nitrogen (N) status are essential tools for optimizing N fertilization input and reducing the environmental impact of agricultural N management, especially in green tea cultivation, which is notably problematic. Previously, hyperspectral indices for chlorophyll (Chl) estimation, namely a green peak and red edge in the visible region, have been identified and used for N estimation because leaf N content closely related to Chl content in green leaves. Herein, datasets of N and Chl contents, and visible and near-infrared hyperspectral reflectance, derived from green leaves under various N nutrient conditions and albino yellow leaves were obtained. A regression model was then constructed using several machine learning algorithms and preprocessing techniques. Machine learning algorithms achieved high-performance models for N and Chl content, ensuring an accuracy threshold of 1.4 or 2.0 based on the ratio of performance to deviation values. Data-based sensitivity analysis through integration of the green and yellow leaves datasets identified clear differences in reflectance to estimate N and Chl contents, especially at 1325-1575 nm, suggesting an N content-specific region. These findings will enable the nondestructive estimation of leaf N content in tea plants and contribute advanced indices for nondestructive tracking of N status in crops.

Tissue ionome response to rhizosphere pH and aluminum in tea plants (Camellia sinensis L.), a species adapted to acidic soils.

The growth of tea plants (Camellia sinensis L.) is promoted by the presence of aluminum (Al), a beneficial element under acidic conditions, but the influence of rhizosphere pH on this interaction is not known. To understand the mechanisms underlying the adaptation to acidic rhizosphere conditions, we evaluated ionome profiles and the effect of pH on tea growth in hydroponic culture. The optimum pH for tea growth was around pH 4.2, and growth was inferior under a pH less than 3.8 or higher than 5.0. Under the optimum pH growth and Al accumulation were markedly stimulated by Al treatment. Al content and accumulation in new and mature leaves and new roots (the predominant tissues that accumulate minerals in tea plants) gradually declined with decrease in pH, especially in new roots. Ionome profiles drastically altered Al treatment, but changes were more pronounced in new roots than in new or mature leaves and did not depend on pH. Although the uptake of most cationic minerals in new roots was decreased by Al treatment, cationic mineral contents in new and mature leaves were not decreased by Al. In contrast to other plant species, the content and accumulation of manganese, despite it being a cationic nutrient, were significantly increased by Al treatment. These results indicated that one role of Al as a beneficial element was to maintain the shoot nutrient status by effectively utilizing Al-limited elements in the roots.

Genomic predictions and genome-wide association studies based on RAD-seq of quality-related metabolites for the genomics-assisted breeding of tea plants.

Effectively using genomic information greatly accelerates conventional breeding and applying it to long-lived crops promotes the conversion to genomic breeding. Because tea plants are bred using conventional methods, we evaluated the potential of genomic predictions (GPs) and genome-wide association studies (GWASs) for the genetic breeding of tea quality-related metabolites using genome-wide single nucleotide polymorphisms (SNPs) detected from restriction site-associated DNA sequencing of 150 tea accessions. The present GP, based on genome-wide SNPs, and six models produced moderate prediction accuracy values (r) for the levels of most catechins, represented by ( -)-epigallocatechin gallate (r = 0.32-0.41) and caffeine (r = 0.44-0.51), but low r values for free amino acids and chlorophylls. Integrated analysis of GWAS and GP detected potential candidate genes for each metabolite using 80-160 top-ranked SNPs that resulted in the maximum cumulative prediction value. Applying GPs and GWASs to tea accession traits will contribute to genomics-assisted tea breeding.

Integrated Metabolome and Transcriptome Analyses Reveal Etiolation-Induced Metabolic Changes Leading to High Amino Acid Contents in a Light-Sensitive Japanese Albino Tea Cultivar.

Plant albinism causes the etiolation of leaves because of factors such as deficiency of chloroplasts or chlorophylls. In general, albino tea leaves accumulate higher free amino acid (FAA) contents than do conventional green tea leaves. To explore the metabolic changes of etiolated leaves (EL) in the light-sensitive Japanese albino tea cultivar "Koganemidori," we performed integrated metabolome and transcriptome analyses by comparing EL with green leaves induced by bud-sport mutation (BM) or shading treatments (S-EL). Comparative omics analyses indicated that etiolation-induced molecular responses were independent of the light environment and were largely influenced by the etiolation itself. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and pathway analyses revealed the downregulation of genes involved in chloroplast development and chlorophyll biosynthesis and upregulation of protein degradation-related pathways, such as the ubiquitin-proteasome system and autophagy in EL. Metabolome analysis showed that most quantified FAAs in EL were highly accumulated compared with those in BM and S-EL. Genes involved in the tricarboxylic acid (TCA) cycle, nitrogen assimilation, and the urea cycle, including the drastically downregulated Arginase-1 homolog, which functions in nitrogen excretion for recycling, showed lower expression levels in EL. The high FAA contents in EL might result from the increased FAA pool and nitrogen source contributed by protein degradation, low N consumption, and stagnation of the urea cycle rather than through enhanced amino acid biosynthesis.

Phenotypic Markers Reflecting the Status of Overstressed Tea Plants Subjected to Repeated Shade Cultivation.

Shade cultivation is a traditional Japanese tea cultivation method in which the shoot buds are shaded for several weeks. This technique is increasingly used for green tea production because it produces tea of high quality (as indicated by umami and nutritional content) and commands high prices. However, given that shaded tea plants are grown under low-light stress, concerns exist regarding damage to tea plants caused by repeated shade cultivation. To understand basic physiological responses and accumulative changes in photosynthetic ability and metabolites of tea plants subjected to repeated shading, we performed a pot experiment on immature tea plants grown in a growth chamber subjected to repeated shading treatments. The results demonstrated that shade cultivation caused a decrease in non-structural carbohydrate content and an increase of several degrees in leaf surface temperature, reflecting transpiration through the leaf stomata, as a result of a reduction in photosynthetic ability. An increase of several degrees in canopy temperature and a reduction in photosynthetic ability in the field in the mid-summer season was also observed in overstressed tea plants subjected to repeated shading. Metabolomic analysis identified several candidate biomarkers, such as citrulline and glycine betaine, that were significantly changed in individuals affected by shade cultivation. These physiological changes may be an indicator of the stress status of tea plants grown under repeated shade cultivation.

Comparative transcriptomic characterization of aluminum, sodium chloride, cadmium and copper rhizotoxicities in Arabidopsis thaliana.

BACKGROUND: Rhizotoxic ions in problem soils inhibit nutrient and water acquisition by roots, which in turn leads to reduced crop yields. Previous studies on the effects of rhizotoxic ions on root growth and physiological functions suggested that some mechanisms were common to all rhizotoxins, while others were more specific. To understand this complex system, we performed comparative transcriptomic analysis with various rhizotoxic ions, followed by bioinformatics analysis, in the model plant Arabidopsis thaliana. RESULTS: Roots of Arabidopsis were treated with the major rhizotoxic stressors, aluminum (Al) ions, cadmium (Cd) ions, copper (Cu) ions and sodium (NaCl) chloride, and the gene expression responses were analyzed by DNA array technology. The top 2.5% of genes whose expression was most increased by each stressor were compared with identify common and specific gene expression responses induced by these stressors. A number of genes encoding glutathione-S-transferases, peroxidases, Ca-binding proteins and a trehalose-synthesizing enzyme were induced by all stressors. In contrast, gene ontological categorization identified sets of genes uniquely induced by each stressor, with distinct patterns of biological processes and molecular function. These contained known resistance genes for each stressor, such as AtALMT1 (encoding Al-activated malate transporter) in the Al-specific group and DREB (encoding dehydration responsive element binding protein) in the NaCl-specific group. These gene groups are likely to reflect the common and differential cellular responses and the induction of defense systems in response to each ion. We also identified co-expressed gene groups specific to rhizotoxic ions, which might aid further detailed investigation of the response mechanisms. CONCLUSION: In order to understand the complex responses of roots to rhizotoxic ions, we performed comparative transcriptomic analysis followed by bioinformatics characterization. Our analyses revealed that both general and specific genes were induced in Arabidopsis roots exposed to various rhizotoxic ions. Several defense systems, such as the production of reactive oxygen species and disturbance of Ca homeostasis, were triggered by all stressors, while specific defense genes were also induced by individual stressors. Similar studies in different plant species could help to clarify the resistance mechanisms at the molecular level to provide information that can be utilized for marker-assisted selection.

Quantitative trait loci controlling resistance to cadmium rhizotoxicity in two recombinant inbred populations of Arabidopsis thaliana are partially shared by those for hydrogen peroxide resistance.

To understand mechanisms of cadmium (Cd) tolerance variation associated with root elongation in Arabidopsis thaliana, quantitative trait loci (QTLs) and epistasis were analyzed using relative root length (RRL: % of the root length in +Cd to -Cd) as a tolerant index. Using the composite interval mapping method, three major QTLs (P < 0.05) were detected on chromosomes 2, 4 and 5 in the recombinant inbred population derived from a cross between Landsberg erecta (Ler-0) and Columbia (Col-4). The highest logarithm of odds (LOD) of 5.6 was detected with the QTL on chromosome 5 (QTL5), which is linked to the genetic marker CDPK9 and explained about 26% of the Cd tolerance variation. There was no significant difference in Cd-translocation ratio from roots to shoots between tolerant and sensitive recombinant inbreed lines (RILs), while greater accumulations of reactive oxygen species were observed in the roots of sensitive RILs. This suggested that accumulation of ROS would explain Cd tolerance variation of the Ler/Col RILs, which is mainly controlled by the QTL on chromosome 5. The QTL5 in Ler/Col population was also detected as one of the major QTLs controlling tolerances to hydrogen peroxide and to copper, which is another ROS generating rhizotoxic metal. The same chromosomal position was detected as a common major QTL for Cd and hydrogen peroxide tolerances in a different recombinant inbreed (RI) population derived from a cross of Col-gl1 and Kashmir (Kas-1). These data, along with a multitraits QTL analysis in both sets of RILs, suggest that peroxide damage depends on the genotype at a major Cd-tolerant locus on the upper part of chromosome 5.

Association mapping of cadmium, copper and hydrogen peroxide tolerance of roots and translocation capacities of cadmium and copper in Arabidopsis thaliana.

Association mapping analysis of Cd, Cu and H (2)O (2) tolerance, judged by relative root length (RRL: % of root length in stress condition relative to that in control condition), and Cd and Cu translocation ratios (amount of metal in the shoot to the total) were performed using 90 accessions of Arabidopsis thaliana. Using 140 SNPs that were distributed across the genome, association mapping analysis was performed with a haploid setting by the Q + K method, which minimizes detection of false associations by combining the Q-matrix of the structured association (Q) with kinship (K) to control for the population structure. Six, five and five significant (-log (10)P-value is 1.3 > or =) linkages were detected between the SNPs and Cd, Cu and H(2)O(2) resistant RRLs, respectively. In addition, six significant linkages were identified with translocation capacities of Cd and Cu. Among those detected loci, two each of Cu and Cd tolerance RRLs were collocated with those of H(2)O(2) tolerance RRL, while one locus each was detected by Cu and Cd tolerance RRLs that collocated with their translocation ratios. These results suggested that these factors might partly explain the phenotypic variation of tolerance RRLs to Cd and Cu of Arabidopsis thaliana. Finally, using a different approach to analyze interactions between individual phenotypes, namely clustering analysis, we found an expected segregation of resistant SNPs (single-nucleotide polymorphisms) of the multiple RRLs in the typical accession groups carrying multiple traits. Almost none of the loci detected by association mapping analysis were linked to the loci of previously identified critical genes regulating the traits, suggesting that this could be useful to identify complex architecture of genetic factors determining variation among multiple accessions.