Genome-wide association study of leaf photosynthesis using a high-throughput gas exchange system in rice.

Enhancing leaf photosynthetic capacity is essential for improving the yield of rice (Oryza sativa L.). Although the exploitation of natural genetic resources is considered a promising approach to enhance photosynthetic capacity, genomic factors related to the genetic diversity of leaf photosynthetic capacity have yet to be fully elucidated due to the limitation of measurement efficiency. In this study, we aimed to identify novel genomic regions for the net CO2 assimilation rate (A) by combining genome-wide association study (GWAS) and the newly developed rapid closed gas exchange system MIC-100. Using three MIC-100 systems in the field at the vegetative stage, we measured A of 168 temperate japonica rice varieties with six replicates for three years. We found that the modern varieties exhibited higher A than the landraces, while there was no significant relationship between the release year and A among the modern varieties. Our GWAS scan revealed two major peaks located on chromosomes 4 and 8, which were repeatedly detected in the different experiments and in the generalized linear modelling approach. We suggest that high-throughput gas exchange measurements combined with GWAS is a reliable approach for understanding the genetic mechanisms underlying photosynthetic diversities in crop species.

Metabolomic analysis of rice brittle culm mutants reveals each mutant- specific metabolic pattern in each organ.

INTRODUCTION: Plant cell walls play an important role in providing physical strength and defence against abiotic stress. Rice brittle culm (bc) mutants are a strength-decreased mutant because of abnormal cell walls, and it has been reported that the causative genes of bc mutants affect cell wall composition. However, the metabolic alterations in each organ of bc mutants have remained unknown. OBJECTIVES: To evaluate the metabolic changes in rice bc mutants, comparative analysis of the primary metabolites was conducted. METHODS: The primary metabolites in leaves, internodes, and nodes of rice bc mutants and wild-type control were measured using CE- and LC-MS/MS. Multivariate analyses using metabolomic data was performed. RESULTS: We found that mutations in each bc mutant had different effects on metabolism. For example, higher oxalate content was observed in bc3 and bc1 bc3 mutants, suggesting that surplus carbon that was not used for cell wall components might be used for oxalate synthesis. In addition, common metabolic alterations such as a decrease of sugar nucleotides in nodes were found in bc1 and Bc6, in which the causative genes are involved in cellulose accumulation. CONCLUSION: These results suggest that metabolic analysis of the bc mutants could elucidate the functions of causative gene and improve the cell wall components for livestock feed or bioethanol production.

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.

QTLs maintaining grain fertility under salt stress detected by exome QTL-seq and interval mapping in barley.

Enhancing salt stress tolerance is a key strategy for increasing global food production. We previously found that long-term salinity stress significantly reduced grain fertility in the salt-sensitive barley (Hordeum vulgare) accession, 'OUC613', but not in the salt-tolerant accession, 'OUE812', resulting in large differences in grain yield. Here, we examined the underlying causes of the difference in grain fertility between these accessions under long-term treatment with 150 or 200 mM NaCl from the seedling stage to harvest and identified quantitative trait loci (QTLs) for maintaining grain fertility. In an artificial pollination experiment of the two accessions, grain fertility was significantly reduced only in OUC613 plants produced using pollen from plants grown under NaCl stress, suggesting that the low grain fertility of OUC613 was mainly due to reduced pollen fertility. Using QTL-seq combined with exome-capture sequencing and composite interval mapping of recombinant inbred lines derived from a cross between OUE812 and OUC613, we identified a QTL (qRP-2Hb) for grain fertility on chromosome 2H. The QTL region includes two genes encoding an F-box protein and a TIFY protein that are associated with male sterility, highlighting the importance of this region for maintaining grain fertility under salt stress.

Leaf photosynthetic rate and mesophyll cell anatomy changes during ontogenesis in backcrossed indica × japonica rice inbred lines.

The high-yielding indica rice variety, 'Takanari', has the high rate of leaf photosynthesis compared with the commercial japonica varieties. Among backcrossed inbred lines from a cross between 'Takanari' and a japonica variety, 'Koshihikari', two lines, BTK-a and BTK-b, showed approximately 20% higher photosynthetic rate than that of 'Takanari' for a flag leaf at full heading. This is a highest recorded rate of rice leaf photosynthesis. Here, the timing and cause of the increased leaf photosynthesis in the BTK lines were investigated by examining the photosynthesis and related parameters, as well as mesophyll cell anatomy during ontogenesis. Their photosynthetic rate was greater than that of 'Takanari' in the 13th leaf, as well as the flag leaf, but there were no differences in the 7th and 10th leaves. There were no consistent differences in the stomatal conductance, or the leaf nitrogen and Rubisco contents in the 13th and flag leaves. The total surface area of mesophyll cells per leaf area (TAmes) in the 13th and flag leaves increased significantly in the BTK lines due to the increased number and developed lobes of mesophyll cells compared with in 'Takanari'. The mesophyll conductance (g m) became greater in the BTK lines compared with 'Takanari' in the flag leaves but not in the 10th leaves. A close correlation was observed between TAmes and g m. We concluded that the increased mesophyll conductance through the development of mesophyll cells during the reproductive period is a probable cause of the greater photosynthetic rate in the BTK lines.

Introgression of two chromosome regions for leaf photosynthesis from an indica rice into the genetic background of a japonica rice.

Increases in rates of individual leaf photosynthesis (P n) are critical for future increases of rice yields. A previous study, using introgression lines derived from a cross between indica cultivar Habataki, with one of the highest recorded values of P n, and the Japanese elite cultivar Koshihikari, identified four QTLs (qCAR4, qCAR5, qCAR8, and qCAR11) that affect P n. The present study examined the combined effect of qCAR4 and qCAR8 on P n in the genetic background of Koshihikari. The pyramided near-isogenic line NIL(qCAR4+qCAR8) showed higher P n than both NIL(qCAR4) and NIL(qCAR8), equivalent to that of Habataki despite being due to only two out of the four QTLs. The high P n of NIL(qCAR4+qCAR8) may be attributable to the high leaf nitrogen content, which may have been inherited from NIL(qCAR4), to the large hydraulic conductance due to the large root surface area from NIL(qCAR4), and to the high hydraulic conductivity from NIL(qCAR8). It might be also attributable to high mesophyll conductance, which may have been inherited from NIL(qCAR4). The induction of mesophyll conductance and the high leaf nitrogen content and high hydraulic conductivity could not be explained in isolation from the Koshihikari background. These results suggest that QTL pyramiding is a useful approach in rice breeding aimed at increasing P n.

The mesophyll anatomy enhancing CO2 diffusion is a key trait for improving rice photosynthesis.

Increases in rates of individual leaf photosynthesis (P(n)) are critical for future increases in yields of rice plants. Although many efforts have been made to improve rice P(n) with transgenic technology, the desired increases in P(n) have not yet been achieved. Two rice lines with extremely high values of P(n) were identified among the backcrossed inbred lines derived from the indica variety Takanari, one of the most productive varieties in Japan, and the elite japonica variety Koshihikari (Koshihikari/Takanari//Takanari). The P(n) values of the two lines at an ambient CO(2) concentration of 370µmol mol(-1) as well as at a saturating concentration of CO(2) were 20-50% higher than those of the parental varieties. Compared with Takanari, these lines had neither a higher content nor a higher activity of ribulose 1,5-bisphosphate carboxylase/oxygenase when the leaf nitrogen contents were similar, but they did have high mesophyll conductance with respect to CO(2) flux due to their higher density and more highly developed lobes of mesophyll cells. These lines also had higher electron transport rates. The plant growth rates of these lines were higher than that of Takanari. The findings show that it is possible to increase P(n) significantly, both at the current atmospheric concentration of CO(2) and at the increased concentration of CO(2) expected in the future, using appropriate combinations of genetic resources that are available at present.

Identification of Novel Quantitative Trait Loci for Culm Thickness of Rice Derived from Strong-Culm Landrace in Japan, Omachi.

Increasing the lodging resistance of rice through genetic improvement has been an important target in breeding. To further enhance the lodging resistance of high-yielding rice varieties amidst climate change, it is necessary to not only shorten culms but strengthen them as well. A landrace rice variety, Omachi, which was established more than 100 years ago, has the largest culm diameter and bending moment at breaking in the basal internodes among 135 temperate japonica accessions. Using unused alleles in such a landrace is an effective way to strengthen the culm. In this study, we performed quantitative trait locus (QTL) analysis to identify the genetic factors of culm strength of Omachi using recombinant inbred lines (RILs) derived from a cross between Omachi and Koshihikari, a standard variety in Japan. We identified three QTLs for the culm diameter of the 5th internode on chromosomes 3 (qCD3) and 7 (qCD7-1, qCD7-2). Among them, qCD7-2 was verified by QTL analysis using the F2 population derived from a cross between one of the RILs and Koshihikari. RNA-seq analysis of shoot apex raised 10 candidate genes underlying the region of qCD7-2. The increase in culm strength by accumulating Omachi alleles of qCD3, qCD7-1 and qCD7-2 was 25.0% in 2020. These QTLs for culm diameter pleiotropically increased spikelet number per panicle but did not affect days to heading or culm length. These results suggest that the Omachi alleles of qCD3, qCD7-1 and qCD7-2 are useful for breeding to increase lodging resistance and yield.

Identification of Genomic Regions for Deep-Water Resistance in Rice for Efficient Weed Control with Reduced Herbicide Use.

Deep-water (DW) management in rice fields is a promising technique for efficient control of paddy weeds with reduced herbicide use. Maintaining a water depth of 10-20 cm for several weeks can largely suppress the weed growth, though it also inhibits rice growth because the DW management is usually initiated immediately after transplanting. Improving the DW resistance of rice during the initial growth stage is essential to avoid suppressing growth. In this study, we demonstrate a large genetic variation in the above-ground biomass (AGB) after the end of DW management among 165 temperate japonica varieties developed in Japan. Because the AGB closely correlated with plant length (PL) and tiller number (TN) at the early growth stage, we analyzed genomic regions associated with PL and TN by conducting a genome-wide association study. For PL, a major peak was detected on chromosome 3 (qPL3), which includes a gene encoding gibberellin biosynthesis, OsGA20ox1. The rice varieties with increased PL had a higher expression level of OsGA20ox1 as reported previously. For TN, a major peak was detected on chromosome 4 (qTN4), which includes NAL1 gene associated with leaf morphological development and panicle number. Although there was less difference in the expression level of NAL1 between genotypes, our findings suggest that an amino acid substitution in the exon region is responsible for the phenotypic changes. We also found that the rice varieties having alternative alleles of qPL3 and qTN4 showed significantly higher AGB than the varieties with the reference alleles. Our results suggest that OsGA20ox1 and NAL1 are promising genes for improving DW resistance in rice.

Pyramiding of multiple strong-culm genes originating from indica and tropical japonica to the temperate japonica rice.

Severe lodging has recurrently occurred at strong typhoon's hitting in recent climate change. The identification of quantitative trait loci and their responsible genes associated with a strong culm and their pyramiding are important for developing high-yielding varieties with a superior lodging resistance. To evaluate the effects of four strong-culm genes on lodging resistance, the temperate japonica near isogenic line (NIL) with the introgressed SCM1 or SCM2 locus of the indica variety, Habataki and the other NIL with the introgeressed SCM3 or SCM4 locus of the tropical japonica variety, Chugoku 117 were developed. Then, we developed the pyramiding lines with double,triple and quadruple combinations derived from step-by-step crosses among NIL-SCM1-NIL-SCM4. Quadruple pyramiding line (NIL-SCM1 + 2 + 3 + 4) showed the largest culm diameter and the highest culm strength among the combinations and increased spikelet number due to the pleiotropic effects of these genes. Pyramiding of strong culm genes resulted in much increased culm thickness, culm strength and spikelet number due to their additive effect. SCM1 mainly contributed to enhance their pyramiding effect. These results in this study suggest the importance of identifying the combinations of superior alleles of strong culm genes among natural variation and pyramiding these genes for improving high-yielding varieties with a superior lodging resistance.