Real-time emulation of future global warming reveals realistic impacts on the phenological response and quality deterioration in rice.

Decreased production of crops due to climate change has been predicted scientifically. While climate-resilient crops are necessary to ensure food security and support sustainable agriculture, predicting crop growth under future global warming is challenging. Therefore, we aimed to assess the impact of realistic global warming conditions on rice cultivation. We developed a crop evaluation platform, the agro-environment (AE) emulator, which generates diverse environments by implementing the complexity of natural environmental fluctuations in customized, fully artificial lighting growth chambers. We confirmed that the environmental responsiveness of rice obtained in the fluctuation of artificial environments is similar to those exhibited in natural environments by validating our AE emulator using publicly available meteorological data from multiple years at the same location and multiple locations in the same year. Based on the representative concentration pathway, real-time emulation of severe global warming unveiled dramatic advances in the rice life cycle, accompanied by a 35% decrease in grain yield and an 85% increase in quality deterioration, which is higher than the recently reported projections. The transcriptome dynamism showed that increasing temperature and CO2 concentrations synergistically changed the expression of various genes and strengthened the induction of flowering, heat stress adaptation, and CO2 response genes. The predicted severe global warming greatly alters rice environmental adaptability and negatively impacts rice production. Our findings offer innovative applications of artificial environments and insights for enhancing varietal potential and cultivation methods in the future.

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.

Deciphering transcriptomic signatures explaining the phenotypic plasticity of nonheading lettuce genotypes under artificial light conditions.

Elucidating the mechanisms and pathways involved in genotype-environment (G×E) interactions and phenotypic plasticity is critical for improving plant growth. Controlled environment agricultural systems allow growers to modulate the environment for particular genotypes. In this study, we evaluated the effects of interactions among 14 genotypes and four artificial light environments on leaf lettuce phenotypes and dissected the underlying molecular mechanism via transcriptome-based modeling. Variations in morphological traits and phytochemical concentrations in response to artificial light treatments revealed significant G×E interactions. The appropriate genotype and artificial light combinations for maximizing phenotypic expression were determined on the basis of a joint regression analysis and the additive main effect and multiplicative interaction model for these G×E interactions. Transcriptome-based regression modeling explained approximately 50%-90% of the G×E variations. Further analyzes indicated Red Lettuce Leaves 4 (RLL4) regulates UV-B and blue light signaling through the effects of the HY5-MBW pathway on flavonoid biosynthesis and contributes to natural variations in the light-responsive plasticity of lettuce traits. Our study represents an important step toward elucidating the phenotypic variations due to G×E interactions in nonheading lettuce under artificial light conditions.

Prediction of heading date, culm length, and biomass from canopy-height-related parameters derived from time-series UAV observations of rice.

Unmanned aerial vehicles (UAVs) are powerful tools for monitoring crops for high-throughput phenotyping. Time-series aerial photography of fields can record the whole process of crop growth. Canopy height (CH), which is vertical plant growth, has been used as an indicator for the evaluation of lodging tolerance and the prediction of biomass and yield. However, there have been few attempts to use UAV-derived time-series CH data for field testing of crop lines. Here we provide a novel framework for trait prediction using CH data in rice. We generated UAV-based digital surface models of crops to extract CH data of 30 Japanese rice cultivars in 2019, 2020, and 2021. CH-related parameters were calculated in a non-linear time-series model as an S-shaped plant growth curve. The maximum saturation CH value was the most important predictor for culm length. The time point at the maximum CH contributed to the prediction of days to heading, and was able to predict stem and leaf weight and aboveground weight, possibly reflecting the association of biomass with duration of vegetative growth. These results indicate that the CH-related parameters acquired by UAV can be useful as predictors of traits typically measured by hand.

Fine mapping of Rf5 region for a sorghum fertility restorer gene and microsynteny analysis across grass species.

Cytoplasmic male sterility (CMS) is widely used to control pollination in the production of commercial F1 hybrid seed in sorghum. So far, 6 major fertility restorer genes, Rf1 to Rf6, have been reported in sorghum. Here, we fine-mapped the Rf5 locus on sorghum chromosome 5 using descendant populations of a 'Nakei MS-3A' × 'JN43' cross. The Rf5 locus was narrowed to a 140-kb region in BTx623 genome (161-kb in JN43) with 16 predicted genes, including 6 homologous to the rice fertility restorer Rf1 (PPR.1 to PPR.6). These 6 homologs have tandem pentatricopeptide repeat (PPR) motifs. Many Rf genes encode PPR proteins, which bind RNA transcripts and modulate gene expression at the RNA level. No PPR genes were detected at the Rf5 locus on the corresponding homologous chromosome of rice, foxtail millet, or maize, so this gene cluster may have originated by chromosome translocation and duplication after the divergence of sorghum from these species. Comparison of the sequences of these genes between fertile and CMS lines identified PPR.4 as the most plausible candidate gene for Rf5.

Genetic effects of Red Lettuce Leaf genes on red coloration in leaf lettuce under artificial lighting conditions.

Some cultivars of lettuce accumulate anthocyanins, which act as functional food ingredients. Leaf lettuce has been known to be erratic in exhibiting red color when grown under artificial light, and there is a need for cultivars that more stably exhibit red color in artificial light cultivation. In this study, we aimed to dissect the genetic architecture for red coloring in various leaf lettuce cultivars grown under artificial light. We investigated the genotype of Red Lettuce Leaf (RLL) genes in 133 leaf lettuce strains, some of which were obtained from publicly available resequencing data. By studying the allelic combination of RLL genes, we further analyzed the contribution of these genes to producing red coloring in leaf lettuce. From the quantification of phenolic compounds and corresponding transcriptome data, we revealed that gene expression level-dependent regulation of RLL1 (bHLH) and RLL2 (MYB) is the underlying mechanism conferring high anthocyanin accumulation in red leaf lettuce under artificial light cultivation. Our data suggest that different combinations of RLL genotypes cause quantitative differences in anthocyanin accumulation among cultivars, and some genotype combinations are more effective at producing red coloration even under artificial lighting.

Remote-Sensing-Combined Haplotype Analysis Using Multi-Parental Advanced Generation Inter-Cross Lines Reveals Phenology QTLs for Canopy Height in Rice.

High-throughput phenotyping systems with unmanned aerial vehicles (UAVs) enable observation of crop lines in the field. In this study, we show the ability of time-course monitoring of canopy height (CH) to identify quantitative trait loci (QTLs) and to characterise their pleiotropic effect on various traits. We generated a digital surface model from low-altitude UAV-captured colour digital images and investigated CH data of rice multi-parental advanced generation inter-cross (MAGIC) lines from tillering and heading to maturation. Genome-wide association studies (GWASs) using the CH data and haplotype information of the MAGIC lines revealed 11 QTLs for CH. Each QTL showed haplotype effects on different features of CH such as stage-specificity and constancy. Haplotype analysis revealed relationships at the QTL level between CH and, vegetation fraction and leaf colour [derived from UAV red-green-blue (RGB) data], and CH and yield-related traits. Noticeably, haplotypes with canopy lowering effects at qCH1-4, qCH2, and qCH10-2 increased the ratio of panicle weight to leaf and stem weight, suggesting biomass allocation to grain yield or others through growth regulation of CH. Allele mining using gene information with eight founders of the MAGIC lines revealed the possibility that qCH1-4 contains multiple alleles of semi-dwarf 1 (sd1), the IR-8 allele of which significantly contributed to the "green revolution" in rice. This use of remote-sensing-derived phenotyping data into genetics using the MAGIC lines gives insight into how rice plants grow, develop, and produce grains in phenology and provides information on effective haplotypes for breeding with ideal plant architecture and grain yield.

Investigation of the Genetic Diversity of a Rice Core Collection of Japanese Landraces using Whole-Genome Sequencing.

The Rice Core Collection of Japanese Landraces (JRC) consisting of 50 accessions was developed by the genebank at the National Agriculture and Food Research Organization (NARO) in 2008. As a Japanese landrace core collection, the JRC has been used for many research projects, including screening for different phenotypes and allele mining for target genes. To understand the genetic diversity of Japanese Landraces, we performed whole-genome resequencing of these 50 accessions and obtained a total of 2,145,095 single nucleotide polymorphism (SNPs) and 317,832 insertion-deletions (indels) by mapping against the Oryza sativa ssp. japonica Nipponbare genome. A JRC phylogenetic tree based on 1,394 representative SNPs showed that JRC accessions were divided into two major groups and one small group. We used the multiple genome browser, TASUKE+, to examine the haplotypes of flowering genes and detected new mutations in these genes. Finally, we performed genome-wide association studies (GWAS) for agronomical traits using the JRC and another core collection, the World Rice Core Collection (WRC), comprising 69 accessions also provided by the NARO genebank. In leaf blade width, a strong peak close to NAL1, a key gene for the regulation of leaf width, and, in heading date, a peak near HESO1 involved in flowering regulation were observed in GWAS using the JRC. They were also detected in GWAS using the combined JRC + WRC. Thus, JRC and JRC + WRC are suitable populations for GWAS of particular traits.

Genetic Elucidation for Response of Flowering Time to Ambient Temperatures in Asian Rice Cultivars.

Climate resilience of crops is critical for global food security. Understanding the genetic basis of plant responses to ambient environmental changes is key to developing resilient crops. To detect genetic factors that set flowering time according to seasonal temperature conditions, we evaluated differences of flowering time over years by using chromosome segment substitution lines (CSSLs) derived from japonica rice cultivars "Koshihikari" × "Khao Nam Jen", each with different robustness of flowering time to environmental fluctuations. The difference of flowering times in 9 years' field tests was large in "Khao Nam Jen" (36.7 days) but small in "Koshihikari" (9.9 days). Part of this difference was explained by two QTLs. A CSSL with a "Khao Nam Jen" segment on chromosome 11 showed 28.0 days' difference; this QTL would encode a novel flowering-time gene. Another CSSL with a segment from "Khao Nam Jen" in the region around Hd16 on chromosome 3 showed 23.4 days" difference. A near-isogenic line (NIL) for Hd16 showed 21.6 days' difference, suggesting Hd16 as a candidate for this QTL. RNA-seq analysis showed differential expression of several flowering-time genes between early and late flowering seasons. Low-temperature treatment at panicle initiation stage significantly delayed flowering in the CSSL and NIL compared with "Koshihikari". Our results unravel the molecular control of flowering time under ambient temperature fluctuations.

Haplotype analysis from unmanned aerial vehicle imagery of rice MAGIC population for the trait dissection of biomass and plant architecture.

Unmanned aerial vehicles (UAVs) are popular tools for high-throughput phenotyping of crops in the field. However, their use for evaluation of individual lines is limited in crop breeding because research on what the UAV image data represent is still developing. Here, we investigated the connection between shoot biomass of rice plants and the vegetation fraction (VF) estimated from high-resolution orthomosaic images taken by a UAV 10 m above a field during the vegetative stage. Haplotype-based genome-wide association studies of multi-parental advanced generation inter-cross (MAGIC) lines revealed four quantitative trait loci (QTLs) for VF. VF was correlated with shoot biomass, but the haplotype effect on VF was better correlated with that on shoot biomass at these QTLs. Further genetic characterization revealed the relationships between these QTLs and plant spreading habit, final shoot biomass and panicle weight. Thus, genetic analysis using high-throughput phenotyping data derived from low-altitude, high-resolution UAV images during early stages of rice growing in the field provides insights into plant growth, architecture, final biomass, and yield.

Analysis of quantitative trait loci for fertility restoration in seven F2 populations derived from sorghum F1 hybrids bred in Japan.

To clarify the genetic mechanisms of fertility restoration in sorghum F1 hybrids produced in Japan ('Ryokuryu', 'Hazuki', 'Haretaka', 'Natsuibuki', 'Hanaaoba', 'Akidachi' and 'Kazetachi'), we analyzed QTLs for fertility restoration using seven F2 populations derived from those hybrids. By QTL mapping with a series of SSR markers, we detected three major QTLs for fertility restoration. These data and the results of haplotype analysis of known fertility restorer (Rf) genes showed that qRf5, corresponding to the Rf5 locus, was the most widely used Rf gene for fertility restoration of sorghum F1 hybrids among the lines tested. Other major Rf genes detected were qRf8, corresponding to Rf1, and qRf2, corresponding to Rf2. QTLs for grain weight also corresponded to these Rf loci. A minor QTL, qRf3, may also affect restoration of fertility. Our data show that three major Rfs-Rf1, Rf2, and Rf5-were used in F1 hybrid sorghum production in Japan. This knowledge can be used to improve the efficiency of the F1 sorghum breeding program.

Surveillance of panicle positions by unmanned aerial vehicle to reveal morphological features of rice.

Rice plant architecture affects biomass and grain yield. Thus, it is important to select rice genotypes with ideal plant architecture. High-throughput phenotyping by use of an unmanned aerial vehicle (UAV) allows all lines in a field to be observed in less time than with traditional procedures. However, discrimination of plants in dense plantings is difficult, especially during the reproductive stage, because leaves and panicles overlap. Here, we developed an original method that relies on using UAV to identify panicle positions for dissecting plant architecture and to distinguish rice lines by detecting red flags attached to panicle bases. The plant architecture of recombinant inbred lines derived from Japanese cultivars 'Hokuriku 193' and 'Mizuhochikara', which differ in plant architecture, was assessed using a commercial camera-UAV system. Orthomosaics were made from UAV digital images. The center of plants was plotted on the image during the vegetative stage. The horizontal distance from the center to the red flag during the reproductive stage was used as the panicle position (PP). The red flags enabled us to recognize the positions of the panicles at a rate of 92%. The PP phenotype was related to but was not identical with the phenotypes of the panicle base angle, leaf sheath angle, and score of spreading habit. These results indicate that PP on orthomosaics could be used as an index of plant architecture under field conditions.

Next generation long-culm rice with superior lodging resistance and high grain yield, Monster Rice 1.

During late 1960s Green Revolution, researchers utilized semidwarf 1 (sd1) to improve the yield and lodging resistance in rice (Oryza sativa L.). However, sd1 has a negative effect to culm strength and biomass production. To increase yield dramatically in 21th century, development of next generation long-culm rice for non-lodging and high grain yield independent of sd1 has been needed. The present study developed Monster Rice 1, a long-culm and heavy-panicle type of rice line and compared it with Takanari, a high-yielding semidwarf rice variety about yield and lodging resistance associated traits. Brown rice yield and bending moment at breaking of the basal elongated internode were higher in Monster Rice 1 than those in Takanari due to a large number of spikelets per panicle and thicker culm. Furthermore, to identify QTLs with superior alleles for these traits, QTL and haplotype analyses were performed using F2 population and recombinant inbred lines derived from a cross between Monster Rice 1 and Takanari. The results from this study suggest that long-culm and heavy-panicle type of rice with a superior lodging resistance by culm strength can perform its high yield potential by using these identified QTLs contributing yield and lodging resistance.

A follow-up study for biomass yield QTLs in rice.

The biomass yield (plant weight) of rice fluctuates from year to year. In a previous study, we demonstrated that six quantitative trait loci (QTLs) contribute to the variation in the plant weight of recombinant inbred lines (RILs) of high-yielding Japanese rice cultivars. However, it remains unclear whether the effects of those QTLs are stable over multiple years. Therefore, we evaluated the effect of the alleles on the plant weight of RILs over multiple years, including a change of fertilization level (i.e., in different environments). Even though the biomass yields of all RILs fluctuated among environments, RILs that were selected on the basis of the genotypes of the detected QTLs had a stable rank order of plant weight that corresponded to their genotypes. This multiple-environment experiment reveals the highly significant contribution of both genotypic and environmental variances to the observed variance in plant weight. A marginally significant QTL-environment interaction was detected at only one of the six QTLs, with a subtle contribution. These results support the idea that the biomass yield of rice can be improved through QTL-based allele selection.

Discovery of QTL Alleles for Grain Shape in the Japan-MAGIC Rice Population Using Haplotype Information.

A majority of traits are determined by multiple quantitative trait loci (QTL) that can have pleiotropic effects. A multi-parent advanced generation inter-cross (MAGIC) population is well suited for genetically analyzing the effects of multiple QTL on traits of interest because it contains a higher number of QTL alleles than a biparental population. We previously produced the JAPAN-MAGIC (JAM) population, derived from eight rice (Oryza sativa L.) cultivars with high yield and biomass in Japan, and developed the method of genome-wide association study (GWAS) using haplotype information on the JAM lines. This method was effective for identifying major genes such as Waxy for eating quality and Sd1 for culm length. Here, we show that haplotype-based GWAS is also effective for the evaluation of multiple QTL with small effects on rice grain shape in the JAM lines. Although both the haplotype- and SNP-based GWAS identified multiple QTL for grain length and width, the sum of the estimated trait values of each allele for the QTL detected by haplotype-based GWAS had higher correlation with observed values than those detected by SNP-based GWAS, indicating high-accuracy QTL detection in the haplotype-based GWAS. Furthermore, the study revealed pleiotropic effects of some QTL regions in regulation of grain shape, suggesting that the haplotype-based GWAS using the JAM lines is an effective means to evaluate the main and side effects of haplotypes at each QTL. Information on the pleiotropic effects of haplotypes on various traits will be useful for designing ideal lines in a breeding program.

Transcriptional switch for programmed cell death in pith parenchyma of sorghum stems.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

Haplotype-based allele mining in the Japan-MAGIC rice population.

Multi-parent advanced generation inter-cross (MAGIC) lines have broader genetic variation than bi-parental recombinant inbred lines. Genome-wide association study (GWAS) using high number of DNA polymorphisms such as single-nucleotide polymorphisms (SNPs) is a popular tool for allele mining in MAGIC populations, in which the associations of phenotypes with SNPs are investigated; however, the effects of haplotypes from multiple founders on phenotypes are not considered. Here, we describe an improved method of allele mining using the newly developed Japan-MAGIC (JAM) population, which is derived from eight high-yielding rice cultivars in Japan. To obtain information on the haplotypes in the JAM lines, we predicted the haplotype blocks in the whole chromosomes using 16,345 SNPs identified via genotyping-by-sequencing analysis. Using haplotype-based GWAS, we clearly detected the loci controlling the glutinous endosperm and culm length traits. Information on the alleles of the eight founders, which was based on the effects of mutations revealed by the analysis of next-generation sequencing data, was used to narrow down the candidate genes and reveal the associations between alleles and phenotypes. The haplotype-based allele mining (HAM) proposed in this study is a promising approach to the detection of allelic variation in genes controlling agronomic traits in MAGIC populations.

Genomic adaptation of flowering-time genes during the expansion of rice cultivation area.

The diversification of flowering time in response to natural environments is critical for the spread of crops to diverse geographic regions. In contrast with recent advances in understanding the molecular basis of photoperiodic flowering in rice (Oryza sativa), little is known about how flowering-time diversification is structured within rice subspecies. By analyzing genome sequencing data and a set of 429 chromosome segment substitution lines (CSSLs) originating from 10 diverse rice accessions with wide distributions, we revealed diverse effects of allelic variations for common flowering-time quantitative trait loci in the recipient's background. Although functional variations associated with a few loci corresponded to standing variations among subspecies, the identified functional nucleotide polymorphisms occurred recently after rice subgroup differentiation, indicating that the functional diversity of flowering-time gene sequences was not particularly associated with phylogenetic relationship between rice subspecies. Intensive analysis of the Hd1 genomic region identified the signature of an early introgression of the Hd1 with key mutation(s) in aus and temperate japonica accessions. Our data suggested that, after such key introgressions, new mutations were selected and accelerated the flowering-time diversity within subspecies during the expansion of rice cultivation area. This finding may imply that new genome-wide changes for flowering-time adaptation are one of the critical determinants for establishing genomic architecture of local rice subgroups. In-depth analyses of various rice genomes coupling with the genetically confirmed phenotypic changes in a large set of CSSLs enabled us to demonstrate how rice genome dynamics has coordinated with the adaptation of cultivated rice during the expansion of cultivation area.

A genomic region harboring the Pl1 allele from the Peruvian cultivar JC072A confers purple cob on Japanese flint corn (Zea mays L.).

Purple corn is a maize variety (Zea mays L.) with high anthocyanin content. When purple corn is used as forage, its anthocyanins may mitigate oxidative stresses causing lower milk production in dairy cows. In this study, we analyzed quantitative trait loci (QTLs) for anthocyanin pigmentation of maize organs in an F2 population derived from a cross between the Peruvian cultivar 'JC072A' (purple) and the inbred line 'Ki68' (yellowish) belonged to Japanese flint. We detected 17 significant QTLs on chromosomes 1-3, 6, and 10. Because the cob accounts for most of the fresh weight of the plant ear, we focused on a significant QTL for purple cob on chromosome 6. This QTL also conferred pigmentation of anther, spikelet, leaf sheath, culm, and bract leaf, and was confirmed by using two F3 populations. The gene Pl1 (purple plant 1) is the most likely candidate gene in this QTL region because the amino acid sequence encoded by Pl1-JC072A is similar to that of an Andean allele, Pl-bol3, which is responsible for anthocyanin production. The markers designed for the Pl1 alleles will be useful for the breeding of F1 lines with anthocyanin pigmentation in cobs.

Fine Mapping of Carbon Assimilation Rate 8, a Quantitative Trait Locus for Flag Leaf Nitrogen Content, Stomatal Conductance and Photosynthesis in Rice.

Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice (Oryza sativa). Exploiting the natural variation in CO2 assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.