OsCPK12 phosphorylates OsCATA and OsCATC to regulate H2O2 homeostasis and improve oxidative stress tolerance in rice.

Calcium-dependent protein kinases (CPKs), the best-characterized calcium sensors in plants, regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. However, how CPKs regulate the antioxidant defense system remains largely unknown. We previously found that impaired function of OsCPK12 leads to oxidative stress in rice, with more H2O2, lower catalase (CAT) activity, and lower yield. Here, we explored the roles of OsCPK12 in oxidative stress tolerance in rice. Our results show that OsCPK12 interacts with and phosphorylates OsCATA and OsCATC at Ser11. Knockout of either OsCATA or OsCATC leads to an oxidative stress phenotype accompanied by higher accumulation of H2O2. Overexpression of the phosphomimetic proteins OsCATAS11D and OsCATCS11D in oscpk12-cr reduced the level of H2O2 accumulation. Moreover, OsCATAS11D and OsCATCS11D showed enhanced catalase activity in vivo and in vitro. OsCPK12-overexpressing plants exhibited higher CAT activity as well as higher tolerance to oxidative stress. Our findings demonstrate that OsCPK12 affects CAT enzyme activity by phosphorylating OsCATA and OsCATC at Ser11 to regulate H2O2 homeostasis, thereby mediating oxidative stress tolerance in rice.

Photoperiod and gravistimulation-associated Tiller Angle Control 1 modulates dynamic changes in rice plant architecture.

KEY MESSAGE: TAC1 is involved in photoperiodic and gravitropic responses to modulate rice dynamic plant architecture likely by affecting endogenous auxin distribution, which could explain TAC1 widespread distribution in indica rice. Plants experience a changing environment throughout their growth, which requires dynamic adjustments of plant architecture in response to these environmental cues. Our previous study demonstrated that Tiller Angle Control 1 (TAC1) modulates dynamic changes in plant architecture in rice; however, the underlying regulatory mechanisms remain largely unknown. In this study, we show that TAC1 regulates plant architecture in an expression dose-dependent manner, is highly expressed in stems, and exhibits dynamic expression in tiller bases during the growth period. Photoperiodic treatments revealed that TAC1 expression shows circadian rhythm and is more abundant during the dark period than during the light period and under short-day conditions than under long-day conditions. Therefore, it contributes to dynamic plant architecture under long-day conditions and loose plant architecture under short-day conditions. Gravity treatments showed that TAC1 is induced by gravistimulation and negatively regulates shoot gravitropism, likely by affecting auxin distribution. Notably, the tested indica rice containing TAC1 displayed dynamic plant architecture under natural long-day conditions, likely explaining the widespread distribution of TAC1 in indica rice. Our results provide new insights into TAC1-mediated regulatory mechanisms for dynamic changes in rice plant architecture.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7.

INTRODUCTION: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. OBJECTIVES: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s-OsELF3-1-OsLUX complex directly regulates flowering-related genes to mediate rice heading. METHODS: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s-OsELF3-1-OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s-OsELF3-1-OsLUX) complex. RESULTS: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s-OsELF3-1-OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. CONCLUSION: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s-OsELF3-1-OsLUX)-Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

Mapping and Validation of qHD7b: Major Heading-Date QTL Functions Mainly under Long-Day Conditions.

Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.

STRIPE3, encoding a human dNTPase SAMHD1 homolog, regulates chloroplast development in rice.

Chloroplast is an important organelle for photosynthesis and numerous essential metabolic processes, thus ensuring plant fitness or survival. Although many genes involved in chloroplast development have been identified, mechanisms underlying such development are not fully understood. Here, we isolated and characterized the stripe3 (st3) mutant which exhibited white-striped leaves with reduced chlorophyll content and abnormal chloroplast development during the seedling stage, but gradually produced nearly normal green leaves as it developed. Map-based cloning and transgenic tests demonstrated that a splicing mutation in ST3, encoding a human deoxynucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 homolog, was responsible for st3 phenotypes. ST3 is highly expressed in the third leaf at three-leaf stage and expressed constitutively in root, stem, leaf, sheath, and panicle, and the encoded protein, OsSAMHD1, is localized to the cytoplasm. The st3 mutant showed more severe albino leaf phenotype under exogenous 1-mM dATP/dA, dCTP/dC, and dGTP/dG treatments compared with the control conditions, indicating that ST3 is involved in dNTP metabolism. This study reveals a gene associated with dNTP catabolism, and propose a model in which chloroplast development in rice is regulated by the dNTP pool, providing a potential application of these results to hybrid rice breeding.

Tiller Angle Control 1 Is Essential for the Dynamic Changes in Plant Architecture in Rice.

Plant architecture is dynamic as plants develop. Although many genes associated with specific plant architecture components have been identified in rice, genes related to underlying dynamic changes in plant architecture remain largely unknown. Here, we identified two highly similar recombinant inbred lines (RILs) with different plant architecture: RIL-Dynamic (D) and RIL-Compact (C). The dynamic plant architecture of RIL-D is characterized by 'loosetiller angle (tillering stage)-compact (heading stage)-loosecurved stem (maturing stage)' under natural long-day (NLD) conditions, and 'loosetiller angle (tillering and heading stages)-loosetiller angle and curved stem (maturing stage)' under natural short-day (NSD) conditions, while RIL-C exhibits a compact plant architecture both under NLD and NSD conditions throughout growth. The candidate locus was mapped to the chromosome 9 tail via the rice 8K chip assay and map-based cloning. Sequencing, complementary tests, and gene knockout tests demonstrated that Tiller Angle Control 1 (TAC1) is responsible for dynamic plant architecture in RIL-D. Moreover, TAC1 positively regulates loose plant architecture, and high TAC1 expression cannot influence the expression of tested tiller-angle-related genes. Our results reveal that TAC1 is necessary for the dynamic changes in plant architecture, which can guide improvements in plant architecture during the modern super rice breeding.

Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice.

KEY MESSAGE: The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

Fine mapping and candidate gene analysis of qRN5a, a novel QTL promoting root number in rice under low potassium.

KEY MESSAGE: qRN5a, a novel QTL for increasing root number under low K in rice, was fine mapped to a 48.8-kb region on chromosome 5, and LOC_Os05g27980 is the most likely candidate gene. Potassium (K) is a mineral nutrient essential for plant growth and development, but the molecular mechanism for low-K (LK) tolerance in rice remains poorly understood. In our previous study, the quantitative trait locus (QTL) qRN5a for root number (RN) under LK was identified in the chromosome segment substitution line CSSL35 carrying segments from XieqingzaoB in the genetic background of Zhonghui9308 (ZH9308). CSSL35 developed more roots than ZH9308 under LK at the seedling stage, and qRN5a was initially located within a 1,023-kb genomic region. In this study, to understand the molecular basis of qRN5a, a large F2:3 (BC5F2:3) population obtained from crossing CSSL35 and ZH9308 was constructed for fine mapping. High-resolution linkage analysis narrowed down qRN5a to a 48.8-kb interval flanked by markers A99 and A139. Seven putative candidate genes were annotated in the delimited region, and three genes (Os05g0346700, LOC_Os05g27980, and LOC_Os05g28000) had nonsynonymous single-nucleotide polymorphisms in the coding sequence between the two parents. Expression analysis suggests that LOC_Os05g27980, which encodes a LATERAL ORGAN BOUNDARIES domain-containing protein, is a positive regulator of RN under LK and is the most likely candidate gene for qRN5a. Moreover, we found that qRN5a promotes expression of OsIAA23 and represses OsHAK5 expression in root tissues to promote root initiation in CSSL35 under LK conditions. Additional investigations on OsHAK5 in rice are needed to elucidate the basis of changing root architecture under different K+ concentrations. qRN5a is useful for marker-assisted selection to develop an ideotype with improved root architecture in rice under K deficiency.

Characterization and genetic analysis of the oshpl3 rice lesion mimic mutant showing spontaneous cell death and enhanced bacterial blight resistance.

Plant lesion mimic mutants have been used as ideal materials for studying pathogen defense mechanisms due to their spontaneous activation of defense responses in plants. Here, we report the identification and characterization of a rice lesion mimic mutant, oshpl3. The oshpl3 mutant initially displayed white spots on leaves of 7-day-old seedlings, and the white spots gradually turned into large brown spots during plant development, accompanied by poor metrics of major agronomic traits. Histochemical analysis showed that spontaneous cell death and H2O2 hyperaccumulation occurred in oshpl3. Defense responses were induced in the oshpl3 mutant, such as enhanced ROS signaling activated by recognition of pathogen-associated molecular patterns, and also upregulated expression of genes involved in pathogenesis and JA metabolism. These defense responses enhanced resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae. The mutated gene was identified as OsHPL3 (LOC_Os02g02000) by map-based cloning. A G1006A mutation occurred in OsHPL3, causing a G-to-D mutation of the 295th amino acid in the transmembrane region of OsHPL3. OsHPL3 localized to the chloroplast, cytoplasm, and another unknown organelle, while the mutated protein OsHPL3G295D was not obviously observed in the chloroplast, suggesting that the G295D mutation affected its chloroplast localization. Based on our findings, the G295D mutation in OsHPL3 is most likely responsible for the phenotypes of the oshpl3 mutant. Our results provide new clues for studying the function of the OsHPL3 protein.

UMP Kinase Regulates Chloroplast Development and Cold Response in Rice.

Pyrimidine nucleotides are important metabolites that are building blocks of nucleic acids, which participate in various aspects of plant development. Only a few genes involved in pyrimidine metabolism have been identified in rice and the majority of their functions remain unclear. In this study, we used a map-based cloning strategy to isolate a UMPK gene in rice, encoding the UMP kinase that phosphorylates UMP to form UDP, from a recessive mutant with pale-green leaves. In the mutant, UDP content always decreased, while UTP content fluctuated with the development of leaves. Mutation of UMPK reduced chlorophyll contents and decreased photosynthetic capacity. In the mutant, transcription of plastid-encoded RNA polymerase-dependent genes, including psaA, psbB, psbC and petB, was significantly reduced, whereas transcription of nuclear-encoded RNA polymerase-dependent genes, including rpoA, rpoB, rpoC1, and rpl23, was elevated. The expression of UMPK was significantly induced by various stresses, including cold, heat, and drought. Increased sensitivity to cold stress was observed in the mutant, based on the survival rate and malondialdehyde content. High accumulation of hydrogen peroxide was found in the mutant, which was enhanced by cold treatment. Our results indicate that the UMP kinase gene plays important roles in regulating chloroplast development and stress response in rice.

WB1, a Regulator of Endosperm Development in Rice, Is Identified by a Modified MutMap Method.

Abnormally developed endosperm strongly affects rice (Oryza sativa) appearance quality and grain weight. Endosperm formation is a complex process, and although many enzymes and related regulators have been identified, many other related factors remain largely unknown. Here, we report the isolation and characterization of a recessive mutation of White Belly 1 (WB1), which regulates rice endosperm development, using a modified MutMap method in the rice mutant wb1. The wb1 mutant develops a white-belly endosperm and abnormal starch granules in the inner portion of white grains. Representative of the white-belly phenotype, grains of wb1 showed a higher grain chalkiness rate and degree and a lower 1000-grain weight (decreased by ~34%), in comparison with that of Wild Type (WT). The contents of amylose and amylopectin in wb1 significantly decreased, and its physical properties were also altered. We adopted the modified MutMap method to identify 2.52 Mb candidate regions with a high specificity, where we detected 275 SNPs in chromosome 4. Finally, we identified 19 SNPs at 12 candidate genes. Transcript levels analysis of all candidate genes showed that WB1 (Os04t0413500), encoding a cell-wall invertase, was the most probable cause of white-belly endosperm phenotype. Switching off WB1 with the CRISPR/cas9 system in Japonica cv. Nipponbare demonstrates that WB1 regulates endosperm development and that different mutations of WB1 disrupt its biological function. All of these results taken together suggest that the wb1 mutant is controlled by the mutation of WB1, and that the modified MutMap method is feasible to identify mutant genes, and could promote genetic improvement in rice.

Premature leaf senescence 3, encoding a methyltransferase, is required for melatonin biosynthesis in rice.

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H2 O2 ) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence-associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O-methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild-type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild-type rice. Moreover, overexpression of OsMTS1 in the wild-type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.

The rice CONSTANS-like protein OsCOL15 suppresses flowering by promoting Ghd7 and repressing RID1.

The photoperiodic flowering pathway is one of the most important regulatory networks controlling flowering time in rice (Oryza sativa L.). Rice is a facultative short-day (SD) plant; flowering is promoted under inductive SD conditions and delayed under non-inductive long-day (LD) conditions. In rice, flowering inhibitor genes play an important role in maintaining the trade-off between reproduction and yield. In this study, we identified a novel floral inhibitor, OsCOL15, which encodes a CONSTANS-like transcription factor. Consistent with a function in transcriptional regulation, OsCOL15 localized to the nucleus. Moreover, OsCOL15 had transcriptional activation activity, and the central region of the protein between the B-box and CCT domains was required for this activity. We determined that OsCOL15 is most highly expressed in young organs and exhibits a diurnal expression pattern typical of other floral regulators. Overexpression of OsCOL15 resulted in a delayed flowering phenotype under both SD and LD conditions. Real-time quantitative RT-PCR analysis of flowering regulator gene expression suggested that OsCOL15 suppresses flowering by up-regulating the flowering repressor Grain number, plant height and heading date 7 (Ghd7) and down-regulating the flowering activator Rice Indeterminate 1 (RID1), thus leading to the down-regulation of the flowering activators Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T1. These results demonstrate that OsCOL15 is an important floral regulator acting upstream of Ghd7 and RID1 in the rice photoperiodic flowering-time regulatory network.

ES7, encoding a ferredoxin-dependent glutamate synthase, functions in nitrogen metabolism and impacts leaf senescence in rice.

Glutamate synthase (GOGAT) is a key enzyme for nitrogen metabolism and ammonium assimilation in plants. In this study, an early senescence 7 (es7) mutant was identified and characterized. The leaves of the es7 mutant begin to senesce at the tillering stage about 60day after sowing, and become increasingly senescent as the plants develop at the heading stage. When es7 plants are grown under photorespiration-suppressed conditions (high CO2), the senescence phenotype and chlorophyll content are rescued. qRT-PCR analysis showed that senescence- associated genes were up-regulated significantly in es7. A map-based cloning strategy was used to identify ES7, which encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). ES7 was expressed constitutively, and the ES7 protein was localized in chloroplast. qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7. Further, we also demonstrated that chlorophyll synthesis-associated genes were significantly down-regulated in es7. In addition, when seedlings are grown under increasing nitrogen concentrations (NH4NO3) for 15days, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly lower in es7. Our results demonstrated that ES7 is involved in nitrogen metabolism, effects chlorophyll synthesis, and may also associated with photorespiration, impacting leaf senescence in rice.

OsCOL16, encoding a CONSTANS-like protein, represses flowering by up-regulating Ghd7 expression in rice.

Flowering time is an important agronomic trait that coordinates the plant life cycle with regional adaptability and thereby impacts yield potentials for cereal crops. The CONSTANS (CO)-like gene family plays vital roles in the regulation of flowering time. CO-like proteins are typically divided into four phylogenetic groups in rice. Several genes from groups I, III, and IV have been functionally characterized, though little is known about the genes of group II in rice. We report the functional characterization in rice of a constitutive floral inhibitor, OsCOL16, encoding a group-II CO-like protein that delays flowering time and increases plant height and grain yield. Overexpression of OsCOL16 resulted in late heading under both long-day and short-day conditions. OsCOL16 expression exhibits a diurnal oscillation and serves as a transcription factor with transcriptional activation activity. We determined that OsCOL16 up-regulates the expression of the floral repressor Ghd7, leading to down-regulation of the expression of Ehd1, Hd3a, and RFT1. Moreover, genetic diversity and evolutionary analyses suggest that remarkable differences in flowering times correlate with two major alleles of OsCOL16. Our combined molecular biology and phylogeographic analyses revealed that OsCOL16 plays an important role in regulating rice photoperiodic flowering, allowing for environmental adaptation of rice.

Genetic dissection of yield traits in super hybrid rice Xieyou9308 using both unconditional and conditional genome-wide association mapping.

With the development and application of super rice breeding, elite rice hybrids with super high-yielding potential have been widely developed in last decades in China. Xieyou9308 is one of the most famous super hybrid rice varieties. To uncover the genetic mechanism of Xieyou9308's high yield potential, a recombinant inbred line (RIL) population derived from cross of XieqingzaoB and Zhonghui9308 was re-sequenced and investigated on the grain yield (GYD) and its three component traits, number of panicles per plant (NP), number of filled grains per panicle (NFGP), and grain weight (GW). Unconditional and conditional genome-wide association analysis, based on a linear mixed model with epistasis and gene-environment interaction effects, were conducted, using ~0.7 million identified SNPs. There were six, four, seven, and seven QTSs identified for GYD, NP, NFGP, and GW, respectively, with accumulated explanatory heritability varying from 43.06% to 48.36%; additive by environment interactions were detected for GYD, some minor epistases were detected for NP and NFGP. Further, conditional genetic mapping analysis for GYD given its three components revealed several novel QTSs associated with yield than that were suppressed in our unconditional mapping analysis.

Detection of Epistatic and Gene-Environment Interactions Underlying Three Quality Traits in Rice Using High-Throughput Genome-Wide Data.

With development of sequencing technology, dense single nucleotide polymorphisms (SNPs) have been available, enabling uncovering genetic architecture of complex traits by genome-wide association study (GWAS). However, the current GWAS strategy usually ignores epistatic and gene-environment interactions due to absence of appropriate methodology and heavy computational burden. This study proposed a new GWAS strategy by combining the graphics processing unit- (GPU-) based generalized multifactor dimensionality reduction (GMDR) algorithm with mixed linear model approach. The reliability and efficiency of the analytical methods were verified through Monte Carlo simulations, suggesting that a population size of nearly 150 recombinant inbred lines (RILs) had a reasonable resolution for the scenarios considered. Further, a GWAS was conducted with the above two-step strategy to investigate the additive, epistatic, and gene-environment associations between 701,867 SNPs and three important quality traits, gelatinization temperature, amylose content, and gel consistency, in a RIL population with 138 individuals derived from super-hybrid rice Xieyou9308 in two environments. Four significant SNPs were identified with additive, epistatic, and gene-environment interaction effects. Our study showed that the mixed linear model approach combining with the GPU-based GMDR algorithm is a feasible strategy for implementing GWAS to uncover genetic architecture of crop complex traits.

Genetic mapping of a QTL controlling source-sink size and heading date in rice.

Source size, sink size and heading date (HD) are three important classes of traits that determine the productivity of rice. In this study, a set of recombinant inbred lines (RILs) derived from the cross between an elite indica line Big Grain1 (BG1) and a japonica line Xiaolijing (XLJ) were used to map quantitative trait loci (QTLs) for source-sink size and heading date. Totally, thirty-one QTLs for source size, twenty-two for sink size, four for heading date and seven QTL clusters which included QTLs for multiple traits were identified in three environmental trials. Thirty QTLs could be consistently detected in at least two trials and generally located in the clusters. Using a set of BC4F2 lines, the QTL cluster in C5-1-C5-2 on chromosome 5 was validated to be a major QTL pleiotropically affecting heading date, source size (flag leaf area) and panicle type (neck length of panicle, primary branching number and the ratio of secondary branching number to primary branching number), and was narrowed down to a 309.52Kb region. QTL clusters described above have a large effect on source-sink size and/or heading date, therefore they should be good resources to improve the adaptability and high yield potential of cultivars genetically.

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions.

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14-18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC3F3 recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC3F3 recombinants, the root lengths of their BC3F4 progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.

Identification of transcriptome SNPs for assessing allele-specific gene expression in a super-hybrid rice Xieyou9308.

Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE) in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq). We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.