QTL mapping by GWAS and functional analysis of OsbZIP72 for cold tolerance at rice seedling stage

Rice is a major crop susceptible to chilling stress.The identification of quantitative trait loci and genes for cold tolerance is crucial for the rice breeding.Of 30 quantitative-trait loci affecting seedling cold tolerance identified in a genome-wide association study of 540 rice accessions,OsbZIP72 was assigned as the causative gene for one,qCTS9.1.A single-nucleotide polymorphism in its promoter accounted for variation in expression between indica and japonica subspecies.The favorable haplotype of OsbZIP72 originated in wild rice and contributed to the expansion of japonica rice to colder habitats.OsbZIP72 positively regulates genes coding reactive oxygen species(ROS)-scavenging proteins and maintains intracellular ROS homeostasis.These findings not only enhanced our understanding of environmental adaptation but also provide novel genetic resources and potential targets for molecular design breeding for cold tolerance in rice.

Genomics-based plant germplasm research(GPGR)

Plant germplasm underpins much of crop genetic improvement. Millions of germplasm accessions have been collected and conserved ex situ and/or in situ, and the major challenge is now how to exploit and utilize this abundant resource. Genomics-based plant germplasm research （GPGR） or ＂Cenoplasmics＂ is a novel cross-disciplinary research field that seeks to apply the principles and techniques of genomics to germplasm research. We describe in this paper the concept, strategy, and approach behind GPGR, and summarize current progress in the areas of the definition and construction of core collections, enhancement of germplasm with core collections, and gene discovery from core collections. GPGR is opening a new era in germplasm research. The contribution, progress and achievements of GPGR in the future are predicted.

QTL mapping and QTL × environment interaction analysis of multi-seed pod in cultivated peanut(Arachis hypogaea L.)

To dissect the genetic mechanism of multi-seed pod in peanut, we explored the QTL/gene controlling multi-seed pod and analyzed the interaction effect of QTL and environment. Two hundred and forty eight recombinant inbred lines(RIL) from cross Silihong × Jinonghei 3 were used as experimental materials planted in 8 environments from 2012 to 2017. Three methods of analysis were performed. These included individual environment analysis, joint analysis in multiple environments, and epistatic interaction analysis for multi-seed pod QTL. Phenotypic data and best linear unbiased prediction(BLUP) value of the ratio of multi-seed pods per plant(RMSP) were used for QTL mapping. Seven QTL detected by the individual environmental mapping analysis and were distributed on linkage groups 1, 6, 9, 14, 19(2), and 21. Each QTL explained 4.42%–11.51% of the phenotypic variation in multi-seed pod, and synergistic alleles of5 QTL were from the Silihong parent. One QTL, explaining 4.93% of the phenotypic variation was detected using BLUP data, and this QTL mapped in the same interval as q RMSP19.1 detected in the individual environment analysis. Seventeen additive QTL were identified by joint analysis across multiple environments. A total of 43 epistatic QTL were detected by ICIM-EPI mapping in the multiple environment trials(MET) module, and involved 57 loci. Two main-effect QTL related to multi-seed pod in peanut were filtered. We also found that RMSP had a highly significant positive correlation with pod yield per plant(PY), and epistatic effects were much more important than additive effects. These results provide theoretical guidance for the genetic improvement of germplasm resources and further fine mapping of related genes in peanut.

GNP6,a novel allele of MOC1,regulates panicle and tiller development in rice

The yield of rice is mostly affected by three factors,namely,panicle number,grain number and grain weight.Variation in panicle and grain numbers is mainly caused by tiller and panicle branches generated from axillary meristems(AMs).MOC1 encodes a putative GRAS family nuclear protein that regulates AM formation.Although several alleles of MOC1 have been identified,its variation in germplasm resources remains unclear.In the present study we characterized a novel mocl allele named gnp6 which has a thymine insertion in the coding sequence of the SAW motif in the GRAS domain.This mutation causes arrested branch formation.The SAW motif is necessary for nuclear localization of GNP6/MOC1 where it functions as a transcription factor or co-regulator.Haplotype analysis showed that the coding region of GNP6/MOC1 was conserved without any non-synonymous mutations in 240 rice accessions.However,variation in the promoter region might affect the expression of it and its downstream genes.Joint haplotype analysis of GNP6/MOC1 and MOC3 showed that haplotype combinations H9,H10 and H11,namely MOC1-Hap1 in combination with MOC3-Hap3,MOC3-Hap4 or MOC3-Hap5 could be bred to promote branch formation.These findings will enrich the genetic resources available for rice breeders.

Genetic control of panicle architecture in rice

Rice panicle architecture affects grain number per panicle and thereby grain yield.Many genes involved in control of panicle architecture have been identified in the past decades.According to their effect on phenotype,these genes are divided into three categories:panicle branch and lateral spikelets,multifloret spikelets,and panicle type.We review these genes,describe their genetic regulatory network,and propose a strategy for using them in rice breeding.These findings on rice panicle architecture may facilitate related studies in other crops.

OsADR3 increases drought stress tolerance by inducing antioxidant defense mechanisms and regulating OsGPX1 in rice(Oryza sativa L.)

The C (Cys) 2H (His) 2-type transcription factor is one of the most important transcription factors in plants and plays a regulatory role in the physiological responses of rice to abiotic stresses.A novel rice C2H2-type zinc finger protein,abscisic acid (ABA)-drought-reactive oxygen species (ROS) 3 (OsADR3),was found to confer drought stress tolerance by enhancing antioxidant defense and regulating Os GPX1.Overexpression of OsADR3 in rice increased tolerance to drought stress by increasing ROS scavenging ability and ABA sensitivity.In contrast,CRISPR/Cas9-mediated knockout of osadr3 increased the sensitivity of rice to drought and oxidative stress.An exogenous ROS-scavenging reagent restored the droughtstress tolerance of osadr3-CRISPR plants.Global transcriptome analysis suggested that OsADR3 increased the expression of Os GPX1 under drought stress.Electrophoretic mobility shift,yeast one-hybrid,and dualluciferase reporter assays revealed that OsADR3 modified the expression of Os GPX1 by directly binding to its promoter.Knockdown of Os GPX1 repressed ROS scavenging ability under drought stress in OsADR3-overexpression plants.These findings suggest that OsADR3 plays a positive regulatory role in droughtstress tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice.

Design of the poloidal field system for KTX

The design of the poloidal field （PF） system includes the ohmic heating field system and the equilibrium （EQ） field system, and is the basis for the design of a magnetic confinement fusion device. A coupling between the poloidal and plasma currents, especially the eddy current in the stabilizing shell, yields design difficulties. The effects of the eddy current in the stabilizing shell on the poloidal magnetic field also cannot be ignored. A new PF system design is thus proposed. By using a low-μ material （μ = 0.001, ε = 1） instead of a conductive shell, an electromagnetic model is established that can provide a continuous eddy current distribution on the conductive shell. In this model, a 3D time-domain problem with shells translates into a 2D magnetostatic problem, and the accuracy of the calculation is improved. Based on these current distributions, we design the PF system and analyze how the EQ coils and conductive shell affect the plasma EQ when the plasma ramps up. To meet the mainframe design requirements and achieve an efficient power-supply design, the position and connection of the poloidal coils are optimized further.

Assessing indica-japonica differentiation of improved rice varieties using microsatellite markers

To assess the indica-japonica differentiation of improved rice varieties, a total of 512 modem varieties including 301 indica and 211 japonica accessions were analyzed using 36 microsatellites. The Fst coefficients ranged from 0.002 to 0.730 among the loci with an average of 0.315. Significant differentiation was detected at 94.4% of the loci studied （P 〈 0.05, pairwise Fst tests）, indicating that there was a high level of indica-japonica differentiation within the improved varieties. At 18 loci, about 74%-98% of the alleles of indica and japonica accessions were distributed in two ranges of amplicon length. Linkage disequilibrium analysis showed that the distribution trends were significantly nonrandomly associated. Using the differentiation trends at the 18 loci, microsatellite index （MI） was proposed for discrimination of the two subspecies. When rice accessions with MI value greater than zero were classified as indica, and those with MI value smaller than zero were classified as japonica, about 96.1% of the accessions could be classified. This result agrees with the classification based on morphological-physiological characters, indicating that this method is feasible and effective.

Comparison and rapid prediction of lignocellulose and organic elements of a wide variety of rice straw based on near infrared spectroscopy

Rice straw is a major kind of biomass that can be utilized as lignocellulosic materials and renewable energy.Rapid prediction of the lignocellulose(cellulose,hemicellulose,and lignin)and organic elements(carbon,hydrogen,nitrogen,and sulfur)of rice straw would help to decipher its growth mechanisms and thereby improve its sustainable usages.In this study,364 rice straw samples featuring different rice subspecies(japonica and indica),growing seasons(early-,middle-,and late-season),and growing environments(irrigated and rainfed)were collected,the differences among which were examined by multivariate analysis of variance.Statistic results showed that the cellulose content exhibited significant differences among different growing seasons at a significant level(p<0.01),and the contents of cellulose and nitrogen had significant differences between different growing environments(p<0.01).Near infrared reflectance spectroscopy(NIRS)models for predicting the lignocellulosic and organic elements were developed based on two algorithms including partial least squares(PLS)and competitive adaptive reweighted sampling-partial least squares(CARS-PLS).Modeling results showed that most CARS-PLS models are of higher accuracy than the PLS models,possibly because the CARS-PLS models selected optimal combinations of wavenumbers,which might have enhanced the signal of chemical bonds and thereby improved the predictive efficiency.As a major contributor to the applications of rice straw,the nitrogen content was predicted precisely by the CARS-PLS model.Generally,the CARS-PLS models efficiently quantified the lignocellulose and organic elements of a wide variety of rice straw.The acceptable accuracy of the models allowed their practical applications.

Development of upland rice introgression lines and identification of QTLs for basal root thickness under different water regimes

Introgression lines （ILs） are valuable materials for identifying quantitative trait loci （QTLs）, evaluating genetic interactions, and marker assisted breeding. A set of 430 ILs （BC5F3） containing segments from upland tropical japonica cultivar IRAT109 in a lowland temperate japonica cultivar Yuefu background were developed. One hundred and seventy-six polymorphic markers were used to identify introgressed segments. No segment from IRAT109 was found in 160 lines. Introgressed segments of the other 270 lines covered 99.1% of the donor genome. The mean number of introgressed donor segments per individual was 3.3 with an average length of 14.4 cM. QTL analysis was conducted on basal root thickness （BRT） of the 270 ILs grown under irrigated lowland, upland and hydroponic conditions. A total of 22 QTLs affecting BRT were identified, six QTLs （qBRT3.1, qBRT3.2, qBRT6.1, qBRTS.2, qBRT9.1, and qBRT9.2） were consistently expressed under at least two environments （location and water regime）, and qBRT7.2 was a new BRT QTL identified under lowland conditions. IL255 containing qBRT9.1 showed an increase of 10.09% and 7.07% BRT over cultivar Yuefu when grown under upland and lowland conditions, respectively. Using a population of 304 F2：3 lines derived from the cross IL255 Yuefu, qBRT9.1 was validated and mapped to a 1.2 cM interval between RM24271 and RM566. The presence of qBRT9.1 explained 12% of BRT variation. The results provide upland rice ILs and BRT QTLs for analyzing the genetic basis of drought resistance, detecting favorable genes from upland rice, and rice drought resistance breeding.

The OsNAC41-RoLe1-OsAGAP module promotes root development and drought resistance in upland rice

Drought is a major environmental stress limiting crop yields worldwide.Upland rice(Oryza sativa)has evolved complex genetic mechanisms for adaptative growth under drought stress.However,few genetic variants that mediate drought resistance in upland rice have been identified,and little is known about the evolution of this trait during rice domestication.In this study,using a genome-wide association study we identified ROOT LENGTH 1(RoLe1)that controls rice root length and drought resistance.We found that a G-to-T polymorphism in the RoLe1 promoter causes increased binding of the transcription factor OsNAC41 and thereby enhanced expression of RoLe1.We further showed that RoLe1 interacts with OsAGAP,an ARF-GTPase activating protein involved in auxin-dependent root development,and interferes with its function to modulate root development.Interestingly,RoLe1 could enhance crop yield by increasing the seed-setting rate under moderate drought conditions.Genomic evolutionary analysis revealed that a newly arisen favorable allelic variant,proRoLe1−526T,originated from the midwest Asia and was retained in upland rice during domestication.Collectively,our study identifies an OsNAC41-RoLe1-OsAGAP module that promotes upland rice root development and drought resistance,providing promising genetic targets for molecular breeding of drought-resistant rice varieties.

Natural variation in MORE GRAINS 1 regulates grain number and grain weight in rice

Grain yield is determined mainly by grain number and grain weight.In this study,we identified and characterized MORE GRAINS1(MOG1),a gene associated with grain number and grain weight in rice(Oryza sativa L.),through map-based cloning.Overexpression of MOG1 increased grain yield by 18.6%-22.3%under field conditions.We determined that MOG1,a bHLH transcription factor,interacts with OsbHLH107 and directly activates the expression of LONELY GUY(LOG),which encodes a cytokinin-activating enzyme and the cell expansion gene EXPANSIN-LIKE1(EXPLA1),positively regulating grain number per panicle and grain weight.Natural variations in the promoter and coding regions of MOG1 between Hap-LNW and Hap-HNW alleles resulted in changes in MOG1 expression level and transcriptional activation,leading to functional differences.Haplotype analysis revealed that Hap-HNW,which results in a greater number and heavier grains,has undergone strong selection but has been poorly utilized in modern lowland rice breeding.In summary,the MOG1-OsbHLH107 complex activates LOG and EXPLA1 expression to promote cell expansion and division of young panicles through the cytokinin pathway,thereby increasing grain number and grain weight.These findings suggest that Hap-HNW could be used in strategies to breed high-yielding temperate japonica lowland rice.

Natural Variation in OsPRR37 Regulates Heading Date and Contributes to Rice Cultivation at a Wide Range of Latitudes

Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus （QTL） mapping and candidate gene analysis using whole- genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 （OsPRR37; hereafter PRR37） is respon- sible for the Early heading7-2 （EH7-2）/Heading date2 （Hd2） QTL which was identified from a cross of late-heading rice ＇Milyang23 （M23）＇ and early-heading rice ＇H143＇. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT （CONSTANS, CO-like, and TOC1） domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day condi- tions, and are adapted to the northernmost regions of rice cultivation, up to 53~ N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and GhdT/ Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions.

Variations in OsSPL10 confer drought tolerance by directly regulating OsNAC2 expression and ROS production in rice

Drought is a major factor restricting the production of rice(Oryza sativa L.).The identification of natural variants for drought stress-related genes is an important step toward developing genetically improved rice varieties.Here,we characterized a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)family,OsSPL10,as a transcription factor involved in the regulation of drought tolerance in rice.OsSPL10 appears to play a vital role in drought tolerance by controlling reactive oxygen species(ROS)production and stomatal movements.Haplotype and allele frequency analyses of OsSPL10 indicated that most upland rice and improved lowland rice varieties harbor the OsSPL10Hap1allele,whereas the OsSPL10Hap2allele was mainly present in lowland and landrace rice varieties.Importantly,we demonstrated that the varieties with the OsSPL10Hap1allele showed low expression levels of OsSPL10 and its downstream gene,OsNAC2,which decreases the expression of OsAP37 and increases the expression of OsCOX11,thus preventing ROS accumulation and programmed cell death(PCD).Furthermore,the knockdown or knockout of OsSPL10 induced fast stomatal closure and prevented water loss,thereby improving drought tolerance in rice.Based on these observations,we propose that OsSPL10 confers drought tolerance by regulating OsNAC2 expression and that OsSPL10Hap1could be a valuable haplotype for the genetic improvement of drought tolerance in rice.

Rice SPL10 positively regulates trichome development through expression of HL6 and auxin-related genes

Trichomes function in plant defenses against biotic and abiotic stresses;examination of glabrous lines,which lack trichomes,has revealed key aspects of trichome development and function.Tests of allelism in 51 glabrous rice(Oryza sativa)accessions collected worldwide identified OsSPL10 and OsWOX3B as regulators of trichome development in rice.Here,we report that OsSPL10 acts as a transcriptional regulator controlling trichome development.Haplotype and transient expression analyses revealed that variation in the approximately 700-bp OsSPL10 promoter region is the primary cause of the glabrous phenotype in the indica cultivar WD-17993.Disruption of OsSPL10 by genome editing decreased leaf trichome density and length in the NIL-HL6 background.Plants with genotype OsSPL10^(WD-17993)/HL6 generated by crossing WD-17993 with NIL-HL6 also had fewer trichomes in the glumes.HAIRY LEAF6(HL6)encodes another transcription factor that regulates trichome initiation and elongation,and OsSPL10 directly binds to the HL6 promoter to regulate its expression.Moreover,the transcript levels of auxin-related genes,such as OsYUCCA5 and OsPIN-FORMED1b,were altered in OsSPL10 overexpression and RNAi transgenic lines.Feeding tests using locusts(Locusta migratoria)demonstrated that non-glandular trichomes affect feeding by this herbivore.Our findings provide a molecular framework for trichome development and an ecological perspective on trichome functions.

Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts

Background:MicroRNA-101(miR-101)is a tumor suppressor microRNA(miRNA)and its loss is associated with the occurrence and progression of various diseases.However,the biological function and target of miR-101 in the pathogenesis of hypertrophic scars(HS)remains unknown.Methods:We harvested HS and paired normal skin(NS)tissue samples from patients and cultured their fibroblasts(HSF and NSF,respectively).We used quantitative reverse transcriptase polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),enzyme-linked immunosorbent assays(ELISA)and Western blot analyses to measure mRNA levels and protein expression of miR-101,enhancer of zeste homolog 2(EZH2),collagen 1 and 3(Col1 and Col3)andα-smooth muscle actin(α-SMA)in different in vitro conditions.We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets.We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo.Results:We found low expression of miR-101 in HS and HSF compared to NS and NSF.Overexpressing miR-101 decreased Col1,Col3 andα-SMA expression in HSF.We detected high expression of EZH2 in HS and HSF.Knockdown of EZH2 decreased Col1,Col3 andα-SMA in HSF.Mechanistically,miR-101 targeted the 3-untranslated region(3UTR)of EZH2,as indicated by the decreased expression of EZH2.Overexpressing EZH2 rescued miR-101-induced collagen repression.MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model.Conclusions:Our data reveal that miR-101 targets EZH2 in HS collagen production,providing new insight into the pathological mechanisms underlying HS formation.

Identification and classification of subspecies of Asian cultivated rice and their hybrids

Having reviewed the major classification systems proposed by various scholars across the world, it is found that \%indica\% and \%japonica\% under \%O.sativa\% L. are two major directions thoroughly differentiated from the Asian cultivated rice, forming the framework of the classification structure. A system with \%indica\% and \%japonica\% as the only two subspecies is therefore reiterated.There are various ways to determine the indica_japonica identity of hybrid rice, but the "combined morphological trait index"(CMT index) method is more efficient and easier to handle, although the isozyme analysis, molecular marker analysis and grain quality assay methods are also feasible.