Showing papers in "Rice in 2019"

Molecular Mechanisms of Root Development in Rice

Abstract: Roots are fundamentally important for growth and development, anchoring the plant to its growth substrate, facilitating water and nutrient uptake from the soil, and sensing and responding to environmental signals such as biotic and abiotic stresses. Understanding the molecular mechanisms controlling root architecture is essential for improving nutrient uptake efficiency and crop yields. In this review, we describe the progress being made in the identification of genes and regulatory pathways involved in the development of root systems in rice (Oryza sativa L.), including crown roots, lateral roots, root hairs, and root length. Genes involved in the adaptation of roots to the environmental nutrient status are reviewed, and strategies for further study and agricultural applications are discussed. The growth and development of rice roots are controlled by both genetic factors and environmental cues. Plant hormones, especially auxin and cytokinin, play important roles in root growth and development. Understanding the molecular mechanisms regulating root architecture and response to environmental signals can contribute to the genetic improvement of crop root systems, enhancing their adaptation to stressful environmental conditions.

Identification of genes for salt tolerance and yield-related traits in rice plants grown hydroponically and under saline field conditions by genome-wide association study

Abstract: Soil salinity is one of the main environmental conditions that affects rice production. Identifying the genetic loci that affect rice salt tolerance (ST)-related traits at the seedling stage, especially under saline field conditions, is crucial for ST rice breeding by pyramiding ST genes that act at different developmental stages. Large phenotypic variations were observed in 708 rice accessions, and yield and its related traits were considerably limited when exposed to salt stress. In a genome-wide association study (GWAS), 2255 marker-trait association signals were detected for all measured traits, and the significant SNPs were distributed in 903 genes. Of these, 43 genes processed same functional annotation, and the gene ontology terms “biological processes” and “molecular function” with the known genes responsive to salt stress in rice. Further haplotype analysis detected 15 promising candidates significantly associated with the target traits, including five known genes and 10 novel genes. We identified seven accessions carrying favorable haplotypes of four genes significantly associated with grain yield that performed well under saline stress conditions. Using high density SNPs within genes to conduct GWAS is an effective way to identify candidate genes for salt tolerance in rice. Five known genes (OsMYB6, OsGAMYB, OsHKT1;4, OsCTR3, and OsSUT1) and two newly identified genes (LOC_Os02g49700, LOC_Os03g28300) significantly associated with grain yield and its related traits under saline stress conditions were identified. These promising candidates provide valuable resources for validating potential ST-related genes and will facilitate rice breeding for salt tolerance through marker-assisted selection.

CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv. oryzae.

Abstract: Genome editing tools are important for functional genomics research and biotechnology applications. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system for gene knockout has emerged as the most effective genome-editing tool. It has previously been reported that, in rice plants, knockdown of the Os8N3 gene resulted in enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), while displaying abnormal pollen development. The CRISPR/Cas9 system was employed to knockout rice Os8N3, in order to confer enhanced resistance to Xoo. Analysis of the genotypes and edited Os8N3 in T0, T1, T2, and T3 transgenic rice plants showed that the mutations were transmitted to subsequent generations, and homozygous mutants displayed significantly enhanced resistance to Xoo. Stable transmission of CRISPR/Cas9-mediated Os8N3 gene editing without the transferred DNA (T-DNA) was confirmed by segregation in the T1 generation. With respect to many investigated agronomic traits including pollen development, there was no significant difference between homozygous mutants and non-transgenic control plants under greenhouse growth conditions. Data from this study indicate that the CRISPR/Cas9-mediated Os8N3 edition can be successfully employed for non-transgenic crop improvements.

Overexpression of a microRNA-targeted NAC transcription factor improves drought and salt tolerance in Rice via ABA-mediated pathways.

Abstract: The NAC (NAM, AFAT, and CUC) transcription factors play critical roles in rice (Oryza sativa) development and stress regulation. Overexpressing a microRNA (miR164b)-resistant OsNAC2 mutant gene, which generates transcripts that cannot be targeted by miR164b, improves rice plant architecture and yield; however, the performance of these mOsNAC2-overexpressing lines, named ZUOErN3 and ZUOErN4, under abiotic stress conditions such as drought have not yet been fully characterized. In this study, we showed that the germination of ZUOErN3 and ZUOErN4 seeds was delayed in comparison with the wild-type (WT) seeds, although the final germination rates of all lines were over 95%. The quantification of the endogenous ABA levels revealed that the germinating mOsNAC2-overexpressing seeds had elevated ABA levels, which resulted in their slower germination. The mOsNAC2-overexpressing plants were significantly more drought tolerance than the WT plants, with the survival rate increasing from 11.2% in the WT to nearly 70% in ZUOErN3 and ZUOErN4 plants after a drought treatment. Salt (NaCl) tolerance was also increased in the ZUOErN3 and ZUOErN4 plants due to significantly increased ABA levels. A reverse transcription quantitative PCR (RT-qPCR) analysis showed a significant increase in the expression of the ABA biosynthesis genes OsNCED1 and OsNCED3 in the mOsNAC2-overexpressing lines, and the expression levels of the stress-responsive genes OsP5CS1, OsLEA3, and OsRab16 were significantly increased in these plants. Moreover, OsNAC2 directly interacted with the promoters of OsLEA3 and OsNCED3 in yeast one-hybrid assays. Taken together, our results show that OsNAC2 plays a positive regulatory role in drought and salt tolerance in rice through ABA-mediated pathways.

Development of a core SNP arrays based on the KASP method for molecular breeding of rice

Abstract: The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method. Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319–9–1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average FST value for the three subgroups was 0.3501; the FST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously. The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.

Genome-wide association study and candidate gene analysis of alkalinity tolerance in japonica rice germplasm at the seedling stage

Abstract: Salinity-alkalinity stress is one of the major factors limiting rice production. The damage caused by alkaline salt stress to rice growth is more severe than that caused by neutral salt stress. At present, the genetic resources (quantitative trait loci (QTLs) and genes) that can be used by rice breeders to improve alkalinity tolerance are limited. Here, we assessed the alkalinity tolerance of rice at the seedling stage and performed a genome-wide association study (GWAS) based on genotypic data including 788,396 single-nucleotide polymorphisms (SNPs) developed by re-sequencing 295 japonica rice varieties. We used the score of alkalinity tolerance (SAT), the concentrations of Na+ and K+ in the shoots (SNC and SKC, respectively) and the Na+/K+ ratio of shoots (SNK) as indices to assess alkalinity tolerance at the seedling stage in rice. Based on population structure analysis, the japonica rice panel was divided into three subgroups. Linkage disequilibrium (LD) analysis showed that LD decay occurred at 109.77 kb for the whole genome and varied between 13.79 kb and 415.77 kb across the 12 chromosomes, at which point the pairwise squared correlation coefficient (r2) decreased to half of its maximum value. A total of eight QTLs significantly associated with the SAT, SNC and SNK were identified by genome-wide association mapping. A common QTL associated with the SAT, SNC and SNK on chromosome 3 at the position of 15.0 Mb, which explaining 13.36~13.64% of phenotypic variation, was selected for further analysis. The candidate genes were filtered based on LD decay, Gene Ontology (GO) enrichment, RNA sequencing data, and quantitative real-time PCR (qRT-PCR) analysis. Moreover, sequence analysis revealed one 7-bp insertion/deletion (indel) difference in LOC_Os03g26210 (OsIRO3) between the alkalinity-tolerant and alkalinity-sensitive rice varieties. OsIRO3 encodes a bHLH-type transcription factor and has been shown to be a negative regulator of the Fe-deficiency response in rice. Based on these results, OsIRO3 maybe a novel functional gene associated with alkalinity tolerance in japonica rice. This study provides resources for improving alkalinity tolerance in rice, and the functional molecular marker could be verified to breed new rice varieties with alkalinity tolerance via marker-assisted selection (MAS).

Marker Assisted Breeding to Develop Multiple Stress Tolerant Varieties for Flood and Drought Prone Areas

Abstract: Climate extremes such as drought and flood have become major constraints to the sustainable rice crop productivity in rainfed environments. Availability of suitable climate-resilient varieties could help farmers to reduce the grain yield losses resulting from the climatic extremities. The present study was undertaken with an aim to develop high-yielding drought and submergence tolerant rice varieties using marker assisted introgression of qDTY1.1, qDTY2.1, qDTY3.1 and Sub1. Performance of near isogenic lines (NILs) developed in the background of Swarna was evaluated across 60 multi-locations trials (MLTs). The selected promising lines from MLTs were nominated and evaluated in national trials across 18 locations in India and 6 locations in Nepal. Grain yield advantage of the NILs with qDTY1.1 + qDTY2.1 + qDTY3.1 + Sub1 and qDTY2.1 + qDTY3.1 + Sub1 ranged from 76 to 2479 kg ha− 1 and 396 to 2376 kg ha− 1 under non-stress (NS) respectively and 292 to 1118 kg ha− 1 and 284 to 2086 kg ha− 1 under reproductive drought stress (RS), respectively. The NIL, IR96322–34-223-B-1-1-1-1 having qDTY1.1 + qDTY2.1 + qDTY3.1 + Sub1 has been released as variety CR dhan 801 in India. IR 96321–1447-651-B-1-1-2 having qDTY1.1 + qDTY3.1 + Sub 1 and IR 94391–131–358-19-B-1-1-1 having qDTY3.1 + Sub1 have been released as varieties Bahuguni dhan-1′ and ‘Bahuguni dhan-2’ respectively in Nepal. Background recovery of 94%, 93% and 98% was observed for IR 96322–34-223-B-1-1-1-1, IR 96321–1447-651-B-1-1-2 and IR 94391–131–358-19-B-1-1-1 respectively on 6 K SNP Infinium chip. The drought and submergence tolerant rice varieties with pyramided multiple QTLs can ensure 0.2 to 1.7 t ha− 1 under reproductive stage drought stress and 0.1 to 1.0 t ha− 1 under submergence conditions with no yield penalty under non-stress to farmers irrespective of occurrence of drought and/or flood in the same or different seasons.

Evaluating rice for salinity using pot-culture provides a systematic tolerance assessment at the seedling stage.

Abstract: Rice (Oryza sativa L.) is one of the major staple food crops consumed globally. However, rice production is severely affected by high salinity levels, particularly at the seedling stage. A good solution would be the development of an efficient screening methodology to identify genotypes possessing genes for salt tolerance. A new salinity tolerance screening technique using rice seedlings in pot-culture was tested. This method controls soil heterogeneity by using pure sand as a growth medium and minimizes unexpected extreme weather conditions with a movable shelter. Seventy-four rice genotypes were screened at three salinity treatments including high salt stress (electrical conductivity (EC) 12 dSm− 1), moderate salt stress (EC 6 dSm− 1), and control (no salt stress), imposed 1 week after emergence. Several shoot and root morpho-physiological traits were measured at 37 days after sowing. A wide range of variability was observed among genotypes for measured traits with root traits being identified as the best descriptors for tolerance to salt stress conditions. Salt stress response indices (SSRI) were used to classify the 74 rice genotypes; 7 genotypes (9.46%) were identified as salt sensitive, 27 (36.48%) each as low and moderately salt tolerant, and 13 (17.57%) as highly salt tolerant. Genotypes FED 473 and IR85427 were identified as the most salt tolerant and salt sensitive, respectively. These results were further confirmed by principal component analysis (PCA) for accuracy and reliability. Although tolerant genotypes still need to be confirmed in field studies and tolerance mechanisms identified at the molecular level, information gained from this study could help rice breeders and other scientists to accelerate breeding by selecting appropriate donor parents, progenies and potential genotypes at early growth stages necessary for salinity tolerance research.

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants.

Abstract: Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.

Genetic Interactions Among Ghd7, Ghd8, OsPRR37 and Hd1 Contribute to Large Variation in Heading Date in Rice

Abstract: Heading date is crucial for rice reproduction and geographic expansion. Many heading date genes are sensitive to photoperiod and jointly regulate flowering time in rice. However, it is not clear how these genes coordinate rice heading. Here, we performed a genetic interaction analysis among four major rice heading date genes Ghd7, Ghd8, OsPRR37/Ghd7.1 (hereafter PRR37) and Hd1 in the near-isogenic background under both natural long-day (NLD) and natural short-day (NSD) conditions. The 4-gene segregating population exhibited a large heading date variation with more than 95 days under NLD and 42 days under NSD conditions. Tetragenic, trigenic and digenic interactions among these four genes were observed under both conditions but more significant under NLD conditions. In the functional Hd1 backgrounds, the strongest digenic interaction was Ghd7 by Ghd8 under NLD but was Ghd7 by PRR37 under NSD conditions. Interestingly, PRR37 acted as a flowering suppressor under NLD conditions, while it functioned alternatively as an activator or a suppressor under NSD conditions depending on the status of the other three genes. Based on the performances of 16 homozygous four-gene combinations, a positive correlation between heading date and spikelets per panicle (SPP) was found under NSD conditions, but changed to a negative correlation when heading date was over 90 days under NLD conditions. These results demonstrate the importance of genetic interactions in the rice flowering regulatory network and will help breeders to select favorable combinations to maximize rice yield potential for different ecological areas.

Nitrogen application and different water regimes at booting stage improved yield and 2-acetyl-1-pyrroline (2AP) formation in fragrant rice.

Abstract: It was highlighted that the original article (Mo et al. 2019) contained an error in Fig. 1f which revealed the biosynthesis pathway of 2AP. This Correction article shows the correct Fig. 1 and incorrect Fig. 1. The original article has been updated.

Genome-wide Association Study of a Panel of Vietnamese Rice Landraces Reveals New QTLs for Tolerance to Water Deficit During the Vegetative Phase

Abstract: Drought tolerance is a major challenge in breeding rice for unfavorable environments. In this study, we used a panel of 180 Vietnamese rice landraces genotyped with 21,623 single-nucleotide polymorphism markers to perform a genome-wide association study (GWAS) for different drought response and recovery traits during the vegetative stage. These landraces originate from different geographical locations and are adapted to different agrosystems characterized by contrasted water regimes. Vietnamese landraces are often underrepresented in international panels used for GWAS, but they can contain original genetic determinants related to drought resistance. The panel of 180 rice varieties was phenotyped under greenhouse conditions for several drought-related traits in an experimental design with 3 replicates. Plants were grown in pots for 4 weeks and drought-stressed by stopping irrigation for an additional 4 weeks. Drought sensitivity scores and leaf relative water content were measured throughout the drought stress. The recovery capacity was measured 2 weeks after plant rewatering. Several QTLs associated with these drought tolerance traits were identified by GWAS using a mixed model with control of structure and kinship. The number of detected QTLs consisted of 14 for leaf relative water content, 9 for slope of relative water content, 12 for drought sensitivity score, 3 for recovery ability and 1 for relative crop growth rate. This set of 39 QTLs actually corresponded to a total of 17 different QTLs because 9 were simultaneously associated with two or more traits, which indicates that these common loci may have pleiotropic effects on drought-related traits. No QTL was found in association with the same traits in both the indica and japonica subpanels. The possible candidate genes underlying the quantitative trait loci are reviewed. Some of the identified QTLs contain promising candidate genes with a function related to drought tolerance by osmotic stress adjustment.

High-resolution mapping and breeding application of a novel brown planthopper resistance gene derived from wild rice ( Oryza. rufipogon Griff)

Abstract: The brown planthopper (Nilaparvata lugens Stal; BPH), one of the most destructive pests of rice, has proven to be a substantial threat, conferring enormous production losses in Asia and becoming a difficult challenge to manipulate and control under field conditions. The continuous use of insecticides promotes the resurgence of BPH, which results in resistant varieties adapting through the upgrading of new BPH biotypes. To overcome resistance acquired by BPH against resistance varieties, different forms of novel resistant gene fusions act as functional domains for breeding to enhance insect resistance. The current study reports on the novel BPH resistance gene Bph36 derived from two introgression lines (RBPH16 and RBPH17) developed from wild rice GX2183 which was previously reported to be resistant to BPH. Using two F2 crossing populations (Kangwenqizhan × RBPH16 and Huanghuazhan × RBPH17) in a bulked segregant analysis (BSA) for identification of resistant genes and QTL analysis, two QTLs for BPH resistance were generated on the long and short arms of chromosome 4, which was further confirmed by developing BC1F2:3 populations by backcrossing via marker assisted selection (MAS) approach. One BPH resistance locus on the short arm of chromosome 4 was mapped to a 38-kb interval flanked by InDel markers S13 and X48, and then was named Bph36, whereas another locus on the long arm of chromosome 4 was also detected in an interval flanked by RM16766 and RM17033, which was the same as that of Bph27. An evaluation analysis based on four parameters (BPH host selection, honeydew weight, BPH survival rate and BPH population growth rate) shows that Bph36 conferred high levels antibiosis and antixenosis to BPH. Moreover, Bph36 pyramided with Bph3, Bph27, and Bph29 through MAS into elite cultivars 9311 and MH511 (harbored Xa23), creating different background breeding lines that also exhibited strong resistance to BPH in the seedling or tillering stage. Bph36 can be utilized in BPH resistance breeding programs to develop high resistant rice lines and the high-resolution fine mapping will facilitate further map-based cloning and marker-assisted gene pyramiding of resistant gene. MAS exploited to pyramid with Bph3, Bph27, Bph29, and Xa23 was confirmed the effectiveness for BPH resistance breeding in rice and provided insights into the molecular mechanism of defense to control this devastating insect.

A novel approach to carotenoid accumulation in rice callus by mimicking the cauliflower Orange mutation via genome editing.

Abstract: β-carotene (provitamin A) is an important target for biofortification of crops as a potential solution to the problem of vitamin A deficiency that is prevalent in developing countries. A previous report showed that dominant expression of splicing variants in the Orange (Or) gene causes β-carotene accumulation in cauliflower curd. In this study, we focused on a putative orthologue of the cauliflower or gene in rice, Osor, and attempt to accumulate β-carotene in rice callus by modification of the Osor gene via genome editing using CRISPR/Cas9. CRISPR/Cas9 vectors for the Osor gene were constructed and transformed into rice calli. Some transformed calli showed orange color due to β-carotene hyper-accumulation. Molecular analyses suggest that orange-colored calli are due to an abundance of in-frame aberrant Osor transcripts, whereas out-of-frame mutations were not associated with orange color. We demonstrate that directed gene modification of the Osor gene via CRISPR/Cas9-mediated genome editing results in β-carotene fortification in rice calli. To date, golden rice, which accumulates β-carotene in rice endosperm, has been developed by conventional transgenic approaches. Our results suggest an alternative approach to enhancing β-carotene accumulation in crops.

A unique bZIP transcription factor imparting multiple stress tolerance in Rice

Abstract: Rice productivity is adversely affected by environmental stresses. Transcription factors (TFs), as the regulators of gene expression, are the key players contributing to stress tolerance and crop yield. Histone gene binding protein-1b (OsHBP1b) is a TF localized within the Saltol QTL in rice. Recently, we have reported the characterization of OsHBP1b in relation to salinity and drought tolerance in a model system tobacco. In the present study, we over-express the full-length gene encoding OsHBP1b in the homologous system (rice) to assess its contribution towards multiple stress tolerance and grain yield. We provide evidence to show that transgenic rice plants over-expressing OsHBP1b exhibit better survival and favourable osmotic parameters under salinity stress than the wild type counterparts. These transgenic plants restricted reactive oxygen species accumulation by exhibiting high antioxidant enzyme activity (ascorbate peroxidase and superoxide dismutase), under salinity conditions. Additionally, these transgenic plants maintained the chlorophyll concentration, organellar structure, photosynthesis and expression of photosynthesis and stress-related genes even when subjected to salinity stress. Experiments conducted for other abiotic stresses such as drought and high temperature revealed improved tolerance in these transgenic plants with better root and shoot growth, better photosynthetic parameters, and enhanced antioxidant enzyme activity, in comparison with WT. Further, the roots of transgenic lines showed large cortical cells and accumulated a good amount of callose, unlike the WT roots, thus enabling them to penetrate hard soil and prevent the entry of harmful ions in the cell. Collectively, our results show that rice HBP1b gene contributes to multiple abiotic stress tolerance through several molecular and physiological pathways and hence, may serve as an important gene for providing multiple stress tolerance and improving crop yield in rice.

Comprehensive evaluation of resistance effects of pyramiding lines with different broad-spectrum resistance genes against Magnaporthe oryzae in rice (Oryza sativa L.).

Abstract: Broad-spectrum resistance gene pyramiding helps the development of varieties with broad-spectrum and durable resistance to M. oryzae. However, detailed information about how these different sources of broad-spectrum resistance genes act together or what are the best combinations to achieve broad-spectrum and durable resistance is limited. Here a set of fifteen different polygene pyramiding lines (PPLs) were constructed using marker-assisted selection (MAS). Using artificial inoculation assays at seedling and heading stage, combined with natural induction identification under multiple field environments, we evaluated systematically the resistance effects of different alleles of Piz locus (Pigm, Pi40, Pi9, Pi2 and Piz) combined with Pi1, Pi33 and Pi54, respectively, and the interaction effects between different R genes. The results showed that the seedling blast and panicle blast resistance levels of PPLs were significantly higher than that of monogenic lines. The main reason was that most of the gene combinations produced transgressive heterosis, and the transgressive heterosis for panicle blast resistance produced by most of PPLs was higher than that of seedling blast resistance. Different gene pyramiding with broad-spectrum R gene produced different interaction effects, among them, the overlapping effect (OE) between R genes could significantly improve the seedling blast resistance level of PPLs, while the panicle blast resistance of PPLs were remarkably correlated with OE and complementary effect (CE). In addition, we found that gene combinations, Pigm/Pi1, Pigm/Pi54 and Pigm/Pi33 displayed broad-spectrum resistance in artificial inoculation at seedling and heading stage, and displayed stable broad-spectrum resistance under different disease nursery. Besides, agronomic traits evaluation also showed PPLs with these three gene combinations were at par to the recurrent parent. Therefore, it would provide elite gene combination model and germplasms for rice blast resistance breeding program. The development of PPLs and interaction effect analysis in this study provides valuable theoretical foundation and innovative resources for breeding broad-spectrum and durable resistant varieties.

Identification of new rice cultivars and resistance loci against rice black-streaked dwarf virus disease through genome-wide association study

Abstract: The rice black-streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Eastern China and other East Asian countries. Breeding resistant cultivars is the most economical and effective strategy to control the disease. However, few varieties and QTLs for RBSDV resistance have been identified to date. In this study, we conducted a genome-wide association study (GWAS) on RBSDV resistance using the rice diversity panel 1 (RDP1) cultivars that were genotyped by a 44,000 high-density single nucleotide polymorphism (SNP) markers array. We found that less than 15% of these cultivars displayed resistance to RBSDV when tested under natural infection conditions at two locations with serious RBSDV occurrence. The aus, indica and tropical japonica sub-populations displayed higher RBSDV resistance than the aromatic and temperate japonica sub-populations. In particular, we identified four varieties that displayed stable levels of RBSDV resistance at all testing locations. GWAS identified 84 non-redundant SNP loci significantly associated with RBSDV resistance at two locations, leading to the identification of 13 QTLs for RBSDV resistance. Among them, qRBSDV-4.2 and qRBSDV-6.3 were detected at both locations, suggesting their resistance stability against environmental influence. Field disease evaluations showed that qRBSDV-6.3 significantly reduces RBSDV disease severity by 20%. Furthermore, introgression of qRBSDV-6.3 into two susceptible rice cultivars by marker-assisted selection demonstrated the effectiveness of qRBSDV-6.3 in enhancing RBSDV resistance. The new resistant cultivars and QTLs against RBSDV disease identified in this study provide important information and genetic materials for the cloning of RBSDV resistance genes as well as developing RBSDV resistant varieties through marker-assisted selection.

Dissecting molecular mechanisms underlying salt tolerance in rice: a comparative transcriptional profiling of the contrasting genotypes.

Abstract: Salinity expansion in arable land is a threat to crop plants. Rice is the staple food crop across several countries worldwide; however, its salt sensitive nature severely affects its growth under excessive salinity. FL478 is a salt tolerant indica recombinant inbred line, which can be a good source of salt tolerance at the seedling stage in rice. To learn about the genetic basis of its tolerance to salinity, we compared transcriptome profiles of FL478 and its sensitive parent (IR29) using RNA-seq technique. A total of 1714 and 2670 genes were found differentially expressed (DEGs) under salt stress compared to normal conditions in FL478 and IR29, respectively. Gene ontology analysis revealed the enrichment of transcripts involved in salinity response, regulation of gene expression, and transport in both genotypes. Comparative transcriptome analysis revealed that 1063 DEGs were co-expressed, while 338/252 and 572/908 DEGs were exclusively up/down-regulated in FL478 and IR29, respectively. Further, some biological processes (e.g. iron ion transport, response to abiotic stimulus, and oxidative stress) and molecular function terms (e.g. zinc ion binding and cation transmembrane transporter activity) were specifically enriched in FL478 up-regulated transcripts. Based on the metabolic pathways analysis, genes encoding transport and major intrinsic proteins transporter superfamily comprising aquaporin subfamilies and genes involved in MAPK signaling and signaling receptor kinases were specifically enriched in FL478. A total of 1135 and 1894 alternative splicing events were identified in transcripts of FL478 and IR29, respectively. Transcripts encoding two potassium transporters and two major facilitator family transporters were specifically up-regulated in FL478 under salt stress but not in the salt sensitive genotype. Remarkably, 11 DEGs were conversely regulated in the studied genotypes; for example, OsZIFL, OsNAAT, OsGDSL, and OsELIP genes were up-regulated in FL478, while they were down-regulated in IR29. The achieved results suggest that FL478 employs more efficient mechanisms (especially in signal transduction of salt stress, influx and transport of k+, ionic and osmotic homeostasis, as well as ROS inhibition) to respond to the salt stress compared to its susceptible parent.

Aeration Increases Cadmium (Cd) Retention by Enhancing Iron Plaque Formation and Regulating Pectin Synthesis in the Roots of Rice ( Oryza sativa ) Seedlings

Abstract: Aeration and water management increasing rhizosphere oxygen amount significantly promote rice (Oryza sativa) growth and yield, but the effect of root aeration on cadmium (Cd) toxicity and accumulation in rice seedlings under hydroponic culture remains unclear. Results showed that aeration promoted rice seedling growth and alleviated Cd toxicity. Transverse section discovered that Cd accelerated root mature and senescence while aeration delayed the mature and senescence of roots. Non-invasive Micro-test Technology (NMT) showed that aeration increased net O2 and Cd2+ influxes on the surface of roots while decreased net Cd2+ influx in xylem. Perls blue staining showed that aeration and Cd treatments increased iron plaque formation on the surface of roots. Results of metal concentration analysis showed that besides increasing Cd retention in iron plaque, aeration also increasing Cd retention in the cell wall of rice roots. Cell wall component analysis showed that aeration not only increased pectin content but also decreased pectin methylesterification degree (PMD) by increasing pectin methylesterase (PME) activity. All of these results indicate that aeration not only delays root mature and senescence but also increases Cd retention in roots by enhancing iron plaque formation and regulating pectin synthesis in the roots of rice seedlings.

Assessing trait contribution and mapping novel QTL for salinity tolerance using the Bangladeshi rice landrace Capsule

Abstract: Salinity is one of the most widespread abiotic stresses affecting rice productivity worldwide. The purpose of this study was to establish the relative importance of different traits associated with salinity tolerance in rice and to identify new quantitative trait loci (QTL) conferring tolerance to salinity at seedling stage. A total of 231 F2:3 plants derived from a cross between a sensitive variety BRRI dhan29 (BR29 hereafter) and Capsule, a salt tolerant Bangladeshi indica landrace, were evaluated under salt stress in a phytotron. Out of the 231 F2 plants, 47 highly tolerant and 47 most sensitive lines were selected, representing the two extreme tails of the phenotypic distribution. These 94 plants were genotyped for 105 simple sequence repeat (SSR) and insertion/deletion (InDel) markers. A genetic linkage map spanning approximately 1442.9 cM of the 12 linkage groups with an average marker distance of 13.7 cM was constructed. QTL were identified on the long arm of chromosome 1 for Na+ concentration, K+ concentration, Na+-K+ ratio and survival; chromosome 3 for Na+ concentration, survival and overall phenotypic evaluation using the Standard Evaluation system (SES); and chromosome 5 for SES. A total of 6 pairwise epistatic interactions were also detected between QTL-linked and QTL-unlinked regions. Graphical genotyping indicated an association between the phenotypes of the extreme families and their QTL genotypes. Path coefficient analysis revealed that Na+ concentration, survival, Na+-K+ ratio and the overall phenotypic performance (SES score) are the major traits associated with salinity tolerance of Capsule. Capsule provides an alternative source of salinity tolerance aside from Pokkali and Nona Bokra, the two Indian salt tolerant landraces traditionally used for breeding salt tolerant rice varieties. Pyramiding the new QTL identified in this study with previously discovered loci, such as Saltol, will facilitate breeding varieties that are highly tolerant of salt stress.

1k-RiCA (1K-Rice Custom Amplicon) a novel genotyping amplicon-based SNP assay for genetics and breeding applications in rice.

Abstract: While a multitude of genotyping platforms have been developed for rice, the majority of them have not been optimized for breeding where cost, turnaround time, throughput and ease of use, relative to density and informativeness are critical parameters of their utility. With that in mind we report the development of the 1K-Rice Custom Amplicon, or 1k-RiCA, a robust custom sequencing-based amplicon panel of ~ 1000-SNPs that are uniformly distributed across the rice genome, designed to be highly informative within indica rice breeding pools, and tailored for genomic prediction in elite indica rice breeding programs. Empirical validation tests performed on the 1k-RiCA showed average marker call rates of 95% with marker repeatability and concordance rates of 99%. These technical properties were not affected when two common DNA extraction protocols were used. The average distance between SNPs in the 1k-RiCA was 1.5 cM, similar to the theoretical distance which would be expected between 1,000 uniformly distributed markers across the rice genome. The average minor allele frequencies on a panel of indica lines was 0.36 and polymorphic SNPs estimated on pairwise comparisons between indica by indica accessions and indica by japonica accessions were on average 430 and 450 respectively. The specific design parameters of the 1k-RiCA allow for a detailed view of genetic relationships and unambiguous molecular IDs within indica accessions and good cost vs. marker-density balance for genomic prediction applications in elite indica germplasm. Predictive abilities of Genomic Selection models for flowering time, grain yield, and plant height were on average 0.71, 0.36, and 0.65 respectively based on cross-validation analysis. Furthermore the inclusion of important trait markers associated with 11 different genes and QTL adds value to parental selection in crossing schemes and marker-assisted selection in forward breeding applications. This study validated the marker quality and robustness of the 1k-RiCA genotypic platform for genotyping populations derived from indica rice subpopulation for genetic and breeding purposes including MAS and genomic selection. The 1k-RiCA has proven to be an alternative cost-effective genotyping system for breeding applications.

Mapping the ‘early salinity response’ triggered proteome adaptation in contrasting rice genotypes using iTRAQ approach

Abstract: To delineate the adaptive mechanisms operative under salinity stress, it is essential to study plant responses at the very early stages of stress which are very crucial for governing plant survival and adaptation. We believe that it is the initial perception and response phase which sets the foundation for stress adaptation in rice seedlings where plants can be considered to be in a state of osmotic shock and ion buildup. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze the pre-existing differences as well as the very early salt shock responsive changes in the proteome of seedlings of contrasting rice genotypes, viz salt-sensitive IR64 and salt-tolerant Pokkali. In response to a quick salt shock, shoots of IR64 exhibited hyperaccumulation of Na+, whereas in Pokkali, these ions accumulated more in roots. Interestingly, we could find 86 proteins to be differentially expressed in shoots of Pokkali seedlings under non-stress conditions whereas under stress, 63 proteins were differentially expressed in Pokkali shoots in comparison to IR64. However, only, 40 proteins under non-stress and eight proteins under stress were differentially expressed in Pokkali roots. A higher abundance of proteins involved in photosynthesis (such as, oxygen evolving enhancer proteins OEE1 & OEE3, PsbP) and stress tolerance (such as, ascorbate peroxidase, superoxide dismutase, peptidyl-prolyl cis-trans isomerases and glyoxalase II), was observed in shoots of Pokkali in comparison to IR64. In response to salinity, selected proteins such as, ribulose bisphosphate carboxylase/oxygenase activase, remained elevated in Pokkali shoots. Glutamate dehydrogenase - an enzyme which serves as an important link between Krebs cycle and metabolism of amino acids was found to be highly induced in Pokkali in response to stress. Similarly, other enzymes such as peroxidases and triose phosphate isomerase (TPI) were also altered in roots in response to stress. We conclude that Pokkali rice seedlings are primed to face stress conditions where the proteins otherwise induced under stress in IR64, are naturally expressed in high abundance. Through specific alterations in its proteome, this proactive stress machinery contributes towards the observed salinity tolerance in this wild rice germplasm.

The Initiation of Inferior Grain Filling is Affected by Sugar Translocation Efficiency in Large Panicle Rice

Abstract: Large panicle rice has a large sink capacity, but inferior spikelet filling is poor in this variety of rice due to asynchronous grain filling. The understanding of the factors that cause asynchronous grain filling will help to propose a model for how to regulate the rice inferior spikelets grain filling. In this study, two large panicle rice varieties, W1844 and CJ03, with the same sink capacity but with differences in asynchronous grain filling were used. The difference in the grain filling rate between superior and inferior spikelets in W1844 was much smaller than that in CJ03. We found that superior spikelet filling was initiated earlier in W1844 than in CJ03. The source-to-sink translocation rate of sucrose during the grain filling stage was more efficient in W1844 than in CJ03, and the gene expression levels of sucrose transporters (OsSUTs) were higher in W1844 functional leaves than in those of CJ03. In addition, carbon output, the transport ratio, and the contribution rate from the stem and sheath to the panicle were much higher at the early filling stage than at later filling stages in W1844. Efficient sugar translocation can satisfy high sink strength, and also the strong sink activity can facilitate the sugar unloading in spikelets. All the above results indicate that an efficient sugar translocation rate at the early grain filling stage can improve sink strength and inferior grain filling initiation. Strategies to limit asynchronous grain filling in rice were also discussed based on our findings.

Competition between Granule Bound Starch Synthase and Starch Branching Enzyme in Starch Biosynthesis.

Abstract: Starch branching enzymes (SBE) and granule-bound starch synthase (GBSS) are two important enzymes for starch biosynthesis. SBE mainly contributes to the formation of side branches, and GBSS mainly contributes for the synthesis of amylose molecules. However, there are still gaps in the understanding of possible interactions between SBE and GBSS. Nineteen natural rice varieties with amylose contents up to 28% were used. The molecular structure, in the form of the chain-length distribution (CLDs, the distribution of the number of monomer units in each branch) was measured after enzymatic debranching, using fluorophore-assisted carbohydrate electrophoresis for amylopectin and size- exclusion chromatography for amylose. The resulting distributions were fitted to two mathematical models based on the underlying biosynthetic processes, which express the CLDs in terms of parameters reflecting relevant enzyme activities. Finding statistically valid correlations between the values of these parameters showed that GBSSI and SBEI compete for substrates during rice starch biosynthesis, and synthesis of amylose short chains involves several enzymes including GBSSI, SBE and SSS (soluble starch synthase). Since the amylose CLD is important for a number of functional properties such as digestion rate, this knowledge is potentially useful for developing varieties with improved functional properties.

The Rice G Protein γ Subunit DEP1/qPE9-1 Positively Regulates Grain-Filling Process by Increasing Auxin and Cytokinin Content in Rice Grains.

Abstract: Heterotrimeric G protein-mediated signal transduction is one of the most important and highly conserved signaling pathways in eukaryotes, which involves in the regulation of many important biological processes. As compared with those in mammals and Arabidopsis thaliana, the functions of rice heterotrimeric G protein and their molecular mechanisms are largely unknown. The rice genome contains a single Gα (RGA1) and Gβ (RGB1), and five Gγ (RGG1, RGG2, GS3, DEP1/qPE9–1, and GGC2) subunits. Recent genetic studies have shown that DEP1/qPE9–1, an atypical putative Gγ protein, is responsible for the grain size as well as the dense and erect panicles, but the biochemical and molecular mechanisms underlying the control of grain size are not well understood. Here, we report that rice plants carrying DEP1/qPE9–1 have more endosperm cells per grain than plants contain the dep1/qpe9–1 allele. The DEP1/qPE9–1 line has a higher rate and more prolonged period of starch accumulation than the dep1/qpe9–1 line. Additionally, the expression of several essential genes encoding enzymes catalyzing sucrose metabolism and starch biosynthesis is higher in the DEP1/qPE9–1 line than in the dep1/qpe9–1 line, especially from the mid to late grain-filling stage. Grains of the DEP1/qPE9–1 line also have higher contents of three phytohormones, ABA, auxin and cytokinin. Exogenous application of auxin or cytokinin enhanced the starch accumulation and the expression of genes encoding grain-filling-related enzymes in the grains of dep1/qpe9–1, whereas ABA produced no effects. Based on these results, we conclude that DEP1/qPE9–1 positively regulates starch accumulation primarily through auxin and cytokinin, which enhance the expression of genes encoding starch biosynthesis during the mid to late grain-filling stage, resulting in increased duration of the grain-filling process.

Genome-wide association mapping of resistance against rice blast strains in South China and identification of a new Pik allele

Abstract: Effective management of rice blast, caused by the fungus Magnaporthe oryzae, requires an understanding of the genetic architecture of the resistance to the disease in rice. Rice resistance varies with M. oryzae strains, and many quantitative trait loci (QTLs) affecting rice blast resistance have been mapped using different strains of M. oryzae from different areas. However, little is known about the genetic architecture of rice resistance against the M. oryzae population in Hunan Province, which is a main rice production area in South China. In this study, we used three isolates from Hunan Province and the rice diversity panel 1 to perform a genome-wide association study (GWAS) of blast resistance in rice. A total of 56 QTLs were identified. One of the QTLs is localized with the resistance gene Pik locus which confers resistance to all three isolates. Genomic sequence analysis of the resistant cultivars led to the identification of a new Pik allele, which we named Pikx. Yeast two-hybrid and co-immunoprecipitation assays between AvrPiks and Pikx confirmed that Pikx is a new allele at the Pik locus. Our GWAS has identified many new blast resistance QTLs. The identified new Pik allele Pikx will be useful for breeding cultivars with high resistance to blast in Hunan and other South China provinces. Further research on the relationship between AvrPiks and Pikx will provide new insights into the molecular mechanism of rice resistance to M. oryzae.

Characterization of Jasmonoyl-Isoleucine (JA-Ile) Hormonal Catabolic Pathways in Rice upon Wounding and Salt Stress.

Abstract: Jasmonate (JA) signaling and functions have been established in rice development and response to a range of biotic or abiotic stress conditions. However, information on the molecular actors and mechanisms underlying turnover of the bioactive jasmonoyl-isoleucine (JA-Ile) is very limited in this plant species. Here we explored two gene families in rice in which some members were described previously in Arabidopsis to encode enzymes metabolizing JA-Ile hormone, namely cytochrome P450 of the CYP94 subfamily (CYP94, 20 members) and amidohydrolases (AH, 9 members). The CYP94D subclade, of unknown function, was most represented in the rice genome with about 10 genes. We used phylogeny and gene expression analysis to narrow the study to candidate members that could mediate JA-Ile catabolism upon leaf wounding used as mimic of insect chewing or seedling exposure to salt, two stresses triggering jasmonate metabolism and signaling. Both treatments induced specific transcriptional changes, along with accumulation of JA-Ile and a complex array of oxidized jasmonate catabolites, with some of these responses being abolished in the JASMONATE RESISTANT 1 (jar1) mutant. However, upon response to salt, a lower dependence on JAR1 was evidenced. Dynamics of CYP94B5, CYP94C2, CYP94C4 and AH7 transcripts matched best the accumulation of JA-Ile catabolites. To gain direct insight into JA-Ile metabolizing activities, recombinant expression of some selected genes was undertaken in yeast and bacteria. CYP94B5 was demonstrated to catalyze C12-hydroxylation of JA-Ile, whereas similarly to its Arabidopsis bi-functional homolog IAR3, AH8 performed cleavage of JA-Ile and auxin-alanine conjugates. Our data shed light on two rice gene families encoding enzymes related to hormone homeostasis. Expression data along with JA profiling and functional analysis identifies likely actors of JA-Ile catabolism in rice seedlings. This knowledge will now enable to better understand the metabolic fate of JA-Ile and engineer optimized JA signaling under stress conditions.

Unraveling the Genetic Elements Involved in Shoot and Root Growth Regulation by Jasmonate in Rice Using a Genome-Wide Association Study

Abstract: Due to their sessile life style, plant survival is dependent on the ability to build up fast and highly adapted responses to environmental stresses by modulating defense response and organ growth. The phytohormone jasmonate plays an essential role in regulating these plant responses to stress. To assess variation of plant growth responses and identify genetic determinants associated to JA treatment, we conducted a genome-wide association study (GWAS) using an original panel of Vietnamese rice accessions. The phenotyping results showed a high natural genetic variability of the 155 tested rice accessions in response to JA for shoot and root growth. The level of growth inhibition by JA is different according to the rice varieties tested. We conducted genome-wide association study and identified 28 significant associations for root length (RTL), shoot length (SHL), root weight (RTW), shoot weight (SHW) and total weight (TTW) in response to JA treatment. Three common QTLs were found for RTL, RTW and SHL. Among a list of 560 candidate genes found to co-locate with the QTLs, a transcriptome analysis from public database for the JA response allows us to identify 232 regulated genes including several JA-responsive transcription factors known to play a role in stress response. Our genome-wide association study shows that common and specific genetic elements are associated with inhibition of shoot and root growth under JA treatment suggesting the involvement of a complex JA-dependent genetic control of rice growth inhibition at the whole plant level. Besides, numerous candidate genes associated to stress and JA response are co-located with the association loci, providing useful information for future studies on genetics and breeding to optimize the growth-defense trade-off in rice.

Genome-Wide Association Analysis of the Genetic Basis for Sheath Blight Resistance in Rice.

Abstract: Sheath blight (ShB), caused by Rhizoctonia solani Kuhn, is one of the most destructive rice diseases. Developing ShB-resistant rice cultivars represents the most economical and environmentally sound strategy for managing ShB. To characterize the genetic basis for ShB resistance in rice, we conducted association studies for traits related to ShB resistance, namely culm length (CL), lesion height (LH), and relative lesion height (RLH). Combined a single locus genome-wide scan and a multi-locus method using 2,977,750 single-nucleotide polymorphisms to analyse 563 rice accessions, we detected 134, 562, and 75 suggestive associations with CL, LH, and RLH, respectively. The adjacent signals associated with RLH were merged into 27 suggestively associated loci (SALs) based on the estimated linkage disequilibrium blocks. More than 44% of detected RLH-SALs harboured multiple QTLs/genes associated with ShB resistance, while the other RLH-SALs were putative novel ShB resistance loci. A total of 261 ShB resistance putative functional genes were screened from 23 RLH-SALs according to bioinformatics and haplotype analyses. Some of the annotated genes were previously reported to encode defence-related and pathogenesis-related proteins, suggesting that quantitative resistance to ShB in rice is mediated by SA- and JA-dependent signalling pathways. Our findings may improve the application of germplasm resources as well as knowledge-based ShB management and the breeding of ShB-resistant rice cultivars.

Rice transcription factor OsMADS57 regulates plant height by modulating gibberellin catabolism

Abstract: The MADS-box transcription factors mainly function in floral organ organogenesis and identity specification. Few research on their roles in vegetative growth has been reported. Here we investigated the functions of OsMADS57 in plant vegetative growth in rice (Oryza sativa). Knockdown of OsMADS57 reduced the plant height, internode elongation and panicle exsertion in rice plants. Further study showed that the cell length was remarkably reduced in the uppermost internode in OsMADS57 knockdown plants at maturity. Moreover, OsMADS57 knockdown plants were more sensitive to gibberellic acid (GA3), and contained less bioactive GA3 than wild-type plants, which implied that OsMADS57 is involved in gibberellin (GA) pathway. Expectedly, the transcript levels of OsGA2ox3, encoding GAs deactivated enzyme, were significantly enhanced in OsMADS57 knockdown plants. The level of EUI1 transcripts involved in GA deactivation was also increased in OsMADS57 knockdown plants. More importantly, dual-luciferase reporter assay and electrophoretic mobility shift assay showed that OsMADS57 directly regulates the transcription of OsGA2ox3 as well as EUI1 through binding to the CArG-box motifs in their promoter regions. In addition, OsMADS57 also modulated the expression of multiple genes involved in GA metabolism or GA signaling pathway, indicating the key and complex regulatory role of OsMADS57 in GA pathway in rice. These results indicated that OsMADS57 acts as an important transcriptional regulator that regulates stem elongation and panicle exsertion in rice via GA-mediated regulatory pathway.