Comparative transcriptomic analysis of germinating rice seedlings to individual and combined anaerobic and cold stress.

BACKGROUND: Rice is one of the most important cereals consumed worldwide. Two major abiotic factors affecting rice plants in different growth stages are flooding stress and cold stress. These abiotic stresses can take place independently or simultaneously and significantly affect rice plants during germination and seedling growth. Fortunately, a wide array of phenotypic responses conferring flooding stress and chilling stress tolerance exist within the rice germplasm, indicating the presence of different molecular mechanisms underlying tolerance to these stresses. Understanding these differences may assist in developing improved rice cultivars having higher tolerance to both stresses. In this study, we conducted a comparative global gene expression analysis of two rice genotypes with contrasting phenotypes under cold stress, anaerobic stress, and combined cold and anaerobic stress during germination. RESULTS: The differential gene expression analysis revealed that 5571 differentially expressed genes (DEGs), 7206 DEGs, and 13279 DEGs were identified under anaerobic stress, cold stress, and combined stress, respectively. Genes involved in the carbohydrate metabolic process, glucosyltransferase activity, regulation of nitrogen compound metabolic process, protein metabolic process, lipid metabolic process, cellular nitrogen compound biosynthetic process, lipid biosynthetic process, and a microtubule-based process were enriched across all stresses. Notably, the common Gene Ontology (GO) analysis identified three hub genes, namely Os08g0176800 (similar to mRNA-associated protein mrnp 41), Os11g0454200 (dehydrin), and OS10g0505900 (expressed protein). CONCLUSION: A large number of differentially expressed genes were identified under anaerobic, cold conditions during germination and the combination of the two stress conditions in rice. These results will assist in the identification of promising candidate genes for possible manipulation toward rice crops that are more tolerant under flooding and cold during germination, both independently and concurrently.

Genetic factors underlying anaerobic germination in rice: Genome-wide association study and transcriptomic analysis.

The success of rice (Oryza sativa L.) germination and survival under submerged conditions is mainly determined by the rapid growth of the coleoptile to reach the water surface. Previous reports have shown the presence of genetic variability within rice accessions in the levels of flooding tolerance during germination or anaerobic germination (AG). Although many studies have focused on the physiological mechanisms of oxygen stress, few studies have explored the breadth of natural variation in AG tolerance-related traits in rice. In this study, we evaluated the coleoptile lengths of a geographically diverse rice panel of 241 accessions, including global accessions along with elite breeding lines and released cultivars from the United States, under the normal and flooded conditions in laboratory and greenhouse environments. A genome-wide association study (GWAS) was performed using a 7K single-nucleotide polymorphism (SNP) array and the phenotypic data of normal coleoptile length, flooded coleoptile length, flooding tolerance index, and survival at 14 d after seeding (DAS). Out of the 30 significant GWAS quantitative trait loci (QTL) regions identified, 14 colocalized with previously identified candidate genes of AG tolerance, whereas 16 were potentially novel. Two rice accessions showing contrasting phenotypic responses to AG stress were selected for the transcriptomics study. The combined approach of GWAS and transcriptomics analysis identified 77 potential candidate genes related to AG tolerance. The findings of our study may assist rice improvement programs in developing rice cultivars with robust tolerance under flooding stress during germination and the early seedling stage.

Phenotypic variation and genome-wide association studies of main culm panicle node number, maximum node production rate, and degree-days to heading in rice.

BACKGROUND: Grain yield is a complex trait that results from interaction between underlying phenotypic traits and climatic, edaphic, and biotic variables. In rice, main culm panicle node number (MCPNN; the node number on which the panicle is borne) and maximum node production rate (MNPR; the number of leaves that emerge per degree-day > 10°C) are primary phenotypic plant traits that have significant positive direct effects on yield-related traits. Degree-days to heading (DDTH), which has a significant positive effect on grain yield, is influenced by the interaction between MCPNN and MNPR. The objective of this research is to assess the phenotypic variation of MCPNN, MNPR, and DDTH in a panel of diverse rice accessions, determine regions in the rice genome associated with these traits using genome-wide association studies (GWAS), and identify putative candidate genes that control these traits. RESULTS: Considerable variation was observed for the three traits in a 220-genotype diverse rice population. MCPNN ranged from 8.1 to 20.9 nodes in 2018 and from 9.9 to 21.0 nodes in 2019. MNPR ranged from 0.0097 to 0.0214 nodes/degree day > 10°C in 2018 and from 0.0108 to 0.0193 nodes/degree-day > 10°C in 2019. DDTH ranged from 713 to 2,345 degree-days > 10°C in 2018 and from 778 to 2,404 degree-days > 10°C in 2019. Thirteen significant (P < 2.91 x 10-7) trait-single nucleotide polymorphism (SNP) associations were identified using the multilocus mixed linear model for GWAS. Significant associations between MCPNN and three SNPs in chromosome 2 (S02_12032235, S02_11971745, and S02_12030176) were detected with both the 2018 and best linear unbiased prediction (BLUP) datasets. Nine SNPs in chromosome 6 (S06_1970442, S06_2310856, S06_2550351, S06_1968653, S06_2296852, S06_1968680, S06_1968681, S06_1970597, and S06_1970602) were significantly associated with MNPR in the 2019 dataset. One SNP in chromosome 11 (S11_29358169) was significantly associated with the DDTH in the BLUP dataset. CONCLUSIONS: This study identifies SNP markers that are putatively associated with MCPNN, MNPR, and DDTH. Some of these SNPs were located within or near gene models, which identify possible candidate genes involved in these traits. Validation of the putative candidate genes through expression and gene editing analyses are necessary to confirm their roles in regulating MCPNN, MNPR, and DDTH. Identifying the underlying genetic basis for primary phenotypic traits MCPNN and MNPR could lead to the development of fast and efficient approaches for their estimation, such as marker-assisted selection and gene editing, which is essential in increasing breeding efficiency and enhancing grain yield in rice. On the other hand, DDTH is a resultant variable that is highly affected by nitrogen and water management, plant density, and several other factors.

Reference-Guided De Novo Genome Assembly to Dissect a QTL Region for Submergence Tolerance Derived from Ciherang-Sub1.

Global climate change has increased the number of severe flooding events that affect agriculture, including rice production in the U.S. and internationally. Heavy rainfall can cause rice plants to be completely submerged, which can significantly affect grain yield or completely destroy the plants. Recently, a major effect submergence tolerance QTL during the vegetative stage, qSub8.1, which originated from Ciherang-Sub1, was identified in a mapping population derived from a cross between Ciherang-Sub1 and IR10F365. Ciherang-Sub1 was, in turn, derived from a cross between Ciherang and IR64-Sub1. Here, we characterize the qSub8.1 region by analyzing the sequence information of Ciherang-Sub1 and its two parents (Ciherang and IR64-Sub1) and compare the whole genome profile of these varieties with the Nipponbare and Minghui 63 (MH63) reference genomes. The three rice varieties were sequenced with 150 bp pair-end whole-genome shotgun sequencing (Illumina HiSeq4000), followed by performing the Trimmomatic-SOAPdenovo2-MUMmer3 pipeline for genome assembly, resulting in approximate genome sizes of 354.4, 343.7, and 344.7 Mb, with N50 values of 25.1, 25.4, and 26.1 kb, respectively. The results showed that the Ciherang-Sub1 genome is composed of 59-63% Ciherang, 22-24% of IR64-Sub1, and 15-17% of unknown sources. The genome profile revealed a more detailed genomic composition than previous marker-assisted breeding and showed that the qSub8.1 region is mostly from Ciherang, with some introgressed segments from IR64-Sub1 and currently unknown source(s).

Transcriptome profiling of two rice genotypes under mild field drought stress during grain-filling stage.

Drought is one of the most critical abiotic stresses that threaten crop production worldwide. This stress affects the rice crop in all stages of rice development; however, the occurrence during reproductive and grain-filling stages has the most impact on grain yield. Although many global transcriptomic studies have been performed during the reproductive stage in rice, very limited information is available for the grain-filling stage. Hence, we intend to investigate how the rice plant responds to drought stress during the grain-filling stage and how the responses change over time under field conditions. Two rice genotypes were selected for RNA-seq analysis: '4610', previously reported as a moderately tolerant breeding line, and Rondo, an elite indica rice cultivar susceptible to drought conditions. Additionally, 10 agronomic traits were evaluated under normal irrigated and drought conditions. Leaf tissues were collected during grain-filling stages at two time points, 14 and 21 days after the drought treatment, from both the drought field and normal irrigated field conditions. Based on agronomic performances, '4610' was less negatively affected than Rondo under mild drought conditions, and expression profiling largely aligned with the phenotypic data. The transcriptomic data indicated that, in general, '4610' had much earlier responses than its counterpart in mitigating the impact of drought stress. Several key genes and gene families related to drought stress or stress-related conditions were found differentially expressed in this study, including transcription factors, drought tolerance genes and reactive oxygen species scavengers. Furthermore, this study identified novel differentially expressed genes (DEGs) without function annotations that may play roles in drought tolerance-related functions. Some of the important DEGs detected in this study can be targeted for future research.

Hybrid Rice Outperforms Inbred Rice in Resistance to Sheath Blight and Narrow Brown Leaf Spot.

Sheath blight (ShB; caused by Rhizoctonia solani AG1-1A) and narrow brown leaf spot (NBLS; Cercospora janseana) are among the most important diseases affecting rice production in Texas and other southern parts of the United States. Because of the high yield potential, hybrid rice acreage is continually increasing. Understanding the relative levels of resistance to ShB and NBLS in hybrids compared to those of inbreds is important for effective disease management; however, this information remains largely unknown. A comparison of the performance of hybrid rice and inbred rice was performed involving 173 hybrid and 155 inbred genotypes (cultivars and elite breeding lines) over the course of five crop seasons (2016 to 2020) and two locations in Texas. The results showed that genotype, cultivar type (hybrid or inbred), location, and their interactions had significant effects on the severity of ShB and NBLS. The ShB severity in hybrid genotypes was significantly lower than that in inbred genotypes, with an average reduction of 27% in disease severity during the 5-year, two-location evaluation. Most (53%) of the hybrid genotypes were rated moderately resistant, whereas almost all (97%) of the inbred genotypes ranged from very susceptible to moderately susceptible. Similarly, NBLS severity in hybrid genotypes is significantly lower than that in inbred genotypes. All but four hybrid genotypes exhibit immune reactions to NBLS. In contrast, 77% of inbred genotypes exhibit NBLS symptoms, with disease resistance reactions ranging from susceptible to resistant. The results demonstrate that hybrid rice is generally less susceptible to sheath blight and has a higher level of resistance against NBLS compared with inbred rice.

An improved 7K SNP array, the C7AIR, provides a wealth of validated SNP markers for rice breeding and genetics studies.

Single nucleotide polymorphisms (SNPs) are highly abundant, amendable to high-throughput genotyping, and useful for a number of breeding and genetics applications in crops. SNP frequencies vary depending on the species and populations under study, and therefore target SNPs need to be carefully selected to be informative for each application. While multiple SNP genotyping systems are available for rice (Oryza sativa L. and its relatives), they vary in their informativeness, cost, marker density, speed, flexibility, and data quality. In this study, we report the development and performance of the Cornell-IR LD Rice Array (C7AIR), a second-generation SNP array containing 7,098 markers that improves upon the previously released C6AIR. The C7AIR is designed to detect genome-wide polymorphisms within and between subpopulations of O. sativa, as well as O. glaberrima, O. rufipogon and O. nivara. The C7AIR combines top-performing SNPs from several previous rice arrays, including 4,007 SNPs from the C6AIR, 2,056 SNPs from the High Density Rice Array (HDRA), 910 SNPs from the 384-SNP GoldenGate sets, 189 SNPs from the 44K array selected to add information content for elite U.S. tropical japonica rice varieties, and 8 trait-specific SNPs. To demonstrate its utility, we carried out a genome-wide association analysis for plant height, employing the C7AIR across a diversity panel of 189 rice accessions and identified 20 QTLs contributing to plant height. The C7AIR SNP chip has so far been used for genotyping >10,000 rice samples. It successfully differentiates the five subpopulations of Oryza sativa, identifies introgressions from wild and exotic relatives, and is useful for quantitative trait loci (QTL) and association mapping in diverse materials. Moreover, data from the C7AIR provides valuable information that can be used to select informative and reliable SNP markers for conversion to lower-cost genotyping platforms for genomic selection and other downstream applications in breeding.

Genome-Wide Association Mapping to Identify Genetic Loci for Cold Tolerance and Cold Recovery During Germination in Rice.

Low temperature significantly affects rice growth and yield. Temperatures lower than 15°C are generally detrimental for germination and uniform seedling stand. To investigate the genetic architecture underlying cold tolerance during germination in rice, we conducted a genome-wide association study using a novel diversity panel of 257 rice accessions from around the world and the 7K SNP marker array. Phenotyping was conducted in controlled growth chambers under dark conditions at 13°C. The rice accessions were measured for low-temperature germinability, germination index, coleoptile length under cold stress, plumule length at 4-day recovery, and plumule length recovery rate. A total of 51 QTLs were identified at p < 0.001 and 17 QTLs were identified using an FDR < 0.05 across the different chilling indices with the whole panel of accessions. At the threshold of p < 0.001, a total of 20 QTLs were identified in the subset of japonica accessions, while 9 QTLs were identified in the subset of indica accessions. Considering the recurring SNPs and linked SNPs across different chilling indices, we identified 31 distinct QTL regions in the whole panel, 13 QTL regions in the japonica subset, and 7 distinct QTL regions in the indica subset. Among these QTL regions, three regions were common between the whole panel and japonica, three regions were common between the whole panel and indica, and one region was common between indica and japonica. A subset of QTL regions was potentially colocalized with previously identified genes and QTLs, including 10 from the japonica subset, 4 from the indica subset, and 6 from the whole panel. On the other hand, a total of 21 potentially novel QTL regions from the whole panel, 10 from the japonica subset, and 1 from the indica subset were identified. The results of our study provide useful information on the genetic architecture underlying cold tolerance during germination in rice, which in turn can be used for further molecular study and crop improvement for low-temperature stressed environments.

Resistant starch: Variation among high amylose rice varieties and its relationship with apparent amylose content, pasting properties and cooking methods.

Resistant starch (RS), which is not hydrolyzed in the small intestine, has proposed health benefits. We evaluated 40 high amylose rice varieties for RS content in cooked rice and a 1.9-fold difference was found. Some varieties had more than two-fold greater RS content than a US long-grain intermediate-amylose rice. The high amylose varieties were grouped into four classes according to paste viscosity and gelatinization temperature based on genetic variants of the Waxy and Starch Synthase IIa genes, respectively. RS content was not different between the four paste viscosity-gelatinization temperature classes. Multiple linear regression analysis showed that apparent amylose content and pasting temperature were strong predictors of RS within each class. Two cooking methods, fixed water-to-rice ratio/time and in excess-water/minimum-cook-time, were compared using six rice varieties that were extremes in RS in each of the genetic variant classes, no difference in RS content due to cooking method was observed.

Evaluation of Host-Plant Resistance of Selected Rice Genotypes to the Rice Water Weevil (Coleoptera: Curculionidae).

The rice water weevil, Lissorhoptrus oryzophilus Kuschel, is the most important insect pest of rice in the United States. Management of L. oryzophilus mainly depends upon the use of insecticides due to the lack of effective alternative management tactics. A 3-yr field study was conducted to determine if difference exists among rice genotypes and cultivars of inbred tropical japonica subspecies commercially grown in the southern United States [Cocodrie (PI 606331), CL171, and CL151 (PI 654463)] and the germplasm lines of indica subspecies adapted to tropical climates of Asia [WC 4644 (PI 312777), TNI (PI 495830), Rondo (PI 615022), 4612 (PI 615039), TeQing (PI 536047), and 4593 (PI 615031)] for resistance to L. oryzophilus Experiments were established as a split-plot design with cultivars as main plots and insecticide treatment as subplots. No significant differences were observed in number of L. oryzophilus larvae recovered across cultivars and genotypes, indicating no significant variation in their preference to L. oryzophilus oviposition. Insecticide treatment had a significant impact on L. oryzophilus larval density. However, grain yield did not vary significantly between treated and untreated plots for any of the cultivars and genotypes. The amount of yield loss in response to L. oryzophilus infestation did not vary significantly across genotypes and cultivars, indicating no variation among these genotypes for resistance to L. oryzophilus.

Soil Texture and Cultivar Effects on Rice (Oryza sativa, L.) Grain Yield, Yield Components and Water Productivity in Three Water Regimes.

The objective of this study was to determine the effects of water regime/soil condition (continuous flooding, saturated, and aerobic), cultivar ('Cocodrie' and 'Rondo'), and soil texture (clay and sandy loam) on rice grain yield, yield components and water productivity using a greenhouse trial. Rice grain yield was significantly affected by soil texture and the interaction between water regime and cultivar. Significantly higher yield was obtained in continuous flooding than in aerobic and saturated soil conditions but the latter treatments were comparable to each other. For Rondo, its grain yield has decreased with soil water regimes in the order of continuous flooding, saturated and aerobic treatments. The rice grain yield in clay soil was 46% higher than in sandy loam soil averaged across cultivar and water regime. Compared to aerobic condition, saturated and continuous flooding treatments had greater panicle numbers. In addition, panicle number in clay soil was 25% higher than in sandy loam soil. The spikelet number of Cocodrie was 29% greater than that of Rondo, indicating that rice cultivar had greater effect on spikelet number than soil type and water management. Water productivity was significantly affected by the interaction of water regime and cultivar. Compared to sandy loam soil, clay soil was 25% higher in water productivity. Our results indicated that cultivar selection and soil texture are important factors in deciding what water management option to practice.