Genetic gains in IRRI's rice salinity breeding and elite panel development as a future breeding resource.

KEY MESSAGE: Estimating genetic gains and formulating a future salinity elite breeding panel for rice pave the way for developing better high-yielding salinity tolerant lines with enhanced genetic gains. Genetic gain is a crucial parameter to check the breeding program's success and help optimize future breeding strategies for enhanced genetic gains. To estimate the genetic gains in IRRI's salinity breeding program and identify the best genotypes based on high breeding values for grain yield (kg/ha), we analyzed the historical data from the trials conducted in the IRRI, Philippines and Bangladesh. A two-stage mixed-model approach accounting for experimental design factors and a relationship matrix was fitted to obtain the breeding values for grain yield and estimate genetic trends. A positive genetic trend of 0.1% per annum with a yield advantage of 1.52 kg/ha was observed in IRRI, Philippines. In Bangladesh, we observed a genetic gain of 0.31% per annum with a yield advantage of 14.02 kg/ha. In the released varieties, we observed a genetic gain of 0.12% per annum with a 2.2 kg/ha/year yield advantage in the IRRI, Philippines. For the Bangladesh dataset, a genetic gain of 0.14% per annum with a yield advantage of 5.9 kg/ha/year was observed in the released varieties. Based on breeding values for grain yield, a core set of the top 145 genotypes with higher breeding values of > 2400 kg/ha in the IRRI, Philippines, and > 3500 kg/ha in Bangladesh with a reliability of > 0.4 were selected to develop the elite breeding panel. Conclusively, a recurrent selection breeding strategy integrated with novel technologies like genomic selection and speed breeding is highly required to achieve higher genetic gains in IRRI's salinity breeding programs.

Extreme trait GWAS (Et-GWAS): Unraveling rare variants in the 3,000 rice genome.

Identifying high-impact, rare genetic variants associated with specific traits is crucial for crop improvement. The 3,010 rice genome (3K RG) dataset offers a valuable resource for discovering genomic regions with potential applications in crop breeding. We used Extreme Trait GWAS (Et-GWAS), employing bulk pooling and allele frequency measurement to efficiently extract rare variants from the 3K RG. This innovative approach facilitates the detection of associations between genetic variants and target traits, concentrating and quantifying rare alleles. In our study, on grain yield under drought stress, Et-GWAS successfully identified five key genes (OsPP2C11, OsK5.2, OsIRO2, OsPEX1, and OsPWA1) known for enhancing yield under drought. In addition, we examined the overlap of our results with previously reported qDTY-QTLs and observed that OsUCH1 and OsUCH2 genes were located within qDTY2.2 We compared Et-GWAS with conventional GWAS, finding it effectively capturing most candidate genes associated with the target trait. Validation with resistant starch showed similar results. To enhance user-friendliness, we developed a GUI for Et-GWAS; https://et-gwas.shinyapps.io/Et-GWAS/.

Screening of potential donors for anaerobic stress tolerance during germination in rice.

The rising cost of transplanting rice has made direct seeding an affordable alternative for rice establishment, particularly in Africa. However, direct seeding, while cost-effective, faces crop establishment challenges due to flooding. Uncontrolled water, driven by erratic rains in low-lying areas or uneven fields, limit germination. Rice possesses the unique ability of anaerobic germination, enabling it to sprout and emerge in oxygen-deprived conditions. Understanding rice's response to anaerobic stress during germination is crucial for resilience breeding. Africa, although relying on direct seeding, has made limited progress in addressing flooding during germination compared to Asia. Anaerobic stress tolerance ensures successful crop emergence even in oxygen-limited environments and can help suppress weeds, a significant challenge in direct-seeded rice cultivation. This study aims to contribute by screening for potential rice donors exhibiting anaerobic stress tolerance. We screened 200 rice genotypes at Sokoine University of Agriculture (SUA) in Morogoro, Tanzania, primarily focusing on landraces with untapped potential. Using an alpha lattice design, we conducted two anaerobic experiments in September and October 2022, adding 7 cm of standing water immediately after dry seeding for flooded and maintaining a 2 cm water level after germination in the control for duration of 21 days. We identified potential donors based on selection index computed from genomic estimated breeding values (GEBVs) using eight variables: germination at 14 DAS, germination at 21 DAS, seedling height at 14 DAS, seedling height at 21 DAS, shoot dry matter at 21 DAS, root dry matter at 21 DAS, culm diameter at 21 DAS, and root length at 21DAS. Ten genotypes emerged as the most promising, exhibiting at least 70% germination in floodwater at 21 DAS and greater selection indices. These genotypes were like: Afaa Mwanza 1/159, Rojomena 271/10, Kubwa Jinga, Wahiwahi, Magongo ya Wayungu, Mpaka wa Bibi, Mwangaza, Tarabinzona, IB126-Bug 2013A, and Kanamalia with respective percentages of 75, 74, 71, 86, 75, 80, 71, 80, 70, and 73. These findings contribute to global efforts to mitigate the impacts of flooding during germination. These donors, will be potential to enrich the gene pool for anaerobic germination, providing valuable resources for breeding for flooding tolerance.

Development and validation of a mobile application for point of care evaluation of growth failure.

OBJECTIVES: Lack of systematic evaluation of short stature results in unnecessary work-up on one hand while missing pathology on the other. We have developed a mobile application that guides work-up based on age, auxology (height, BMI, and corrected standard deviation score), and skeletal maturation with an aim of reducing the diagnostic errors. Aim of this study is to develop and validate a mobile application for point of care evaluation of short stature. METHODS: The application was developed (n=400) and validated (n=412) on children and adolescents (2-18 years of age) presenting to our Pediatric Endocrinology Clinic with short stature. Height standard deviation score thresholds determining the need for workup were derived from Receiver Operating Characteristics (ROC) curve. Student's t-test and ROC curves were used to identify the most appropriate parameter differentiating constitutional delay of growth and puberty (CDGP) from pathological and nutritional from endocrine causes. The validation of the application involved comparing the application predicted and clinical diagnosis at each step of the algorithm. RESULTS: The mobile application diagnosis was concordant with clinical diagnosis in 408 (99.0%) with discordance in four (two with CDGP labeled as growth hormone deficiency [GHD] and two with GHD labeled as CDGP). CONCLUSIONS: Mobile application guided short stature assessment has a high concordance with the clinical diagnosis and is expected to help point of care short stature evaluation.

Breeding rice for a changing climate by improving adaptations to water saving technologies.

Climate change is expected to increasingly affect rice production through rising temperatures and decreasing water availability. Unlike other crops, rice is a main contributor to greenhouse gas emissions due to methane emissions from flooded paddy fields. Climate change can therefore be addressed in two ways in rice: through making the crop more climate resilient and through changes in management practices that reduce methane emissions and thereby slow global warming. In this review, we focus on two water saving technologies that reduce the periods lowland rice will be grown under fully flooded conditions, thereby improving water use efficiency and reducing methane emissions. Rice breeding over the past decades has mostly focused on developing high-yielding varieties adapted to continuously flooded conditions where seedlings were raised in a nursery and transplanted into a puddled flooded soil. Shifting cultivation to direct-seeded rice or to introducing non-flooded periods as in alternate wetting and drying gives rise to new challenges which need to be addressed in rice breeding. New adaptive traits such as rapid uniform germination even under anaerobic conditions, seedling vigor, weed competitiveness, root plasticity, and moderate drought tolerance need to be bred into the current elite germplasm and to what extent this is being addressed through trait discovery, marker-assisted selection and population improvement are reviewed.

Genetic Mapping by Sequencing More Precisely Detects Loci Responsible for Anaerobic Germination Tolerance in Rice.

Direct seeded rice (DSR) is a mainstay for planting rice in the Americas, and it is rapidly becoming more popular in Asia. It is essential to develop rice varieties that are suitable for this type of production system. ASD1, a landrace from India, possesses several traits desirable for direct-seeded fields, including tolerance to anaerobic germination (AG). To map the genetic basis of its tolerance, we examined a population of 200 F2:3 families derived from a cross between IR64 and ASD1 using the restriction site-associated DNA sequencing (RAD-seq) technology. This genotyping platform enabled the identification of 1921 single nucleotide polymorphism (SNP) markers to construct a high-resolution genetic linkage map with an average interval of 0.9 cM. Two significant quantitative trait loci (QTLs) were detected on chromosomes 7 and 9, qAG7 and qAG9, with LOD scores of 7.1 and 15.0 and R2 values of 15.1 and 29.4, respectively. Here, we obtained more precise locations of the QTLs than traditional simple sequence repeat and low-density SNP genotyping methods and may help further dissect the genetic factors of these QTLs.

Enriched-GWAS and Transcriptome Analysis to Refine and Characterize a Major QTL for Anaerobic Germination Tolerance in Rice.

Tolerance of anaerobic germination (AG) is a key trait in the development of direct seeded rice. Through rapid and sustained coleoptile elongation, AG tolerance enables robust seedling establishment under flooded conditions. Previous attempts to fine map and characterize AG2 (qAG7.1), a major centromere-spanning AG tolerance QTL, derived from the indica variety Ma-Zhan Red, have failed. Here, a novel approach of "enriched haplotype" genome-wide association study based on the Ma-Zhan Red haplotype in the AG2 region was successfully used to narrow down AG2 from more than 7 Mb to less than 0.7 Mb. The AG2 peak region contained 27 genes, including the Rc gene, responsible for red pericarp development in pigmented rice. Through comparative variant and transcriptome analysis between AG tolerant donors and susceptible accessions several candidate genes potentially controlling AG2 were identified, among them several regulatory genes. Genome-wide comparative transcriptome analysis suggested differential regulation of sugar metabolism, particularly trehalose metabolism, as well as differential regulation of cell wall modification and chloroplast development to be implicated in AG tolerance mechanisms.

An epigenetic pathway in rice connects genetic variation to anaerobic germination and seedling establishment.

Rice production is shifting from transplanting seedlings to direct sowing of seeds. Following heavy rains, directly sown seeds may need to germinate under anaerobic environments, but most rice (Oryza sativa) genotypes cannot survive these conditions. To identify the genetic architecture of complex traits, we quantified percentage anaerobic germination (AG) in 2,700 (wet-season) and 1,500 (dry-season) sequenced rice genotypes and performed genome-wide association studies (GWAS) using 693,502 single nucleotide polymorphisms. This was followed by post-GWAS analysis with a generalized SNP-to-gene set analysis, meta-analysis, and network analysis. We determined that percentage AG is intermediate-to-high among indica subpopulations, and AG is a polygenic trait associated with transcription factors linked to ethylene responses or genes involved in metabolic processes that are known to be associated with AG. Our post-GWAS analysis identified several genes involved in a wide variety of metabolic processes. We subsequently performed functional analysis focused on the small RNA and methylation pathways. We selected CLASSY 1 (CLSY1), a gene involved in the RNA-directed DNA methylation (RdDm) pathway, for further analyses under AG and found several lines of evidence that CLSY1 influences AG. We propose that the RdDm pathway plays a role in rice responses to water status during germination and seedling establishment developmental stages.

Genomics-assisted breeding for successful development of multiple-stress-tolerant, climate-smart rice for southern and southeastern Asia.

Rice (Oryza sativa L.) in rainfed marginal environments is prone to multiple abiotic and biotic stresses, which can occur in combination in a single cropping season and adversely affect rice growth and yield. The present study was undertaken to develop high-yielding, climate-resilient rice that can provide tolerance to multiple biotic and abiotic stresses. An assembled first-crossing scheme was employed to transfer 15 quantitative trait loci (QTL) and genes-qDTY1.1 , qDTY2.1 , qDTY3.1 , qDTY12.1 (drought), Sub1 (submergence), Gm4 (gall midge), Pi9, Pita2 (blast), Bph3, Bph17 (brown plant hoppers), Xa4, xa5, xa13, Xa21, and Xa23 (bacterial leaf blight)-from eight different parents using genomics-assisted breeding. A funnel mating design was employed to assemble all the targeted QTL and genes into a high-yielding breeding line IR 91648-B-1-B-3-1. Gene-based linked markers were used in each generation from intercrossing to the F6 generation for tracking the presence of desirable alleles of targeted QTL and genes. Single-plant selections were performed from F2 onwards to select desirable recombinants possessing alleles of interest with suitable phenotypes. Phenotyping of 95 homozygous F6 lines carrying six to 10 QTL and genes was performed for nonstress, reproductive-stage (RS) drought, blast, bacterial leaf blight (BLB), gall midge (GM), and for grain quality parameters such as chalkiness, amylose content (AC), gelatinization temperature (GT), and head rice recovery (HRR). Finally, 56 F7 homozygous lines were found promising for multiple-location evaluation for grain yield (GY) and other traits. These multiple-stress-tolerant lines with the desired grain quality profiling can be targeted for varietal release in southern and southeastern Asia through national release systems.

Comparative Transcriptomics and Co-Expression Networks Reveal Tissue- and Genotype-Specific Responses of qDTYs to Reproductive-Stage Drought Stress in Rice (Oryza sativa L.).

Rice (Oryza sativa L.) is more sensitive to drought stress than other cereals. To dissect molecular mechanisms underlying drought-tolerant yield in rice, we applied differential expression and co-expression network approaches to transcriptomes from flag-leaf and emerging panicle tissues of a drought-tolerant yield introgression line, DTY-IL, and the recurrent parent Swarna, under moderate reproductive-stage drought stress. Protein turnover and efficient reactive oxygen species scavenging were found to be the driving factors in both tissues. In the flag-leaf, the responses further included maintenance of photosynthesis and cell wall reorganization, while in the panicle biosynthesis of secondary metabolites was found to play additional roles. Hub genes of importance in differential drought responses included an expansin in the flag-leaf and two peroxidases in the panicle. Overlaying differential expression data with allelic variation in DTY-IL quantitative trait loci allowed for the prioritization of candidate genes. They included a differentially regulated auxin-responsive protein, with DTY-IL-specific amino acid changes in conserved domains, as well as a protein kinase with a DTY-IL-specific frameshift in the C-terminal region. The approach highlights how the integration of differential expression and allelic variation can aid in the discovery of mechanism and putative causal contribution underlying quantitative trait loci for drought-tolerant yield.

Responses of AG1 and AG2 QTL introgression lines and seed pre-treatment on growth and physiological processes during anaerobic germination of rice under flooding.

Rice seeds germinating in flooded soils encounter hypoxia or even anoxia leading to poor seed germination and crop establishment. Introgression of AG1 and AG2 QTLs associated with tolerance of flooding during germination, together with seed pre-treatment via hydro-priming or presoaking can enhance germination and seedling growth in anaerobic soils. This study assessed the performance of elite lines incorporating AG1, AG2 and their combination when directly seeded in flooded soils using dry seeds. The QTLs were in the background of two popular varieties PSB Rc82 and Ciherang-Sub1, evaluated along with the donors Kho Hlan On (AG1) and Ma-Zhan Red (AG2) and recipient parents PSB Rc82 and Ciherang-Sub1. In one set of experiments conducted in the greenhouse, seedling emergence, growth, and carbohydrate mobilization from seeds were assessed. Metabolites associated with reactive oxygen species (ROS) scavenging including malondialdehyde (MDA) as a measure of lipid peroxidation, ascorbate, total phenolic concentration (TPC), and activities of ROS scavenging enzymes were quantified in seeds germinating under control (saturated) and flooded (10 cm) soils. In another set of experiments conducted in a natural field with 3-5 cm flooding depths, control and pretreated seeds of Ciherang-Sub1 introgression lines and checks were used. Flooding reduced seedling emergence of all genotypes, though emergence of AG1 + AG2 introgression lines was greater than the other AG lines. Soluble sugars increased, while starch concentration decreased gradually under flooding especially in the tolerant checks and in AG1 + AG2 introgression lines. Less lipid peroxidation and higher α-amylase activity, higher ascorbate (RAsA) and TPC were observed in the tolerant checks and in the AG1 + AG2 introgression lines. Lipid peroxidation correlated negatively with ascorbate, TPC, and with ROS scavengers. Seed hydro-priming or pre-soaking increased emergence by 7-10% over that of dry seeds. Introgression of AG2 and AG1 + AG2 QTLs with seed pretreatment showed 101-153% higher emergence over dry seeds of intolerant genotypes in the field. Lines carrying AG1 + AG2 QTLs showed higher α-amylase activity, leading to rapid starch degradation and increase in soluble sugars, ascorbate, and TPC, together leading to higher germination and seedling growth in flooded soils. Seed hydro-priming or pre-soaking for 24 h also improved traits associated with flooding tolerance. Combining tolerance with seed management could therefore, improve crop establishment in flooded soils and encourage large-scale adoption of direct seeded rice system.

Trait-based mapping to identify the genetic factors underlying anaerobic germination of rice: Phenotyping, GXE, and QTL mapping.

BACKGROUND: Anaerobic germination is one of the most important traits for rice under direct-seeded conditions. The trait reduces risk of crop failure due to waterlogged conditions after seeding and allows water to be used as a means of weed control. The identification of QTLs and causal genes for anaerobic germination will facilitate breeding for improved direct-seeded rice varieties. In this study, we explored a BC1F2:3 population developed from a cross between BJ1, an indica landrace, and NSIC Rc222, a high-yielding recurrent parent. The population was phenotyped under different screening methods (anaerobic screenhouse, anaerobic tray, and aerobic screenhouse) to establish the relationship among the methods and to identify the most suitable screening method, followed by bulk segregant analysis (BSA) to identify large-effect QTLs. RESULTS: The study showed high heritability for survival (SUR) under all three phenotyping conditions. Although high correlation was observed within screening environments between survival at 14 and 21 days after seeding, the correlation across environments was low. Germination under aerobic and anaerobic conditions showed very low correlation, indicating the independence of their genetic control. The results were further confirmed through AMMI analysis. Four significant markers with an effect on anaerobic germination were identified through BSA. CIM analysis revealed qAG1-2, qAG6-2, qAG7-4, and qAG10-1 having significant effects on the trait. qAG6-2 and qAG10-1 were consistent across screening conditions and seedling age while qAG1-2 and qAG7-4 were specific to screening methods. All QTLs showed an effect when survival across all screening methods was analyzed. Together, the QTLs explained 39 to 55% of the phenotypic variation for survival under anaerobic conditions. No QTL effects were observed under aerobic conditions. CONCLUSIONS: The study helped us understand the effect of phenotyping method on anaerobic germination, which will lead to better phenotyping for this trait in future studies. The QTLs identified through this study will allow the improvement of breeding lines for the trait through marker-assisted selection or through forward breeding approaches such as genomic selection. The high frequency of the BJ1 allele of these QTLs will enhance the robustness of germination under anaerobic conditions in inbred and hybrid rice varieties.

Deciphering Genetics Underlying Stable Anaerobic Germination in Rice: Phenotyping, QTL Identification, and Interaction Analysis.

Anaerobic germination (AG) is an important trait for direct-seeded rice (DSR) to be successful. Rice usually has low germination under anaerobic conditions, which leads to a poor crop stand in DSR when rain occurs after seeding. The ability of rice to germinate under water reduces the risk of poor crop stand. Further, this allows the use of water as a method of weed control. The identification of the genetic factors leading to high anaerobic germination is required to develop improved DSR varieties. In the present study, two BC1F2:3 mapping families involving a common parent with anaerobic germination potential, Kalarata, an indica landrace, and two recurrent parents, NSIC Rc222 and NSIC Rc238, were used. Phenotyping was done under two environmental conditions and genotyping was carried out through the KASP SNP genotyping platform. A total of 185 and 189 individuals genotyped with 170 and 179 polymorphic SNPs were used for QTL analysis for the two populations, Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222, respectively. A total of five QTLs on chromosomes 3, 5, 6, 7, and 8 for survival (SUR) and four QTLs on chromosomes 1, 3 (two locations), and 7 for the trait seedling height (SH) across the populations and over the screening conditions were identified. Except for the QTLs on chromosomes 5 and 8, the parent with AG potential, Kalarata, contributed all the other QTLs. Among the five QTLs for SUR, the second-largest QTL (qSUR6-1) was novel for AG potential in rice, showing a stable expression in terms of genetic background and screening conditions explaining 11.96% to 16.01% of the phenotypic variation. The QTL for SH (qSH1-1) was also novel. Considering different genetic backgrounds and different screening conditions, the QTLs identified for the trait SUR explained phenotypic variation in the range of 57.60% to 73.09% while that for the trait SH ranged from 13.53% to 34.30%.

Epistatic interactions of major effect drought QTLs with genetic background loci determine grain yield of rice under drought stress.

Epistatic interactions of QTLs with the genetic background and QTL-QTL interaction plays an important role in the phenotypic performance of introgression lines developed through genomic-assisted breeding (GAB). In this context, NIL pairs developed with various drought QTL (qDTY) combinations in the genetic background of IR64, TDK1-Sub1 and Savitri backgrounds were utilized to study the interactions. Multi-season phenotyping of NIL pairs harboring similar qDTY combinations provided contrasting performance for grain yield under drought (RS) (classified as high and low yielding NILs) but nearly similar performance under non-stress(NS) conditions. Genome wide genotyping data revealed a total of 16, 5 and 6 digenic interactions were detected under RS conditions in low yielding NILs of IR64, TDK1-Sub1 and Savitri respectively while no significant interaction was found in high yielding NILs under RS and NS conditions in any of the genetic backgrounds used in this study. It is evident from this study that existence of epistatic interactions between QTLs with genetic background, QTL-QTL interaction and interactions among background markers loci itself on different chromosomes influences the expression of a complex trait such as grain yield under drought. The generated information will be useful in all the GAB program of across the crops for precise breeding.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Stomatal conductance responses to evaporative demand conferred by rice drought-yield quantitative trait locus qDTY12.1.

Rice quantitative trait locus (QTL) qDTY12.1 is a major-effect drought yield QTL that was identified from a cross of Vandana (recipient parent) and Way Rarem (donor parent) through breeding efforts to improve rice yield under upland drought stress conditions. The two main physiological effects previously observed to be related to the presence of qDTY12.1 were (i) increased lateral root growth, and (ii) increased transpiration efficiency. Since relatively more progress has thus far been made on characterising the lateral root growth response related to qDTY12.1, the present study focussed on characterising how qDTY12.1 confers higher transpiration efficiency under upland drought stress in the Vandana background. In a series of field experiments in which stomatal conductance was measured across different times of day in four qDTY12.1 near isogenic lines (NILs), the NILs and Way Rarem showed consistently higher stomatal conductance than Vandana under conditions of low vapour pressure deficit (VPD) and low photosynthetically active radiation (PAR), and consistently lower stomatal conductance than Vandana under high VPD and high PAR. Leaf δ18O was higher in the qDTY12.1 NIL than in Vandana, and although this trend was previously observed for leaf δ13C it appeared to be more consistent across measurement dates and treatments for leaf δ18O. The qDTY12.1 NILs and Way Rarem tended to show greater large vein to small vein interveinal distance and mesophyll area than Vandana, also consistent across treatments. In terms of aquaporin-related plant hydraulics, variation among NILs in terms of aquaporin inhibition of root hydraulic conductivity (Lpr) was observed, with the highest-yielding NIL showing a lack of Lpr inhibition similar to Way Rarem. The results reported here suggest that the effects of qDTY12.1 are in response not only to soil moisture, but also to atmospheric conditions. An interaction among multiple mechanisms including leaf anatomy and aquaporin function appear to confer the transpiration efficiency effect of qDTY12.1.

Physiological mechanisms contributing to the QTL qDTY3.2 effects on improved performance of rice Moroberekan x Swarna BC2F3:4 lines under drought.

BACKGROUND: Traditional rice (Oryza sativa) varieties are valuable resources for the improvement of drought resistance. qDTY3.2 is a drought-yield quantitative trait locus that was identified in a population derived from the traditional variety Moroberekan and the drought-susceptible variety Swarna. In this study, our aim was to characterize the physiological mechanisms associated with qDTY3.2. Our approach was to phenotype fifteen BC2F3:4 lines for shoot and root drought resistance-related traits as compared to Swarna in the field under well-watered and drought stress conditions. Four BC2F3:4 lines contrasting for yield under drought were selected for detailed characterization of shoot morphology, water use related traits, flowering time and root system architecture in the field as well as in controlled environments (lysimeters in a greenhouse, and gel imaging platform in a growth chamber). RESULTS: Across five field experiments, grain yield correlated significantly with root growth along the soil profile, flowering time, and canopy temperature under drought conditions. The four selected BC2F3:4 lines showed earlier flowering time, reduced distribution of root growth to shallow soil layers which resulted in lower water uptake (between 0 and 30 cm) and drought-induced increased distribution of root growth to deep soil layers (between 30 and 60 cm) as compared to Swarna in the field. Root system architecture phenotypes were confirmed in whole root systems in lysimeters, and corresponded to higher numbers of root tips in a gel imaging platform, highlighting the potential stability of some root traits across different growth stages and systems. CONCLUSIONS: We conclude that earlier flowering time, reduced shallow root growth, and drought-induced increased deep root growth are associated with the presence of qDTY3.2 since these phenotypes were consistently observed in the selected QTL lines with full introgression of qDTY3.2. We hypothesize that the qDTY3.2 associated RSA phenotypes led to better use of water and metabolic resources which, combined with earlier flowering time, improved yield under drought.

Marker-assisted selection strategy to pyramid two or more QTLs for quantitative trait-grain yield under drought.

BACKGROUND: Marker-assisted breeding will move forward from introgressing single/multiple genes governing a single trait to multiple genes governing multiple traits to combat emerging biotic and abiotic stresses related to climate change and to enhance rice productivity. MAS will need to address concerns about the population size needed to introgress together more than two genes/QTLs. In the present study, grain yield and genotypic data from different generations (F3 to F8) for five marker-assisted breeding programs were analyzed to understand the effectiveness of synergistic effect of phenotyping and genotyping in early generations on selection of better progenies. RESULTS: Based on class analysis of the QTL combinations, the identified superior QTL classes in F3/BC1F3/BC2F3 generations with positive QTL x QTL and QTL x background interactions that were captured through phenotyping maintained its superiority in yield under non-stress (NS) and reproductive-stage drought stress (RS) across advanced generations in all five studies. The marker-assisted selection breeding strategy combining both genotyping and phenotyping in early generation significantly reduced the number of genotypes to be carried forward. The strategy presented in this study providing genotyping and phenotyping cost savings of 25-68% compared with the traditional marker-assisted selection approach. The QTL classes, Sub1 + qDTY 1.1  + qDTY 2.1 + qDTY 3.1 and Sub1 + qDTY 2.1 + qDTY 3.1 in Swarna-Sub1, Sub1 + qDTY 1.1 + qDTY 1.2 , Sub1 + qDTY 1.1 + qDTY 2.2 and Sub1 + qDTY 2.2 + qDTY 12.1 in IR64-Sub1, qDTY 2.2 + qDTY 4.1 in Samba Mahsuri, Sub1 + qDTY 3.1 + qDTY 6.1 + qDTY 6.2 and Sub1 + qDTY 6.1 + qDTY 6.2 in TDK1-Sub1 and qDTY 12.1 + qDTY 3.1 and qDTY 2.2 + qDTY 3.1 in MR219 had shown better and consistent performance under NS and RS across generations over other QTL classes. CONCLUSION: "Deployment of this procedure will save time and resources and will allow breeders to focus and advance only germplasm with high probability of improved performance. The identification of superior QTL classes and capture of positive QTL x QTL and QTL x background interactions in early generation and their consistent performance in subsequent generations across five backgrounds supports the efficacy of a combined MAS breeding strategy".

Positive interactions of major-effect QTLs with genetic background that enhances rice yield under drought.

To improve the grain yield of the lowland-adapted popular rice variety Samba Mahsuri under reproductive-stage drought (RS) and to understand the interactions between drought QTLs, two mapping populations were developed using marker-assisted selection (MAS) and marker-assisted recurrent selection (MARS). The mean grain yield of pyramided lines (PLs) with qDTY 2.2 + qDTY 4.1 in MAS is significantly higher under RS and irrigated control than lines with single QTLs. Among MARS PLs, lines with four qDTYs (qDTY 1.1 + qDTY 2.1 + qDTY 3.1 + qDTY 11.1 ) and two QTLs (qDTY 1.1 + qDTY 11.1 ) yielded higher than PLs with other qDTY combinations. The selected PLs showed a yield advantage of 0.3-2.0 t ha-1 under RS. An allelic profile of MAS PLs having same qDTY combination but with different yields under drought was studied. Hierarchical clustering grouped together the selected lines with high yield under drought. Epistasis test showed the interaction of qDTY 4.1 and qDTY 9.1 loci with qDTY 7.1 significantly increased yield under drought and all the lines with higher yield under drought possessed the conserved region of qDTY 7.1 on chromosome 7. The positive interactions among QTLs, effectiveness of QTLs in different backgrounds, introgression of DTY QTLs together with resistance to biotic stresses shall help enhance grain yield under RS.

Combining drought and submergence tolerance in rice: marker-assisted breeding and QTL combination effects.

TDK1 is a popular rice variety from the Lao PDR. Originally developed for irrigated conditions, this variety suffers a high decline in yield under drought conditions. Studies have identified three quantitative trait loci (QTLs) for grain yield under drought conditions, qDTY3.1 , qDTY6.1 , and qDTY6.2 , that show a high effect in the background of this variety. We report here the pyramiding of these three QTLs with SUB1 that provides 2-3 weeks of tolerance to complete submergence, with the aim to develop drought- and submergence-tolerant near-isogenic lines (NILs) of TDK1. We used a tandem approach that combined marker-assisted backcross breeding with phenotypic selection to develop NILs with high yield under drought stress and non-stress conditions and preferred grain quality. The effect of different QTL combinations on yield and yield-related traits under drought stress and non-stress conditions is also reported. Our results show qDTY3.1 to be the largest and most consistent QTL affecting yield under drought conditions, followed by qDTY6.1 and qDTY6.2 , respectively. QTL class analysis also showed that lines with a combination of qDTY3.1 and qDTY6.1 consistently showed a higher tolerance to drought than those in which one of these QTLs was missing. In countries such as Lao PDR, where large areas under rice cultivation suffer vegetative-stage submergence and reproductive-stage drought, these lines could ensure yield stability. These lines can also serve as valuable genetic material to be used for further breeding of high-yielding, drought- and submergence-tolerant varieties in local breeding programs.