Genetic enhancement of reproductive stage drought tolerance in RPHR-1005R and derivative rice hybrids through marker-assisted backcross breeding in rice (Oryza sativa L.).

BACKGROUND: Drought stress is considered as one of the major production constraints in rice. RPHR-1005R is a restorer line (R-Line) with a popular, medium-slender grain type, and is the male parent of the popular Indian rice hybrid, DRR-H3. However, both the hybrid and its restorer are highly vulnerable to the drought stress, which limits the adoption of the hybrid. Therefore, the selection of the restorer line RPHR-1005R has been made with the objective of enhancing drought tolerance. METHODS AND RESULTS: In this study, we have introgressed a major QTL for grain yield under drought (qDTY 1.1) from Nagina22 through a marker-assisted backcross breeding (MABB) strategy. PCR based SSR markers linked to grain yield under drought (qDTY1.1 - RM431, RM11943), fertility restorer genes (Rf3-DRRM-Rf3-10, Rf4-RM6100) and wide compatibility (S5n allele) were deployed for foreground selection. At BC2F1, a single plant (RPHR6339-4-16-14) with target QTL in heterozygous condition and with the highest recurrent parent genome recovery (85.41%) and phenotypically like RPHR-1005R was identified and selfed to generate BC2F2. Fifty-eight homozygous lines were advanced to BC2F4 and six promising restorer lines and a hybrid combination (APMS6A/RPHR6339-4-16-14-3) were identified. CONCLUSIONS: In summary, the six improved restorer lines could be employed for developing heterotic hybrids possessing reproductive stage drought tolerance. The hybrid combination (APMS6A/RPHR6339-4-16-14-3) was estimated to ensure stable yields in drought-prone irrigated lowlands as well as in directly seeded aerobic and upland areas of India.

Improvement of bacterial blight resistance of the high yielding, fine-grain, rice variety, Gangavati sona through marker-assisted backcross breeding.

Gangavati sona (GS) is a high-yielding, fine-grain rice variety widely grown in the Tungabhadra command area in Karnataka, India; however, it is susceptible to bacterial blight (BB). Therefore, the present study was conducted to improve the GS variety for BB resistance. Three BB-resistant genes (xa5, xa13, and Xa21) were introgressed into the genetic background of susceptible cultivar GS through marker-assisted backcrossing (MABB) by using Improved samba Mahsuri (ISM), a popular, high-yielding, bacterial blight resistant rice variety as a donor parent. Foreground selection was carried out using gene-specific markers, viz., xa5FM (xa5), xa13prom (xa13), and pTA248 (Xa21), while background selection was carried out using well-distributed 64 polymorphic microsatellite markers. The true heterozygote F1 was used as the male parent for backcrossing with GS to obtain BC1F1. The process was repeated in BC1F1 generation, and a BC2F1 plant (IGS-5-11-5) possessing all three target genes along with maximum recurrent parent genome (RPG) recovery (86.7%) was selfed to obtain BC2F2s. At BC2F2, a single triple gene homozygote plant (IGS-5-11-5-33) with 92.6% RPG recovery was identified and advanced to BC2F5 by a pedigree method. At BC2F5, the seven best entries were selected, possessing all three resistance genes with high resistance levels against bacterial blight, yield level, and grain quality features equivalent to better than GS. The improved versions of GS will immensely benefit the farmers whose fields are endemic to BB.

Genome-wide association studies in rice germplasm reveal significant genomic regions for root and yield-related traits under aerobic and irrigated conditions.

The development of nutrient-use efficient rice lines is a priority amidst the changing climate and depleting resources viz., water, land, and labor for achieving sustainability in rice cultivation. Along with the traditional transplanted irrigated system of cultivation, the dry direct-seeded aerobic system is gaining ground nationwide. The root-related traits play a crucial role in nutrient acquisition, adaptation and need to be concentrated along with the yield-attributing traits. We phenotyped an association panel of 118 rice lines for seedling vigour index (SVI) traits at 14 and 21 days after sowing (DAS), root-related traits at panicle initiation (PI) stage in polythene bags under controlled aerobic condition, yield and yield-related traits under the irrigated condition at ICAR-IIRR, Hyderabad, Telangana; irrigated and aerobic conditions at ARS, Dhadesugur, Raichur, Karnataka. The panel was genotyped using simple sequence repeats (SSR) markers and genome-wide association studies were conducted for identifying marker-trait associations (MTAs). Significant correlations were recorded for root length, root dry weight with SVI, root volume at the PI stage, number of productive tillers per plant, spikelet fertility, the total number of grains per panicle with grain yield per plant under irrigated conditions, and the total number of grains per panicle with grain yield per plant under aerobic condition. The panel was divided into three sub-groups (K = 3) and correlated with the principal component analysis. The maximum number of MTAs were found on chromosomes 2, 3, and 12 with considerable phenotypic variability. Consistent MTAs were recorded for SVI traits at 14 and 21 DAS (RM25310, RM80, RM22961, RM1385), yield traits under irrigated conditions (RM2584, RM5179, RM410, RM20698, RM14753) across years at ICAR-IIRR, grain yield per plant (RM22961, RM1146) under the aerobic condition, grain yield per plant at irrigated ICAR-IIRR and SVI (RM5501), root traits at PI stage (RM2584, RM80, RM410, RM1146, RM18472). Functionally relevant genes near the MTAs through in-silico expression analysis in root and panicle tissues viz., HBF2 bZIP transcription factor, WD40 repeat-like domain, OsPILS6a auxin efflux carrier, WRKY108, OsSCP42, OsMADS80, nodulin-like domain-containing protein, amino acid transporter using various rice expression databases were identified. The identified MTAs and rice lines having high SVI traits (Langphou, TI-128, Mouli, TI-124, JBB-631-1), high yield under aerobic (Phouren, NPK-43, JBB-684, Ratnamudi, TI-112), irrigated conditions (KR-209, KR-262, Phouren, Keibi-Phou, TI-17), robust root traits like root length (MoirangPhou-Angouba, Wangoo-Phou, JBB-661, Dissi, NPK-45), root volume (Ratnachudi, KJ-221, Mow, Heimang-Phou, PUP-229) can be further employed in breeding programs for the targeted environments aimed at improving seedling vigour, yield-related traits under irrigated condition, aerobic condition as adaptability to water-saving technology.

Rice biofortification: breeding and genomic approaches for genetic enhancement of grain zinc and iron contents.

Rice is a highly consumed staple cereal cultivated predominantly in Asian countries, which share 90% of global rice production. Rice is a primary calorie provider for more than 3.5 billion people across the world. Preference and consumption of polished rice have increased manifold, which resulted in the loss of inherent nutrition. The prevalence of micronutrient deficiencies (Zn and Fe) are major human health challenges in the 21st century. Biofortification of staples is a sustainable approach to alleviating malnutrition. Globally, significant progress has been made in rice for enhancing grain Zn, Fe, and protein. To date, 37 biofortified Fe, Zn, Protein and Provitamin A rich rice varieties are available for commercial cultivation (16 from India and 21 from the rest of the world; Fe > 10 mg/kg, Zn > 24 mg/kg, protein > 10% in polished rice as India target while Zn > 28 mg/kg in polished rice as international target). However, understanding the micronutrient genetics, mechanisms of uptake, translocation, and bioavailability are the prime areas that need to be strengthened. The successful development of these lines through integrated-genomic technologies can accelerate deployment and scaling in future breeding programs to address the key challenges of malnutrition and hidden hunger.

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice.

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC1F1 generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC1F1 generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC1F3 generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC1F5 generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC1F5 generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.

DRR Dhan 58, a Seedling Stage Salinity Tolerant NIL of Improved Samba Mahsuri Shows Superior Performance in Multi-location Trials.

BACKGROUND: Improved Samba Mahsuri (ISM) is an elite, high-yielding, bacterial blight resistant, fine-grained rice variety with low glycaemic index. It is highly sensitive to salt stress, particularly at seedling stage, which significantly reduces its yield potential in coastal areas. A salinity tolerant QTL, Saltol, associated with seedling stage tolerance was previously mapped on chromosome 1 (10.6-11.5 Mb) from the Indian landrace, Pokkali and is effective in different genetic backgrounds. The objective of this study was to enhance salinity tolerance of ISM by incorporating the Saltol QTL through marker-assisted backcross breeding using the breeding line, FL478 (Pokkali/IR29). RESULTS: Foreground selection was carried out at each generation using five Saltol-specific markers and three bacterial blight resistance genes, Xa21, xa13 and xa5. Background selection was conducted using 66 well distributed polymorphic SSR markers and at the BC3F2 generation, a single plant with maximum recurrent parent genome recovery (95.3%) was identified and advanced to the BC3F4 generation. Based on bacterial blight resistance, seedling stage salinity tolerance and resemblance to ISM, four advanced breeding lines were selected for testing in replicated experiments near Hyderabad, India. A promising near-isogenic line, DRR Dhan 58, was evaluated in multi-location trials-coastal salinity and it showed significant salinity tolerance, resistance to bacterial blight disease, high yield and excellent grain quality during the 2019 and 2020 trials. DRR Dhan 58 was 95.1% similar to ISM based on genotyping with the 90 K SNP chip. Whole genome resequencing analysis of Pokkali and FL478 which were salinity tolerant checks, ISM and DRR Dhan 58 showed a high degree of relatedness with respect to the candidate gene loci for Saltol and OsSKC1 (Shoot K+ Concentration 1). CONCLUSION: DRR Dhan 58, possessing Saltol and three bacterial blight resistance genes (Xa21, xa13 and xa5) in the genetic background of the Indian mega-variety of rice, Samba Mahsuri, was developed for potential cultivation in areas prone to seedling stage salinity, as well as areas with endemic bacterial blight disease. This entry had a 24% yield advantage over the recurrent parent ISM under coastal saline conditions in multi-location trials and was recently released for commercial cultivation in India.

Evaluation of genotype by environment interaction and adaptability in lowland irrigated rice hybrids for grain yield under high temperature.

Recent predictions on climate change indicate that high temperature episodes are expected to impact rice production and productivity worldwide. The present investigation was undertaken to assess the yield stability of 72 rice hybrids and their parental lines across three temperature regimes over two consecutive dry seasons using the additive main effect and multiplicative interaction (AMMI), genotype and genotype × environment interaction (GGE) stability model analysis. The combined ANOVA revealed that genotype × environment interaction (GEI) were significant due to the linear component for most of the traits studied. The AMMI and GGE biplot explained 57.2% and 69% of the observed genotypic variation for grain yield, respectively. Spikelet fertility was the most affected yield contributing trait and in contrast, plant height and tiller numbers were the least affected traits. In case of spikelet fertility, grain yield and other yield contributing traits, male parent contributed towards heat tolerance of the hybrids compared to the female parent. The parental lines G74 (IR58025B), G83 (IR40750R), G85 (C20R) and hybrids [G21 (IR58025A × KMR3); G3 (APMS6A × KMR3); G57 (IR68897A × KMR3) and G41 (IR79156A × RPHR1005)] were the most stable across the environments for grain yield. They can be considered as potential genotypes for cultivation under high temperature stress after evaluating under multi location trials.

Novel major QTLs associated with low soil phosphorus tolerance identified from the Indian rice landrace, Wazuhophek.

With an objective of mapping novel low soil P (Phosphorus) tolerance loci in the non-Pup1 type donor rice line, Wazuhophek, we screened a recombinant inbred line (RIL) mapping population consisting of 330 lines derived from the cross Wazuhophek x Improved Samba Mahsuri (which is highly sensitive to low soil P) in a plot with low soil P for tolerance associated traits. Molecular mapping with SSR markers revealed a total of 16 QTLs (seven major and nine minor QTLs), which are associated with low soil P tolerance related traits. Interestingly, a QTL hotspot, harbouring 10 out of 16 QTLs were identified on the short arm of chromosome 8 (flanked by the makers RM22554 and RM80005). Five major QTLs explaining phenotypic variance to an extent of 15.28%, 17.25%, 21.84%, 20.23%, and 18.50%, associated with the traits, plant height, shoot length, the number of productive tillers, panicle length and yield, respectively, were located in the hotspot. Two major QTLs located on chromosome 1, associated with the traits, total biomass and root to shoot ratio, explaining 15.44% and 15.44% phenotypic variance, respectively were also identified. Complex epistatic interactions were observed among the traits, grain yield per plant, days to 50% flowering, dry shoot weight, and P content of the seed. In-silico analysis of genomic regions flanking the major QTLs revealed the presence of key putative candidate genes, possibly associated with tolerance.

Grain Fe and Zn content, heterosis, combining ability and its association with grain yield in irrigated and aerobic rice.

Genetic improvement of rice for grain micronutrients, viz., iron (Fe) and zinc (Zn) content is one of the important breeding objectives, in addition to yield improvement under the irrigated and aerobic ecosystems. In view of developing genetic resources for aerobic conditions, line (L) × tester (T) analysis was conducted with four restorers, four CMS lines and 16 hybrids. Both hybrids and parental lines were evaluated in irrigated and aerobic field conditions for grain yield, grain Fe and Zn content. General Combining Ability (GCA) effects of parents and Specific Combining Ability (SCA) effects of hybrids were observed to be contrasting for the micronutrient content in both the growing environments. The grain Fe and Zn content for parental lines were negatively correlated with grain yield in both the contrasting growing conditions. However, hybrids exhibited positive correlation for grain Fe and Zn with grain yield under limited water conditions. The magnitude of SCA mean squares was much higher than GCA mean squares implying preponderance of dominance gene action and also role of complementary non-allelic gene(s) interaction of parents and suitability of hybrids to the aerobic system. The testers HHZ12-SAL8-Y1-SAL1 (T1) and HHZ17-Y16-Y3-Y2 (T2) were identified as good combiners for grain Zn content under irrigated and aerobic conditions respectively.

Molecular mapping of QTLs for yield related traits in recombinant inbred line (RIL) population derived from the popular rice hybrid KRH-2 and their validation through SNP genotyping.

The study was undertaken to identify the quantitative trait loci (QTLs) governing yield and its related traits using a recombinant inbred line (RIL) population derived from the popular rice hybrid, KRH-2 (IR58025A/KMR3R). A genetic map spanning 294.2 cM was constructed with 126 simple sequence repeats (SSR) loci uniformly distributed across the rice genome. QTL analysis using phenotyping and genotyping information identified a total of 22 QTLs. Of these, five major effect QTLs were identified for the following traits: total grain yield/plant (qYLD3-1), panicle weight (qPW3-1), plant height (qPH12-1), flag leaf width (qFLW4-1) and panicle length (qPL3-1), explaining 20.23-22.76% of the phenotypic variance with LOD scores range of 6.5-10.59. Few genomic regions controlling several traits (QTL hotspot) were identified on chromosome 3 for total grain yield/plant (qYLD3-1) and panicle length (qPL3-1). Significant epistatic interactions were also observed for total grain yield per plant (YLD) and panicle length (PL). While most of these QTLs were observed to be co-localized with the previously reported QTL regions, a novel, major QTL associated with panicle length (qPL3-1) was also identified. SNP genotyping of selected high and low yielding RILs and their QTL mapping with 1,082 SNPs validated most of the QTLs identified through SSR genotyping. This facilitated the identification of novel major effect QTLs with much better resolution and precision. In-silico analysis of novel QTLs revealed the biological functions of the putative candidate gene (s) associated with selected traits. Most of the high-yielding RILs possessing the major yield related QTLs were identified to be complete restorers, indicating their possible utilization in development of superior rice hybrids.

Expression profiling of immature florets of IR58025A, a wild-abortive cytoplasmic male sterile line of rice and its cognate, isonuclear maintainer line, IR58025B.

Interaction between gene products encoded by the cytoplasm and nucleus form the core of wild abortive cytoplasmic male sterile (WA-CMS) system of hybrid breeding in rice. Gaining insights into such interactions can be helpful in the development of better three-line rice hybrids and also identify novel male sterility systems. In the present study, the whole transcriptome profiles of immature florets of IR58025A, a WA-CMS line and its isonuclear maintainer line, IR58025B, collected at pre-anthesis stage were compared to delineate the pathways involved in pollen abortion and male sterility. Among the 774 differentially expressed transcripts (DETs), 496 were down regulated and 278 were up regulated in IR58025A compared to IR58025B. The genes associated with oxidative stress response, defense response, etc. were significantly up-regulated, while those associated with respiration, cell wall modifications, pectinesterase activity, etc. were significantly down-regulated in the WA-CMS line. Gene ontology and pathway enrichment analyses revealed the down-regulation of both nuclear and organellar genes involved in key metabolic processes of cell respiration, photosynthesis and other energy yielding metabolites in IR58025A, relative to IR58025B, indicating a general shift toward conservation of energy and other key resources in the florets of WA-CMS line. The data derived from RNA-Seq analysis were validated through qRT-PCR analysis. Based on the results obtained, it can be hypothesized that pollen abortion principally occurs due to up-regulation of pathways leading to oxidative stress leading to energy starvation conditions in consonance with reduced expression of genes associated with the cell wall formation, respiration, and other key metabolic processes.

Studies on root anatomy, morphology and physiology of rice grown under aerobic and anaerobic conditions.

With the changing climate and rainfall abrasions, there is a gradual shift in the system of rice cultivation from traditional transplanted anaerobic to aerobic system. Studies on the root anatomical and morpho-physiological traits provide insights about the adaptation under aerobic conditions. We investigated the root anatomical and morpho-physiological traits in anaerobic (BPT 5204) and aerobic (CR Dhan 202) adapted rice genotypes grown under anaerobic and aerobic conditions. It was observed that the formation of fewer aerenchyma, thickened root and larger xylem area were critical anatomical traits associated with aerobic adaptation as compared to anaerobic conditions. The root length of CR Dhan 202 significantly increased under aerobic condition which may be attributed to its aerobic adaptation in terms of water acquisition. The photosynthetic rate was significantly higher in CR Dhan 202 as compared to that of BPT 5204 under the aerobic condition. The morpho-physiological results showed that the root length, total dry weight and photosynthetic rate are the key parameters for imparting aerobic adaptation. These root anatomical and morpho-physiological traits associated with the adaptation can be used as screening criteria for phenotyping and selection of genotypes suitable for aerobic system of cultivation. Such study is expected to expedite the development of rice aerobic varieties in aerobic breeding programmes.

Genes encoding membrane proteins showed stable expression in rice under aerobic condition: novel set of reference genes for expression studies.

A set of reference genes expressing stably under aerobic and anaerobic conditions in rice is essential to execute omics studies relating to aerobic adaptations. Stability of expression of ten rice reference genes, viz. Actin, eEF-1a, eIF-5C, Exp1, Exp2, Memp, SKP1A, TF-SUI1, TPH, and UBQ5 was validated across six experimental sets in shoot and root tissues at seedling, tillering, and panicle initiation stages. Comprehensively, Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) were revealed as the most stable set with acceptable M and V value according to the gold standards of qRT-PCR using various algorithms/tools. The identified set of reference genes was validated using root trait genes, which showed concurrence with the functional expression patterns in the aerobic and anaerobic adapted cultivars. The Memp (Membrane protein), TPH (Tumor protein homolog), and Exp1 (Expressed protein) genes are the most stable reference genes across the root and shoot at various developmental stages under aerobic and anaerobic conditions in rice. This is the first study for accurate and reliable relative gene expression analysis in rice grown in aerobic and anaerobic conditions.

Diverse Rice Landraces of North-East India Enables the Identification of Novel Genetic Resources for Magnaporthe Resistance.

North-East (NE) India, the probable origin of rice has diverse genetic resources. Many rice landraces of NE India were not yet characterized for blast resistance. A set of 232 landraces of NE India, were screened for field resistance at two different hotspots of rice blast, viz., IIRR-UBN, Hyderabad and ICAR-NEH, Manipur in two consecutive seasons. The phenotypic evaluation as well as gene profiling for 12 major blast resistance genes (Pitp, Pi33, Pi54, Pib, Pi20, Pi38, Pita2, Pi1, Piz, Pi9, Pizt, and Pi40) with linked as well as gene-specific markers, identified 84 resistant landraces possessing different gene(s) either in singly or in combinations and also identified seven resistant landraces which do not have the tested genes, indicating the valuable genetic resources for blast resistance. To understand the molecular diversity existing in the population, distance and model based analysis were performed using 120 SSR markers. Results of both analyses are highly correlated by forming two distinct subgroups and the existence of high diversity (24.9% among the subgroups; 75.1% among individuals of each subgroup) was observed. To practically utilize the diversity in the breeding program, a robust core set having an efficiency index of 0.82 which consists of 33 landraces were identified through data of molecular, blast phenotyping, and important agro-morphological traits. The association of eight novel SSR markers for important agronomic traits which includes leaf and neck blast resistance was determined using genome-wide association analysis. The current study focuses on identifying novel resources having field resistance to blast as well as markers which can be explored in rice improvement programs. It also entails the development of a core set which can aid in representing the entire diversity for efficiently harnessing its properties to broaden the gene pool of rice.