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Rice is staple food in many countries of Africa and a major part of the diet in many others. However, Africa’s demand for rice exceeds production with the deficit of 40% being imported. One way to improve Africa’s rice production is through breeding high yielding varieties suitable for the different environment conditions. This study was conducted to assess the genetic variability and stability performance of 48 lowland rice genotypes including 37 interspecific (Oryza glaberrima × Oryza sativa ssp. indica) and 11 intraspecific (O. sativa ssp. indica × O. sativa ssp. indica) in 12 environments in Nigeria, Benin Republic and Togo using Additive Main Effect and Multiplicative Interaction (AMMI) and Genotype+ Genotype x Environment (GGE) biplot models. The combined analysis of variance revealed significant differences (P

To screen for new sources of salinity tolerance, 688 traditional rice varieties from the Philippines and Bangladesh were obtained, and their tolerance to hypersaline conditions at the seedling stage was examined. A total of 29 Philippine lines and 15 Bangladeshi lines were scored as salt-tolerant. Morphological assessment (plant height, biomass and Na-K ratio) revealed that among the 44 salt-tolerant accessions, Casibon, Kalagnon and Ikogan had significantly higher relative shoot length difference, relative shoot growth reduction and shoot Na-K ratio than the tolerant check FL478. Additionally, AC and Akundo exhibited significantly higher Na-K ratios than the other genotypes. The genetic diversity of the 44 genotypes was assessed using 34 simple sequence repeat markers. A total of 133 alleles were detected across all loci. Cluster analysis showed that AC, Akundo and Kuplod were clustered along with FL478, indicating a strong genetic relatedness between these genotypes. IR29 (susceptible check) was singly separated. The haplotype analysis revealed that none of the 44 genotypes had a similar allele combination as FL478. These accessions are of interest since each genotype might be different from the classical salinity-tolerant Pokkali. Keywords: rice, simple sequence repeat, Na-K ratio, salinity tolerance, traditional landrace

The phenomenon of transgressive segregation, where a small minority of recombinants are outliers relative to the range of parental phenotypes, is commonly observed in plant breeding populations. While this phenomenon has been attributed to complementation and epistatic effects, the physiological and developmental synergism involved have not been fully illuminated by the QTL mapping approach alone, especially for stress-adaptive traits involving highly complex interactions. By systems-level profiling of the IR29 × Pokkali recombinant inbred population of rice, we addressed the hypothesis that novel salinity tolerance phenotypes are created by reconfigured physiological networks due to positive or negative coupling-uncoupling of developmental and physiological attributes of each parent. Real-time growth and hyperspectral profiling distinguished the transgressive individuals in terms of stress penalty to growth. Non-parental network signatures that led to either optimal or non-optimal integration of developmental with stress-related mechanisms were evident at the macro-physiological, biochemical, metabolic, and transcriptomic levels. Large positive net gain in super-tolerant progeny was due to ideal complementation of beneficial traits while shedding antagonistic traits. Super-sensitivity was explained by the stacking of multiple antagonistic traits and loss of major beneficial traits. The synergism uncovered by the phenomics approach in this study supports the modern views of the Omnigenic Theory, emphasizing the synergy or lack thereof between core and peripheral components. This study also supports a breeding paradigm rooted on genomic modeling from multi-dimensional genetic, physiological, and phenotypic profiles to create novel adaptive traits for new crop varieties of the 21st century.

Wild relatives of rice in the genus Oryza (composed of 24 species with 11 different genome types) have been significantly contributing to the varietal improvement of rice (Oryza sativa). More than 4000 accessions of wild rice species are available and they are regarded as a “genetic reservoir” for further rice improvement. DNA markers are essential tools in genetic analysis and breeding. To date, genome-wide marker sets for wild rice species have not been well established and this is one of the major difficulties for the efficient use of wild germplasm. Here, we developed 541 genome-wide InDel markers for the discrimination of alleles between the cultivated species O. sativa and the other seven AA-genome species by positional multiple sequence alignments among five AA-genome species with four rice varieties. The newly developed markers were tested by PCR-agarose gel analysis of 24 accessions from eight AA genome species (three accessions per species) along with two representative cultivars (O. sativa subsp. indica cv. IR24 and subsp. japonica cv. Nipponbare). Marker polymorphism was validated for 475 markers. The number of polymorphic markers between IR24 and each species (three accessions) ranged from 338 (versus O. rufipogon) to 416 (versus O. longistaminata) and the values in comparison with Nipponbare ranged from 179 (versus O. glaberrima) to 323 (versus O. glumaepatula). These marker sets will be useful for genetic studies and use of the AA-genome wild rice species.

Transgressive segregation is common in plant breeding populations, where a small minority of recombinants are outliers relative to parental phenotypes. While this phenomenon has been attributed to complementation and epistatic effects, the physiological, biochemical, and molecular bases have not been fully illuminated. By systems-level scrutiny of the IR29 x Pokkali recombinant inbred population of rice, we addressed the hypothesis that novel salt tolerance phenotypes are created by positive or negative coupling or uncoupling effects and novel regulatory networks. Hyperspectral profiling distinguished the transgressive individuals in terms of stress penalty to growth. Non-parental network signatures that led to either optimal or non-optimal integration of developmental with stress-related mechanisms were evident at the macro-physiological, biochemical, metabolic, and transcriptomic levels. The large positive net gain in super-tolerant progeny was due to ideal complementation of beneficial traits, while shedding antagonistic traits. Super-sensitivity was explained by the stacking of multiple antagonistic traits and loss of major beneficial traits. The mechanisms elucidated in this study are consistent with the Omnigenic Theory, emphasizing the synergy or lack thereof between core and peripheral components. This study supports a breeding paradigm based on genomic modeling to create the novel adaptive phenotypes for the crops of the 21stcentury.

To elucidate the mechanisms of salt acclimation, physiological parameters of 70 rice varieties were compared under control and salt stress conditions after the acclimation treatment. The results indicated that some rice varieties had the ability to acclimatize to salt stress, exhibiting improved growth following the acclimation treatment under subsequent salinity stress compared to those without acclimation treatment. Conversely, some varieties exhibited reduced growth both with and without acclimation treatment under subsequent salinity stress. Acclimatized varieties had differential patterns of Na

food security, COVID-19, Philippines, Southeast Asia

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

Breeding for salt tolerance is the most promising approach to enhance the productivity of saline prone areas. However, polygenic inheritance of salt tolerance in rice acts as a bottleneck in conventional breeding for salt tolerance. Hence, we set our goals to construct a single nucleotide polymorphism (SNP)-based molecular map employing high-throughput SNP marker technology and to investigate salinity tolerant QTLs with closest flanking markers using an elite rice background. Seedling stage salinity responses were assessed in a population of 281 recombinant inbred lines (RILs) derived from the cross between At354 (salt tolerant) and Bg352 (salt susceptible), by 11 morpho-physiological indices under a hydroponic system. Selected extreme 94 RILs were genotyped using Illumina Infinium rice 6K SNP array and densely saturated molecular map spanning 1460.81 cM of the rice genome with an average interval of 1.29 cM between marker loci was constructed using 1135 polymorphic SNP markers. The results revealed 83 significant QTLs for 11 salt responsive traits explaining 12.5-46.7 % of phenotypic variation in respective traits. Of them, 72 QTLs responsible for 10 traits were co-localized together forming 14 QTL hotspots at 14 different genomic regions. The all QTL hotspots were flanked less than 1 Mb intervals and therefore the SNP loci associated with these QTL hotspots would be important in candidate gene discovery for salt tolerance.

African rice (Oryza glaberrima Steud.) is a cereal crop species closely related to Asian rice (Oryza sativa L.) but was independently domesticated in West Africa ∼3,000 years ago. African rice is rarely grown outside sub-Saharan Africa but is of global interest because of its tolerance to abiotic stresses. Here we describe a map of 2.32 million SNPs of African rice from whole-genome resequencing of 93 landraces. Population genomic analysis shows a population bottleneck in this species that began ∼13,000-15,000 years ago with effective population size reaching its minimum value ∼3,500 years ago, suggesting a protracted period of population size reduction likely commencing with predomestication management and/or cultivation. Genome-wide association studies (GWAS) for six salt tolerance traits identify 11 significant loci, 4 of which are within ∼300 kb of genomic regions that possess signatures of positive selection, suggesting adaptive geographical divergence for salt tolerance in this species.

Genotype × environment (G × E) interaction effects are of special interest for identifying the most suitable genotypes with respect to target environments, representative locations and other specific stresses. Twenty-two advanced breeding lines contributed by the national partners of the Salinity Tolerance Breeding Network (STBN) along with four checks were evaluated across 12 different salt affected sites comprising five coastal saline and seven alkaline environments in India. The study was conducted to assess the G × E interaction and stability of advanced breeding lines for yield and yield components using additive main effects and multiplicative interaction (AMMI) model. In the AMMI1 biplot, there were two mega-environments (ME) includes ME-A as CARI, KARAIKAL, TRICHY and NDUAT with winning genotype CSR 2K 262; and ME-B as KARSO, LUCKN, KARSA, GOA, CRRI, DRR, BIHAR and PANVE with winning genotypes CSR 36. Genotypes CSR 2K 262, CSR 27, NDRK 11-4, NDRK 11-3, NDRK 11-2, CSR 2K 255 and PNL 1-1-1-6-7-1 wer...

Productivity of rice is adversely affected by salinity and water logging stresses in coastal zones, and affected areas are progressively expanding as consequences of climate change and overexploitation of natural resources to meet the needs of increasing populations. Several varieties were developed and commercialized in this region in recent years, including some salt and stagnant flood (SF, medium deep, 25–50 cm water depth) tolerant varieties. However, suitability of these varieties based on adaptation to local conditions across the region and farmers’ preferences have not been sufficiently evaluated. Genotype by environment analysis leads to identification of varieties with stable performance, or suitability to specific regions. Two frequently used statistical analyses are additive main effects and multiplicative interaction (AMMI); and the genotype main effects and genotype × environment interaction effects (GGE). These models were used in this study to identify stable rice varieties for the wet and dry seasons in South Bangladesh. Experiments were conducted at six sites over four years (2010–2013) in coastal Bangladesh, using eight and five varieties for wet and dry season, respectively. The trials were laid in a randomized complete block design (RCBD) with three replications. Genotypic contribution was much higher than the environmental effect on grain yield in the wet season, while the environmental contribution was much higher than genotypic effect during the dry season. SF stress affected yield of wet season rice; but in the dry season, salinity stress had major effect on grain yield. The variety BRRI dhan54 was identified as the most suitable genotype with wider adaptability in the region during the wet season, followed by BRRI dhan40 and BRRI dhan53, while BRRI dhan47 was the most stable variety followed by BRRI dhan61 and BINA dhan8 for the dry season across all sites. However, when these varieties were evaluated based on farmers’ preferences in participatory varietal selection, BRRI dhan53; a short duration variety with medium slender grains was the most preferred, followed by BRRI dhan54 and BRRI dhan40 for wet season, and in the dry season BRRI dhan47 was the most popular variety followed by BRRI dhan61, which agreed with ranking based on yield. Modern varieties were far superior over traditional varieties based on grain yield and farmers’ preferences, and should be targeted for large-scale seed production and dissemination.

To elucidate the mechanisms of salt acclimation, physiological parameters of 70 rice varieties were compared under control and salt stress conditions after the acclimation treatment. The results indicated that some rice varieties had the ability to acclimatize to salt stress, exhibiting improved growth following the acclimation treatment under subsequent salinity stress compared to those without acclimation treatment. Conversely, some varieties exhibited reduced growth both with and without acclimation treatment under subsequent salinity stress. Acclimatized varieties had differential patterns of Na+ accumulation in the leaf blades because some varieties reduced Na+ accumulation under salinity stress, whereas others did not. Under salt stress, the acclimatized varieties with low Na+ accumulation in the leaf blades highly induced the expression of the OsHKT1;5 gene in the roots, which may contribute to Na+ exclusion from the shoots. On the other hand, the acclimatized varieties with high Na+ accumulation in the leaf blades exhibited higher induction of the OsNHX1 gene, whose gene product participates in the compartmentalization of Na+ into vacuoles. Thus, rice develops different mechanisms of salinity acclimation using two Na+ transport systems, and active regulation of Na+ transport at the transcription level may be involved in the salt acclimation process and enhance salinity tolerance.

This article describes the growth of 18 acclimatized and 11 non-acclimatized rice varieties grown in a hydroponic nutrient solution in a glasshouse. Four plants from each variety were grown under control conditions, salinity stress following control conditions (salinity), and salinity stress following acclimation (salinity/acclimation) conditions. Sampling was performed at the end of the salinity treatment (36 days of growth). Growth traits such as shoot and root biomass accumulation and lengths were measured for each variety, and the average was calculated using four replicates. This dataset may aid interested researchers in making comparisons with their data and further advance the research on the salinity acclimation process in rice.

Zinc deficiency is a widespread soil constraint in rice production. Here, we present QTL/candidate genes associated with Zn deficiency tolerance identified through bi-parental QTL mapping and genome-wide association analysis. Zinc (Zn) deficiency is a widespread soil constraint in rice production. Despite several physiological studies elucidating Zn deficiency tolerance mechanisms, little is known about genetic factors conferring tolerance. To identify QTL associated with root development, biomass accumulation, and grain yield under Zn deficiency, we combined bi-parental QTL mapping in a population of 200 backcross inbred (BC1F6) lines and genome-wide association analysis using 247 k SNP markers across 140 accessions of an indica diversity panel. Three QTLs for Zn deficiency tolerance on chromosomes 3, 6, and 12 co-localized in both approaches and the association analysis detected two additional strong QTL on chromosomes 1 and 9 not present in the bi-parental population. Based on haplotype analysis of the indica panel, biomass consistently increased due to the minor ‘tolerance’ haplotypes, which had frequencies between 13 and 34%. By utilizing the previous transcript data collected from the same Zn-deficient field, we identified one putative candidate gene within the chromosome 6-QTL, which was associated with all traits in both analyses. Gene Os06g44220 was barely expressed under +Zn conditions but strongly upregulated in both root and shoot under stress and consistently more so in the tolerant genotype. Os06g44220 is an uncharacterized gene with expression previously detected only under salinity stress. Four SNP alterations within the promoter region distinguish the two alleles identified and a genotype tolerant to Zn deficiency shares the same allele as salinity tolerant varieties, lending support to the hypothesis that this gene may confer tolerance to both stresses.

Rice production needs to increase in the future in order to meet increasing demands. The development of new improved and higher yielding varieties more quickly will be needed to meet this demand. However, most rice breeding programmes in the world have not changed in several decades. In this article, we revisit the evidence in favour of using rapid generation advance (RGA) as a routine breeding method. We describe preliminary activities at the International Rice Research Institute (IRRI) to re-establish RGA on a large scale as the main breeding method for irrigated rice breeding. We also describe experiences from the early adoption at the Bangladesh Rice Research Institute. Evaluation of RGA breeding lines at IRRI for yield, flowering time and plant height indicated transgressive segregation for all traits. Some RGA lines were also higher yielding than the check varieties. The cost advantages of using RGA compared to the pedigree method were also empirically determined by performing an economic analysis. This indicated that RGA is several times more cost effective and advantages will be realized after 1 year even if facilities need to be built. Based on our experience, and previous independent research empirically testing the RGA method in rice, we recommend that this method should be implemented for routine rice breeding in order to improve breeding efficiency.

Salinity is a major abiotic stress that limits rice production across rice areas as high-yielding modern rice varieties are generally sensitive to salt stress. The study was conducted to deduce heritability and combining ability estimates of rice for various morphological and physiological traits using a 7 × 7 full-diallel-cross analysis at seedling and reproductive stages. The salinity stress treatment was 12 dS m−1 at the seedling stage and 8 dS m−1 at the reproductive stage. Diallel analysis revealed high for salinity tolerance scores and shoot height, moderate for shoot dry weight and root dry weight and low for Na+ and K+ concentrations and K+/Na+ ratio. The low-to-moderate narrow-sense heritability for number of panicles, number of fertile spikelets, grain weight, spikelet fertility and K+/Na+ ratio suggests a large breeding population and delayed selection for tolerance until later generations. Significant maternal effects indicate that selection of the female parent is very important for desired trait development. The results of this study confirmed that salinity tolerance at the seedling and reproductive stages is regulated by a different set of genes that could be pyramided using different donors to enhance the level of tolerance.

Salinity is a major constraint affecting rice productivity in rainfed and irrigated agro-ecosystems. Understanding salinity effects on rice production at the reproductive stage could improve adaptation for this trait. Identifying quantitative trait loci (QTLs) controlling adaptation to salinity may also accelerate breeding rice germplasm for environments prone to this stress. We used the salt tolerant landrace ‘Hasawi’ as a donor parent to generate three F2 offspring (consisting each of 500 individuals) with three African cultivars (‘NERICA-L-19’, ‘Sahel 108’ and ‘BG90-2’) used as recipient parents (RP). The F2s and F2:3s were evaluated for grain yield and other traits in saline fields. Salinity caused reduction in all measured traits across the F2-derived offspring, e.g. grain yield reduced between 65 and 73 %, but some offspring had twice the RP’s grain yield. QTL analysis revealed 75 QTLs for different traits in all 3 genetic backgrounds (GBs): 24 of them were common among all the 3 GBs while 31 were noted in 2 GBs, and 17 in one GB. ‘Hasawi’ contributed on average 49 % alleles to these QTLs. Two yield and yield related QTLs (qGY11 and qTN11) common in all 3 GBs were mapped on the same chromosomal segment suggesting these QTLs might be stable across different GBs. Four other QTLs were strongly associated with salinity tolerance with peak marker RM419, representing a potential candidate for MAS due to high LOD score and relatively large effect QTLs.

In Asia, a significant area under rice is affected by salinity. Salt stress can affect growth of crops as well as weeds. A study was conducted in a greenhouse to determine the effect of salinity (electrical conductivity [EC] of 1, 6, 12, 18, and 24 dS m−1) on growth of barnyardgrass, horse purslane, junglerice, and rice. Growth variables were analyzed using regression analysis. The tested levels of EC influenced leaf production of barnyardgrass and junglerice but not that of horse purslane. As compared with the control treatment (EC of 1 dS m−1), shoot biomass of barnyardgrass decreased by only 24% at 12 dS m−1, whereas rice biomass declined by 59% at this level of EC. At EC of 24 dS m−1, barnyardgrass still produced 4% of the biomass of the control treatment, whereas rice did not survive at this level of EC. Junglerice shoot biomass decreased by 73% at 18 dS m−1EC compared with the control treatment, whereas rice shoot biomass declined by more than 86% at 18 dS m−1EC. An EC of 10 dS m−1was required to inhibit 50% shoot biomass of rice, whereas the EC to inhibit 50% shoot biomass of barnyardgrass and junglerice was 15 and 13 dS m−1, respectively. Shoot biomass of horse purslane was not influenced by the tested levels of EC. At the highest EC (24 dS m−1), at which rice did not survive, horse purslane shoot biomass was similar to that of the control treatment. In all weed species, data for root biomass showed trends similar to those of shoot biomass. The results of this study suggest that weeds were more tolerant to salt than rice, and horse purslane was the most tolerant species among the weeds.

Salt stress is a complex physiological trait affecting plants by limiting growth and productivity. Rice, one of the most important food crops, is rated as salt-sensitive. High-throughput screening methods are required to exploit novel sources of genetic variation in rice and further improve salinity tolerance in breeding programmes. To search for genotypic differences related to salt stress, we genotyped 392 rice accessions by EcoTILLING. We targeted five key salt-related genes involved in mechanisms such as Na+/K+ ratio equilibrium, signalling cascade and stress protection, and we found 40 new allelic variants in coding sequences. By performing association analyses using both general and mixed linear models, we identified 11 significant SNPs related to salinity. We further evaluated the putative consequences of these SNPs at the protein level using bioinformatic tools. Amongst the five nonsynonymous SNPs significantly associated with salt-stress traits, we found a T67K mutation that may cause the destabilization of one transmembrane domain in OsHKT1;5, and a P140A alteration that significantly increases the probability of OsHKT1;5 phosphorylation. The K24E mutation can putatively affect SalT interaction with other proteins thus impacting its function. Our results have uncovered allelic variants affecting salinity tolerance that may be important in breeding. (Resume d'auteur)

Environmental Gene Regulatory Influence Networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental and developmental signals. EGRINs encompass many layers of regulation, which culminate in changes in the level of accumulated transcripts. Here we infer EGRINs for the response of five tropical Asian rice cultivars to high temperatures, water deficit, and agricultural field conditions, by systematically integrating time series transcriptome data (720 RNA-seq libraries), patterns of nucleosome-free chromatin (18 ATAC-seq libraries), and the occurrence of known cis-regulatory elements. First, we identify 5,447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes with known cis-regulatory motifs in nucleosome-free chromatin regions proximal to transcriptional start sites (TSS) of genes. We then use network component analysis to estimate the regulatory activity for these TFs from the expression of these putative target genes. Finally, we inferred an EGRIN using the estimated TFA as the regulator. The EGRIN included regulatory interactions between 4,052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of a large TF family, including a putative regulatory connection between abiotic stress and the circadian clock, as well as specific regulatory functions for TFs in the drought response. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference and that supplementing data from controlled experimental conditions with data from outdoor field conditions increases the resolution for EGRIN inference.

This manuscript reports the identification and characterization of five transcription factors binding to the promoter of OsNHX1 in a salt stress tolerant rice genotype (Hasawi). Although NHX1 encoding genes are known to be highly regulated at the transcription level by different abiotic stresses, namely salt and drought stress, until now only one transcription factor (TF) binding to its promoter has been reported. In order to unveil the TFs regulating NHX1 gene expression, which is known to be highly induced under salt stress, we have used a Y1H system to screen a salt induced rice cDNA expression library from Hasawi. This approach allowed us to identify five TFs belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) binding to the OsNHX1 gene promoter. We have also shown that these TFs act either as transcriptional activators (OsPCF2, OsNIN-like4) or repressors (OsCPP5, OsNIN-like2) and their encoding genes are differentially regulated by salt and PEG-induced drought stress in two rice genotypes, Nipponbare (salt-sensitive) and Hasawi (salt-tolerant). The transactivation activity of OsNIN-like3 was not possible to determine. Increased soil salinity has a direct impact on the reduction of plant growth and crop yield and it is therefore fundamental to understand the molecular mechanisms underlying gene expression regulation under adverse environmental conditions. OsNHX1 is the most abundant K+-Na+/H+ antiporter localized in the tonoplast and its gene expression is induced by salt, drought and ABA. To investigate how OsNHX1 is transcriptionally regulated in response to salt stress in a salt-tolerant rice genotype (Hasawi), a salt-stress-induced cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsNHX1 promoter as bait. Five transcription factors (TFs) belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) were identified as binding to OsNHX1 promoter. Transactivation activity assays performed in Arabidopsis and rice protoplasts showed that OsPCF2 and OsNIN-like4 are activators of the OsNHX1 gene expression, while OsCPP5 and OsNIN-like2 act as repressors. The transactivation activity of OsNIN-like3 needs to be further investigated. Gene expression studies showed that OsNHX1 transcript level is highly induced by salt and PEG-induced drought stress in both shoots and roots in both Nipponbare and Hasawi rice genotypes. Nevertheless, OsNHX1 seems to play a particular role in shoots in response to drought. Most of the TFs binding to OsNHX1 promoter showed a modest transcriptional regulation under stress conditions, however, in response to most of the conditions studied, the OsPCF2 was induced earlier than OsNHX1, indicating that OsPCF2 may activate OsNHX1 gene expression. In addition, although the OsNHX1 response to salt and PEG-induced drought stress in either shoots or roots was quite similar in both rice genotypes, the expression of OsPCF2 in roots under salt stress and the OsNIN-like4 in roots subjected to PEG was mainly up-regulated in Hasawi, indicating that these TFs may be associated with the salt and drought stress tolerance observed for this genotype.

Sheath lesions, grain sterility and grain discolouration of rice caused by Pseudomonas fuscovaginae can cause yield losses of up to 100 %. The most sustainable method of managing this disease is the use of host plant resistance. To identify sources of resistance an inoculation method that is practical, rapid and reliable is needed. We compare three different inoculation methods. Results showed that the pin-prick method is appropriate for identifying sources of resistance to P. fuscovaginae, while the spray method could be useful for mass screening of rice genotypes. The seed-soaking method was also evaluated and has showed potential in detection of early disease resistance. A total of 16 Multiparent Advanced Generation Inter-Crosses and 20 OryzaSNP set varieties from the International Rice Research Institute were evaluated using the pin-prick and seed-soaking methods. All growth stages were susceptible to the pathogen and the 107 cfu mL−1 inoculum concentration was optimal for discriminating between resistant and susceptible genotypes. For the pin-prick method, a single point assessment of disease severity at 14 days post-inoculation could be used instead of the AUDPC values to classify genotypes. An index of reduction in seedling height 10 days after seed soaking was established for the classification of the genotypes reaction to the disease. Resistant varieties identified using both the pin-prick and seed-soaking methods could be verified for use in disease resistance breeding programs. Of the 36 genotypes evaluated 22 were found to be resistant at the late booting or early panicle exsertion stage by pin-prick method, while 25 were resistant at the seed to germination stage. No correlation was found between the resistance classification of varieties between the two inoculation methods, indicating that there could be different mechanisms of resistance to P. fuscovaginae in rice.

Rice, with its wide geographic distribution extending from 50° Nt o 35°S, is expected to be the most vulnerable cultivated crop to future changing climates. Among the different abiotic stresses, extreme temperatures coinciding with critical developmental stages, increasingly frequent floods and drought spells, and worsening sea water inundation are some of the major threats to sustainable rice productivity. Following the successful implementation of molecular markerassisted backcrossing to introgress large-effect QTL for submergence tolerance in rice mega varieties, rice breeding for drought, salinity and, recently, heat tolerance is employing the same approach. Although tolerance for combined submergence and salinity has been achieved, developing rice varieties with multiple tolerance for other abiotic and biotic stresses and finding the appropriate agronomic package to exploit their performance remain a challenge. The major bottleneck is the lack of unidentified large-effect QTL for other abiotic stresses that are strongly influenced by genotype 9 environment (G 9 E) interaction. Rapid advances in the use of molecular tools, including a plethora of SNP markers, are expected to facilitate the development of major abiotic stress-tolerant rice. In response to the actual farmer field situation, progress achieved in understanding and developing independent abiotic stress tolerance is being exploited to combine tolerances (for example, heat and drought; salinity and submergence) to address emerging environmental problems across a wide range of rice ecosystems.

Traditional rice varieties harbour a large store of genetic diversity with potential to accelerate rice improvement. For a long time, this diversity maintained in the International Rice Genebank has not been fully used because of a lack of genome information. The publication of the first reference genome of Nipponbare by the International Rice Genome Sequencing Project (IRGSP) marked the beginning of a systematic exploration and use of rice diversity for genetic research and breeding. Since then, the Nipponbare genome has served as the reference for the assembly of many additional genomes. The recently completed 3000 Rice Genomes Project together with the public database (SNP-Seek) provides a new genomic and data resource that enables the identification of useful accessions for breeding. Using disease resistance traits as case studies, we demonstrated the power of allele mining in the 3,000 genomes for extracting accessions from the GeneBank for targeted phenotyping. Although potentially useful landraces can now be identified, their use in breeding is often hindered by unfavourable linkages. Efficient breeding designs are much needed to transfer the useful diversity to breeding. Multi-parent Advanced Generation InterCross (MAGIC) is a breeding design to produce highly recombined populations. The MAGIC approach can be used to generate pre-breeding populations with increased genotypic diversity and reduced linkage drag. Allele mining combined with a multi-parent breeding design can help convert useful diversity into breeding-ready genetic resources.

With 2 figures and 5 tables Abstract A population of BC3F4 rice lines derived from a cross of salt-tolerant Pokkali and sensitive IR29 was developed for validation of the Saltol QTL and comparison of this QTL effect with background QTLs. Specific BC3F4 progenies from the advanced backcross population were found to have similar salinity tolerance at the seedling stage even without the Saltol alleles. Therefore, additional QTLs responsible for seedling-stage salinity tolerance were investigated by screening and genotyping with markers at multiple loci throughout the 12 rice chromosomes. Single marker regression (SMR) using Q-Gene detected seven QTLs above the threshold logarithm of the odds of 3.0. Single marker analysis using IciMapping detected the same markers linked to the seven QTLs identified by SMR. Likelihood ratio test-based on stepwise regression (LRT-RSTEP) analysis using IciMapping, more suitable for BC3F4 introgression lines located six QTLs, of which two were common with SMR and included two significant segments within Saltol. The new non-Saltol QTLs represent targets for further study to evaluate their usefulness in breeding for higher levels of seedling-stage salinity tolerance in rice.

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F1 plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F2 populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.

Background Climate change is affecting rice production in many countries. Developing new rice varieties with heat tolerance is an essential way to sustain rice production in future global warming. We have previously reported four quantitative trait loci (QTLs) responsible for rice spikelet fertility under high temperature at flowering stage from an IR64/N22 population. To further explore additional QTL from other varieties, two bi-parental F2 populations and one three-way F2 population derived from heat tolerant variety Giza178 were used for indentifying and confirming QTLs for heat tolerance at flowering stage. Results Four QTLs (qHTSF1.2, qHTSF2.1, qHTSF3.1 and qHTSF4.1) were identified in the IR64/Giza178 population, and two other QTLs (qHTSF6.1 and qHTSF11.2) were identified in the Milyang23/Giza178 population. To confirm the identified QTLs, another three-way-cross population derived from IR64//Milyang23/Giza178 was genotyped using 6K SNP chips. Five QTLs were identified in the three-way-cross population, and three of those QTLs (qHTSF1.2, qHTSF4.1 and qHTSF6.1) were overlapped with the QTLs identified in the bi-parental populations. The tolerance alleles of these QTLs were from the tolerant parent Giza178 except for qHTSF3.1. The QTL on chromosome 4 (qHTSF4.1) is the same QTL previously identified in the IR64/N22 population. Conclusion The results from different populations suggest that heat tolerance in rice at flowering stage is controlled by several QTLs with small effects and stronger heat tolerance could be attained through pyramiding validated heat tolerance QTLs. QTL qHTSF4.1 was consistently detected across different genetic backgrounds and could be an important source for enhancing heat tolerance in rice at flowering stage. Polymorphic SNP markers in these QTL regions can be used for future fine mapping and developing SNP chips for marker-assisted breeding. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0199-7) contains supplementary material, which is available to authorized users.

Selection of new rice (Oryza sativa L.) variety tolerant to soil salinity is one of the most important issues to avoid salt stress in rice production. In this study, the morphological characterization was performed on 5 rice genotypes under two salt stress treatments EC8 and 12 dS/m at seedling, EC 12 dS/m at vegetative and EC8 dS/m at reproductive stages. Morphological characters and Na+/K+ uptake ratio were surveyed to evaluate salt stress effects. At vegetative stage, all the genotypes have survived. However, at reproductive stage all the genotypes were affected by salt stress except for the Binadhan-10 that survived the whole life cycle. A total of 160 SSR markers were used which revealed 209 alleles among the 5 rice genotypes. Interestingly, 4 SSR markers RM105, RM125, RM178 and RM549 with highest PIC (0.67) value have scored the highest level of genetic diversity value (0.72). These markers could be used to pyramiding the major and minor salt stress related genes via marker assisted selection in rice. The present investigation on variant salt stress responsiveness of 5 promising rice genotypes will be of great value in rice breeding.

Salt stress affects about 25% of the 4.4 million ha of irrigated and lowland systems for rice cultivation in West Africa (WA). A major quantitative trait locus (QTLs) on chromosome 1 (Saltol) that enhances tolerance to salt stress at the vegetative stage has enabled the use of marker-assisted selection (MAS) to develop salt-tolerant rice cultivar(s) in WA. We used 3 cycles of backcrossing with selection based on DNA markers and field-testing using 'FL478' as tolerant donor and the widely grown 'Rassi' as recurrent parent. In the BC3F2 stage, salt-tolerant lines with over 80% Rassi alleles except in the region around Saltol segment were selected. 429 introgression lines (Saltol-ILs) were identified as tolerant at vegetative stage, of which 116 were field-tested for four seasons at the reproductive stage. Sixteen Saltol-ILs had less yield loss (3-26% relative to control trials), and 8 Saltol-ILs showed high yield potential under stress and non-stress conditions. The 16 Saltol-ILs had been included for further African-wide testing prior to release in 6 WA countries. MAS reduced the time for germplasm improvement from at least 7 to about 4 years. Our objective is to combine different genes/QTLs conferring tolerance to stresses under one genetic background using MAS.

Genome-wide association mapping studies (GWAS) are frequently used to detect QTL in diverse collections of crop germplasm, based on historic recombination events and linkage disequilibrium across the genome. Generally, diversity panels genotyped with high density SNP panels are utilized in order to assay a wide range of alleles and haplotypes and to monitor recombination breakpoints across the genome. By contrast, GWAS have not generally been performed in breeding populations. In this study we performed association mapping for 19 agronomic traits including yield and yield components in a breeding population of elite irrigated tropical rice breeding lines so that the results would be more directly applicable to breeding than those from a diversity panel. The population was genotyped with 71,710 SNPs using genotyping-by-sequencing (GBS), and GWAS performed with the explicit goal of expediting selection in the breeding program. Using this breeding panel we identified 52 QTL for 11 agronomic traits, including large effect QTLs for flowering time and grain length/grain width/grain-length-breadth ratio. We also identified haplotypes that can be used to select plants in our population for short stature (plant height), early flowering time, and high yield, and thus demonstrate the utility of association mapping in breeding populations for informing breeding decisions. We conclude by exploring how the newly identified significant SNPs and insights into the genetic architecture of these quantitative traits can be leveraged to build genomic-assisted selection models.

This study fine mapped and validated a QTL on rice chromosome 4 that increases spikelet fertility under high temperature (over 37 °C) at the flowering stage. Climate change has a negative effect on crop production and food security. Understanding the genetic mechanism of heat tolerance and developing heat-tolerant varieties is essential to cope with future global warming. Previously, we reported on a QTL (qHTSF4.1) from an IR64/N22 population responsible for rice spikelet fertility under high-temperature stress at the flowering stage. To further fine map and validate the effect of qHTSF4.1, PCR-based SNP markers were developed and used to genotype BC2F2, BC3F2, BC3F3, and BC5F2 populations from the same cross. The interval of the QTL was narrowed down to about 1.2 Mb; however, further recombination was not identified even with a large BC5F2 population that was subsequently developed and screened. The sequence in the QTL region is highly conserved and a large number of genes in the same gene family were observed to be clustered in the region. The QTL qHTSF4.1 consistently increased spikelet fertility in all of the backcross populations. This was confirmed using 24 rice varieties. Most of the rice varieties with the QTL showed a certain degree of heat tolerance under high-temperature conditions. In a BC5F2 population with clean background of IR64, QTL qHTSF4.1 increased spikelet fertility by about 15 %. It could be an important source for enhancing heat tolerance in rice at the flowering stage. PCR-based SNP markers developed in this study can be used for QTL introgression and for pyramiding with other agronomically important QTLs/genes through marker-assisted selection.

We have established marker-aided selection strategies for the two major Rf genes (Rf3 and Rf4) governing fertility restoration of␣cytoplasmic-genetic male sterility (CMS) in rice. Polymorphisms between restorer and non-restorer␣lines were observed using RG140/PvuII for Rf3 located on chromosome 1 and S10019/BstUI for Rf4 located on chromosome 10. DNA polymorphisms associated with these two loci in restorer lines of wild abortive (WA), Dissi, and Gambiaca cytoplasm are conserved, suggesting that similar biological processes control pollen fertility in this diverse cytoplasm. Because of their close linkage to Rf genes and distinct banding patterns, STS markers RG140/PvuII and S10019/BstUI are well suited for marker-aided selection, enhanced backcross procedures, and pyramiding of Rf genes in agronomically superior non-restorer lines. The combined use of markers associated with these two loci improved the efficiency of screening for putative restorer lines from a set of elite lines. Positional analyses of Rf4 and the inheritance pattern of the polymorphism in S10019/BstUI suggest that Rf4, governing fertility restoration in WA-CMS in rice, is likely to be the same gene governing fertility restoration in BT- and HL-CMS that has a gametophytic effect, which explains why 100% pollen fertility in hybrids is impossible to attain.

Low and unstable rice productivity in many areas of Asia is associated with many abiotic and biotic stresses such as drought, salinity, anaerobic conditions during germination, submergence, phosphorus and zinc deficiency, etc. To develop rice varieties with tolerance to these stresses, we undertook a large backcross (BC) breeding effort for the last 6 years, using three recurrent elite rice lines and 203 diverse donors, which represent a significant portion of the genetic diversity in the primary gene pool of rice. Significant progress has been made in the BC breeding program, which resulted in development of large numbers ofintrogression lines with improved tolerance to these stresses. Promising lines have been developed with excellent tolerances (extreme phenotypes) to salinity, submergence and zinc deficiency; resistance to brown plant hopper, ability to germinate under the anaerobic condition and low temperature. Our results indicated that there exist tremendous amounts of ‘hidden’ diversity for abiotic and biotic stress tolerances in the primary gene pool of rice. Furthermore, we demonstrated that despite the complex genetics and diverse physiological mechanisms underlying the abiotic stress tolerances, introgression of genes from a diverse source of donors into elitegenetic backgrounds through BC breeding and efficient selection (careful screening under severestress) is a powerful way to exploit this hidden diversity for improving abiotic stress tolerances of rice. We have developed three large sets of introgression lines, which not only provide an unique platform of breeding materials for developing new rice cultivars with superior yield and stability by trait/gene pyramiding, but also represent unique genetic stocks for a large-scale discovery of genes/alleles underlying the abiotic and biotic stress tolerances of rice using genomic tools. # 2005 Elsevier B.V. All rights reserved.

Breeding for tolerance to iron (Fe) toxicity in rice (Oryza sativa L.) is hindered by lack of a suitable screening technique. In the culture solution methods used to date, a major difficulty has been maintaining an excess level of iron concentration in order to reveal toxicity symptoms. Experimental results showed that this problem was solved by lowering the solution temperature to around 20°C, at which leaf discoloration in susceptible cultivars became more pronounced. At 20°C Fe uptake was increased from a threshhold content of 300 mg/kg of dry weight of shoot for toxic symptom to more than 1000mg/kg in susceptible and in tolerant cultivars. Concentrations of other related minerals in the plant tissue, i.e., potassium (K) and phosphorus (P), were not affected by the low solution temperature itself but by Fe content, which can be inferred on the basis of their response curves to the excess Fe treatment. Using the proposed screening method makes it possible to obtain reproducible results in scree...

Iron toxicity is one of the major constraints for lowland rice production in highly weathered soils, which are widely distributed in tropics and subtropics and often lack macronutrients such as potassium (K) and phosphorus (P). To analyze the genetic factors for excess iron accumulation under K or P deficiency, a set of seedlings in F3 and F8 generations from an Oryza sativa cross between a japonica cultivar ‚Gimbozu’ and an indica cultivar ‚Kasalath’ were raised and exposed to nutritional stresses in a short period under nutritional solutions. In the F8 lines, contents of K, P, Fe, and Mg in dried shoots were measured. Quantitative trait loci (QTL) for the iron accumulation and related mineral contents in each plant were analyzed with composite interval mapping. QTLs for the Fe, P and Mg content in shoots were compared in the maps of F3 and F8. The QTLs for the Fe content in shoots varied in three types of nutritional conditions, but consistently indicated two overlapping regions on chromosome 3 and 4. The obtained QTLs were crosschecked with those reported before. Some of these QTLs were indicative of iron excluding the power of the root, which was expressed under reduced P content in solution.

The success of salt tolerance breeding programs employing traditional screening and selection has been limited in the past decades. This study was designed to characterize the genetic diversity within a subset of rice germplasm with different adaptations to saline soils using microsatellit e markers. Salt tolerance was then analyzed among molecularly characterized genotypes. Plants of 33 genotypes were grown in sand tanks under greenhouse condition and irrigated with Yoshida nutrient solution. Two salt treatments were imposed with electrical conductivities of 0.9 dS m −1 (control) and 6.5 dS m −1 (6:1 molar ratio of NaCl and CaCl2). A total of 123 alleles were generated at 25 microsatellite loci among the 33 genotypes. Genotypes of japonica rice grouped into three clusters and those of indica rice grouped into two clusters based on microsatellite markers. Thirty percent of the alleles detected in 20 breeding lines were not identified in the cultivars analyzed. These alleles may provide favorable allelic combinations if the breeding lines are used for intercrosses. Physiological and morphological characters under salt stress were significantly ( P = 0.05) different among microsatellite clusters. There was a highly significant correlation ( r =− 0.25; P = 0.005) between the matrices of Jaccard genetic similarity based on microsatellite markers and taxonomic distance based on ion data. These results indicate that the adaptation of rice to saline soils is different among genotypes with diverse genetic backgrounds. Implications for engineering salt tolerance are: (1) Improving salt tolerance can be achieved by selecting parental genotypes prior to intercrossing based on microsatellite markers. (2) Phenotypic variation of ion contents in segregating populations can be increased by selecting parental genotypes prior to intercrossing based on microsatellite markers. (3) Different salt tolerance components can be combined into a cultivar by intercrossing genotypes from different microsatellite clusters with diverse salt tolerance mechanisms. © 2003 Elsevier Ireland Ltd. All rights reserved.

The use of physiological characters as selection criteria in salt tolerance breeding requires the identification of the contribution each individual character makes to salt tolerance. Rice genotypes were evaluated for salt tolerance in terms of grain yield and physiological characters. Plants of twelve genotypes were grown in sand tanks in a greenhouse and irrigated with Yoshida nutrient solution. Sodium chloride and calcium chloride (5:1 molar ratio) were added at two concentrations to give moderate (4.5 dS m-1) and high (8.3 dS m-1) salinity treatments. One set of plants was harvested at 635 °Cċd (accumulative thermal time) after planting to determine LAI and mineral ion concentrations. Another set of plants was allowed to grow to maturity. High genotypic diversity for LAI and shoot ion contents was observed. LAI contributed the most to the variation of the grain yield under salt stress. Significant correlations between LAI and yield components in both salt-tolerant and-sensitive genotypes further confirmed the significant contribution of LAI to grain yield. K-Na selectivity increased with increasing salinity. Conversely, Na-Ca selectivity decreased with increasing salinity. Significant correlations were identified between grain yield and both Na-Ca and K-Na selectivity. Highly significant (p

Seventy-nine di-haploid lines were produced in rice through anther culture of the cross of two indica breeding lines (IR5657-33-2×IR4630-22-2-5-1-3). The cross was designed to combine the high-yielding ability of IR5657-33-2 with the salinity tolerance of IR4630-22-2-5-1-3. The anther culture (AC)-derived lines were evaluated in the greenhouse, field, and salinity affected areas. Several lines showed desirable traits such as high yield, salinity tolerance, early maturity, good plant type, and resistance to pests and diseases. IR51500-AC9-7 and IR51500-AC11-1 selected from evaluations were included in the National Cooperative Trials (NCT) for saline-prone areas conducted by the Rice Varietal Improvement Group (RVIG) of the National Seed Industry Council (NSIC) of the Philippines. Trials conducted from the 1990 wet season to 1994 dry season at nine sites revealed that IR51500-AC11-1 performed better than other cultivars grown in saline-prone lands. In 1995, the NSIC named IR51500-AC11-1 as PSBRc50 “Bicol” and recommended it for commercial cultivation in salt-affected rice lands. This is the first F 1 AC-derived line from an indica/indica cross to be released as a cultivar for cultivation in saline-prone areas.