Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands.

Introduction: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease. Methods: Pyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB. Results and discussion: Both BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses.

Genome wide association study of MAGIC population reveals a novel QTL for salinity and sodicity tolerance in rice.

The present study was conducted to identify the novel QTLs controlling salinity and sodicity tolerance using indica MAGIC rice population. Phenotyping was carried out in salinity (EC ~ 10 dS/m) and sodicity (pH ~ 9.8) at the seedling stage. Among 391 lines, 43 and 98 lines were found tolerant and moderately tolerant to salinity. For sodicity condition, 2 and 45 lines were showed tolerance and moderately tolerance at seedling stage. MAGIC population was genotyped with the help of genotyping by sequencing (GBS) and filtered 27041SNPs were used for genome wide marker trait association studies. With respect to salinity tolerance, 25 SNPs were distributed on chromosomes 1, 5, 11 and 12, whereas 18 SNPs were mapped on chromosomes 6, 4 and 11 with LOD value of > 3.25 to sodicity tolerance in rice. The candidate gene analysis detected twelve causal genes including SKC1 gene at Saltol region for salinity and six associated genes for sodic stress tolerance. The significant haplotypes responsible for core histone protein coding gene (LOC_Os12g25120) and three uncharacterized protein coding genes (LOC_Os01g20710, LOC_Os01g20870 and LOC_Os12g22020) were identified under saline stress. Likewise, five significant haplotypes coding for ribose 5-phosphate isomerise (LOC_Os04g24140), aspartyl protease (LOC_Os06g15760), aluminum-activated malate transporter (LOC_Os06g15779), OsFBX421-Fbox domain containing protein (LOC_Os11g32940) and one uncharacterized protein (LOC_Os11g32930) were detected for sodic stress tolerance. The identified novel SNPs could be the potential candidates for functional characterization. These candidate genes aid to further understanding of genetic mechanism on salinity and sodicity stress tolerance in rice. The tolerant line could be used in future breeding programme to enhance the salinity and sodicity tolerance in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01174-8.

Marker aided introgression of 'Saltol', a major QTL for seedling stage salinity tolerance into an elite Basmati rice variety 'Pusa Basmati 1509'.

Marker assisted backcross breeding was used to transfer Saltol, a major QTL for seedling stage salinity tolerance from the donor FL478 to Pusa Basmati 1509 (PB 1509), a high yielding and early maturing Basmati rice variety. Foreground selection was carried out using three markers namely, AP3206f, RM3412b and RM10793, linked to Saltol. In addition, 105 genome-wide SSR markers polymorphic between FL478 and PB 1509 were used in background selection. Among the BC3F4 near isogenic lines (NILs) developed, recurrent parent genome recovery ranged from 96.67 to 98.57%. Multi-season evaluation identified some of the NILs showing significantly higher yield with grain and cooking quality comparable to PB 1509. All the NILs exhibited tolerance to salinity with significantly higher relative water content, membrane stability index and proline content as compared to PB 1509. The root and shoot concentration of Na+, K+ and Na+/K+ in NILs was at par with FL478 under stress conditions. The gene OsHKT1;5 located in the Saltol region showed higher expression levels under stress indicating its role in conferring salinity tolerance. Salt tolerant NILs of PB 1509 will be useful in stabilizing production in salt affected areas.

Introgressed Saltol QTL Lines Improves the Salinity Tolerance in Rice at Seedling Stage.

Rice is a staple food crop in Asia and plays a crucial role in the economy of this region. However, production of rice and its cultivating areas are under constant threat of soil salinity. A major QTL, Saltol, responsible for salinity tolerance at seedling stage has been mapped on chromosome 1 using Pokkali/IR29 Recombinant Inbred Lines (RIL) population. The present study was aimed to incorporate Saltol Quantitative Trait Loci (QTL) in two high yielding mega rice varieties i.e. Pusa44 and Sarjoo52 through Marker Assisted Backcross Breeding (MABB). To improve the seedling stage salinity tolerance in these cultivars, we introgressed the Saltol QTL from donor parent FL478 a derivative of Pokkali. A total of three backcrosses (BC3) followed by selfing have led to successful introgression of Saltol QTL. Foreground selection at each breeding cycle was done using micro-satellite markers RM3412 and AP3206 to confirm Saltol QTL. The precise transfer of Saltol region was established using recombinant selection through flanking markers RM493 and G11a. Finally, 10 Saltol near isogenic lines (NILs) of Pusa44 and eight NILs of Sarjoo52 were successfully developed. These NILs (BC3F4) were evaluated for seedling stage salinity under hydroponic system. The NILs PU99, PU176, PU200, PU215, PU229, PU240, PU241, PU244, PU252, PU263 of Pusa44 and SAR17, SAR23, SAR35, SAR39, SAR77, SAR87, SAR123, SAR136 NILs of Sarjoo52 confirmed tolerance to salinity with low salt injury score of 3 or 5. Ratio of Na+/K+ content of Saltol NILs ranged from 1.26 to 1.85 in Pusa44 and 1.08 to 1.69 in Sarjoo52. The successfully developed NILs were further phenotyped stringently for morphological traits to estimate Phenotypic Recovery. Background selection of NILs along with parents was carried out with 50K SNP chip and recovered 94.83-98.38% in Pusa44 NILs and 94.51 to 98.31% in Sarjoo52 NILs of recurrent genome. The present study of MAB has accelerated the development of salt tolerant lines in the genetic background of Pusa44 and Sarjoo52. These NILs could be used for commercial cultivation in saline affected area.

Enhancing trehalose biosynthesis improves yield potential in marker-free transgenic rice under drought, saline, and sodic conditions.

Edaphic factors such as salinity, sodicity, and drought adversely affect crop productivity, either alone or in combination. Despite soil sodicity being reported as an increasing problem worldwide, limited efforts have been made to address this issue. In the present study, we aimed to generate rice with tolerance to sodicity in conjunction with tolerance to salinity and drought. Using a fusion gene from E. coli coding for trehalose-6-phosphate synthase/phosphatase (TPSP) under the control of an ABA-inducible promoter, we generated marker-free, high-yielding transgenic rice (in the IR64 background) that can tolerate high pH (~9.9), high EC (~10.0 dS m-1), and severe drought (30-35% soil moisture content). The transgenic plants retained higher relative water content (RWC), chlorophyll content, K+/Na+ ratio, stomatal conductance, and photosynthetic efficiency compared to the wild-type under these stresses. Positive correlations between trehalose overproduction and high-yield parameters were observed under drought, saline, and sodic conditions. Metabolic profiling using GC-MS indicated that overproduction of trehalose in leaves differently modulated other metabolic switches, leading to significant changes in the levels of sugars, amino acids, and organic acids in transgenic plants under control and stress conditions. Our findings reveal a novel potential technological solution to tackle multiple stresses under changing climatic conditions.

Harmonizing technological advances in phenomics and genomics for enhanced salt tolerance in rice from a practical perspective.

Half of the global human population is dependent on rice as a staple food crop and more than 25% increase in rice productivity is required to feed the global population by 2030. With increase in irrigation, global warming and rising sea level, rising salinity has become one of the major challenges to enhance the rice productivity. Since the loss on this account is to the tune of US$12 billion per annum, it necessitates the global attention. In the era of technological advancement, substantial progress has been made on phenomics and genomics data generation but reaping benefit of this in rice salinity variety development in terms of cost, time and precision requires their harmonization. There is hardly any comprehensive holistic review for such combined approach. Present review describes classical salinity phenotyping approaches having morphological, physiological and biochemical components. It also gives a detailed account of invasive and non-invasive approaches of phenomic data generation and utilization. Classical work of rice salinity QLTs mapping in the form of chromosomal atlas has been updated. This review describes how QTLs can be further dissected into QTN by GWAS and transcriptomic approaches. Opportunities and progress made by transgenic, genome editing, metagenomics approaches in combating rice salinity problems are discussed. Major aim of this review is to provide a comprehensive over-view of hitherto progress made in rice salinity tolerance research which is required to understand bridging of phenotype based breeding with molecular breeding. This review is expected to assist rice breeders in their endeavours by fetching greater harmonization of technological advances in phenomics and genomics for better pragmatic approach having practical perspective.

Heterologous Expression of Serine Hydroxymethyltransferase-3 From Rice Confers Tolerance to Salinity Stress in E. coli and Arabidopsis.

Among abiotic stresses, salt stress adversely affects growth and development in rice. Contrasting salt tolerant (CSR27), and salt sensitive (MI48) rice varieties provided information on an array of genes that may contribute for salt tolerance of rice. Earlier studies on transcriptome and proteome profiling led to the identification of salt stress-induced serine hydroxymethyltransferase-3 (SHMT3) gene. In the present study, the SHMT3 gene was isolated from salt-tolerant (CSR27) rice. OsSHMT3 exhibited salinity-stress induced accentuated and differential expression levels in different tissues of rice. OsSHMT3 was overexpressed in Escherichia coli and assayed for enzymatic activity and modeling protein structure. Further, Arabidopsis transgenic plants overexpressing OsSHMT3 exhibited tolerance toward salt stress. Comparative analyses of OsSHMT3 vis a vis wild type by ionomic, transcriptomic, and metabolic profiling, protein expression and analysis of various traits revealed a pivotal role of OsSHMT3 in conferring tolerance toward salt stress. The gene can further be used in developing gene-based markers for salt stress to be employed in marker assisted breeding programs. HIGHLIGHTS: - The study provides information on mechanistic details of serine hydroxymethyl transferase gene for its salt tolerance in rice.

Participatory evaluation guides the development and selection of farmers' preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia.

Rice is the staple food and provides livelihood for smallholder farmers in the coastal delta regions of South and Southeast Asia. However, its productivity is often low because of several abiotic stresses including high soil salinity and waterlogging during the wet (monsoon) season and high soil and water salinity during the dry season. Development and dissemination of suitable rice varieties tolerant of these multiple stresses encountered in coastal zones are of prime importance for increasing and stabilizing rice productivity, however adoption of new varieties has been slow in this region. Here we implemented participatory varietal selection (PVS) processes to identify and understand smallholder farmers' criteria for selection and adoption of new rice varieties in coastal zones. New breeding lines together with released rice varieties were evaluated in on-station and on-farm trials (researcher-managed) during the wet and dry seasons of 2008-2014 in the Indian Sundarbans region. Significant correlations between preferences of male and female farmers in most trials indicated that both groups have similar criteria for selection of rice varieties. However, farmers' preference criteria were different from researchers' criteria. Grain yield was important, but not the sole reason for variety selection by farmers. Several other factors also governed preferences and were strikingly different when compared across wet and dry seasons. For the wet season, farmers preferred tall (140-170 cm), long duration (160-170 d), lodging resistant and high yielding rice varieties because these traits are required in lowlands where water stagnates in the field for about four months (July to October). For the dry season, farmers' preferences were for high yielding, salt tolerant, early maturing (115-130 d) varieties with long slender grains and good quality for better market value. Pest and disease resistance was important in both seasons but did not rank high. When farmers ranked the two most preferred varieties, the ranking order was sometimes variable between locations and years, but when the top four varieties that consistently ranked high were considered, the variability was low. This indicates that at least 3-4 of the best-performing entries should be considered in succeeding multi-location and multi-year trials, thereby increasing the chances that the most stable varieties are selected. These findings will help improve breeding programs by providing information on critical traits. Selected varieties through PVS are also more likely to be adopted by farmers and will ensure higher and more stable productivity in the salt- and flood-affected coastal deltas of South and Southeast Asia.

Identification of mega-environments and rice genotypes for general and specific adaptation to saline and alkaline stresses in India.

In the present study, a total of 53 promising salt-tolerant genotypes were tested across 18 salt-affected diverse locations for three years. An attempt was made to identify ideal test locations and mega-environments using GGE biplot analysis. The CSSRI sodic environment was the most discriminating location in individual years as well as over the years and could be used to screen out unstable and salt-sensitive genotypes. Genotypes CSR36, CSR-2K-219, and CSR-2K-262 were found ideal across years. Overall, Genotypes CSR-2K-219, CSR-2K-262, and CSR-2K-242 were found superior and stable among all genotypes with higher mean yields. Different sets of genotypes emerged as winners in saline soils but not in sodic soils; however, Genotype CSR-2K-262 was the only genotype that was best under both saline and alkaline environments over the years. The lack of repeatable associations among locations and repeatable mega-environment groupings indicated the complexity of soil salinity. Hence, a multi-location and multi-year evaluation is indispensable for evaluating the test sites as well as identifying genotypes with consistently specific and wider adaptation to particular agro-climatic zones. The genotypes identified in the present study could be used for commercial cultivation across edaphically challenged areas for sustainable production.

Marker Aided Incorporation of Saltol, a Major QTL Associated with Seedling Stage Salt Tolerance, into Oryza sativa 'Pusa Basmati 1121'.

Pusa Basmati 1121 (PB1121), an elite Basmati rice cultivar is vulnerable to salinity at seedling stage. A study was undertaken to impart seedling-stage salt tolerance into PB1121 by transferring a quantitative trait locus (QTL), Saltol, using FL478 as donor, through marker assisted backcrossing. Sequence tagged microsatellite site (STMS) marker RM 3412, tightly linked to Saltol was used for foreground selection. Background recovery was estimated using 90 genome-wide STMS markers. Systematic phenotypic selection helped in accelerated recovery of recurrent parent phenome (RPP). A set of 51 BC3F2 lines homozygous for Saltol were advanced to develop four improved near isogenic lines (NILs) of PB1121 with seedling stage salt tolerance. The background genome recovery in the NILs ranged from 93.3 to 99.4%. The improved NILs were either similar or better than the recurrent parent PB1121 for yield, grain and cooking quality and duration. Biochemical analyses revealed significant variation in shoot and root Na+ and K+ concentrations. Correlation between shoot and root Na+ concentration was stronger than that between root and shoot K+ concentration. The effect of QTL integration into the NILs was studied through expression profiling of OsHKT1;5, one of the genes present in the Saltol region. The NILs had significantly higher OsHKT1;5 expression than the recurrent parent PB1121, but lower than FL478 on salt exposure validating the successful introgression of Saltol in the NILs. This was also confirmed under agronomic evaluation, wherein the NILs showed greater salt tolerance at seedling stage. One of the NILs, Pusa1734-8-3-3 (NIL3) showed comparable yield and cooking quality to the recurrent parent PB1121, with high field level seedling stage salinity tolerance and shorter duration. This is the first report of successful introgression of Saltol into a Basmati rice cultivar.

From QTL to variety-harnessing the benefits of QTLs for drought, flood and salt tolerance in mega rice varieties of India through a multi-institutional network.

Rice is a staple cereal of India cultivated in about 43.5Mha area but with relatively low average productivity. Abiotic factors like drought, flood and salinity affect rice production adversely in more than 50% of this area. Breeding rice varieties with inbuilt tolerance to these stresses offers an economically viable and sustainable option to improve rice productivity. Availability of high quality reference genome sequence of rice, knowledge of exact position of genes/QTLs governing tolerance to abiotic stresses and availability of DNA markers linked to these traits has opened up opportunities for breeders to transfer the favorable alleles into widely grown rice varieties through marker-assisted backcross breeding (MABB). A large multi-institutional project, "From QTL to variety: marker-assisted breeding of abiotic stress tolerant rice varieties with major QTLs for drought, submergence and salt tolerance" was initiated in 2010 with funding support from Department of Biotechnology, Government of India, in collaboration with International Rice Research Institute, Philippines. The main focus of this project is to improve rice productivity in the fragile ecosystems of eastern, northeastern and southern part of the country, which bear the brunt of one or the other abiotic stresses frequently. Seven consistent QTLs for grain yield under drought, namely, qDTY1.1, qDTY2.1, qDTY2.2, qDTY3.1, qDTY3.2, qDTY9.1 and qDTY12.1 are being transferred into submergence tolerant versions of three high yielding mega rice varieties, Swarna-Sub1, Samba Mahsuri-Sub1 and IR 64-Sub1. To address the problem of complete submergence due to flash floods in the major river basins, the Sub1 gene is being transferred into ten highly popular locally adapted rice varieties namely, ADT 39, ADT 46, Bahadur, HUR 105, MTU 1075, Pooja, Pratikshya, Rajendra Mahsuri, Ranjit, and Sarjoo 52. Further, to address the problem of soil salinity, Saltol, a major QTL for salt tolerance is being transferred into seven popular locally adapted rice varieties, namely, ADT 45, CR 1009, Gayatri, MTU 1010, PR 114, Pusa 44 and Sarjoo 52. Genotypic background selection is being done after BC2F2 stage using an in-house designed 50K SNP chip on a set of twenty lines for each combination, identified with phenotypic similarity in the field to the recipient parent. Near-isogenic lines with more than 90% similarity to the recipient parent are now in advanced generation field trials. These climate smart varieties are expected to improve rice productivity in the adverse ecologies and contribute to the farmer's livelihood.

Effect of salinity and use of stress indices of morphological and physiological traits at the seedling stage in rice.

Rice (Oryza sativa L.) is the most important cereal crop and a major staple food for majority of the human populations worldwide. Rice crop is sensitive to salinity. In spite of large number of studies on salinity tolerance of rice, our knowledge on the overall effect of salinity on rice seedling growth is limited. Improvement in salt tolerance of crop plants remains indescribable, largely due to the fact that salinity is a complex trait which affects almost every aspect of the physiology, biochemistry and genomics of plants. The present investigation was conducted to establish the relationship between various morphological, physiological traits and stress indices. A set of 131 rice accessions was evaluated in two levels namely, non-stress (EC ~ 1.2 dS/m) and saline stress (EC ~ 10 dS/m) in hydroponics at seedling stage. Root length and shoot lengths were reduced by 52 and 50%, respectively in saline stress compared to non-stress conditions. There was a significant correlation between various morphological and physiological parameters in non-saline in addition to saline stress as well as non-stress. The effect of the increased Na+ concentration in the medium is detrimental to root length and shoot length as observed by reduction in root length and a concomitant reduction in shoot length. Increased concentration of Na+ led to augmented Na+/K+ ratio with increased stress in the medium and decreased expression of traits. A significant positive correlation (r=0.60) was noticed between stress tolerance index (STI) of root and shoot length. The stress susceptibility index (SSI) for root length was expressed significant positive correlation with SSI for shoot length (r=0.43). SSI for K+ content was registered significant negative correlation with STI for Na+ content (r=-0.43). The three accessions namely, IC 545004, IC 545486 and IC 545215 were found to be the best performers adjudged on the morphological and physiological criteria in saline stress situation. These three rice accessions could be used as a donor parent or for genotypic studies in future breeding programs.

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa).

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na(+)/K(+) ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5-18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na(+)/K(+) ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.