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Abstract Rice blast disease is the most devastating disease constraining crop productivity. Vertical resistance to blast disease is widely studied despite its instability. Clusters of genes or QTLs conferring blast resistance that offer durable horizontal resistance are important in resistance breeding. In this study, we aimed to refine the reported QTLs and identify stable meta-QTLs (MQTLs) associated with rice blast resistance. A total of 435 QTLs were used to project 71 MQTLs across all the rice chromosomes. As many as 199 putative rice blast resistance genes were identified within 53 MQTL regions. The genes included 48 characterized resistance gene analogs and related proteins, such as NBS–LRR type, LRR receptor-like kinase, NB-ARC domain, pathogenesis-related TF/ERF domain, elicitor-induced defense and proteins involved in defense signaling. MQTL regions with clusters of RGA were also identified. Fifteen highly significant MQTLs included 29 candidate genes and genes characterized for blast resistance, such as Piz, Nbs-Pi9, pi55-1, pi55-2, Pi3/Pi5-1, Pi3/Pi5-2, Pikh, Pi54, Pik/Pikm/Pikp, Pb1 and Pb2. Furthermore, the candidate genes (42) were associated with differential expression (in silico) in compatible and incompatible reactions upon disease infection. Moreover, nearly half of the genes within the MQTL regions were orthologous to those in O. sativa indica, Z. mays and A. thaliana, which confirmed their significance. The peak markers within three significant MQTLs differentiated blast-resistant and susceptible lines and serve as potential surrogates for the selection of blast-resistant lines. These MQTLs are potential candidates for durable and broad-spectrum rice blast resistance and could be utilized in blast resistance breeding.

Abstract:: Grain size plays a significant role in rice, starting from affecting yield to consumer preference, which is the driving force for deep investigation and improvement of grain size characters. Quantitative inheritance makes these traits complex to breed on account of several alleles contributing to the complete trait expression. We employed genome-wide association study in an association panel of 88 rice genotypes using 142 new candidate gene based SSR (cgSSR) markers, derived from yield-related candidate genes, with the efficient mixed-model association coupled mixed linear model for dissecting complete genetic control of grain size traits. A total of 10 significant associations were identified for four grain size-related characters (grain weight, grain length, grain width, and length-width ratio). Among the identified associations, seven marker trait associations explain more than 10% of the phenotypic variation, indicating major putative QTLs for respective traits. The allelic variations at genes OsBC1L4, SHO1 and OsD2 showed association between 1000-grain weight and grain width, 1000-grain weight and grain length, and grain width and length-width ratio, respectively. The cgSSR markers, associated with corresponding traits, can be utilized for direct allelic selection, while other significantly associated cgSSRs may be utilized for allelic accumulation in the breeding programs or grain size improvement. The new cgSSR markers associated with grain size related characters have a significant impact on practical plant breeding to increase the number of causative alleles for these traits through marker aided rice breeding programs.

Abstract High grain number is positively correlated with grain yield in rice, but it is compromised because of poor filling of basal spikelets in dense panicle bearing numerous spikelets. The phenomenon that turns the basal spikelets of compact panicle sterile in rice is largely unknown. In order to understand the factor(s) that possibly determines such spikelet sterility in compact panicle cultivars, QTLs and candidate genes were identified for spikelet fertility and associated traits like panicle compactness, and ethylene production that significantly influences the grain filling using recombinant inbred lines developed from a cross between indica rice cultivars, PDK Shriram (compact, high spikelet number) and Heera (lax, low spikelet number). Novel QTLs, qSFP1.1, qSFP3.1, and qSFP6.1 for spikelet fertility percentage; qIGS3.2 and qIGS4.1 for panicle compactness; and qETH1.2, qETH3.1, and qETH4.1 for ethylene production were consistently identified in both kharif seasons of 2017 and 2018. The comparative expression analysis of candidate genes like ERF3, AP2-like ethylene-responsive transcription factor, EREBP, GBSS1, E3 ubiquitin-protein ligase GW2, and LRR receptor-like serine/threonine-protein kinase ERL1 associated with identified QTLs revealed their role in poor grain filling of basal spikelets in a dense panicle. These candidate genes thus could be important for improving grain filling in compact-panicle rice cultivars through biotechnological interventions.

Ranidhan is a popular late-maturing rice variety of Odisha state, India. The farmers of the state suffer heavy loss in years with flash floods as the variety is sensitive to submergence. Bacterial blight (BB) disease is a major yield-limiting factor, and the variety is susceptible to the disease. BB resistance genes Xa21, xa13, and xa5, along with the Sub1 QTL, for submergence stress tolerance were transferred into the variety using marker-assisted backcross breeding approach. Foreground selection using direct and closely linked markers detected the progenies carrying all four target genes in the BC1F1, BC2F1, and BC3F1 generations, and the positive progenies carrying these genes with maximum similarity to the recipient parent, Ranidhan, were backcrossed into each segregating generation. Foreground selection in the BC1F1 generation progenies detected all target genes in 11 progenies. The progeny carrying all target genes and similar to the recipient parent in terms of phenotype was backcrossed, and a total of 321 BC2F1 seeds were produced. Ten progenies carried all target genes/QTL in the BC2F1 generation. Screening of the BC3F1 progenies using markers detected 12 plants carrying the target genes. A total of 1270 BC3F2 seeds were obtained from the best BC3F1 progeny. Foreground selection in the BC3F2 progenies detected four plants carrying the target genes in the homozygous condition. The bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight pathogen. These BB pyramided lines were submergence-tolerant and similar to Ranidhan in 13 agro-morphologic and grain quality traits; hence, they are likely to be adopted by farmers.

Low light intensity affects several physiological parameters during the different growth stages in rice. Plants have various regulatory mechanisms to cope with stresses. One of them is the differential and temporal expression of genes, which is governed by post-transcriptional gene expression regulation through endogenous miRNAs. To decipher low light stress-responsive miRNAs in rice, miRNA expression profiling was carried out using next-generation sequencing of low-light-tolerant (Swarnaprabha) and -sensitive (IR8) rice genotypes through Illumina sequencing. Swarnaprabha and IR8 were subjected to 25% low light treatment for one day, three days, and five days at the active tillering stage. More than 43 million raw reads and 9 million clean reads were identified in Swarnaprabha, while more than 41 million raw reads and 8.5 million clean reads were identified in IR8 after NGS. Importantly, 513 new miRNAs in rice were identified, whose targets were mostly regulated by the genes involved in photosynthesis and metabolic pathways. Additionally, 114 known miRNAs were also identified. Five novel (osa-novmiR1, osa-novmiR2, osa-novmiR3, osa-novmiR4, and osa-novmiR5) and three known (osa-miR166c-3p, osa-miR2102-3p, and osa-miR530-3p) miRNAs were selected for their expression validation through miRNA-specific qRT-PCR. The expression analyses of most of the predicted targets of corresponding miRNAs show negative regulation. Hence, miRNAs modulated the expression of genes providing tolerance/susceptibility to low light stress. This information might be useful in the improvement of crop productivity under low light stress.

Abstract Background Rice plants show yellowing, stunting, withering, reduced tillering and utimately low productivity in susceptible varieties under low temperature stress. Comparative transcriptome analysis was performed to identify novel transcripts, gain new insights into different gene expression and pathways involved in cold tolerance in rice. Results Comparative transcriptome analyses of 5 treatments based on chilling stress exposure revealed more down regulated genes in susceptible and higher up regulated genes in tolerant genotypes. A total of 13930 and 10599 differentially expressed genes (DEGs) were detected in cold susceptible variety (CSV) and cold tolerant variety (CTV), respectively. A continuous increase in DEGs at 6, 12, 24 and 48 h exposure of cold stress was detected in both the genotypes. Gene ontology (GO) analysis revealed 18 CSV and 28 CTV term significantly involved in molecular function, cellular component and biological process. GO classification showed a significant role of transcription regulation, oxygen, lipid binding, catalytic and hydrolase activity for tolerance response. Absence of photosynthesis related genes, storage products like starch and synthesis of other classes of molecules like fatty acids and terpenes during the stress were noticed in susceptible genotype. However, biological regulations, generation of precursor metabolites, signal transduction, photosynthesis, regulation of cellular process, energy and carbohydrate metabolism were seen in tolerant genotype during the stress. KEGG pathway annotation revealed more number of genes regulating different pathways resulting in more tolerant. During early response phase, 24 and 11 DEGs were enriched in CTV and CSV, respectively in energy metabolism pathways. Among the 1583 DEG transcription factors (TF) genes, 69 WRKY, 46 bZIP, 41 NAC, 40 ERF, 31/14 MYB/MYB-related, 22 bHLH, 17 Nin-like 7 HSF and 4C3H were involved during early response phase. Late response phase showed 30 bHLH, 65 NAC, 30 ERF, 26/20 MYB/MYB-related, 11 C3H, 12 HSF, 86 Nin-like, 41 AP2/ERF, 55 bZIP and 98 WRKY members TF genes. The recovery phase included 18 bHLH, 50 NAC, 31 ERF, 24/13 MYB/MYB-related, 4 C3H, 4 HSF, 14 Nin-like, 31 bZIP and 114 WRKY TF genes. Conclusions Transcriptome analysis of contrasting genotypes for cold tolerance detected the genes, pathways and transcription factors involved in the stress tolerance.

The introgression of wild chromosomal segments into popular rice varieties is one of the potential approaches for developing varieties for drought stress condition. Sixteen genotypes, including nine indica, two tropical japonica and five chromosome segments substitution lines (CSSLs) with different levels of tolerance/susceptibility to drought stress, were selected for diversity study. Sixty-three microsatellite markers were utilized for assessing genetic diversity. A total of 95 alleles were amplified, and out of them, 60 were polymorphic. Six unique alleles, amplified by the microsatellite loci RM276, RM472, RM488, RM537, RM541 and RM28089, were identified in six genotypes, namely FR13A, Brahamanakhi, RUF44, Swarna-sub1, Brahamanakhi and Satyabhama. The highest genetic similarity was found among CSSLs. Polymorphism information content (PIC) value varied from 0 to 1.00 with an average of 0.66 per locus. Twenty-eight microsatellites were found to be polymorphic, which could be used in marker-assisted selection programme. All the sixteen genotypes were grouped into two major clusters at genetic similarity of 0.64. In the cluster I, five CSSLs identified as diverse genotypes had wild ancestor segments responsible for drought tolerance, and hence they could be utilized as potential donors. The popular Indian varieties, Swarna-sub1 and IR64-sub1, could be used as recurrent parents in the future breeding program for developing varieties for abiotic stresses such as submergence and drought. Keywords: Genetic diversity, Marker-assisted selection, Microsatellite marker, Rice, Drought stress, Submergence, Chromosome segment substitution line

The bacterial blight (BB) disease of rice is a major disease that reduces yield heavily in susceptible varieties. Ranidhan is a late maturing popular rice variety but shows high susceptibility to the disease. Two BB resistance genes were transferred into the variety through a marker-assisted backcross breeding approach. Tightly linked molecular markers were deployed to track the BB resistance genes in the plants carrying the target genes in each backcross generation. Foreground screening detected 17, 16 and 15 progenies to carry the 3 BB resistance genes in BC1F1, BC2F1 and BC3F1 generations, respectively. The selected BC3F1 plant was selfed and three different combinations of BB resistance genes were tracked in homozygous state in seven BC3F2 plants. The pyramided lines carrying three resistance genes in homozygous conditions were evaluated for BB disease resistance by inoculating with eight virulent Xoo strains. Five pyramided lines carrying two resistance gene combinations (Xa21+xa13 and Xa21+xa5) exhibited enhanced resistance against the BB pathogens. The disease resistance was in the order of Xa21+xa5 < Xa21+xa13 < xa13+xa5 gene combinations in conferring the resistance. The developed pyramided lines were similar to the recipient parent for the majority of the important agro-morphologic and grain quality traits.

Bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae is a major biotic constraint on obtaining higher grain yields in rice. Marker-assisted backcross breeding (MABB) was performed by the pyramiding of Xa4, xa5, xa13 and Xa21 resistance genes in the popular variety, Ranidhan. A foreground selection in BC1F1, BC2F1, and BC3F1 progenies detected all the target genes in 12, 7 and 16 progenies by using the closely linked markers from a population size of 446, 410, and 530, respectively. The BB-positive progenies carrying the target genes with a maximal similarity to the recipient parent was backcrossed in each backcross generation. A total of 1784 BC3F2 seeds were obtained from the best BC3F1 progeny. The screening of the BC3F2 progenies for the four target genes resulted in eight plants carrying all the four target genes. A bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight disease. In addition, these pyramided lines were similar to Ranidhan in 16 morpho-quality traits, namely, plant height, filled grains/panicle, panicles/plant, grain length, grain breadth, grain weight, milling, head rice recovery, kernel length after cooking, water uptake, the volume expansion ratio, gel consistency, alkali-spreading value, and the amylose content.

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant.

A panel of 60 genotypes comprising New Plant Types (NPTs) along with indica, tropical and temperate japonica genotypes was phenotypically evaluated for four seasons in irrigated situation for grain yield per se and component traits. Twenty NPT genotypes were found promising with an average grain yield varying from 5.45 to 8.8 t/ha. A total of 85 SSR markers were used in the study to identify QTLs associated with grain yield per se and related traits. Sixty-six (77.65%) markers were found to be polymorphic. The PIC values varied from 0.516 to 0.92 with an average of 0.704. A moderate level of genetic diversity (0.39) was detected among genotypes. Variation to the tune of 8% within genotypes, 68% among the genotypes within the population and 24% among the populations were observed (AMOVA). This information may help in identification of potential parents for development of transgressive segregants with very high yield. The association analysis using GLM and MLM models led to the identification of 30 and 10 SSR markers associated with 70 and 16 QTLs, respectively. Thirty novel QTLs linked with 16 SSRs were identified to be associated with eleven traits, namely tiller number (qTL-6.1, qTL-11.1, qTL-4.1), panicle length (qPL-1.1, qPL-5.1, qPL-7.1, qPL-8.1), flag leaf length (qFLL-8.1, qFLL-9.1), flag leaf width (qFLW-6.2, qFLW-5.1, qFLW-8.1, qFLW-7.1), total no. of grains (qTG-2.2, qTG-a7.1), thousand-grain weight (qTGW-a1.1, qTGW-a9.2, qTGW-5.1, qTGW-8.1), fertile grains (qFG-7.1), seed length-breadth ratio (qSlb-3.1), plant height (qPHT-6.1, qPHT-9.1), days to 50% flowering (qFD-1.1) and grain yield per se (qYLD-5.1, qYLD-6.1a, qYLD-11.1).Some of the SSRs were co-localized with more than two traits. The highest co-localization was identified with RM5709 linked to nine traits, followed by RM297 with five traits. Similarly, RM5575, RM204, RM168, RM112, RM26499 and RM22899 were also recorded to be co-localized with more than one trait and could be rated as important for marker-assisted backcross breeding programs, for pyramiding of these QTLs for important yield traits, to produce new-generation rice for prospective increment in yield potentiality and breaking yield ceiling.

A total of 17 simple sequence repeat (SSR) markers linked to QTLs (qDTYs) governing grain yield under reproductive stage (RS) drought stress were used to assess the genetic relationship and prospecting new donors for qDTYs among 32 popular upland rice genotypes. These SSR markers generated a total of 36 alleles with an average allele count of 2.1 per locus. Polymorphic information content value of the markers ranged from 0.376 to 0.662 with an average value of 0.484. The expected heterozyogosity ranged from 0.381 to 0.632. STRUCTURE analysis divided the 32 genotypes into three sub-populations. Subsequent phenotyping revealed that all the tolerant genotypes were grouped into one sub-population, whereas the moderately tolerant and susceptible genotypes were grouped into separate sub-populations. Phylogenetic tree constructed by the unweighted neighbour-joining method also divided the genotypes into three clusters. The grouping pattern of genotypes into the clusters was similar to that into the STRUCTURE analysis, on the basis of drought tolerance level. The average value of genetic dissimilarity coefficient among the genotypes was observed to be 0.486. Furthermore, by combining genotyping data with phenotyping data, 16 new donors for 6 qDTYs were identified. Keywords: drought screening, genetic relationship, population structure, qDTY donor, upland rice, grain yield

Grain number is an important trait for yield in rice. Several genes have been identified controlling grain and panicle number, which has direct or indirect effect on yield. Some genes play a key role for panicle formation and number of panicles per plant. The number of panicles per plant is directly regulating the grain number per plant. In present study, in silico approach was adopted for identifying differentially expressed genes associated with grain number and panicle number. Further, pathway enrichment analysis of these genes performed. The microarray data, GSE51616, downloaded from the GEO database originally submitted by Wang et al. (2014).Young leaves in vegetative stage (35-days old) and developing panicles (0.1cm) from field-grown OX-Ghd7HJ19 transgenic and wild-type plants with two biological replicates were used to isolate RNA for chip analysis. Background correction and normalization of raw microarray data was carried out using the Robust Multichip Averaging (RMA) method of affy packages of R (v. 3.1.3). The linear regression model package, limma was utilized to classify chips into two groups. The Bayes method (Benjamini and Hochberg) was used to correct for multiple testing. Adjusted P-value < 0.01 and |logFC| > 2 was used as a cut-off to identify differentially expressed genes. We identified 393 differentially expressed genes, which mainly belongs to either Phosphatidylethanolamine-binding protein family or DUF3778 domain family. Proteins of these two families regulate formation of high grain number as well as panicle number. These genes like Ghd7, Ehd1, Dep1, Os10g0463400, Os03g0752800, Os03g0215400, Os06g0157700 and Os06g0157500 function to integrate the dynamic environmental inputs with phase transition, architecture regulation, and stress response to maximize the reproductive success of the rice plant. Thus, these genes can act as potential target protein for increasing grain and panicle number in rice plant, which will lead to increase in yield of rice grain.

Panicle number is directly associated with grain number in rice. As the panicle number increases, it affects the total yield of rice. We examined the evolution of genes associated with panicle number formation in Oryza sativa. Intramural program written in JAVA script and fastPHASE software used for the generation of genotype and haplotype file of SNPs of 11 individual genes associated with panicle number formation utilizing VCF file obtained from RiceCAP project (USDA/CSREES http://www.uark.edu/ua/ricecap/).Tests for natural selection executed on these genes using the Haplotype data. Tajima’s D and Fu Li’s D* analysis were performed using DNASP v4.0. Rates of non-synonymous Vs synonymous changes were calculated according to the dN/dS algorithm of Nei and Gojobori.dN/dS calculation compared with the ancestral (Oryza meridionalis) sequence individually showed that out of 11, almost all genes responsible for grain number formation, Os01g0746400, Os03g0203200, Os03g0706500, Os04g0550600, Os06g0127800, Os06g0154200, Os06g0610350, Os06g0660200, Os08g0162100 and Os11g0528700 are negatively selected throughout evolution., Although Tajima’s D was not found significant, the negative value for 8 genes, Os01g0746400, Os03g0123300, Os03g0706500, Os06g0127800, Os06g0610350, Os06g0660200, Os08g0162100 and Os11g0528700 indicated that low frequency variants are more in number than high frequency variants. For Fu Li’s D*, the significantly negative values in most of the genes, Os01g0746400, Os03g0123300, Os03g0706500, Os06g0127800, Os06g0610350, Os08g0162100 and Os11g0528700, indicated that the high frequency variants detected through Tajima’s D are predominantly singletons. Thus, result from dN/dS, Tajima’s D and Fu Li indicated that negative (purifying) selection acts on genes responsible for panicle number formation.These results will be useful for further investigation on how the genes associated with panicle number and how purifying selection result in stabilizing selection through the purging of deleterious variations that arise.