Identification and Functional Characterization of Two Major Loci Associated with Resistance against Brown Planthoppers (Nilaparvata lugens (Stål)) Derived from Oryza nivara.

The brown planthopper (BPH) is a highly destructive pest of rice, causing significant economic losses in various regions of South and Southeast Asia. Researchers have made promising strides in developing resistance against BPH in rice. Introgression line RPBio4918-230S, derived from Oryza nivara, has shown consistent resistance to BPH at both the seedling and adult stages of rice plants. Segregation analysis has revealed that this resistance is governed by two recessive loci, known as bph39(t) and bph40(t), contributing to 21% and 22% of the phenotypic variance, respectively. We later mapped the genes using a backcross population derived from a cross between Swarna and RPBio4918-230S. We identified specific marker loci, namely RM8213, RM5953, and R4M17, on chromosome 4, flanking the bph39(t) and bph40(t) loci. Furthermore, quantitative expression analysis of candidate genes situated between the RM8213 and R4M17 markers was conducted. It was observed that eight genes exhibited up-regulation in RPBio4918-230S and down-regulation in Swarna after BPH infestation. One gene of particular interest, a serine/threonine-protein kinase receptor (STPKR), showed significant up-regulation in RPBio4918-230S. In-depth sequencing of the susceptible and resistant alleles of STPKR from Swarna and RPBio4918-230S, respectively, revealed numerous single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) mutations, both in the coding and regulatory regions of the gene. Notably, six of these mutations resulted in amino acid substitutions in the coding region of STPKR (R5K, I38L, S120N, T319A, T320S, and F348S) when compared to Swarna and the reference sequence of Nipponbare. Further validation of these mutations in a set of highly resistant and susceptible backcross inbred lines confirmed the candidacy of the STPKR gene with respect to BPH resistance controlled by bph39(t) and bph40(t). Functional markers specific for STPKR have been developed and validated and can be used for accelerated transfer of the resistant locus to elite rice cultivars.

Mapping QTLs for yield and photosynthesis-related traits in three consecutive backcross populations of Oryza sativa cultivar Cottondora Sannalu (MTU1010) and Oryza rufipogon.

MAIN CONCLUSION: Identification of trait enhancing QTLs for yield and photosynthesis-related traits in rice using interspecific mapping population and chromosome segment substitution lines derived from a cross between Oryza sativa and Oryza rufipogon. Wild rice contains novel genes which can help in improving rice yield. Common wild rice Oryza rufipogon is a known source for enhanced photosynthesis and yield-related traits. We developed BC2F2:3:4 mapping populations using O. rufipogon IC309814 with high photosynthetic rate as donor, and elite cultivar MTU1010 as recurrent parent. Evaluation of 238 BC2F2 families for 13 yield-related traits and 208 BC2F2 families for seven photosynthesis-related physiological traits resulted in identification of significantly different lines which performed better than MTU1010 for various yield contributing traits. 49 QTLs were identified for 13 yield traits and 7 QTLs for photosynthesis-related traits in BC2F2. In addition, 34 QTLs in BC2F3 and 26 QTLs in BC2F4 were also detected for yield traits.11 common QTLs were identified in three consecutive generations and their trait-increasing alleles were derived from O. rufipogon. Significantly, one major effect common QTL qTGW3.1 for thousand grain weight with average phenotypic variance 8.1% and one novel QTL qBM7.1 for biomass were identified. Photosynthesis-related QTLs qPN9.1, qPN12.1, qPN12.2 qSPAD1.1 and qSPAD6.1 showed additive effect from O. rufipogon. A set of 145 CSSLs were identified in BC2F2 which together represented 87% of O. rufipogon genome. In addition, 87 of the 145 CSSLs were significantly different than MTU1010 for at least one trait. The major effect QTLs can be fine mapped for gene discovery. CSSLs developed in this study are a good source of novel alleles from O. rufipogon in the background of Cottondora Sannalu for rapid improvement of any trait in rice.

Whole-Genome Sequencing of KMR3 and Oryza rufipogon-Derived Introgression Line IL50-13 (Chinsurah Nona 2/Gosaba 6) Identifies Candidate Genes for High Yield and Salinity Tolerance in Rice.

The genomes of an elite rice restorer line KMR3 (salinity-sensitive) and its salinity-tolerant introgression line IL50-13, a popular variety of coastal West Bengal, India, were sequenced. High-quality paired-end reads were obtained for KMR3 (147.6 million) and IL50-13 (131.4 million) with a sequencing coverage of 30X-39X. Scaffolds generated from the pre-assembled contigs of each sequenced genome were mapped separately onto the reference genome of Oryza sativa ssp. japonica cultivar Nipponbare to identify genomic variants in terms of SNPs and InDels. The SNPs and InDels identified for KMR3 and IL50-13 were then compared with each other to identify polymorphic SNPs and InDels unique and common to both the genomes. Functional enrichment analysis of the protein-coding genes with unique InDels identified GO terms involved in protein modification, ubiquitination, deubiquitination, peroxidase activity, and antioxidant activity in IL50-13. Linoleic acid metabolism, circadian rhythm, and alpha-linolenic acid metabolism pathways were enriched in IL50-13. These GO terms and pathways are involved in reducing oxidative damage, thus suggesting their role in stress responses. Sequence analysis of QTL markers or genes known to be associated with grain yield and salinity tolerance showed polymorphism in 20 genes, out of which nine were not previously reported. These candidate genes encoded Nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC) domain-containing protein, cyclase, receptor-like kinase, topoisomerase II-associated protein PAT1 domain-containing protein, ion channel regulatory protein, UNC-93 domain-containing protein, subunit A of the heteromeric ATP-citrate lyase, and three conserved hypothetical genes. Polymorphism was observed in the coding, intron, and untranslated regions of the genes on chromosomes 1, 2, 4, 7, 11, and 12. Genes showing polymorphism between the two genomes were considered as sequence-based new candidates derived from Oryza rufipogon for conferring high yield and salinity tolerance in IL50-13 for further functional studies.

Mapping of QTLs for Yield Traits Using F2:3:4 Populations Derived From Two Alien Introgression Lines Reveals qTGW8.1 as a Consistent QTL for Grain Weight From Oryza nivara.

Wild introgressions play a crucial role in crop improvement by transferring important novel alleles and broadening allelic diversity of cultivated germplasm. In this study, two stable backcross alien introgression lines 166s and 14s derived from Swarn/Oryza nivara IRGC81848 were used as parents to generate populations to map quantitative trait loci (QTLs) for yield-related traits. Field evaluation of yield-related traits in F2, F3, and F4 population was carried out in normal irrigated conditions during the wet season of 2015 and dry seasons of 2016 and 2018, respectively. Plant height, tiller number, productive tiller number, total dry matter, and harvest index showed a highly significant association to single plant yield in F2, F3, and F4. In all, 21, 30, and 17 QTLs were identified in F2, F2:3, and F2:4, respectively, for yield-related traits. QTLs qPH6.1 with 12.54% phenotypic variance (PV) in F2, qPH1.1 with 13.01% PV, qTN6.1 with 10.08% PV in F2:3, and qTGW6.1 with 15.19% PV in F2:4 were identified as major effect QTLs. QTLs qSPY4.1 and qSPY6.1 were detected for grain yield in F2 and F2:3 with PV 8.5 and 6.7%, respectively. The trait enhancing alleles of QTLs qSPY4.1, qSPY6.1, qPH1.1, qTGW6.1, qTGW8.1, qGN4.1, and qTDM5.1 were from O. nivara. QTLs of the yield contributing traits were found clustered in the same chromosomal region. qTGW8.1 was identified in a 2.6 Mb region between RM3480 and RM3452 in all three generations with PV 6.1 to 9.8%. This stable and consistent qTGW8.1 allele from O. nivara can be fine mapped for identification of causal genes. From this population, lines C212, C2124, C2128, and C2143 were identified with significantly higher SPY and C2103, C2116, and C2117 had consistently higher thousand-grain weight values than both the parents and Swarna across the generations and are useful in gene discovery for target traits and further crop improvement.

Detecting CSSLs and yield QTLs with additive, epistatic and QTL×environment interaction effects from Oryza sativa × O. nivara IRGC81832 cross.

Chromosome segment substitution lines (CSSLs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action and interaction in inter-specific crosses. In this study, a set of 90 back cross lines at BC2F8 generation derived from Swarna x Oryza nivara IRGC81832 was evaluated for yield traits under irrigated conditions in wet seasons of 3 consecutive years. We identified a set of 70 chromosome segment substitution lines, using genotyping data from 140 SSR markers covering 94.4% of O. nivara genome. Among these, 23 CSSLs were significantly different for 7 traits. 22 QTLs were detected for 11 traits with 6.51 to 46.77% phenotypic variation in 90 BILs. Three pleiotropic genomic regions associated with yield traits were mapped on chromosomes 1, 8 and 11. The marker interval RM206-RM144 at chromosome 11 was recurrently detected for various yield traits. Ten QTLs were identified consistently in the three consecutive years of testing. Seventeen pairs of significant epistatic QTLs (E-QTLs) were detected for days to flowering, days to maturity and plant height. Chromosome segments from O. nivara contributed trait enhancing alleles. The significantly improved lines and the stable QTLs identified in this study are valuable resource for gene discovery and yield improvement.

Genome-wide transcriptome profile of rice hybrids with and without Oryza rufipogon introgression reveals candidate genes for yield.

In this study, we compared genome-wide transcriptome profile of two rice hybrids, one with (test hybrid IR79156A/IL50-13) and the other without (control hybrid IR79156A/KMR3) O. rufipogon introgressions to identify candidate genes related to grain yield in the test hybrid. IL50-13 (Chinsurah Nona2 IET21943) the male parent (restorer) used in the test hybrid, is an elite BC4F8 introgression line of KMR3 with O. rufipogon introgressions. We identified 2798 differentially expressed genes (DEGs) in flag leaf and 3706 DEGs in panicle. Overall, 78 DEGs were within the major yield QTL qyld2.1 and 25 within minor QTL qyld8.2. The DEGs were significantly (p < 0.05) enriched in starch synthesis, phenyl propanoid pathway, ubiquitin degradation and phytohormone related pathways in test hybrid compared to control hybrid. Sequence analysis of 136 DEGs from KMR3 and IL50-13 revealed 19 DEGs with SNP/InDel variations. Of the 19 DEGs only 6 showed both SNP and InDel variations in exon regions. Of these, two DEGs within qyld2.1, Phenylalanine ammonia- lyase (PAL) (Os02t0626400-01, OsPAL2) showed 184 SNPs and 11 InDel variations and Similar to phenylalanine ammonia- lyase (Os02t0627100-01, OsPAL4) showed 205 SNPs and 13 InDel variations. Both PAL genes within qyld2.1 and derived from O. rufipogon are high priority candidate genes for increasing grain yield in rice.

Phenotyping and Genotype × Environment Interaction of Resistance to Leaffolder, Cnaphalocrocis medinalis Guenee (Lepidoptera: Pyralidae) in Rice.

Rice leaffolder, Cnaphalocrocis medinalis is one of the key foliage feeding insects of great concern throughout Asia as it results in significant yield losses. High visibility of damage is triggering farmers to apply toxic pesticides for its management. Therefore, it is vital to identify new stable sources of resistance for leaffolder. Phenotyping of 160 recombinant inbred lines (RILs) of a cross between a resistant parent, W1263 and a susceptible parent, TN1 using a rapid field screening method for three seasons resulted in identification of nine RILs as stable sources of resistance to rice leaffolder. Phenotypic frequency distributions were found continuous indicating that the resistance is a quantitative trait governed by polygenes. Phenotypic data for three seasons were analyzed using Genotype and Genotype × Environment Interaction (GGE) analysis for identification of stable resistant lines. Additive main effect and multiplicative interaction (AMMI) analysis showed that 86.41% of the total sum of square of damaged leaf area was attributed to genotype (GEN) effect; 0.48% to environment (ENV) effects and 5.68% to genotype by environment (G × E) interaction effects. Damage area, damage score and leaf length showed very high broad-sense heritability across three environments. However, leaf width had low heritability indicating higher environment influence. Phylogenetic analysis grouped these 160 RILs and parents into five clusters based on resistant reaction. AMMI and GGE biplot analysis revealed that stable genotypes G8 (MP114) and G3 (MP108) with lower damage area and damage score can be utilized in developing cultivars with leaffolder resistance.

Development and use of chromosome segment substitution lines as a genetic resource for crop improvement.

KEY MESSAGE: CSSLs are a complete library of introgression lines with chromosomal segments of usually a distant genotype in an adapted background and are valuable genetic resources for basic and applied research on improvement of complex traits. Chromosome segment substitution lines (CSSLs) are genetic stocks representing the complete genome of any genotype in the background of a cultivar as overlapping segments. Ideally, each CSSL has a single chromosome segment from the donor with a maximum recurrent parent genome recovered in the background. CSSL development program requires population-wide backcross breeding and genome-wide marker-assisted selection followed by selfing. Each line in a CSSL library has a specific marker-defined large donor segment. CSSLs are evaluated for any target phenotype to identify lines significantly different from the parental line. These CSSLs are then used to map quantitative trait loci (QTLs) or causal genes. CSSLs are valuable prebreeding tools for broadening the genetic base of existing cultivars and harnessing the genetic diversity from the wild- and distant-related species. These are resources for genetic map construction, mapping QTLs, genes or gene interactions and their functional analysis for crop improvement. In the last two decades, the utility of CSSLs in identification of novel genomic regions and QTL hot spots influencing a wide range of traits has been well demonstrated in food and commercial crops. This review presents an overview of how CSSLs are developed, their status in major crops and their use in genomic studies and gene discovery.

Increased Catalase Activity and Maintenance of Photosystem II Distinguishes High-Yield Mutants From Low-Yield Mutants of Rice var. Nagina22 Under Low-Phosphorus Stress.

An upland rice variety, Nagina22 (N22) and its 137 ethyl methanesulfonate (EMS)-induced mutants, along with a sensitive variety, Jaya, was screened both in low phosphorus (P) field (Olsen P 1.8) and in normal field (Olsen P 24) during dry season. Based on the grain yield (YLD) of plants in normal field and plants in low P field, 27 gain of function (high-YLD represented as hy) and 9 loss of function (low-YLD represented as ly) mutants were selected and compared with N22 for physiological and genotyping studies. In low P field, hy mutants showed higher P concentration in roots, leaves, grains, and in the whole plant than in ly mutants at harvest. In low P conditions, F v/F m and qN were 24% higher in hy mutants than in ly mutants. In comparison with ly mutants, the superoxide dismutase (SOD) activity in the roots and leaves of hy mutants in low P fields was 9% and 41% higher at the vegetative stage, respectively, but 51% and 14% lower in the roots and leaves at the reproductive stage, respectively. However, in comparison with ly mutants, the catalase (CAT) activity in the roots and leaves of hy mutants in low P fields was 35% higher at the vegetative stage and 15% and 17% higher at the reproductive stage, respectively. Similarly, hy mutants in low P field showed 20% and 80% higher peroxidase (POD) activity in the roots and leaves at the vegetative stage, respectively, but showed 14% and 16% lower POD activity at the reproductive stage in the roots and leaves, respectively. Marker trait association analysis using 48 simple sequence repeat (SSR) markers and 10 Pup1 gene markers showed that RM3648 and RM451 in chromosome 4 were significantly associated with grain YLD, tiller number (TN), SOD, and POD activities in both the roots and leaves in low P conditions only. Similarly, RM3334 and RM6300 in chromosome 5 were associated with CAT activity in leaves in low P conditions. Notably, grain YLD was positively and significantly correlated with CAT activity in the roots and shoots, F v/F m and qN in low P conditions, and the shoots' P concentration and qN in normal conditions. Furthermore, CAT activity in shoots was positively and significantly correlated with TN in both low P and normal conditions. Thus, chromosomal regions and physiological traits that have a role in imparting tolerance to low P in the field were identified.

Characterization of backcross introgression lines derived from Oryza nivara accessions for photosynthesis and yield.

Improvement of photosynthetic traits is a promising strategy to break the yield potential barrier of major food crops. Leaf photosynthetic traits were evaluated in a set of high yielding Oryza sativa, cv. Swarna × Oryza nivara backcross introgression lines (BILs) along with recurrent parent Swarna, both in wet (Kharif) and dry (Rabi) seasons in normal irrigated field conditions. Net photosynthesis (P N) ranged from 15.37 to 23.25 µmol (CO2) m-2 s-1 in the BILs. Significant difference in P N was observed across the seasons and genotypes. Six BILs showed high photosynthesis compared with recurrent parent in both seasons. Chlorophyll content showed minimum variation across the seasons for any specific BIL but significant variation was observed among BILs. Significant positive association between photosynthetic traits and yield traits was observed, but this association was not consistent across seasons mainly due to contrasting weather parameters in both seasons. BILs 166s and 248s with high and consistent photosynthetic rate exhibited stable high yield levels in both the seasons compared to the recurrent parent Swarna. There is scope to exploit photosynthetic efficiency of wild and weedy rice to identify genes for improvement of photosynthetic rate in cultivars.

Down regulation of a heavy metal transporter gene influences several domestication traits and grain Fe-Zn content in rice.

Biofortification of rice (Oryza sativa L.) would alleviate iron and zinc deficiencies in the target populations. We identified two alleles 261 and 284 of a Gramineae-specific heavy metal transporter gene OsHMA7 by analyzing expression patterns and sequences of genes within QTLs for high Fe & Zn, in Madhukar x Swarna recombinant inbred lines (RILs) with high (HL) or low (LL) grain Fe & Zn. Overexpression of 261 allele increased grain Fe and Zn but most of the transgenic plants either did not survive or did not yield enough seeds and could not be further characterized. Knocking down expression of OsHMA7 by RNAi silencing of endogenous gene resulted in plants with altered domestication traits such as plant height, tiller number, panicle size and architecture, grain color, shape, size, grain shattering, heading date and increased sensitivity to Fe and Zn deficiency. However, overexpression of 284 allele resulted in transgenic lines with either high grain Fe & Zn content (HL-ox) and tolerance to Fe and Zn deficiency or low grain Fe & Zn content (LL-ox) and phenotype similar to RNAi-lines. OsHMA7 transcript levels were five-fold higher in the HL-ox plants whereas LL-ox and RNAi plants showed 2-3 fold reduced levels compared to Kitaake control. Spraying LL-ox and RNAi lines with Fe & Zn at grain filling stage resulted in increased grain yield, significant increase in Fe & Zn content and brown pericarp. Altered expression of OsHMA7 influenced transcript levels of iron-responsive genes indicating cellular Fe-Zn homeostasis and also several domestication-related genes in rice. Our study shows that a novel heavy metal transporter gene influences yield and grain Fe & Zn content and has potential to improve rice production and biofortification.

Genotype × Environment interactions of Nagina22 rice mutants for yield traits under low phosphorus, water limited and normal irrigated conditions.

Multi environment testing helps identify stable genotypes especially for adverse abiotic stress situations. In the era of climate change and multiple abiotic stresses, it becomes important to analyze stability of rice lines under both irrigated and stress conditions. Mutants are an important genetic resource which can help in revealing the basis of natural variation. We analyzed 300 EMS induced mutants of aus rice cultivar Nagina22 (N22) for their G × E interaction and stability under low phosphorus (P), water limited and irrigated conditions. Environmental effect and interaction were more significant than genotypic contribution on grain yield (GY), productive tillers (TN) and plant height (PH) under these three environmental conditions in dry season, 2010. GY and TN were more affected by low P stress than by water limited condition, but PH was not significantly different under these two stresses. Mutants G17, G209, G29, G91, G63 and G32 were stable for GY in decreasing order of stability across the three environments but G254 and G50 were stable only in low P, G17 and G45 only in water limited and G295 and G289 only in normal irrigated condition. We then selected and evaluated 3 high yielding mutants, 3 low yielding mutants and N22 for their stability and adaptability to these 3 environments in both wet and dry seasons for six years (2010-2015). The most stable lines based on the combined analysis of 12 seasons were G125 (NH210) under normal condition, G17 (NH686), G176 (NH363) and G284 (NH162) in low P condition and G176 (NH363) under water limited condition. G176 was the best considering all 3 conditions. When screened for 15 Pup1 gene-specific markers, G176 showed alleles similar to N22. While two other low-P tolerant lines G17 and G65 showed N22 similar alleles only at k-1 and k-5 but a different allele or null allele at 13 other loci. These stable mutants are a valuable resource for varietal development and to discover genes for tolerance to multiple abiotic stresses.

Whole Genome Characterization of a Few EMS-Induced Mutants of Upland Rice Variety Nagina 22 Reveals a Staggeringly High Frequency of SNPs Which Show High Phenotypic Plasticity Towards the Wild-Type.

The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1-2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22).

Characterization of the loss-of-function mutant NH101 for yield under phosphate deficiency from EMS-induced mutants of rice variety Nagina22.

In earlier studies at IIRR, Hyderabad, screening of ∼2000 EMS mutants of the rice variety Nagina22 (N22) resulted in the identification of 11 loss-of-function mutants with zero grain yield in Pi-deprived soil under field condition. Among these mutants, NH101 was selected for comparative analyses with N22 for various morphophysiological and/or molecular traits during growth in a hydroponic system (7 d) and in a pot soil (50% flowering) under different Pi regime. The total length of the seminal and adventitious roots, agronomic traits (panicle length and unfilled spikelet/panicle), activities of the antioxidant enzymes (SOD, POD, and APX), and the relative expression levels of the genes involved in the maintenance of Pi homeostasis (MPH) i.e., OsPHR2, SPX1/2 OsPT4, 6, and 8 showed significant increase in the Pi-deprived mutant compared with N22. Whereas, some of the traits showed significant reduction in NH101 than N22 such as number of tillers and filled spikelets/panicle, yield, contents of Pi and externally secreted APase, activity of CAT, and the relative expression levels of MPH genes i.e., OsmiR399a, OsPHO1;2, OsIPS1, OsPAP10a, OsPT2, 9, and 10. The study highlighted wide spectrum differential effects of the mutation in NH101 on various traits that play important roles governing the maintenance of Pi homeostasis. This mutant thus provides a rich repository of genetic material amenable for the identification of the genes that are pivotal for Pi use efficiency.

Trait and Marker Associations in Oryza nivara and O. rufipogon Derived Rice Lines under Two Different Heat Stress Conditions.

Wild species and derived introgression lines (ILs) are a good source of genes for improving complex traits such as heat tolerance. The effect of heat stress on 18 yield traits was studied in four treatments in two seasons, under field conditions by subjecting 37 ILs and recurrent parents Swarna and KMR3, N22 mutants, and wild type and 2 improved rice cultivars to heat stress using polycover house method in wet season and late sowing method in dry season. Normal grown unstressed plants were controls. Both correlation and path coefficient analysis showed that the major contributing traits for high yield per plant (YPP) under heat stress conditions were tiller number, secondary branches in panicle, filled grain number, and percent spikelet fertility. Three ILs, K-377-24, K-16-3, and S-148 which gave the highest YPP of 12.30-32.52 g under heat stress in both the seasons were considered the most heat tolerant. In contrast, K-363-12, S-75, and Vandana which gave the least YPP of 5.36-10.84 g were considered heat susceptible. These lines are a good genetic resource for basic and applied studies on heat tolerance in rice. Genotyping using 49 SSR markers and single marker analysis (SMA) revealed 613 significant marker- trait associations in all four treatments. Significantly, nine markers (RM243, RM517, RM225, RM518, RM525, RM195, RM282, RM489, and RM570) on chromosomes 1, 2, 3, 4, 6, and 8 showed association with six traits (flag leaf spad, flag leaf thickness, vegetative leaf temperature, plant height, panicle number, and tiller number) under heat stress conditions in both wet and dry seasons. Genes such as heat shock protein binding DnaJ, Hsp70, and temperature-induced lipocalin-2 OsTIL-2 close to these markers are candidates for expression studies and evaluation for use in marker assisted selection for heat tolerance.

Identification of Major Effect QTLs for Agronomic Traits and CSSLs in Rice from Swarna/Oryza nivara Derived Backcross Inbred Lines.

Backcross inbred lines (BILs) derived from elite x wild crosses are very useful for basic studies and breeding. The aim of this study was to map quantitative trait loci (QTLs) associated with yield and related traits and to identify chromosomal segment substitution lines (CSSLs) from unselected BC2F8 BILs of Swarna/Oryza nivara IRGC81848. In all, 94 BILs were field evaluated in 2 years (wet seasons, 2014 and 2015) for nine traits; days to 50% flowering, days to maturity (DM), plant height (PH), number of tillers, number of productive tillers, panicle weight, yield per plant, bulk yield, and biomass. BILs were genotyped using 111 polymorphic simple sequence repeats distributed across the genome. Fifteen QTLs including 10 novel QTLs were identified using composite interval mapping, Inclusive composite interval mapping and multiple interval mapping (MIM). O. nivara alleles were trait-enhancing in 26% of QTLs. Only 3 of 15 QTLs were also reported previously in BC2F2 of the same cross. These three included the two major effect QTLs for DM and PH detected in both years with 13 and 20% phenotypic variance. Further, a set of 74 CSSLs was identified using CSSL Finder and 22 of these showed significantly higher values than Swarna for five yield traits. CSSLs, 220S for panicle weight and 10-2S with consistent high yield in both years are worthy of large scale field evaluation. The major QTLs and 22 significantly different CSSLs are a useful resource for rice improvement and dissecting yield related traits.

Genome-wide changes in microRNA expression during short and prolonged heat stress and recovery in contrasting rice cultivars.

MicroRNAs (miRNAs) are known to regulate expression of genes under stress. We report here the deep sequencing of small RNAs expressed during control, short and prolonged heat stress and recovery. Genome-wide identification of miRNAs in tolerant (Nagina 22) and susceptible (Vandana) rice cultivars was performed in 16 samples representing root and shoot of 13-day-old seedlings. The expression profile of miRNAs was analysed in 36 pairwise combinations to identify the genotype-, treatment- and tissue-dependent expression of miRNAs. Small-RNA sequencing of 16 libraries yielded ~271 million high-quality raw sequences; 162 miRNA families were identified. The highly expressed miRNAs in rice tissues were miR166, miR168, miR1425, miR529, mR162, miR1876, and miR1862. Expression of osa-miR1436, osa-miR5076, osa-miR5161, and osa-miR6253 was observed only in stressed tissue of both genotypes indicating their general role in heat stress response. Expression of osa-miR1439, osa-miR1848, osa-miR2096, osa-miR2106, osa-miR2875, osa-miR3981, osa-miR5079, osa-miR5151, osa-miR5484, osa-miR5792, and osa-miR5812 was observed only in Nagina 22 during high temperature, suggesting a specific role of these miRNAs in heat stress tolerance. This study provides details of the repertoire of miRNAs expressed in root and shoot of heat susceptible and tolerant rice genotypes under heat stress and recovery.

Genotype × Environment Interactions of Yield Traits in Backcross Introgression Lines Derived from Oryza sativa cv. Swarna/Oryza nivara.

Advanced backcross introgression lines (BILs) developed from crosses of Oryza sativa var. Swarna/O. nivara accessions were grown and evaluated for yield and related traits. Trials were conducted for consecutive three seasons in field conditions in a randomized complete block design with three replications. Data on yield traits under irrigated conditions were analyzed using the Additive Main Effect and Multiplicative Interaction (AMMI), Genotype and Genotype × Environment Interaction (GGE) and modified rank-sum statistic (YSi) for yield stability. BILs viz., G3 (14S) and G6 (166S) showed yield stability across the seasons along with high mean yield performance. G3 is early in flowering with high yield and has good grain quality and medium height, hence could be recommended for most of the irrigated locations. G6 is a late duration genotype, with strong culm strength, high grain number and panicle weight. G6 has higher yield and stability than Swarna but has Swarna grain type. Among the varieties tested DRRDhan 40 and recurrent parent Swarna showed stability for yield traits across the seasons. The component traits thousand grain weight, panicle weight, panicle length, grain number and plant height explained highest genotypic percentage over environment and interaction factors and can be prioritized to dissect stable QTLs/ genes. These lines were genotyped using microsatellite markers covering the entire rice genome and also using a set of markers linked to previously reported yield QTLs. It was observed that wild derived lines with more than 70% of recurrent parent genome were stable and showed enhanced yield levels compared to genotypes with higher donor genome introgressions.

Cytokinin delays dark-induced senescence in rice by maintaining the chlorophyll cycle and photosynthetic complexes.

The phytohormone cytokinin (CK) is known to delay senescence in plants. We studied the effect of a CK analog, 6-benzyl adenine (BA), on rice leaves to understand the possible mechanism by which CK delays senescence in a drought- and heat-tolerant rice cultivar Nagina22 (N22) using dark-induced senescence (DIS) as a surrogate for natural senescence of leaves. Leaves of N22-H-dgl162, a stay-green mutant of N22, and BA-treated N22 showed retention of chlorophyll (Chl) pigments, maintenance of the Chl a/b ratio, and delay in reduction of both photochemical efficiency and rate of oxygen evolution during DIS. HPLC analysis showed accumulation of 7-hydroxymethyl chlorophyll (HmChl) during DIS, and the kinetics of its accumulation correlated with progression of senescence. Transcriptome analysis revealed that several plastid-localized genes, specifically those associated with photosystem II (PSII), showed higher transcript levels in BA-treated N22 and the stay-green mutant leaves compared with naturally senescing N22 leaves. Real-time PCR analyses showed that genes coding for enzymes associated with Chl a/b interconversion and proteins associated with light-harvesting complexes maintained higher transcript levels up to 72h of DIS following BA treatment. The pigment-protein complexes analyzed by green gel remained intact in both N22-H-dgl162 and BA-treated N22 leaves even after 96h of DIS. Thus, CK delays senescence by accumulation of HmChl and up-regulating genes in the Chl cycle, thereby maintaining the Chl a/b ratio. Also, CK treatment retains higher transcript levels of PSII-related genes, resulting in the stability of photosynthetic pigment complexes and functional stay-greenness in rice.

Genetic characterization and population structure of Indian rice cultivars and wild genotypes using core set markers.

Genetic diversity among 23 rice genotypes including wild species and cultivars of indica, japonica, aus and aromatic type was investigated using 165 genomewide core set microsatellite (SSR) markers. This genotypic characterization was undertaken to know the genetic similarity among the parental lines to be used in developing a set of chromosome segment substitution lines. In all, 253 alleles were identified using 77 polymorphic SSRs, and polymorphism information content ranged from 0.31 to 0.97 with a mean of 0.79. Cluster analysis grouped the genotypes into three clusters at a genetic similarity of 0.26-0.75. Wild accessions grouped together in cluster-I, indica cultivars formed cluster-II, and aromatic, japonica and aus types came under cluster-III. Principal component analysis also showed similar results. The genotypic data was analyzed using STRUCTURE, and genotypes were grouped into four populations. RM1018 on chromosome 4, RM8009 on chromosome 7, and RM273 on chromosome 12 amplified alleles specific to wild accessions. The information obtained from core set markers would help in selecting diverse parents including wild accessions and for tracking alleles in mapping or breeding populations.