N. Sarla

ISSR markers based on GA and AG repeats reveal genetic relationship among rice varieties tolerant to drought, flood, or salinity

Drought, flood, salinity, or a combination of these limits rice production. Several rice varieties are well known for their tolerance to specific abiotic stresses. We determined genetic relationship among 12 rice varieties including 9 tolerant to drought, flood, or salinity using inter-simple sequence repeat (ISSR) markers. Based on all markers, the nine tolerant varieties formed one cluster distinct from the cluster of three control varieties. The salt-tolerant varieties were closest to two flood-tolerant varieties, and together they were distinct from the drought-tolerant varieties. (GA)8YG was the most informative primer, showing the highest polymorphic information content (PIC) and resolving power (Rp). The drought-, flood-, and salt-tolerant varieties grouped in three distinct clusters within the group of tolerant varieties, when (GA)8YG was used. Sabita was the only exception. The two aus varieties, Nagina22 and FR13A, were separated and grouped with the drought- and flood-tolerant varieties, respectively, but they were together in dendrograms based on other primers. The results show that ISSR markers associated with (GA)8YG delineated the three groups of stress-tolerant varieties from each other and can be used to identify genes/new alleles associated with the three abiotic stresses in rice germplasm.

Mapping and introgression of QTL for yield and related traits in two backcross populations derived from Oryza sativa cv. Swarna and two accessions of O. nivara

Advanced backcross QTL (AB-QTL) analysis was carried out in two Oryza nivara-derived BC2F2 populations. For nine traits, we identified 28 QTL in population 1, and 26 QTL in population 2. The two most significant yield-enhancing QTL, yldp9.1 and yldp2.1, showed an additive effect of 16 and 7 g per plant in population 1, while yld2.1 and yld11.1 showed an additive effect of 11 and 10 g per plant in population 2. At least one O. nivara-derived QTL with a phenotypic variance of more than 15% was detected for seven traits in population 1 and three traits in population 2. The O. nivara-derived QTL ph1.1, nt12.1, nsp1.1, nfg1.1, bm11.1,yld2.1, and yld11.1 were conserved at the same chromosomal locations in both populations. Two major QTL clusters were detected at the marker intervals RM488–RM431 and RM6–RM535 on chromosomes 1 and 2, respectively. The co-location of O. nivara-derived yield QTL with yield meta-QTL on chromosomes 1, 2, and 9 indicates their accuracy and consistency. The major-effect QTL reported in this study are useful for marker-assisted breeding and are also suitable for further fine mapping and candidate gene identification.

Molecular Mapping of QTLs for Yield and Yield-Related Traits in Oryza sativa cv Swarna × O. nivara (IRGC81848) Backcross Population

Advanced backcross QTL analysis was used to identify QTLs for seven yield and yield-related traits in a BC2F2 population from the cross between a popular Oryza sativa cv Swarna and O. nivara IRGC81848. Transgressive segregants with more than 15% increased effect over Swarna were observed for all the traits except days to heading and days to 50% flowering. Thirty QTLs were detected for seven yield and yield-related traits using interval and composite interval mapping. Enhancing alleles at 13 (45%) of these QTLs were derived from O. nivara, and enhancing alleles at all the QTLs for stem diameter and rachis diameter were derived from O. nivara. Three stem diameter QTLs, two rachis diameter QTLs and one number of secondary branches QTL identified by both Interval and composite interval mapping contributed more than 15% of the total phenotypic variance. The QTL epistasis was significant for stem diameter and plot yield. The most significant QTLs qSD7.2, qSD8.1 and qSD9.1 for stem diameter, qRD9.1 for rachis diameter and qNSB1.1 for number of secondary branches are good targets to evaluate their use in marker-assisted selection. O. nivara is a good source of novel alleles for yield related traits and reveals major effect QTLs suitable for marker-assisted selection.

A substitution mutation in OsCCD7 cosegregates with dwarf and increased tillering phenotype in rice

Dwarf plant height and tillering ability are two of the most important agronomic traits that determine the plant architecture, and have profound influence on grain yield in rice. To understand the molecular mechanism controlling these two traits, an EMS-induced recessive dwarf and increased tillering1 (dit1) mutant was characterized. The mutant showed proportionate reduction in each internode as compared to wild type revealing that it belonged to the category of dn-type of dwarf mutants. Besides, exogenous application of GA3 and 24-epibrassinolide, did not have any effect on the phenotype of the mutant. The gene was mapped on the long arm of chromosome 4, identified through positional candidate approach and verified by cosegregation analysis. It was found to encode carotenoid cleavage dioxygenase7 (CCD7) and identified as an allele of htd1. The mutant carried substitution of two nucleotides CC to AA in the sixth exon of the gene that resulted in substitution of serine by a stop codon in the mutant, and thus formation of a truncated protein, unlike amino acid substitution event in htd1. The new allele will facilitate further functional characterization of this gene, which may lead to unfolding of newer signalling pathways involving plant development and architecture.

Hydroponic experiment for identification of tolerance traits developed by rice Nagina 22 mutants to low-phosphorus in field condition

Development of phosphate (P)-deficiency tolerant rice cultivars is constrained by lack of suitable, reproducible, and consistent seedling stage screening methods in breeding programs. This study reports the screening and characterization of M5 mutants derived from an ethyl methane sulfonate treated population of rice cv. Nagina 22 (N22) in low-P field (soil Olsen P 1.94–2.01 mg kg−1; alkaline Vertisol; pH 7.94) for high yield. The present study showed that seedling growth responses such as increase in root weight, root length, root/shoot weight, and dry weight in P-deficient medium can be taken as indices of low-P tolerance in mature plants in field. Total phosphorus content in seedlings showed an inverse relationship with total phosphorus content and low-P tolerance in mature plants in the field. But, phosphorus content in seeds and acid phosphatase activity in the seedling stage were positively correlated with survival and seed set in low-P field. In low-P field, plant height correlated most with yield per plant, and the number of productive tillers in mature plants was highly correlated with tiller number at vegetative stage. These mutants (NH776, NH710, and NH719) have agronomic importance because of their ability to grow and give higher yield than N22 in P-deficient field.

Comparative Study of Susceptible and Tolerant Genotype Reveals Efficient Recovery and Root System Contributes to Heat Stress Tolerance in Rice

In changing climate scenario, heat stress caused by increased atmospheric CO2 is a major concern for rice productivity. There is a need to decipher the mechanisms of heat stress susceptibility and tolerance response of rice cultivars considering that high temperature is detrimental to growth and development of rice crop. The present study was designed to understand the heat stress response in heat-susceptible (Vandana) and heat-tolerant (N22) cultivars of rice. Rice seedlings were subjected to short-duration (24 h, SDS) and long-duration (5 days, LDS) heat stress (42 °C/36 °C, day/night). Besides the heat stress, recovery response (REC) of both the cultivars was also studied. Physiological parameters (chlorophyll content and membrane thermostability) and root/shoot length analysis revealed that N22 has better efficiency in recovering from heat stress. In particular, root tissue of N22 showed increased thermotolerance during SDS and LDS when compared with Vandana. In addition to physiological studies, gene expression pattern of 13 genes including heat shock transcription factors and heat shock proteins and 9 microRNAs (miRNAs) was analyzed in root and shoot of both the genotypes during various treatments. Gene and miRNA expression studies showed that root tissue of N22 was more responsive during SDS and LDS, suggesting important function of roots in heat stress tolerance. Further, during recovery, root tissue of both the genotypes showed more significant change in gene expression than shoot which signifies the vital role of plant root system in heat stress recovery response. Very high expression of an unknown iron-sulfur cluster-binding protein OsFd involved in electron transport activity was observed in root tissue of N22 during all the stress treatments. This study shows that better recovery and efficient root system play an important role in heat tolerance trait of N22.

Prediction and Expression Analysis of miRNAs Associated with Heat Stress in Oryza sativa

Abstract Computational prediction of potential microRNAs (miRNAs) and their target genes was performed to identify the miRNAs and genes associated with temperature response in rice. The data of temperature-responsive miRNAs of Arabidopsis, and miRNAs and the whole genome data of rice were used to predict potential miRNAs in Oryza sativa involved in temperature response. A total of 55 miRNAs were common in both the species, and 27 miRNAs were predicted at the first time in rice. Target genes were searched for these 27 miRNAs in rice genome following stringent criteria. Real time PCR based on expression analysis of nine miRNAs showed that majority of the miRNAs were down regulated under heat stress for rice cultivar Nagina 22. Furthermore, miR169, miR1884 and miR160 showed differential expression in root and shoot tissues of rice. Identification and expression studies of miRNAs during heat stress will advance the understanding of gene regulation under stress in rice.

Field level evaluation of rice introgression lines for heat tolerance and validation of markers linked to spikelet fertility

Rice lines derived from wild species and mutants can serve as a good resource for favorable alleles for heat tolerance. In all, 48 stable lines including 17 KMR3/O. rufipogon introgression lines (KMR3 ILs), 15 Swarna/O. nivara ILs (Swarna ILs) along with their parents, Nagina 22 (N22) and its 4 EMS induced mutants and 7 varieties were evaluated for heat tolerance under irrigated conditions under field in two seasons, wet season 2012 using poly cover house method and dry season 2013 using late sown method. Spikelet fertility (SF), yield per plant (YP) and heat susceptibility index (HSI) for these two traits were considered as criteria to assess heat tolerance compared to control. Four KMR3 ILs and eight Swarna ILs were identified as heat tolerant based on SF and YP and their HSIs in both wet and dry seasons. S-65 and S-70 showed low SF and high YP consistently in response to heat in both seasons. We provide evidence that SF alone may not be the best criterion to assess heat tolerance and including YP is important as lines with low SF but high YP and vice versa were identified under heat stress. Out of 49 SSR markers linked to spikelet fertility, 18 were validated for five traits. RM229 in wet season and RM430 and RM210 in dry season were significantly associated with both SF and its HSI under heat stress. RM430 was also significantly associated with both YP and its HSI in dry season. Thirty two candidate genes were identified close to nine markers associated with traits under heat stress.

Expression profiling of iron deficiency responsive microRNAs and gene targets in rice seedlings of Madhukar x Swarna recombinant inbred lines with contrasting levels of iron in seeds

AimsTo test if microRNAs are involved in iron (Fe) homeostasis in Oryza sativa.MethodsRecombinant inbred lines (RILs) of rice with contrasting levels of iron in seeds (high iron line HL, low iron line LL) and parent Swarna were grown in Fe sufficient (+Fe) and deficient (−Fe) environment. miRNAs whose target genes underlie the QTLs mapped for iron concentration (mapped in our previous study) were identified using bioinformatics. The expression analysis of these miRNAs and their targets along with few other miRNAs involved in nutrient homeostasis was done in root and shoot tissue. Real time PCR was used to study the relative expression of miRNAs and their target genes.ResultsOut of nine miRNAs used in this study, 7 miRNAs-miR156, 168, 172, 162, 167, 171, and 398 showed down-regulation under Fe deficiency in root and shoot of high iron line when compared with Fe sufficient condition. Further, most of the miRNAs showed down-regulation while their target genes showed up-regulation under Fe deficiency in roots of all three genotypes (HL, LL and Swarna) suggesting roots are more responsive to Fe deficiency. Important role of miRNAs in iron homeostasis was analyzed by comparing the expression of these miRNAs in HL, LL and Swarna under + Fe and –Fe.ConclusionMicroRNAs showed differential expression in + Fe and –Fe environment. Further, their expression is more effectively regulated in root under Fe deficiency. This indicates that miRNAs might be playing regulatory roles in iron homeostasis in rice. This study suggests that Fe deficiency responsive miRNAs are involved in cross talk between other nutrients stress.

Transcriptomics of Heat Stress in Plants

High-temperature stress is a major abiotic stress that affects various biological processes of plants such as biochemical and physiological response, growth, development, and yield. High-temperature stress has critical effects at cellular and molecular levels also. The increased concentration of regulatory proteins such as heat shock transcription factors (Hsfs) is a major molecular response that occurs during heat stress. These regulatory proteins in turn regulate the expression of heat shock protein (HSP) genes that act as critical players during stress to maintain cell homeostasis. Besides HSPs, the other metabolic and regulatory genes, signaling compounds, compatible osmolytes, and antioxidants too play an important role during heat stress in plants. Apart from the protein-coding genes, recent studies have shown that noncoding microRNAs (miRNAs) also play a key role during heat stress by modulating the gene expression at the transcription and post-transcriptional level. The transcriptome approaches are important to understand the molecular and cellular changes occurring in response to heat stress. The approaches rely mostly by adopting the traditional methods like Northern blot/RNA blot and reverse transcription PCR (RT-PCR), where the expression of the genes can be studied in different tissues and cells, whereas the extent of their expression can be achieved by quantitative PCR or real time PCR. Further, the genome-wide expression profiling tools such as microarray analysis, next-generation sequencing, and RNA sequencing offer a great potential in this direction. This chapter primarily provides the current understanding on the role of regulatory genes (transcription factors), HSP genes, metabolic genes, signaling compounds, osmolytes, reactive oxygen species, and miRNAs as well as other small RNAs of plants under high temperature. In addition, it gives a brief account of various transcriptome approaches to study the expression profiling of genes during heat stress.