Surekha Agarwal

Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar×Swarna RILs.

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from Madhukar×Swarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224 ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified.

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains

BACKGROUND Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe+ and Fe- medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe- conditions. RESULTS HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe- condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna. CONCLUSION HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.

MicroRNAs and Their Role in Salt Stress Response in Plants

MicroRNAs (miRNAs) are a subset of endogenous approximate 22 nucleotide (nt) small non-coding regulatory RNA molecules that regulate gene expression post-transcriptionally, by mediating mRNA degradation or translational repression in a sequence specific manner. These small regulatory molecules are involved in regulating the intrinsic normal growth of cells and development of organisms as well as in maintaining the integrity of genomes. The plant miRNA research gained momentum, 2002 onwards, which was accompanied by the discovery of plant proteins involved in miRNA biogenesis. Early discovery of miRNAs has been implicated in the regulation of developmental processes. Since then much has been discovered about their involvement in plant responses to adverse environmental conditions, including abiotic stress. Various approaches of miRNAs discovery such as cloning, deep sequencing and prediction using bioinformatic tools have been adapted to learn more about the miRNA expression patterns during stress. The master regulators such as miRNAs having important role in salt stress response are very much crucial to understand the molecular regulation of stress adaptation. Many target genes of miRNAs encode transcription factors, each of which further regulates a set of downstream genes and affect physiological responses. This chapter contains a concise account on historical importance of miRNAs discovery. The miRNA biogenesis pathway and the associated proteins are also discussed along with the tools of miRNAs prediction and identification. In addition, the role of plant miRNAs and their target in plant metabolism and in particular salt stress is elaborated. With the growing knowledge on salt responsive miRNAs, the efforts to develop salt stress tolerance using miRNAs are also given.

RNAi: Machinery and Role in Pest and Disease Management

RNA interference (RNAi) is a homology-dependent gene silencing technology that is initiated by double stranded RNA (dsRNA). A multitude of small RNAs accumulate in plant tissues, although heterogeneous in size, sequence, genomic distribution, biogenesis, and action, most of these molecules mediate repressive gene regulation through RNA silencing. Micro and small interfering RNAs represent small RNA families that are recognized as critical regulatory species across the eukaryotes. Besides their roles in developmental patterning and maintenance of genome integrity, small RNAs are also integral components of plant responses to adverse environmental conditions, including biotic stress. Recent studies broaden the role of RNAi, and many successful examples have described the application of RNAi for engineering plant resistance against a range of prokaryotic and eukaryotic organisms. Expression of dsRNA directed against suitable pathogen and insect genes in transgenic plants showed protection against pests, opening the way for a new generation of pest and disease resistant crops. Here, current knowledge on the uptake mechanisms of dsRNA in plant pests and the potential of RNAi to control pest and pathogen is described. Concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in pest and disease management have been discussed. Further, the progress of RNAi-based transgenic plant resistance against eukaryotic pests, as well as future challenges and prospects are addressed.

Genetic variation of coat protein gene among the isolates of Rice tungro spherical virus from tungro-endemic states of the India

Rice tungro disease, one of the major constraints to rice production in South and Southeast Asia, is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). The present study was undertaken to determine the genetic variation of RTSV population present in tungro endemic states of Indian subcontinent. Phylogenetic analysis based on coat protein sequences showed distinct divergence of Indian RTSV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Coimbatore (Tamil Nadu), and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic study were further supported with the SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. In addition, sequence difference count matrix revealed 2–68 nucleotides differences among all the Indian RTSV isolates taken in this study. However, at the protein level these differences were not significant as revealed by Ka/Ks ratio calculation. Sequence identity at nucleotide and amino acid level was 92–100% and 97–100%, respectively, among Indian isolates of RTSV. Understanding of the population structure of RTSV from tungro endemic regions of India would potentially provide insights into the molecular diversification of this virus.

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.

Quantitative trait loci and candidate genes for yield and related traits in Madhukar x Swarna RIL population of rice

Yield of popular rice varieties such as Swarna grown in rainfed lowlands and Madhukar grown in flood prone areas needs to be continuously improved. Recombinant inbred lines (RILs) were developed from the cross between two indica cultivars Madhukar and Swarna. QTLs were mapped using 110 markers in 168 RILs. In all, 26 QTLs were mapped for yield and five related traits on chromosomes 1, 2, 3, 6, 7, 8, 10, 11, and 12. QTL for plant height and days to flowering were co-located between RM23147 — RM337 on chromosome 8. RM251, RM314, and RM1135 were significantly associated with plant height and OsYSL17 was significantly linked with grain yield. Epistatic interaction was detected for plant height and number of tillers. Several candidate genes reported for yield and related traits underlie the QTL regions.

Breeding and Transgenic Approaches for Development of Abiotic Stress Tolerance in Rice

Abiotic stresses such as drought, salt, heat, and cold are serious threats to the sustainability of rice yield. As rice is considered one of the major food crops, the development of enhanced abiotic stress tolerance using breeding and transgenic approaches will undoubtedly have a serious impact on global food security. Significant genetic variation in rice germplasm for resistance/tolerance to abiotic stresses makes plant breeding as a viable option of stress tolerance development. Though high-yielding rice cultivars with enhanced tolerance to abiotic stresses have been released, the progress made by conventional breeding was considerably slow due to multigenic nature of the traits and other associated problems. Biotechnology tools like molecular breeding and genetic engineering have accelerated the efforts of developing abiotic stress-tolerant crops. Further, the progress in identification of new genes and QTLs involved in abiotic stress response has supplemented the breeding and transgenic approaches of stress tolerance. The transgenic approach offers an alternative to breeding approaches for the genetic improvement of rice germplasm. In recent years, a number of stress-related genes have been identified and transferred in rice to improve its tolerance against abiotic stresses. Several transgenic rice lines with enhanced abiotic stress tolerance have been generated and evaluated. This chapter highlights the updates of breeding and transgenic approaches for the development of abiotic stress tolerance in rice with illustrations from research targeted at drought, salinity, heat, cold, and submergence.

Advances, Challenges and Prospects in Small RNA Mediated Approaches of Virus Resistance in Plants

RNA interference (RNAi) is a mechanism of small RNA-guided regulation of gene expression in which small RNAs inhibit the expression of genes with complementary nucleotide sequences. RNAi has emerged as a powerful modality for battling challenging viruses and provides a natural defense against viral pathogens. In plants, RNAi has been successfully used to express cognate dsRNAs for viral transcripts in order to initiate the process of viral gene silencing. Despite the wide applicability of RNAi for achieving viral resistance, there are some challenges and constraints that must to be addressed to develop RNAi as a more effective tool for virus resistance. The present review provides an update on different approaches of using RNAi for virus resistance development in various crop plants. The factors influencing RNAi-mediated virus resistance and the major constraints are also discussed in detail.

Physical interaction of RTBV ORFI with D1 protein of Oryza sativa and Fe/Zn homeostasis play a key role in symptoms development during rice tungro disease to facilitate the insect mediated virus transmission

Rice tungro disease is caused by the combined action of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The RTBV is involved in the development of symptoms while RTSV is essential for virus transmission. We attempted to study the mode of action of RTBV in the development of symptoms. The tungro disease symptoms were attributed to viral interference in chlorophyll and carotenoids biosynthesis, photosynthesis machinery, iron/zinc homeostasis, and the genes encoding the enzymes associated with these biological processes of rice. The adverse effects of virus infection in photosystem II (PSII) activity was demonstrated by analyzing the Fv/Fm ratio, expression of psbA and cab1R genes, and direct interaction of RTBV ORF I protein with the D1 protein of rice. Since ORF I function is not yet known in the RTBV life cycle, this is the first report showing its involvement in regulating host photosynthesis process and symptoms development.