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Professor Jayashankar Telangana State Agricultural University, Hyderabad (Telangana State)
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ThesisItem Open Access TRANSCRIPTOMIC ANALYSIS TO IDENTIFY KEY GENES INVOLVED IN RICE- Rhizoctonia solani INTERACTION AND THEIR FUNCTIONAL CHARACTERIZATION(PROFFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2022-07-22) Naresh Babu, P; Durga Rani, Ch. V.Rice (Oryza sativa L.) crop is one of the primary food sources in Asia and a large part of the human population worldwide. Rice crop is affected by both biotic and abiotic stresses thereby reducing production and productivity. Many disease-causing pathogens like bacteria, fungi and viruses inflict heavy damage to rice plants. Sheath blight (ShB) of rice is one of the major fungal diseases caused by Rhizoctonia solani is considered as one of the most economically important diseases of rice all over the regions of the world. No major sheath blight resistance genes are identified in rice because the low inherent level of resistance and R.solani resistance is polygenic in nature. A comprehensive approach- transcriptomic analysis was used to identify the key genes involved in rice-R.solani and to study molecular mechanisms involved in susceptible (TN1, BPT5204, Vandana and N22) and moderately tolerant (Tetep and Pankaj) rice genotypes. In our current investigation we identified and used PR-8, class III chitinase1 (Oschib1) to develop sheath blight tolerance in Taipei309. The differentially expressed genes (DEGs) in all the 6 genotypes in infected and control were studied. The commonly expressed DEGs in all the six genotypes are 352. Based on the PlantTFDB database 507 TFs were identified in six genotypes. 33 TFs were expressed in all the six genotypes and heat maps were visualized using Rstudio. Further, the cell wall genes induced in the six genotypes were identified manually based on the transcriptomic data. Total 57 cell wall genes were induced during the necrotrophic phase of R.solani in the six rice genotypes. We identified 15 major DEGs uniquely present in all six genotypes. Five chitinases were induced to R.solani infection in rice genotypes which usually don’t induced in plant system under controlled conditions. Over all in our rice genotypes a good number of cell wall degrading genes were induced. The GO and KEGG analysis provided the enrichment of the pathways expression levels of the genes in rice genotypes under sheath blight incidence. KEGG analysis identified the genes responsible for metabolic pathways and biosynthesis of secondary metabolites was highly expressed due to sheath blight incidence. Identification of changes in the expression levels of the genes in these pathways can provides an insight for the regulatory mechanism of host defensive responsive mechanism. Better understanding of pathogen genomics and gene regulatory networks are also critical to devise alternate strategies for developing resistance against this noxious pathogen. In this study, miRNA-like RNAs (milRNAs) of an Indian strain of R. solani were identified by deep sequencing of small RNAs. We identified 128 known and 22 novel milRNAs from 20,963,123 sequence reads. These milRNAs showed 1725 target genes in the fungal genome which include genes associated with growth, development, pathogenesis and virulence of R. solani. Notably, these fungal milRNAs showed their target genes in host (rice) genome also which were later verified by qRT-PCR. The detailed expression analysis of 17 milRNAs by qRT-PCR was analysed during infection at different time points of inoculation, at different growth stages of the host, in four different genotypes of the host, and also in four different strains of fungi which revealed differential regulation of milRNAs associated with pathogenesis and virulence. This study highlights several important findings on fungal milRNAs which need to be further studied and characterized to decipher the gene expression and regulation of this economically important phytopathogen. This study suggested that Oschib1 is a potential targets for genetic manipulation and may play an important role for resistance/tolerance to sheath blight either directly or indirectly. This study unravelled the molecular mechanisms of rice-R.solani interaction through the RNA-seq approach. Transcriptome sequence analysis showed the differentially expressed genes (DEGs) during R.solani infection in six rice genotypes, identified many TFs and cell wall genes and visualised using heatmaps. 352 DEGs were commonly expressed in all the six rice genotypes. From 352 DEGs identified 23DEGs were selected based on their functional annotation for expression analysis to know the expression of the genes during interaction of R.solani with rice. Chitinase 1 (Oschib1) gene was highly up regulated during fungal infection in both tolerant, Tetep and susceptible, TN1 genotypes. Validation of the Oschib1 was done by overexpression of the gene by mobilising into Taipei309. These experimental and bioinformatic advances will provide a powerful toolbox for fully characterizing the transcriptome as it relates to basic biological questions. The main purposes of this work is to the identification of candidate genes for sheath blight resistance/tolerance, which could further be exploited in manipulating tolerance to other biotic stresses in rice. Additional experiments are needed to understand the precise role of identified candidate genes for resistance/tolerance for sheath blight in rice.ThesisItem Open Access MOLECULAR MAPPING OF GENETIC LOCI ASSOCIATED WITH TOLERANCE TO LOW SOIL PHOSPHORUS IN RICE (Oryza sativa L.)(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2019) KALE RAVINDRA RAMRAO; SUNDARAM, R MRice is one of the most important cereal crops and occupies second prominent position in global agriculture. A significant proportion of rice growing soils across the world and in India is either phosphorus (P)-deficient or has a high P-fixing capacity resulting in reduced productivity levels. The basic mechanism plants follow for P utilization are P acquisition efficiency and internal P–use efficiency. Presence of genetic variability for P uptake has been reported in rice and a major QTL called Pup1 conferring tolerance to low soil P has been identified earlier. The present study was taken up in order to characterize available germplasm lines for low P tolerance and QTL mapping for low soil P tolerance in recombinant inbred line (RIL) mapping population developed by crossing, Wazuhophek (which has significant tolerance to low soil P condition and completely devoid of Pup1 genomic region) and Improved Samba Mahsuri (ISM; Which is highly sensitive to low soil P). In the first objective, i.e. a set of 56 germplasm lines of rice were analyzed for their genotype with respect to markers spanning the Pup1 locus along with phenotypic characterization for low soil P tolerance. Significant genetic variability was observed with respect to plant height, number of productive tillers, root volume, dry root weight, dry shoot weight and grain yield under low P condition. Cluster analysis based on the stress indices and low soil P tolerance ability, grouped the genotypes into three groups, viz., highly tolerant, tolerant and sensitive. Genotyping of the rice lines with a set of Pup1 specific markers (viz., K46-1, K46-2, K52 and 46CG-1), revealed the presence of partial or complete Pup1 genomic region in most of the tolerant genotypes. A total of 15 genotypes showed tolerance to low soil P based on yield reduction in comparison to the tolerant and sensitive checks. Interestingly, the tolerant genotype line, IC216831 was observed to be completely devoid of Pup1, and hence it can be explored for identification and characterization of new loci underlying low soil P tolerance. The second objective focused on SSR marker-based molecular mapping of genomic regions associated with low soil P tolerance in the non-Pup1 type, low P tolerant rice line from North-eastern part of India, Wazuhophek. Quantitative trait loci (QTLs) related to low soil P tolerance were identified in recombinant inbred lines (RILs), developed from cross Improved Samba Mahsuri/Wazuhophek. A total of 16 QTLs related to yield and other physio- morphological traits were identified under low soil P condition. Out of them, 10 QTLs were found on chromosome 8, spanning a 4.49 Mb region in the genomic interval of RM22554-RM8005. Two QTLs were identified on chromosome 7, spanning a 2.71 Mb (RM5847-RM22031) region and 11.31 Mb (RM21521-RM21103) region for root volume and P content of grains, respectively. Four QTLs were identified on chromosome 1, spanning a 9.2 Mb region in between the markersWR1.7-RM8105. In silico analysis of identified genomic regions spanning the identified QTLs was carried out. A total of 538 genes were predicted in the QTL region on chromosome 8, of which eight genes related to phosphorus uptake or utilization, including genes related to auxin signalling, phosphatase activity and zinc figure transcription factor appear to be of interest. A total of 1339 genes were predicted in the QTL region on chromosome 1, of which 14 genes related to phosphorus uptake or utilization, including auxin responsive genes and genes involved in maintenance of Pi homeostasis are of interest. A total of 712 genes were predicted in the QTL region controlling root volume, located on chromosome 7. Among them, 12 genes related to phosphorus uptake or utilization, including auxin responsive genes and transcription factors involved in regulation of stress related genes appear to be of interest. Similarly, in silico analysis of another QTL for P content in seed located on chromosome 7, predicated 570 genes. Among them, 16 were found to be possibly related to phosphorus uptake or utilization. All the QTLs detected in the present study, appear to be novel and did not overlap with the already reported genes/QTL. The novel low P tolerant rice line, IC216831, which is devoid of Pup1 can be subjected for genetic and molecular marker analysis to identify novel low P tolerance genes/QTLs. Further, the novel QTLs associated with low soil P tolerance identified from Wazuhophek, after their fine-mapping and validation, can be transferred into elite rice varieties and parental lines, which are sensitive to low soil P levels through marker-assisted breeding.ThesisItem Open Access MARKER ASSISTED SELECTION FOR DURABLE GALL MIDGE (Orseolia oryzae) RESISTANCE IN RICE(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2018) MAHESH DURISETI; DURGA RANI, Ch.VBiotic stresses such as gall midge (GM) and bacterial blight (BB) account for significant economic losses annually in rice. The major resistance genes viz., Gm4, gm3, Gm8 for GM and xa13, Xa21 for BB offer resistance to these stresses. Marker assisted pedigree selection was carried out at Institute of Biotechnology, PJTSAU, Hyderabad to develop rice lines with resistance to these stresses. For this three parents involved in a crossing programme viz., NIL of MTU1010 (Gm4, xa13, Xa21), RP2068- 18-3-5 (gm3) and Aganni (Gm8) were used. During Rabi 2014-15 two crosses were made involving NIL of MTU1010 as female parent and RP2068-18-3-5 and Aganni as male parents. F1 seeds obtained in these crosses were raised in Kharif 2015. Functional markers (LRR del for Gm4, gm3del3 for gm3, PRP for Gm8, xa13 promo for xa13 gene) and another closely gene linked marker (pTA248 for Xa21) were used to confirm hybridity of the two crosses. One plant from each cross were intercrossed to get ICF1 seeds. During Rabi 2015-16, ICF1 plants were raised, genotyped using aforesaid markers. One plant with desired gene combination (Gm4gm4Gm3gm3Gm8gm8xa13xa13Xa21Xa21) was selected and forwarded to ICF2 generaton. During Kharif 2016 ICF2 plants were genotyped and 7 distinct gene combinations (Gm4Gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21, Gm4Gm4gm3gm3gm8gm8xa13xa13Xa21Xa21, Gm4Gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21, gm4gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21, Gm4Gm4Gm3Gm3gm8gm8xa13xa13Xa21Xa21, gm4gm4gm3gm3gm8gm8xa13xa13Xa21Xa21 and gm4gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21) representing five gene, four gene and three gene combinations were selected and forwarded to ICF3 generation. During Rabi 2016-17 ICF3 progenies obtained from selected ICF2 plants were checked for uniformity of genes using aforesaid functional markers. Single plant selections were forwarded to ICF4 generation for phenotyping studies. A total of 69 ICF4 lines comprising of 51 five gene pyramids (Gm4Gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21), 9 four gene pyramids (Gm4Gm4gm3gm3gm8gm8xa13xa13Xa21Xa21, Gm4Gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21 and ii gm4gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21) and another 9 three gene pyramids (Gm4Gm4Gm3Gm3gm8gm8xa13xa13Xa21Xa21, gm4gm4gm3gm3gm8gm8xa13xa13Xa21Xa21, gm4gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21) were subjected to phenotyping studies at RARS, Warangal where natural occurrence of GMB4M is prevalent. Out of 69 ICF4 lines, 12 lines scored ‘0’ with zero percent incidence of silvershoot. Among the 12, ten are five gene pyramids (Gm4Gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21) and two are four gene pyramids (Gm4Gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21). 37 lines scored ‘1’ with less than 1% incidence of silvershoots. Out of 37 lines, 36 are five gene pyramids (Gm4Gm4gm3gm3Gm8Gm8xa13xa13Xa21Xa21) and one is four gene pyramid (Gm4Gm4Gm3Gm3Gm8Gm8xa13xa13Xa21Xa21). Hence these 49 progenies with 0 and 1 scores were considered as resistant. Phenotyping was done in these 49 progenies for gall midge resistance against GMB1 biotype at ICAR-IIRR, Hyderabad under green house condition. Results showed that out of the 49 lines except three five gene pyramid ICF4 lines remaining scored ‘0’ hence considered as resistant. Therefore a total of 46 lines considered to be showing resistance against both the gall midge biotypes. As a whole five gene pyramids performed better followed by four gene pyramids, indicating the complementary/additive nature of GM resistance genes. BB screening in these 46 lines shown most of them scored ‘1’ and a few scored ‘3’ hence all these lines were considered to be resistant for BB. There is no antagonistic nature was observed between gall midge and BB genes. Agronomic evaluation also shown 12 lines performed better when compared with standard check MTU1010. Out of 12, five lines (F2-646-14, F2-701-11, F2-701-15, F2-735-12 and F2-899-1) registered significant yield superiority to MTU1010, while 7 lines (F2-598-12, F2-646-8, F2-646-18, F2-811- 16, F2-899-9, F2-998-2 and F2-36-11) showed numerical superiority over MTU1010 along with durable GM and BB resistance. In the present study phenotypic correlation coefficient analysis revealed that plant height, productive tillers, panicle length and number of seeds/panicle components contributed to the plant yield. Through pedigree method of breeding with marker assisted selection, we have successfully combined GM and BB resistance genes against two important biotic stresses i.e. gall midge and bacterial blight. Key words: Gall midge, Bacterial Blight, functional markers, gene pyramid, Gm4, gm3, Gm8, xa13 and Xa21 genes.ThesisItem Open Access MOLECULAR AND PHYSIOLOGICAL INSIGHTS INTO THE RESPONSE OF RICE TO AEROBIC CONDITIONS(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2018) PHULE AMOL SARJERAO; ANANDA KUMAR, PIn view of a paradigm shift in the system of rice cultivation from anaerobic to aerobic condition, it is very much essential to identify the transcripts and understand the mechanism of aerobic adaptation. To get deeper insights of aerobic adaptation in rice, RNA-seq analysis of root and shoot i.e. developing panicle tissues at panicle initiation stage in two cultivars adapted to traditional transplanted anaerobic (BPT 5204) and aerobic (CR Dhan 202) systems of cultivation under aerobic and anaerobic conditions was performed. The physiological and anatomical observations showed that root length, total dry weight, fewer aerenchyma formation and larger xylem area were critical to aerobic adaptation. The RNA-seq data emanated in 1.65 billion clean reads with approximately 37 million reads per sample. The number of differentially expressed transcripts was higher for root (1535, 492) than the shoot (441, 726) under both aerobic and anaerobic conditions. Differentially expressed transcripts (DETs) of root under aerobic condition were classified based on GO-enriched categories. Among those, transcripts related to stress (GO:0006950), oxidoreductase activity (GO:0016491) and cytoplasmic membrane-bounded vesicle (GO:0016023) were majorly found in the main categories of biological processes, molecular function and cellular components respectively. Similarly, in shoot under aerobic situation, among the DETs, transcripts related to water deprivation (GO:0009414), nucleic xix acid binding, transcription factor activity (GO:0001071) and extracellular region (GO:0005576) were significantly enriched. The transcription factors viz. MADS 15, MADS 24, MADS26, HOX 12, HOX 14, DREB1F and DREB1C were exclusively expressed in adapted cultivar under aerobic condition specifying its role in adaptation. In addition to that, various transporter families viz. bidirectional sugar transporter (SWEET 16, 3a and 4), metal-nicotianamine transporter (YSL2 and YSL13), inorganic phosphate transporter (PHT1;6), cation transporter (HKT8), phosphate transporter (PHO1;2), potassium transporter (HAK1) and ABC transporters were highly expressed exclusively in CR Dhan 202, which suggested that the transporters play a major role in aerobic adaptation. Among the hormones, Ethylene and Abscisic acid seem to be mainly involved in imparting aerobic adaptation. The root traits related genes (MT2B, MT2c, MT4A, MT1g, PHT1;6, HKT8, HAK1, YSL2, YSL13, PIP2;3, TIP1;2 and NIP2;1) were also highly expressed in root tissue of CR Dhan 202 under aerobic condition, which suggested the involvement and sensitivity of roots to water limiting conditions. Highly differentially expressed transcripts were also mapped to the reported QTLs responsible for root related traits, yield under drought etc. Many of the transcripts fall within the reported QTLs, which further strengthened the involvement of those genes in aerobic adaptation. The validation of the results of RNA-seq and qRT-PCR analysis was carried out. Reference genes suitable for the specific condition were not reported for this analysis. Hence, identification of the reference genes was carried out. Selection of stable reference genes across the tissue samples at various developmental stages or at specific stress conditions is very much essential to accurately assess the relative temporal and spatial gene expression. The expression stability of ten rice genes viz. Actin, eEF-1a, eIF-5C, Exp1, Exp2, Memp, SKP1A, TF-SUI1, TPH and UBQ5 across six experimental sets in shoot and root tissues at seedling, tillering and panicle initiation stages revealed Memp (Membrane protein), TPH (Tumor protein homolog) and Exp1 (Expressed protein) as the most stable genes with acceptable M and V value according to the gold standards of qRT-PCR. Eleven DETs in aerobic and anaerobic conditions were selected for validation through qRT-PCR using newly identified reference genes which revealed high correlation between the RNA seq and qRT-PCR. xx It is inferred that high metabolic activity may occur in aerobic condition by high expression of various TFs, transporters and root traits related genes in root compared to shoot which might play a role in adaptation mechanism in CR Dhan 202. The presence of alternative splicing (AS) events also lay a concrete foundation that alternative splice variants are generated as a result of adaptation which might play important role in transcript generation. The adaptation under aerobic condition can be bestowed by the combined hormonal signaling and response mechanism through transcription factors and transporters.ThesisItem Open Access ISOLATION AND MOLECULAR CHARACTERIZATION OF ABIOTIC STRESS TOLERANT PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR) FROM DIFFERENT RHIZOSPHERIC SOILS OF TELANGANA(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, 2018) DAMODARA CHARI, K; SUBHASH REDDY, Rn rain fed agro-ecosystems is predominance of abiotic stresses like high temperature, salinity and drought where the applied bioinoculants survival and viability is a major issue in Indian conditions. In the results of laboratory conditions with bioinoculants and field could be variable due to various abiotic stresses that prevail under farmers’ field conditions for microbial inoculants to establish and to show the efficient desirable effect. Such type of problems can be overcome by sound screening programme for efficient stress tolerant plant growth promoting rhizobacteria from different rhizospheric soils for effective management of abiotic stress in crop plants. Evolving efficient, low cost, easily adaptable methods for the abiotic stress management is a major challenge. Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that actively colonize plant root and increase plant growth by production of various plant growth hormones, phosphate solubilizing activity, nitrogen fixation and biocontrol activity. Few strains from genera such as Pseudomonas, Azospirillium, Azotobacter, Bacillus, Burkholderia, Enterobacter, Rhizobium, Erwinia and Flavobacterium are well known PGPR. They aid in improving plant stress tolerance to drought, salinity and metal toxicity. Present investigation was carried out to identify plant growth promoting rhizobacterial isolates for abiotic stress tolerance. To achieve this bacterial isolates were isolated from different rhizospheric soils of Telanagana and screened for plant growth promoting properties and tolerance to different abiotic stresses such as pH, temperature, salt, drought and heavy metals. Such PGPR will be helpful for efficient management of abiotic stresses in crop production. Rhizospheric soils from normal, salt affected, drought affected and bulk soils were collected from different places of Telangana state. From all soil samples, about 142 different isolates were selected and purified on respective media. Based on cultural, morphological and biochemical characterization, it was found that forty four were of Bacillus spp, forty were of Pseudomonas spp, twenty one were of Rhizobium spp and thirty seven were of Azotobacter spp. All the bacterial isolates (Bacillus, Pseudomonas, Rhizobium and Azotobacter) were subjected to further studies under in vitro conditions to understand their multiple beneficial activities such as plant growth promoting properties, biocontrol activity and tolerance to abiotic stress. Among the forty four Bacillus isolates, twenty eight isolates were showing plant growth promoting properties. Isolate BJRB-18 showed plant growth promoting properties and abiotic stress tolerance to pH, temperature, salt, drought and heavy metals (Arsenic and Cadmium). Among the Pseudomonas isolates twenty eight were showed plant growth promoting properties. KRP-1 showed efficient plant growth promoting properties followed by BJPP-15. For biocontrol properties such as HCN production, siderophore production and antifungal activity with Rhizoctonia solani, efficient isolates were KTP-3, CAPP-40. For plant growth promoting properties and abiotic stress, BJPP-15 was found efficient. No single isolate showed combination of plant growth promoting properties, biocontrol activity and tolerance to abiotic stress conditions. Isolates KTB-3 and KRP-1 could be a good combination for this purpose. Similarly no single rhizobial isolate had shown positive for combination of plant growth promoting properties, biocontrol activity and tolerance to abiotic stress conditions. However, Rhizobium isolates KLSR-10 and PGR-14 could be used in combination to meet the requirement of plant growth promoting properties, biocontrol and abiotic stress conditions. Among the Azotobacter isolates, KRAZ-1 was found suitable for use commonly for plant growth promoting properties, biocontrol and abiotic stress. In the present study, the sequenced PCR products of effective bacterial isolates were matched with the available sequences in the Gene Bank database. BLAST Search results through NCBI showed 97% similarity of BJRB-18 with Paenibacillus lautus, 99% similarity of KLSR-10 with Bradyrhizobium japonicum, 99% similarity of BJPP-15 with Pseudomonas fluorescens, 99 % similarity of KRP-1 with Pseudomonas fluorescens. All of them had shown tolerance to different abiotic stress conditions indicating their potential for their use as bioinoculants.ThesisItem Open Access Isolation, Characterisation, Immunology And Pathogenicity Of Mycoplasma And Chlamysia From Pneumonia In Lambs(College Of Veterinary Science ; Apau ; Tirupati, 1984) Sreeramulu, PiedyThesisItem Open Access Protective Antigen (Pa) Of Bacillus Anthracis(College Of Veterinary Science ; Apau ; Tirupati, 1986) Panduranga Rao, V.ThesisItem Open Access Studies On Egg Drop Syndrome Virus (Eds-76) In Chickens In Andhra Pradesh(Sri Venkateswara Agricultural College ;Tirupati, 1985) Satyanarafna Chetty Mitta
