1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Morphological, biochemical and molecular characterization of Trichoderma isolates from tuber crop ecosystems(Department of Plant Biotechnology, College of Agriculture,Vellayani, 2019) Linet K Joseph; Veena, S SElephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson) is an important tuber crop popular as a food security crop and as a remunerative cash crop. Collar rot caused by Sclerotium rolfsii is the most destructive and predominant disease causing great crop loss in elephant foot yam. Application of Trichoderma spp is being recommended as the eco-friendly strategy to combat the crop loss. The present study, ―Morphological, biochemical and molecular characterization of Trichoderma isolates from tuber crop ecosystem‖ was conducted at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram during October 2018 – August 2019. The study was performed to evaluate 43 Trichoderma isolates obtained from tuber crop ecosystem for their bio-control potential against S. rolfsii, to characterize the isolates using morphological, biochemical and molecular approaches and to analyse the molecular diversity. The differential antagonistic potential of the isolates were assessed by adopting three in vitro screening methods. The screening methods executed were dual culture/ direct confrontation method, antibiosis test for production of diffusible metabolites and volatiles. Based on the additive effect of each mode of inhibition, it was concluded that the isolates viz., T38, T36, T32, T40 and T6 have excellent antagonistic potential. Twenty six best isolates were selected for further study based on the ranking of additive result. The efficiency of 26 isolates for induction of chitinase and β-1,3-glucanase was studied against the cell wall of S. rolfsii as carbon source. There was no direct correlation observed between antagonistic potential of isolates and induction of chitinase enzyme. Whereas, positive correlation was observed between antagonistic potential and induction of β-1,3-glucanase enzyme. The effect of volatile organic compounds (VOCs) on plant growth was studied using mustard seeds. The isolates 94 showed differential response to various growth parameters like fresh weight, number of leaves, root length, shoot length and number of shoot lets. For morphological characterization, the macro and micro morphological characters such as growth rate, colony color, reverse colony color, odor of culture and branching pattern of conidiophore, size of conidia and phialides of isolates were studied. Morphological identification of Trichoderma isolates up to species level was found difficult due to the overlapped expression of these characters. The molecular characterization was done by amplifying and analyzing the sequences of ITS gene 1 and 2 and tef1 gene. The six different species identified are T. asperellum, T. virens, T. hamatum, T. reesei, T. longibrachiatum and T. erinaceum. The variability was studied using SSR markers and it was found that Jaccard’s similarity coefficient of 10 SSR primer banding patterns varies from 0.31 to 1.00. Fourteen T. asperellum isolates (T1, T2, T3, T5, T11, T13, T15, T16, T17, T18, T19, T20, T42, T43) were clustered in cluster A and the remaining three T. asperellum isolates (T32, T34 and T41) were clustered into cluster B. It showed the significant variability even within the same species. Considering the antagonistic potential, high chitinase and glucanase production and plant growth promotion, isolates T2, T15, T32, T34 (T. asperellum) and T40 (T. erinaceum) were selected as effective bio-control agents. The present study helped in identifying the Trichoderma isolate with high antagonistic potential against S. rolfsii and the entire process ensured more précised and targeted application of Trichoderma isolate in field condition. The outcome of the study will be a key factor in developing appropriate management strategy to mitigate collar rot disease in elephant foot yam.Item Identification and characterisation of virus responsive miRNAs in banana musa (AAB) Nendran(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Athira Subramanian; Soni, K BThe study entitled “Identification and characterization of virus responsive miRNAs in banana Musa (AAB) ‘Nendran’” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to identify the miRNAs associated with Banana Bract Mosaic Virus (BBrMV) infection in banana var. Nendran from the expression profile of selected miRNAs. Five miRNAs were selected from the 52 computationally predicted miRNAs in a previous study conducted in the Department of Plant Biotechnology. Selection was based on the function of their target genes i.e. their role in biotic stress conditions. The miRNAs selected were miR-3900-5p (targets: Plant viral response family protein and Putative disease resistance protein genes), miR-2172-5p (target: Putative ethylene responsive transcription factor1gene), miR-6928-5p (target: Flavin adenine dinucleotide dependent oxidoreductase gene), miR-5417 (target: Stress endoplasmic reticulum protein 2 gene) and miR-971-5p (targets: Argonaute protein and Transport inhibitor response-1 like protein genes). For studying the expression of miRNAs, three month old in vitro raised banana var. Nendran plants were infected with BBrMV through viruliferous aphids (Pentalonia nigronervosa) by acquisition feeding method. Fifteen to twenty aphids reared in healthy banana suckers were transferred to the BBrMV infected sucker for 2 hour acquisition feeding, after starving for 30 min. These aphids were released on to the leaf axils of the tissue culture plants for a period of 24 h. After this time period the aphids were killed using an insecticide. Infection was confirmed by PCR using replicase specific primers and the results showed the presence of BBrMV specific amplicons from 24h onwards in all the samples. For studying the expression of these miRNAs, leaf samples were collected from the infected plants 24 h, 48 h, 1 wk and 2 wk after infection. RNA was isolated using CTAB method, reverse transcribed to cDNA and PCR was done. PCR analysis confirmed the presence of all the five computationally predicted miRNAs in banana. The expression profiles of the miRNAs and their target genes were studied by RT-qPCR. The results showed upregulation of miR-3900-5p, miR-2172-5p, miR-6928-5p and miR-971-5p (1.2, 2.0, 1.27, 2.0 fold respectively) 24h after BBrMV infection. Among them miR-3900-5p, miR-2172-5p and miR-971-5p maintained higher expression upto one week compared to uninfected control. miR-6928-5p showed a 1.72 fold increase in expression 48 h after infection. Expression of miR-5417 was down regulated by BBrMV infection at 24 h of infection. The two targets of miR-3900-5p (Plant viral response family protein and Putative disease resistance protein genes) showed contrasting trends in their expression after BBrMV infection. While Putative disease resistance protein showed a drastic increase (13 fold) 24 h after infection, Plant viral response family protein expression showed a continuous reduction throughout the period of observation. miR-2172- 5p and its target Putative Ethylene-responsive transcription factor 1gene (3.39 fold) were found upregulated during the infection. While miR-6928-5p showed maximum expression at 48 h, its target FAD (Flavin adenine dinucleotide) dependent oxidoreductase gene showed maximum expression at 24 h after infection and maintained a higher level upto 48 h. Among the two targets of miR-971-5p, Transport inhibitor response-1like protein gene showed a quick response to virus infection with a 6.15 fold expression at 24 h, while Argonaute protein gene showed a lower expression upto 48h and a drastic increase upto 2 fold as the infection progressed. The study suggested that miR3900-5p, miR2172-5p, miR6928-5p and miR971-5p were associated with BBrMV infection in banana var. Nendran. Their targets viz., putative disease resistance protein gene, putative ethylene responsive transcription factor 1gene, FAD dependent oxidoreductase gene and transport inhibitor response-1 like protein gene showed significant increase immediately after the infection. Suppression of the targets viz., Plant viral response family protein and Argonaute protein genes during BBrMV infection suggested the possible role of miR-3900-5p and miR-971-5p in regulating the infection process.Item Identification and characterization of suppressor of over expression of constans1 (SOC1) gene in black pepper (Piper nigrum L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2016) Manu K Venu; Lekha SreekantanThe present study entitled“Identification and characterization of Suppressor of Overexpression of Constans1 gene in Black Pepper (Piper nigrum L.)”was conducted at the Integrated Biotechnology Block, College of Agriculture, Vellayani, during 2015-2016.The study envisagedisolation and sequencing ofSOC1, a flowering integrator gene in black pepper (variety - Karimunda) and functional characterization of the gene by studying the expression patterns. Degenerate primers were designed for the above said gene based on the gene sequences from NCBI database (SOC1 forward and reverse primers) which were used to isolate and identify the gene. Total RNA of black pepper was isolated using modified CTAB method followed by synthesis of cDNA using AMV RT (Avian myeloblastosis virus reverse transcriptase). PCR (Polymerase chain reaction) with degenerate primers was done using cDNA as the template. However no amplifications were observed after the first reactions. Therefore nested PCR reactions were done using the PCR products of the first reaction as the template. Two bands of size 640 bp and 330 bp were produced in the nested reactions. Sequencing of the product yielded four sequences with each of the sequence showing similarity to the SOC1 gene, when done sequence analysis, thus making it the first flowering integrator gene to be identified in black pepper. Microscopy studies were carried out to see the floral characters of black pepper in detail. Microscopy studies were done using FAA fluid as fixative, sectioning the tissues and staining with safranin and fast green were carried out to see the changes occurring in different development stages of spikes from immature spike to complete spike with berries.Item Characterization and validation of microsatelite markers for resistance to vascular streak dieback disease in cocoa (Theobroma cacao L.)(Centre for Plant Biotechnolgy and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Waghmare Sandesh Tulshiram; Deepu MathewCocoa is the third important plantation crop next to coffee and tea. The global production and consumption of cocoa is 27.00 lakh MT. Among the fungal diseases, Vascular Streak Dieback (VSD) caused by Ceratobasidium theobromae is the main constraint in cocoa growing countries, causing heavy losses in mature trees as well as seedlings. The VSD disease cannot be effectively controlled by chemicals and hence breeding for the development of resistant varieties is the best strategy to tackle the disease. In order to confirm the transfer of a desired gene into the offspring, conventional breeding methods rely on the field screening which will be highly influenced by the environmental factors. Marker assisted selection is an alternate where the tightly linked molecular markers will be employed to confirm the presence of the gene of interest in the selected plants. Five ISSR and one SSR markers linked to VSD resistance were identified at Kerala Agricultural University (Chandrakant, 2014). The present study was undertaken with the objective of validating the identified SSR and ISSR markers and to characterize the ISSR markers to identify the corresponding SSR markers. For validation and characterization, twenty VSD resistant hybrids and four susceptible clones were used. For molecular analyses, good quality genomic DNA was isolated from twenty four genotypes and ISSR markers UBC 811, UBC 815, UBC 826, UBC 857, UBC 866 and SSR marker mTcCIR 42 were screened. ISSR analysis had shown that all the primers are capable to differentiate resistant and susceptible genotypes. The SSR assay has also differentiated the resistant and susceptible genotypes. The distinct markers generated in resistant genotypes using UBC 811, UBC 826 and UBC 857 were eluted, cloned to pGEMT vector and sequenced. The nucleotide sequences were annotated using BLAST, ORF finder and SSR finder. The BLASTn of UBC811A and UBC811D nucleotide sequence have shown that this resistance locus lie in the chromosome V of Theobroma cacao genome. BLASTn of UBC826A, UBC826B and UBC857 has positioned these loci in chromosome III. ORF1 and ORF3 in UBC811D are shown to code for aflatoxin biosynthesis regulatory protein and NAD(P)H dehydrogenase quinine, respectively. ORF1 in UBC826B and ORF5 in UBC857-2 code for potassium transporter 27 (0sHAK-27) and structural polyprotein precursor of VP2, capsid protein VP2, respectively. All these proteins are identified to have definite roles in defence pathways. The frequency and distribution of SSR motifs, dimmers to decamers, in these ISSR markers and the corresponding primers were identified. The reported ISSR and SSR markers were validated and found to be successful in differentiating resistant and susceptible genotypes of cocoa; thereby these markers can be used in marker assisted breeding for VSD resistance.Item Validation of temperature induction response (TIR) technique for inducing drought and heat stress tolerence in rice (Oryza sativa L.)(Department of Plant Physiology, College of Agriculture, Vellayani, 2018) Reshma Mohan; Beena, RItem Physiological and molecular analysles of flowering responses in amaranthus (amaranthus spp.) and cowpea (vigna spp.) under elevated CO2 environment(Department of Plant Physiology, College of Agriculture, Vellayani, 2018) Ghade Rameshwar Pandurang; Manju, R VItem Tagging of phytophthor pod rot disease resistance gene in cocoa (Theobroma cacao L.) using ISSR markers(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Jeughale Kishor, Pundlik; Minimol, J SCocoa (Theobroma cacao L.) known as ‘Chocolate tree’, is a major cash crop in tropical countries. Cocoa production is seriously affected by pod rot diseases caused by many Phytophthora species. Among these, the pod rot caused by Phytophthora palmivora has been reported in India. Yearly losses to the cocoa growers around the world from Phytophthora diseases were assessed at 30 per cent of the total yield loss. Disease resistance can be scored using a number of morphological and physiological characters. However, the morpho-physiological characters greatly depend upon the environment which ultimately affect the experimental data. Hence, confirmation of transfer of genes by tagging with the help of a strong tool is of utmost importance in crop breeding. Molecular markers such as Inter simple sequence repeats (ISSRs) have already proven to be a good tool to detect and tag the genes of interest and will help to reduce the breeding cycle. In this context, the present study was taken up with an objective to develop a strategy to tag gene(s) for Phytophthora pod rot (PPR) resistance in cocoa using ISSR markers. Morphological characterization of 28 hybrid progenies of SVI 1.26 × PII 12.11 was carried out by recording five pod and bean characters. High variability was observed for characters viz., pod weight, pod length and breadth, wet bean weight per pod and single dry bean weight among the progeny of the same cross. Detached pod inoculation technique was adopted to classify the hybrids into resistant and susceptible ones. The wide variability was also recorded for disease reaction among the progenies. Based on the resistance score, three resistant and three susceptible hybrids were selected from the segregating progeny. The eight accessions were screened with fifty ISSR and 15 SSR primers to observe polymorphism between resistance and susceptible genotypes. Polymorphism was observed in 11 ISSR primers and from these, six primers viz., UBC 810, UBC 826, UBC 827, UBC 857, Oligo ISSR 04 and Oligo ISSR 08 were eluted and cloned. Plasmid DNA was isolated from clones and sequenced. Though various SSR primer sets screened were found to yield polymorphism, none of them was successful to give a clear distinction among the resistant and susceptible hybrids. This may be due to the fact that, Quantitative trait loci (QTLs) associated with these reported SSR primers may be absent in the genotypes considered for the study. BLASTn analysis specific to plants was done for all six sequences. Upon analysis, Oligo ISSR 04561 had shown 98 per cent identity with Predicted: T. cacao histidine-containing phosphotransfer protein 1 (HPt). HPts play an important role in propagating cytokinin signal transduction. Cytokinins are instrumental in mediating disease resistance by generating a green island around the infection zones, exhibiting delayed leaf senescence and upregulating the expression of the pathogenesis related (PR) gene/s. In addition to this, the auxin-cytokinin antagonism that occurs as part of a complex hormonal interplay, exerts a critical influence on the core SA-JA/ET plant immunity pathways. The BLASTn analysis of marker UBC 810877 resulted in 99 per cent sequence identity with Predicted: T. cacao phospholipid: diacylglycerol acyltransferase (PDAT) 1 mRNA. This protein regulates the synthesis of triacylglycerol, which is a building component of oils in the plant. Accumulation of oil content in plant cells could impart resistance against the pathogen. UBC 827571 had shown 73 per cent sequence identity with T. cacao clone TCC_BA049P20 complete sequence and it is reported to be QTL rich region associated with different traits of T. cacao. Moreover, ISSR markers UBC 810877, UBC 826535 and UBC 857839 are located on chromosome nine, six and four respectively as inferred from NCBI Genome Data Viewer tool through BLASTn annotations. These markers are found to be located in PPR resistance regions rich in defense associated genes. Further validation and exploitation of polymorphic amplicons or markers in response to PPR would be required. The linkage of Oligo ISSR 04561 and UBC 810877 with HPts and PDAT correspondingly have to be validated to elucidate the association and role of cytokinin and triacylglycerol with PPR disease resistance. If validated, UBC 810877, UBC 826535 and UBC 857839 and Oligo ISSR 04561 could be employed as a marker in PPR resistance breeding programmes in cocoa.Item Comparative and functional genomics analysis of starch biosynthesis pathways in cassava(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Pooja Harshan; Sreekumar, JItem Evaluation of systemic acquired resistance and induced systemic resistance on the suppression of foliar blight disease of amaranthus (Amaranthus tricolor L.)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Athira Babu, B M; Anith, K NItem Analysis of capsanthin capsorubin synthase gene in byadagi chilli (Capsicum Annuum L.) and elucidation of carotenoid metabolic pathway(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Naresh, S; Shylaja, M RByadagi chilli is famous for its deep red colour and negligible or zero pungency. Demand for Byadagi chilli has increased enormously as a source of natural red colour in food industry, confectionaries, cosmetics, beverages, pharmaceuticals and even as a dye in textile industries. Byadagi chilli is mainly exported as oleoresin which serves as a substitute for paprika oleoresin.The red color of chilli fruits is due to several related carotenoid pigments. The most important pigments are capsanthin and its isomer capsorubin. The present study was conducted to analyze Capsanthin-capsorubin synthase gene (Ccs) in Byadagi chilli and to elucidate the carotenoid metabolic pathway for production of capsanthin and capsorubin. The studies were focused on seven genetically distinct chilli varieties /accessions of three different Capsicum spp. based on colour at fully ripe fruit stage. The accessions selected were ByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha (Capsicum annuum), Vellayani Samrudhi (Capsicum frutescens), Vellayani Thejus and CC8-1 (Capsicum chinense). Genomic DNA was isolated from tender leaves of one month old plants by CTAB method. Two chilli Ccs gene specific SSR primers viz. Ccs Cds and Ccs promoter were used to amplify the Ccs gene.The amplified PCR products obtained with Ccs Cds and Ccs promoter were sequenced by outsourcing and sequence data analyzed using bioinformatics tools. The Ccs gene was found amplified in all the genotypes including the yellow fruitedaccession CC8-1. Size of amplified product was 1.5kb with Ccs cds primer in all the genotypes. For Ccs promoter, amplified product was920bp inByadagiKaddi, Byadagi Dabbi, Ujwala, Anugraha, Vellayani Samrudhi and 1200bp in Vellayani Thejus and CC8-1 BLASTn analysis of the Ccs gene amplified with Ccs cds primer showed 99 -100 per cent similarity with the reference nucleotide sequence in all the genotypes. BLASTx analysis of Ccs gene sequence amplified with Ccs cds primer showed 99-100 per cent similarity with the reference amino acid sequence in the seven genotypes studied. Analysis of conserved domains revealed that lycopene beta cyclase was the conserved domain in Capsicum annuum and C. chinense genotypes while in C. frutescens NADB super family protein was the conserved domain. The number of ORFs in the Ccs sequence amplified with Ccs cds primer ranged from six to seven in the genotypes studied and the number of amino acids coded ranged from 463-469 in C. annuum, 298 in C. frutescens and 217 in C. chinense. Multiple sequence alignment of the sequences revealed SNP variations in the genotypes studied and SNP variation caused change in amino acid coded. SNP variations were observed in five genotypes viz. Byadagi Kaddi, Byadagi Dabbi, Vellayani Samrudhi, Vellayani Thejus and CC8-1 while no SNP variations were seen in the varities Ujwala and Anugraha Byadgi kaddi had two SNPs leading to change in amino acids at 43rd and 425th position of Capsanthin capsorubin synthase peptide. Tyrosine (Y) was found replaced by Phenyl alanine (F) in the 43rd position and Lysine (K) was found replaced by Glutamic acid (E) in the 425th position. Byadagi dabbi also had the same amino acid change at 425th position, Lysine (K) was replaced by Glutamic acid (E). PrematureStop codon UAG was observed in yellow fruited variety CC8-1 at 200th position BLASTn analysis of Ccs gene sequence amplified with Ccs promoter primer in seven genotypes showed 90-99 per cent similarity with the reference nucleotide sequence. Multiple sequence alignment of the promoter region could see structural changes in the sequences. Several SNPs in the sequences, a tandem repeat structure, insertion, deletions and various cis regulatory elements like heat stress related cis-elements (HSE), Myb binding site (MYBPZM) and light responsive elements, TATA box, and CAAT box could be observed in the promoter region. Ccs gene was located in Chromosome six of Capsicum annuum and in the genome map of chilli it was seen in between 9497216 – 9500911kb. Ccs cds gene specific primer was seen to bind 18bp downstream region of the sequence. The Ccs promoter was seenupstream of the protein coding region. Elucidation of carotenoid metabolic pathway in Capsicum annuum revealed that 17 enzymes were present in the carotenoid biosynthesis pathway. Gene regulatory network analysis, using cytoscape showed that network contained 94 nodes and many of the genes were associated with carotenoid biosynthesis processes. The main seventeen carotenoid metabolic pathway genes, some transcription factors and transferase/transport proteins were densely connected. Among the pathway genes, Phytoene synthase had the highest number (30 No.) of interactive proteins. The identified SNPs in the present study have to be further characterized and validated, transcriptome analysis of Ccs gene in the different genotypes, homology modeling the Ccs enzyme and prediction of active sites could derive more information on the identified SNPs