1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item DNA barcoding in Abelmoschus spp.(Malvaceae)(Department of Vegetable Science, College of Agriculture,Vellanikkara, 2025) Oliviya Regi.The genus Abelmoschus, a member of the Malvaceae family, is reported to have 14 species worldwide. Of these, twelve viz., A. esculentus (L.) Moench, A. moschatus Medik., A. ficulneus (Linn.) Wight & Arn., A. manihot (L.) Medik., A. tetraphyllus (Roxb. ex Hornem.) Borss., A. crinitus Wall., A. tuberculatus Pal & Singh, A. caillei (A. Chev.) Stevels, A. angulosus Wall. ex Wight & Arn., A. enbeepeegeearensis, A. palianus and A. odishae, are reported in India, either as native or introduced species. The A. esculentus and A. callei are cultivated for edible pods whereas A. manihot is primarily grown for its consumable leaves, and A. moschatus is valued for the muskscented seeds. Taxonomic classification and species identification in this genus is often challenging due to their polyploid nature, morphological plasticity and intermediate crossability, all resulted through long cultivation and global dispersion. Accurate identification of species is extremely important in crop breeding programmes. DNA barcoding accurately differentiates the closely related taxa based on the nucleotide polymorphisms characteristically conserved for each in the candidate locus. The matK is one of the most extensively studied loci in plants, and has been shown efficient in many horticultural crops including Momordica, Luffa, and Mangifera. This study, "DNA barcoding in Abelmoschus spp. (Malvaceae)" was conducted at Centre for Plant Biotechnology and Molecular Biology, Kerala Agricultural University, and ICAR-National Bureau of Plant Genetic Resources, Regional Station, Thrissur, with the objective of generation of characteristic barcodes for different species in genus Abelmoschus using identified chloroplast genomic locus/ loci. The study included eighteen Abelmoschus accessions representing six species: A. esculentus, A. caillei, A. tetraphyllus, A. tuberculatus, A. moschatus, and A. enbeepeegeearensis. Morphological traits of each accession were also studied in detail to support the species identifications. The accessions were raised in the open field at ICAR-NBPGR, Regional Station, Thrissur during from mid-August 2023 to February 2024. Morphological characterization of six Abelmoschus species, using seventeen traits, revealed diversity in leaf area, pod length, trichome density, seed characteristics, and epicalyx persistence. These traits, assessed using IBPGR descriptors, provided insights into species differentiation and relationships within the genus. Among the species, A. caillei had the largest leaf area, while A. enbeepeegeearensis had the smallest leaves. The A. esculentus and A. caillei produced larger pods, contrasting with A. tetraphyllus and A. tuberculatus, which had shorter pods of less than 7 cm length. Trichome characteristics varied: A. esculentus and A. caillei had pubescent fruit trichomes, while they were short and prickly in A. moschatus, A. tuberculatus, and A. tetraphyllus, and soft and medium in A. enbeepeegeearensis. Seed morphology was distinctive in A. tuberculatus due to trichomes. The epicalyx was non-persistent in A. esculentus, A. caillei, and A. tetraphyllus, adding further differentiation. DNA obtained through manual extraction protocols has failed to amplify the matK gene in PCR reactions and hence good quality DNA was extracted from the tender leaves and seeds using commercial kit (CTAB plant DNA extraction kit). The matK gene from all the accessions was PCR amplified using a universal primer combination designed in this study. PCR products were cleaned up and paired-end sequenced on Sanger platform, sequence contigs generated using CAP3, submitted to the Barcode of Life Data System (BOLD) and barcodes were generated. Alignment of the sequences using MAFFT has shown species-specific nucleotide polymorphisms, with unique variations observed in A. enbeepeegeearensis and A. tetraphyllus. In A. enbeepeegeearensis, all the accessions had specific base substitutions at two positions: adenine (A) replacing thymine (T) at position 54, and cytosine (C) substituting for adenine (A) at position 289. Similarly, A. tetraphyllus exhibited a consistent variation at position 154, where C was replaced with A. These unique polymorphisms in these species are keys to precisely identify them, highlighting their genetic distinction, contributing valuable data to support future taxonomic and conservation efforts. To identify the unique barcodes to differentiate the remaining species of Abelmoschus, alternate chloroplast genes have to be studied. This is the first study on DNA barcoding in this genus and the findings have contributed to its taxonomic resolution. The findings underscore the utility of combining morphological and DNA barcoding methods for accurate species identification and conservation planning.Item Evaluvation of new insecticides against major pests of okra, Abelmoschus esculentus(Department of Agriculural Entomology,Padanakkad, 2019) Reshma Chandran; Ramesha, BAn experiment was conducted on ‘Evaluation of new insecticides against major pests of okra, Abelmoschus esculentus L.’ at College of Agriculture, Padannakkad and RARS Pilicode sub centre, Karuvachery in two seasons viz., rabi season (September to December 2018) and summer season (January to April 2019).The objective of the study was ‘to study the efficacy of different new insecticides against major pests of okra, viz., shoot and fruit borer, gram pod borer, leaf roller and leafhopper’. The variety selected for the study was Varsha Uphar released by Haryana Agricultural University, Hisar. The experiment was laid out in Randomized Block Design with 8 treatments and 3 replications. The different treatments were; T1: Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC (Voliam flexi®) @ 0.7ml/l; T2: Lambda cyhalothrin 4.6 per cent + Chlorantraniliprole 9.3 per cent ZC (Ampligo®) @ 0.4 ml/l; T3: Thiamethoxam 12.6 per cent + Lambda cyhalothrin 9.5 per cent ZC (Alika®) @ 0.5 ml/l; T4: Flubendiamide 19.92 per cent w/w + Thiacloprid 19.92 per cent w/w (Belt-expert®) @ 0.4ml/l; T5: Novaluron 10 EC @ 2ml/l; T6: Chlorantraniliprole 18.5 SC @ 0.3 ml/l (check); T7: Thiamethoxam 25 WG @ 0.3g/l (check); T8: Absolute control. Treatments were applied one at vegetative and one at reproductive stage after the incidence of pest. The observations like damage symptoms were recorded at seven and fourteen days after treatment and population of jassids were recorded at one, three, five, seven and fourteen days after treatment. Biometric observations and yield parameters were recorded during each harvest. The data recorded from field experiment was tabulated and statistical analysis was performed using analysis of variance (ANOVA). Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC (T1 -Voliam flexi) @ 0.7 ml/l of water significantly reduced the percentage of shoot and fruit damage during both rabi and summer season. No infestation was recorded at seven and fourteen days after treatment. It was on par with the standard check Chlorantraniliprole 18.5 SC. The damage caused by leaf roller was significantly reduced by Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC @ 0.7 ml/l of water. There was no incidence of leaf roller after seven and fourteen days after first spray until the end of the crop period during both rabi and summer season. Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC @ 0.7 ml/l of water was very effective against the population of leafhopper. The population of adults and nymphs of leafhopper was drastically reduced by Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC and three days after spray no hoppers were found on plots treated with Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC. It showed non-significant difference with standard check Thiamethoxam 25 WG @ 0.3 g/l of water. Alika (Thiamethoxam 12.6 per cent + Lamda cyhalothrin 9.5 per cent ZC) @ 0.5ml/l of water significantly reduced the population of leaf hopper after Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC. Hopper population was negligible at fifth and seventh day after treatment while it was increased by fourteenth day. Length of the fruit was inversely proportional to the percentage of damage caused by Earias vitella. Maximum fruit length of 14.85 cm and 15.99 cm was recorded from Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC treated plots during both rabi and summer season respectively. This indicates higher efficacy of Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC against E. vitella. The highest total yield was recorded from Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC treated plots during rabi and summer season with yield of 469.86 and 594.31 g/plant respectively. Chlorantraniliprole 8.8 per cent + Thiamethoxam 17.5 per cent SC also showed high benefit-cost ratio of 2.42 and 3.12during rabi and summer season respectively.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 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.