PG Thesis

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    DNA barcoding in Abelmoschus spp.(Malvaceae)
    (Department of Vegetable Science, College of Agriculture,Vellanikkara, 2025) Oliviya Regi.; Deepu Mathew
    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.
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    In-situ characterization and DNA profiling of selected genotypes of tamarind (Tamarindus indica L.)
    (Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2025-04-16) Mrithyunjay Nandakumar; Asna, A C
    Tamarind (Tamarindus indica L.), belonging to the family Leguminosae, is widely utilized in food, traditional medicine, cosmetics, and other industries. It is particularly valued for its acidic pulp as a souring agent. Despite its economic and ecological significance, research on its genetic variability, remains limited, especially in Kerala, the region harbouring unique primitive cultivars such as ‘valanpuli’ and sweet types like ‘madhurapuli’ and ‘thenpuli’. The present study which forms a part of the ongoing state plan was taken up in this background at COA, Vellanikkara during 2022-2025 with the objectives of in-situ characterization and DNA profiling of selected genotypes of tamarind. Thirty steady bearing tamarind genotypes located from three revenue blocks of Palakkad district of Kerala formed the material for the study. These genotypes were subjected to morphological, biochemical and molecular evaluation. The morphological evaluation based on 11 qualitative and 17 quantitative characters was done using the descriptors from established studies (Patil, 2004; Singh, 2010) and Protection of Plant Varieties and Farmer's Rights Authority (PPVFRA,2017) guidelines. TSS, total sugar, titratable acidity, vitamin C, total antioxidant capacity and seed protein were estimated following standard procedures for biochemical characterization. Wide variability was observed among the accessions for all the quantitative traits. Variations expressed by the genotypes in terms of both pod and seed quantitative traits was also high. Among the genotypes, KTJ 166 was having the highest pod weight and pod length. The highest pulp percentage was observed in KTJ 162, while the highest real pulp value (RPV) was recorded in KTJ 164 (10.83). The biochemical characters viz., TSS, total sugar, titratable acidity, vitamin C, total antioxidant capacity and seed protein ranged from 46.35 to 85.97°B, 13.96-41 per cent, 7.59 - 13.27 per cent, 5.12 - 20.93 mg/100 g, 26.50 to 58.36 mg AAE/g and14.43 g/100 g to 19.87 g/100 g, respectively. The sugar-to-acid ratio in tamarind is a good way to differentiate between sweet and sour types. Two genotypes, KTJ 60 and KTJ 48, exhibiting acidity levels below 8 per cent, with total sugar content varying between 36.30-41.00 per cent showed a high sugar acid ratio representing sweet types. Based on organoleptic evaluation, the maximum score for overall acceptability was also obtained for KTJ 48 followed by KTJ 88 and KTJ 66. Agglomerative hierarchical clustering based on quantitative and biochemical characters resulted in five clusters. Clustering clearly separated KTJ 77 and KTJ 162 from other genotypes by placing them in cluster II and III, respectively. High heritability coupled with high genetic gain was observed for pod weight, pod length, pulp percentage, fibre percentage, seed weight, number of seeds per pod, TSS, titratable acidity, ascorbic acid, total antioxidant capacity and total sugar. Pod weight, a crucial determinant of yield, demonstrated significant positive correlation with pod length (r = 0.94), RPV (r= 0.90) seed weight per pod (r = 0.94), and number of seeds per pod (r = 0.71). RPV demonstrated positive correlations with pod weight, pod length, pulp percentage, seed weight per pod and number of seeds per pod. TSS demonstrated a strong positive correlation with total antioxidant capacity. Conversely, titratable acidity exhibited a significant negative correlation with antioxidant capacity (r = -0.735). Principal Component Analysis (PCA), done for the purpose of clustering the accessions, revealed that the first three components explained 88.93 per cent of total variation. From the comprehensive evaluation of PCA analysis and clustering, four genotypes—KTJ 84, KTJ 164, KTJ 166, and KTJ 174—were identified as plus trees with high RPV, while KTJ 48 (41%), KTJ 60 (36.3%), KTJ 77 (39.52%), and KTJ 172 (36.5%) stood out for notable total sugar content. All 30 genotypes were initially screened with 39 random primers, of which all produced reproducible bands except ISSR-835, ISSR-815, (CT)₈T, and (GT)₈GC. The number of bands per primer ranged from 3 to 13, averaging 6.32. ISSR-29 generated the highest number of amplicons (368), while ISSR-22 produced the least (80). The highest polymorphism was observed in ISSR-22 (62.5%), followed by ISSR-18 (56.83%), and ISSR-2 (47.98%), whereas ISSR-30 had the lowest (4.11%). The UPGMA dendrogram generated via NTSYS-pc software using Jaccard similarity coefficients indicated high genetic similarity among accessions. At 60 per cent similarity, 30 genotypes clustered into five groups. KTJ 169 exhibited the greatest genetic distinction, forming an independent branch, indicating some genetic divergence. Among 18 shortlisted DNA markers, seven (ISSR-1(800bp) ISSR-2 (1000bp), ISSR-17 (600bp), ISSR-18 (525bp), ISSR-25 (625bp, 725bp), ISSR-26 (900bp), ISSR-34 (325bp)) effectively detected polymorphism, producing unique bands in KTJ 174, KTJ 51, KTJ 162, KTJ 172, KTJ 169, KTJ 164, and KTJ 89. ISSR 1 (550bp) differentiated KTJ 72 and KTJ 77, while ISSR-1 (700bp) and ISSR-6 (675bp) uniquely identified KTJ 48 and KTJ 60. ISSR-18 (625bp) separated KTJ 48, KTJ 60, and KTJ 170, and ISSR-24 distinguished KTJ 48, KTJ 60, and KTJ 174. ISSR-25 (525bp) effectively differentiated KTJ 48, KTJ 84, KTJ 164, and KTJ 168. The study provides a comprehensive assessment of tamarind genetic diversity through morphological, biochemical, and molecular characterization. Molecular markers identified in this study can facilitate further genetic studies and marker assisted selection in tamarind improvement programs. The findings of this research are expected to contribute to the conservation and improvement of tamarind germplasm for future breeding programs.
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    Characterization of Mycosphaerella spp. causing sigatoka leaf spot disease complex of banana in Kerala and its management
    (Department of Plant Pathology College of Horticulture, Vellanikkara, 2019) Milsha George; KAU; Anita Cherian, K
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    Hybridization using promising second generation inbred and molecular characterization of a the third generation inbreds of WCT coconut
    (Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2018) Hassain, N; Sujatha, R
    The research project entitled “Hybridization using promising second generation inbred and molecular characterization of the third generation inbreds of WCT coconut (Cocos nucifera L.)” was carried out in the Department of Plant Breeding & Genetics, College of Agriculture, Padannakkad during the year 2016-2018. The major objectives of this study include development of D x T hybrids using promising palms in second generation (S2) inbreds of West Coast Tall (WCT) as male parent and Malayan Yellow Dwarf (MYD) as female parent and characterization of third generation (S3) inbred seedlings of superior S2 palms of WCT using RAPD analysis. For hybridisation, the male parent was selected from the superior inbred family based on the reports on morphological evaluation and estimation of inbreeding depression during the period 2013-2015 (Chethana, 2016). The family IIIS2-1 showed lowest inbreeding depression for yield related traits among 12 families followed by VS2-2. The palm 313 (IIIS2-1) and 225 (VS2-2) were selected as male parents. Superior MYD palm to be used as the female parent was selected from among the MYD palms available at CoA, Padannakkad based on the general features of mother palm selection (Nair et al., 2008). Observations were recorded from these parental palms which included 24 morphological characters. The male parents showed inbreeding depression for several vegetative characters such as stem girth, functional leaves etc and reproductive characters such as number of female flowers per inflorescence, setting percent and nut yield per palm per year, compared to WCT. The results showed that inbreeding depression for these characters was more in palm 313 (developed by selfing) than palm 225 (developed by sibmating). Total five nuts were harvested from MYD (two nuts by using pollen from palm 313 and three nuts using pollen from palm 225) during May 2018 and were sown in the nursery. Molecular characterization of the 15nos of S3 inbred seedlings of IIIS2 family planted in the field in replicated plots were done using RAPD. Genomic DNA was isolated from 15 seedlings of IIIS3 family among which 10 were produced by selfing the S2 palms (IIIS3-1) and 5 were produced by sibmating the S2 palms (IIIS3-2) along with COD, CGD and WCT. After DNA isolation, samples were subjected to RAPD analysis using 10 decamer primers namely OPAU02, OPBA3, OPAW14, OPAW19, OPAW12, OPAW13, OPAW13, OPAW09, OPAU03, OPAW08 and OPAW15 selected based on screening 30 primers. Using primer OPAW08, a band of size of ~400bp was specifically found in inbreds alone while it was absent in dwarfs and WCT seedlings. In OPAU 03 primer, band size of ~1200bp was absent in WCT but present in all inbreds and CGD. The sibmated inbred NL1 showed a distict pattern when amplified with OPAW 09 compared to all other inbreds. The primer produced a specific band of size ~1050bp only in the sibmated inbred CL1 and was absent in all other samples. Amplification with OPAW19 primer produced a banding pattern in which it mainly distinguished CGD from all other seedlings where there were only two bands while others were having six bands. Primer OPAU 02 produced two unique amplicons: 1100bp present in selfed inbred I1M1 TH2 and MYD; 1000bp present in COD and MYD. Genetic similarity among the 15 IIIS3 seedlings, CGD, COD, MYD and WCT was estimated by using of software DARwin (Darwin 6.Ink). The dendrogram indicated three major clusters in which the grouping of several inbreds along with WCT, COD and CGD may be an indication supporting the hypothesis that the dwarfs might have been originated from the talls during the course of evolution followed by selection. The two dwarfs present in the group are COD and CGD which are from Chavakkad area in Kerala and WCT is the most common cultivar of Kerala. As a future line of study, further evaluation of these S3 inbreds regarding the flowering time and yield characters has to be carried out to find out the genetic potential of these inbreds as breeding lines for producing superior hybrids. The seedlings to be emerged from the hybrid nuts produced in the present study, though limited in number, needs to be evaluated for important traits and the hybridisation programme using superior palms in S2 generation needs to be continued for production of more number of hybrid nuts.
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    Development of molecular markers for blight disease resistance in taro using bioinformatics tools
    (Department of Plant Biotechnology College of Agriculture, Vellayani, 2018) Athul, V S; Sreekumar, J
    Development of molecular markers using sequential information publicly available in the biological databases has enhanced their credibility over the years. The study entitled “Development of Molecular markers for blight disease resistance in taro using bioinformatics tools” was conducted at the Central Tuber Crop Research Institute (CTCRI) during 2017-2018. The objectives of the study included the development and evaluation of various Single Nucleotide Polymorphism (SNP) and Simple Sequence Repeats (SSR) prediction pipelines, computational prediction and validation of the molecular markers for blight disease resistance in taro. The preliminary data set for the study was obtained from the Sequence Read Archive (SRA) section of NCBI. A total of 6,479,882 sequences obtained initially were reduced to 6,319,834 after pre-processing. The processed sequences were reduced to 79,608 sequences after de novo assembly and were finally assembled to 8547 contigs and 59,242 singlets. The contigs were then processed with various prediction pipelines to predict SSRs and SNPs. The tools, QualitySNP and AutoSNP were employed to detect the SNPs present within the contig sequences. The efficiency of these tools in determining the number of synonymous and non-synonymous SNPs was also analyzed. The tools, MISA and SSRIT were used to detect the SSRs within the sequences. The efficiency in predicting more number and types of reliable repeats were considered. The analysis was done with a wide range of repeats such as mono-, di-, tri-, tetra-, penta-, hexa-, and poly repeats and their numbers. QualitySNP identified 518 synonymous and 44 non-synonymous SNPs from the 8547 contigs. MISA identified 967 mono-, 1484 di-, 558 tri-, 14 tetra-, 2 penta-, 9 hexa-, and 393 compound SSRs. Five SNP and SSR primers were designed and synthesized from the contigs containing SSRs and SNPs. The synthesized SNP and SSR primers were then validated against tolerant and susceptible varieties of taro leaf blight. Among the primers synthesized the SSR primer CeSSR4 and SNP primer CeSNP3 were capable of differentiating leaf blight resistant and susceptible varieties. The markers need to be analyzed further with a large number of samples to develop them as a marker for taro leaf blight. Once analyzed, they could be used in marker-assisted selection and breeding programmes of taro.