PG Thesis
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Item Genetic diversity analysis of gladiolus genotypes (Gladiolus grandiflorus L.) using molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022) Karthika Nair, A S; Beena ThomasCharacterization of plant genotypes based on crop genetic diversity is important for effective usage and conservation. This is generally achieved by either morphological tools or molecular tools or by using both. This study entitled “Genetic diversity analysis of gladiolus genotypes (Gladiolus grandiflorus L.) using molecular markers” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram during 2020-2021 with an objective to analyse genetic diversity in the gladiolus genotypes using ISSR as well as morphological markers. Gladiolus (Gladiolus sp.) is a genus of perennial herbaceous cormous flowering plants in the family Iridaceae which is of high economic importance. Fifteen varieties of gladiolus including twelve varieties from IIHR, Bangalore and three varieties from NBRI, Lucknow were selected for this study. The study was divided into two parts- morphological characterization and molecular characterization. Morphological characterization was done by analysing both vegetative and floral characters. Different tools such as analysis of variance, co-variance, correlations, PCA and dendrogram were used for statistically analysing the recorded data. The dendrogram divided the genotypes into two principal clusters at a distance of 0.10. The major variables that contributed to the clustering of gladiolus genotypes were plant height, number of leaves per shoot, length of leaf, width of leaf, internode length, number of florets open at one time and number of florets per spike as revealed by PCA analysis. For molecular characterization using ISSR markers the genomic DNA was isolated using CTAB method of DNA isolation with little modifications. Ten ISSR primers were used for screening fifteen gladiolus genotypes. After the final PCR with selected primers, the amplicons were resolved in 2% agarose gel and polymorphic bands were obtained. Primers showed 94.56% polymorphism and the number of bands obtained ranged from 3(UBC 857) to 14 (UBC 890) with a mean value of 8.7 polymorphic bands per primer. A total of 87 polymorphic bands were obtained. The data analysed using NTSYS PC 2.02 program created a dendrogram, which grouped 113 the genotypes based on Jaccard’s similarity coefficient in which the fifteen genotypes were separated into two principal clusters. The first principal cluster consisted of most of the genotypes (12 genotypes). The second principal cluster comprised of ‘Arka Naveen’, ‘Archana’ and ‘Arka Gold’ with ‘Archana’ as an outlier. In molecular characterization, least similarity of 34% was observed between G3 (Arka Sapna) and G9 (Archana) whereas, maximum genetic similarity of 82% was observed between G6 (Arka Amar) and G10 (Arka Kumkum). The highest morphological similarity was also observed between G6 (Arka Amar) and G10 (Arka Kumkum) at a distance of 0.83 in UPGMA dendrogram based on Jaccard’s coefficient. Though some similarity in results existed between the morphological and molecular tools used for identifying the genetic relationships among selected gladiolus varieties in this study, it also revealed that the varieties were grouped as separate clusters based on morphological dendrogram. This may be due to the dependence of morphological expression on the physiological state of the individual plant along with environmental influence. Self-incompatibility, along with the outcrossing nature together might have contributed to the high variation observed among the gladiolus genotypes. Being a commercial cut flower crop, based on different floral parameters considered ‘Arka Sapna’, ‘Arka Nazrana’, Arka Darshan’, ‘Arka Amar’ and ‘Arka Poonam’ are recommended as the gladiolus genotypes that showed best performance in Kerala conditions. Tags from this library: No tags from this library for this title.Item Identification of microsatellite markers associated with root traits for drought tolerance in rice (Oryza sativa L.)(Department of Plant Technology, College of Agriculture, Vellayani, 2017) Rejeth, R; Beena, RItem Characterisation and systematic evaluation of genetic resources of the genus Vigna(Department of Plant Breeding and Genetics, College of Horticulture,Vellanikkara, 2010) Latha, M; Pushpalatha, K TVigna belonging to the family Leguminoseae is a large genus comprising of seven sub-genera and over 150 species. The two sub-genera Vigna and Ceratotropis contain the most important cultivated species. The taxonomical identification of many of these species is still confusing. The closely related wild species serve as a source of many desirable genes that can be utilised in the interspecific hybridisation programmes. This is possible only when the relationships among the different Vigna species are well understood. In this context, the present study “Characterisation and systematic evaluation of genetic resources of the genus Vigna” was undertaken in the Department of Plant Breeding and Genetics, College of Horticulture at Vellanikkara. Investigations were undertaken to characterise the accessions of Vigna germplasm available at National Bureau of Plant Genetic Resources (NBPGR) Regional Station, Vellanikkara, Thrissur using morphological markers and to confirm the results using biochemical and molecular markers in distinct variants belonging to different taxa as well as to prepare a key for the identification of different Vigna taxa. The 150 accessions available at NBPGR Regional Station, Vellanikkara were subjected to morphological, biochemical and molecular characterisation. For morphological characterisation 48 qualitative and 24 quantitative characters were taken. The biochemical characterisation of the selected distinct variants from each taxa was done by isozymes, peroxidase and poly-phenol oxidase. The molecular characterisation was done with Inter Sequence Repeat Analysis using 10 different primers. The clustering patterns based on all three characterisation were compared and key for identification of different taxa of Vigna was developed. Among the qualitative characters evaluated, type of seed germination, nature of attachment of primary leaves, size of stipule, shape of stipule, presence of ligule, shape of bracteole, nature of pod attachment to peduncle, curvature of pod, shape of seed and shape of hilum were distinct for each taxa. Variability was observed in size and shape of stipules and bracteoles. Based on the qualitative characters the 150 accessions were reclassified into 22 taxa. One accession originally classified as V.radiata var.sublobata was found to be distinct taxa of Vigna and hence regrouped as distinct taxa. All the 24 quantitative characters studied exhibited wide range of variability. The keel pocket was present in all taxa except V.unguiculata, V.marina and V.pilosa. The length of keel pocket also varied from taxa to taxa. Cluster analysis based on qualitative, quantitative, biochemical and molecular characters resulted in 10, 5, 4 and 12 clusters respectively. A statistical methodology was worked out to compare the parallelism among the different clustering patterns. The result showed that there existed a similarity between clusters formed based on quantitative and qualitative characters, with majority of accessions of each taxa in a qualitative cluster falling in the same quantitative cluster. The accessions taken for isozyme and molecular study were distinct. Accessions of same taxa which fell in same clusters based on isozyme and molecular markers fell in different clusters based on quantitative characters and vice-versa, indicating the differences and similarities among these accessions at isozyme and molecular level. Key quantitative characters were also identified for each taxa based on weighted average. Based on morphological, biochemical and molecular characters, a dichotomous key was developed to identify different taxa. The key that is now proposed is different from the existing one which is based on floral and fruit characters alone.Item Identification of the population genetic structure of Carcharhinus longimanus (oceanic white tip shark or brown Milbert's shark) using mitochondrial DNA markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Sreelekshmi, S; Sandhya SukumaranEven though sharks are the largest fishes in the world with their size varying size and behaviour, they were over exploited and most of them were at the fear of extinction. Among these Carcharhinus longimanus, an epipelagic bottomless shark considered as at the point of extinction were IUCN Red list points out this shark as a “vulnerable” species at global level. In order to implement the management measures for these species which require the information regarding its population in interoceanic regions. Population genetics can be characterized as the study of how hereditary variance is dispersed among the species and population on a very basic level (Hansen, 2003). Assessment of genetic makeup and variability of fish stock is important for scientific management of fishery, conservation and rejuvenation of endangered species. Mitochondrial DNA (mtDNA), which in general possess a five to ten times greater variability than single copy nuclear genes hence, served as a powerful tool for elucidating population structures studies. Among the 150 specimens of C. longimanus sequenced, we obtained sequences ranging from 720 base pairs were obtained 12 polymorphic sites yielding 13 haplotypes. Genetic differentiation among the populations of C. longimanus from Indian Ocean was revealed as a non-significant statistical analysis. Vital insights were gained from this study indicating lack of significant substructuring and its capability to migrate across large expanses of Open Ocean. The capability to migrate may provide it with some buffering against habitat loss and climate change, but excessive fishing is a danger to its populations. Globally sharks are in danger due to their inherent vulnerabilities like long gestation time and reduced number of offsprings coupled with over fishing. Our study also corroborated the findings of shark decline, as decline in genetic diversity is an indicator of decrease in resilience capacity. The present study calls for restrictions on its fishery so that populations will get sufficient time to replenish and consequently their resilience is ensured in the face of changing oceans.Item Molecular characterization and in vitro conservation of taro (Colocasia esculenta (L.)Schott)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Sreevidya, M R; Asha Devi, AItem 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 Evaluation and characterisation of promising hybrids of long pepper (Piper longum L.)(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2017) Sruthy, K; Sujatha, V SPiper longum L., commonly known as long pepper, belongs to family Piperaceae. The species has originated in South Asia. Piper longum is an important medicinal plant used in more than 300 ayurvedic preparations. Inspite of the importance of the species, „Viswam‟ is the only variety released so far. As a part of a KSCSTE funded project, hybridization studies were carried out at the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara to develop high yielding types in Piper longum. In the preliminary evaluation trials, four hybrids were found promising. The present study entitled „evaluation and characterisation of promising hybrids of long pepper (Piper longum L.)‟ was conducted with the objective of evaluating these promising hybrids at different shade levels for growth, yield and quality and also to characterise them using molecular markers. The research was conducted in three experiments viz., evaluation of hybrids in pots at different shade levels, field evaluation of selected hybrids and molecular characterisation of promising hybrids and parents. Morphological characterisation of the accessions was done using IPGRI descriptor for Piper nigrum with necessary modifications. Variations were observed among accessions in shape of leaf, immature and mature spike color as well as shape of spike. Characters like plant height, number of primary branches per plant and time taken for production of first lateral were significantly different among hybrids. Flowering and fruit set were higher at zero per cent and 25 per cent shade compared to 50 per cent shade level. Field planted genotypes of P. longum showed significant difference in number of primary branches, internodal length of orthotropic as well as plagiotropic shoots and leaf area. Characters like pedicel length, spike length, spike girth and yield in terms of number of spikes per plant, fresh weight of spikes per plant and dry spike yield per plant also differed significantly. Among the hybrids evaluated in the field, Pl 9 followed by Pl 63 were found to be promising. They were significantly higher yielders compared to other hybrids, female parent and Viswam. Essential oil content was found to be uniform (0.8 per cent) in all the accessions except Pl 141 (0.83 per cent). Pl 9 showed maximum oleoresin (15.2 per cent) and piperine (3.47 per cent) content than other genotypes. For molecular characterisation using RAPD, 30 decamer primers were screened. From these ten best primers were selected. Six primers showed polymorphism between the male and female parents. The hybrids Pl 9 and Pl 63 were closely related with 92 per cent similarity. Pl 140 was found different from the rest of the three hybrids and it was grouped along with the parents. Among the accessions studied, Viswam showed highest variability from others. Among the hybrids evaluated, Pl 9 and Pl 63 were found to be promising in terms of yield. Pl 9 was superior in quality. These hybrids could be further evaluated in multi-location trials to explore the possibility of releasing as high yielding hybrids in future.Item Characterization of selected accessions of cassava germplasm using morphological and molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2017) Anjali sabu, C; Asha, K ICassava (Manihot esculenta Crantz), a perennial shrub, is an important crop in many parts of the tropics.This research work attempts morphological and molecular characterization of 27 cassava germplasm collected from Southern India. In the present study27 accessions of cassava maintained in the field genebank of ICAR- Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram were characterized based on 20 qualitative and 10 quantitative traits including the major yield components. There were no duplicate accessions identified based on morphological classification and it can be maintained as core collection. The genetic diversity on molecular basis was evaluated using 10 SSR primers. Molecular markers are proved to be valuable tools in the characterization and evaluation of genetic diversity within and between species and population. All the SSR primers in the study showed the polymorphism. The SSR primers on an average produced 9 polymorphic alleles with mean observed hetrozygosity and values of Polymorphism Information Content (PIC) 0.8293 and 0.8091 respectively. The value of heterozygosity here ranged from 0.2975 (SSRY 148) to 0.8293 (SSRY 9). The PIC value ranged from 0.2533 (SSRY 148) to 0.8091 (SSRY 9). The hetrozigosity and average PIC content observed in SSRY 9. Clustering based on morphological descriptors and molecular markers was done. In morphological clustering, Cluster-I consists of 7 accessions was further subdivided into two sub clusters I A and I B. Cluster-I A consisted of four accessions while Cluster-I Bof three accessions. The bigger cluster Cluster-II consisting of 20 accessions was found to have two subgroups namely II A with 12 accessions and II B with 8 accessions. Clustering based on SSR marker analysis grouped the genotypes into 2 Clusters. Cluster-I contain 6 accessions was further sub grouped into I A and I B with 3 accessions each. Cluster II with 21 accessions was found to have two subgroups II A with 13accessions and II B with 8 accessions. By comparing the morphological and molecular clusters, In Cluster II of each dendrogram have 6 similar accessions of cassava (TCR-5, TCR-10,TCR-15,TCR- 45,TCR-79,TCR-69,). Clustering and Principal Componant Analysis of the data validated the variation among the cassava accessions. In PCA of morphological characters the percentage variation obtained in PC component 1 (54.22). Mantel’s test proves that there is no correlation between the morphological and molecular data. The present results indicated that the primers selected for the present study will be useful for future genetic variability studies and would provide breeders with a genetic base for selection of diverse parents for crop improvement programmes in cassava.Item Bulked segregant analysis for heat tolerance in segregating generation of rice (Oryza sativa L.) using SSR markers(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Waghmare Swapnil Gorakh; Sindhumole, P