1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Real-time PCR assay for p-1,3- glucanasc gene expression in black pepper (Piper nigrum L.)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2009) Dukare Kiran Shankar; Nazeem, P AItem Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex Poir)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Sahna Hamsa, N H; Girija, DThe study entitled “Molecular characterization of geminivirus causing yellow vein mosaic in pumpkin (Cucurbita moschata Duch. Ex poir.)” was carried out in the Molecular Biology Laboratory of Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2009-2011. The objectives of the study included the molecular characterization of geminivirus causing yellow vein mosaic disease in pumpkin and developing a PCR based diagnostic kit. Yellow vein mosaic infected pumpkin leaf sample was collected from the field of Olericulture Department, College of Horticulture, Vellanikkara and total genomic DNA was extracted by CTAB method. Specific primers for coat protein and movement protein genes were designed based on the sequences of geminiviruses infecting vegetables, obtained from the NCBI database. PCR amplification was carried out using these primers. Amplicons of size ~900bp and ~700bp were obtained for coat protein and movement protein genes respectively. The purified PCR products were ligated in pGEMT plasmid vector and cloned. The recombinant E. coli cells were selected based on blue white screening on LB agar containing ampicillin layered with X-gal and IPTG. After confirmation of the inserts by colony PCR, the clones were sequenced. Sequences of 891bp and 702 bp were obtained for coat protein and movement protein genes respectively. Sequence analysis was carried out with standard bioinformatics tools. On blastn analysis both coat protein and movement protein sequences showed maximum similarity to Squash leaf curl China virus (SLCCV) from Coimbatore. For coat protein gene, full length ORF of 771bp was obtained and the ORF of movement protein was partial. Primers MP1F and MP1R with expected amplicon size of 1363bp were designed to get full length ORF (846bp) of movement protein gene. The technique was validated with DNA from 15 PYVM infected and 4 healthy pumpkin leaf samples collected from Palakkad, Thrissur and Malappuaram districts. The virus was detected only in the diseased samples. Hence these primers could be used in developing a molecular diagnostic tool to detect the virus. PCR amplifications were carried out in weed plants like Emilia sonchifolia, Ageratum conyzoides, Hibiscus surattensis and Synedrella nodiflora and crop plants like okra and ash gourd with yellow vein symptom to check whether these plants serve as the collateral hosts of the virus. PCR amplification was also performed in bitter gourd with distortion mosaic symptom. No amplifications were obtained in plants other than pumpkin. Using the primers PYVMV was detected in apparently healthy mature leaves of infected pumpkin. Hence, these primers could be used to detect latent infection. Sequence and phylogenetic analysis of coat protein and movement protein gene sequences showed maximum similarity to bipartite Squash leaf curl china virus (SLCCV) from Coimbatore. Hence Pumpkin yellow vein mosaic virus (PYVMV) from Kerala can be considered as a strain of SLCCV.Item Sex determination in nutmeg (Myristica fragrans Houtt.) through molecular and biochemical markers(Centre for Plant Biotechnology, College of Horticulture, Vellanikkara, 2010) Maddela Sudhamayee; Valsala, P ANutmeg (Myristica fragrans Houtt.) is an important tree spice of southern India, Malaysia and Indonesia yielding two products of commercial value, ‘nutmeg’ and ‘mace’. Nutmeg of commerce is the dried seed and mace is the dried aril. It is a spreading evergreen tree of the family Myristicaceae and is dioecious with long pre bearing period of 5 to 7 years. Presently, the dioecy in nutmeg is overcome by vegetative propagation or top working. Even though several vegetative methods have been reported, the large scale adoption of these methods is constrained due to insufficient number of orthotrops. So seedling continues to be the major propagating material. In the present study, an attempt was made to identify sex in nutmeg through molecular and biochemical markers so as to identify sex at seedling stage itself. Molecular marker used was RAPD and biochemical marker was isozymes of acid phosphatase and glutamate oxaloacetate transaminase (GOT). Based on bearing pattern and floral morphology, five typical male and female plants of age 10-15 years were selected for RAPD analysis. DNA isolation technique was standardised using CTAB method. Good quality DNA with UV absorbance ratio (A260/A280) 1.80- 1.84 was used for analysis. Sixty seven decamer primers were screened and four primers showing the polymorphism has been selected for further RAPD analysis. PCR amplification with selected primers viz. OPD 15, OPA 27, OPF 05 and OPK 01 was carried out with samples of bulk DNA from five male and female, DNA of individual male and female and negative control. Among them OPK 01 amplified reproducible female specific band (1.1 kb) in bulked and individual samples. The polymorphic female specific band amplified by OPK 01 primer was eluted and cloned in pDrive vector and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed white clones were sequenced at Bioserve, Hyderabad. The sequence obtained after VecScreen (Nut seq 816 bp) was analysed with various bioinformatic tools like Blastn, Blastp, GenScan, ORF Finder, Transeq, GOR and Protparam. Three open reading frames identified in the sequence Nut seq816 sing NCBI tool “ORF Finder”. The +2 ORF strand encodes two domains for amino asparate transaminase (GOT) and cystathionine beta- lyases with E value 3.6. The other ORFs didn’t encode any domain. GenScan predicted no gene in the Nut seq816. Based on sequence information two pairs (SP1 and SP2) of SCAR primers (24 bp) were designed using primer3 programme. The efficiency of SCAR primer to distinguish male and female plants was tested by PCR amplification of DNA from five male and females. The SCAR primers amplified around 300 bp single band in five females. SCAR primer was again checked with four occasional fruiting males and it amplified 300 bp band in one occasional fruiting male. Expression of SCAR primers was tested in ten seedlings and got amplified in four samples. Leaf samples from identified male and female trees were used for the biochemical marker analysis. Polyacrylamide gel electrophoresis (PAGE) for isozyme assay was standardized with standard protein and nutmeg leaf protein. Acid phosphatase assay recorded four zones of activity and all were monomorphic. Glutamate oxaloacetate transaminase showed polymorphism and need protocol refinement. Accuracy of SCAR primer SP1 to distinguish male, female and occasional fruiting male has to be done with more samples. Commercialization of the technique can be explored based on the accuracy and cost benefit analysis.Item Beta lactoglobulin polymorphism in goats of Kerala(Department of Animal Breeding, Genetics and Biostatistics, College of Veterinary and Animal Sciences, Mannuthy, 2010) Sudina, K; Bindu, K A