1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Development of scar marker for authentication of gender in kodampuli (Garcinia gummi-gutta var.gummigutta)(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2010) Shelke Sunil Marotarao; Rajendran, P CThe diabetes and cardiovascular disease are the two serious obesity related life-style diseases, spreading at alarming rate throughout the world, especially in thickly populated third world countries in which India occupies the prime position. The fleshy fruit rind of Kodampuli (Garcinia gummi-gutta) is the richest natural source of anti-obesity metabolite hydroxyl citric acid (HCA). Which inhibit the conversion of carbohydrate to fats without affecting Kreb’s cycle through an enzyme ATP citrate lyase. Since Kodampuli is a polygamodioecious tree, it takes 8 to 12 years to identify the female trees. No significant reports are available for sex determination in Kodampuli on the basis of physiological, biochemical or molecular characters. Sex identification, lack of orthotropic shoots for grafting, prolonged seed dormancy, poor seed germination and lack of awareness of its pharmaceutical significance are hindering the extensive cultivation of this backyard companion crop in Kerala and other coastal regions of country. In the present study, an attempt was made to develop simple PCR based technique which can use for gender diagnostic in this plant. DNA samples were extracted from field grown 15 to 20 years old 25 male and 25 female trees and were bulked to 5 samples each by sex type. Earlier reported RAPD primers viz. Kit C1, Kit C8 and Kit C9 were screened but no significant polymorphism was observed. So a total of random 46 decamer primers were tested and six primers were selected for further analysis. On rescreening of the six selected primes viz. RN 5, RN 9, RN 10, RY 5, RY 18 and OPAH 12 only OPAH 12 reproduce male specific band in bulked and individual samples. Random amplified polymorphic DNA (RAPD) fragments were generated in the both bulks in order to identify markers that were polymorphic between male and female plants. A 550 base-pair (bp) male-specific DNA fragment generated with the OPAH-12 primer was identified. The polymorphic male specific band produced by OPAH 12 primer was eluted and cloned in pGEM-T vector, and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed one was sent for sequencing The sequence obtained after vector screening was subjected to nucleotide blast search and ORF finder. It does not reveal any significant levels of homology and reading frame. Two pairs of SCAR primer were designed on the basis of sequence. These SCAR primers were checked for male and female samples but no polymorphic band was observed. The future line of work can be to screen the male and female genotypes with more number of primers to obtain larger base pair polymorphic band. That can used to convert this dominant marker to co-dominant one like SCAR marker. SCAR marker would be successfully employed in breeding experiments for Marker Assisted Selection.Item Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Choudhary Rakeshkumar Sheshrao; Kesavachandran, RThe present investigation on “Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers” was undertaken in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2009-2011 with an aim to characterize the variability in plantain ecotypes of banana (Musa spp.) using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Twelve plantain ecotypes collected from BRS, Kannara was used for the study. Standardisation of DNA was done with the CTAB method. Optimum PCR conditions for both RAPD and ISSR were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 60 RAPD and 40 ISSR primers were screened against genomic DNA of two plantain ecotypes (Big Ebanga and Njockkon) for their ability to amplify DNA fragments. Of these, 16 RAPD and 14 ISSR primers were selected for further detailed RAPD and ISSR profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on 1.4 per cent and 1.6 per cent agarose gel respectively stained with ethidium bromide. A total of 138 bands were obtained by using 16 RAPD primers. The number of bands produced by the primers varied from 5 (OPS 40) to 14 (OPS 31 and 37) and the molecular weight of bands varied from 2.876 to 0.564 Kb. The average number of bands was 8.63 and average percentage of polymorphism was 3.25. The total percentage of polymorphism was 37.68. The Polymorphic Information Content (PIC) value for 16 primers varied between 0.79 (OPS 40) and 0.92 (OPS 37) with mean of 0.87. The Resolving power (Rp) of the random primers ranged between 9.33 (OPS 12) and 24.33 (OPS 37) with an average of 15.49. The Marker Indices (MI) of primers varied from 0.83 (OPS 7) to 7.28 (OPS 31) with a mean of 2.86. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Njockkon and Changalikodan, occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. A total of 111 bands were obtained by using 14 ISSR primers. The number of bands produced by the primers varied from 5 (UBC 835, 820) and 11 (UBC 857) and the molecular weight of bands varied from 1.584 to 0.564 Kb. The average number of bands was 7.93 and average percentage of polymorphism was 4.14. The total percentage of polymorphism was 52.25. The PIC value for 14 primers varied between 0.77 (UBC 835) and 0.90 (ISSR 6) with an average of 0.85. The resolving power of the ISSR primers ranged between 7.83 (UBC 820) and 16.83 (ISSR 6) with an average of 12.49. The Marker Indices (MI) of primers ranged from 0.77 (UBC 835) to 8.01 (UBC 857) with a mean of 3.55. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Changalikodan and Zanzibar; Manjeri Nendran (a) and Manjeri Nendran (b) occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. The combined dendrogram was also derived from pooled data from RAPD and ISSR analysis and morphological data. The dendrogram generated revealed grouping of plantain ecotypes into clusters more or similar to earlier dendrogram with a few exceptions. The study revealed that variability exists among the plantain ecotypes of banana.