Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers.

Abstract

The 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.