MSc

Banana is one of the important fruit crops of India. It is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production are Fusarium wilt or Panama wilt caused by
Fusariumoxysporumf. sp. cubense (Foc). Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by
conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Cloning and characterization of Fusarium wilt resistance gene analogs in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2013- 2015 with an objective to PCR amplify the genomic DNA from Fusarium wilt resistant banana genotype with primers specific to ‘R’ genes of TIR-NBS-LRR class for cloning and characterization of resistant gene analogs. 105 Palayankodanresistant (Mysore Poovan AAB) and Poovan
susceptible(Rasthali AAB)varieties of banana were used for the present study.Fusarium culture was isolated from roots of infected banana plant and cultured inPDA (Potato Dextrose Agar) media. Fungal spore was suspended in sterile water
and filled in small polythene bag. Artificial inoculation – root feeding of inoculum of water suspension was done. The symptoms of Fusarium wilt was observed two months after infection. Results confirmed resistance of Palayankodan and
susceptibility of Poovan The DNA was isolated from Palayankodan (resistant) and Poovan (susceptible)genotypesand isolated DNA was subjected to RNase treatment. Quality checking was done using 0.8% agarose gel electrophoresis and quantity analysis was
done using nanodrop ND-1000 spectrophotometer. Thirty five ng of DNA was used
as template DNA for PCR amplification using reported degenerate primers. Twenty five primer combinations were made using five pair of degenerate primers. Among those combinations only one primer combination F9(F)+F6(R) showed polymorphic band of 700bp which was eluted, purified and cloned in pGEMT easy vector system.The presence of insert was confirmed by colony PCR and the eluted product was sequenced by outsourcing. The sequence obtained was subjected Blastn, Blastx and Blastp analysis and was compared with NCBI database. The sequence showed similarity with NBS-LLR resistant gene of Musa spp. Open reading frames (ORFs)
were also identified using ORF finder software and four ORFs were identified For further validation, new primers were designed from 675 bp region of NBS-LLR class of resistant gene using primer3 plus software with an expected band size of 430bp. The DNA from both infected and healthy samples were amplified with designed primers and the expected bands were obtained in DNA samples of healthy 106 (resistant) plants where as it was absent in DNA samples of infected (susceptible) plants.
The degenerate primer F9(F)+F6(R) and designed primer FTGAGCAGCATCGCCTA. R- GCCTGACACCAGTGAAGC can be used for Fusarium wilt disease diagnostics. Sequence information with respect to above primers amplicons can be used for synthesis of gene construct for genetic engineering.