Cloning and Analysis of Fusarium Wilt Resistance Gene Analogs in ‘Goldfinger’ Banana

Abstract

Based on the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in cloned wilt resistance genes, the polymerase chain reaction with degenerate primers was employed to isolate resistance gene analogues (RGAs) from the genomic DNA of wilt resistance germplasm ‘Goldfinger’ (AAAB) banana. As a result, twenty fragments of RGAs were isolated, which were of expected size (about 530 bp). Analysis of the deduced amino acids of these RGAs show that they share the NB-ARC domain and belong to the non-TIR-NBS class resistance gene candidates, containing 4 conservative amino acid domains, i.e. P-loop (GMGGVGKTT), Kinase-2 (LLVLDDIW), RNBS-B (CKVLFTTRS), and hydrophobic amino acids GLPL (GLPLALKVL). Other results reveal that sequence identity among the 20 RGAs rang from 41.1% to 99.3%, while identity of the deduced amino acid sequences range from 33.2% to 96.3%. The phylogenetic analysis of the RGA nucleotide sequences and the deduced amino acids showed that the 20 sequences could be divided into 5 distinct types. All of the amino acids deduced from the RGAs share a homology of 28%~54% with those deduced from the known wilt resistance genes such as Fom-2, I2C-1, I2C-2 and I2. This result to some degree indicates the conservation of disease resistance gene evolution. Technically, these RGAs isolated in the present study would lay a base for the further cloning of wilt resistance genes in banana, which could also be used as molecular markers for screening candidate wilt resistance genes in banana.