Molecular Characterization of Resistance Banana Cultivars to Panama Wilt Disease Caused by Fusarium oxysporum f.sp. cubense

Abstract

Sustainability of banana and plantain in Indonesia encounters the development of banana pests and diseases that dramatically decreased national banana production. Panama wilt disease caused by Fusarium oxysporum f.sp. cubense (FOC) is one of major banana diseases that infected almost all of banana plantation in Indonesia. A series of experiments were carried out to isolate, characterize resistance gene analogues (RGAs) and defense gene analogues (DGAs), and to develop molecular markers for disease resistance against fusarium wilt. The experiment was started by the assessment of resistance banana cultivars against FOC tropical race-4 (TR4), and selected two cultivars showed resistant to FOC i.e. Calcuta-4 and Klutuk Wulung. From those selected cultivars and one resistant cultivar, Rejang, 17 RGAs were isolated and characterized and showed high sequence identity to NBS-LRR. The RGA sequences were designated as MNBS1-MNBS17. Based on phylogenetic analysis, the RGAs were classified into four groups. First group contained 14 RGA sequences (MNBS1-MNBS14), and the other three groups contained one sequence MNBS15, MNBS16, and MNBS17, respectively. The isolation and characterization of DGA (chitinase and β-1,3-glucanase genes) were carried out using local banana cultivars. Eight putative chitinase sequences (586 bp in length) were isolated from Rejang, Klutuk Wulung, Kepok, Ambon Hijau and Barangan. The sequences were showed high identity (90 %) to banana class II chitinase gene and coded by MaChi. Four putative β-1,3- glucanase sequences (788 bp in length) were isolated and characterized from Rejang, Klutuk Wulung, Ambon Hijau and Barangan. The sequences shared 99 % identity to banana β-1,3-glucanase, and designated as MaGlu. Based on SNP identification, it was revealed that RGA sequences from Rejang (MNBS2-MNBS5) and Calcuta-4 (MNBS6-MNBS14) contained 16 and 9 putative SNPs, respectively. Based on SNP analysis, RGA sequences of Rejang and Calcuta-4 generated 4 and 8 haplotypes, respectively. Twenty two SNPs were identified from 8 chitinase fragments and generated 8 haplotypes, while 8 putative SNPs were identified from 4 β-1,3-glucanase fragments and generated 4 haplotypes. SNAP markers were developed based on non synonymous SNPs identified from RGA (MNBS) and DGA (chitinase and β-1,3-glucanase) sequences. Using PCR technique and allel specific primers approaches, 7 loci based on RGA and DGA sequences were selected as SNAP markers for FOC resistance banana cultivars, there were SNP4_MNBS, SNP2_MChi, SNP6_MChi, SNP8_MChi, SNP10_MChi, SNP11_MChi, and SNP1_MGlu. However, the use of SNP1_MGlu locus can be omitted, because without SNP1_MGlu primers, the grouping of banana cultivar base on FOC resistance will be better.