Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

Robert N G Miller,Paulo C Alves,Candice M R Santos,Franc C Baurens,Roberto C Togawa,David J Bertioli,Natalia F Martins,Georgios J Pappas,Manoel T Souza

doi:10.1186/1471-2229-8-15

Abstract

BackgroundMany commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.ResultsA computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.ConclusionThis is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 – ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

Highlights

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background
In order to enrich the fraction of resistance gene analogs (RGAs) candidates in Musa recoverable by PCR, an in silico protocol was devised to facilitate design of degenerate primers derived from monocotyledon sequences and targeting nucleotide-binding site (NBS) and additional domains
We report the first large scale analysis of NBS-leucine-rich repeat (LRR) RGAs in this cultivar, using a degenerate primer design strategy devised for targeted RGA amplification across monocotyledon genomes

Summary

Introduction

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. In the case of Fusarium wilt, chemical control is ineffective For these reasons, the development of new disease resistant varieties is of paramount importance for the Musa industry. Whilst wild species are generally fertile, many of today's commercial cultivars are sterile triploids or diploids, with fruit development via parthenocarpy. This translates to seedless fruits, or fruits which contain mostly non-viable seeds. As such cultivars have largely evolved via asexual vegetative propagation, their genetic base is narrow, with diversity dependent upon somatic mutation Such limited genetic variation has resulted in a commercial crop that lacks resistance to pests and disease, as observed in cultivars such as Gros Michel and Grande Naine [2]

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