Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Abstract

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-010-1395-5) contains supplementary material, which is available to authorized users.

Introduction

“…TILLING has been applied to over 20 plant and animal species, and similar approaches have been used to characterize naturally occurring nucleotide variation, known as Ecotilling Jankowicz-Cieslak et al 2011). While TILLING and Ecotilling have been primarily used in seed crops, the methods work well in vegetatively (clonally) propagated and polyploid species such as banana and cassava (Jankowicz-Cieslak et al 2012;Till et al 2010). The PBGL has developed low-cost methods for the extraction of enzymes from a variety of plant materials, including easily obtainable weedy plants.…”

Targeted mutation breeding of horticultural plants

“…translation initiation factor 4E). Till et al, 2010Till et al, , 2011 Candidate gene sequence from the target species is needed for genetic screening. Sequenced genomes of over 30 plant species are currently available, at least in draft form, and genomic analysis of more than 100 other plant species is in progress (NCBI Genome Project database; http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi).…”

“…An EcoTILLING analysis of 80 banana accessions identified 870 novel alleles in 14 genes (Till et al, 2010). Variants in eIF4E that conferred virus resistance were discovered by the genetic screening of 113 melon accessions and 233 pepper accessions (Nieto et al, 2007;Ibiza et al, 2010).…”

A Protocol for Benchtop Extraction of Single-Strand-Specific Nucleases for Mutation Discovery

“…The use of crude enzyme extracts was shown to result in accurate recovery of DNA variation. In studies of both human samples and highly heterozygous polyploid bananas, false-negative and false-positive discovery errors were reported below 5 % (Till et al 2006a, b;Till et al 2010). While many TILLING and Ecotilling projects have utilized fluorescence-based detection of DNA to increase sensitivity and throughput, the development of a more simple and low-cost method employing PCR and standard gel electrophoresis has made mutation discovery suitable for any laboratory equipped for basic molecular biology (Dong et al 2009;Uauy et al 2009;Lee et al 2014).…”