Targeted Next-Generation Sequencing Identification of Mutations in Disease Resistance Gene Analogs (RGAs) in Wild and Cultivated Beets

Piergiorgio Stevanato,Bahram Heidari,Luca Pajola,Claudia Chiodi,Elide Formentin,Lee Panella,Chris Richards,Giuseppe Concheri,Filippo Biscarini,Chiara Broccanello,Mahdi Hassani

doi:10.3390/genes8100264

Abstract

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets (Beta vulgaris L. subsp. maritima) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8_184910_pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet.

Highlights

Sugar beet is an important crop that supplies around 20% of the sugar consumed worldwide.It is cultivated in over 50 countries [1]
Domestication is associated with the loss of resistance to biotic and abiotic stresses, it can be regarded as the selection associated with the loss of resistance to biotic and abiotic stresses, it can be regarded as the selection of of suitable wild accessions according to agriculturally relevant phenotypes [28]
The initial domestication domestication event did sample the full range of natural variation in the wild progenitor populations event did sample the full range of natural variation in the wild progenitor populations and the and the characterization of such diversity at the functional locus level can result in more efficient characterization of such diversity at the functional locus level can result in more efficient preservation preservation of plant genetic resources (PGR)

Summary

Introduction

Sugar beet is an important crop that supplies around 20% of the sugar consumed worldwide. Ion Torrent Ampliseq designer is a new technology with the ability to resequence genes and search for associations based on the Disease Research Area database [20] It has superior quality, specificity and coverage, which supports short (140–175 bp), medium (275 bp) and long (375 bp) sized PCR amplicons [21]. Ion Torrent sequencing technology with the personal genome machine (PGM) was used to detect genetic mutations inside 21 selected RGAs for rhizomania and nematode infections in wild and cultivated beets. This is the first report of using this accurate and efficient technology in sugar beet research.

Plant Material

DNA Isolation

Library Preparation and Sequencing

Data Analysis

Results

Discussion