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Title: Targeted next-generation sequencing identification of mutations in disease resistance gene anologs (RGAs) in wild and cultivated beets

Author
item STEVENATO, PIERGIORGIO - Universita Di Padova
item BROCENELLO, CHIRARA - Universita Di Padova
item PAJOLA, FILIPPO - Universita Di Padova
item Richards, Christopher
item PANELLA, LEONARD - Colorado State University
item HASSANI, MAHDI - Shiraz University
item FORMENTIN, ELIDE - Universita Di Padova
item CHIODI, CLAUDIA - Universita Di Padova
item CONCHERI, GIUSEPE - Universita Di Padova
item HEIDARI, BAHRAM - Shiraz University

Submitted to: Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2017
Publication Date: 10/11/2017
Citation: Stevenato, P., Brocenello, C., Pajola, F., Richards, C.M., Panella, L.W., Hassani, M., Formentin, E., Chiodi, C., Concheri, G., Heidari, B. 2017. Targeted next-generation sequencing identification of mutations in disease resistance gene anologs (RGAs) in wild and cultivated beets. Genes. 8(10):264. doi:10.3390/genes8100264.

Interpretive Summary: Sugarbeet is damaged by biotic and abiotic stresses and the development of new varieties that are tolerant under adverse conditions is one of the main breeding challenges. Rhizomania and nematode-infections are the most widespread diseases, respectively induced by Beet Necrotic Yellow Vein Virus and beet-cyst nematode (Heterodera schachtii Schm.). The only efficient and cost-effective strategy to control these diseases is to introgress resistance genes into commercial sugar beet varieties. Molecular marker technologies greatly facilitate introgression of disease resistance traits into sugar beet breeding programs. In the present study we evaluated genetic mutations inside 21 selected candidate disease resistance loci to rhizomania and nematode infections in wild accessions of seabeet to find variation not present in the cultivated varieties. Comparison of wild and domesticated beets can assist in the identification of novel sources of disease resistance; alleles that never were part of the domesticated lineages. Cataloging the variation at these disease loci will be useful in allele mining of wild germplasm diversity for breeding improvement.

Technical 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 singlenucleotide 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.