ddRADseq Applications for Petunia × hybrida Clonal Line Breeding: Genotyping and Variant Identification for Target-Specific Assays

Molecular genotyping is a key factor for plant breeding programming and Plant Variety Protection. However, its potential still remains to be elucidated when considering ornamental plants like Petunia × hybrida. In this study, a Petunia breeding clone collection, including sister line groups, was genotyped through double digest Restriction-site Associated DNA sequencing (ddRADseq), and its genetic diversity and structure were studied. In addition to estimate the high genetic similarity observed among sister lines, this approach allowed the unique discrimination of each clone too. Molecular results agreed with genealogy data, assessing genotyping effectiveness. In addition, the minimal number of variants able to correctly genotype the population was investigated. The variant number could be reduced to a hundred with minor adjustments in genotyping results. Moreover, analysis of the chromosomal distribution of variants revealed a predominant accumulation in distal regions. Eventually, genetic analyses were repeated considering only variants located in coding sequences and results were in agreement with what previously observed, disclosing the potential of the expressed regions for genotyping purposes. Our findings encourage the adoption of high-throughput and cost-effective sequencing techniques for plant genotyping, to enhance the technological advancement of breeding programs, in particular for ornamental species.