Single nucleotide polymorphism (SNP) discovery through genotyping-by-sequencing (GBS) and genetic characterization of Dendrobium mutants and cultivars

Dendrobium plants are the most important orchids used in horticultural industries and oriental medicine. Recently, several Dendrobium mutant genotypes with improved horticultural traits have been developed using various mutagens such as gamma irradiation, somaclonal variation, and aerospace mutagenesis. This study investigated genetic diversity and variations among 7 Dendrobium mutant genotypes and 11 commercial Dendrobium cultivars using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS) analysis. In addition, the SNPs were converted into a competitive allele-specific PCR (KASP) assay to discriminate the Dendrobium genotypes. A total of 67,669,394 clean data reads were generated, with 3.7 million reads per genotype on average, and 38,168,915 (56.4%) mapped reads. A total of 517,660 SNPs were detected, which included 443,305 homozygous and 74,355 heterozygous loci. The frequency of transition substitutions was shown to be higher than that of transversions, and the transition:transversion ratio is 1.34. A set of 37,721 filtered SNPs was used to perform a phylogenetic analysis, which showed that there were clear differences among the Dendrobium genotypes based on their species. A total of 18 SNPs were used for the development of KASP assay sets, of which 10 were successfully applied to distinguish among Dendrobium genotypes. This study demonstrated that the SNPs and KASP assay sets are an economically efficient tool for mutant screening and for selection of elite genotypes in Dendrobium breeding programs.