Development of 50 InDel-based barcode system for genetic identification of tartary buckwheat resources

Abstract

Tartary buckwheat (Fagopyrum tataricum Gartn.) is a highly functional crop that is poised to be the target of many future breeding efforts. The reliable ex situ conservation of various genetic resources is essential for the modern breeding of tartary buckwheat varieties. We developed PCR-based co-dominant insertion/deletion (InDel) markers to discriminate tartary buckwheat genetic resources. First, we obtained the whole genome from 26 accessions across a superscaffold-scale reference genome of 569.37 Mb for tartary buckwheat cv. “Daegwan 3–7.” Next, 171,926 homogeneous and 53,755 heterogeneous InDels were detected by comparing 26 accessions with the “Daegwan 3–7” reference sequence. Of these, 100 candidate InDels ranging from 5–20 bp in length were chosen for validation, and 50 of them revealed polymorphisms between the 26 accessions and “Daegwan 3–7.” The validated InDels were further tested through the assessment of their likelihood to give rise to a single or a few PCR products in 50 other accessions, covering most tartary buckwheat genome types. The major allele frequencies ranged from 0.5616 at the TB42 locus to 0.9863 at the TB48 locus, with the average PIC value of 0.1532 with a range of 0.0267–0.3712. To create a user-friendly system, the homology of the genotypes between and among the accessions were visualized in both one- (1D) and two-dimensional (2D) barcode types by comparing amplicon polymorphisms with the reference variety, “Daegwan 3–7.” A phylogenetic tree and population structure of the 76 accessions according to amplicon polymorphisms for the 50 InDel markers corresponded to those using non-synonymous single nucleotide polymorphism variants, indicating that the barcode system based on the 50 InDels was a useful tool to improve the reliability of identification of tartary buckwheat accessions in the germplasm stocks.