Genome-wide analysis-based single nucleotide polymorphism marker sets to identify diverse genotypes in cabbage cultivars (Brassica oleracea var. capitata).

Plant variety protection is essential for breeders' rights granted by the International Union for the Protection of New Varieties of Plants. Distinctness, uniformity, and stability (DUS) are necessary for new variety registration; to this end, currently, morphological traits are examined, which is time-consuming and laborious. Molecular markers are more effective, accurate, and stable descriptors of DUS. Advancements in next-generation sequencing technology have facilitated genome-wide identification of single nucleotide polymorphisms. Here, we developed a core set of single nucleotide polymorphism markers to identify cabbage varieties and traits of test guidance through clustering using the Fluidigm assay, a high-throughput genotyping system. Core sets of 87, 24, and 10 markers are selected based on a genome-wide association-based approach. All core markers could identify 94 cabbage varieties and determine 17 DUS traits. A genotypes database was validated using the Fluidigm platform for variety identification, population structure analysis, cabbage breeding, and DUS testing for plant cultivar protection.

Genome-wide core sets of SNP markers and Fluidigm assays for rapid and effective genotypic identification of Korean cultivars of lettuce (Lactuca sativa L.).

Lettuce is one of the economically important leaf vegetables and is cultivated mainly in temperate climate areas. Cultivar identification based on the distinctness, uniformity, and stability (DUS) test is a prerequisite for new cultivar registration. However, DUS testing based on morphological features is time-consuming, labor-intensive, and costly, and can also be influenced by environmental factors. Thus, molecular markers have also been used for the identification of genetic diversity as an effective, accurate, and stable method. Currently, genome-wide single nucleotide polymorphisms (SNPs) using next-generation sequencing technology are commonly applied in genetic research on diverse plant species. This study aimed to establish an effective and high-throughput cultivar identification system for lettuce using core sets of SNP markers developed by genotyping by sequencing (GBS). GBS identified 17 877 high-quality SNPs for 90 commercial lettuce cultivars. Genetic differentiation analyses based on the selected SNPs classified the lettuce cultivars into three main groups. Core sets of 192, 96, 48, and 24 markers were further selected and validated using the Fluidigm platform. Phylogenetic analyses based on all core sets of SNPs successfully discriminated individual cultivars that have been currently recognized. These core sets of SNP markers will support the construction of a DNA database of lettuce that can be useful for cultivar identification and purity testing, as well as DUS testing in the plant variety protection system. Additionally, this work will facilitate genetic research to improve breeding in lettuce.

High-throughput SNP markers for authentication of Korean wheat cultivars based on seven complete plastomes and the nuclear genome.

Wheat (Triticum aestivum) has diverse uses in the food industry, and different cultivars have unique properties; therefore, it is important to select the optimal cultivar for the intended end use. Here, to establish an identification system for Korean wheat cultivars, we obtained the complete plastome sequences of seven major Korean cultivars. Additionally, the open access database CerealsDB was queried to discover single-copy genomic single-nucleotide polymorphisms (SNPs) in the hexaploid wheat genome. Ten SNPs were developed into allele-specific PCR (ASP) markers, and eight of the SNPs used for ASP markers were converted into TaqMan high-throughput genotyping markers. Phylogenetic analysis using SNP genotypes revealed the genetic diversity and relationships among 137 wheat lines from around the world, including 35 Korean cultivars. This research thus presents a high-throughput authentication system for Korean wheat cultivars that may promote food industry uses of Korean wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01043-w.

Genetic Diversity Assessment and Cultivar Identification of Cucumber (Cucumis sativus L.) Using the Fluidigm Single Nucleotide Polymorphism Assay.

Genetic diversity analysis and cultivar identification were performed using a core set of single nucleotide polymorphisms (SNPs) in cucumber (Cucumis sativus L.). For the genetic diversity study, 280 cucumber accessions collected from four continents (Asia, Europe, America, and Africa) by the National Agrobiodiversity Center of the Rural Development Administration in South Korea and 20 Korean commercial F1 hybrids were genotyped using 151 Fluidigm SNP assay sets. The heterozygosity of the SNP loci per accession ranged from 4.76 to 82.76%, with an average of 32.1%. Population genetics analysis was performed using population structure analysis and hierarchical clustering (HC), which indicated that these accessions were classified mainly into four subpopulations or clusters according to their geographical origins. The subpopulations for Asian and European accessions were clearly distinguished from each other (FST value = 0.47), while the subpopulations for Korean F1 hybrids and Asian accessions were closely related (FST = 0.34). The highest differentiation was observed between American and European accessions (FST = 0.41). Nei's genetic distance among the 280 accessions was 0.414 on average. In addition, 95 commercial F1 hybrids of three cultivar groups (Baekdadagi-, Gasi-, and Nakhap-types) were genotyped using 82 Fluidigm SNP assay sets for cultivar identification. These 82 SNPs differentiated all cultivars, except seven. The heterozygosity of the SNP loci per cultivar ranged from 12.20 to 69.14%, with an average of 34.2%. Principal component analysis and HC demonstrated that most cultivars were clustered based on their cultivar groups. The Baekdadagi- and Gasi-types were clearly distinguished, while the Nakhap-type was closely related to the Baekdadagi-type. Our results obtained using core Fluidigm SNP assay sets provide useful information for germplasm assessment and cultivar identification, which are essential for breeding and intellectual right protection in cucumber.

Genome-wide SNP discovery and core marker sets for assessment of genetic variations in cultivated pumpkin (Cucurbita spp.).

Three pumpkin species Cucurbita maxima, C. moschata, and C. pepo are commonly cultivated worldwide. To identify genome-wide SNPs in these cultivated pumpkin species, we collected 48 F1 cultivars consisting of 40 intraspecific hybrids (15 C. maxima, 18 C. moschata, and 7 C. pepo) and 8 interspecific hybrids (C. maxima x C. moschata). Genotyping by sequencing identified a total of 37,869 confident SNPs in this collection. These SNPs were filtered to generate a subset of 400 SNPs based on polymorphism and genome distribution. Of the 400 SNPs, 288 were used to genotype an additional 188 accessions (94 F1 cultivars, 50 breeding lines, and 44 landraces) with a SNP array-based platform. Reliable polymorphisms were observed in 224 SNPs (78.0%) and were used to assess genetic variations between and within the four predefined populations in 223 cultivated pumpkin accessions. Both principal component analysis and UPGMA clustering found four major clusters representing three pumpkin species and interspecific hybrids. This genetic differentiation was supported by pairwise Fst and Nei's genetic distance. The interspecific hybrids showed a higher level of genetic diversity relative to the other three populations. Of the 224 SNPs, five subsets of 192, 96, 48, 24, and 12 markers were evaluated for variety identification. The 192, 96, and 48 marker sets identified 204 (91.5%), 190 (85.2%), and 141 (63.2%) of the 223 accessions, respectively, while other subsets showed <25% of variety identification rates. These SNP markers provide a molecular tool with many applications for genetics and breeding in cultivated pumpkin.