Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers

Authors: Driskill, Mandie; PARDEE, KATIE - Former ARS Employee; Hummer, Kim; ZURN, JASON - Kansas State University; AMUNDSEN, KEENAN - University Of Nebraska; WILES, ANNETTE - Midwest Hop Producers; WIEDOW, CLAUDIA - Plant And Food Research; PATZAK, JOSEF - Hop Research Center; Henning, John; Bassil, Nahla

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2022
Publication Date: 4/14/2022
Citation: Driskill, M.J., Pardee, K., Hummer, K.E., Zurn, J., Amundsen, K., Wiles, A., Wiedow, C., Patzak, J., Henning, J.A., Bassil, N.V. 2022. Two fingerprinting sets for Humulus lupulus based on KASP and microsatellite markers. PLoS ONE. 17(4). Article e0257746. https://doi.org/10.1371/journal.pone.0257746.
DOI: https://doi.org/10.1371/journal.pone.0257746

Interpretive Summary: Cultivated hops are derived from the European hop, though other subspecies have contributed to breeding genepools. Breeding programs at the USDA-ARS Forage Seed and Cereal Research Unit as well as the USDA-ARS National Clonal Germplasm Repository maintain hop collections that are vegetatively propagated and used to create new varieties that meet industry needs. Accurate and economic DNA-based tools to confirm cultivar identity are needed for breeding and germplasm management and for hop identification in nurseries and grower fields. Our objective was to develop two fingerprinting sets from different marker systems: one based on tandemly repeated DNA sequences (referred to as simple sequence repeat, SSR); and the other one based on variations at a single nucleotide base (referred to as single nucleotide polymorphism, SNP). The resulting 9-SSR set was used to genotype 629 hop samples that included cultivated and wild accessions. Parentage and sibling relationship analyses were used to identify true-to-type cultivars. The SNP assay consisted of 25 markers. Comparison of marker assays across 190 hop accessions demonstrated that both systems distinguished unique genotypes of the cultivated European hop accessions while only the 25 SNP assay was unable to differentiate WNA accessions. The 9-SSR assay is cost efficient when genotyping a small number of wild and cultivated hop samples (<96) while the SNP assay is easy to interpret and cost efficient for genotyping a large number of cultivated samples (multiples of 96). Both sets are useful tools for identity certification in hops.

Technical Abstract: Verification of clonal identity of hop (Humulus lupulus L.) cultivars within breeding programs and germplasm collections is vital to conserving genetic resources. Accurate and economic DNA-based tools are needed in dioecious hop to confirm identity and parentage, neither of which can be reliably determined from morphological observations. In this study, we developed two fingerprinting sets for hop: a 9-SSR fingerprinting set containing high-core repeats that can be run in a single PCR reaction and a kompetitive allele specific PCR (KASP) assay of 25 single nucleotide polymorphisms (SNPs). The SSR set contains a sex-linked primer pair, HI-AGA7, and was used to genotype 629 hop accessions from the US Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR), the USDA Forage Seed and Cereal Research (FSCR), and the University of Nebraska-Lincoln (UNL) collections. The SSR set identified unique genotypes except for 89 sets of synonymous samples. These synonyms included: cultivars with different designations, the same cultivars from different sources, heat-treated clones, and clonal variants. Population structure analysis clustered accessions into wild North American (WNA) and cultivated groups. Diversity was slightly higher in the cultivated samples due to larger sample size. Parentage and sib-ship analyses were used to identify true-to-type cultivars. HI-AGA7 generated two male- and nine female-specific alleles among the cultivated and WNA samples. The SSR and KASP fingerprinting sets were compared for 190 samples consisting of cultivated and WNA accession for ability to confirm identity and assess diversity and population structure. The SSR fingerprinting set distinguished cultivars, selections and WNA accessions while the KASP assays were unable to distinguish the WNA samples and had lower diversity estimates than the SSR set. Both fingerprinting sets are valuable tools for identity confirmation and parentage analysis in hop for different purposes. The 9-SSR assay is cost efficient when genotyping a small number of wild and cultivated hop samples (<96) while the KASP assay is easy to interpret and cost efficient for genotyping a large number of cultivated samples (multiples of 96).