Simple SNP-based minimal marker genotyping for Humulus lupulus L. identification and variety validation

Abstract

BackgroundHop is an economically important crop for the Pacific Northwest USA as well as other regions of the world. It is a perennial crop with rhizomatous or clonal propagation system for varietal distribution. A big concern for growers as well as brewers is variety purity and questions are regularly posed to public agencies concerning the availability of genotype testing. Current means for genotyping are based upon 25 microsatellites that provides relatively accurate genotyping but cannot always differentiate sister-lines. In addition, numerous PCR runs (25) are required to complete this process and only a few laboratories exist that perform this service. A genotyping protocol based upon SNPs would enable rapid accurate genotyping that can be assayed at any laboratory facility set up for SNP-based genotyping. The results of this study arose from a larger project designed for whole genome association studies upon the USDA-ARS hop germplasm collection consisting of approximately 116 distinct hop varieties and germplasm (female lines) from around the world.ResultsThe original dataset that arose from partial sequencing of 121 genotypes resulted in the identification of 374,829 SNPs using TASSEL-UNEAK pipeline. After filtering out genotypes with more than 50 % missing data (5 genotypes) and SNP markers with more than 20 % missing data, 32,206 highly filtered SNP markers across 116 genotypes were identified and considered for this study. Minor allele frequency (MAF) was calculated for each SNP and ranked according to the most informative to least informative. Only those markers without missing data across genotypes as well as 60 % or less heterozygous gamete calls were considered for further analysis. Genetic distances among individuals in the study were calculated using the marker with the highest MAF value, then by using a combination of the two markers with highest MAF values and so on. This process was reiterated until a set of markers was identified that allowed for all genotypes in the study to be genetically differentiated from each other. Next, we compared genetic matrices calculated from the minimal marker sets [(Table 2; 6-, 7-, 8-, 10- and 12-marker set matrices] and that of a matrix calculated from a set of markers with no missing data across all 116 samples (1006 SNP markers). The minimum number of markers required to meet both specifications was a set of 7-markers (Table 3). These seven SNPs were then aligned with a genome assembly, and DNA sequence both upstream and downstream were used to identify primer sequences that can be used to develop seven amplicons for high resolution melting curve PCR detection or other SNP-based PCR detection methods.ConclusionsThis study identifies a set of 7 SNP markers that may prove useful for the identification and validation of hop varieties and accessions. Variety validation of unknown samples assumes that the variety under question has been included a priori in a discovery panel. These results are based upon in silica studies and markers need to be validated using ...

Development of model web-server for crop variety identification using throughput SNP genotyping data

“…In development of any crop variety identification methodology/system, the smallest number of markers discriminating all or maximum number of varieties in the panel are most desirable. Such approach of SNP minimization reduces the cost of genotyping without compromising the identification accuracy 11 , 12 . In fact 2–3 SNP/per chromosome are enough for variety identification 13 .…”

Methods for verifying the authenticity of hops - an effective tool against falsification

“…Unknown sequences in molecular hybridization system have also been used in Diversity Arrays Technology (DArT) method (Howard et al, 2011) but better identifi cation and determination of hop cultivars is achieved when using DNA sequence specifi c methods such as the previously mentioned STS methods in structural genes (Patzak et al, 2007;Bassil et al, 2008;Castro et al, 2008;Venger et al, 2015). Microsatellite nucleotide (di-or tri-) repeats are the most useful for molecular genetic analysis of variability and evaluation of biodiversity in different plant zeny nové molekulární markery typu jednoduchých sekvenčních repetic v exprimovaných sekvenčních úsecích (EST-SSR) (Patzak a Matoušek, 2011;Jakše et al, 2011;Koelling et al, 2012;Singh et al, 2012) a jedno-nukleotidového polymorfi smu (SNP) (Matthews et al, 2013;Yamauchi et al, 2014;Henning et al, 2015). Nedávno byla vydána studie představující efektivní markerovací systém pro genotypování a kontrolu autenticity českých odrůd chmele založe-ný na EST-SSR, který byl implementován do systému identifi kace odrůd chmele a kontroly čistoty sadbového materiálu (Patzak and Matoušek, 2013a;2013b).…”

“…The increasing information by Next Generation Sequencing (NGS) about transcriptome (Nagel et al, 2008;Clark et al, 2013;Xu et al, 2013) and whole genome sequences (Natsume et al, 2015) complemented hop DNA gene sequences in GeneBank EST databases, which have provided the possibility to look for new gene specifi c molecular markers. From this information, a new type of molecular markers was derived such as Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) (Patzak and Matoušek, 2011;Jakše et al, 2011;Koelling et al, 2012;Singh et al, 2012) and Single Nucleotide Polymorphism (SNP) markers (Matthews et al, 2013;Yamauchi et al, 2014;Henning et al, 2015). Recently, we reported on an effi cient marker system for genotyping and authenticity control of Czech hop cultivars based on EST-SSR, which was implemented into the identifi cation of hop genotypes and purity control of cultivar rootstocks (Patzak and Matoušek, 2013a;.…”

Study on the identification ofPolygonatum kingianumand its adulterants by PCR amplification of specific alleles

“…Site specific PCR requires only a single PCR reaction, and then we can judge the genuine from whether there is amplification, is very simple and fast. Today, the site-specific PCR technology has been applied in Panax ginseng [7], Humulus lupulus L. [8], Sarcandra glabra and Chloranthus [9], Amomum villosum Lour. [10], Mentha haplocalyx and Mentha spicata [11].…”