3D Molecular Cytology of Hop (Humulus lupulus) Meiotic Chromosomes Reveals Non-disomic Pairing and Segregation, Aneuploidy, and Genomic Structural Variation

Hop (Humulus lupulus L.) is an important crop worldwide, known as the main flavoring ingredient in beer. The diversifying brewing industry demands variation in flavors, superior process properties, and sustainable agronomics, which are the focus of advanced molecular breeding efforts in hops. Hop breeders have been limited in their ability to create strains with desirable traits, however, because of the unusual and unpredictable inheritance patterns and associated non-Mendelian genetic marker segregation. Cytogenetic analysis of meiotic chromosome behavior has also revealed conspicuous and prevalent occurrences of multiple, atypical, non-disomic chromosome complexes, including those involving autosomes in late prophase. To explore the role of meiosis in segregation distortion, we undertook 3D cytogenetic analysis of hop pollen mother cells stained with DAPI and FISH. We used telomere FISH to demonstrate that hop exhibits a normal telomere clustering bouquet. We also identified and characterized a new sub-terminal 180 bp satellite DNA tandem repeat family called HSR0, located proximal to telomeres. Highly variable 5S rDNA FISH patterns within and between plants, together with the detection of anaphase chromosome bridges, reflect extensive departures from normal disomic signal composition and distribution. Subsequent FACS analysis revealed variable DNA content in a cultivated pedigree. Together, these findings implicate multiple phenomena, including aneuploidy, segmental aneuploidy, or chromosome rearrangements, as contributing factors to segregation distortion in hop

Identification of tandem repeat families from long-read sequences of Humulus lupulus

Hop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding the hop genome represents a considerable challenge, requiring additional resources. In order to facilitate investigations into the transmission genetics of hop, we report here a tandem repeat discovery pipeline developed using k-mer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. From this we identified 17 new and distinct tandem repeat sequence families, which represent candidates for FISH probe development. For two of these candidates, HuluTR120 and HuluTR225, we produced oligonucleotide FISH probes from conserved regions of and demonstrated their utility by staining meiotic chromosomes from wild hop, var. neomexicanus to address, for example, questions about hop transmission genetics. Collectively, these tandem repeat sequence families represent new resources suitable for development of additional cytogenomic tools for hop research

Next Generation Sequencing for a Plant of Great Tradition: Application of NGS to SNP Detection and Validation in Hops (Humulus lupulus L.)

Application of next generation DNA sequencing technology to hops yielded an unprecedented, large number of novel single nucleotide polymorphisms (17, 128 SNPs). The markers were detected and then validated for use in genotyping and control of quality for hops. By using genotyping-by-sequencing (GBS) and a universal network-enabled analysis kit (UNEAK) designed for species with no "reference genome", we generated a set of molecular markers with a genome-wide distribution. Validation of the markers was accomplished by observation of metrics of sequencing quality, by marker behavior in genetic segregation and by application to genetic distance and hierarchical cluster analyses across a set of commonly known cultivars. The SNPs were characterized by average read depth of 3.7 and a call rate across 178 diverse individuals of 0.82. Many SNP alleles segregated with near test cross ratios of 1 : 3 or 3 : 1 and intercross ratios of 0.50 among 103 full-siblings. Erroneous SNPs, with unusually high or low allele segregation ratios were detected at a rate of 4.1 % and could be removed from further analyses. Filtering of SNPs for potentially higher quality was accomplished by selection of call rate thresholds above 0.5, 0.75 and 0.90 or, alternatively, by selection of markers with minimal segregation distortion. Genetic distance matrices and dendrograms for marker subgroups were similar as shown by Mantel's Z-tests and cophenetic correlation coefficients. Bootstrapping generated an exceptionally well-supported tree for genetic relationships among the hop cultivars. Descriptors: genotyping-by-sequencing, single nucleotide polymorphisms, genetic distance analysis, Humulus lupulus L., hop quality Introduction Hop is a difficult species for genetic dissection and for application of molecular crop improvement methods, such as marker-assisted selection (MAS). In contrast to species with inbred lines; i.e., maize or rice; Humulus lupulus L. is dioecious and, thus, an outcrosser. Outcrossing portends a heterozygous species; which makes molecular marker isolation, validation and application more complex. Additionally, the hop genome is large, about 2.9 pg (2.8 GiB) [43]; suggesting, perhaps, two ancient genome-wide duplications or extensive repetitive element content Applications of molecular markers in the hop growing, handling and brewing industries includes many important utilities, including (1) trueness-to-type genotyping for identification of hop propagation material in reference to commonly known cultivars (2) purity assessment of putatively mixed fields or lots of processed raw hop products (3) genetic similarity (nearest neighbor) analysis for guidance in substitution of hop cultivars (4) and development of novel, improved and agronomically sustainable hop varieties by MAS. DNA-based molecular markers have the greatest potential; because, unlike chemical markers, such as essential oils, molecular markers are insulated from sampling and environmental effects and only require very small samples of tissue; such as a single bract, a shoot tip, a plumule or a leaf fragment. A number of technologies have been applied to increase the number of molecular markers for hops. Pillay and Kenny were the first to Massively parallel nucleic acid sequencing technologies, often called "next generation sequencing" (NGS) platforms have enabled rapid development of gene expression measurements and genome-wide SNP detection in hops. Next generation sequencing relies on massively parallel sequencing reactions produced in short reads of 20 to 250 nucleotides length in a microfluidics flowcell monitored by high-density charged couple devices. A single instrument "run" can produce 100-600 billion sequenced nucleotides in about two weeks. Excellent, current reviews of the revolutionary technologies in historical context of sequencing innovations Materials and Methods A plant collection of 178 distinct genotypes maintained by Hopsteiner, S.S. Steiner, Inc. was employed. All accessions are available from the United States Department of Agriculture, Agricultural Resource Service, Germplasm Resource Information Network (USDA-ARS-GRIN) (www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?19415) or are cultivars available from Hopsteiner (www.hopsteiner.com). Only commonly known cultivars (CKCs) were used, since the purpose of the study is validation of GBS TASSEL UNEAK generated SNPs over common commercial germplasm: no "wild hops" or other species of Humulus are included in this study. Additionally, a full-sib family of 103 individuals from a mating of Zenith X DG0117m was included to validate SNP segregation. The triploids Alpha Aroma, Crystal, Green Bullet, Liberty, Mt. Hood, Pacific Gem, Sticklebract, Willamette and Ultra the tetraploid USDA21055 and the remaining diploids are named in Total DNA (genomic, chloroplast, mitochondrial, and contaminants) was extracted from 50 mg of young, expanding hop leaves with a Qiagen 96-well DNAEasy™. DNA extracts were assayed on 0.7 % argarose gels for amount (more than 1 ug) and median molecular mass (> 30 kb Results and Discussion Detection of SNPs The number, quality and distribution of molecular markers produced by the GBS UNEAK TASSEL pipeline depends on DNA quality, genome size, sequencing density and the natural distribution of restriction sites within the genome. Read depth and call rate are interdependent and useful general characteristics for SNP quality. Distribution of number of sampled sites depends on the restriction enzyme used for genome reduction Further validation of marker performance was accomplished by assessing trends in allele frequencies in subpopulations of the cultivars. The SNPs from the admixture of populations were subgrouped into individual families and panels. We also included a consideration of ploidy-level. The association panel was mostly diploids, but one tetraploid and 7 triploid hops were initially included. Exclusion or inclusion of the polyploidy hops did not affect the summary statistics or cluster analyses of SNPs presented here (data not shown). Nevertheless, the polyploids were removed from analyses of allele frequency distribution. The distribution of the SNPs across categories of minor allele frequencies (MAF) is shown for the diploid association panel and the full-sib family in fi gure 3 (see next page). In the association panel We tested the hyptothesis that more stringent fi ltering for subgroups of SNPs would not signifi cantly affect the performance of the markers for genetic distance analyses. To provide a reduced set of more stringently fi ltered sets of SNPs, we fi ltered by three minimum call rates (0.5, 0.75, 0.90) within the diploid association panel. Filtering resulted in reduced sets of 16, 106; 10, 243 and 3,068 SNPs with a call rate => 0.5, => 0.75 and => 0.90, respectively. As an independent approach to fi ltering for high value SNPs suggested by Lu et al. [21], we also fi ltered and then grouped the SNPs based on MAF within the full-sib family of near 0.20-0.30, Our success with SNP subgroups indicate that fewer markers may be enough for some studies and also suggest cost-effi ciency might come from lower-coverage sequencing. Greater economy for sequencing and less coverage is accomplished by increasing the number of uniquely indexed genotypes per sequencing lane. A pilot run of 12 independent Saaz cultivars sequenced in a 384-plex lane of Illumnia HiSeq 2000™ generated an average of 11, 544 +/-3578 SNPs per individual with an average read depth of 0.80 +/-0.25, with our stringent UNEAK setting of c = 8 (minimum call rate = 0.67). While the average read depth of 0.80 is signifi cantly lower than that for markers validated in this study (average read depth = 3.7), it is comparable to several successful, low-coverage studies that do not depend on imputation of missing data Conclusions We report the fi rst application of next generation sequencing technology to simple and cost-effective molecular markers for hop research and for hop production quality control. More than an order-of-magnitude increase in the number of molecular markers for hops research potentially solves most previously reported problems. The power, ease and utility of SNP markers for true-nessto-type determination, population genetic studies, and markerassisted breeding in hops suddenly emerged from a combination of sequencing and computing technological advances. Additionally, SNPs markers, in contrast to other marker types, are superior in reproducibility, genome-wide distribution, co-dominance and "transfer-ability" to other laboratories and to other types of assay technologies. The sequenced-based markers can be recreated from the method without transfer of complicated sequence knowledge. The pipeline for marker production for "non-reference" genomes, GBS UNEAK TASSEL, has recently been validated for use in a several important, specialty crops; such as We showed the UNEAK pipeline with stringent settings produced a large number of markers that require little to no further fi ltering for applications to genetic distance analyses and, thus, also to true-ness-to-type determination. Bootstrap analysis with 17, 128 SNPs produced a tree of the cultivar's relatedness with unprecedented support. Modifi cations to the GBS UNEAK TASSEL pipeline as implemented here are relatively easy to accomplish to match different sequenc

Non-Mendelian Single-Nucleotide Polymorphism Inheritance and Atypical Meiotic Configurations are Prevalent in Hop

Hop ( L.) breeding programs seek to exploit genetic resources for bitter flavor, aroma, and disease resistance. However, these efforts have been thwarted by segregation distortion including female-biased sex ratios. To better understand the transmission genetics of hop, we genotyped 4512 worldwide accessions of hop, including cultivars, landraces, and over 100 wild accessions using a genotyping-by-sequencing (GBS) approach. From the resulting ∼1.2 million single-nucleotide polymorphisms (SNPs), prequalified GBS markers were validated by inferences in population structures and phylogeny. Analysis of pseudo-testcross (Pt) mapping data from F families revealed mixed patterns of Mendelian and non-Mendelian segregation. Three-dimensional (3D) cytogenetic analysis of late meiotic prophase nuclei from two wild and two cultivated hop revealed conspicuous and prevalent occurrences of multiple, atypical, nondisomic chromosome complexes including autosomes. We used genome-wide association studies (GWAS) and fixation index (F) analysis to demonstrate selection mapping of genetic loci for key traits including sex, bitter acids, and drought tolerance. Among the possible mechanisms underlying the observed segregation distortion from the genomic data analysis, the cytogenetic analysis points to meiotic chromosome behavior as one of the contributing factors. The findings shed light on long-standing questions on the unusual transmission genetics and phenotypic variation in hop, with major implications for breeding, cultivation, and the natural history of

Identification of tandem repeat families from long-read sequences of Humulus lupulus.

Hop (Humulus lupulus L.) is known for its use as a bittering agent in beer and has a rich history of cultivation, beginning in Europe and now spanning the globe. There are five wild varieties worldwide, which may have been introgressed with cultivated varieties. As a dioecious species, its obligate outcrossing, non-Mendelian inheritance, and genomic structural variability have confounded directed breeding efforts. Consequently, understanding the hop genome represents a considerable challenge, requiring additional resources. In order to facilitate investigations into the transmission genetics of hop, we report here a tandem repeat discovery pipeline developed using k-mer filtering and dot plot analysis of PacBio long-read sequences from the hop cultivar Apollo. From this we identified 17 new and distinct tandem repeat sequence families, which represent candidates for FISH probe development. For two of these candidates, HuluTR120 and HuluTR225, we produced oligonucleotide FISH probes from conserved regions of and demonstrated their utility by staining meiotic chromosomes from wild hop, var. neomexicanus to address, for example, questions about hop transmission genetics. Collectively, these tandem repeat sequence families represent new resources suitable for development of additional cytogenomic tools for hop research