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

Abstract

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.

New Tools for Hop Cytogenomics: Identification of Tandem Repeat Families from Long-Read Sequences ofHumulus lupulus

“…Male panicles were collected before pollen shedding and fixed in Farmer's fluid as previously described (Zhang et al, 2017;Easterling et al, 2018). Wild hops, H. lupulus var.…”

“…Humulus lupulus (hop) is a dioecious twining bine in the Cannabaceae family of flowering plants with a long history of cultivation (Neve, 1991;Moir, 2000) for various uses including medicine (as reviewed by Ososki and Kennelly, 2003;Bolton et al, 2019) and animal fodder (Siragusa et al, 2008), but is most commonly known as a flavoring agent in the brewing industry. The quest for complex taste and aromas in the rapidly expanding craft brewing industry has placed increasing demands on breeders to produce new varieties of plants with specific desirable traits as well as disease resistance (Kavalier et al, 2011;Easterling et al, 2018;Yan et al, 2019).…”

“…Cytogenetic analysis of male meiosis in hop has revealed a tendency for unusual meiotic configurations such as multivalent chromosomal complexes (Sinotô, 1929;Winge, 1929;Shephard et al, 2000;Zhang et al, 2017). Recent work with 3D molecular cytology has shown that pervasive whole chromosome or segmental aneuploidy exists in hop and is exacerbated by passage through meiosis (Easterling et al, 2018). To date, there are limited cytological tools for assessing segregation patterns and establishing hop karyotypes (9 autosomes, XY).…”

“…To date, there are limited cytological tools for assessing segregation patterns and establishing hop karyotypes (9 autosomes, XY). These tools have included telomere, 5S rDNA, HSR1 (Humulus subtelomeric repeat 1) (Karlov et al, 2003;Divashuk et al, 2011), and more recently HSR0 (Humulus subtelomeric repeat 0) (Easterling et al, 2018). Despite these advances, most genomes of model hop varieties remain to be sequenced, assembled, and fully annotated, except for unannotated, partial assemblies of Shinshu Wase, H lupulus var.…”

“…Among the most useful tools for cytological chromosome analysis are FISH probes (Jiang, 2019). Of these, tandem repeat FISH probes are the most robust and have proven particularly useful for karyotyping and distinguishing chromosomes as illustrated for various eudicot and monocot plant species (Kato et al, 2004;Divashuk et al, 2011;Zuccolo et al, 2011;Weiss-Schneeweiss et al, 2015;Razumova et al, 2016;Novák et al, 2017;Easterling et al, 2018;Sun et al, 2018;Majtánová et al, 2019;Vondrak et al, 2019). Tandem repeat FISH probes specific for telomeres, centromeres, and knobs have also proven especially informative in 3D microscopic studies of meiosis (Bass et al, 1997;Birchler et al, 2007;Zuccolo et al, 2011;Howe et al, 2013;Higgins et al, 2014;Easterling et al, 2018;Majtánová et al, 2019).…”