Recipient Organization
HUDSONALPHA INSTITUTE FOR BIOTECHNOLOGY
601 GENOME WAY
HUNTSVILLE,AL 358062908
Performing Department
(N/A)
Non Technical Summary
Hops are perennial bines, best known for their role in beer production. The goal of breeding programs is to debut new varieties with distinct flavor profiles, disease resistance, and/or suitability to a new environment. What is lacking in hops, that has accelerated research for other crops, is quality genome references. Importantly, in the hop genus, Humulus, only females develop the valuable "hop cones" that are enriched in the bitter acids and other essential oils used in beer production. This suggests there are genomic differences between females and males that control hop cone development. Identifying the genes involved in initiating female hop cone development versus male pollen development in Humulus would reduce labor and crop loss from unintended crosses. Additionally, superior markers for early identification of females and males across all cultivars and varieties in breeding programs would reduce resource usage by early identification of the males that have no economic value. With this research, I will use cutting-edge technologies to generate a genomic toolkit to help sustainable accelerate hop breeding programs. To accomplish this, I will first generate high-quality genome references for the five varieties of Humulus lupulus and its sister species H. japonicus that I will make publically available. I will also examine differences in genes expressed in hop cones and other tissues to determine those involved in hop cone development. I will also develop markers for early identification of females, as well as variety and isolate-specific markers across Humulus. Finally, I will establish the Southeastern Hop Alliance, where our community of hop scientists can organize germplasm, keep track of crosses, and communicate research findings.
Animal Health Component
(N/A)
Research Effort Categories
Basic
10%
Applied
(N/A)
Developmental
90%
Goals / Objectives
The major goal of the USDA NIFA AFRI postdoctoral research I will perform is to provide foundational genomic tools for accelerating breeding programs for the economically valuable hop cone. Firstly, because there are limited genomic resources, like high-quality genome references available for hops, the ones I develop for all five H. lupulus varieties, which are adapted to unique environments, will be ideal for developing marker-assisted breeding for disease resistance, conservation, and other critical areas of improvement. Secondly, the sex-specific tools I develop will better identify and control sex, which will help more sustainably grow hops and reduce crop loss. Moreover, the comparative genomic analyses will uncover other key genes for hop cone development, that will be novel targets for improving hops and expanding to new locations like the southeast. Indeed, we are working to establish hops in Alabama, and in doing so will use the genomic tools presented here.My objectives are as follows:Objective 1. Generate ultra-contiguous male references across Humulus and examine differences in genome architecture.Objective 2. Identify the sex-determining genes across Humulus.Objective 3. Generate Humulus sex and variety-specific markers for breeding
Project Methods
Genome assembly and annotation. I will collaborate with hop breeders at the University of Minnesota, who have wellmaintained accessions for the proposed analyses. To generate reference genomes, I will sequence male isolates using PacBio HiFi and Dovetail Hi-C. I will assemble scaffolds using hifiasm and generate pseudomolecules representing chromosomes using JUICER. To annotate the genomes I will identify repeats using RepeatMasker and genes using root, stem, leaf, and flower mRNA (in triplicate) using BRAKER2.Identifying the sex chromosome non-recombining region. To identify the sex chromosomes for each genome reference, and the non-recombining region within them, I will use whole-genome sequencing data from three females and three males for each variety/species to identify the location of male Y-specific k-mers, Y-specific read coverage, and high heterozygosity. To determine which genes are shared on the sex chromosomes across Humulus, I will use gene tree analyses. Finally, I will test whether sex-linkage has affected protein evolution, and whether these patterns differ across varieties/species.Comparative genome evolution. To examine relationships within Humulus to use as a backbone for genome evolution, I will leverage the genome annotations and existing data in the Rosales to build a phylogenetic tree. I will first identify single-copy genes using Orthofinder2 and build a bootstrapped maximum-likelihood tree using RAxML. To identify homologous chromosomes and begin characterizing structural variation (SV), I will first do synteny analyses using GENESPACE. For finer characterization of SVs, including insertions, deletions, and translocations, I will use the var. lupulus assembly as a reference and align PacBio data for the remaining five using NGMLR and call SVs using Sniffles. To examine gene family evolution for traits of interest, including flower development and secondary metabolites, I will test for gene family expansions or contractions using CAFE. Finally, I will reconstruct the ancestral structure of the autosomes and sex chromosomes in the Cannabaceae family.Identifying the hop sex-determining genes. The candidate sex-determining genes for hops will be located in the region of suppressed recombination of the Y chromosome, exclusively expressed in the male flowers, and ideally have known floral functions. To identify the sex-determining genes, I will guide mapping and assembly of transcripts expressed using HISAT2 and StringTie, respectively, using stem, leaf, and root mRNA from Aim 1, and three additional stages of female (hop cone) and male flower development. These stages will include Stage 3: floral meristem initiation; Stage 5/6: sepal/carpel initiation and elongation; and Stage 9: when lupulin glands form on bracteoles in females. I will use Orthofinder2 to identify which genes are known to function in the floral pathway. To examine gene regulatory networks throughout flower development, I will first construct gene co-expression modules using WGCNA, or another state-of-the-art tool. I will compare differences in these modules by examining the hub genes and overlap these with translocations to the sex chromosomes, gene family expansions, or other SVs.Developing sex, variety, and isolate-specific markers. I will use the genome references and resequence data and additionally generate whole-genome sequencing for 60 cultivars across the hop pedigree. I will map readss, call SNPs, and use the Integrative Genomics Viewer to visually identify regions that are conserved on the Y to identify males, but are different enough from the X to identify females. I will extend this logic to finding regions that are variety- and isolate-specific.Efforts. I will make all data and results resulting from this research available to the public. All sequence data will be deposited in the NCBI SRA. To make thes genome assemblies and annotations openly available, I will upload them to NCBI, Phytozome, and CoGe. I will deposit all other results and metadata in Dryad or a similar archive. To disseminate research findings, I will publish all results in a peer-reviewed journal in a timely manner and I will present results at scientific conferences. To share these things with the greater hop community, I will establish the Southeastern Hop Alliance. I will create a project-specific website for which I will provide links to all of these resources. Finally, Iwill use Twitter to communicate major milestones broadly and will generate a Linkedin profile to document my career progress.