A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is highly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. We identified extensive sequence divergence between alleles of CsFT1 from photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences, especially in introns. Furthermore, we observed higher expression of one of the CsFT1 copies found in the photoperiod-insensitive cultivar. Genotyping of several mapping populations and a diversity panel confirmed a correlation between CsFT1 alleles and photoperiod response, affirming that at least two independent loci involved in the photoperiodic control of flowering, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

Genetic Mapping, Identification, and Characterization of a Candidate Susceptibility Gene for Powdery Mildew in Cannabis sativa.

Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, and two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with PM and surveyed for disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single-nucleotide polymorphism array, and a consensus genetic map was produced. A major effect quantitative trait locus on C. sativa chromosome 1 (Chr01) and other smaller-effect quantitative trait loci (QTL) on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8-kb insertion in FL 58, relative to TJ's CBD, of which 846 bp are typically spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM-resistant and PM-susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea.

The Salicaceae, including Populus and Salix, are dioecious perennials that utilize different sex determination systems. This family provides a useful system to better understand the evolution of dioecy and sex chromosomes. Here, a rare monoecious genotype of Salix purpurea, 94003, was self- and cross-pollinated and progeny sex ratios were used to test hypotheses on possible mechanisms of sex determination. To delimit genomic regions associated with monoecious expression, the 94003 genome sequence was assembled and DNA- and RNA-Seq of progeny inflorescences was performed. Based on alignments of progeny shotgun DNA sequences to the haplotype-resolved monoecious 94003 genome assembly and reference male and female genomes, a 1.15 Mb sex-linked region on Chr15W was confirmed to be absent in monecious plants. Inheritance of this structural variation is responsible for the loss of a male-suppressing function in what would otherwise be genetic females (ZW), resulting in monoecy (ZWH or WWH ), or lethality, if homozygous (WH WH ). We present a refined, two-gene sex determination model for Salix purpurea, mediated by ARR17 and GATA15 that is different from the single-gene ARR17-mediated system in the related genus Populus.

De Novo Assembly and Annotation of 11 Diverse Shrub Willow (Salix) Genomes Reveals Novel Gene Organization in Sex-Linked Regions.

Poplar and willow species in the Salicaceae are dioecious, yet have been shown to use different sex determination systems located on different chromosomes. Willows in the subgenus Vetrix are interesting for comparative studies of sex determination systems, yet genomic resources for these species are still quite limited. Only a few annotated reference genome assemblies are available, despite many species in use in breeding programs. Here we present de novo assemblies and annotations of 11 shrub willow genomes from six species. Copy number variation of candidate sex determination genes within each genome was characterized and revealed remarkable differences in putative master regulator gene duplication and deletion. We also analyzed copy number and expression of candidate genes involved in floral secondary metabolism, and identified substantial variation across genotypes, which can be used for parental selection in breeding programs. Lastly, we report on a genotype that produces only female descendants and identified gene presence/absence variation in the mitochondrial genome that may be responsible for this unusual inheritance.

Comparative transcriptomics and eQTL mapping of response to Melampsora americana in selected Salix purpurea F2 progeny.

BACKGROUND: Melampsora spp. rusts are the greatest pathogen threat to shrub willow (Salix spp.) bioenergy crops. Genetic resistance is key to limit the effects of these foliar diseases on host response and biomass yield, however, the genetic basis of host resistance has not been characterized. The addition of new genomic resources for Salix provides greater power to investigate the interaction between S. purpurea and M. americana, species commonly found in the Northeast US. Here, we utilize 3' RNA-seq to investigate host-pathogen interactions following controlled inoculations of M. americana on resistant and susceptible F2 S. purpurea genotypes identified in a recent QTL mapping study. Differential gene expression, network analysis, and eQTL mapping were used to contrast the response to inoculation and to identify associated candidate genes. RESULTS: Controlled inoculation in a replicated greenhouse study identified 19 and 105 differentially expressed genes between resistant and susceptible genotypes at 42 and 66 HPI, respectively. Defense response gene networks were activated in both resistant and susceptible genotypes and enriched for many of the same defense response genes, yet the hub genes of these common response modules showed greater mean expression among the resistant plants. Further, eight and six eQTL hotspots were identified at 42 and 66 HPI, respectively. The combined results of three analyses highlight 124 candidate genes in the host for further analysis while analysis of pathogen RNA showed differential expression of 22 genes, two of which are candidate pathogen effectors. CONCLUSIONS: We identified two differentially expressed M. americana transcripts and 124 S. purpurea genes that are good candidates for future studies to confirm their role in conferring resistance.

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae.

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.

Genetic mapping of sexually dimorphic volatile and non-volatile floral secondary chemistry of a dioecious willow.

Secondary chemistry often differs between sexes in dioecious plant species, a pattern attributed to its possible role in the evolution and/or maintenance of dioecy. We used GC-MS to measure floral volatiles emitted from, and LC-MS to quantitate non-volatile secondary compounds contained in, female and male Salix purpurea willow catkins from an F2 family. Using the abundance of these chemicals, we then performed quantitative trait locus (QTL) mapping to locate them on the genome, identified biosynthetic candidate genes in the QTL intervals, and examined expression patterns of candidate genes using RNA-seq. Male flowers emitted more total terpenoids than females, but females produced more benzenoids. Male tissue contained greater amounts of phenolic glycosides, but females had more chalcones and flavonoids. A flavonoid pigment and a spermidine derivative were found only in males. Male catkins were almost twice the mass of females. Forty-two QTL were mapped for 25 chemical traits and catkin mass across 16 of the 19 S. purpurea chromosomes. Several candidate genes were identified, including a chalcone isomerase associated with seven compounds. A better understanding of the genetic basis of the sexually dimorphic chemistry of a dioecious species may shed light on how chemically mediated ecological interactions may have helped in the evolution and maintenance of dioecy.

The Melampsora americana Population on Salix purpurea in the Great Lakes Region Is Highly Diverse with a Contributory Influence of Clonality.

Shrub willows (Salix spp.) are emerging as a viable lignocellulosic, second-generation bioenergy crop with many growth characteristics favorable for marginal lands in New York State and surrounding areas. Willow rust, caused by members of the genus Melampsora, is the most limiting disease of shrub willow in this region and remains extremely understudied. In this study, genetic diversity, genetic structure, and pathogen clonality were examined in Melampsora americana over two growing seasons via genotyping-by-sequencing to identify single-nucleotide polymorphism markers. In conjunction with this project, a reference genome of rust isolate R15-033-03 was generated to aid in variant discovery. Sampling between years allowed regional and site-specific investigation into population dynamics, in the context of both wild and cultivated hosts within high-density plantings. This work revealed that this pathogen is largely panmictic over the sampled areas, with few sites showing moderate genetic differentiation. These data support the hypothesis of sexual recombination between growing seasons because no genotype persisted across the two years of sampling. Additionally, clonality was determined as a driver of pathogen populations within cultivated fields and single shrubs; however, there is also evidence of high genetic diversity of rust isolates in all settings. This work provides a framework for M. americana population structure in the Great Lakes region, providing crucial information that can aid in future resistance breeding efforts.

First Report of Downy Mildew caused by Pseudoperonospora cannabina on Cannabis sativa in New York.

Hemp (Cannabis sativa <0.3% tetrahydrocannabinol) is an emerging crop used for grain, fiber, and cannabinoid production (Fike et al. 2020). In New York, hemp is grown both in controlled environment facilities, including greenhouses, and as a field crop. In August 2020, downy mildew-like symptoms were observed on leaves and inflorescence of hemp plants in a field research trial in Ithaca, NY. Several cultivars, including 'Auto CBD', were affected. Disease was severe with some plants reaching 75% disease severity at the individual plant level. In the most severely affected plots, there was no marketable yield. The disease was characterized by chlorotic and necrotic lesions producing sporangiophores under high humidity. Pigmented sporangia were produced on branched sporangiophores. On artificially inoculated leaves incubated at 18°C, 80% humidity, 12h light for 5d, sporangiophores produced 8-19 pigmented, lemon-shaped sporangia with mean ± SD dimensions of 25.2 ± 3.0 (18.9 to 30.4) x 18.2 ± 2.1 (14.6 to 23.2) µm (n=50). Each sporangium produced 2-5 zoospores after less than 45 min in water at room temperature (22°C). Sporangia were collected from sporulating lesions and DNA was extracted as outlined in Crowell et al. (2020). Fragments of the ribosomal internal transcribed spacer (ITS) region (White et al. 1990), the beta-tubulin ras-associated ypt1 gene (Moorman et al. 2002), and the mitochondrial cytochrome B oxidase subunit 2 (cox2) gene (Hudspeth et al. 2000) were amplified by PCR and sequenced bidirectionally. Sequences were deposited in GenBank under accession numbers OK086084, OM867581, and OM867580, respectively. BLAST searches using the amplified ITS and cox2 sequences resulted in 100% identity to Pseudoperonospora cannabina (HM636051.1, HM636003.1) with ypt1 aligning at 97.95% identity (382/390 bp) with P. cannabina (KJ651402.1). The molecular characterization identified the causal agent as P. cannabina. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070922. Sporangia were rinsed from detached leaves and used to confirm pathogenicity on whole plants. Ten 4-week-old 'Anka' plants were spray-inoculated until run off with a suspension of 1x104 sporangia mL-1. Ten control plants were sprayed with water. After inoculation, plants were placed in a 19˚C growth chamber with a 12-h photoperiod and misted for 30 min twice daily to maintain humidity above 80%. Sporangia and previously described symptoms were observed 7 days post-inoculation, while control plants were asymptomatic. The pathogen was reisolated onto detached leaves of 'Anka' from inoculated leaves where both sporangia and oospores were observed. The reisolated pathogen was confirmed morphologically and molecularly, through PCR amplification and bidirectional sequencing of the ITS, cox2, and ypt1 genes, as P. cannabina. To our knowledge, this is the first report of P. cannabina causing hemp downy mildew in New York. Depending on the severity and timing of infections, this disease could pose a significant threat to hemp production in the state. Other members of the genus, P. cubensis and P. humuli cause downy mildew on cucurbits and hops, respectively. As these can cause devastating diseases on their hosts, P. cannabina must be monitored with vigilance as an emerging pathogen (Purayannur et al. 2021; Savory et al. 2011). Literature Cited: Crowell, C. R., et al.2020. Plant Dis. 104:2949. DOI 10.1094/PDIS-04-20-0718-RE Fike, J. H., et al. 2020. Page 89 In: Sustainable Agriculture Reviews, vol 42. Springer, Cham, Switzerland. DOI 10.1007/978-3-030-41384-2_3 Hudspeth, D. S. S., et al. 2000. Mycologia 92:674. DOI 10.2307/3761425 Moorman, G. W., et al. 2002. Plant Dis. 86:1227. DOI 10.1094/PDIS.2002.86.11.1227 Purayannur, S., et al. 2021. Mol. Plant Pathol. 22:755. DOI 10.1111/mpp.13063 Savory, E. A., et al. 2011. Mol. Plant Pathol. 12:217. DOI 10.1111/j.1364-3703.2010.00670.x White, T. J., et al. 1990. Page 315 In: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI 10.1016/B978-0-12-372180-8.50042-1.

Morphometric relationships and their contribution to biomass and cannabinoid yield in hybrids of hemp (Cannabis sativa).

The breeding of hybrid cultivars of hemp (Cannabis sativa L.) is not well described, especially the segregation and inheritance of traits that are important for yield. A total of 23 families were produced from genetically diverse parents to investigate the inheritance of morphological traits and their association with biomass accumulation and cannabinoid yield. In addition, a novel classification method for canopy architecture was developed. The strong linear relationship between wet and dry biomass provided an accurate estimate of final dry stripped floral biomass. Of all field and aerial measurements, basal stem diameter was determined to be the single best selection criterion for final dry stripped floral biomass yield. Along with stem diameter, canopy architecture and stem growth predictors described the majority of the explainable variation of biomass yield. Within-family variance for morphological and cannabinoid measurements reflected the heterozygosity of the parents. While selfed populations suffered from inbreeding depression, hybrid development in hemp will require at least one inbred parent to achieve uniform growth and biomass yield. Nevertheless, floral phenology remains a confounding factor in selection because of its underlying influence on biomass production, highlighting the need to understand the genetic basis for flowering time in the breeding of uniform cultivars.

Sex determination through X-Y heterogamety in Salix nigra.

The development of non-recombining sex chromosomes has radical effects on the evolution of discrete sexes and sexual dimorphism. Although dioecy is rare in plants, sex chromosomes have evolved repeatedly throughout the diversification of angiosperms, and many of these sex chromosomes are relatively young compared to those found in vertebrates. In this study, we designed and used a sequence capture array to identify a novel sex-linked region (SLR) in Salix nigra, a basal species in the willow clade, and demonstrated that this species has XY heterogamety. We did not detect any genetic overlap with the previously characterized ZW SLRs in willows, which map to a different chromosome. The S. nigra SLR is characterized by strong recombination suppression across a 2 MB region and an excess of low-frequency alleles, resulting in a low Tajima's D compared to the remainder of the genome. We speculate that either a recent bottleneck in population size or factors related to positive or background selection generated this differential pattern of Tajima's D on the X and autosomes. This discovery provides insights into factors that may influence the evolution of sex chromosomes in plants and contributes to a large number of recent observations that underscore their dynamic nature.

Sexual dimorphism in the dioecious willow Salix purpurea.

PREMISE: The evolution of sex chromosomes is driven by sexual dimorphism, yet it can be challenging to document sexually dimorphic traits in dioecious plant species. At the genetic level, sexual dimorphism can be identified through sequence variation between females and males associated with sexually antagonistic traits and different fitness optima. This study aims to examine sexual dimorphism for 26 traits in three populations of Salix purpurea (a diversity panel and F1 and F2 populations) and determine the effect of the traits on biomass yield, a key trait in Salix bioenergy crops across multiple years, locations, and under manipulated growth conditions. METHODS: Sexual dimorphism was evaluated for morphological, phenological, physiological, and wood composition traits in a diversity panel of unrelated S. purpurea accessions and in full-sib F1 and F2 families produced through controlled cross pollinations and grown in replicated field trials. RESULTS: We observed sexual dimorphism in the timing of development for several traits that were highly predictive of biomass yield across three populations of S. purpurea. Across all populations and years surveyed, males had significantly shallower branching angle. Male plants highly predictive of biomass yield across three populations of S. purpurea also accumulated more nitrogen under fertilizer amendment as measured by SPAD in the diversity panel and had greater susceptibility to the rust fungus Melampsora americana in the F2 family. Allometric modelling of biomass yield showed an effect of sex and of location on the interaction between yield and stem height. CONCLUSIONS: These results provide evidence of sexual dimorphism for certain traits in S. purpurea that may be involved in sex chromosome evolution.

Phylogenomics of the genus Populus reveals extensive interspecific gene flow and balancing selection.

Phylogenetic analysis is complicated by interspecific gene flow and the presence of shared ancestral polymorphisms, particularly those maintained by balancing selection. In this study, we aimed to examine the prevalence of these factors during the diversification of Populus, a model tree genus in the Northern Hemisphere. We constructed phylogenetic trees of 29 Populus taxa using 80 individuals based on re-sequenced genomes. Our species tree analyses recovered four main clades in the genus based on consensus nuclear phylogenies, but in conflict with the plastome phylogeny. A few interspecific relationships remained unresolved within the multiple-species clade because of inconsistent gene trees. Our results indicated that gene flow has been widespread within each clade and also occurred among the four clades during their early divergence. We identified 45 candidate genes with ancient polymorphisms maintained by balancing selection. These genes were mainly associated with mating compatibility, growth and stress resistance. Both gene flow and selection-mediated ancient polymorphisms are prevalent in the genus Populus. These are potentially important contributors to adaptive variation. Our results provide a framework for the diversification of model tree genus that will facilitate future comparative studies.

Differential Susceptibility of Diverse Salix spp. to Melampsora americana and Melampsora paradoxa.

Melampsora spp. willow rust is the most serious disease of shrub willow bioenergy production in the northeastern United States. Recent phylogenetic studies have identified several Melampsora spp. present on willow in the Northeast; however, in-depth understanding of Melampsora spp. host susceptibility remain unresolved. In this study, a panel of 82 rust isolates collected from the northeastern United States were genotyped via ribosomal DNA sequencing and a subset of these isolates were assayed for host susceptibility. This work revealed that Melampsora americana is the most prevalent species in the sampled geographic region and that there is potential for rust resistance breeding using the Salix spp. taxa assayed. Additionally, leaf morphology traits of these Salix spp. hosts were quantified for correlation analysis, revealing that trichome density and stomata density are possible contributors to resistance. This work provides foundational rust pathology information, which is crucial for M. americana resistance breeding.

Joint linkage and association mapping of complex traits in shrub willow (Salix purpurea L.).

BACKGROUND AND AIMS: Increasing energy demands and the necessity to reduce greenhouse gas emissions are key motivating factors driving the development of lignocellulosic crops as an alternative to non-renewable energy sources. The effects of global climate change will require a better understanding of the genetic basis of complex adaptive traits to breed more resilient bioenergy feedstocks, like willow (Salix spp.). Shrub willow is a sustainable and dedicated bioenergy crop, bred to be fast-growing and high-yielding on marginal land without competing with food crops. In a rapidly changing climate, genomic advances will be vital for the sustained improvement of willow and other non-model bioenergy crops. Here, joint genetic mapping was used to exploit genetic variation garnered from both recent and historical recombination events in S. purpurea. METHODS: A panel of North American naturalized S. purpurea accessions and full-sib F2S. purpurea population were genotyped and phenotyped for a suite of morphological, physiological, pest and disease resistance, and wood chemical composition traits, collected from multi-environment and multi-year replicated field trials. Controlling for population stratification and kinship in the association panel and spatial variation in the F2, a comprehensive mixed model analysis was used to dissect the complex genetic architecture and plasticity of these important traits. KEY RESULTS: Individually, genome-wide association (GWAS) models differed in terms of power, but the combined approach, which corrects for yearly and environmental co-factors across datasets, improved the overall detection and resolution of associated loci. Although there were few significant GWAS hits located within support intervals of QTL for corresponding traits in the F2, many large-effect QTL were identified, as well as QTL hotspots. CONCLUSIONS: This study provides the first comparison of linkage analysis and linkage disequilibrium mapping approaches in Salix, and highlights the complementarity and limits of these two methods for elucidating the genetic architecture of complex bioenergy-related traits of a woody perennial breeding programme.

Characterization of a large sex determination region in Salix purpurea L. (Salicaceae).

Dioecy has evolved numerous times in plants, but heteromorphic sex chromosomes are apparently rare. Sex determination has been studied in multiple Salix and Populus (Salicaceae) species, and P. trichocarpa has an XY sex determination system on chromosome 19, while S. suchowensis and S. viminalis have a ZW system on chromosome 15. Here we use whole genome sequencing coupled with quantitative trait locus mapping and a genome-wide association study to characterize the genomic composition of the non-recombining portion of the sex determination region. We demonstrate that Salix purpurea also has a ZW system on chromosome 15. The sex determination region has reduced recombination, high structural polymorphism, an abundance of transposable elements, and contains genes that are involved in sex expression in other plants. We also show that chromosome 19 contains sex-associated markers in this S. purpurea assembly, along with other autosomes. This raises the intriguing possibility of a translocation of the sex determination region within the Salicaceae lineage, suggesting a common evolutionary origin of the Populus and Salix sex determination loci.

Early selection of novel triploid hybrids of shrub willow with improved biomass yield relative to diploids.

BACKGROUND: Genetic improvement of shrub willow (Salix), a perennial energy crop common to temperate climates, has led to the development of new cultivars with improved biomass yield, pest and disease resistance, and biomass composition suitable for bioenergy applications. These improvements have largely been associated with species hybridization, yet little is known about the genetic mechanisms responsible for improved yield and performance of certain willow species hybrids. RESULTS: The top performing genotypes in this study, representing advanced pedigrees compared with those in previous studies, were mostly triploid in nature and outperformed current commercial cultivars. Of the genotypes studied, the diploids had the lowest mean yield of 8.29 oven dry Mg ha-1 yr-1, while triploids yielded 12.65 Mg ha-1 yr-1, with the top five producing over 16 Mg ha-1 yr-1. Triploids had high stem area and height across all three years of growth in addition to greatest specific gravity. The lowest specific gravity was observed among the tetraploid genotypes. Height was the early trait most correlated with and the best predictor of third-year yield. CONCLUSIONS: These results establish a paradigm for future breeding and improvement of Salix bioenergy crops based on the development of triploid species hybrids. Stem height and total stem area are effective traits for early prediction of relative yield performance.

Composition and functional properties of hemp seed protein isolates from various hemp cultivars.

Hemp seed protein isolates (HPI) were extracted from seven commercial hemp cultivars, a Cornell breeding line, and a commercial hemp heart product, and their composition and functional properties were investigated. HPI contained different ratios of edestin, vicilin, and albumin proteins, which affected protein solubility and functionality. Higher protein solubility was associated with cultivars that contained more vicilin and albumin, which influenced the subsequent functional properties of HPI. Significant differences in water holding capacity (0.83-1.05 g water/g protein isolate), oil holding capacity (1.28-1.81 g oil/g protein isolate), foam capacity (52.9%-84.9%), and foam stability (68.1%-89.4%) were observed across HPI. The Cornell hemp breeding line exhibited the highest protein solubility at pH 7.0 and was uniquely capable of forming an emulsion. The relationship identified between hemp seed protein composition and functionality, in conjunction with the demonstration of an on-going hemp breeding line, suggest that continued, targeted development of hemp cultivars can improve its seed protein functional properties for ingredient utilization in plant-based foods.

Correlations among morphological and biochemical traits in high-cannabidiol hemp (Cannabis sativa L.).

Cannabis sativa is cultivated for multiple uses including the production of cannabinoids. In developing improved production systems for high-cannabinoid cultivars, scientists and cultivators must consider the optimization of complex and interacting sets of morphological, phenological, and biochemical traits, which have historically been shaped by natural and anthropogenic selection. Determining factors that modulate cannabinoid variation within and among genotypes is fundamental to developing efficient production systems and understanding the ecological significance of cannabinoids. Thirty-two high-cannabinoid hemp cultivars were characterized for traits including flowering date and shoot-tip cannabinoid concentration. Additionally, a set of plant architecture traits, as well as wet, dry, and stripped inflorescence biomass were measured at harvest. One plant per plot was partitioned post-harvest to quantify intra-plant variation in inflorescence biomass production and cannabinoid concentration. Some cultivars showed intra-plant variation in cannabinoid concentration, while many had a consistent concentration regardless of canopy position. There was both intra- and inter-cultivar variation in architecture that correlated with intra-plant distribution of inflorescence biomass, and concentration of cannabinoids sampled from various positions within a plant. These relationships among morphological and biochemical traits will inform future decisions by cultivators, regulators, and plant breeders.