HASCH - A high-throughput amplicon-based SNP-platform for medicinal cannabis and industrial hemp genotyping applications.

BACKGROUND: Cannabis sativa is seeing a global resurgence as a food, fiber and medicinal crop for industrial hemp and medicinal Cannabis industries respectively. However, a widespread moratorium on the use and research of C. sativa throughout most of the 20th century has seen the development of improved cultivars for specific end uses lag behind that of conventional crops. While C. sativa research and development has seen significant investments in the recent past, resulting in a suite of publicly available genomic resources and tools, a versatile and cost-effective mid-density genotyping platform for applied purposes in breeding and pre-breeding is lacking. Here we report on a first mid-density fixed-target SNP platform for C. sativa. RESULTS: The High-throughput Amplicon-based SNP-platform for medicinal Cannabis and industrial Hemp (HASCH) was designed using a combination of filtering and Integer Linear Programming on publicly available whole-genome sequencing and RNA sequencing data, supplemented with in-house generated genotyping-by-sequencing (GBS) data. HASCH contains 1,504 genome-wide targets of high call rate (97% mean) and even distribution across the genome, designed to be highly informative (> 0.3 minor allele frequency) across both medicinal cannabis and industrial hemp gene pools. Average numbers of mismatch SNP between any two accessions were 251 for medicinal cannabis (N = 116) and 272 for industrial hemp (N = 87). Comparing HASCH data with corresponding GBS data on a collection of diverse C. sativa accessions demonstrated high concordance and resulted in comparable phylogenies and genetic distance matrices. Using HASCH on a segregating F2 population derived from a cross between a tetrahydrocannabinol (THC)-dominant and a cannabidiol (CBD)-dominant accession resulted in a genetic map consisting of 310 markers, comprising 10 linkage groups and a total size of 582.7 cM. Quantitative Trait Locus (QTL) mapping identified a major QTL for CBD content on chromosome 7, consistent with previous findings. CONCLUSION: HASCH constitutes a versatile, easy to use and cost-effective genotyping solution for the rapidly growing Cannabis research community. It provides consistent genetic fingerprints of 1504 SNPs with wide applicability genetic resource management, quantitative genetics and breeding.

From dawn 'til dusk: daytime progression regulates primary and secondary metabolism in Cannabis glandular trichomes.

Cannabis sativa L. glandular trichomes (GTs) synthesise large amounts of secondary metabolites, predominantly cannabinoids and terpenoids. The associated demand for carbon and energy makes GTs strong sink tissues with indications that their secondary metabolism is coupled to the availability of photoassimilates. Many metabolites show diurnal patterns of flux, but it is unknown whether cannabinoids and terpenoids are regulated by time of day. We quantified cannabinoids, terpenoids and the GT proteome over a 12-hour light period in flowers of Hindu Kush, a high-tetrahydrocannabinol (THC) cultivar. Major cannabinoids changed significantly over the course of day, resulting in an increase in total measured cannabinoids. Major terpenoids also changed, with sesquiterpenes generally decreasing with day progression. While monoterpenes generally did not decrease, the second most abundant, α-pinene, increased. The GT proteome changed the most within the first six hours of the day and analysis of differentially abundant proteins indicated upregulation of primary metabolism. Surprisingly, key cannabinoid biosynthetic enzymes decreased with daytime progression despite increases in cannabinoid content, which indicate that daytime increases of photoassimilates are the main driver of cannabinoid regulation. This first reporting of variability of cannabinoid and terpenoid biosynthesis over the course of the day has implications for Cannabis research and production.

Overexpression of a wheat α-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination.

Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0-437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development.

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling.

Importance of Media Composition and Explant Type in Cannabis sativa Tissue Culture.

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications.

Why not XY? Male monoecious sexual phenotypes challenge the female monoecious paradigm in Cannabis sativa L.

Monoecy in Cannabis sativa L. has long been considered an industrially important trait due to the increased uniformity it offers and was thought to be exclusively associated with XX females. The isolation and characterisation of a monoecious individual with XY chromosomes sourced from non-proprietary germplasm is reported for the first time. The chromosomal make up of this trait was confirmed through inflorescence structure, growth habit, PCR analysis and sexual phenotypes of progeny from a series of targeted crosses. The identification of an XY monoecious phenotype widens our understanding of monoecy in Cannabis and has important implications for breeding, particularly for producing F1-hybrid seed.

Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay.

BACKGROUND: A crucial step in the evaluation of newly produced transgenic plants is the selection of homozygous plants. Here we describe an efficient and highly flexible real-time PCR-based method for the development of homozygous lines in plant models with complex (multiple) genomes and/or relatively long generation times (>3 months) using direct copy number determinations. RESULTS: An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses. Highly specific and efficient primer pairs were developed for a bread wheat reference gene (Epsilon Cyclase) and for standard sequence elements in the gene cassette routinely used for cereal transformations (an intron bridge and the Nopaline Synthase terminator). The real-time PCR assay reliably distinguished wheat plants with a single copy of the transgene from individuals with multiple copies or those lacking the transgene. To obtain homozygous lines carrying a unique insertion event as efficiently as possible, T0 plants (plants raised from transformed callus) with a single copy of the transgene were selected and their progeny screened for homozygous plants. Finally, the assay was adapted to work on rice. CONCLUSIONS: The ability to quickly, easily and accurately quantify the construct copy numbers, as provided by the real-time PCR assay, greatly improved the efficiency and reliability of the selection of homozygous transgenic plants in our case study. We were able to select homozygous plants in early generations, avoiding time-consuming methods such as large scale analysis of segregation patterns of descendants and/or Southern blotting. Additionally, the ability to specifically develop homozygous lines carrying a unique insertion event could be important in avoiding gene silencing due to co-suppression, and if needed assist in the selection of lines suitable for future deregulation. The same primer pairs can be used to quantify many different wheat transgenic events because the construct-specific primer pairs are targeted to standard sequence elements of the cereal gene cassettes, making the method widely applicable in wheat GM research. Moreover, because all procedures described here are standardized, the method may easily be adapted to vectors lacking the target regions used here and/or to other plant models.

Using a global diversity panel of Cannabis sativa L. to develop a near InfraRed-based chemometric application for cannabinoid quantification.

C. sativa has gained renewed interest as a cash crop for food, fibre and medicinal markets. Irrespective of the final product, rigorous quantitative testing for cannabinoids, the regulated biologically active constituents of C. sativa, is a legal prerequisite across the supply chains. Currently, the medicinal cannabis and industrial hemp industries depend on costly chromatographic analysis for cannabinoid quantification, limiting production, research and development. Combined with chemometrics, Near-InfraRed spectroscopy (NIRS) has potential as a rapid, accurate and economical alternative method for cannabinoid analysis. Using chromatographic data on 12 therapeutically relevant cannabinoids together with spectral output from a diffuse reflectance NIRS device, predictive chemometric models were built for major and minor cannabinoids using dried, homogenised C. sativa inflorescences from a diverse panel of 84 accessions. Coefficients of determination (r2) of the validation models for 10 of the 12 cannabinoids ranged from 0.8 to 0.95, with models for major cannabinoids showing best performance. NIRS was able to discriminate between neutral and acidic forms of cannabinoids as well as between C3-alkyl and C5-alkyl cannabinoids. The results show that NIRS, when used in conjunction with chemometrics, is a promising method to quantify cannabinoids in raw materials with good predictive results.

Moving Away from 12:12; the Effect of Different Photoperiods on Biomass Yield and Cannabinoids in Medicinal Cannabis.

The standard practice to initiate flowering in medicinal cannabis involves reducing the photoperiod from a long-day period to an equal duration cycle of 12 h light (12L)/12 h dark (12D). This method reflects the short-day flowering dependence of many cannabis varieties but may not be optimal for all. We sought to identify the effect of nine different flowering photoperiod treatments on the biomass yield and cannabinoid concentration of three medicinal cannabis varieties. The first, "Cannatonic", was a high cannabidiol (CBD)-accumulating line, whereas the other two, "Northern Lights" and "Hindu Kush", were high Δ9-tetrahydrocannabinol (THC) accumulators. The nine treatments tested, following 18 days under 18 h light/6 h dark following cloning and propagation included a standard 12L:12D period, a shortened period of 10L:14D, and a lengthened period of 14L:10D. The other six treatments started in one of the aforementioned and then 28 days later (mid-way through flowering) were switched to one of the other treatments, thus causing either an increase of 2 or 4 h, or a decrease of 2 or 4 h. Measured parameters included the timing of reproductive development; the dry weight flower yield; and the % dry weight of the main target cannabinoids, CBD and THC, from which the total g cannabinoid per plant was calculated. Flower biomass yields were highest for all lines when treatments started with 14L:10D; however, in the two THC lines, a static 14L:10D photoperiod caused a significant decline in THC concentration. Conversely, in Cannatonic, all treatments starting with 14L:10D led to a significant increase in the CBD concentration, which led to a 50-100% increase in total CBD yield. The results show that the assumption that a 12L:12D photoperiod is optimal for all lines is incorrect as, in some lines, yields can be greatly increased by a lengthened light period during flowering.

Over-Expression of a Wheat Late Maturity Alpha-Amylase Type 1 Impact on Starch Properties During Grain Development and Germination.

The hydrolysis of starch is a complex process that requires synergistic action of multiple hydrolytic enzymes, including α-amylases. Wheat over-expression of TaAmy1, driven by seed specific promoter, resulted in a 20- to 230-fold total α-amylase activity in mature grains. Ectopic expression of TaAmy1 showed a significant elevated α-amylase activity in stem and leaf without consequences on transitory starch. In mature grain, overexpressed TaAMY1 was mainly located in the endosperm with high expression of TaAmy1. This is due to early developing grains having effect on starch granules from 18 days post-anthesis (DPA) and on soluble sugar accumulation from 30 DPA. While accumulation of TaAMY1 led to a high degree of damaged starch in grain, the dramatic alterations of starch visco-properties caused by the elevated levels of α-amylase essentially occurred during processing, thus suggesting a very small impact of related starch damage on grain properties. Abnormal accumulation of soluble sugar (α-gluco-oligosaccharide and sucrose) by TaAMY1 over-expression reduced the grain dormancy and enhanced abscisic acid (ABA) resistance. Germination study in the presence of α-amylase inhibitor suggested a very limited role of TaAMY1 in the early germination process and starch conversion into soluble sugars.

Characterization of the Cannabis sativa glandular trichome proteome.

Cannabis sativa has been cultivated since antiquity as a source of fibre, food and medicine. The recent resurgence of C. sativa as a cash crop is mainly driven by the medicinal and therapeutic properties of its resin, which contains compounds that interact with the human endocannabinoid system. Compared to other medicinal crops of similar value, however, little is known about the biology of C. sativa. Glandular trichomes are small hair-like projections made up of stalk and head tissue and are responsible for the production of the resin in C. sativa. Trichome productivity, as determined by C. sativa resin yield and composition, is only beginning to be understood at the molecular level. In this study the proteomes of glandular trichome stalks and heads, were investigated and compared to the proteome of the whole flower tissue, to help further elucidate C. sativa glandular trichome biochemistry. The data suggested that the floral tissue acts as a major source of carbon and energy to the glandular trichome head sink tissue, supplying sugars which drive secondary metabolite biosynthesis. The trichome stalk seems to play only a limited role in secondary metabolism and acts as both source and sink.

Does Late Maturity Alpha-Amylase Impact Wheat Baking Quality?

Late maturity α-amylase (LMA) and pre-harvest sprouting (PHS) are both recognized as environmentally induced grain quality defects resulting from abnormally high levels of α-amylase. LMA is a more recently identified quality issue that is now receiving increasing attention worldwide and whose prevalence is now seen as impeding the development of superior quality wheat varieties. LMA is a genetic defect present in specific wheat genotypes and is characterized by elevated levels of the high pI TaAMY1 α-amylase, triggered by environmental stress during wheat grain development. TaAMY1 remains present in the aleurone through the harvest, lowering Falling Number (FN) at receival, causing a down-grading of the grain, often to feed grade, thus reducing the farmers' income. This downgrading is based on the assumption within the grain industry that, as for PHS, a low FN represents poor quality grain. Consequently any wheat line possessing low FN or high α-amylase levels is automatically considered a poor bread wheat despite there being no published evidence to date, to show that LMA is detrimental to end product quality. To evaluate the validity of this assumption a comprehensive evaluation of baking properties was performed from LMA prone lines using a subset of tall non-Rht lines from a multi-parent advanced generation inter-cross (MAGIC) wheat population grown at three different sites. LMA levels were determined along with quality parameters including end product functionality such as oven spring, bread loaf volume and weight, slice area and brightness, gas cell number and crumb firmness. No consistent or significant phenotypic correlation was found between LMA related FN and any of the quality traits. This manuscript provides for the first time, compelling evidence that LMA has limited impact on bread baking end product functionality.

Fast and Efficient Screening for Wheat Loss-of-Gene Mutants Using Multiplexed Melt Curve Analyses.

This study describes a new approach in the screening for loss-of-gene mutants in Heavy Ion Bombardment (HIB) mutant populations of genetically complex organisms such as hexaploid bread wheat using multiplexed single-color (SYBR Green) melt curve analyses. The assay was set up for three target genes to test its validity and applicability. For each gene, three genome-specific primer pairs (one for each genome) with distinct melt curves were developed and multiplexed. This allowed screening for "single null mutants" (plants with the target gene deleted in one of the three genomes) for all three genomes in a single reaction. The first two genes (α-Amylase 3 and Epsilon Cyclase) were used to test the approach as HIB null lines for all three genomes were already available for these. The third assay was successfully applied to identify new single null lines of the target gene α-Amylase 2 in an in-house HIB wheat collection. The use of SYBR Green greatly reduced the time and/or cost investment compared to other techniques and the approach proved highly suitable for high-throughput applications.

Vibrio zinc-metalloprotease causes photoinactivation of coral endosymbionts and coral tissue lesions.

BACKGROUND: Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on a Vibrio zinc-metalloprotease causing rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to non-inhibited and EDTA-inhibited supernatants derived from coral white syndrome pathogens. CONCLUSION/SIGNIFICANCE: These findings demonstrate a common virulence factor from four phylogenetically related coral pathogens, suggesting that zinc-metalloproteases may play an important role in Vibrio pathogenicity in scleractinian corals.

The roles and interactions of symbiont, host and environment in defining coral fitness.

BACKGROUND: Reef-building corals live in symbiosis with a diverse range of dinoflagellate algae (genus Symbiodinium) that differentially influence the fitness of the coral holobiont. The comparative role of symbiont type in holobiont fitness in relation to host genotype or the environment, however, is largely unknown. We addressed this knowledge gap by manipulating host-symbiont combinations and comparing growth, survival and thermal tolerance among the resultant holobionts in different environments. METHODOLOGY/PRINCIPAL FINDINGS: Offspring of the coral, Acropora millepora, from two thermally contrasting locations, were experimentally infected with one of six Symbiodinium types, which spanned three phylogenetic clades (A, C and D), and then outplanted to the two parental field locations (central and southern inshore Great Barrier Reef, Australia). Growth and survival of juvenile corals were monitored for 31-35 weeks, after which their thermo-tolerance was experimentally assessed. Our results showed that: (1) Symbiodinium type was the most important predictor of holobiont fitness, as measured by growth, survival, and thermo-tolerance; (2) growth and survival, but not heat-tolerance, were also affected by local environmental conditions; and (3) host population had little to no effect on holobiont fitness. Furthermore, coral-algal associations were established with symbiont types belonging to clades A, C and D, but three out of four symbiont types belonging to clade C failed to establish a symbiosis. Associations with clade A had the lowest fitness and were unstable in the field. Lastly, Symbiodinium types C1 and D were found to be relatively thermo-tolerant, with type D conferring the highest tolerance in A. millepora. CONCLUSIONS/SIGNIFICANCE: These results highlight the complex interactions that occur between the coral host, the algal symbiont, and the environment to shape the fitness of the coral holobiont. An improved understanding of the factors affecting coral holobiont fitness will assist in predicting the responses of corals to global climate change.