All Cannabis germplasm is completely interfertile, suggesting that the genus consists of a single species, with two subspecies and subsp. indica . Through DNA analysis, evidence pointed towards C. sativa, C. indica, C. ruderalis populations of Cannabis as being distinct subspecies of Cannabis sativa . Common vernacular terms, sativa and indica, have been used to describe different types of marijuana, causing much confusion for consumers who believe that these different forms of Cannabis happen to function in different ways. Generally, sativa types are plants with tall and slender morphology, narrow leaflets, and late maturation while indica types have shortened stature, broad leaflets, and early maturation . Whatever true population differences there may have been between indica and sativa types of C. sativa subsp. indica have become lost over generations of repeated hybridization events , although clearer population differentiation between hemp and marijuana still exist . Thus, industrial hemp is simply classified taxonomically as Cannabis sativa L. with low amounts of THC . One way to classify genotypes or populations of C. sativa is through their chemotypes, or chemical phenotypes, based on the predominant cannabinoids in the plant, in particular, cannabidiol , cannabigerol , and tetrahydrocannabinol . C. sativa is divided into five different chemotypes: THC dominant, referred to as the drug-type, CBD dominant, referred to as fiber-type, and one which THC and CBD are present in equal proportion. Two other chemotypes, CBG dominant and those which contain low concentrations of cannabinoids, are less frequently used in scientific literature . The classification of C. sativa germplasm with chemotypes enables scientists to easily classify individuals for breeding for certain uses like pharmacology, fiber, or seed . Cannabis sativa L. is a dioecious plant with individuals from the male and female sexes having distinct morphological differences .
Male individuals are described as slenderer in stature than their female counterparts and have less reproductive biomass compared to a females’ dense inflorescence . Differences in morphology, how to cure cannabis specifically between the sexes of hemp plants, only become apparent after the seedling stage . The genetic basis for sex in Cannabis is determined by the inheritance of either an X or Y chromosome from the male parent. Female plants have a sticky inflorescence that captures wind-dispersed pollen from male plants . Male flowers, within their hanging panicles, have a perianth of five sepals that surround the androecium; the anthers at maturity split lengthwise, releasing the pollen grains . Female flowers develop as thick clusters called racemes, and receive the pollen grains through insect, wind, or mechanical dispersion onto the pistils. In production settings, formation of seeds is undesirable if the use of hemp is the extraction of essential oils or the sale of the flowers themselves; consequently, most hemp producers prefer to grow only female individuals and avoid fertilization from male plants . Grandular trichomes, a form of sessile trichomes, cover the surface of female flowering tissues and produce cannabinoid oils . The secreted oils which burst from these trichome sacs coat the surface with a sticky resin, which results in flowers that are waxy in texture . Cannabinoids likely serve multiple purposes within the Cannabis plant, such as a defense response against herbivory from insects and the dissipation of heat stress in the environment. Cannabinoids are produced in substantially higher quantities when exposed to UV-B radiation , suggesting they act as a barrier against the damaging effects of UV-B radiation .
There are over 180 different cannabinoids present in C. sativa, with the primary cannabinoids being THC, also called Δ9-THC, CBD, and CBC . These cannabinoids coexist along with their acid-precursors: tetrahydrocannabinolic acid , cannabidiolic acid , and cannabichromenic acid . The acid forms of these cannabinoids change into their decarboxylated forms primarily from the application of lightand/or heat onto the harvested crop, but the decarboxylated forms still exist at certain levels within the flowering tissue before harvesting . All acid forms of cannabinoids come from a primary precursor phytocannabinoid called cannabigerolic acid . CBGA is a product of two metabolites, olivetolic acid and geranyl diphosphate, which are formed from the polyketide pathway and plastidial deoxyxylulose phosphate/methyl-erythritol phosphate pathway , respectively . Cannabinoids have been used as a component of human therapeutic medicine for thousands of years . Specific cannabinoids have been used to minimize chronic pain, improve sleep quality, and treat a wide variety of other ailments. C. sativa has obtained more attention over the last 30 years as a source of medicine in America after California passed the Compassionate Use Act of 1996, a bill which allowed the state to provide patients with access to medical marijuana “in the treatment of cancer, anorexia, AIDS, chronic pain, spasticity, glaucoma, arthritis, migraine, or any other illness for which marijuana provides relief” . Since then, there has been increasing interest in the use of cannabinoids as a way of minimizing pain, a malady affecting 1 in 5 Americans on a daily basis . CBD is of particular interest because it can provide pain relief without the psychoactive effects that come with other cannabinoids like THC .
The global value of CBD products is 2.8 billion dollars as of 2020 and is expected to increase on the order of 20-23% year-to-year over the next five years . In America, Cannabis was a widely grown crop for many years during the 18th and 19th centuries before being replaced by cotton as the predominant crop used for textiles . With increased interest in the crop medically for its oils, it has also renewed interest in its fiber , oil, and seed . Hemp bast, the long fibers from the outer stem of hemp, can be used to make carpets, shoes, diapers, insulation, yarn, composite materials, and plastics . The hurd, or inner core fibers of the hemp plant, can be used to create hempcrete, animal bedding, potting mix, and soil amendments . With a wide variety of uses, industrial hemp fibers, as of 2019, have a current market value of $4.46 billion with an expected compound annual growth rate of 33% through 2027 to total $43.8 billion . With the value of industrial hemp between its fiber and CBD products currently being valued at over $7.2 billion, there is increasing interest in agronomic improvements for the crop. Hemp is typically grown in field settings but can be grown in greenhouses when growers are focusing on greater pest control, year-round growth, and control of lighting regiment. In field settings, hemp generally is grown on well-drained soil which is high in fertility . Planting density varies among varieties and the grower’s intended use for the crop. Hemp grown for oil extraction requires wider spacing to promote branching and flower development; planting densities for the purpose of harvesting its fiber is typically double that of oil seed varieties, however, in general the architecture of the plant itself is strongly associated with the density of planting, nutritionally availability within the soil, and the length of day that the plant is exposed too during its life cycle . Hemp is either planted by direct seeding or through the transplantation of seedlings or clones . When hemp is grown for medicinal oil extraction, the field typically consists of only female plants. Usually this is accomplished by using “feminized” seed, which is produced from female plants that have been induced to produce male gametes and seed using either chemical or environmental stress which results in seeds which will produce seeds which result mostly in female plants . This process of masculizing female hemp plants on an industrial scale is done through foliar applications of silver thiosulfate . The rationale for producing feminized seeds, outside of maintaining genetically identical inbred lines for commercial sale, is to minimize the production of seeds within the flowering tissue of female plants due to consumer, grower, and processor preferences. Hemp improvement can be done through the use of phenotypic recurrent selection, aka mass-selection, by selecting the best individual plants based on field performance and using their seed for the next evaluation cycle . Once an elite variety has been developed, back crossing can be used to incorporate new traits from undesirable germplasm . For traits such as fiber quantity and quality, with high trait heritability, trimming cannabis mass-selection can work well . Other breeding practices have been employed for the purpose of either increasing variation, specifically when crossbreeding individuals, or for fixing a trait through inbreeding to produce inbred lines and/or to capitalize on heterosis of F1 hybrid cultivars .Advances in biotechnology can accelerate the incorporation of a trait into an existing population with high accuracy and speed using next generation sequencing and marker assisted selection .
Marker assisted selection works to track the phenotype of an individual plant by associating genetic polymorphisms with trait variation, enabling selection on seedling plants without having to grow the individual to maturity . Recent advances in sequencing technology, specifically with the development of next-generation sequencing , has reduced the price of sequencing whole genomes of individual plants , making marker identification and use more tractable. Day neutrality is a trait which is present in many agricultural crops such as soybeans, wheat, barley, rice, tomatoes, strawberries, and alfalfa . Several genes involved light sensing contribute to differences among genotypes in time of flower and in day neutrality; the major genes appear to be conserved across species. For instance, day-neutrality is controlled by CONSTANS , a gene which encodes a transcription factor involved in the transduction of light signals, promoting the expression of other genes downstream . The genetic basis for the change from vegetative growth to flowering within hemp cultivars is mostly unknown. Petit et al. , identified six QTLs related to genes which control the perception and transduction of light and their associated transcription factors. However, hemp germplasm has a wide range of flowering times, affected by both genetics and the environment . Hemp, Cannabis sativa L. is a valuable medicinal, fiber, seed, and oil crop. Understanding and manipulating the flowering time of hemp could facilitate cultivar development for diverse environments and cropping systems. The time at which hemp transitions from vegetative growth to flowering is critical for the development and quality of the final harvested product. Most hemp germplasm requires short days to begin flowering and producing seed, oil, and cannabinoid products . Before the transition to flowering can occur, a period of vegetative growth stage controlled by the accumulation of thermal time in the environment is necessary . Vegetative growth for hemp is optimal around 30o C and continues to a maximum temperature about 42o C. Around 300 to 600 units of cumulated thermal time over 1o C must occur for the plant to be able to initiate flowering if the critical daylength is reached . While typically a short-day plant, hemp germplasm has considerable variation in TOF . Some hemp germplasm has day-neutral flowering, which means that regardless of photoperiod, they will flower after a certain amount of thermal time has accumulated . Day-neutral Cannabis varieties can go from seed to flowering in a soon as four weeks, and some varieties can be harvested within 100 days after seeding. The trait for day-neutrality originated from the ruderalis variety of Cannabis sativa in parts of Southern Russia. These plants are described as being short and stalkyin nature, while producing small amounts of flower with low concentrations of THC . Despite its undesirable attributes, ruderalis has been used to introgress day neutrality into high yielding flower and oil varieties. The day neutral trait can provide significant agronomic benefits by standardizing harvest time and potentially enabling growers to have more harvest cycles in one year compared to those growing day-sensitive varieties of hemp which takes much longer to flower in most environments. The genetic basis for day neutrality, specifically in hemp, is currently unknown. We hypothesized that at least one major locus controls day neutral flowering with multiple minor loci affecting the variation in time to flower within day neutral or daylength sensitive germplasm. To test this hypothesis, we hybridized day neutral and daylength sensitive accessions of hemp and evaluated the progeny for flowering time across three field trials over two years in order and genotyped them using next generation sequencing technology to identify genetic loci involved in flowering time. Hemp seeds were placed into 72-cell flats containing potting soil mix, composed of mostly perlite, and were then covered in vermiculite and watered daily.