The sexual differentiation of Cannabis sativa L.: A morphological and molecular study

Summary Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova.Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment.Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.     

The inheritance of chemical phenotype in Cannabis sativa L. (II): Cannabigerol predominant plants

This paper aims to clarify the genetic mechanism that is responsible for the accumulation of cannabigerol (CBG) in certain phenotypes of Cannabis sativa L. CBG is the direct precursor of the cannabinoids CBD, THC and CBC. Plants strongly predominant in CBG have been found in different fibre hemp accessions. Inbred offspring derived from one such individual were crossed with true breeding THC predominant- and CBD predominant plants, respectively. The segregations in the cross progenies indicate that CBG accumulation is due to the homozygous presence of a minimally functional allele, tentatively called B₀, at the single locus B that normally controls the conversion of CBG into THC (allele B_T) and/or CBD (allele B_D). The fact that CBG accumulating plants have so far been found in European fibre hemp populations that are generally composed of B_D/B_D plants, and the observation that the here investigated B₀ allele possesses a residual ability to convert small amounts of CBG into CBD, make it plausible that this B₀ is a mutation of normally functional B_D. Therefore, B₀ is considered as a member of the B_D allelic series encoding a CBD synthase isoform with greatly weakened substrate affinity and/or catalytic capacity.     

The CPRO Cannabis germplasm collection

Summary A collection of more than a 150 Cannabis accessions is being established as a source for evaluation and breeding experiments. Origin of the accessions and maintenance of the collection are described.     

Copper localization in Cannabis sativa L. grown in a copper-rich solution

Summary With the aim to examine its potential as a renewable resource to decontaminate polluted soils, electron microscopy combined with X-ray microanalysis was used to investigate the localization of copper in Cannabis sativa grown in hydroponic copper-rich culture. Cu was found to accumulate preferentially in the upper leaf epidermal cells; it was also detected in spiculae and in abaxial trichomes too. Primary bast fibres seem to be not involved in copper accumulation.     

DNA polymorphisms in Oryza sativa L. and Lactuca sativa L. amplified by arbitrary primed PCR

Summary Thirty-five rice (Oryza sativa L.) varieties, including 18 japonica, 5 javanica and 12 indica subspecies and 12 lettuce (Lactuca sativa L.) varieties were identified taxonomically, using PCR with originally designed 21 RAPD (Random Amplified Polymorphic DNA) primers and 8 sequence-specific primers, used for amplifying four specific DNA fragments. Use of these primers revealed polymorphisms among varieties in rice and lettuce and facilitates DNA fingerprinting. Dendrograms of both species based on polymorphisms were constructed and genetical relationships were established. In rice, half the number of amplified bands were polymorphic and almost all varieties differentiated. However, differentiation of minor genetic alterations among somaclonal variants or mutants and their mother varieties was not feasible. In L. sativa, 47% of the amplified fragments were polymorphic and all 12 varieties were differentiated. Some of the PCR fragments were variety or type specific, which could be used for indicators for type-selection. The dendrogram obtained showed differentiated clusters of crisphead, leaf and butterhead type, findings in good accord with the classification based on the genetic background.     

Characterisation of Cannabis accessions with regard to cannabinoid content in relation to other plant characters

Summary Ninety seven Cannabis accessions were evaluated for cannabinoid content and non-chemical plant characters. Variation within populations for cannabinoid content, and consistency of chemical characters at the population level were investigated. The relationship between chemical and other plant characters was very limited. Leaflet width and phenological data can be used for a rough prediction of the chemical phenotype on a population level. Various combinations of cannabinoid content and other economic plant characters were observed, thus a breeding programme will not be hampered by strict linkage. For a selection programme a direct analysis of cannabinoids will be inevitable.     

Predicting the chemical composition of fibre and core fraction of hemp (Cannabis sativa L.)

Summary Fibre formation in hemp (Cannabis sativa L.) is a dynamic process. In order to follow this process, the chemical composition of the hemp stem was studied during plant development using the acid and neutral detergent fibre and lignin methods. Additionally, near infra-red spectroscopy was carried out. To predict the chemical composition of the stem samples partial least square (PLS) analysis was carried out. The developed PLS models can predict the level of %ADF and %NDF in the hemp bast and core samples. For %ADL, the models are only suited for either core or bast tissues and can not be used for both tissues simultaneously.     

Evaluation and verification of resistance to Meloidogyne hapla Chitwood in a Cannabis germplasm collection

A collection of Cannabis accessions was evaluated for resistance to the root-knot nematode Meloidogyne hapla Chitwood in a seedling test. After inoculation with a larval suspension, significant variation was found for the number of galls and egg masses on the seedling roots. These parameters were considered as estimates for nematode infection and larval multiplication, respectively. A subset of the tested accessions was grown on a naturally infested arable field to study the relation between the test results and host characteristics in the field. The ranking order of accessions for the number of galls in the seedling test agreed well with that for the number of root galls on juvenile plants in the field. Therefore the test provides a useful indication for nematode infection. The number of egg masses in the seedling test and the population density of Meloidogyne measured in post-treatment soil samples of the field trial, showed a comparable ranking of accessions. Differences between accessions in the field were however smaller and not statistically significant.     

Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes

The oil content, the tocopherol composition, the plastochromanol-8 (P-8) content and the fatty acid composition (19 fatty acids) of the seed of 51 hemp (Cannabis sativa L.) genotypes were studied in the 2000 and 2001 seasons. The oil content of the hemp seed ranged from 26.25% (w/w) to 37.50%. Analysis of variance revealed significant effects of genotype, year and of the interaction (genotype × year) on the oil content. The oil contents of the 51 genotypes in 2000 and 2001 were correlated (r = 0.37**) and averaged 33.19 ± 1.45% in 2000 and 31.21 ± 0.96% in 2001. The -tocopherol, -tocopherol, -tocopherol, P-8- and -tocopherol contents of the 51 genotypes averaged 21.68 ± 3.19, 1.82 ± 0.49, 1.20 ± 0.40, 0.18 ± 0.07 and 0.16 ± 0.04 mg 100g^–1 of seeds, respectively (2000 and 2001 data pooled). Hierarchical clustering of the fatty acid data did not group the hemp genotypes according to their geographic origin. The -linolenic acid yield of hemp (3–30 kg ha^–1) was similar to the -linolenic acid yield of plant species that are currently used as sources of -linolenic acid (borage (19–30 kg ha^–1), evening primrose (7–30 kg ha^–1)). The linoleic acid yield of hemp (129–326 kg ha^–1) was similar to flax (102–250 kg ha^–1), but less than in sunflower (868–1320 kg ha^–1). Significant positive correlations were detected between some fatty acids and some tocopherols. Even though the average content of P-8 in hemp seeds was only 1/120^th of the average -tocopherol content, P-8 content was more closely correlated with the unsaturated fatty acid content than -tocopherol or any other tocopherol fraction. The average broad-sense heritabilities of the oil content, the antioxidants (tocopherols and P-8) and the fatty acids were 0.53, 0.14 and 0.23, respectively. The genotypes Fibrimon 56, P57, Juso 31, GB29, Beniko, P60, FxT, Félina 34, Ramo and GB18 were capable of producing the largest amounts of high quality hemp oil.     

Optimisation of breeding for agronomic traits in fibre hemp (Cannabis sativa L.) by study of parent-offspring relationships

Summary In the framework of a comprehensive Dutch research project the feasibility of hemp cultivation for paper pulp production is being investigated. This project also includes a hemp breeding programme which was initiated at CPRO-DLO in 1990. Hemp breeding is primarily aimed at improving bast fibre production since bast fibre is qualitatively superior to woody core fibre for paper pulp production. The progress of the ongoing breeding programme is hampered by the lack of knowledge on the inheritance of agronomic traits.The following traits were examined on 252 parental plants in 1991 and subsequently on their progenies in 1992: fibre content, plant height, date of flowering, stem diameter and cannabinoid contents. For each trait heritabilities were estimated and direct and indirect effects of artificial selection were studied.The heritability of bast fibre content was high and mass selection proved to be an efficient method causing no undesirable influence on other characters. Characters not directly related to bast fibre yield such as date of flowering, plant height and stem diameter were shown to have disadvantages as selection criteria for the improvement of bast fibre yield.The cannabinoids THC and CBD were studied, as the acceptance of hemp cultivation requires a low level. The content of THC, the psychoactive component can be successfully reduced by mass selection, but it is not certain that mass selection is the most efficient method.     

Independent segregation of two isozyme markers and inter-plant differences in nuclear DNA content in the interspecific backcross (Allium fistulosum L. × A. cepa L.) × A. cepa L.

Summary From backcrosses of three interspecific hybrids (A. fistulosum x A. cepa) with a. cepa 14 diploid and 2 triploid plants were recovered.In this BC1 population introgression of A. fistulosum genetic material into the A. cepa nuclear genome was studied using two isozyme markers: Got-1 and Got-2. Both loci carried two alleles. A. cepa was monomorphic for both markers. A. fistulosum was polymorphic for Got-2. Based on their Got isozyme pattern seven out of the 14 diploid BC1 plants had a recombinant genotype. The loci appeared to be unlinked. Differences were observed in nuclear DNA contents between the diploid BC1 plants, indicating that gametes produced by the interspecific hybrids contained different combinations of chromosomal material from A. cepa and A. fistulosum.Abbreviations Adh alcohol dehydrogenase - Got glutamate oxaloacetate transaminase - Idh isocitrate dehydrogenase - Pgi phosphoglucoisomerase - Pgm phosphoglucomutase     

Molecular Marker based Genetic Diversity Analysis in Rice (Oryza sativa L.) using RAPD and SSR markers

The availability of an array of molecular marker systems allowed comparing the efficiency of two of these marker systems to estimate the relationships among various taxa. The objective of this study was to assess the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. Out of 38 SSR primer pairs used, only one locus viz., RM115 was monomorphic. The average Polymorphism Information Content (PIC) value was 0.578 and it ranged from a low of zero (RM 115) to a high of 0.890 (RM 202). The Mantel matrix correspondence test was used to compare the similarity matrices and the correlation coefficient was 0. 582. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

The inheritance of chemical phenotype in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. (III): variation in cannabichromene proportion

The mechanism that controls the proportion of cannabichromene (CBC), a potential pharmaceutical, in the cannabinoid fraction of Cannabis sativa L. is explored. As with tetrahydrocannabinol (THC) and cannabidiol (CBD), CBC is an enzymatic conversion product of the precursor cannabigerol (CBG). CBC is reported to dominate the cannabinoid fraction of juveniles and to decline with maturation. This ontogeny was confirmed in inbred lines with different mature chemotypes. A consistent CBC presence was found in early leaves from a diverse clone collection, suggesting that CBC synthase is encoded by a fixed locus. Morphological variants possessing a ‘prolonged juvenile chemotype’ (PJC), a substantial proportion of CBC persisting up to maturity, are presented. PJC is associated with a reduced presence of floral bracts, bracteoles, and capitate-stalked trichomes. Genetic factors causing these features were independent of the allelic chemotype locus B that was previously postulated and regulates THC and CBD synthesis and CBG accumulation. In contrast to previously described Cannabis chemotypes, the cannabinoid composition of PJCs showed plasticity in that reduced light levels increased the CBC proportion. The ability of PJC plants to enable the production of pharmaceutical raw material with high CBC purity is demonstrated.     

RAPD markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Groundnut rust (Puccinia arachidis Speg.) is an important air borne pathogen, which causes substantial losses in groundnut yield and quality. Although large numbers of accessions were identified as rust resistant in wild, interspecific derivative and cultivated groundnut species, transfer of resistance to well-adapted cultivars is limited due to linkage drag, which worsens yield potential and market acceptance. A F₂ mapping population comprising 117 individuals was developed from a cross between the rust resistant parent VG 9514 and rust susceptible parent TAG 24. Rust resistance was governed by single dominant gene in this cross. We identified 11 (out of 160) RAPD primers that exhibited polymorphism between these two parents. Using a modified bulk segregant analysis, primer J7 (5′CCTCTCGACA3′) produced a single coupling phase marker (J7₁₃₅₀) and a repulsion phase marker (J7₁₃₀₀) linked to rust resistance. Screening of the entire F₂ population using primer J7 revealed that the coupling phase marker J7₁₃₅₀ was linked with the rust resistance gene at a distance of 18.5 cM. On the other hand, the repulsion phase marker J7₁₃₀₀ was completely linked with rust resistance. Additionally, both J7₁₃₀₀ (P = 0.00075) and J7₁₃₅₀ (P < 0.00001) were significantly associated with the rust resistance. Marker J7₁₃₀₀ identified all homozygous rust resistant genotypes in the F₂ population and was present in all the eight susceptible genotypes tested for validation. Thus, J7₁₃₀₀ should be applicable for marker-assisted selection (MAS) in the groundnut rust resistance breeding programme in India. To the best of our knowledge this is the first report on the identification of RAPD markers linked to rust resistance in groundnut.     

Time course of cannabinoid accumulation and chemotype development during the growth of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L

The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gas-chromatographic analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R × 15R) × NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103 days after sowing, and consistent with the ratios determined on mature inflorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80 days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.     

Genotypic control of chromosome pairing in amphiploids involving the cultivated oat Avena sativa L.

Summary A genotype of the diploid species Avena longiglumis (Cw 57) has been shown to modify the genetic control of diploid-like chromosome pairing in the cultivated oat, A. sativa (2n=6x=42) leading to increased homoeologous chromosome pairing in 4x hybrids between the two species (Rajhathy & Thomas, 1974). The Cw 57 genotype has a similar effect in increasing homoeologous chromosome pairing in amphiploids combining diploid and hexaploid genomes including associations between alien chromosomes and their corresponding pairs in hexaploid species. The effect of the Cw 57 genotype is probably in altering the specificity of chromosome pairing in the early stages of meiosis. The use of the Cw 57 genotype to induce homoeologous chromosome pairing as a technique for the transfer of desirable alien variation into the cultivated oat is discussed.     

Random amplified polymorphic DNA (RAPD) markers variability among cultivars and landraces of common beans (Phaseolus vulgaris L.) of south-Brazil

To evaluate the variability among cultivars and landraces of common bean(Phaseolus vulgaris L.), 15 cultivars and 18 landraces of common bean (Phaseolus vulgarisL.), a undefined species of Phaseolus,two landraces of Vigna angularis L., and a landrace of soybean (Glycine maxL.), were screened with fifteen oligonucleotide primers in PCR reactions. An average of 20.3 RAPD bands were scored per primer. A total of 304 amplification products were scored of which 88.8% were polymorphic among Phaseolus genotypes. Based on the RAPD markers, four major clusters were formed. Three clusters corresponded to the soybean, to the two Vigna angularis landraces, and to the Phaseolus sp. landrace, respectively. The fourth cluster include all the landraces and cultivars of Phaseolus vulgaris. This large group could be separated into three subgroups that were correlated with the phaseolin patterns and the average seed weight of the genotypes. The analysis shows that most of the landraces collected in South Brazil (17 out of 18) belong to the Andean gene pool, and most of the cultivars (13 out of 15) belong to the Middle American gene pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of sex in Carica papaya L. using RAPD markers

Brazil is currently the worlds largest producer of papaya (Carica papayaL.), producing fruits for both the domestic market and export. Only fruits from hermaphrodite plants are marketed because they have the necessary commercial characteristics, i.e. they are pear-shaped and have thicker flesh and a smaller internal cavity. Increased papaya yield has been limited mainly by the ratio of female to hermaphrodite (1: 2) plants normally occurring in orchards. This ratio causes great losses to papaya producers and the identification of the sex of seedlings during the nursery stage would be an important advance. In our study random amplified polymorphic DNA (RAPD) analysis was used to differentiate between the sexual forms of three commercial C. papaya cultivars belonging to the Solo group. RAPD assays using the BC210 primer were able to detect hermaphrodites in all of the cultivars tested. The BC210₄₃₈molecular marker was much better at papaya sex differentiation than other markers described in the literature. This revised version was published online in August 2006 with corrections to the Cover Date.     

RAPD markers linked to resistance to downy mildew disease in soybean

To determine and utilize RAPD markers linked to resistance to downymildew incited by Peronospora manshurica in soybean, a resistantcultivar `AGS129' was crossed to a susceptible cultivar `Nakhon Sawan 1'(NS1). F₂ and BC₁ populations were advanced from the F₁ and evaluatedfor resistance to the disease. ²-test demonstrated that the resistancewas controlled by a single dominant gene (Rpmx). Near-isogenic lines(NILs) and bulked segregant analysis (BSA) were used to identify RAPDmarkers linked to the gene. Six DNA bulks namely F₅(R), F₅(S),BC₆F₃(R), BC₆F₃(S), F₂(R) and F₂(S) were set up by pooling equalamount of DNA from 8 randomly selected plants of each disease responsetype. A total of 180 random sequence decamer oligonucleotide primerswere used for RAPD analysis. Primer OPH-02 (5 TCGGACGTGA 3 andOPP-10 (5 TCCCGCCTAC 3) generated OPH-02₁₂₅₀ and OPP-10₈₃₁fragments in donor parent and resistant bulks, but not in the recurrentparent and susceptible ones. Co-segregation analysis using 102 segregatingF₂ progenies confirmed that both markers were linked to the Rpmxgene controlling downy mildew disease resistance with a genetic distance of4.9 cm and 23.1 cm, respectively. Marker OPH-02₁₂₅₀ was presentin 13 of 16 resistant soybean cultivars and absent in susceptible cultivars,thus confirming a potential for MAS outside the mapping population.     

工业大麻产量和品质性状的对应分析

对4个工业大麻品种的产量性状和纤维品质性状进行对应分析。结果表明,在本试验范围内,影响大麻纤维产量的主要性状是鲜茎重和干皮重;影响大麻纤维品质的性状主要是纤维支数、断裂功和含胶率,其次是断裂强度和断裂伸长率;汾麻3号较其余品种具有明显的纤维产量高和纤维支数高的特点。