Assessment of the Cannabinoid Content from Different Varieties of Cannabis sativa L. during the Growth Stages in Three Regions

Hemp (Cannabis sativa L.) belongs to the Cannabaceae family. It is very rich in chemical constituents, especially the cannabinoids which has not been reported in any other plant, and has broad pharmacological properties. Hemp as a multi-purpose crop is a good source of fibers, seed, fixed and volatile oil. It is known that the cannabinoid content of hemp is related to genetic factors, as well as plant's growth stages and environmental factors such as latitude, altitude, weather, particularly moisture availability and nutrient supply during the growing season. The present study was designed to produce hemp that contains allowable concentration of THC (<3 %) by comparing different varieties of hemp, different stages of plant growth, and different geographical locations where it was planted. To achieve this, seeds of two native populations from Iran (Fars and Yazd Provinces) and one foreign variety from France (Fedora17, as an industrial hemp cultivar) with its progenies (Fedora17-2) were cultivated in 3 research fields (Gilan, Golestan and Alborz provinces) in Iran. The following plant materials were extracted with methanol/chloroform and analyzed by HPLC: foliage in the vegetative stage, inflorescent in the flowering stage, inflorescent of seeds in the seeding stage and the mature seed. The THC concentration of Fedora17 (Fed17) in all three geographical locations was found to be under 0.03 % or even non-detectable. Same result was also observed in its progenies (Fed17-2), indicating stability of the trait in this cultivar. The THC concentration of the Yazd variety that was planted in Alborz and Gilan regions was less than 0.080 % in all growth stages. The female flowers planted in Golestan, showed a THC concentration of 1.029 % which was more than the allowed THC concentration of <3 %. The THC concentration in all growth stages of all of the different varieties planted varied from 0 to 1.392 %. The above results indicates that the type of cannabinoid produced depends on the difference in genetic prosperities of the different seed types as well as the growth stage in which the plant material was extracted. On the other hand, the climate and the region in which the seeds were planted had little influence on the THC concentration.     

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

Introduction – Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low‐cost, high‐throughput analytical methods for quality control. Objective – The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. Methodology – The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Results – Δ⁹‐THC gives linear calibration curves in the range of 50–500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r² = 0.9968. Conclusion – Results have shown that the HPTLC method is reproducible and accurate for the quantification of Δ⁹‐THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars. Copyright © 2009 John Wiley & Sons, Ltd.     

大麻种质资源中大麻素化学型及基因型鉴定与评价

大麻素(cannabinoids)是大麻植物中特有的次生代谢产物,主要成分为四氢大麻酚(THC,tetrahydrocannabinol)和大麻二酚(CBD,cannabidiol)。本研究通过对我国不同来源地的23份大麻种质资源共69个单株材料中THC和CBD含量特征及其合成关键酶基因多态性进行分析,鉴定了我国大麻种质资源的大麻素化学型及基因型。结果显示,69个单株中大麻素含量差异显著,THC含量均值为0.56%,范围为0.01%~2.45%;CBD含量均值为0.53%,范围为0~2.24%;根据CBD/THC含量比值,大麻资源可划分为毒品型(占44.93%)、中间型(占20.29%)和纤维型(占34.78%)3种大麻素化学型,毒品型、中间型中分别有93.5%和71.4%的植株中THC含量0.3%,纤维型植株中THC含量≤0.08%。3种化学型遗传位点(共显性位点B)的基因型分别为BT/BT、BT/BD和BD/BD;BT等位基因(THCAS)存在10个变异位点,氨基酸序列有4处变异,BD等位基因(CBDAS)存在4个变异位点,均为同义突变。根据THCAS和CBDAS基因多态性,设计了一个共显性复合PCR分子标记,可准确鉴定出大麻3种化学型。研究结果揭示了我国大麻种质资源中大麻素含量、化学型和基因型三者之间的关系,可为大麻素遗传研究与利用提供理论依据。     

大麻染色体行为分析

以大麻不同性别的植株为材料,常规压片法观察细胞染色体行为规律。核型分析结果表明:大麻雌雄株的体细胞染色体数目均为2n=20,核型公式分别为雌株2n=2x=20=18m 2sm,雄株2n=2x=20=18m 2sm(1SAT)。雌株体细胞中有2条X染色体,而雄株只有一条X染色体和一条具有大随体的Y染色体。雌雄株核型均为2A型,为较对称核型。这一结果可为进一步研究大麻性别的分化机制提供细胞遗传学理论依据。     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Flavonoid glycosides and cannabinoids from the pollen of Cannabis sativa L

Chemical investigation of the pollen grain collected from male plants of Cannabis sativa L. resulted in the isolation for the first time of two flavonol glycosides from the methanol extract, and the identification of 16 cannabinoids in the hexane extract. The two glycosides were identified as kaempferol 3-O-sophoroside and quercetin 3-O-sophoroside by spectroscopic methods including high-field two-dimensional NMR experiments. The characterisation of each cannabinoid was performed by GC-FID and GC-MS analyses and by comparison with both available reference cannabinoids and reported data. The identified cannabinoids were delta9-tetrahydrocannabiorcol, cannabidivarin, cannabicitran, delta9-tetrahydrocannabivarin, cannabicyclol, cannabidiol, cannabichromene, delta9-tetrahydrocannabinol, cannabigerol, cannabinol, dihydrocannabinol, cannabielsoin, 6a, 7, 10a-trihydroxytetrahydrocannabinol, 9, 10-epoxycannabitriol, 10-O-ethylcannabitriol, and 7, 8-dehydro-10-O-ethylcannabitriol.     

Isolation of microsatellite markers in Cannabis sativa L. (marijuana)

We have identified 15 variable microsatellite loci in Cannabis sativa. In 48 samples from five fibre crop seed accessions, we detected an average of 10 alleles per locus (range 2–28) with mean heterozygosity of 0.68 (range 0.28–0.94). Significant genetic differentiation was found between accessions (F_ST = 0.12, P < 0.001). These markers have utility for characterizing genetic diversity in cultivated and naturalized Cannabis populations.     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

Chemical Characterization of Leaves,Male and Female Flowers from Spontaneous Cannabis (Cannabis sativa L.) Growing in Hungary

Spontaneous forms of hemp (Cannabis sativa L., often reported as Cannabis sativa var. spontanea Vavilov ) with a low content of psychoactive cannabinoids can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this article, we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analyzed using GC/MS, NMR and LC‐DAD‐MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists, (E)‐caryophyllene (28.3 %) and cannabidiol (CBD; 24.9 %), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC/MS allowed to quantify cannabidiol (CBD) and cannabidiolic acid (CBD?A) in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9 %, and 0.2, 0.3 and 0.4 %, respectively. Flavonoids of this spontaneous form of hemp were formed by C‐glycosides and glucuronic acids of kaempferol and apigenin with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, spontaneous C. sativa may represent an important source of CB₂ agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.     

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex‐linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex‐reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress‐induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North‐Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female‐to‐male sex‐reversed adults had similar body mass as normal males. We recorded no events of male‐to‐female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human‐induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex‐reversed individuals surviving to adulthood may participate in breeding.     

棉铃虫性染色体两种分子标记的克隆及序列分析

为了建立棉铃虫Helicoverpa armigera性染色体的特异性分子标记,利用RAPD-PCR技术对雌雄棉铃虫基因组DNA进行筛选,从500种随机引物中筛选到1 条引物（Operon编号为AF-18）,可扩增出1条约450 bp 的雌性特异片段。经克隆测序并合成特异引物进行验证,表明该片段为棉铃虫雌性特异分子标记,位于W染色体上。利用家蚕、果蝇等昆虫Kettin基因序列,克隆了棉铃虫的同源基因HaKettin片段,并采用荧光定量PCR技术,以棉铃虫的DH-PBAN基因为参照基因,检测棉铃虫雌雄不同个体间HaKettin基因与DH-PBAN基因的拷贝数之比,结果表明：雄体HaKettin∶DH-PBAN=1.0,雌体HaKettin∶DH-PBAN=0.5,据此推断HaKettin基因位于棉铃虫Z染色体上。     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

The red bayberry genome and genetic basis of sex determination

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high‐quality genome for both a female and a male individual of red bayberry. The genome size was 313‐Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole‐genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59‐Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59‐Kb female‐specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non‐recombinant in the female‐specific region. Sex‐specific molecular markers developed from candidate genes co‐segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999     

PlantGM: a database for genetic markers in rice (Oryza sativa) and Chinese cabbage (Brassica rapa)

The Plant Genetic Map Database (PlantGM) has been developed as a web-based system which provides information about genetic markers in rice (Oryza sativa) and Chinese cabbage (Brassica rapa). The database has three major parts and functions; (1) Map Search, (2) Marker Search, and (3) QTL Search. At present, the database provides characterization information for about 3258 genetic markers. It has 2800 RFLP and 112 QTL markers related to rice in addition to 321 RFLP and 25 PCR-based markers for Chinese cabbage. In addition, a genetic linkage map was also constructed by using 1,054 markers from 2,912 markers in rice.

Availability

The database is available for free at http://www.niab.go.kr/nabic/PlantGM     

与大白菜霜霉病抗性主效QTL连锁的分子标记开发

霜霉病是危害大白菜的三大病害之一,该病的发生会严重影响大白菜的产量及品质,因而研究与霜霉病抗性QTL紧密连锁的分子标记对大白菜抗病新品种培育具有重要意义。该研究在前期工作的基础上,选用高感霜霉病株系91-112、高抗霜霉病株系T12-19以及由二者为双亲构建的DH群体为实验材料,针对大白菜霜霉病抗性主效QTL——BrDW所在的标记区间,利用已有的大白菜基因组信息发展与抗性QTL紧密连锁的分子标记,通过Blast和IMap分析,将与BrDW连锁的RAPD标记K14-1030定位于大白菜KBrB058M10上(位于Contig214上),根据KBrB058M10附近的BAC及BAC-end序列设计引物,结合限制性内切酶酶切及HRM分析方法,筛选得到5个与BrDW连锁的分子标记,包括1个Indel标记Brb062-Indel230,3个CAPS标记Brb094-DraⅠ787、Brb094-AatⅡ666和Brb043-BglⅡ715,1个SNP标记Brh019-SNP137;同时,通过筛选与目标区域具有同源性的Unigene序列得到了1个与BrDW紧密连锁的SSR标记bru1209。标记Brb062-Indel230、Brb094-DraⅠ787、Brb094-AatⅡ666、Brb043-BglⅡ715、Brh019-SNP137和bru1209与RAPD标记K14-1030之间的遗传距离分别为4.3 cM、1.7 cM、5.9 cM、5.9 cM、4.6 cM和0.8 cM,在对DH群体中的抗性株系选择上准确率分别为69.7%、70.9%、72.4%、72.4%、58.3%和74.2%,可应用于分子标记辅助选择,为霜霉病抗性分子育种奠定了良好基础。     

Development and validation of L allele-specific markers in Capsicum

Tobamovirus is one of most destructive viruses in Capsicum. Accordingly, the L locus, a resistance gene against tobamoviruses, has been used for pepper breeding programs. Previously, the L ³ gene, one of the L alleles, was isolated through map-based cloning, and a L ⁴ gene candidate was isolated by homology-based PCR methods. Here, the L4segF&R marker was developed based on the leucine-rich repeat (LRR) region of the L ⁴ candidate, and co-segregation analysis was performed using two L ⁴ -segregating F2 populations derived from the commercial cultivars Special and Myoung-sung. The L4segF&R marker was located within 0.3?cM of the L ⁴ gene but did not completely co-segregate with the L ⁴ gene, indicating that the candidate is not actually L ⁴ . To confirm the mapping result, L4segF&R genotypes of L ⁴ -containing breeding lines from three different seed companies were analyzed, resulting in the identification of several recombinants in the breeding lines. Based on these results, we postulate several genetic models that show different introgression histories and genetic structures for the L ⁴ -containing segment in different breeding lines. All of the models demonstrate that resistance conferred by the L ⁴ segment could not be explained by the L ⁴ gene candidate alone. Although the presence of the L ⁴ gene candidate could not fully explain the L ⁴ resistance, we were able to develop allele-specific markers for the L locus using the candidate sequence. To develop allele-specific markers for the L locus, HRM analysis was performed using primer pairs based on the LRR sequence of the L ⁴ gene candidate. When commercial breeding lines homozygous for L ⁰ , L ¹ , L ² , L ³ or L ⁴ were analyzed, L4RP-3F/L4RP-3R correctly detected the L allele in 90 out of 91 lines. We believe that the L allele-specific marker developed in the study provides a solution for pepper breeders developing improved resistance lines against tobamoviruses.     

Genetics and Marker-assisted Selection of the Chemotype in Cannabis sativa L.

Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp breeding. In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements. In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible applications in the forensic and pharmaceutical fields.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.