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.     

The inheritance of chemical phenotype in <Emphasis Type="Italic">Cannabis</Emphasis><Emphasis Type="Italic">sativa</Emphasis> L. (IV): cannabinoid-free plants

A genetic factor that blocks the cannabinoid biosynthesis in Cannabis sativa has been investigated. Crosses between cannabinoid-free material and high content, pharmaceutical clones were performed. F₁s were uniform and had cannabinoid contents much lower than the mean parental value. Inbred F₂ progenies segregated into discrete groups: a cannabinoid-free chemotype, a chemotype with relatively low cannabinoid content and one with relatively high content, in a monogenic 1:2:1 ratio. In our model the cannabinoid knockout factor is indicated as a recessive allele o, situated at locus O, which segregates independently from previously presented chemotype loci. The genotype o/o underlies the cannabinoid-free chemotype, O/o is expressed as an intermediate, low content chemotype, and O/O is the genotype of the high content chemotype. The data suggests that locus O governs a reaction in the pathway towards the phenolic cannabinoid precursors. The composition of terpenoids and various other compound classes of cannabinoid-free segregants remains unaffected. Backcrossing produced cannabinoid-free homologues of pharmaceutical production clones with potential applications in pharmacological research. A new variant of the previously presented allele ‘B ₀’, that almost completely obstructs the conversion of CBG into CBD, was also selected from the source population of the cannabinoid knockout factor.     

Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.

DNA from female and male hemp (Cannabis sativa L.) plants belonging to nine different varieties were screened with180 RAPD primers in a search for sex-associated DNA markers. About 1500bands were produced in total, nine primers were found yielding one or two DNA bands amplified in all nine male DNA bulks and absent in all female DNA bulks. These putatively male-associated markers were then scored in three different F1progenies, deriving from a cross between a common male parent and three different female plants. The sex of the progeny was accurately scored on the basis of the floral phenotype, and the presence of the nine male-associated markers was verified by RAPD analysis. In all three progenies examined, all the male plants showed the DNA markers previously identified by bulk segregant analysis (BSA) on the hemp varieties, while all the female plants lacked them. The fact that the association between these markers and the staminate phenotype is found when examining male plants of distantly related varieties, and that such linkage is never broken when different progenies are examined, strongly supports the hypothesis that the markers found are physically located on the Y chromosome, in a region excluded from recombination during meiosis. Another marker was shown to be present in the male parent, in all the male plants of each progeny, and in 50% of the female progenies, while it was absent in the female parent; the possible occurrence of markers deriving from multiple amplification sites of the genome is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

The inheritance of nitrate content in lettuce (Lactuca sativa L.)

Summary The inheritance of nitrate content in lettuce was analysed using 16 F₂ populations and three F₃ populations. Frequency distributions of nitrate content in F₂ and F₃ populations were unimodal and symmetrical, indicating a quantitative inheritance. Both significant positive and negative deviations of the F₂ mean from the mid-parent value were found, indicating dominance or epistasis. Deviations towards low nitrate content were more frequent than deviations in the other direction. Estimates of heritabilities for nitrate content in the F₂ populations ranged from 18% to 69% and were in most cases above 50%. Crosses between low nitrate cultivars did not have lower estimates of heritability in the F₂ than crosses between cultivars with larger differences in nitrate content. In one case a genotype x experiment interaction for nitrate content of parental cultivars was found.Three F₃ populations of crosses between cultivars with low nitrate content were analysed. Estimates of heritabilities for F₃ line means ranged from 78% to 91% and estimates of the genetic standard deviation of nitrate content in unselected advanced generations ranged from 0.24–0.33 g kg^–1. The estimates of heritabilities and of genetic variation in advanced generations offer good prospects of selection of low nitrate genotypes in lettuce. A comparison of efficiency of selection in the F₂ generation and F₃ line selection is made.     

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.     

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.     

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.     

Research on the inheritance of seed dormancy in lettuce (Lactuca sativa L.) and selection for non-dormancy

Summary Inheritance of dormancy and the results of selection of non-dormant genotypes in segregating populations of lettuce were investigated. Diallel crosses were therefore carried out between two dormant (DOR) and two non-dormant (NDOR) cultivars. F₁, F₂ and F₃ populations were analysed.Environmental variation for dormancy usually was large. The mean germination time (GT) of F₁ seeds from the NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses was often intermediate to the GT of the NDOR and DOR parent. The mean GT of F₁ seeds from DOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses equalled the mean GT of the parents; the same applies for the NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]NDOR crosses. No differences between reciprocals were observed and neither were such differences found for F₂ populations. F₂ populations from DOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses showed no significant segregation of rapidly germinating seeds and in F₂ populations from NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses no accumulation of genes for long GT occurred. In F₂ populations from NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses usually 40–60% of the seeds germinated as rapidly as the seeds of the NDOR parents. Only one gene (D) could be responsible for the difference in dormancy behaviour of the DOR and NDOR cultivars. The behaviour of the F₃ lines from various NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses supports this hypothesis. A regression of F₃ means on the value of F₂ seeds for GT of various NDOR % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabaGaciaacaqabeaadaqaaqaaaKaaafaafaqabeGaba% aajqgaa+FaaiaadIhaaeaacaWG4baaaaaa!3B90!\[\begin{array}{*{20}c}x \\x \\\end{array}\]DOR crosses showed that h²-narrow usually was rather high for most crosses implying that selection for non-dormancy can be carried out in F₂ populations.     

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.     

Recurrent selection programs for white clover (Trifolium repens L.) using self-compatible plants. I. Selection of self-compatible plants and inheritance of a self-compatibility factor

Summary When white clover plants were self-pollinated, 75% of a 143-plant population did not set seed. Plants which did set small numbers of seed probably did so as a result of pseudo-self-compatibility. One highly self-compatible plant was selected from the breeding materials. The S₂ progeny of this plant was autogamous and genetically fixed for morphology and isozyme patterns. Genetic analyses of self-compatibility were conducted as far as the F₃ progeny. It was concluded from these data that self-compatibility was inherited in a simple Mendelian way and that plants homozygous for self-compatibility were obtained. There was no correlation between presence or absence of self-compatibility and growth vigor. The possibility of application of self-compatible plants to recurrent selection procedures is outlined and the utilization of self-compatible plants in white clover breeding programs is discussed.     

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.     

Multifactor inheritance of seedcoat colour in greengram (Vigna Radiata (L.) Wilczek.)

Summary Seedcoat colour in greengram (Vigna radiata (L.) Wilczek.) is a useful marker for genetic studies and varietal identification. Its mode of inheritance was examined in five crosses among nine parents which differed for seedcoat colour. Four of the parents had sap green seedcoat colour while the others had raw sienna, brownish green, densely black spotted, black and greenish yellow seedcoat colour, respectively. At the F₂ generation, no more than 20 different colour classes were observed. The segregation in F₃ and backcross generations indicated that at least five major genes were involved in seedcoat colour inheritance. Sap green seemed to be dominant over raw sienna. The segregation ratios further indicated the role of non-allelic gene interactions (epistasis) in inheritance of seedcoat colour. Gene symbols were assigned to each colour and genotypes to each parent.     

Research on the inheritance of fasciation in lettuce (Lactuca sativa L.)

Summary Inheritance of fasciation was investigated in reciprocal crosses between two lettuce varieties: Suzan (non-fasciated) and Noran (fasciated). In glasshouses parental plants, F₁, F₂, and F₃ populations were assessed for fasciation according to a scale from 0 (non-fasciated)-9 (extremely fasciated).The observed environmental variation for fasciation of the homozygous parents was very low or absent, but the environmental variation for the F₁ populations was large. On the basis of this large variation of the heterozygous F₁ plants we supposed that such a variation also occurred in F₂ and F₃ populations.Considerable mortality was observed in all generations, which supposedly occurred at random in parents and F₁ but was non-random in the segregating F₂ and F₃ populations. So, means and variances of F₂ populations will be biased and therefore they were not used for genetical analyses of fasciation.From F₁ and F₃ populations and from some F₂ populations it appeared that no differences occurred between reciprocals. The differences for fasciation between reciprocal F₂ populations in the 1977 A experiment may result from non-random plant survival. A regression of F₃-means on the values for fasciation of parental F₂ plants, adjusted for their inbreeding, resulted in a realized h²-narrow of 0.4. This indicates that in the surviving plants of the F₂ populations still additive genetic variation was present to select successfully for non-fasciated plants.     

Maternal inheritance of cytoplasmic organelles in intergeneric hybrids of Carica papaya L. and Vasconcellea spp. (Caricaceae Dumort., Brassicales)

The mode of inheritance of chloroplast and mitochondrial DNA has been determined in intergeneric hybrids between C. papaya and four different Vasconcellea species by employing a restriction fragment length polymorphism analysis of a PCR-amplified chloroplast and mitochondrial DNA region. Artificial F₁ hybrids were produced between a female specimen of C. papaya and male specimens of either V. parviflora, V. goudotiana, V. cundinamarcensis or V. quercifolia. The hybridization patterns of all hybrids correspond in all cases with that of the C. papaya mother, and are different from that of the paternal Vasconcellea species, thus indicating the maternal inheritance of cpDNA and mtDNA in intergeneric hybrids between C. papaya and wild relatives of the Vasconcellea genus.     

Identification and inheritance of male sterility in groundnut (Arachis hypogaea L.)

Summary Some plants without pods but with gynophores were observed in two F₄ progenies of two crosses of goundnut (Arachis hypogaea L.). The flowers on these plants had translucent white anthers with no or a few sterile pollen grains. Three such plants in the succeeding generation were hand pollinated with pollen from a short-duration Indian cv. JL 24. The resulting F₁ hybrid plants (male sterile x JL 24) were normal. Chi-square tests for segregation for male fertile and male sterile plants in F₂ and F₃ generations indicated that the male sterility in these crosses of groundnut is governed by two recessive genes. We designate these genes as ms₁ and ms₂ with ms₁ms₁ms₂ms₂ being a male sterile genotype.Submitted as ICRISAT J. A. No. 1812.     

Studies of the inheritance of mature fruit color in Capsicum annuum L.

Summary Crosses were made among a permanent white accession of pepper, genetically yyc₁c₁c₂c₂ and parents having the following mature fruit color: orange, orange-yellow, lemon-yellow, permanent green and brown. The F₂ and BC generations from these combinations were studied and several models of inheritance were suggested. The discrepancies between the present findings and previous studies were considered, with an emphasis on the inheritance of the white color.     

Rate and inheritance of cross-fertilization in faba bean (Vicia faba L.)

Summary To assess the feasibility for synthetic varieties of Vicia faba L., 108 faba bean genotypes were screened for cross-fertilization rate in five field experiments carried out in 1989 and 1990 at Wageningen.Significant genotypic differences were observed for outcrossing rate, which ranged from 1 to 55%. These percentages, however, are thought to be not high enough for a commercially feasible production of synthetic varieties.The inheritance of cross-fertilization rate, determined by testing the F₄ generation of a cross between two inbreds, both with high outcrossing rates, appeared to be of a polygenic nature, as was indicated by the wide variation in, and the continous distribution of, the cross-fertilization rates. Among the F₄ lines tested some had a significantly higher cross-fertilization rate than the parents. However, this difference was not found when these lines were retested, indicating that possibilities for successful selection for higher outcrossing rates might be limited.     

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.     

Inheritance of groat protein percentage in Avena sativa L. × A. Fatua L. Crosses

Summary Gene action and heritability of groat protein percentage were determined in F₁, F₂, and F₃ generations of nine crosses between three Avena sativa L. cultivars and three A. fatua L. selections. Relationships among groat protein percentage, grain yield, and 100-seed weight also were evaluated. The three A. sativa parents were Dal (high grain yield and intermediate groat protein percentage), Goodland (low grain yield and high groat protein percentage), and Stout (high grain yield and low groat protein percentage). The three A. fatua parents were chosen for the study on the basis of vigorous plant growth and high groat protein percentage. The study was conducted at Madison, Wisconsin in 1979 and 1980.There was partial dominance towards low groat protein percentage. Narrow sense heritability estimates for groat protein percentage were low in Dal and Goodland crosses and intermediate in Stout crosses. In the F₂ generation, groat protein percentage was significantly higher in shattering than in nonshattering plants in 1979, but not in 1980. There were significant, positive correlations between groat protein percentage, 100-seed weight, and grain yield in F₁ and F₂ generations, but they were not large numerically. Relationships among these traits were either negative or nonsignificant in the F₃ generation. Although our results indicated that selection for higher groat protein percentage is possible when a low protein A. sativa cultivar is used, most of our simple cross progenies from A. sativa x A. fatua crosses had weak straw and were susceptible to crown rust (Puccinia coronata Cda. var. avenae Fraser and Led.).