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.     

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.     

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.     

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.     

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.     

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.     

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.     

Novel male-specific molecular markers (MADC5, MADC6) in hemp

Decamer RAPD primers were tested on dioecious and monoecious hemp cultivars to identify sex-specific molecular markers. Two primers (OPD05 and UBC354) generated specific bands in male plants. These two DNA fragments were isolated, cloned and sequenced. Both markers proved to be unique, since no sequence with significant homology to OPD05₉₆₁ and UBC354₁₅₁ markers were found in databases. These markers were named MADC3 (OPD05₉₆₁) and MADC4 (UBC354₁₅₁) (Male-Associated DNA from Cannabis sativa). The markers were converted into sequence-characterized amplified region (SCAR) markers. The SCAR markers correlated with the sex of the segregating F₂ population and proved the tight linkage to the male phenotype. Results of F₂ plant population analysis suggest these markers are to be linked to the Y chromosome. This revised version was published online in August 2006 with corrections to the Cover Date.     

A gametocidal factor of Oryza glaberrima Steud. in O. sativa L.

Summary Recurrent backcrossing has been carried out with a view to transfer a gene for non-glutinous endosperm from two strains of O. glaberrima (Wx _g /Wx _g ) to glutinous japonica and indica varieties (wx/wx) of Oryza sativa. In the course of backcrosses Wx _g /wx segregants were crossed with each of the two glutinous varieties of sativa as the respective recurrent male parent. The wx/wx and Wx _g /wx segregants in the successive generations were consistently fully fertile and semi-sterile, respectively. The semi-sterility of Wx _g /wx plants was attributable to abortion of most of the pollen grains carrying the gene wx. The nucleus but not cytoplasm was related to the semi-sterility. The Wx _g /Wx plants having the gene for non-glutinous endosperm of a glaberrima strain and a japonica variety of sativa were also semi-sterile. Both wx- and Wx-megaspores in the plants heterozygous for the gene Wx _g were deleteriously affected. The results could be explained by assuming that a factor tightly linked with the gene Wx _g of glaberrima sterilizes gametes not carrying it in the heterozygotes and that the gametocidal action is exerted when combined with the sativa nucleus by the recurrent backcross method.     

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.     

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.).     

Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv. Adriana)

Summary In alfalfa (Medicago sativa) regeneration is genotype-specific. In order to study the genetic control of somatic embryogenesis and to constitute a synthetic cultivar characterized by its high regeneration ability, 2 embryogenic plants selected from the cv. Adriana were selfed, intercrossed and also crossed in both directions with 5 non-embryogenic genotypes of the same cultivar.Progenies of all crosses were scored for their regeneration ability and results indicate that somatic embryogenesis is under the control of 2 dominant loci. However some non-embryogenic genotypes prevent regeneration when crossed with embryogenic ones and this characteristic is not under the control of a single dominant gene.When plants chosen for their capacity to regenerate within F₁ and S₁ progenies were freely intercrossed the regeneration efficiency dropped to 2% (1 plant out of 50). This result indicates that if the genetic background of the population is changed the regeneration is greatly affected and therefore some other mechanism could play a role in determining plant regeneration.     

Modelling approach for the quantitative variation of sex expression in monoecious hemp (Cannabis sativa L.)

Sex expression is of primary importance for the genetic improvement and production of monoecious hemp: masculinized phenotypes are associated with higher fungal sensitivity, and feminized phenotypes with higher seed yields. However, sex expression varies quantitatively among plants and nodes and with time. Here, we developed eight variables that characterize the sex expression in monoecious hemp to dissect its genetic determinism. The monoecy degree (MD), ranging from 1 (mostly male flowers) to 5 (mostly female flowers), was recorded for each node of 167 plants, at 6 times at 1‐week intervals. Two types of longitudinal variables were constructed: ‘synthesis’ (mean MD and percentages of nodes of each MD) and ‘structure’. The latter consisted of the parameters of a logistic curve describing MD as a function of the node position. An r‐square of 0.97 was obtained between the estimated and observed MD values, and the logistic parameters were weakly correlated with each other and with the synthesis variables. Therefore, we conclude that the present modelling approach is relevant for characterizing the sex expression in monoecious hemp.     

Inbreeding depression and heterosis in autotetraploids with application to Medicago sativa L.

Inbreeding depression observed in Medicago sativa L. is greater in the early generations of self-fertilization than is expected for an autotetraploid based upon the coefficient of inbreeding (F). Forage yield often is reduced by 30 percent or more after one generation of selfing, while F is only one-sixth, =0. A high frequency of loci containing 3 or 4 different alleles is postulated as a basis for this rapid loss of vigor. A formula is developed that permits computation of the proportions of loci with 1, 2, 3 and 4 different alleles, along with estimates of additive and non-additive values of genes.Forage yield data for backcross and sib-mated populations indicate that quantitative characters are inherited tetrasomically.Journal Paper No. J-5190 of the Iowa Agricultural and Home Economics Experiment Station. Project 1048.Graduate Assistant and Professor of Agronomy, Department of Agronomy, Iowa State University, Ames, Iowa.     

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.     

The potential of selection and breeding for improved salt tolerance in lucerne (Medicago sativa L.)

Summary Four cultivars of Medicago sativa L. were subjected to selection for improved salt tolerance using a salinized solution culture technique at 250 mM NaCl. Selections were made after two weeks growth, based upon seedling shoot length differences. High (shoot lengths 14–31 mm) and low (7–12 mm) selection lines were established. Unselected shoot lengths ranged from 0–11 mm. Selection intensities ranged from 0.17% to 0.22% for the high selection lines, and from 0.30% to 1.05% for the low selection lines.Eleven selected plants from cv. CUF 101, and 10 from Local Syria were grown on and polycrossed. A sample of progeny seed from each was grown at 8 NaCl concentrations in solution cultures. Selected line seedlings produced shoots at 225, 250, 260, and 275 mM NaCl, whereas unselected material failed to grow above 225 mM NaCl. A second cycle of selection at 280 mM NaCl with these two cultivars achieved selection intensities of 0.21% and 0.45% for high and low selection lines, respectively. Selected lines grew and produced shoots at 300 mM NaCl.Seedlings derived from 1st and 2nd selection cycles and unselected control material were grown for 6 weeks at 0, 150, 175, and 200 mM NaCl in a sand culture experiment. The high selection line produced significantly greater shoot fresh weight, dry weight, and % live shoot weight than the unselected control material. Clearly selection as practised here can isolate individual seedlings having enhanced genetically based tolerance to NaCl, which is manifest in mature plants derived from polycrossing those selected individuals.     

Interspecific cytoplasm substitutions of an Indica strain of Oryza sativa L. and O. Glaberrima Steud

Summary To study differential nucleus-cytoplasm interactions between the two cultivated rice species, Oryza sativa and O. glaberrima, cytoplasmic substitution lines were made by using a glaberrima strain (G) and an Indica strain of sativa (S). The G cytoplasm had no adverse effect on pollen development when combined with the nucleus of S. On the other hand, when the S cytoplasm was combined with the G nucleus, the substitution line showed no seed set because of male sterility although the pollen grains were normally stained with I₂-KI solution. A dominant gene derived from S strain seemed to cause anther indehiscence in the substitution line. Further, a restorer gene (Rf _j)from Akebono of Japonica type was effective on pollen restoration in the male sterile line, suggesting that the S cytoplasm is the same as those of Japonica type in terms of a fertility-restoring system.This paper is Genetic studies of speciation in cultivated rice. 4.     

The indica-japonica classification of Asian rice ecotypes and Japanese lowland and upland rice (Oryza sativa L.)

Summary Rice cultivars (Oryza sativa L.) belonging to five ecotypes (aus, aman, boro, bulu and tjereh) and to two groups of Japanese rice (lowland and upland) are examined with respect to KClO₃ resistance, phenol reaction and apiculus hair length. These characters have been used as available criteria to classify rice into two types indica and japonica, for the last thirty years.The findings of this study are that the aman, boro and tjereh ecotypes should be classified as typical indica; and that the Japanese lowland rice cultivars are mainly typical japonica. Some of the aus, bulu and Japanese upland rice cultivars differ from typical indica and typical japonica, so the respective terms aus type, bulu type and J.u.r. type, are proposed. Aman, boro tjereh and Japanese lowland rice are cultivated in lowland. Some of the aus, bulu and Japanese upland rice cultivars have the characteristics of upland rice. In general, lowland rice cultivars can be clearly classified into indica or japonica, while upland cultivars cannot.Abbreviations Aph dominant gene for apiculus hair length > 0.7 mm - aph recessive gene for apiculus hair length < 0.7 mm - J.u.r. type Japanese upland rice type     

Genotypical differences in nitrate content inLactuca sativa L. and related species and correlation with dry matter content

Summary Nitrate content was measured in 135 genotypes of cultivated lettuce (Lactuca sativa L.) and 21 genotypes of wildLactuca spp. grown in two experiments. In Experiment 1 plants were grown on nutrient film. In Experiment 2 plants were grown in large pots of potting soil. Within cultivated lettuce five plant types were distinguished and within each of them genotypes were found with low nitrate content. The coefficient of variation in the experiment with plants growing in large pots was not larger than in the experiment with plants growing on nutrient film. In butterhead genotypes nitrate content was negatively correlated with dry matter content and positively with plant fresh weight.     

Organelle based molecular analyses of the genetic relatedness of cultivated alfalfa (Medicago sativa L) to Medicago edgeworthii Sirjaev, and Medicago ruthenica (L.) Ledebour

Medicago edgeworthii Sirjaev and M. ruthenica (L.) Ledebour are allogamous, diploid (2n = 2x = 16) perennials with flat pods.Medicago edgeworthii is indigenous to the Himalayas and alpine areas west to Afghanistan, and Medicago ruthenica is found in Siberia, Mongolia, and Manchuria on open hillsides and mixed grass steppes. Because both species have a remarkable ability to survive extreme cold and poor soils, the possibility of hybridizing them with alfalfa (M. sativa L.) is being investigated. The objective of this research was to conduct an organelle based molecular assessment of the genetic relatedness of cultivated alfalfa (2n = 4x = 32) to M. edgeworthii and M. ruthenica. A hypervariable, intergenic region of cpDNA was amplified, and mtDNA was amplified with two primer pairs developed from soybean (Glycine max L.) mtDNA sequences. Mean Nei and Li genetic distances (GDs) between alfalfa and M. edgeworthii and alfalfa and M. ruthenica were 0.56 and 0.48 (mtDNA), and 0.33 and 0.30 (cpDNA), respectively. Intra specific GDs were 0.37 (mtDNA) and 0.25 (cpDNA) for M. edgeworthii; 0.42 (mtDNA) and 0.15 (cpDNA) for M. ruthenica; and 0 = 0.50 (mtDNA) and 0 = 0.23 (cpDNA) for alfalfa. Cluster analyses grouped someM. edgeworthii and M. ruthenica entries with alfalfa entries. There is some chance that alfalfa and M. edgeworthii entries which clustered closely could be hybridized; chances of alfalfa × M. ruthenica hybridizations appear to be more problematic. This revised version was published online in July 2006 with corrections to the Cover Date.