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.     

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.     

Plant Development and Stem Yield of Non-domestic Fibre Hemp (Cannabis sativa L.) Cultivars in Long-day Growth Conditions in Finland

Plant development and stem yield of mono- and dioecious fibre hemp cultivars ( Cannabis sativa L.), introduced to Finland from regions of lower latitude, were studied in field experiments under long-day growth conditions in 1995–96. Plant density, plant mortality rate, stem elongation and stem yield were determined. Plant densities at seedling stage were less than the targeted viable seeds sown m⁻². Seedling densities differed significantly among cultivars, but had no significant effect on plant mortality rate in 1996. Plant mortality during the 1996 growing season averaged 34 plants m⁻². Stem elongation was measured at one-week intervals throughout the growing time. The elongation among cultivars was different and depended on the date of measurement. Rapid elongation began five to six weeks after sowing and elongation was most pronounced in July. Dioecious cultivars were significantly taller than monoecious ones in 1995 but not in 1996 when hemp stands were dense, nitrogen was deficient and an infection of Botrytis vinerea was present. Dioecious cultivars produced higher stem yields than monoecious ones. However, in 1996 the difference in yield was not statistically significant among most of the cultivars, Cultivar Uso 11 was early maturing and produced highest stem yield among monoecious cultivars; it also competed well with the higher yielding dioecious cultivars.     

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 relationship of stem and seed yields to flowering phenology and sex expression in monoecious hemp (Cannabis sativa L.)

Flowering phenology and sexual dimorphism are two major features that affect stem and seed production in hemp (Cannabis sativa L.), a short-day naturally dioecious plant. The sowing time is of primary importance because it affects flowering time and thereby influences stem yield. In spite of their unstable sexual phenotype, monoecious cultivars facilitate the harvest of both stems and seeds by reducing crop heterogeneity. The main objective of this paper was to determine the stem and seed yields for five monoecious hemp cultivars in relation to their flowering phenology and sex expression. Sowing was carried out on five distinct dates during 2007 and 2008 at two sites in Belgium. The duration from sowing to flowering in days, both stem and seed yields and the seed harvest index decreased when sowing was postponed from mid-April to the end of June. The stem and seed yields from the mid-April sowing (approximately 12.5 and 1.9 t ha⁻¹, respectively) were within the ranges that were reported for fibre and both fibre and seeds production, respectively, in monoecious hemp. No interaction was observed between the sowing date and cultivar for both yields. Sex expression varied among cultivars, indicating that it might be selected, and was partly linked to earliness. Stem yields were lowest in the earliest cultivar (Uso 31) and highest in the latest one (Epsilon 68) while seed yields were lowest in the most masculinized and earliest cultivar (Uso 31) and highest in the most feminized and early (Fedora 17) or mid-early (Felina 32) ones. Both stem and seed yields correlated best with the duration from sowing to full female flowering or from sowing to the end of male flowering.According to our results, harvesting the seeds in addition to the stems in monoecious hemp requires early sowing and the selection of feminized early or mid-early cultivars, earliness depending on the climatic conditions in the cultivation area. Therefore, it might be agriculturally valuable to take sex expression into account in addition to earliness during the selection of cultivars that are adapted to a dual purpose.     

工业大麻雄性相关RAPD和SCAR标记的筛选与鉴定

利用42条RAPD（Random Amplified Polymorphic DNA）随机扩增引物分析工业大麻品种“火麻一号”组成的雄性或雌性DNA池（DNA pools）,结果显示,引物OPV-08扩增得到一条大小为869bp与工业大麻雄性相关的特异条带。根据测序结果,合成了两条SCAR（Sequence Characterized Amplified Region）标记引物,该SCAR标记不仅可以对工业大麻雌雄异株材料花期已知性别的雌雄植株进行准确鉴定,还可以对幼苗期未知性别的大麻雌雄植株进行鉴定;也可对雌雄同株材料可能出现的雄化进行早期鉴定。这不仅为工业大麻早期性别鉴定提供基础,且为减少雌雄同株材料的雄化提供支撑。     

雌雄同株黄檗有性繁殖特性研究

一般认为黄檗为严格的雌雄异株植物,但本研究首次发现自然界存在两性植株。为了研究两性植株的有性繁殖能力,以两性植株为试材,雌株和雄株为对照,对两性植株性器官形态和繁殖能力进行了研究。通过2年对5000余朵花的研究发现：（1）两性植株的花粉和柱头与雄株和雌株在形态和发育上没有差异,花粉的萌发率在23%以上,与雄株花粉基本一致;（2）两性植株花粉授粉至雌株后得果率为86.69%,两性植株雌花在接受雄株花粉后的得果率为91.30%,自然受粉条件下的得果率为95.69%,自交授粉的得果率为88.01%。两性植株雄性和雌性都能正常繁殖,也可以成功自交繁殖。本研究为黄檗性别分化研究提供了新方向,为其生物保护提供了新途径。     

Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia

Lavandula angustifolia (lavender) is a small woody perennial grown for essential oil, which is steam distilled from flowers. To potentially improve size of flowers and oil yield we produced and characterised autotetraploid plants. L. angustifolia seed germinated in the presence of the mitotic spindle inhibitor colchicine at concentrations of 125 mg l⁻¹ or less resulted in plants carrying sports with larger flowers. Propagation of two sports gave rise to putative polyploid cultivars C3/2 and C6/24. Direct chromosome counts in root tip cells of seedlings from four common cultivars of L. angustifolia and the seed lot from which C3/2 and C6/24 were derived was 50 whereas C3/2 and C6/24 had greater than 90 chromosomes indicating they were autotetraploid. Ploidy level assessed by flow cytometry (FCM) of nuclei showed that 12 cultivars of L. angustifolia had similar nuclear DNA content whereas C3/2 and C6/24 had double the amount of DNA confirming autotetraploidy. The genome size (1C-value) of a diploid L. angustifolia cultivar was estimated by FCM to be 0.90 (±0.07) pg. Morphological characteristics were measured in autotetraploid and control plants. Autotetraploids had thicker peduncles, larger flowers and larger seeds than diploids. Scanning electron microscopy revealed peltate glandular trichomes were larger in the tetraploids relative to diploids. Both tetraploid and diploid cultivars had complex non-glandular trichomes on leaves and sepals and two different types of capitate glandular trichomes were identified on leaves. Autotetraploid lavenders represent useful germplasm both for commercial oil production and future breeding.     

Development of male-specific markers and identification of sex reversal mutants in papaya

Papaya is a productive and nutritious fruit grown in tropical and sub-tropical regions worldwide. It is polygamous with three sex types: female, male and hermaphrodite. Sex determination in papaya is controlled by an XY sex chromosome system with two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. Comparative analysis of the hermaphrodite-specific region of Y^h chromosome (HSY) and male-specific region of Y chromosome (MSY) revealed 99.6% sequence identity, which explains why DNA markers that amplify for both males and hermaphrodites have easily been developed, but not for the male trait specifically. We examined the 0.4% sequence differences, and found 1887 indels and 21,088 SNPs between MSY and HSY. The vast majority of indels are single nucleotide or few base pairs. A large male-specific retrotransposon insertion of 8396 bp was used to develop two papaya male-specific markers, PMSM1 and PMSM2 that amplify 585 and 548 bp fragments, respectively. These two markers were tested in 11 gynodioecious and four dioecious varieties along with autosomal DNA marker 71E and male/hermaphrodite marker W11, and the results showed clear separation of male from hermaphrodite and female. PMSM1 and PMSM2 were also used to test the sex type of six sex male-to-hermaphrodite reversal mutants which are crucial materials for validating candidate genes for sex determination in papaya. Our result showed all six mutants were positive for the male-specific markers. These male-specific markers can be used to distinguish gynodioecious and dioecious cultivars in papaya seed market, and facilitate genetic and genomic research for papaya improvement.     

Diversity in ploidy levels and nuclear DNA amounts in Korean Miscanthus species

Because of its high biomass productivity and nutrient-use efficiency, Miscanthus spp. have emerged as promising bioenergy crops. Polyploidism in Miscanthus species is important because it allows non-native countries to cultivate sterile types, such as triploid M. × giganteus, in order to prevent ecosystem disturbance. Although M. sacchariflorus and M. sinensis are considered to be native to Korea, China, and Japan, accurate information describing the ploidy levels of these species has not yet been well established. To evaluate rough ploidy levels, 215 accessions of Miscanthus species were estimated by relative fluorescence intensities of DAPI-stained nuclei. After evaluation of rough ploidy levels, 20 plants were randomly selected by species and ploidy levels, and ploidy levels were examined by counting chromosomes, estimating propidium iodide-stained DNA content with flow cytometry, and measuring sizes of stomata and caryopsis. Among the 20 plants examined, 3 were diploid and 6 were tetraploid M. sacchariflorus (4.56 ± 0.01 and 8.90 ± 0.14 pg/2C nuclear DNA content, respectively), while 6 were diploid and 3 were triploid M. sinensis (5.40 ± 0.18 and 8.29 ± 0.33 pg/2C nuclear DNA content, respectively). One plant was a putative triploid M. × giganteus having 7.31 pg/2C nuclear DNA content, which was similar to an M. × giganteus plant from Illinois, USA having 7.23 pg/2C nuclear DNA content. We confirmed the occurrence of diploid and tetraploid M. sacchariflorus and M. sinensis plants as well as a putative triploid M. × giganteus native to Korea. Thus, measurement of the size of stomata and caryopsis allowed prediction of ploidy levels in the field and laboratory.     

Application of AFLP for the detection of sex-specific markers in hemp

Two dioecious hemp accessions (Can18 and Can17) were tested by bulked segregant analysis for polymorphisms between male and female bulks with amplified fragment length polymorphisms. Thirty‐nine primer combinations were tested and 20 of these yielded one to three male‐specific bands. In contrast, no female‐specific band was detected. Eight of these primer combinations were used for testing 80 progeny plants from a cross between two plants from Can18 and 30 plants from Can17. A total of 16 and 17 male‐specific fragments were obtained for Can 18 and Can 17, respectively. Eleven fragments exhibited the same fragment size in both accessions. All male plants, but not one female plant, showed the respective polymorphic band with each of the eight primer combinations. Problems regarding sex determination under field conditions were successfully overcome by testing plants that had been grown in small pots in a greenhouse. The abundant number of potential markers for the male sex, their complete cosegregation with male plants and the absence of markers for the female sex support the presence of a male sex chromosome in hemp.     

The effect of in vivo and in vitro applications of ethrel and GA<Subscript>3</Subscript> on sex expression in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

Bitter melon (Momordica Charantia L.) is an important vegetable crop with nutritional and medicinal qualities. As a member of cucurbitaceae it is monoecious with varying proportions of staminate and pistillate flowers. This study was undertaken to determine the effects of various applications of ethrel and gibberellic acid (GA₃) on sex modification in M. charantia. In the first set of experiments, various concentrations of hormones were added to the seed germination medium, in the second, adult plants growing in the field were sprayed with aqueous solutions of ethrel or GA₃ three times at three-day intervals. The number and sex of open flowers was recorded daily for 60 days after the first flower opened and total number of staminate and pistillate flowers was calculated at the end of the experiment. The highest frequency (29.5%) of pistillate flowers was observed in plants treated with 500 ppm ethrel at germination. Similarly, spraying of adult plants with 100 ppm GA₃ increased the proportion of pistillate flowers to 26% relative to 15% in untreated controls. Both ethrel and GA₃ induced significantly higher number of pistillate flowers than control. In vitro hormone application during seed germination was much more successful than spraying of field grown plants.     

Gene flow in common bean (Phaseolus vulgaris L.)

This study was conducted in Brazil in order to assess the potential risk posed by gene escape from transgenic into non-transgenic plants and wild populations. A new methodology was applied to evaluate the gene flow between common bean cultivars, by means of a specially delineated experiment in two stages. The first stage consisted of the planting of one cultivar with violet flowers (BB) as pollen source (‘Diamante Negro’), and a receiver (‘Talismã’) with white flowers (bb), at different distances. The source was sown in the center of the area. The pollen receiver cultivar was sown, in concentrical squares around it. At maturity, the rows were sampled at varied distances from the source in the four cardinal directions. In the second stage, the sampled seeds of the previous stage were sown, and the percentage of outcrossing was evaluated during flowering through the presence of violet flowers (Bb). The highest frequency of natural hybrids, 0.136%, occurred at a distance of 0.5 m between the cultivars. The natural outcrossing rate was practically zero beyond a distance of 3.25 m.     

Using tube rhizotrons to measure variation in depth penetration rate among modern North-European winter wheat (Triticum aestivum L.) cultivars

Deeper plant root systems are desired for improved water and nitrogen uptake in leaching environments. However, phenotyping for deep roots requires methods that enable plants to develop deep roots under realistic conditions. Winter cereals raise further complications as early growth occurs under low light and temperature during autumn and winter—conditions not met in standard glasshouse facilities. This study used tube rhizotrons of 2 m length, positioned outdoor under a rainout shelter to screen for depth penetration rates (DPR) of roots. Rooting depths of 1 to 1.5 m were achieved with 23 widely grown North European winter wheat cultivars in two autumn/winter and two summer experiments and nine of the cultivars were represented in two or more experiments. Heritability of DPR of roots was only consistent in autumn/winter experiments (27 %) signifying the importance of phenotyping in relevant seasons and environments. Depth penetration rate of roots varied significantly within the tested cultivars, from 1.39 (±0.35) mm °C^?1 day^?1 for cv. Tuareg to 2.07 (±0.34) mm °C^?1 day^?1, for cv. Mercedes. This study documented consistent differences of DPR among North-European winter wheat cultivars in long tube rhizotrons under semi-natural conditions, which may form part of future phenotyping facilities for deep rooting traits.     

Generation and validation of unique male sex-specific sequence tagged sites (STS) marker from diverse genotypes of dioecious Jojoba-Simmondsia chinensis (Link) Schneider

DNA fingerprinting studies have been carried out with the physiologically mature male and female plants of Jojoba using 80 ISSR primers with a view to generate sex-linked markers. After bulk segregant analysis, two unique ISSR markers, viz. ISSR848₁₅₀₀ and VIS11₁₃₁₇ have been developed which can be used for determining the sex at the seedling stage. Of the eighty primers tested on the pooled male DNA and pooled female DNA samples, six ISSR primers were found to be associated with sex expression. Of the six, only two primers ISSR848 and VIS11 generated unique male sex specific bands of ~1,500 and ~1,300 bp which were consecutively present in all the male genotypes and absent in all the respective female genotypes. The remaining four primers when tried on individuals of different genotypes were confined to their sex specificity in only two female genotypes and absent in their male counterparts. One of the male-sex specific markers, VIS11₁₃₁₇ has also been cloned and sequenced which showed homology with a sex linked gene, DD44 from dioecious Silene species. Furthermore, VIS11₁₃₁₇ was converted into a male sex-specific sequence tagged sites (STS) marker of 584 bp. The male specific STS marker thus developed has been verified and validated on 100 populations of male and female individuals from ten different genotypes of Jojoba to endorse the diagnostic reliability of the STS marker. This can gainfully be employed for screening of sex at seedling stage which would be quite helpful for uprooting the undesired plants, thereby, saving resources like labor, water, fertilizers and space for highly desirable female plants.     

Genomic constitution of Festulolium cultivars released in the Czech Republic

Genomic in situ hybridization (GISH) was used to characterize the chromosome constitutions of individual plants from a set of tetraploid and hexaploid cultivars of Festulolium developed and released in the Czech Republic from hybrids of Lolium multiflorum with Festuca pratensis and F. arundinacea. A simplified GISH protocol readily discriminated parental genomes in the hybrids and facilitated the screening of large numbers of plants per accession. The contribution of parental genomes in the cultivars tested ranged from predominance of chromatin from one of the parents to a more balanced contribution from both parents. However, in none of the cultivars were equal proportions of chromatin from both parents present. The parental contribution to the hybrids was both in the form of complete chromosomes or as chromosome translocations. In hexaploid cultivars from (L. multiflorum × F. arundinacea) × F. arundinacea hybrids the average numbers of complete L. multiflorum chromosomes ranged from 4.95 to 7.5 and the numbers of translocations from 6.33 to 10.21. Two tetraploid cultivars from (L. multiflorum × F. arundinacea) × L. multiflorum hybrids showed a strong prevalence of L. multiflorum chromatin and intergeneric translocations were rare. In the tetraploid cultivar ‘Perun’ of the L. multiflorum × F. pratensis hybrid there were 11.7 chromosomes of L. multiflorum and 14.7 recombined chromosomes on average. Reasons for the domination of one of the parental genomes in hybrid cultivars are not clear and are only partially explained by breeding history. Recombination rates of individual genomes in hybrids involving F. arundinacea were evaluated in double hybridization experiments. The results indicated a strong affinity of the L. multiflorum genome for the F. pratensis genome present in F. arundinacea and little affinity for the F. glaucescens genome. This suggests that introgressions from F. arundinacea into L. multiflorum are primarily limited to the F. pratensis genome which can be more readily accessed in L. multiflorum × F. pratensis hybrids.     

A SCAR marker for the sex types determination in Colombian genotypes of Carica papaya

Sex type determination in papaya (Carica papaya L.) is very important for crop improvement processes because it accelerates the identification of the fruitful plants. The use of molecular technology provides a quick and reliable identification of sex types in plantlets growing in seedbeds. Random amplified polymorphic DNA (RAPD) markers were used to determine the sex types of Colombian cultivars of dioecious papaya genotypes. This species has three sex types (male, female and hermaphrodite) determined by a multiallelic locus. There are no morphological differences at the chromosome level; therefore the identification of sex types by chromosomal dimorphism is not possible. A RAPD marker of 900 bp was found in male plants, but not in females or hermaphrodites. From this RAPD marker a sequence characterized amplified region (SCAR) was developed and it was possible to amplify fragments from the genomes of male and hermaphrodite plants, but not the female ones. The results indicate that this new SCAR marker will be valuable to determine the sex type of papaya plants.     

Changed sex expression and possibilities for F1-hybrid seed production in some cucurbits by application of Ethrel and Alar (B-995)

Summary Experiments were performed to study the effects of the ethylene releasing compound Ethrel on sex expression in cucumbers and squash, and of Alar (B-995) plus Ethrel in muskmelons. As a result of foliage sprays with one or both of the above compounds normally monoecious plants produced female flowers only, for the first 2–3 weeks of flowering. The optimum treatments for cucumbers were two foliage sprays with Ethrel 250 ppm or 500 ppm applied at the second and the fourth true leaf stages. The optimum treatments for squash were Ethrel 250 ppm and 500 ppm applied at the first and the third true leaf stages.High doses (1000 ppm) or repeated applications of Ethrel retarded growth of muskmelons and cucumbers. Applications of B-995 (5000 ppm) plus Ethrel (500 ppm) at the second true leaf stage inhibited male flowering for 2–3 weeks of the flowering period. F₁-hybrid seeds of muskmelons were experimentally produced in large isolation cages in the field, using two monoecious lines as female parents. The merits and some of the problems associated with the production of F₁-hybrid seeds by the above methods are discussed.     

Amplified fragment length polymorphism analysis in diploid cultivars of rhodesgrass

The amplified fragment length polymorphism (AFLP) technique was applied to detect genetic variation in a sample of 47 plants representing 12 diploid cultivars of rhodesgrass. In this analysis, 50±91 easily scorable fragments could be detected in a single reaction. Each of the individual plants was uniquely identified by a combination of three primer pairs and an 80.2% level of polymorphism was obtained. Large amounts of genetic variation were present within all the cultivars. The results showed that AFLPs could be a robust technique for genome analysis in rhodesgrass with a promising potential as a breeding tool.     

Sex distinction of asparagus by loop-mediated isothermal amplification and observation of seedling phenotypes

Asparagus (Asparagus officinalis L.) is a dioecious plant. In general, male and female plants are used for open-field culture and intensive cultivation, respectively. Farmers distinguish between the sexes by observing the form of the flower organs. However, because flowering begins 2?C3 years after planting, the sexes cannot be differentiated at transplantation by using this method, and planting of an all-male population is not possible. In this study, the usefulness of loop-mediated isothermal amplification (LAMP), a simple method of gene amplification, for sex distinction at the DNA level was determined. In addition, the phenotypic differences in seeds and seedlings of male and female plants were investigated for application as a method of early sex distinction. By using the LAMP method, the sex could be correctly identified in 100% of the seedlings, suggesting that this method is effective for sex distinction at the gene level. Principal component analysis was conducted with 11 selected parameters after investigating the seeds and seedlings of both male and female plants. The results revealed that male plants tend to have many stalks or cladophylls and female plants tend to have large plant forms, suggesting that the sexes can be distinguished by the external appearance of the seedlings before planting. LAMP and observation of the seedling phenotypes could be useful methods of sex distinction for increasing the efficiency of asparagus breeding.