Sex chromosomes and quantitative sex expression in monoecious hemp (Cannabis sativa L.)

Hemp (Cannabis sativa) has a highly variable sexual phenotype. In dioecious hemp, the sex is controlled by heteromorphic sex chromosomes according to an X-to-autosomes equilibrium. However, in monoecious hemp, the sex determinism remains widely unknown and has never been related to a quantitative approach of sex expression. The present paper aims to contribute to the comprehension of the sex determinism in monoecious hemp by assessing the genotypic variability of its sex expression and establishing its sex chromosomes. Five monoecious and one dioecious cultivars were grown in controlled conditions under several photoperiods. The monoecy degree of 194 monoecious plants was recorded at each node by a figure ranging from 0 (male flowers only) to 6 (female flowers only). The genome size of 55 plants was determined by flow cytometry. The DNA of 115 monoecious plants was screened with the male-associated marker MADC2. The monoecy degree varied significantly among monoecious cultivars from 3.36 ± 2.28 in ‘Uso 31’ to 5.70 ± 0.81 in the most feminised ‘Epsilon 68’. The variation of monoecy degree among cultivars remained consistent across trials despite a significant “cultivar × trial” interaction and partly agreed with their earliness. The genome size of monoecious plants (1.791 ± 0.017 pg) was not different from that of females (1.789 ± 0.019 pg) but significantly lower than that of males (1.835 ± 0.019 pg). MADC2 was absent from all monoecious plants. These results strongly support that cultivars of monoecious hemp have the XX constitution and that their sex expression has a genetic basis.     

纤用工业大麻雌雄株主要农艺性状的初步研究

为了明确大麻雌雄株之间的差异及为大麻全雌、全雄、雌雄同株育种提供理论依据,本试验以‘火麻一号’、‘汉麻5号’和‘汉麻2号’为试验材料,分析了大麻雌雄株之间原茎产量和农艺性状的差异及其相关性。结果表明,雌株的原茎产量比雄株高出18.18%~24.56%,差异显著;雄株的株高和节数分别比雌株高出了3.08~5.86 cm、0.28~1.92节,株高差异显著;雌株的茎粗和穗长分别比雄株高出了0.05~0.08 cm、3.35~4.90 cm,穗长差异显著;雌株的根长略高于雄株,而雄株的工艺长度比雌株高出了3.26%~5.55%,差异显著;雌株的茎干重、根干重分别比雄株高出了22.16%~27.06%、21.97%~31.91%,茎干重差异显著;雌雄株的节数、穗长及叶干重与雌雄株单株的原茎重呈负相关关系,雌雄株的株高、茎粗、根长、工艺长度及根干重与雌雄株单株的原茎重呈正相关关系。纤用工业大麻高产栽培技术及高产育种要以雌雄株比值小,叶少、节数少、穗短、株高和茎粗适中、工艺长度长、根干重及单株原茎重大作为主要目标。     

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.     

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。     

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.     

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.     

Validation of a male‐linked gene locus (OGI) for sex identification in persimmon (Diospyros kaki Thunb.) and its application in F1 progeny

It is crucial to develop a rapid technique for identifying sexuality in the seedling stage of persimmon (Diospyros kaki Thunb.), and the elimination of male progeny has been regarded as an important strategy for enhancing breeding efficiency. In this study, phenotype characterization and genotyping of the male‐linked OGI marker were carried out using 205 accessions, including persimmon cultivars, F₁ progeny and nine related Diospyros species. All persimmon cultivars displayed consistent results regarding OGI amplification and sex phenotype. A total of 143 F₁ progeny were derived from 11 crosses, among which 95 individuals had flowered. In the flowering full‐sib families, the amplification of the OGI marker in agreement with the sex phenotype was obtained in 85 plants (89.5%). The segregation of OGI in ‘Huashi 1’ × ‘Luotian Tianshi’ and ‘Huashi 1’ × Male 3 F₁ populations fit a 1 : 1 ratio. Furthermore, high OGI transferability was observed in nine related species. Overall, the results indicated that the OGI locus could be used to distinguish male from female persimmon plants at an early stage.     

分子标记技术在工业大麻性别分化研究中的应用进展

工业大麻性别表达影响着工业大麻种子、纤维和药用成分产量,其性别表达除了受到遗传物质控制外,还受到外界环境条件的影响,这种复杂性使得性别研究一直是工业大麻研究的难点和热点。分子标记技术能在DNA水平上鉴定工业大麻性别,开发与工业大麻性别紧密连锁的分子标记可为大麻性别早期鉴定和特定性别分子辅助选择育种提供理论依据。本文就分子标记在工业大麻性别研究中的应用进展进行了总结,并对应用前景进行了展望。     

Generation and characterisation of colchicine-induced polyploid Lavandula × intermedia

Double podding in cultivated chickpeas (Cicer arietinum L.) can increase yield and yield stability. In the present study, we performed reciprocal crosses of ‘kabuli’ (double podded) and ‘desi’ (single podded) chickpeas to determine (i) the expressivity and penetrance of double podding, (ii) the correlations of yield and yield components, and (iii) the heritability of double podding, flower color, and stem pigmentation in F₂ plants. Reciprocal crosses were performed with two genotypes, AC 2969 (kabuli) and ICC 4969 (desi), to generate F₁ and F₂ plants. The results indicated hybrid vigor (heterosis) for yield in F₁ plants and better performance of F₂ plants. Yield and yield components of some lines in F₂ were superior to the best parent, indicative of transgressive segregation. In particular, the presence of double podding (‘s’ allele) significantly increased yield in some of the transgressive segregants. Expressivity and penetrance of the ‘s’ allele depends on the background of the female parent. Some of the double podding progeny had greater seed yields than those of the single podding progeny and greater seed yields than the best parents. Double podding, stem pigmentation, and pink flowers each appears to be governed by a single recessive gene. Stem pigmentation and pink flowers appear to be linked traits that depend on the genetic background of the crossed chickpeas. Taken together, our studies of reciprocal crosses of kabuli and desi chickpeas clearly showed that yield could be improved by selection for transgressive phenotypes that have double podding.     

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.     

Ethylene and postharvest performance of potted kalanchoë

The postharvest life of different cultivars of kalanchoë plants (Kalanchoë blossfeldiana Poelln.) varied widely. The display life of plants of ‘Nadia’ was much longer than that of plants of ‘Debbie’ (10 weeks compared with 7 weeks). During display life, senescing flowers were replaced by opening buds and the difference in display life was primarily a function of differences in life of the individual flowers in the inflorescences (approximately 45 and 20 days, respectively, for ‘Nadia’ and ‘Debbie’). Approximately 8 h after the start of exposure to ethylene, kalanchoë flowers started to inroll, reaching a minimum diameter after exposure for 24 h. The effects of ethylene were somewhat reversed by returning the plants to air within the first 24 h of exposure. Thereafter the flowers wilted and senesced irreversibly. Ethylene sensitivity varied widely among cultivars; while flowers of ‘Alexandra’ lost 57% of their diameter in response to 1 μl l⁻¹ ethylene treatment, flowers of ‘Debbie’ lost only 2%. Flowers of the new ‘African Queen’ series were particularly sensitive to ethylene. There was no clear relationship between ethylene sensitivity and postharvest life in the absence of ethylene; although ‘Nadia’ had the longest display life of all tested cultivars, ‘Nadia’ flowers showed a 35% diameter reduction in response to treatment with 1 μl l⁻¹ ethylene for 24 h. Treatment with 1-methylcyclopropene (1-MCP) had no effect on the normal life of kalanchoë plants, although 1-MCP has been shown to prevent the response of kalanchoë flowers to ethylene.     

白蜡属植物的开花特性和动态观察

研究花器官形态结构和开花特性，为白蜡授粉受精提供理论依据。本研究选取了3个白蜡品种（‘鲁蜡2号’、‘鲁蜡5号’和‘金箭’），对其开花特性、花器官构造进行观察。结果表明，白蜡品种雄株花期为11～13天，开花时间比雌株早13～15天；雌株‘鲁蜡2号’的花期为5～6天。3个白蜡品种的小花密集，开花后苞片2裂，花萼钟状4深裂，无花冠。雄花开花后露出舟形花药，花丝极短。雌花苞片开裂后露出雌蕊，柱头成熟时两裂，呈粉红色，花柱极短。扫描电镜下的3个白蜡品种苞片和花萼表面均有盾毛分布，不同品种间盾毛的形态有差异；‘金箭’、‘鲁蜡5号’的花药与花粉结构无显著差异；‘鲁蜡2号’整个雌蕊表面都有不规则褶皱分布。本研究可为进一步开展白蜡属植物的育种和种质资源创新利用研究提供参考。     

EMS诱发大豆不同品种M2代与M3代农艺性状变异比较

为了明确甲基磺酸乙酯(EMS)对大豆品种诱变的后代变异情况,利用0.5% EMS溶液处理4个大豆品种种子,对M2、M3代主要农艺性状的遗传变异进行分析比较。结果表明：各世代农艺性状均发生一定程度的变异,与M2代相比,M3代株高、分枝数、单株粒数、单株粒重及百粒重的平均变异系数变小,而主茎节数的平均变异系数变大,单株荚数的变异系数大致相当,然而这种趋势在各品种间却不尽相同;各世代都诱变出早熟或晚熟的变异株,各品种总熟期变异率由高到低M2代为‘黑河43’(0.71%)>‘北豆5’(0.63%)>‘合丰50’(0.57%)>‘合丰55’(0.53%),M3代为‘北豆5’(0.64%)>‘黑河43’(0.5%)>‘合丰55’(0.25%)>‘合丰50’(0.22%),说明熟期的变异至M3已下降。     

西瓜强雌性性状的遗传分析及分子标记研究

为了解西瓜强雌性性状的遗传规律,以强雌性系BG1为母本,分别与不同的西瓜品系杂交、自交及回交,共获得5个F1群体、1个F2群体、1个回交群体BC1R34,以及1个回交转育品系(BC4S3).5个F1群体植株全部表现为正常花性型;经χ2测验F2分离群体中强雌性型与普通花性型的分离符合1∶3的分离比率;与普通花性型回交后代的BC1R34表现为普通花性型;回交转育得到的BC4S3品系表现为强雌性型,雌花率高达77.9%.试验结果表明,该强雌性状主要受单隐性核基因控制,但根据F2单株雌花率的分布情况,认为还可能存在微效基因的作用.对两对近等基因系BG1和BG2及BC4S3和R34进行RAPD分析,共使用333条随机引物,经过进一步F2验证在强雌性系中获得一条稳定扩增的片段s1454-1100.     

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.     

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.     

锥栗开花生物学特性研究

为了解锥栗雌雄花生长和发育规律,掌握其开花生物学特性。以不同锥栗品种为材料,对其雌雄花开花节律、物候期、雌雄花数量、雄花序及结果枝生长节律等问题进行研究。锥栗雄花先于雌花出现,雄花序出现18~26天后雌花开始出现,且雄花先于雌花1~5天开放;不同品种开花物候期有较大差异,早熟品种和晚熟品种初花期相差10天左右;不同品种雌雄花数量差异明显,其雌花与雄花数量比为1:(518~653);不同品种结果枝生长、发育时期大致相近,但长度和粗度差异明显,其中,‘YLZ 7号’结果枝长度和粗度明显大于‘优16号’和‘优22号’。锥栗雌雄异熟,属雄先型植物,不同品种开花物候期、雌雄花数量差异明显,生产上应选择少雄花品种,或采取早期摘除部分雄花序、花期加强肥水管理等措施,以保证雌花生长和发育,提高锥栗产量和经济效益。     

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.