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。     

DNA分子标记技术在农作物种子质量检验中的应用

种子质量是农业生产中的重要元素,它包括种子真实性和品种纯度2个方面。目前检测种子质量大致主要有3种方法：大田鉴定法,生化标记鉴定法和DNA分子标记鉴定法。大田鉴定法和生化标记鉴定法在实践过程中存在着一些无法解决的问题,使得它们的应用受到一定的局限性。DNA分子标记技术是随着分子生物学的发展而发展起来的一种新型的鉴定方法。它具有简便、快速、准确的特点,弥补上述2种传统鉴定法的缺点。本文对几种生产中常用的DNA分子标记技术根据其特点进行了分类,介绍了每种标记方法的基本概念,基本原理,并对其应用过程中优缺点进行了简要概括;主要就每种标记技术在种子真实性和品种纯度检测应用方面对前人的研究进行了详细综述;列举了理想的作物种子真实性和品种纯度鉴定方法应该满足的基本要求。DNA分子标记技术在农作物种子真实性和品种纯度鉴定方面存在的问题主要是,目前还没有建立专门针对杂交种纯度检测的分子标记,这也制约了分子标记应用的普遍性。DNA分子标记技术与大田鉴定法和生化标记鉴定法相比具有独特之处,故在农作物种子真实性和品种纯度检验中成为首选的方法。但是其更为广阔的应用前景还有赖于DNA分子标记技术的进一步发展。     

分子标记在裸子植物应用中的研究进展

分子标记技术应用于裸子植物研究,对裸子植物亲缘关系与遗传多样性研究、分子辅助育种、遗传图谱构建及资源保护利用等研究带来了极大的便利。本文从种质遗传多态性及亲缘关系、遗传图谱构建、目标性状连锁分子标记与辅助育种、种质与性别鉴定等方面展开综述,探讨近年来分子标记在裸子植物应用中的研究进展,并讨论了分子标记在裸子植物研究上存在的问题。     

分子标记在大白菜遗传育种中的应用

大白菜是我国起源的特产蔬菜,利用分子辅助育种手段进行大白菜遗传育种越来越受到育种家的关注。本文对已构建的二十多张大白菜遗传图谱和白菜类蔬菜间的亲缘关系进行了分析,同时也从芜菁花叶病毒病等抗病分子标记、春化、抽薹和雄性不育相关的分子标记、其它农艺性状标记及种子纯度鉴定等方面阐述了分子标记技术在大白菜遗传育种中的应用。分子标记整合、比较与国际接轨及试验材料的选择都可能成为分子标记今后的研究方向。     

野生大麻叶绿体基因组分子多态标记的筛选与开发

以6份不同生态区野生大麻为试验材料,对叶绿体17个高变区的通用引物进行多态性筛选,同时基于已公布的大麻叶绿体基因组全序列,应用生物信息学软件对其进行比较基因组学分析,开发新的多态性引物。结果显示:(1)17对通用引物,通用性达76.5%,但仅ndh F-rpl32引物产物多态性好;(2)比较基因组学研究表明大麻叶绿体基因组多态性比较低,但存在极少的高变区,针对全序列高变区段设计的4对引物(trn L-rpl32~a,rps8-rps11,psa I-acc D,rps16~a)在6份野生大麻群体材料中分别有2~8个变异位点,具有较高的多态性。叶绿体非编码基因片段通用引物ndh F-rpl32及本研究针对大麻开发的trn L-rpl32~a、rps8-rps11、psa I-acc D、rps16~a引物在大麻中具有专一性强、产物多态性高等特点,可作为高效分子标记应用于大麻种质资源遗传多样性与保护、物种起源与遗传进化、种下分类鉴定、工业大麻育种等研究。     

分子标记技术在家畜育种中的应用

为了将分子标记技术更好的应用到家畜育种工作中。本文详细总结了分子标记技术在家畜遗传多样性分析、亲缘关系分析、遗传图谱构建、基因定位、杂种优势预测、性别鉴定和抗病育种等诸多方面的应用研究。提出应将表型性状、系谱和分子标记三者结合应用于家畜遗传育种等方面的工作。     

DNA分子标记在银杏中的应用

DNA分子标记广泛用于生物研究的许多方面。本文简要介绍了RFLP、RAPD、AFLP、SSR及ISSR等几种目前常用分子标记的原理,归纳总结了分子标记在银杏中的应用研究进展。(1)获得了2个雄性特有的RAPD标记和2个雌性特有的AFLP标记,为银杏的早期性别鉴定及相关基因克隆奠定了基础;(2)采用RAPD标记和ISSR标记对我国部分栽培品种进行了分子鉴别和分类的研究,编制了一些品种的DNA指纹检索表;(3)利用RAPD和ISSR标记对一些群体、个体及栽培品种或变异类型进行了遗传分化和遗传多样性研究,结果发现银杏具有较高的遗传多样性,群体间、个体间、栽培品种及类型间都存在不同程度的遗传分化;(4)利用ISSR标记对一些个体的遗传杂合性进行了研究,结果显示个体的平均杂合率为43.53%;(5)构建了包含62个RAPD标记、19个连锁群的银杏分子遗传图谱;(6)探索了银杏优先保护种群的确定。分子标记在银杏其它方面的应用还很少。今后,除了继续对上述方面进行深入系统的研究外,还应充分运用DNA分子标记技术,开展银杏的分子标记辅助选择育种、种质评价与鉴别及保育生物学等方面的研究。     

多元统计分析法在大麻资源评价与农业生产中的应用

为系统阐述大麻的纤维产量与大麻的多项农艺性状以及生育期之间存在的相关性,本研究采用回归分析,并结合聚类分析和主成分分析方法,来研究大麻的遗传多样性,揭示大麻资源变异与环境因素互作的内在规律。结果显示,大麻资源的遗传分布整体上与地理位置相关,其干茎产量是由原茎产量和脱水率决定的,也受栽培条件所影响。本研究将分子标记和细胞学标记的生物学试验结果相结合,能系统全面地剖析大麻资源的遗传多样性。研究结果可为品种引进、杂交育种、种质资源保护,优异大麻新品种选育及配套田间栽培技术研究提供理论依据及指导。     

SNP分子标记及其在作物品种鉴定中的应用

作物品种鉴定是优良品种选育和推广的重要保障,而合适的检测方法是对品种进行准确鉴定的关键。随着分子标记技术的发展,第3代分子标记SNP逐渐应用到品种鉴定领域。本研究概述了SNP分子标记的特点,分析了高分辨率熔解曲线、竞争性等位基因特异性PCR、基因芯片、测序法、靶向测序基因型检测等5种作物研究中常用的高通量检测方法的特点及适用性,梳理总结了SNP标记在品种真实性鉴定、纯度检测和亲缘关系分析与分类等方面的研究与应用情况,以期为后续利用SNP分子标记进行品种鉴定提供技术参考。     

茄子SSR多态性引物的筛选及品种纯度鉴定

为应用分子标记建立一套快速准确的杂交种鉴定方法,利用已有的236对SSR分子标记对3个茄子品种进行多态性标记筛选,并进行待测品种的分子标记纯度鉴定以及田间纯度鉴定,获得特定品种的指纹信息。结果显示,筛选出用于茄子纯度鉴定的多态性引物15对,利用该系列引物进行纯度鉴定,品种1-2010、3-2010和9-2010纯度结果分别为98%,96%和100%,在此基础上获得了对应品种的指纹信息。应用SSR进行茄子品种纯度鉴定与田间检测结果一致,证明分子标记检测茄子杂交种纯度可行且高效,具有一定的理论和应用价值。     

Potential of marker-assisted selection in hemp genetic improvement

Summary The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.     

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.     

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.     

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.     

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.     

工业大麻内生真菌菌群结构及其在植株内的空间分布特征

[目的]研究旨在探讨不同样地和不同部位大麻组织中内生真菌的多样性及其分布特征,[方法][结果]从4个不同样地大麻（云麻1号）的植株不同部位组织中共分离得到内生真菌531株,其中茎分离出138株,叶分离出393株。经形态学观察和ITS序列分子鉴定发现其分属于29个属,其中茎22个属,叶24个属。有18个属普遍存在于大麻茎和叶中,其中炭疽菌属是大麻的优势菌属,优势度为40.11%。大麻茎部的内生真菌分离率显著低于叶部,接近地面部分的大麻茎叶组织内生真菌分离率最高。4个样地大麻内生真菌多样性指数在1.84到2.46之间,不同样地中大麻内生真菌组成的相似系数普遍较高,同一样地中大麻茎与叶的相似性系数较小。[结论]结果表明,工业大麻（云麻1号）内生真菌多样性偏低、宿主专一性较小。     

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.     

Sex-linked SSR markers in hemp

Hemp is a dioecious plant with sex chromosomes X and Y, the male sex being heterogametic. The quality of the fibre depends on the sex type. The sex chromosomes can be characterized by molecular markers. In this report, sex‐linked simple sequence repeat (SSR) markers are described. One SSR marker was polymorphic in both the populations derived from single crosses, two other markers in but one of the two populations. Three alleles were detected for two SSR markers indicating polymorphism not only between X and Y, but also between different X chromosomes. In addition, several sex‐linked RAPD markers were detected in one population. Recombination within the sex chromosomes was observed for nearly all markers.     

Current status and future scenarios of hemp breeding

Summary Hemp is a multi-use crop, able to provide fiber, cellulose, seeds and seed oil, cannabinoid, and biomass. Integrating many agroindustrial chains, Cannabis is considered a crop model in which insights into specific metabolic pathways and biosynthetic processes are valuable for improvement of the plant for all sets of industrial derivatives. In this review the hemp breeding status is elucidated and many aspects are focused: (i) recovering, maintenance and characterization of genetic resources; (ii) widening of germplasm and genetic variability; (iii) marker-assisted selection and development of breeding programs; (iv) sexual differentiation; (v) monitoring of THC content. Modern hemp varieties for fiber and other specific end uses have been developed and new varieties are entering production. The scenario for the breeding advances in hemp relies on basic and applied research which provides insights to identify a strategy for the design of modified plants with enhanced performance. This is accounted by the dissection of traits into components and the modification of single steps of the related metabolic pathways. These advances are provided by genomic techniques and are able: (i) to identify key genes encoding enzymes and regulatory factors participating in cannabinoid, fiber and oil biosynthesis; (ii) to identify the mode of regulation of these genes; (iii) to characterize the function of the selected genes through higher, lower or specific expression incited by specific promoters. The identification of molecular markers for specific traits, gathered in a saturated linkage map, will have a remarkable impact on hemp breeding. The advances in basic and applied research make it possible to design methods for the identification of superior parents and cross combinations and the development of selection schemes that rely on less labour-intensive and time-consuming methods.