SRAP标记技术在甜瓜品种亲缘关系和种子纯度鉴定上的应用

利用SRAP分子标记对12份甜瓜材料进行亲缘关系鉴定和F1代杂交种进行纯度检测。结果表明:从40对引物中筛选出13对多态性好的引物,共扩增出78条带,多态性带的比例为50%;聚类分析结果显示,12份甜瓜材料的相似系数在0.263～0.921之间,可将该12份甜瓜材料分为2大类,进一步细分为5类。利用引物Me15-Em7和Me3-Em16对‘红绿早脆’100S杂交种进行纯度检测,杂交种纯度为77%。研究为甜瓜品种间亲缘关系及种子纯度检测提供了可靠的理论依据。     

中国姜花属基于SRAP分子标记的聚类分析

依据苞片是覆瓦状排列或是卷筒状排列而把姜花属Hedychium分为两个亚属的分类法近年颇受质疑.本文利用30对多态性好的随机引物,对中国姜花属的19种1变种共22份材料进行SRAP分子标记分析.其中最有效的28对引物共扩出152条带,当中135条为多态性带,占88.8%.平均每对4.8条,多态性条带里最多的为引物组合F13R1,达到13条.聚类分析表明:(1)中国姜花属植物可分为三个类群:第1群植株较矮小,主要分布于石灰岩地区;第Ⅱ群与第Ⅰ群每苞片均具2朵以上的小花,但植株较高大且基本上不分布于石灰岩地区;第Ⅲ群每苞片仅具1朵小花.此结果与Wood(2000)用ITS分析得出的结果基本一致.(2)支持吴德邻和Larsen(2000)把H. emeiense Z.Y Zhu归并为峨眉姜花H.flavescens Carey ex Roscoe的观点.(3)盘珠姜花H.panzhuum Z.Y Zhu与黄姜花H.flavum Roxb.是同一种植物,即盘珠姜花是黄姜花的晚出同名.(4)土壤基质可能是导致姜花属物种分化的重要因素.作者认为根据每苞片有花多少的特征,在系统学上可把姜花属分为两个类群:A类群,每苞片仅有1朵小花:B类群,每苞片有2朵以上的小花.     

Genetic molecular analysis of Coffea arabica (Rubiaceae) hybrids using SRAP markers

In Coffea arabica (arabica coffee), the phenotypic as well as genetic variability has been found low because of the narrow genetic basis and self fertile nature of the species. Because of high similarity in phenotypic appearance among the majority of arabica collections, selection of parental lines for inter-varietals hybridization and identification of resultant hybrids at an early stage of plant growth is difficult. DNA markers are known to be reliable in identifying closely related cultivars and hybrids. Sequence Related Amplified Polymorphism (SRAP) is a new molecular marker technology developed based on PCR. In this paper, sixty arabica-hybrid progenies belonging to six crosses were analyzed using 31 highly polymorphic SRAP markers. The analysis revealed seven types of SRAP marker profiles which are useful in discriminating the parents and hybrids. The number of bands amplified per primer pair ranges from 6.13 to 8.58 with average number of seven bands. Among six hybrid combinations, percentage of bands shared between hybrids and their parents ranged from 66.29% to 85.71% with polymorphic bands varied from 27.64% to 60.0%. Percentage of hybrid specific fragments obtained in various hybrid combinations ranged from 0.71% to 10.86% and ascribed to the consequence of meiotic recombination. Based on the similarity index calculation, it was observed that F1 hybrids share maximum number of bands with the female parent compared to male parent. The results obtained in the present study revealed the effectiveness of SRAP technique in cultivar identification and hybrid analysis in this coffee species.     

Genetic identification of 91 poplar cultivars based on SSR markers

Accurate genetic identification and relationship analysis of poplar cultivars is necessary to establish commercial poplar plantations and select suitable breeding strategies. In this study, 91 poplar cultivars belonging to four sections (Aigeiros, Tacamahaca, Populus and Turanga) and inter/intra-sectional hybrids were genotyped using 18 polymorphic simple sequence repeat (SSR) markers. In total, 222 alleles were amplified with an average of 12.3 alleles per marker. The mean polymorphic information content and power of discrimination were 0.706 and 0.813, respectively. Five SSR markers (ORPM_103, ORPM_247, GCPM_1048, GCPM_1255 and LG_X_19) constituted a core fingerprint and were sufficient to identify all the tested cultivars. With some notable exceptions, cultivars of the same species generally clustered together in cluster (UPGMA) and ordination (PCO) analyses. Flow cytometry indicated that 11 poplar cultivars were triploid. Among these, seven had three alleles at some loci, suggesting that SSR markers could indicate the ploidy level to some extent. This study provides useful genetic information for the identification and protection of poplar cultivars in China and offers a guideline for the selection of poplar crossing parents based on ploidy level and genetic relationships.     

利用SRAP和ISSR建立快速鉴定灵芝属菌株的SCAR标记

根据ERIC聚类分析的结果,把152株灵芝属菌株(包括128株来自中国的栽培菌株及24株国外菌株)建成48个DNA池。用SRAP和ISSR引物对48个DNA池进行扩增,筛选获得4个特异性标记,回收特异性条带,经克隆测序后设计了4对SCAR引物,并通过SCAR-PCR扩增验证,从而将SRAP标记和ISSR标记均成功地转化为特异性和稳定性更好的SCAR标记;将得到的4个SCAR标记在构成DNA池的152个菌株上验证,并建立多重PCR体系,最终证实了SCAR特异标记在菌株快速检测鉴定中的可行性和可靠性。     

Genetic diversity analysis of Pinellia ternata based on SRAP and TRAP markers

An assessment of the genetic diversity and variation of Pinellia ternata collected from 43 populations in China was undertaken using SRAP + TRAP markers. A total of 13 SRAP primers in addition to 3 TRAP primer combinations yielded 292 bands in total of which 286 were polymorphic (98.0%), with an average of 16 for each. The PIC value ranged from 0.88 to 0.95, with a mean polymorphic information content (PIC) of 0.92 over all the primers. A cluster analysis was conducted based on the unweighted pair group method with arithmetic average (UPGMA), and a principle coordinate analysis (PCA) was performed relying on Nei's genetic distance, both of which showed similar outcomes in that the results of clustering did not correlate to the geography locations but showed an association with morphology. An analysis of molecular variance (AMOVA) was performed to detect the source of variation; the results indicated that the main variation existed within populations (67.2%), whereas there was still 32.8% differentiation existing among the populations.     

黄牡丹与芍药组间杂交花粉与柱头识别的解剖学研究

分别用TTC染色法和培养液培养法测定黄牡丹花粉的生活力和萌发率;并分别以5个芍药品种为母本,黄牡丹为父本进行人工杂交,用苯胺蓝染色法,在荧光显微镜下观察黄牡丹花粉在母本('墨玉双辉'、'奇花露霜'、'手扶银须'、'金凤'和'美菊')柱头上的黏附、萌发以及花粉管在柱头和花柱中的动态变化.结果显示:(1)黄牡丹离体花粉生活力达64.9%、萌发率达68.7%.(2)授粉后24 h,黄牡丹花粉在'奇花露霜'、'手扶银须'和'金凤'柱头上的黏附数最多分别为134.5、80.9和100.5个,萌发花粉数也达到最多,分别为46.3、69.7和86.6,说明花粉与母本柱头的黏附性较好.(3)授粉后黄牡丹花粉黏附在柱头处及其附近的柱头乳突细胞内产生大量胼胝质;花粉管生长迟缓,且有扭曲、肿胀、结合、分枝等现象发生;少数花粉管能穿过柱头进入花柱,但常有胼胝质沉积其中.从黄牡丹花粉与5个芍药品种柱头的识别作用来看,受精前障碍是影响黄牡丹与芍药组间杂交的主要原因之一.     

Systematic studies on Paeonia sect. Moutan DC. based on RAPD analysis

Plants in Paeonia sect. Moutan DC., whose wild types are endemic to China, are deciduous subshrubs. Taxonomic treatments of most species in this section have long been in dispute. To address this question, both intraspecific and interspecific relationships of the species in this section were analyzed using RAPD markers. The dendrogram constructed by UPGMA showed that the accessions of the same species were always grouped together earlier than those of different species. The intraspecific similarity coefficients ranged from 0.60 to 0.90, grouping precisely those species of the same subsection together. Hence, the seven species under question can be well distinguished from each other. The similarity coefficient between P. delavayi and P. ludlowii was 0.60, and they were clustered in a clade. The similarity coefficients between P. jishanensis and the three species P. rockii, P. ostii, P. qiui, and between P. jishanensis and P. decomposita were both 0.48. These five species were clustered in another clade. These two clades corresponded well to Subsect. Delavayanae and Subsect. Vaginatae. Our results support the taxonomic treatment of Sect. Moutan re- cently proposed by Hong (1998,1997).     

芍药属牡丹组的系统学研究——基于RAPD分析

芍药属牡丹组(Paeonia sect．Moutan DC)是落叶亚灌木,其野生类群为我国特有。长期以来不同 学者根据形态性状对这个组中种的分类处理不断修正,不断有新种描述。我们采用RAPD标记分析了 牡丹组种内与种间遗传关系。从10个RAPD引物获得121个多态位点。用UPGMA方法构建的树系 图表明每个种的所有个体都各自聚为一支,种内的相似性系数为0.60～0.90,因此现有的7个种能很好 地区分开来。P．delavayi与P．ludlowii相似性系数为0.60,聚为一支;P．jishanensis与P．rockii、P. ostii、P．qiui以及P．decomposita之间的相似性系数为0.48,聚为一大支。这两支与肉质花盘亚组和革 质花盘亚组相对应。这些结果与洪德元根据形态性状对该组所做的分类处理基本相符。我们认为RAPD技术用于牡丹基因组分析是灵敏而行之有效的工具。     

基于SRAP标记的墨西哥落羽杉优良单株的遗传多样性分析

采用SRAP标记方法,分析了原产地及种植地不同的18个墨西哥落羽杉(Taxodium mucronatum Tenore)优良单株的遗传多样性,并根据遗传相似系数、采用UPGMA法对它们的遗传关系进行了聚类分析.结果显示:用7对引物组合从18个单株的总DNA中共扩增出87条带,其中多态性条带71条,多态性条带百分率为81.6％.按照原产地和种植地可将18个单株分为4组,它们的总体Nei's基因多样性指数、Shannon信息指数和基因分化系数分别为0.229、0.355和0.479 9,而基因流仅为0.542,说明各组间的基因交流较少.18个单株间的遗传相似系数为0.632 2～0.919 5,平均值为0.753 9.聚类分析结果显示:18个单株可分为3组,其中18号和14号单株分别单独成组,其余16个单株聚为1组.后者可进一步分为8个亚组:1号、7号、12号和15号单株各自单独成亚组;2号、3号、5号、9号、11号和13号单株聚为1个亚组;4号和6号单株、8号和17号单株、10号和16号单株分别聚为3个亚组.研究结果表明:18个优良单株间存在丰富的遗传变异,但它们的遗传关系与原产地及种植地明显不相关.     

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR₁₃₀, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR₁₃₀ was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR₁₃₀ is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.     

多元统计分析方法在万寿菊品种抗旱性评价中的应用

利用主成分分析法、隶属函数法和聚类分析法,对9个万寿菊品种16个生理指标的抗旱性进行综合评价.结果表明： 不同品种万寿菊的16个生理指标变化程度不同,其中脯氨酸(Pro)、H₂O₂和抗坏血酸过氧化物酶(APX)对干旱胁迫的敏感性最大,而且部分抗旱系数之间显著相关;4个主因子代表16个生理指标抗旱性88.6%的数据信息,‘珍妮’和‘金门’、‘鸿运’、‘珍妮’、‘拳王’分别在4个主因子上的抗旱性最强;9个品种的抗旱性综合评价值大小为：‘珍妮’>‘金门’>‘鸿运’>‘拳王’>‘巨人’>‘大英雄’>‘小英雄’>‘迪阿哥’>‘发现’;聚类分析将9个品种分为3类,其中‘金门’、‘珍妮’、‘鸿运’和‘拳王’属于抗旱品种.     

Molecular identification of Greek olive (Olea europaea) cultivars based on microsatellite loci

Olea europaea is one of the oldest species of domesticated trees. We used microsatellite markers for fingerprinting and for evaluation of genetic similarity and structure of 26 Greek olive cultivars, which cover most of the olive cultivation regions of Greece, including previously undescribed denominations from northern Greece. Eighty-one alleles were revealed with six SSR loci that were selected as most informative of 10 SSR primers that were initially investigated. The number of alleles per locus varied from 7 to 20 (mean, 13.5). Heterozygosity ranged from 0.240 at locus DCA-3 to 0.826 at locus UDO99-9, with a mean value of 0.600. Analysis of 104 trees representing 26 denominations (four trees per denomination) revealed 26 distinct SSR profiles, indicating 26 olive cultivars; no intracultivar variability was observed. Genetic and geographic distances were not significantly correlated, based on the Mantel test. These SSR loci allowed unequivocal identification of all the cultivars and will be useful for future breeding and olive germplasm management efforts.     

Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers

Combined randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were used to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. For the RAPD and SRAP analyses, 479 and 282 polymorphic bands were obtained from 20 P. ostreatus strains using 20 and 13 selected primers or primer pairs, respectively. A combined RAPD/SRAP dendrogram grouped the 20 strains into five clades with a coefficient of 0.690. The comparison of RAPD and SRAP was evaluated in the present study. The combined RAPD/SRAP markers provided reliable information regarding the relationships among the P. ostreatus strains.     

Genetic diversity analysis of Perinereis aibuhitensis based on ISSR and SRAP markers of Chinese coast populations

The objective of this study was to obtain an overview of the genetic relationships within Perinereis aibuhitensis using Inter-Simple Sequence Repeat (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) markers that were derived from related populations residing in the Chinese coasts. The percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at the species level. The analysis of molecular variance revealed that 81.22% (ISSR) and 76.29% (SRAP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Nei's genetic differentiation (Gst) (0.2568/0.2876). The gene flow number (Nm) was 1.4470/1.2385, which indicated that there was limited gene exchange between populations. The phylogenetic tree of the ten P. aibuhitensis populations was separated into four major clusters using the neighbor-joining (NJ) method. These results provide a simple and useful basis for P. aibuhitensis germplasm research and aquaculture breeding.     

Genetic variation and conservation of the endangered Chinese endemic herb Dendrobium officinale based on SRAP analysis

Dendrobium officinale (Orchidaceae) is used for traditional medicine and is critically endangered in China. To investigate the genetic structure of this species and to offer some advice on conservation strategies, 84 individuals from nine wild populations of D. officinale were analyzed using the method of sequence-related amplified polymorphism. A high level of genetic diversity was detected (PPB = 88.07%, H _E = 0.2880) at the species level. However, the genetic diversity at the population level was lower (PPB = 51.68%, H _E = 0.1878) in comparison with other species with similar life history characteristics. Based on analysis of molecular variation, there was moderate variation between pairs of populations with Φ _ST values ranging from 0.1327 to 0.4151 and on average 27.05% of the genetic variation occurred among populations. Two main clusters were shown in UPGMA using TFPGA, which is consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is the first advocated and and ex situ should be proposed at the same time to protect the endangered plant and to preserve germplasm resources.     

Molecular characterisation of indigenous Swedish apple cultivars based on SSR and S-allele analysis

Trees of 68 apple cultivars, aimed for preservation by the 'National Program for diversity of cultivated plants' as mandate cultivars, were analysed using a set of 10 SSR (simple sequence repeat) primer pairs and the self-incompatibility (S-)locus to evaluate genetic diversity and reveal inter-cultivar relationships. The 12 polymorphic SSR loci exhibited 2 to 15 alleles, with expected heterozygozity (H(e)) ranging from 0.36 to 0.88 and a mean of 0.74. Numerous alleles were classified as rare or unique (35% and 18% respectively). For the S-locus, a total of 14 alleles were identified in this study. Five alleles, S1-S3, S5 and S7 had frequencies ranging from 11 to 18%, whereas the remaining 9 alleles were below 6%. All sexually obtained cultivars could be distinguished with the set of SSR loci. Sports were identical with their progenitors in two cases, but differed in one SSR allele in a third case. An SSR-based dendrogram, based on Roger's genetic distances, did not reveal any clear pattern of clustering. The genetic distances were, however, correlated with a corresponding matrix obtained in a previously conducted RAPD-based study of the same cultivars. Non-mandate parents of Swedish mandate cultivars together with some other reference cultivars were included in this study to check the accuracy of allele scoring, verify parentage and compare the results of this study with those presented in previously published studies. Some discrepancies in allele sizing were revealed and the possibilities of avoiding this problem are discussed.     

Genome‐wide identification and analysis of heterotic loci in three maize hybrids

Heterosis, or hybrid vigour, is a predominant phenomenon in plant genetics, serving as the basis of crop hybrid breeding, but the causative loci and genes underlying heterosis remain unclear in many crops. Here, we present a large‐scale genetic analysis using 5360 offsprings from three elite maize hybrids, which identifies 628 loci underlying 19 yield‐related traits with relatively high mapping resolutions. Heterotic pattern investigations of the 628 loci show that numerous loci, mostly with complete–incomplete dominance (the major one) or overdominance effects (the secondary one) for heterozygous genotypes and nearly equal proportion of advantageous alleles from both parental lines, are the major causes of strong heterosis in these hybrids. Follow‐up studies for 17 heterotic loci in an independent experiment using 2225 F₂ individuals suggest most heterotic effects are roughly stable between environments with a small variation. Candidate gene analysis for one major heterotic locus (ub3) in maize implies that there may exist some common genes contributing to crop heterosis. These results provide a community resource for genetics studies in maize and new implications for heterosis in plants.     

The identification of poinsettia cultivars by HPLC analysis of their anthocyanin content

The anthocyanins of 28 cultivars of poinsettia were resolved by HPLC. The amount of each of the 5 anthocyanins was calculated, both by integration of the under their HPLC profile peaks and by second derivatives of the peaks. Comparisons of the two methods showed a correlation of 0.986. The amount and proportion oft he anthocyanins were affected by maturity, season of flowering, quality of irradiance and the specific area of the bract sampled. With careful selection of samples from plants grown under uniform conditions, there wre significant differences amongall 28 cultivars in the amount and proportion of the 5 anthocyanins.     

The identification of poinsettia cultivars by HPLC analysis of their flavonol content

The flavonols from each of 38 poinsettia cultivars were quantitatively resolved by HPLC. Careful selection of samples revealed significant quantitative differences in flavonol content. While all cultivars of seedling origin could be differentiated on the basis of their flavonols, several families of sports could not. Most new commercial poinsettia cultivars have been selected from among somatic mutants exhibiting different bract color. Such changes in color were usually the results of change in anthocyanin content rather than flavonol content. The flavonol content increased in color sports with less anthocyanin. Three of the flavonols were absent from a few of the cultivars but most differences between cultivars were only quantitative. Genetic data suggested independent assortment of control of quercetin 3-rhamnoside and quercetin 3-galactoside synthesis but linkage of quercetin 3-rhamnoside and kaempferol 3-rhamnoside synthesis.