卡瓦胡椒SCAR标记的研究

目的：采用6份卡瓦胡椒材料、21份栽培胡椒和野生胡椒材料、1份不同属的草胡椒材料共计28份试验材料,开发1对特异SCAR引物。方法：在对它们进行了RAPD研究的基础上,通过克隆、测序和引物设计进行了SCAR分子标记研究。结果：本研究开发了1对特异SCAR引物P10．1和P10．2,用这对特异引物对本次试验的28份材料进行PCR扩增,结果显示,6份卡瓦胡椒材料扩增出了三条带,三条带离的较近,中间一条为预期494bp特异片段。其它胡椒属材料均扩增出一条494bp的特异带,而不同属的草胡椒无任何扩增。结论：这说明引物P10．1和P10．2适用于卡瓦胡椒的分子鉴定(三条带),也可用于胡椒属植物的分子鉴定(一条带),这对卡瓦胡椒种质资源的真伪鉴定及胡椒属植物的分子分类有一定帮助。     

Effect of Development Stage on the Artemisinin Content and the Sequence Characterized Amplified Region （SCAR） Marker of High-Artemisinin Yielding Strains of Artemisia annua L.

The effects of development states on the artemisinin content of clone S1 of Artemisia anuua L. grown in a greenhouse were investigated in the present study. The artemisinin content increased gradually during the phase of vegetative growth and reached its highest level at 8-9 mg/g dry weight （DW） when the S1 was 6 months old on a long day （LD） photoperiod. Treatment with 9-18 d of short day （SD） photoperiod resulted in the artemisinin content reaching and being maintained at a higher level （2.059-2.289 mg/g DW）, twofold that of control plants and plants of S1 presented at the pro-flower budding and flower-budding stages. The artemisinin content varied in different parts of the plant. The artemisinin content of leaves was higher than that of florets and branches. The artemisinin content in middle leaves was higher than that of bottom leaves, and then top leaves. Different densities of capitate glands （the storage organ of artemisinin） located on the surface of leaves, florets, and branches explained the variations in artemisinin content in these parts of the plant. The correlation coefficient between artemisinin content and density of capitate glands on the surface of different organs was 0.987. The genetic marker for artemisinin content was screened using random amplified polymorphic DNA （RAPD） and sequence characterized amplified region （SCAR） techniques. The random primer OPAl5 （5＇-TTCCGAACCC-3＇） could amplify a specific band of approximately 1 000 bp that was present in all high-artemisinin yielding strains, but absent in all low-yielding strains in three independent replications. This specific band was cloned and its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain a more stable marker.     

Molecular characterization of the SCAR markers tightly linked to the Tm-2 locus of the genus Lycopersicon

The Tm-2 gene and its alleles conferring tomato mosaic virus resistance in tomato originate from Lycopersicon peruvianum, a wild relative of tomato. DNA fragments of several RAPD markers tightly linked with the Tm-2 locus in tomato were successfully cloned and sequenced. Subsequently, the 24-mer oligonucleotide primer pairs of the SCAR markers corresponding to the RAPD markers were designed based on the 5’-endmost sequences. A fragment of the same size as that of a SCAR marker was amplified in the ToMV-susceptible tomato line with no Tm-2, but the digests of the PCR fragments by AccI exhibited polymorphism in fragment length between the two lines. We chose three SCAR markers and three RAPD markers tightly linked with the Tm-2 locus, and examined whether the same-sized fragments corresponding to these markers were also present in three other lines carrying Tm-2a or one of the other Tm-2 alleles. The fragments corresponding to the three SCAR markers were present in all of the three lines, but the other markers (three RAPDs ) were absent in one or two lines, suggesting that the three SCAR markers are closer to Tm-2 than the other markers. Comparison of the nucleotide sequences of these fragments revealed that they are all homologous to the corresponding SCAR markers. Received: 8 November 1999 / Accepted: 15 November 1999     

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria dispar L.是世界性农林害虫, 包含不同的亚种, 其中亚洲舞毒蛾的雌蛾具有较强的飞行能力, 已成为国际性的重要检疫性有害生物。然而, 不同舞毒蛾亚种及种群间形态难辨, 因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明, 所检测的舞毒蛾种群的遗传分化系数G_st为0.7571, 由此推算出的平均有效迁移数（基因流参数）N_em为0.1604, 说明不同舞毒蛾种群间的遗传分化程度较高, 缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上, 筛选出了4个舞毒蛾种群的特异性遗传位点, 然后对这些特异性位点进行了克隆测序、 序列分析和位点特异性引物设计。结果表明, 其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域（SCAR）标记。经验证, 这些标记可被用来鉴别特定的舞毒蛾地理种群, 因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。     

A molecular marker that segregates with sorghum leaf blight resistance in one cross is maternally inherited in another

Leaf blight-resistant sorghum accession SC326-6 was crossed to the susceptible cultivar BTx623 to analyze the genetic basis for resistance. Field scoring of inoculated F₂ progeny revealed that resistance was transmitted as a dominant single-gene trait. By combining the random amplified polymorphic DNA (RAPD) technique with bulked-segregant analysis, it was possible to identify PCR amplification products that␣segregated with disease response. Primer OPD12 amplified a 323-bp band (D12R) that segregated with resistance. Creation of longer primers, or SCARs (sequence characterized amplified regions) for D12R resulted in the amplification of a single major band of the predicted size from all the resistant F₂ progeny and the resistant parent SC326-6, but not from BTx623 or 24 of 29 susceptible F₂ progeny. The SCAR primers also amplified a single band with DNA from IS3620C, the female parent in a cross with BTx623 that has been used to produce a recombinant inbred population for RFLP mapping. An equivalent band was amplified from all 137 recombinant inbred progeny, indicating that organelle DNA is the amplification target in this cross. Received: 31 July 1998 / Accepted: 23 November 1998     

条形柄锈菌33号生理小种的分子标记

采用RAPD-SCAR分子标记技术,从300条RAPD随机引物中筛选到了对条形柄锈菌Puccinia striiformis f. sp. tritici 33号生理小种特异的2条引物,将特异性片段回收、克隆和测序后（GenBank注册号为AB914691和AB914692）,依据其序列设计出了2对引物S261F33/S261R33和S300F33/S300R33,能够特异性地从33号生理小种基因组DNA及发病小麦叶片总DNA中分别扩增出247bp和763bp的片段,其结果与采用常规的鉴别寄主法鉴定的结果一致。因此,这2对引物都可用于条形柄锈菌33号生理小种的快速鉴定与监测。     

三种摇蚊幼虫RAPD扩增条件的优化及在昆虫系统发育分析中的应用

为了研究摇蚊科昆虫种群遗传的多样性,以促进对其资源的合理保护,以萨摩亚摇蚊Chironomus samoensisEdwards基因组DNA为模板,对摇蚊幼虫的RAPD扩增条件进行优化,建立了摇蚊幼虫RAPD扩增反应的最佳体系:按照利用优化的RAPD扩增条件进行研究,实验有着良好的重现性。用16个随机引物对3种摇蚊幼虫类群各10个个体进行RAPD扩增,其中萨摩亚摇蚊共扩增出78个条带,多态座位率为41.03%,Shannon遗传多样性指数为0.2570,群体内相似度为0.8730;红裸须摇蚊Propsilocerus akamusi(Tokunaga)共75个条带,多态座位率为44.0%,Shannon遗传多样性指数为0.2472,群体内相似度为0.8731;刺铗长足摇蚊Tanypus punctipennis(Fabricius)共67个条带,多态座位率为41.79%,Shannon遗传多样性指数为0.1943,群体内相似度为0.9066。聚类分析结果表明,刺铗长足摇蚊与红裸须摇蚊的亲缘关系较近。     

pms3 is the locus causing the original photoperiod-sensitive male sterility mutation of‘Nongken 58S'

Photoperiod-sensitive genic male sterile (PSGMS) rice is a very useful germplasm for hybrid rice development. It was first found as a spontaneous mutant in a japonica cultivar 'Nongken 58'. pms3 on chromosome 12 was determined to be the locus where the original PSGMS mutation occurred, changing the normal cultivar Nongken 58 to PSGMS Nongken 58S. Large amounts of RAPD and AFLP analyses were also conducted for the fine mapping of the pms3 genomic region, which resulted in 4 molecular markers linked to pms3. Although these markers somewhat increased the marker density of this region, the pms3 locus is still located in a marker-sparse region.     

Estimation of Genetic Variability in Plantago ovata Cultivars

Five cultivars of Plantago ovata Forsk. (medicinal plant) have been developed by different agricultural universities in India. Genetic variability of these cultivars was estimated using RAPD markers. The data were correlated to morphological characters and a dendrogram was obtained from Jaccard's coefficient. This revised version was published online in July 2006 with corrections to the Cover Date.     

应用随机PCR方法鉴定一株真养产碱杆菌

采用随机引物PCR技术从新建细胞培养室空气中获得一段长414bp的片段,通过克隆测序及序列分析,结果表明所测序列与真养产碱杆菌主要参考菌株的同源性分别高达79%-83%,由其推导的氨基酸序列与真养产碱杆菌主要参考菌株的同源性高达86.4%-89.1%,从而确定所分离菌株为真养产碱杆菌。     

Bassam和Sanguinetti银染方法在SRAP和TRAP标记中的比较研究

银染作为一种重要的DNA染色方法,对分子标记检测有重要影响.SRAP标记和TRAP标记是两种较为新型的分子标记,近年来得到了广泛应用,尤其在缺少遗传图谱的物种中应用价值更大.以普通烟草种(Nicotiana tabacum L.)烤烟和香料烟品种作为供试材料,对Bassam和Sanguinetti两种银染方法,在标记检测效率、成本及染色效果等方面进行了研究,并对其在SRAP标记和TRAP标记中的应用进行了探讨.结果表明,Sanguinetti银染法比Bassam银染更为简便、经济,染色照片的色阶图说明Sanguinetti染色方法的背景与扩增带区分明显,能够清楚地读带;且该方法能够扩增出很好的SRAP和TRAP标记谱带.因此,推荐在SRAP和TRAP标记检测中采用Sanguinetti银染方法.     

A molecular study of hybridization and homoploid hybrid speciation in Argyranthemum (Asteraceae) on Tenerife,the Canary Islands

Examples of recurrent homoploid hybrid speciation are few. One often‐cited example is Argyranthemum sundingii. This example includes two described species, A. lemsii and A. sundingii, resulting from reciprocal hybridization between A. broussonetii and A. frutescens on Tenerife. The four species and artificial F₁ and F₂ hybrids have previously been investigated morphologically and cytologically. Here, we examine population differentiation based on amplified fragment length polymorphism to get a better understanding of the genetic relationships among the species and the extent of hybridization. We aim to investigate if there is molecular support for treating the hybrid species as one taxon. Seven parental and four hybrid species populations (149 individuals) were analysed and we scored 85 polymorphic markers. A few (2–5) were private to each species but variably present and mostly rare. Our principal coordinate, STRUCTURE and BAPS analyses and AMOVA resulted in a clear separation of the parental species. The hybrid species were genetically less divergent but not identical. Our data indicate that hybridization and introgression are common in all these species on Tenerife and support the hypothesis that homoploid hybrid speciation has occurred repeatedly. Intrinsic post‐zygotic barriers are notoriously weak in Argyranthemum and reproductive isolation and speciation result primarily from strong ecological selection. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 19–31.     

华北地区小丛红景天种群的AFLP遗传多样性

利用扩增片段长度多态性(AFLP)标记, 对分布于华北地区5个山脉的25个小丛红景天(Rhodiola dumulosa)自然种群的776个样品进行了遗传多样性和遗传结构的研究。结果表明: 华北地区小丛红景天种群具有较高的遗传多样性, 4对选择扩增引物共扩增出398条清晰的条带, 其中多态带312条, 种群的平均多态位点百分率为78.46%, 种群总的Nei’s基因多样性为0.364 9, 总Shannon多态性信息指数为0.542 2。华北地区小丛红景天种群间的遗传分化系数G_st = 0.150 7, 基因流Nm = 2.817 9, 表明种群间遗传分化较低, 有一定的基因交流。AMOVA分析结果也表明: 华北地区小丛红景天的遗传变异主要存在于种群内, 地理单元间有一定的遗传分化, 而种群间的遗传分化较低。STRUCTURE的分析和UPGMA聚类分析结果一致, 结果显示地理分布距离相近的种群优先聚在一起。Mantel检验也进一步证实, 华北地区小丛红景天种群的遗传距离与地理距离间呈显著的正相关关系(r = 0.512 9, p < 0.001)。种群的遗传多样性与海拔呈显著的负相关关系(p < 0.05), 而与坡向没有显著相关性。用Dfdist软件分析海拔对遗传多样性的影响, 结果表明没有显著的受选择位点。     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

非损伤性取样在朱?种群遗传研究中的应用

采用非损伤性取样方法提取DNA,利用20条10bp的随机引物对陕西朱?保护观察站的16只朱?个体间遗传距离进行分析研究。获得的DNA可进行RAPD研究分析,18条引物扩增结果稳定,遗传距离指数介于30.52%～10.18%之间;揭示16只朱?间亲缘关系较近。尝试在朱?种群遗传学研究中应用非损伤性方法抽提基因组DNA,以方便对朱?等小种群珍稀濒危生物进行相关研究。 Abstract：Genetic distance of sixteen crested ibises (Nipponia nippon) was analyzed at the DNA level by polymerase chain reaction (PCR) using random primers after DNA was sampled with non-invasive sampling. The amount of DNA was enough to apply t o genetic analysis by RAPD. Eighteen primers had stable effect. The genetic dist ances are between 30.52% and 10.18%,which showed that the relationship between 16 crested ibises was similar. This study tried to apply a non-invasive method to obtain DNA, which is helpful to research on the genetic background of creste d ibises and other tiny population based on DNA level.     

Markers derived from amplified fragment length polymorphism gels for plant ecology and evolution studies

We describe the types of polymerase chain reaction (PCR) markers that we have isolated using amplified fragment length polymorphisms (AFLP) in closely related taxa from diverse plant genera. With these markers, both inter- and intraspecific differences have been identified. The characterization of the nucleotide sequences and fragment length polymorphisms of such AFLP-derived PCR markers is promising for investigating the ecology and evolution of closely related plant taxa.     

卡瓦胡椒SCAR分子标记的研究

目的:采用6份卡瓦胡椒材料、21份栽培胡椒和野生胡椒材料1、份不同属的草胡椒材料共计28份试验材料,开发1对特异SCAR引物。方法:在对它们进行了RAPD研究的基础上,通过克隆、测序和引物设计进行了SCAR分子标记研究。结果:研究开发了1对卡瓦胡椒特异SCAR引物P4.1和P4.2,用这对特异引物对试验的28份材料进行PCR扩增,结果只有6份卡瓦胡椒材料扩增出了预期大小562bp的特异带,其它材料均无任何扩增。结论:这说明引物P4.1和P4.2为卡瓦胡椒特异SCAR引物,可用于卡瓦胡椒的分子鉴定,这对卡瓦胡椒种质资源的真伪鉴定有一定帮助。     

新型标记SRAP在棉花F2分离群体及遗传多样性评价中的适用性分析

将新型分子标记SRAP(Sequence-related Amplified Polymorphism)应用于棉花的遗传研究,并建立了完整的PCR反应体系,此体系稳定可靠、扩增效果好、可重复性强。采用30个SRAP引物组合对海岛棉品种“Pima90”和陆地棉品种“邯郸208”进行比较扩增,29个引物组合可以获得多态性扩增,显示了较高的多态性。对上述两个品种的F2群体进行检测,共产生149个多态性条带,平均每个组合产生5．14个,单引物组合最多可产生13个多态性条带。用SRAP标记对11份陆地棉材料进行遗传多样性检测,30个引物组合中15个组合有多态性,得到22个多态性条带,显示了较高的多态性比率。研究结果表明,SRAP标记可在棉花分子生物学领域中广泛应用。     

条形柄锈菌34号生理小种的分子标记

条形柄锈菌Puccinia striiformis f. sp. tritici 34号生理小种(CYR34)是目前我国毒性谱最宽、毒性最强的生理小种,对小麦生产和抗病品种选育造成了极大的影响。本研究采用RAPD-SCAR分子标记技术,从300条RAPD随机引物中筛选到CYR34的特异引物,通过特异性片段回收、克隆和测序(GenBank登录号为OL907303),依据序列设计出了S2008F34/S2008R34特异性引物,能够从CYR34及接种CYR34的小麦发病叶片总DNA中都扩增出417 bp的目标片段。采用该特异性引物检测2021年陕西渭南、咸阳和宝鸡地区小麦条锈菌CYR34的流行频率分别为8.6%、6.0%和10.8%。该项研究为小麦条锈菌CYR34号生理小种的快速检测提供了技术支撑。