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。     

一条卡瓦胡椒特异RAPD带转化成SCAR标记的研究

采用27份不同来源的胡椒属(Piper)材料和1份不同属的草胡椒(Peperomia pellucida)材料用引物OPQ-03扩增得到一条约400碱基对(bp)卡瓦胡椒特异片段。对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequence characterized amplifiedre-gions,序列特征化扩增区)分子标记。本研究设计出了1对卡瓦胡椒特异SCAR引物P7.1(5′-GGT CAC CTC ACC GCA GCA GGA TGA ACG-3′)和P7.2(5′-GGT CAC CTC AAT GAC ATG GGA TGA ATC-3′),用这对特异引物对本次试验的28份材料进行PCR扩增,结果只有不同属的草胡椒材料无任何扩增,其它材料均扩增出了预期大小440bp的特异带。     

一条卡瓦胡椒特异RAPD 带转化成SCAR 标记的研究

采用27 份不同来源的胡椒属( Piper) 材料和1 份不同属的草胡椒( Peperomia pellucida) 材料用引物OPQ-03 扩增得到一条约400 碱基对( bp) 卡瓦胡椒特异片段。对该片段进行了克隆和序列分析, 并根据序列分析结果将上述RAPD 分子标记转化为重复性和特异性更好的SCAR ( sequence characterized amplified regions, 序列特征化扩增区) 分子标记。本研究设计出了1 对卡瓦胡椒特异SCAR 引物P7. 1 ( 5′-GGT CAC CTC ACC GCA GCA GGA TGA ACG-3′) 和P7 . 2 (5′-GGT CAC CTC AAT GAC ATG GGA TGA ATC-3′) , 用这对特异引物对本次试验的28 份材料进行PCR 扩增, 结果只有不同属的草胡椒材料无任何扩增, 其它材料均扩增出了预期大小440 bp 的特异带。     

卡瓦胡椒及胡椒的RAPD聚类分析

目的：通过RAPD分析搞清楚卡瓦胡椒与胡椒及胡椒属其它近缘野生种的亲缘关系。方法和结果：采用随机引物80条进行筛选,用从80条随机引物中入选的20条引物,对卡瓦胡椒和胡椒属共计28份材料进行RAPD扩增,均能产生清晰的扩增谱带。重复1—2次,结果稳定可靠。20个引物共扩增出170个条带,其中多态性条带有20条,占总扩增条带数的12％。RAPD分析结果显示在相似系数0．36处对28份种质可划分为6个类型,其中卡瓦胡椒被单独聚为一类,说明卡瓦胡椒与胡椒及其它近缘野生种的亲缘关系有一定的距离。     

卡瓦胡椒RAPD反应体系的建立

卡瓦胡椒RAPD分子标记的研究,目前国内外尚未见有报道。该试验通过CTAB法提取卡瓦胡椒基因组DNA,通过对模板DNA用量、Mg^2＋浓度、退火温度、电泳上样量等几个单因子试验来建立RAPD稳定扩增体系和反应条件,RAPD扩增结果重复性好,稳定可靠,为卡瓦胡椒RAPD分子标记的研究打下基础。     

SCAR标记在茶树种质资源鉴定中的应用

SCAR标记是一种在RAPD技术的基础上发展起来的新型分子标记技术,提高了分子标记辅助选择育种的效率,在茶树种质资源的合理开发与利用中具有广阔的应用前景.运用优化后的RAPD反应体系对10个茶树品种的基因组DNA进行遗传差异分析,随机引物S89、S4分别在白毫早和福云6号中扩增得到长度为498 bp、1 622 bp的差异片段,命名为BHZ498、FY1622.根据它们的测序结果分别设计了一对特异引物,BHZ498的特异引物为SB1/SB2;FY1622的特异引物为SC1/SC2,用这两对特异引物对10个茶树品种的基因组DNA进行扩增.引物SB1/SB2和SC1/SC2分别在白毫早和福云6号中扩增出唯一的一条扩增带,而这两对引物在其他供试茶树材料中均无相应的扩增带,结果表明已将BHZ498、FY1622标记成功转化成SCAR标记.     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示:原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示：原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

5种杂交粳稻亲本SCAR标记的建立及其在真伪纯度鉴定中的应用

选用36个随机引物对"寒丰A"、"寒丰B"、"8204A"、"8204B"、"R161"等5份杂交粳稻亲本材料进行RAPD扩增,对其中特异RAPD标记片段进行克隆和测序.根据获得的特异DNA序列设计序列特征扩增区(SCAR)特异的引物,将18个RAPD标记转化成6个稳定的SCAR标记.用这些SCAR标记对亲本和杂种F1代单株进行检测,实验室检测种子纯度的结果与海南田间种植的结果基本一致.此外,应用水稻细胞质雄性不育特异的1对PCR引物,分辨出2对不育系/保持系亲本:"寒丰A"与"寒丰B"、"8204A"与"8204B".     

Study on the RAPD Specific Band of Kava ( Piper,Piperaceae) Transferring to the SCAR Molecular Marker

This paper studied on 28 pepper materials, including 16 cultivated papper materials, 3 wild pepper materials, 2 wild relatives of pepper materials , 6 kava ( Piper methysticum) materials, and 1 Peperomia pellucida materials. According to RAPD analysis, we generate SCAR marker for identifying Kava ( Piper, Piperaceae ) . A Kava-associated fragment with a length ofabout 400 bp was generated with OPQ-03 primer . The fragment was cloned and sequenced . PCR amplification with the specific primers P7 . 1 ( 5′-GGT CAC CTC ACC GCA GCA GGA TGA ACG-3′) and P7 .2 (5′-GGT CAC CTC AAT GAC ATG GGA TGA ATC-3′) was performed to 28 materials , which 27 materials amplified the 440 bp specific band except for Peperomia pellucida Kunth .     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Molecular studies on mungbean (Vigna radiata (L.) Wilczek) and ricebean (Vigna umbellata (Thunb.)) interspecific hybridisation for Mungbean yellow mosaic virus resistance and development of species-specific SCAR marker for ricebean

The aim of this study was to identify the molecular markers (SSR, RAPD and SCAR) associated with Mungbean yellow mosaic virus resistance in an interspecific cross between a mungbean variety, VRM (Gg) 1 X a ricebean variety, TNAU RED. The parental survey was carried out by using 118 markers (including 106 azuki bean primers, seven mungbean primers and five ricebean primers). This study revealed that 42 azuki bean markers (39.62%) and four mungbean markers (54.07%) showed parental polymorphism. These polymorphic markers were surveyed among the 187 F₂ plants and the results showed distorted segregation or chromosomal elimination at all the marker loci (thus, deviating from the expected 1:2:1 segregation). None of the plants harboured the homozygous ricebean allele for the markers surveyed and all of them were skewed towards mungbean, VRM (Gg) 1, allele, except a few plants which were found to be heterozygous for few markers. Among the 42 azuki bean SSR markers surveyed, only 10 markers produced heterozygotic pattern in six F₂ lines viz. 3, 121, 122, 123, 185 and 186. These markers were surveyed in the corresponding F₃ individuals, which too skewed towards the mungbean allele. In this study, one species-specific SCAR marker was developed for ricebean by designing primers from the sequenced putatively species-specific RAPD bands. A single, distinct and brightly resolved band of 400?bp was found amplified only in the resistant parent, TNAU RED, and not in any other six species or in the resistant or the susceptible bulks of the mapping population clearly indicated the identification of SCAR marker specific to the ricebean.     

An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F₁ females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked. Received: 16 August 1999 / Accepted: 27 December 1999