西瓜核心种质的AFLP指纹图谱和SCAR标记

西瓜(Citnllus lanatus (Thunb.) Mansf.)种质资源的鉴定与评价是对其有效利用的基础。以往的研究表明,西瓜是一种遗传资源特别狭窄的作物,在用同工酶、RAPD及SSR技术对西瓜种质资源进行鉴定时,发现很难将品种完全区分开来。本研究利用高效可靠的AFLP技术,对30个西瓜核心种质材料进行了遗传分析,最终建立了这30个材料的DNA指纹图谱。在该图谱中,每个材料均有其独特的“指纹”,材料之间可以相互区分开来。为了进一步利用AFLP分子标记,将重要抗病种质材料“P1296341”的AFLP特异带转化成了生产上可以直接利用的SCAR标记。     

红豆杉种质资源遗传多样性的AFLP分析

目的：通过对5份红豆杉种质资源的AFLP分析,探求各种质间的遗传多样性。方法：采用扩增片段长度多态性（AFLP）标记,在DNA水平上进行遗传多样性研究,筛选了32对选择性扩增引物,将扩增出的条带作为原始矩阵,用NTSYS-PC软件计算并分析了红豆杉种质间的相似度,构建了遗传系统进化树。结果：（1）SDS法提取的红豆杉基因组DNA质量较佳,能够满足AFLP分析的要求;（2）从32对选择性扩增引物中,筛选出10对多态性较强、带型较好、分辨率较高的组合;（3）构建了红豆杉AFLP指纹图谱,将5个红豆杉种质全部区分开来;（4）通过构建进化树,把5个种质分成3类。结论：红豆杉种质资源有丰富的遗传多样性。     

用EST-SSR分子标记技术构建大白菜核心种质及其指纹图谱库

利用EST-SSR分子标记对大白菜种质资源基因库中686份样品所代表的1 900份大白菜种质资源进行分析研究.构建大白菜种质资源的核心种质并且形成核心种质的EST-SSR指纹图谱库.结果表明利用4组鉴定白菜品种的EST-SSR的特异性标记组合,获得近158个EST-SSR多态的标记,对大白菜种质资源基因库中686份样品所代表的1 900份大白菜种质资源进行核心种质的构建提供了分析数据.形成的核心种质包括168份样品,占库存资源的8.8%,它的多态位点百分率保持了原群体的100%.所构建的核心种质涵盖了原资源的绝大部分区域来源的品种,包含了早、中、晚熟品种中所有典型的大白菜类型和其相关的特征特性.并进一步进行了核心种质资源遗传多样性分析.核心种质的EST-SSR指纹图谱库中,每一份样品的指纹都是唯一的,为登记、评价、整理、分发、繁殖等种质资源库的管理和育种者对其材料的利用提供了重要的有价值的信息.EST-SSR标记组合是构建中国大白菜核心种质及其指纹图谱的经济、高效的方法.     

应用SRAP标记绘制88份南瓜属种质资源DNA指纹图谱

为了给南瓜属种质资源鉴定和分类提供分子生物学依据,本研究采用SRAP分子标记技术与DNAMAN指纹图谱绘制软件对88份南瓜属种质资源(包含美洲南瓜、中国南瓜、印度南瓜)进行分子指纹图谱绘制。结果表明:35对SRAP多态性引物共扩增出499条清晰条带,其中多态性条带438条,多态性条带比率高达87.8%。根据扩增出的条带成功绘制出88份南瓜属种质资源的DNA指纹图谱,每一份种质都具有其独特的分子身份证,使得每份种质均可被区别开来。其中,多态性最好的引物是E5EM8,可以同时绘制72份南瓜属种质资源的指纹图谱。所有供试材料用5对多态性SRAP引物即可全部区别开来。研究表明,SRAP分子标记技术可成功地绘制南瓜属种质资源DNA指纹图谱。本研究对南瓜属种质资源鉴别、分子数据库构建及品种权保护具有较重要的意义。     

利用AFLP技术鉴定凤凰单丛古茶树种质资源

本文采用AFLP技术对34个凤凰单丛古茶树资源进行了种质鉴定分析.结果显示,筛选出多态性较高的5对引物中,引物组合E41M42、E41M39和E41M33可鉴别出供试的全部资源,鉴别效率为100%;有23份资源具有特异带,占供试材料的67.6%.由此研究表明,AFLP技术可以通过特异带、特异的谱带类型、不同引物提供谱带的组合将风凰单丛古茶树资源区分开来,这对保护凤凰单丛古茶树资源的育种产权、登录新品种、鉴定和检测其种子与苗木的真实性和纯度具有重要的现实意义.     

水生实验动物剑尾鱼的DNA指纹图谱构建

目的剑尾鱼是由原良种委员会审定并由农业部公布的水生实验动物,在遗传学研究、水环境污染监测、细菌性疾病研究方面显示出较好的应用前景。为了对剑尾鱼选育系进行种质资源监测、区分选育系与非选育以及鉴定近交系纯度,本研究采用微卫星DNA进行水生实验动物剑尾鱼的指纹图谱构建。方法根据相关报道设计合成了50对微卫星引物,对几个剑尾鱼品系的种质资源进行检测,筛选品系间差异性引物;确立剑尾鱼核心引物,用EXCEL散点图绘制DNA指纹图谱模式图;并将数字化指纹数据输入珠江水产研究所鱼类种质鉴定软件V1.0,形成剑尾鱼标准化指纹图谱鉴定数据库。结果共获得剑尾鱼品系间特异标记5个可用于剑尾鱼近交系鉴定,确立46个微卫星标记为核心引物,构建剑尾鱼选育系RR-B系、RW-H系和非选育的野生品种的DNA指纹图谱。结论本研究筛选出的微卫星标记与构建的指纹图谱,可用于剑尾鱼3个品种间的品种鉴定、纯度检测及遗传监测。     

基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立

利用TP-M13-SSR分子标记方法,构建27份中国原产苹果属植物在12个SSR位点的指纹图谱,运用条码技术生成其分子身份证。12对引物共获得251个等位基因,平均21个。引物多态性好,仅用引物CH05b06即可区分全部供试材料。27份苹果材料在12个SSR位点遗传多样性、多态性信息含量和位点杂合度的变化范围为0.6620～0.9455、0.6327～0.9211和0.6538～0.9319。基于CH05b06位点处获得的指纹谱图即可得到每份供试材料独有的分子身份证。TP-M13-SSR分子标记技术适用于苹果属植物种质资源的指纹图谱构建,利于分子基础数据库的积累。基于苹果种质资源TP-M13-SSR指纹图谱可获得每份苹果种质资源独有的分子身份证。     

武夷山及周边地区黄精植物ISSR分子标记鉴定及HPLC指纹图谱研究

采用ISSR分子标记鉴定方法及HPLC色谱方法对武夷山及周边地区5份黄精植物样本进行DNA分子标记鉴定及指纹图谱分析,并通过聚类分析软件分别探讨其遗传相关聚类图谱。ISSR分子标记法能较好地对5份黄精植物样本进行区分,HPLC指纹图谱分析同样表明5份黄精样本化学成分存在一定的差别。DNA分子标记及HPLC指纹图谱分析的遗传相关聚类分析结果相似,表明武夷山及周边地区的野生黄精种质资源存在明显的差别。     

新疆枸杞种质资源遗传多样性分析及DNA指纹图谱构建

利用SCoT分子标记对新疆枸杞种质资源进行遗传多样性分析和DNA指纹图谱构建,为杂交育种和种质鉴定提供理论依据。结果显示:9条SCoT引物扩增出条带256条,其中219条为多态性条带,多态性比率达85.62%,多态性信息含量(PIC)值变化范围在0.77~0.91之间,平均值为0.85,观测等位基因数(Na)、有效等位基因数(Ne)、Nei's基因多样性指数(H)和Shannon信息指数(I)的平均值分别为1.8562、1.4350、0.2611、0.3989,聚类分析表明,遗传相似系数变化范围在0.5938~0.8398之间,在遗传相似系数为0.66和0.71处,可将30份材料分别分为2大类和4个亚类,主坐标分析结果和聚类结果基本一致,同时利用5条多态性SCoT引物构建了30份材料的DNA指纹图谱。新疆枸杞种质资源遗传多样性水平较高,且SCoT分子标记适于新疆枸杞种质资源遗传多样性分析和DNA指纹图谱构建,该研究结果为新疆枸杞种质资源评价、鉴定和新品种选育奠定了基础。     

应用SRAP标记构建山药种质资源DNA指纹图谱

利用30对多态性良好的SRAP引物对90份山药种质资源进行PCR扩增,构建扩增图谱,共扩增到722个位点,多态性位点581个,多态性比例为80.47%,每对引物组合检测多态性位点3~30个,每对引物能鉴别6~51份山药种质资源;采用DNA数据分析软件对扩增出的多态性位点进行分析,构建山药种质资源方框指纹图谱,该图谱清晰地反映出每对引物能扩增的多态位点数、鉴别资源份数及资源具体编号,资源在该引物组合下所能检测得到的多态位点数及所处的具体位置等信息;从10对SRAP引物组合中挑选出的21个多态性位点,根据谱带的有无转化成的1/0字符串编码,形成山药种质资源DNA数字指纹图谱,该图谱可鉴别区分90份山药种质资源中的82份资源。同时这些指纹图谱可为下一步的山药品种鉴定,种质资源评价、利用,分子标记辅助育种及品种权保护提供技术支撑。     

花生黄曲霉侵染抗性的SCAR标记

利用与花生黄曲霉侵染抗性基因紧密连锁的AFLP标记 “E45/M53-440”, 经PAGE凝胶电泳后回收、克隆、测序, 并根据测序结果设计PCR特异引物, 通过对PCR条件的优化, 成功地将AFLP标记“E45/M53-440”转化为实验结果稳定, 操作更简单的SCAR标记“AFs-412”, 标记与花生黄曲霉侵染抗性间的遗传距离为6.5 cM。利用获得的SCAR标记对抗、感黄曲霉的花生种质资源进行了分子鉴定, 结果表明标记与抗性鉴定结果具有较高的一致性, 证实了该标记应用于研究群体之外的育种潜力。SCAR标记的建立为开展花生黄曲霉侵染抗性的标记辅助选择育种提供了简便实用的鉴定技术。     

几种分子标记方法相结合建立的新型分子标记方法

介绍了几种分子标记相结合,产生的分子标记新方法的基本原理、优缺点,如AFLP、SCAR、CAPS以及RMAPD分子标记。其中主要介绍了RMAPD,对RMAPD作为一种新型分子标记进行探讨。     

筛选与尼罗罗非鱼性别相关的AFLP标记

尼罗罗非鱼(Tilapia)隶属于鲈形目(Perciformes)、鲈形亚目(Percoidei)、丽鱼科(Cichildae)的热带性鱼类,是联合国粮农组织向全世界推广的优良养殖鱼类,已成为世界性的主要养殖对象之一。由于繁殖快、成熟早,养殖过程中极易造成繁殖过剩,密度过大,个体过小等不利情况;另外,尼罗罗非鱼的雌雄个体之间具有明显的生长差异,从而影响产量的提高。目前解决这一问题最为理想的措施是单性养殖全雄鱼,这样既可以防止过度繁殖,又可利用雄鱼的生长优势。但是由于缺乏性别或性染色体特异的分子遗传标记,遗传性别的准确鉴定问题也一直是罗非鱼类性别控…     

Development of a species-specific AFLP-based SCAR marker for authentication of black muntjac (Muntiacus crinifrons)

Black muntjac is a rare and endangered deer endemic to eastern China. Due to the economic and pharmaceutical value of the meat, antlers and skin, the species has chronically suffered from poaching though it is regarded as the state key protected animal. To provide an effective molecular method for authentication of tissue specimen (such as meat, skin etc.) of the species, we developed a Sequence Characterized Amplified Region (SCAR) derived from a species specific Amplification Fragment Length Polymorphism (AFLP) marker. Initially, a 707-bp species specific DNA fragment of the animal was detected by a pair of AFLP primers (Ep7/Mp8). Subsequently, a species-specific primer pair (P-F/P1-R) was designed based on the specific AFLP fragment sequence, obtaining a 298-bp SCAR for the species. Finally, the reliability of the SCAR primers was verified by two separate PCRs using the designed SCAR primers and a cyt b universal primer pair. As expected, all black muntjac samples presented two bands but the others failed to produce the SCAR by merely showing one band. Our results indicated that the SCAR primers developed in this study may provide a useful tool for forensic authentication of black muntjac samples though further testing with larger sample sizes is warranted.     

Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations

Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish ( Pelteobagrus fulvidraco ), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62 . Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish.     

Cloning,characterization of two female-specific AFLP markers and development of PCR-based sex identification method for the half-smooth tongue sole Cynoglossus semilaevis

Two female-specific AFLP(amplified fragment length polymorphism)markers(named CseF464 and CseF136)were isolated by using one selective primer combination(E-AGC/M-CTG)from the genomic DNA of 20 females and 20 males of the half-smooth tongue sole Cynoglossus semilaevis.Both the markers were re-amplified,recovered from the agarose gels,cloned and sequenced.Bioinformatics analysis indicated that the length of the two markers were 468 bp and 134 bp,respectively,and the sequences showed no similarity to each othe...     

Identification of AFLP markers in soybean linked to resistance to Meloidogyne javanica and conversion to Sequence Characterized Amplified Regions (SCARs)

Meloidogynejavanica is the most widely spread nematode pest on soybean in SouthAfrica. Only a few registered commercial South African cultivars are poor hostsof this nematode species and there is an urgent need for an efficient breedingprogramme for resistant cultivars of all maturity groups. However, breeding ishampered by laborious screening procedures for selection of poor host cultivarsand/or lines. The objective of this study was to develop an economically viablemolecular marker system for application in selection procedures. BothRestriction Fragment Length Polymorphism (RFLP) and Amplified Fragment LengthPolymorphism (AFLP) screening techniques identified markers linked togall-indexvariation in a segregating population of 60 F₂ progeny from a crossbetween a resistant cultivar (Gazelle) and a highly susceptible variety(Prima).A codominant RFLP marker( B212) was linked significantly to M.javanica resistance and explained 62% of the variation ingall-index.Seven AFLP markers were linked significantly to the resistance trait, of whichfour were linked in repulsion phase and three in coupling phase. All seven AFLPmarkers mapped to LG-F (Linkage Group F) on the public soybean molecular map.The major quantitative trait locus (QTL) for resistance mapped between markersE-ACC/M-CTC2(SOJA6) (linked in coupling phase), B212 and E-AAC/M-CAT1(SOJA7)(linked in repulsion phase). These two AFLP markers bracketing the majorresistance QTL were successfully converted to SCARs (Sequence CharacterizedAmplified Regions). Marker E-ACC/M-CTC2 was converted to a codominant SCARmarker SOJA6, which accounted for 41% of variation in gall-index in the mappingpopulation. Marker E-AAC/M-CAT1 was converted to a dominant SCAR marker (SOJA7)and explained 42% of gall-index variation in the mapping population. These twomarkers mapped approximately 3.8 cM and 2.4 cMrespectively from the resistance QTL. This study represents the first report ofthe development of PCR-based sequence specific markers linked to M.javanica resistance in soybean.     

Mode of reproduction is detected by Parth1 and Sex1 SCAR markers in a wide range of facultative apomictic Kentucky bluegrass varieties

Gametophytic apomixis in Kentucky bluegrass (Poa pratensis L.) involves the parthenogenetic development of unreduced eggs from aposporic embryo sacs. Marker-assisted selection for the mode of reproduction in P. pratensis would avoid costly and time-consuming phenotypic progeny tests. We developed and tested two SCAR primer pairs that are associated with the mode of reproduction in P. pratensis. The SCAR primers identified the apomictic and sexual genotypes among progenies of sexual x apomictic crosses with very low bias. Furthermore, when tested on a wide range of Italian and exotic P. pratensis germplasm, they were able to unequivocally distinguish sexual from apomictic genotypes. This system should, therefore, allow new selection models to be set up in this species.     

Narrowing down the region of the<Emphasis Type="Italic"> Vf</Emphasis> locus for scab resistance in apple using AFLP-derived SCARs

A narrow-down strategy to restrict the Vf region, which controls resistance to the fungal disease apple scab in apple, to a genetic distance of 0.4 cM is presented. Using 11 AFLP-derived SCARs and three RAPD-derived SCARs, all linked to the Vf gene, we subjected 1,412 scab-resistant individuals from 16 mapping populations to genotype analysis. Eleven recombinant individuals were identified within a genetic distance of 0.9 cM around the Vf gene. Using these 11 recombinants, we achieved fine-resolution of several AFLP-derived SCAR markers surrounding the Vf gene, resulting in the following genetic linkage map: ACS-6 and ACS are located left of the Vf gene at genetic distances of 0.2 cM and 0.1 cM, respectively; ACS-7 and ACS-9 are inseparable from the Vf gene; ACS-8, ACS-10, and ACS-4 are located to the right of the Vf gene at genetic distances of 0.1 cM, 0.4 cM, and 0.5 cM, respectively; the remaining five SCARs—ACS-11, ACS-5, ACS-2, ACS-1, and AL07—are inseparable and are located right of the Vf gene at a genetic distance of 0.7 cM. By integrating this linkage data with our previous physical map, we generated a revised map of the narrowed-down region of Vf.Communicated by P. Langridge