应用SRAP标记对莲藕资源的聚类分析

利用一种新的分子标记SRAP技术对17个莲藕品种进行了DNA多态性分析。选取7对引物扩增基因组DNA,共获得168条带,其中159条为多态性条带,每对引物平均提供24个标记信息。由UPMGA方法得到的聚类分析结果表明了17个品种间的遗传关系:(1)亚洲莲与美洲莲之间有明显的差异;(2)在亚洲莲内部,藕莲和花莲有明显的遗传分化,在花莲内部亚洲莲与美洲莲的杂交后代和其它花莲品种之间存在明显的差异;(3)SRAP标记是作分子图谱的好标记,但很难区分遗传关系较近的品种。     

利用ISSR和SRAP标记分析细辛资源遗传多样性与亲缘关系

采用ISSR、SRAP分子标记对61份细辛资源进行遗传多样性与亲缘关系进行分析,结果表明:(1)ISSR标记平均每条引物可获得8.35个DNA片段,多态性比率为86.3%,SRAP标记平均每对引物可获得7.85个DNA片段,多态性比率为86.0%。(2)利用相同数量的引物,ISSR标记揭示的多态性略高于SRAP标记。(3)按照种质间相似系数得出聚类图,可将所有细辛资源分开,在依据ISSR标记聚类分析中,生物学上北细辛和汉城细辛的划分,其作用不如地域来源的效应。SRAP分子标记中,大部分资源的聚类与地域性有关,但有4份汉城细辛优先聚类,SRAP分子标记在揭示基因组差异方面有一定的优势。(4)2种分子标记的聚类图中,来自同一产地的北细辛和汉城细辛优先聚类,其亲缘关系更近。聚类图中未出现北细辛与汉城细辛分别聚类。分子标记分类与传统植物学分类不一致。     

ISSR标记技术在莲藕遗传研究中的运用

利用ISSR技术对 1 2个莲藕品种进行了DNA多态性分析。从 6 5个随机引物中筛选出 7个引物扩增基因组DNA ,共获得 91条带 ,其中 75条为多态性标记 ,每个引物平均提供 1 3个标记信息。由UPMGA方法得到的聚类分析结果表明了1 2个品种间的亲缘关系 ,聚类结果与它们的系谱关系非常吻合     

基于EST-SSR标记的甘薯种质资源DNA指纹图谱构建

采用EST-SSR标记构建了国家种质广州甘薯圃中52份甘薯种质资源的DNA指纹图谱。利用52份供试资源,从早期筛选出的342对候选多态性引物中筛选出16对核心引物,16对引物在52份资源中共扩增出159个等位位点,每对引物的等位位点数为5~19个不等,平均为9.94个,多态性信息含量变化范围为0.6235~0.9593,平均为0.7991。选择带型清晰、重复性好、易于统计且能将52份资源完全区分开的2对引物组合构建供试资源的DNA指纹图谱。NTSYS软件聚类分析表明,EST-SSR标记能正确反映出不同资源间的亲缘关系,为构建甘薯大型DNA指纹图谱数据库奠定了基础。     

RAPD技术分析不同抗旱性苜蓿品种DNA的多态性

分别从不同抗旱性的紫花苜蓿品种中挑选抗旱性强的、抗旱性中等的和抗旱性弱的品种各三个,提取叶片基因组DNA,相同抗旱性苜蓿品种DNA等量混合构建三个池DNA。采用RAPD技术分析不同抗旱性混合DNA的多态性,并筛选出标记多态性的引物。结果表明：13组260个随机引物经五轮筛选,得到48个引物对不同抗旱性苜蓿品种池DNA扩增结果产生多态性,多态性引物占18．5％。其中5个引物能够稳定标记池DNA多态性,且特异性明显。表明不同抗旱性苜蓿品种之间具有明显的遗传多样性。     

虎杖种质资源的分子标记研究

本文应用RAPD、ISSR和SRAP标记对26份虎杖种质资源遗传多样性进行检测.在22个引物中有17个引物(77.3%)扩增产物具多态性,多态性水平相对较高.22个引物共得到98条扩增DNA片段,其中90.8%具有多态性.每个多态性引物平均可扩增出5.24个多态性片段.聚类分析表明,利用BAPD、ISSR和SRAP技术相结合可将全部供试材料区分开,26份材料在Gs值0.54水平上全部聚为一类,以所有材料间的平均遗传相似遗传系数0.71为阈值,将其分为11类.虎杖种质资源在分子水平上确实存在较大遗传差异,RAPD、ISSR和SRAP标记可作为构建虎杖DNA指纹图谱的有效工具.     

棉花2个多标记基因系及其杂交后代AFLP分析

应用AFLP分子标记技术,对陆地棉两个多标记基因系T582和T586及其杂交后代F1等进行了DNA多态性分析。结果表明:在58对EcoRI/MseI引物组合中,筛选出41对引物组合具有多态性,多态性的引物组合占筛选总组合的70.69%。AFLP分子标记具有高度的多态性,非常适于基因组差异较小的(棉花)材料之间的多态性筛选。采用聚丙烯酰胺银染法显带技术,AFLP进行PCR扩增能看到30~80条DNA亮带,且检测灵敏度高,可区别只相差十几个bp甚至几个bp大小的DNA片段。但AFLP标记以显性标记占绝对优势,共显性标记比率极少,故而难以区分种质的杂合和纯合,这是它的惟一不足之处。     

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。     

茄子耐盐种质资源遗传多样性的SRAP和ISSR分析

利用SRAP和ISSR分子标记,研究了14份耐盐茄子种质资源的遗传多样性,结果表明,2种标记均能揭示材料间较高的遗传多样性,其中ISSR标记多态性略高于SRAP标记。在SRAP分析中,每对引物组合可扩增出8-15条DNA片段,平均为12．12条：26对SRAP引物组合共扩增出315条DNA片段,其中263条具有多态性,多态性比率为83．49％;材料间遗传相似系数变化范围为0．212～0．923,平均值为0．755。在ISSR分析中,每个引物可获得5～16条DNA片段,平均为10．87条;15个ISSR引物共扩增出163条DNA片段,其中141条具有多态性,多态性比率为86．50％;材料间遗传相似系数变幅为0．333-0．957,平均值为0．736。聚类分析表明,2种标记都能将供试材料完全区分开来,聚类结果具有一定的相似性,但也存在明显差异。Mantel相关分析表明,SRAP分析与ISSR分析的相关性达到极显著性水平（r=0．904,P〈0．01）。     

油梨基因组DNA提取、SSR-PCR反应体系优化及引物筛选

旨在建立稳定可靠的油梨(Persea americana Mill)叶片DNA的提取方法和SSR-PCR反应体系及筛选出稳定的油梨SSR多态性引物,为开展油梨种质SSR分子标记提供遗传研究的基础。以油梨叶片为试材,比较3种油梨叶片DNA提取方法 ;利用L16(45)正交实验设计对油梨SSR-PCR反应体系进行优化;利用优化的反应体系筛选引物;同时,选取5对多态性引物对45份油梨种质进行PCR扩增,进一步检测该优化体系的稳定性。结果表明,常规2×CTAB法、改良2×CTAB法和植物DNA提取试剂盒法等3种DNA提取方法中,改良2×CTAB法对油梨基因组DNA的提取效果最佳;获得最优反应体系为:20μL总反应体系中,含约40 ng DNA模板、1.5 mmol/L Mg~(2+)、0.15 mmol/L d NTPs、0.5 U Taq DNA聚合酶、0.5μmol/L引物;以此体系为基础进行引物筛选,从73对油梨SSR引物中筛选出了30对扩增条带清晰的多态性引物,说明该反应体系可用于油梨SSR标记的进一步研究;稳定性检测获得的谱带清晰,表明该优化反应体系是稳定可靠的。由此可见,改良的2×CTAB法可用于油梨叶片DNA的大量样本提取,优化后的SSR-PCR反应体系及筛选出的30对多态性引物可用于油梨SSR标记的进一步研究。     

漆树品种的AFLP分析及评价(简报)

漆树(Toxicodendron vernicifluum(stokes)F.A.Barkley)隶属于漆树科(Anacardiaceae)漆树属(Tox- icodendron)的落叶乔木,是我国重要的特用经济林木。漆树栽培与生漆使用在我国已有几千年的历史,在长期栽培过程中形成了许多农家品种,它们具有一定的形态特点,适应一定的生长环境,并具有产漆量高、生漆品质好等特性。     

AFLP 引物组合数量对准确研究竹子系统关系的影响

利用AFLP技术对26个竹子种类进行了多样性分析, 以探索引物组合数量对准确研究竹子类群系统关系的影响。实验共随机选取10对AFLP引物, 并对所得10组AFLP标记数据随机组合后进行Nei氏遗传距离/UPGMA聚类分析。每对AFLP引物 扩增数据为一组, 随着用于聚类统计的AFLP标记数据随机组合数量的增加, 26个竹子种类的聚类关系趋向一致。这提示我们,在系统学研究中, 足够数量的引物组合是获得供试材料间准确聚类关系的基础, 应采用对各AFLP引物组合数据随机累加后进行聚类分析的方法, 以聚类关系为标准来确定用于分析供试品种的最少引物组合数量。     

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.     

Chemiluminescent detection of AFLP markers

Nonradioactive amplified fragment-length polymorphism (AFLP) marker detection, a PCR-based, DNA-fingerprinting technique, was achieved by blotting AFLP products after electrophoresis onto a nylon membrane and subsequently hybridizing the blot with an alkaline phosphatase-labeled AFLP probe. Similar AFLP profiles were obtained by both a nonradioactive, chemiluminescent detection technique and by conventional AFLP marker detection using 32P-labeled AFLP primers. The suitability of the method using different gel systems combined with subsequent chemiluminescent detection of AFLP markers is validated by similar dendrograms that were generated using the unweighted pair group method with arithmetic averages (UPGMA). Moreover, chemiluminescent detection of AFLP markers using a universal AFLP nonradioactive probe has been successfully applied on prokaryotes such as Agrobacterium and eukaryotic genomes such as soybean and fungi.     

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

云斑尖塘鳢AFLP分析技术的建立

以云斑尖塘鳢为对象,对其扩增片段长度多态性（AFLP）体系中的连接、预扩增、选择扩增等关键步骤的参数进行了对比实验。结果表明,在采用磷酸化接头,预扩增PCR增加延伸反应及20倍稀释预扩增产物的条件下,可得到清晰稳定的电泳图谱。所筛选的48个引物组合中,14个引物组合的产物带型稳定清晰,分布均匀,扩增片段为50-70条。本研究获得的AFLP优化参数及引物能够运用于云斑尖塘鳢群体遗传分析及分子辅助育种研究。     

Genetic mapping of sex-linked markers in Salix viminalis L

A total of 88 selective primer combinations were screened using bulked males and females sampled from four families of Salix viminalis. A total of more than 1000 polymorphic fragments was obtained, of which only four cosegregated with sex. These four sex-linked markers were subsequently scored in individuals that were used for bulked sample preparation in additional individuals of the same families, and in individuals in other families. A pair of primers that amplified the sex-linked fragments was constructed from one of the sex-linked amplified fragment length polymorphism (AFLP) fragments. In hybridization of Southern blot filters with the sex-linked DNA fragments, the band was present in females and absent in males, but the opposite pattern of band segregation (a band found in males and no band in females) was never observed in either the AFLP or RFLP experiments. Two of the sex-linked markers were placed on a linkage map. They both map at the same location in a linkage group comprising other markers not segregating with sex. Our data suggest that a single locus governs the sex determination and that nonrecombining sex chromosomes are absent in S. viminalis. A close association was found between skewed sex ratio and segregation distortion at this locus.     

普通枇杷、大渡河枇杷和栎叶枇杷遗传关系研究——基于RAPD和AFLP分析

利用筛选得到的24个随机引物和5对AFLP引物组合对普通枇杷、大渡河枇杷及栎叶枇杷的遗传关系进行RAPD及AFLP分析,结果表明,普通枇杷与栎叶枇杷的相似性系数最小(0.3864和0.5364),而大渡河枇杷介于普通枇杷和栎叶枇杷之间,偏向于栎叶枇杷。分别以普通枇杷、大渡河枇杷和栎叶枇杷作为待定杂种,计算了三者谱带的叠加性,结果表明,无论是RAPD分子标记,还是AFLP分子标记,以大渡河枇杷作待定杂种获得最高叠加性,支持大渡河枇杷可能是普通枇杷与栎叶枇杷的杂交种的结论。     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

扩增片断长度多态性(AFLP)荧光标记和银染技术的比较分析

实验采用AFLP的荧光标记分析技术和常规的AFLP银染技术,对施用农药和未施用农药的狼蛛基因多态性进行分析.用4对引物扩增和银染法共检测出32条带,荧光标记技术共检测出223条带.荧光技术比银染方法在每个位点上多检测到24条带,检测效果更为理想,更适于进行遗传多样性分析和研究;对两种方法的费用和工作效率做了初步分析,AFLP荧光标记技术的检测效率是银染法的690倍,同时对AFLP荧光标记技术中低成本、高通量多重PCR体系的建立及Genescan软件数据分析中出现的一些具体问题进行了探讨.