农作物品种DNA指纹库构建研究进展

构建品种标准DNA指纹库应满足三个基本要求,即标准样品、核心引物和标准程序。本文综述了近20年来国内外农作物品种DNA指纹库构建进展情况,结果显示国外品种标准DNA指纹库构建主要是在UPOV框架下开展植物品种权保护品种的建库,而中国品种标准DNA指纹库构建主要是在农业部品种管理部门的统筹下开展品种权保护品种及区试审定品种的建库。所用建库技术主要为SSR技术,少数作物如玉米、水稻、大豆等已开始采用SNP技术,玉米、水稻、小麦等作物品种建库数量均已达到3 000份以上,其中玉米建库数量已经超过2万份。进一步分析发现,不同作物分子标记开发情况、作物的物种特性、不同国家对不同作物建库需求的迫切程度、所建DNA指纹库的级别是影响不同作物DNA指纹库构建进展的四个主要因素。最后本文对农作物品种未来DNA指纹库构建工作的开展进行展望。     

中棉所48的SSR数字指纹图谱的构建

利用25对核心引物对中棉所48及其亲本进行多态性检测,共有16对引物在2个亲本间具有多态性,其中,有14对引物在两亲本间扩增出大小不同的带,且这些标记位点在F1中均表现为杂合带,为共显性标记;另外2个引物的F1带型与母本或父本一致,为显性标记。以这25对引物在不同材料间扩增得到的01(二进制)数据转换成十进制数据,构建了中棉所48的数字指纹图谱,为杂交种的真伪鉴定和纯度检测提供了方法。为了构建准确、稳定和实用的棉花DNA指纹图谱,指出了需要规范分子标记类型和数量,DNA、Taq酶等试剂数量和质量,统一PCR反应,电泳和染色条件,统一提供标准品种和标准DNA,标准化电泳条带的识别、数据处理方式和编码方式。     

SSR分子标记技术及其在构建玉米DNA指纹库上的应用

SSR分子标记技术是在PCR基础上发展起来的一种DNA多态性检测技术,已开始应用于植物基因组研究的各个领域。本文概述了SSR标记的原理、类型、功能及其引物的来源和利用SSR构建玉米DNA指纹库的进展以及玉米DNA指纹库在品种鉴定、品种保护及品种监测等方面的应用。     

我国玉米品种标准DNA指纹库构建研究及应用进展

介绍了我国玉米品种标准DNA指纹库构建的进展及应用情况。我国玉米品种标准DNA指纹库规模已达到22 089份,经与国内外作物品种DNA指纹库比较,是全球品种数量和建库规模最大的DNA指纹库,并且在区域试验、品种权保护及种子质量管理等方面都得到了广泛应用。至今已开展玉米品种真实性鉴定等达40 000多份次。同时,对DNA指纹库构建未来发展进行预测,建议继续发挥SSR标记的作用、加快推进SNP标记的研发、发掘In Del标记的应用潜力并持续关注测序技术的发展。     

玉米SSR-DNA指纹库构建方案在高粱中的通用性

近年来,随着主要农作物品种SSR分子鉴定技术规程的相继颁布,玉米、水稻已经建立了标准样品SSR-DNA指纹数据库,其它作物正在启动,整合一套稳定性好、通用性高、操作便捷的建库方案对多种作物DNA指纹库的构建具有重要意义。本研究以6份高粱和3份玉米标准样品为例,以高粱和玉米品种真实性检测标准为基础,研究跨作物应用玉米DNA指纹库构建方案的可行性。研究表明将玉米DNA指纹库构建方案应用于高粱中是可行的,这为后续探究其他作物DNA指纹库构建通用性方案研究奠定基础。     

国家芝麻区域试验新品种(系)的DNA指纹分析

以我国近年参加国家芝麻区域试验的43个品种(系)为材料,利用12对SSR核心引物开展DNA指纹分析,初步构建参试品种的DNA指纹图谱,并根据指纹图谱分析了参试品种的特异性和一致性。聚类分析结果表明,12对引物检测到30个标记,可将43个品种完全区分开,来源于同一育种单位的品种遗传基础相近,而不同来源的品种其遗传背景相差较大;以遗传相似系数0.90为划分标准,95%的供试品种具备特异性;以引物位点的一致性为指标,80%的品种具有一致性;分配试验中,86%的单株都能正确地分配回到原来的品种,这些品种的一致性较好,个别品种的单株正确分配率较低,品种一致性差。表明大部分参加国家区试的品种都具有很好的特异性和一致性。     

玉米杂交种DNA指纹图谱及其在亲子鉴定中的应用

采用79对SSR引物分析了86个玉米杂交种的72份亲本自交系的遗传多态性,从中筛选出扩增带型清晰、稳定的50对引物用于构建86个杂交种的DNA指纹图谱数据库;进而确定10对核心引物和判别标准用于亲子鉴定研究。结合玉米SSR分子标记检测技术和单籽粒DNA快速提取方法,DNA指纹数据库和判别标准可有效地用于玉米杂交种的亲子鉴定。     

基于SSR标记新疆陆地棉的DNA指纹图谱构建及遗传多样性分析

【目的】利用Simple sequence repeats(SSR)标记构建新疆近40年间审定的120个陆地棉品种的DNA指纹图谱并进行遗传多样性分析。【方法】从586对候选引物中筛选得到78对多态性高、扩增稳定且均匀分布于棉花染色体上的引物,并用这78对引物构建120个陆地棉品种的DNA指纹图谱。【结果】78对核心引物在120个材料中检测到392个等位位点,其中多态性位点324个,多态性比率达82.7%。24个标记位点在17个品种上具有特征谱带。采用12对引物组合即可鉴定120个棉花品种。聚类分析显示,120个陆地棉品种遗传相似系数变化范围为0.50~0.96,平均为0.73,遗传相似系数偏高,表明新疆陆地棉品种的遗传基础较狭窄。【结论】引物组合法是构建DNA指纹图谱最有效的方法。遗传相似系数矩阵将120个陆地棉品种分为三大类群,与系谱来源较为吻合。     

适用于玉米DNA指纹库构建的SSR核心引物的重新设计与优化

为了确定出适用于玉米DNA指纹库构建的引物,对部分并不是完全符合玉米DNA指纹库构建引物筛选原则的引物进行重新设计与优化,使其基本符合核心引物设计的要求.以umc2105、umc1705和phi299852为例,下载原引物的基因组序列,利用两个常用的引物设计软件Primer Premier 5.0和Oligo 6.57进行引物的重新设计及评估,在Tm值、GC含量、引发效率、错误引发率、3'端G值、引物二聚体等方面对引物进行改善,结果表明,经过对原引物的重新设计和优化,新设计的引物均能清晰的扩增,扩增效果得到了明显的改善,并且均能在统一的条件下进行实验,符合了玉米DNA指纹库构建的要求.     

花生新品种汕油21种子纯度SSR标记鉴定体系的建立和应用

为探索花生品种纯度的分子标记鉴定方法,采用SSR标记构建不同花生品种的指纹图谱。结果表明:利用3对SSR标记引物构建20个花生品种的指纹图谱,该图谱可以将汕油21与其它19个花生品种进行区分,这些品种包括汕油162、汕油212、汕油31、汕油33、汕油71、粤油5号、粤油7号、粤油29、粤油32、汕油27、汕油523、粤油9号、粤油13、粤油14、粤油79、粤油114、J11、仲恺花1号和泉608等;以3对SSR引物对汕油21繁育种子60个样品进行种子纯度检测,共有2对标记引物检测到3个杂株样品,该种子批的纯度为95.0%。因此,利用SSR标记构建花生品种指纹图谱并应用于汕油21品种纯度鉴定是可行的。     

中国棉花主栽品种DNA指纹数据库初步构建研究

 基于多重PCR（Polymerase chain reaction）与毛细管五色荧光检测系统,利用36对核心引物构建了138份棉花主栽品种DNA指纹数据库,采用棉花DNA指纹数据库软件管理系统进行数据统计与分析。36对引物在138份材料中共扩增出143个多态性等位位点,每对引物的等位位点数为2～8个,平均每对引物扩增出3.97个多态性等位位点。抽查的101个主栽品种仅45.6%的位点完全纯合,中棉所系列品种61.6%的位点完全纯合。存在5种非纯合位点表现形式,以2种基因型混杂所占比例最大,其中以5∶1的混杂类型最多。4种类型的同名品种指纹比对分析表明,品种内的遗传变异度均在0～2个差异位点。初步建立了高通量的棉花DNA指纹检测与数据分析平台,提出了不同品种间的差异判别标准。     

棉花DUS测试标准品种的SSR指纹数据库构建

基于SSR核心引物,采用荧光毛细管电泳检测系统与多重PCR技术相结合,构建我国棉花DUS(Distinctness,Uniformity and Stability)测试标准品种的DNA指纹数据库并进行遗传多样性分析。依据多重PCR组合的基本原则与五色荧光检测系统的特点,采用40对核心引物构建了10个4重PCR组合,利用DNA遗传分析仪进行指纹检测与数据采集。40对荧光引物在30份DUS测试标准品种中共扩增产生146个等位变异,每对引物的等位变异数为2~7个不等,平均每对引物产生3.65个等位变异。海7124与陆地棉品种明显划分为2类,来源于新疆的新陆早1号区别于其他陆地棉品种,单独聚为1类。多色荧光检测系统相比常规聚丙烯酰胺凝胶电泳具有高精度、高通量、自动化程度高的优点,尤其适用于大规模指纹数据库的构建,提出了通过构建已知品种DNA指纹数据库,将分子标记技术应用于棉花DUS测试的初步设想。     

甜菜DAMD引物的筛选及不同凝胶系统对扩增产物多态性的影响

为了筛选适宜于甜菜品种纯度鉴定的DAMD引物,笔者利用12 个不同的甜菜品种分别对26 条DAMD引物进行扩增,并分别采用6%聚丙烯酰胺凝胶电泳和2%的琼脂糖凝胶电泳对扩增产物进行检测。结果表明,从26 条DAMD引物中筛选出16 条扩增清晰的引物,这16 条引物共扩增出139 条带,其中多态性条带为122 条,多态性百分比为87.7%,这16 条引物可以作为甜菜品种纯度和真实性鉴定的核心引物,同时发现聚丙烯酰胺凝胶电泳与琼脂糖凝胶电泳的检测结果差异不大,而琼脂糖凝胶具有制作方便、检测简单以及不使用任何有毒药剂等优点,推荐甜菜DAMD扩增产物的检测使用琼脂糖凝胶电泳。     

甜菜SCoT核心引物的筛选

【研究目的】为了筛选出适合甜菜品种遗传多样性分析的SCoT引物,为SCoT引物得以在甜菜中进行深入应用奠定基础,本研究甜菜以8个来自不同国家的甜菜品种为材料对80条SCoT引物进行扩增【方法】扩增方法采用SCoT-PCR最优反应体系包含2.0 μL的10×PCR buffer（含Mg2+）、10 ng的DNA、0.5 U的Taq DNA聚合酶、10 μmol/L的引物2 μL以及0.1 μL的dNTPs (2.5 mmol/L each)。【结果】结果从80条SCoT引物中筛选出20条多态性高的引物,这20条引物最多扩增出16条多态性条带,最少扩增出2条多态性条带,扩增总条带155条,其中多态性条带为134条,多态性条带的百分比为86.5%。【结论】通过对核心引物的有效性验证,表明筛选出的20个核心引物非常适合用于甜菜指纹图谱的构建。     

DNA Fingerprint Construction and Genetic Diversity Analysis Based on SSR Markers for Upland Cotton in Xinjiang

[Objective] The aim of this study was to construct a DNA fingerprinting database of 120 upland cotton cultivars from Xinjiang and to analyze their genetic diversity based on SSR markers. [Methods] Seventy-eight evenly distributed SSR primer pairs with high polymorphism and good repeatability were successfully screened out from 586 candidates to construct the fingerprinting database. [Result] A total of 392 alleles from 120 varieties were screened using 78 pairs of core primers, 324 of which were polymorphic loci with a polymorphism rate of 82.7%. Seventeen cultivars had specific genotypes determined using 24 primer pairs and 120 upland cotton cultivars could be identified by only 12 primer combinations. Cluster analysis indicated that genetic similarity coefficient for the 120 upland cotton cultivars ranged from 0.50 to 0.96, with an average of 0.73, indicating that upland cotton resources possess high genetic similarity and have an accordingly narrow genetic basis. [Conclusion] The primer combination method is one of the most effective methods for constructing DNA fingerprinting. The 120 upland cotton varieties were divided into three types with the genetic similarity coefficient matrix; these groups were strongly consistent with their pedigrees.     

Application of SSR markers and real-time PCR for variety identification in azuki-bean [Vigna angularis (Willd.) Ohwi and Ohashi]

The azuki bean in Korea consists of seven domestic varieties which have been developed and registered for the public during last 25 years. Here, we present a simple but reliable method to screen and identify Korean azuki bean varieties. A method based on simple sequence repeat (SSR) markers is widely used for prominent gene identification and variety discrimination. In molecular biology, real-time polymerase chain reaction (PCR) is a laboratory technique based on the polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule. It enables easy detection of a specific sequence in a DNA sample without performing electrophoresis and further processes. For separation of seven Korean azuki bean varieties, 110 unique azuki bean SSR markers from an (AG)n-enriched library were selected, synthesized and used for polymerase chain reaction (PCR). Data were taken through acrylamide gel electrophoresis and automated multi-capillary electrophoresis system for selection of specific markers and then changed into proper formats for data mining analysis. Ten primer pairs that showed high polymorphism were chosen for the indepth study. These ten primers were re-amplified with real-time PCR and checked the cycle threshold (Ct) and temperature (Tm) for comparison of amplification sequence in seven varieties. Consequently, a total of 20 alleles and 6 SSR primers were detected from the standard PCR amplification. Within these 6 primers, 7 alleles of 3 SSR primers were isolated for variety identification. From real-time PCR results, 3 SSR primers were selected as efficient markers for discrimination of seven Korean azuki bean varieties. The approach described here could be applied in monitoring our varieties and can be adapted in the azuki bean breeding program.     

Extraction of Cotton Fiber DNA and Its Application in Traceability

[Objective] To address the questions of lacking monitoring system for origins of varieties, this study explored a DNA extraction method suitable for cotton fiber, aiming to establish an identification system that uses cotton fiber DNA as a medium to trace the authenticity and cultivar of cotton varieties. [Method] By improving and optimizing the extraction method, the DNAs of cotton fibers at different days post anthesis and lint from different years were extracted and used as templates for routine polymerase chain reaction (PCR) amplification. 13 pairs of simple sequence repeat(SSR) primers were selected for SSR-PCR amplification using the DNA of Lu 1127, Shikang 126 and Ruiza 816. [Result] The DNA extraction method can extract cotton fiber DNAs of different developmental stages and different years. With the development of fiber, although the extracted DNA content is reduced, it can still meet the needs of downstream experiments. The extracted cotton fiber DNAs can be subjected to conventional PCR amplification, the PCR amplified bands were clear; 13 pairs of cotton SSR primers were used for SSR-PCR amplification of cotton fiber DNAs of Lu 1127, Shikang 126 and Ruiza 816, and a clear polymorphic band could be amplified and easily distinguished. [Conclusion] This extraction method is suitable for extracting genomic DNA of cotton fiber, and is satisfied with conventional PCR amplification with SSR markers. It is confirmed that the cotton fiber DNA molecular markers are feasible for tracing the source of cotton varieties, and it is expected to provide technical support for ensuring the safety of cotton industry.     

Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties

Summary In this study, two PCR-based methodologies were evaluated for potential use in the determination of DNA diversity between 20 commercial sugarcane hybrids and 6 outgroup varieties of S. spontaneum, S. officinarum and hybrids from early in the genealogy. The first method involved PCR amplification of sugarcane DNA in the presence of random, decamer primers (RAPDs), while the second protocol utilized specific microsatellite and telomere sequences as primers. A total of 41 RAPD primers (356 loci) were screened across the varieties of which 15 (160 loci) were used in the calculation of DNA diversity (expressed% similarity). This varied from 61 to 95%, with most of the commercial varieties showing more than 80% similarity in their DNA. The RAPD data indicated that there had been a gradual decline in DNA diversity (84% reduction) from the early inter-specific crosses to the commercial hybrids, probably as a result of backcrossing and in-breeding strategies used in the previous 5 to 6 generations of sugarcane breeding. The microsatellite and telomere data produced a much greater range in DNA similarity values (25–91%), probably due to the fact that these primers detect highly variable regions of the genome. It is suggested that these specific primers would not be suitable for determination of DNA diversity, but could be used more effectively in the development of a methodology for routine, rapid identification of sugarcane varieties.     

利用SSR快速鉴定棉花品种真实性和纯度

利用SSR分子标记技术对棉花品种真实性和纯度进行分析研究,旨在为棉花品种提供分子鉴定依据。以具有代表性的棉花品种为材料,经过引物初筛和复筛,确定26对均匀分布于棉花染色体上的SSR核心标记。结果表明,26对核心标记在120份材料中筛选得到138个等位位点,其中129个为多态性位点,多态性比率达93.48%。以标准样品为对照,利用26对核心标记对10份棉花常规种进行真实性鉴定,发现仅有源棉6号与标准品种的DNA图谱高度一致,其他9份常规种与标准品种均存在8~18个差异位点,分子鉴定结果与田间鉴定相符。另外分别筛选5对特征引物作为纯度检测标记,采用单位点平均法统计4份常规棉品种检测结果表明,源棉6号纯度最高,为98.0%,源棉1号最低,为90.5%,检测结果与田间纯度鉴定呈现正相关性。研究表明利用SSR标记技术鉴定棉花品种真实性和纯度的方法完全可行。