SRAP技术在遗传的研究进展

SRAP是一种新型的DNA分子标记,具有简便、稳定、中等产率和容易得到选择条带序列的特点。SRAP利用独特的引物设计对开放读码框(ORFs)进行扩增,上游引物长17bp,对外显子进行特异扩增,下游引物长18bp,对内含子区域、启动子区域进行特异扩增,因个体不同及其物种的内含子、启动子与间隔长度不等而产生多态性。本文阐述SRAP的原理和操作流程,综述了SRAP标记目前在植物遗传图谱构建、遗传多样性、基因定位、基因克隆、杂种优势利用等方面的研究进展及应用前景。     

荔枝SRAP-PCR反应体系的优化

SRAP(sequence-related amplified polymorphism)是基于基因的内含子和外显子区域进行检测的新型分子标记技术,具有多态性高、重复性好、易测序、便于克隆目标片段等特点,在荔枝(Litchi chinensis Sonn)中还没有相关的研究报道.为了建立适合荔枝的SRAP反应体系,对反应体系中的DNA模板、Mg2+、dNTP、引物、Taq DNA聚合酶诸因子的用量进行了探索,确立了适合荔枝的SRAP反应体系为:在20 μL反应体系中,模板DNA为40 ng,Mg2+浓度2.5 mmol/L,dNTP 0.3 mmol/L,引物5 mmol/L,Taq DNA聚合酶1.0 U.扩增程序为:94℃预变性3 min,反应前5个循环在94℃1 min、35℃1 min、72℃ 1 min的条件下运行;随后的35个循环退火温度提高到50℃;最后72℃延伸10 min.利用该反应体系对荔枝10个不同品系进行SRAP反应,用5%的变性聚丙烯酰胺凝胶电泳检测,结果显示:不同品系间DNA谱带多态性丰富.证实该体系稳定可靠,可以用于荔枝的分子标记研究.     

基于SRAP标记的薏苡种质资源遗传多样性及DNA指纹图谱构建

利用SRAP分子标记对从各主要产地收集到的90份薏苡种质进行遗传多样性分析,其中68份收集于福建省,6份来自中国台湾,16份来自浙江、辽宁、山东、河南、云南、江苏、湖南、广东、上海等省(市)。结果表明,从88对SRAP引物组合中筛选出26对引物进行SRAP扩增,共扩增出185条带,其中具有多态性的有157,占总数的84.86%,表明90份薏苡种质表现出丰富的遗传多样性。基于SRAP标记利用系统聚类法将90份薏苡种质资源分为4大类,与形态性状分类结果有一定的相似性;利用16对SRAP引物构建了73份薏苡种质资源的DNA指纹图谱,为薏苡遗传研究、品种选育与资源保护提供了依据。     

辣椒优良自交系间遗传差异的分子分析

作物自交系间遗传差异的分析与评价是杂种优势育种和杂交育种成功的基础。鲜食尖椒类(Capsicum an-nuumvar.longum)品种是我国辣椒生产的主要品种类型之一。针对我国鲜食尖椒的育种目标,以国内外10份尖椒优良自交系为材料,利用相关序列扩增多态性(SRAP)和简单序列重复(SSR)标记技术对其进行了遗传差异分析。结果显示:SRAP技术具有较高的位点和多态性检测能力,平均每次检测的位点数和多态性位点数分别为34个和10个,是SSR的10倍和5倍;辣椒自交系间基于SRAP标记的遗传距离和基于SSR标记的遗传距离之间的相关程度较低(r=0.144);基于SRAP标记和SSR标记联合数据计算的遗传距离,10个尖椒自交系被分为3大类,这种分类结果与辣椒杂种优势育种实践相一致。本研究结果表明,SRAP具有较高的遗传分析效力;基于不同分子标记遗传分析结果的差异与标记间共享位点的多少有关;10个尖椒自交系的分类结果可用于指导育种实践。     

SRAP标记及其在植物遗传育种中的应用

SRAP标记是基于选择性扩增开放性阅读框的新型分子标记,具有简便、高效、重复性好、高共显性等优点,在植物育种中已经得到广泛应用.本文介绍了SRAP标记基本原理和特点,对SRAP标记在遗传多样性研究、遗传连锁图谱构建、比较基因组以及分子标记辅助选择育种等方面的应用进行了综述.     

SRAP结合SCoT标记分析番木瓜种质的遗传多样性

利用SRAP和SCoT两种分子标记相结合对中国22个番木瓜主要栽培品种(系)进行遗传多样性研究。SCoT标记检测获得的遗传多样性参数值和遗传相似系数范围均高于SRAP标记检测的结果,表明SCoT标记检测多态性的能力高于SRAP标记。基于两种标记数据合并后的UPGMA聚类结果显示,番木瓜种质间的遗传相似系数为0.65~0.90,种质间遗传多样性水平较低;在遗传相似系数为0.82时,将所有参试材料划分为3个类群。应用Mantel检测对SRAP和SCoT及两种标记合并进行相关性分析,表明三者之间具有显著的相关性,且相关性很高。聚类结果从分子水平反映出中国番木瓜主要栽培品种(系)的遗传基础狭窄。     

辣椒31个优良自交系的亲本类群分析

以包含我国重要尖椒品种的亲本材料在内的31份优良自交系为材料, 利用SRAP标记和基因型值分析技术开展了辣椒自交系间遗传差异的分析与类群划分研究。结果表明: 在30个引物组合中, 27个引物组合可以 在自交系间扩增出多态性条带, 共扩增出310个多态性条带, 平均每个引物组合产生11.5个多态性条带, 显示出SRAP技术具有较强的分析效率; 基于SRAP标记和Yule相似系数对这些自交系进行的聚类分析中, 可以基本区分辣椒的2个变种(C. annuum var. grossum和C. annuum var. longum), 而且可以反映出自交系间的亲缘及系谱关系; 在相似系数为0.67处, 可将这31个自交系分为4个类群; 基于基因型值和标准Euclidean距离对这些自交系进行的聚类分析可成功地将辣椒的两个变种完全区分; 在遗传距离约4.5处, 可将这31个自交系分为4个类群; 自交系间基于SRAP标记与基因型值的遗传距离存在一定的相关性。     

中国灌木辣椒种质遗传多样性的SRAP和SSR分析

应用SRAP和SSR分子标记对8份辣椒种质进行了遗传多样性分析,结果表明,15对SRAP引物组合共扩增出321条带,平均每对引物扩增出21.40条,多态性位点比率为72.90%;18对SSR引物共扩增出109条带,平均每对引物扩增出6.06条,多态性位点比率为98.17%。与SRAP比较,SSR检测到的Shannon多样性指数(I)、观测等位基因数(Na)和有效等位基因数(Ne)等遗传多样性参数都较大,说明SSR有更高的多态性检测效率。基于SRAP的聚类与基于SSR的聚类之间存在极显著正相关,且都能将中国灌木辣椒种质与美洲灌木辣椒种质及一年生辣椒种质有效区分。     

RSAP、SSR和SRAP分析马铃薯遗传多样性的应用比较

本研究分别利用RSAP、SSR和SRAP对15份马铃薯种质进行遗传多样性分析,比较3种分子标记的分析效力。结果表明,平均每对引物扩增出的多态性位点RSAP(5.75个)SSR(6.33个)SRAP(7.75个);RSAP+SSR+SRAP联合聚类的结果与SRAP和RSAP聚类结果基本一致,相关性分别达到极显著和显著水平,与SSR的聚类结果基本相似,呈显著相关性。在分析马铃薯遗传多样性中,SRAP标记的效果最优,SSR标记效果略优于RSAP标记。RSAP分子标记能较好地分析马铃薯的遗传多样性,而以RSAP+SSR+SRAP联合分析,则能更好地评估种质的遗传多样性和亲缘关系。     

利用小肽控制的基于蛋白质内含子非层析标签制备重组蛋白(英文)

基于蛋白质内含子的蛋白质纯化自我断裂标签已经被广泛使用超过15年之久.但这一系统体内表达过程的提前断裂一直是限制这一技术广泛应用的瓶颈,特别是在需要高温表达和长表达周期的真核表达系统中.本研究介绍了一种利用小肽控制的基于蛋白质内含子和非层析标签ELP(elastin-like polypeptide)的自我断裂系统.在这一系统中,蛋白质内含子的体内外活性严格受到其结构互补小肽控制.在体内表达不含有互补小肽时,蛋白质内含子不具有活性;而在体外添加结构互补小肽,蛋白质内含子结构恢复并发生C端断裂反应释放目的蛋白.由于非层析标签ELP的引入,因此整个纯化过程可以简单地通过几步机械沉淀完成.此外,这一系统反应pH、小肽与前体蛋白之间的摩尔比及断裂速率也一并进行了系统的研究.     

浙南柚类地方资源遗传多样性分析和鉴定

利用SRAP标记对13份浙南柚类地方资源和琯溪蜜柚及芽变进行遗传多样性分析和鉴定。结果表明:平均每个引物组合可扩增出15.7条谱带,14对SRAP引物共产生220条谱带,其中多态性谱带为122条,多态率为55.4%,表明15份材料间检测到的SRAP位点多态性不高,不同引物组合可将11个基因型完全分开。聚类分析结果显示,15份柚类种质在遗传相似系数0.97处可以分为8大类,第1类群为四季柚7个优异株系,第2类群包括琯溪蜜柚及其芽变,而平阳文旦、早香柚、处红柚、红心1号土柚、红心2号土柚、酸柚分别单独为第3、4、5、6、7、8类群。四季柚选系中务城1号、务城3号、马站红心四季柚的指纹图谱有可区分的差异,说明DNA水平发生轻微变异。     

产喜树碱喜树内生真菌的筛选及喜树内生真菌的SRAP分析

生活在宿主植物里的内生真菌是很重要的药用资源。喜树是中国的传统药用植物。从喜树植物中分离得到了大约50种菌株,其中一株产喜树碱的菌株通过形态学鉴定为青霉属,这是首次在喜树植物中发现产喜树碱的青霉属菌株。为研究简单序列重复相关序列扩增多态性(SRAP)方法在喜树内生真菌中应用的可行性,选择了十株喜树内生真菌进行SRAP多态性分析。SRAP引物共扩增出1 295条带,而这些菌株也被分为三大类。这些结果表明,SRAP研究喜树内生真菌具有高效性,是讨论喜树内生真菌的遗传多样性的有效方法。     

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.Communicated by B. Friebe     

Genetic characterization of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> isolates from Turkey using sequence-related amplified polymorphism (SRAP)

Southern root knot nematode Meloidogyne incognita is the most widespread-species, causing serious yield losses in protected vegetables fields in the West Mediterranean region of Turkey. The knowledge of genetic variation within M. incognita is required for disease management and improvement of resistant varieties by breeding programs. In the present study, the isolates were classified into different groups based on sequence-related amplified polymorphism (SRAP) fingerprints. To our knowledge, this is the first study carried out on the characterization of M. incognita isolates using SRAP. The schematic diagram by tested primers to differentiate of M. incognita isolates was formed in discrimination of nematodes as an effective molecular tool since it is cost effective and easiness. Data presents a genetic variation on root-knot nematode species. These selected SRAP markers can be used to follow genetic structure and differentiation on M. incognita isolates in a certain region.     

Assessment of genetic diversity among faba bean genotypes using agro-morphological and molecular markers

Forty faba bean (Vicia faba L.) genotypes were evaluated for their agro-morphological performance and molecular diversity under Central Region of Saudi Arabia conditions during 2010–11 and 2011–12 seasons. Field performance results showed that faba genotypes exhibited a significant amount of variation for their agro-morphological studied parameters. Giza40 recorded the tallest genotype (139.5 cm), highest number of seeds per plants (100.8), and the highest seed yield per plant (70.8 g). The best performing genotypes were Giza40, FLIP03-014FB, Gazira1 and Goff1. Genetic variability among genotypes was determined using Sequence Related Amplified Polymorphism (SRAP) and Amplified Fragment Length Polymorphism (AFLP) markers. A total of 183 amplified fragments (alleles) and 1758 polymorphic fragments (bands) in SRAP and 202 alleles and 716 bands in AFLP were obtained using six SRAP and four AFLP primer combinations respectively. Polymorphism information content (PIC) values for AFLP and SRAP markers were higher than 0.8, indicating the existence of a considerable amount of genetic diversity among faba tested genotypes. The UPGMA based clustering of faba genotypes was largely based on origin and/or genetic background. Result of cluster analysis based on SRAP showed weak and not significant correlation while, it was highly significant based on AFLP analysis with agro-morphological characters (r = 0.01, p > 0.54 and r = 0.26, p < 0.004 respectively). Combined SRAP and AFLP markers proved to be significantly useful for genetic diversity assessment at molecular level. They exhibited high discrimination power, and were able to distinguish the faba bean genotypes with high efficiency and accuracy levels.     

Analysis of Genetic Diversity Among Chinese Auricularia auricula Cultivars Using Combined ISSR and SRAP Markers

DNA polymorphism among 34 Chinese Auricularia auricula cultivars was analyzed using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Thirty ISSR primers amplified a total of 129 DNA fragments of which 125 (96.9%) were polymorphic, whereas 11 SRAP primer combinations amplified 154 fragments of which 148 (96.1%) were polymorphic. Both methods were highly effective in discriminating among the test strains. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method distributed the 34 strains into four or five major groups. Clustering analysis based on all the three data sets indicated a high level of genetic diversity among A. auricula, although the combined ISSR/SRAP data were more concordant with the main agronomic characters of strains and their geographical centers of cultivation. Our findings will facilitate future A. auricula breeding programs and the development of bioactive products from this commercially important medicinal mushroom.