简单重复序列区间(ISSR)引物反应条件优化与筛选-已看
中国兰ISSR—PCR反应体系优化及引物筛选
中国兰ISSR—PCR反应体系优化及引物筛选作者：黄晓慧巫伟峰陈春张毅智汪长水徐建球陈发兴陈孝丑来源：《南方农业学报》2018年第07期摘要：【目的】优化中国兰的ISSR-PCR反应体系，并筛选适用于中国兰ISSR分析的候选引物，为ISSR分子标记在中国兰的辅助育种及亲缘关系和遗传多样性分析等提供技术参考。
【方法】以6个中国兰品种为材料，采集其叶片样品，分别用研钵法和研磨仪法进行破碎研磨，比较两种方法提取DNA的效果，利用L25（53）正交试验和单因素试验对DNA模板量、引物浓度、2×Taq Master Mix添加量、循环数和退火温度进行优化，建立最佳ISSR-PCR 反应体系，并从ISSR分子标记通用引物中筛选适用于中国兰的ISSR分析候选引物。
【结果】研磨仪法提取的DNA浓度明显高于研钵研磨法，但二者提取的DNA质量均较好（OD260/OD280为1.7～2.0）。
对ISSR-PCR反应体系扩增结果的影响程度排序为DNA模板量>引物浓度>2×Taq Master Mix添加量。
综合考虑成本和DNA模板量，最佳ISSR-PCR反应体系（20.0 μL）：DNA模板10.0 ng、引物0.8 μmol/L和2×Taq Master Mix 9.0 μL。
最佳循环数为35，最佳退火温度为49.6 ℃。
基于上述优化结果，从100条引物中共筛选出42条适用于中国兰的ISSR候选引物。
【结论】研磨仪法可有效提高中国兰基因组DNA的提取率和质量，且利用优化后的ISSR-PCR反应体系和扩增程序及筛选出的引物，扩增获得的条带清晰、稳定，多样性好，可用于中国兰的遗传多样性和亲缘关系等分析研究。
关键词：中国兰；ISSR；分子标记；反应；引物筛选中图分类号： S682.310.36 文献标志码：A 文章编号：2095-1191（2018）07-1282-070 引言【研究意义】中国兰又称国兰，是中国传统兰花的统称，为兰科（Orchidaceae）兰属（Cymbidium）植物，其味幽香，花色淡雅，素有花中君子和天下第一香之美称，且具有独特的内涵和意境，观赏和经济价值很高（陈心启，2011）。
细胞遗传学课后题答案
细胞遗传学课后题答案《细胞遗传学》复习题第⼀章染⾊体的结构与功能+第三章染⾊体识别1.什么是花粉直感？花粉直感是怎样发⽣的？作物种⼦的哪些部分会发⽣花粉直感？花粉直感⼜叫胚乳直感，植物在双受精后，在3n胚乳上由于精核的影响⽽直接表现⽗本的某些性状。
由雄配⼦供应的⼀份显性基因能够超过由母本卵核或两个极核隐形基因的作⽤，杂交授粉当代母本植株所结的种⼦表现显性性状。
胚乳和胚性状均具有花粉直感的现象。
2.什么叫基因等位性测验？如何进⾏基因等位性测验？确定两个基因是否为等位基因的测验为基因的等位性测验。
将突变性状个体与已知性状的突变种进⾏杂交，凡是F1表现为已知性状，说明两对基因间发⽣了互补，属于⾮等位基因。
若F1表现为新性状，表明被测突变基因与已知突变基因属于等位基因。
3.原位杂交的原理是什么？原位杂交所确定的基因位置与遗传学上三点测验所确定的基因位置有何本质的不同？根据核酸碱基互补配对原则，将放射性或⾮放射性标记的外源核酸探针，与染⾊体经过变性的单链DNA互补配对，探针与染⾊体上的同源序列杂交在⼀起，由此确定染⾊体特定部位的DNA序列的性质；可将特定的基因在染⾊体上定位。
第⼀步，制备⽤来进⾏原位杂交的染⾊体制⽚；第⼆步，对染⾊体DNA进⾏变性处理；第三步，进⾏杂交；第四步，信号检出和对染⾊体进⾏染⾊；第五步，显微镜检查。
原位杂交是⼀种物理图谱绘制的⽅法，它所确定是特定基因在染⾊体上的物理位置；三点测验是绘制连锁图谱的实验⽅法，它是利⽤三对连锁基因杂合体，通过⼀次杂交和⼀次测交，确定三对基因在同⼀染⾊体上排列顺序以及各个基因的相对距离。
4.什么叫端粒酶(telomerase)？它有什么作⽤？端粒酶是参与真核⽣物染⾊体末端的端粒DNA复制的⼀种核糖核蛋⽩酶,由RNA和蛋⽩质组成，其本质是⼀种逆转录酶。
作⽤：它以⾃⾝的RNA作为端粒DNA复制的模版，合成出富含G的DNA序列后添加到染⾊体的末端并与端粒蛋⽩质结合，从⽽稳定了染⾊体的结构。
分子生药学
分子生药学研究策略
分子遗传标记技术
通过直接分析遗传物质的多态性来诊断生物内在基因排布规律及其 外在性状表现规律的技术。任何生物种或个体都具有特定的DNA 多态性，通过直接诊断分析DNA 的多态性，便能避开遗传特性表 现过程中的环境因素、数量性状遗传或部分与完全显性的干扰，快
速准确地鉴定药材真伪。
分子生药学研究方法
PCR反应的结果。①循环参数
变性 退火 延伸
②反应成分
（3）PCR反应引物的设计 引物的设计在整个PCR扩增中占有十分重要的地位 特异性，扩增性 ①引物的序列应位于基因组DNA的高度保守区，且与非扩增区无同源序列。这样 可以减少引物与基因组的非特异性结合，提高反应的特异性 ②引物长度：15－30nt为宜。引物过短或过长均可使反应的特异性下降。 ③引物的碱基尽可能随机发布，避免出现数个嘌呤或嘧啶的连续排列，G+C碱基
dNTP:一般为50－200μmol/L
Mg2+ 模板：PCR对模板的要求不高，单、双链DNA均可，但
样品中不能混有蛋白酶、核酸酶、DNA聚合酶抑制剂以
及能与 DNA结合的蛋白质。 添加剂：DMSO（二甲基亚枫），提高扩增效率及特异性
（1） 理论上PCR合成产物的数量经过每轮循环都将增加一倍，应按2n-2n 的指数方式递增，PCR反应30轮循环后，PCR扩增应达到230个拷贝，约109个拷 贝。但由于DNA聚合酶的质量、待扩增片段的序列及反应系统的条件等各种因素 的影响，实际扩增效率比预期的要低，一般可达106－107个拷贝。 “平台效应”：PCR反应中，当引物－模板与DNA聚合酶达到一定比值时， DNA聚合酶催化反应趋于饱和，即PCR反应不再增加。 平台效应在PCR反应中是不可避免的，但一般在平台效应出现前，PCR产物 的数量足以满足实验的需要。 （2）PCR反应条件的优化 PCR方法操作简便，但影响因素颇多，因此需要根据不同的DNA模板，摸索最 适条件。主要从： 反应的特异性、敏感性、忠实性、扩增效率等四个方面衡量
简单序列重复(ISSR)多态性及其在植物育种中的应用
Euphytica128:9–17,2002.©2002Kluwer Academic Publishers.Printed in the Netherlands.9Inter simple sequence repeat(ISSR)polymorphism and its application in plant breedingM.Pradeep Reddy,N.Sarla∗&E.A.SiddiqDirectorate of Rice Research,Rajendranagar,Hyderabad–500030,India;(∗author for correspondence,e-mail: nsarla@)Received3July2001;accepted6March2002Key words:anchored primer,DNA marker,genome mapping,gene tagging,genetic diversity,ISSR-PCR SummaryInter simple sequence repeat(ISSR)-PCR is a technique,which involves the use of microsatellite sequences as primers in a polymerase chain reaction to generate multilocus markers.It is a simple and quick method that combines most of the advantages of microsatellites(SSRs)and amplified fragment length polymorphism(AFLP) to the universality of random amplified polymorphic DNA(RAPD).ISSR markers are highly polymorphic and are useful in studies on genetic diversity,phylogeny,gene tagging,genome mapping and evolutionary biology.This review provides an overview of the details of the technique and its application in genetics and plant breeding in a wide range of crop plants.IntroductionDNA markers have proved valuable in crop breed-ing,especially in studies on genetic diversity and gene mapping.The commonly used polymerase chain reaction(PCR)-based DNA marker systems are ran-dom amplified polymorphic DNA(RAPD),amplified fragment length polymorphism(AFLP)and more re-cently simple sequence repeats(SSRs)or microsatel-lites(Staub et al.,1996;Gupta&Varshney,2000).The major limitations of these methods are low reprodu-cibility of RAPD,high cost of AFLP and the need to know theflanking sequences to develop species spe-cific primers for SSR polymorphism.ISSR-PCR is a technique that overcomes most of these limitations (Zietkiewicz et al.,1994;Gupta et al.,1994;Wu et al.,1994;Meyer et al.,1993).It is rapidly being used by the research community in variousfields of plant improvement(Godwin et al.,1997).The technique is useful in areas of genetic diversity,phylogenetic stud-ies,gene tagging,genome mapping and evolutionary biology in a wide range of crop species.In this method SSRs are used as primers to amplify mainly the inter-SSR regions.SSRs or microsatellites are short tandem repeats(STRs)or variable number of tandem repeats (VNTRs)of1–4bases of DNA ubiquitously present in eukaryote genomes(Tautz&Renz,1984).They are dispersed throughout the genome and vary in the number of repeat units.The details of the technique and its major applications are discussed in this review. The techniqueInter simple sequence repeat(ISSR)technique is a PCR based method,which involves amplification of DNA segment present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction.The technique uses mi-crosatellites,usually16–25bp long,as primers in a single primer PCR reaction targeting multiple gen-omic loci to amplify mainly the inter-SSR sequences of different sizes.The microsatellite repeats used as primers can be di-nucleotide,tri-nucleotide,tetra-nucleotide or penta-nucleotide.The primers used can be either unanchored(Gupta et al.,1994;Meyer et al., 1993;Wu et al.,1994)or more usually anchored at3’or5’end with1to4degenerate bases extended into theflanking sequences(Zietkiewicz et al.,1994)(Fig-ure1).The technique combines most of the benefits of10Figure1.ISSR-PCR:A schematic representation of a single primer(AG)8,unanchored(a),3’-anchored(b)and5’-anchored(c)targeting a (TC)n repeat used to amplify inter simple sequence repeat regionflanked by two inversely oriented(TC)n sequences.(a)Unanchored(AG)n primer can anneal anywhere in the(TC)n repeat region on the template DNA leading to slippage and ultimately smear formation(b)(AG)n primer anchored with2nucleotides(NN)at the3’end anneals at specific regions on the template DNA and produces clear bands(c)(AG)n primer anchored with2nucleotides(NN)at the5’end anneals at specific regions and amplifies part of the repeat region also leading to larger bands.11AFLP and microsatellite analysis with the universality of RAPD.ISSRs have high reproducibility possibly due to the use of longer primers(16–25mers)as com-pared to RAPD primers(10-mers)which permits the subsequent use of high annealing temperature(45–60◦C)leading to higher stringency.The studies on reproducibility show that it is only the faintest bands that are not reproducible.About92–95%of the scored fragments could be repeated across DNA samples of the same cultivar and across separate PCR runs when detected using polyacrylamide(Fang&Roose,1997; Moreno et al.,1998).10ng template DNA yielded the same amplification products as did25or50ng per20µl PCR reaction.The annealing temperature depends on the GC content of the primer used and usually ranges from45to65◦C.ISSRs segregate mostly as dominant markers fol-lowing simple Mendelian inheritance(Gupta et al., 1994;Tsumura et al.,1996;Ratnaparkhe et al.,1998; Wang et al.,1998).However,they have also been shown to segregate as co-dominant markers in some cases thus enabling distinction between homozygotes and heterozygotes(Wu et al.,1994;Akagi et al.,1996; Wang et al.,1998;Sankar&Moore,2001).Source of variability/polymorphismThe evolutionary rate of change within microsatellites is considerably higher than most other types of DNA, so the likelihood of polymorphism in these sequences is greater.The source of variability in the ISSRs can be attributed to any one of the following reasons or any combination of these.(a)Template DNASlippage of DNA polymerase during DNA replica-tion and failure to repair mismatches is considered as a mechanism for creation and hypervariability of SSRs(Levinson&Gutman,1987).Mutations at the priming site i.e.SSR could prevent amplifica-tion of a fragment,as also in RAPD markers and thus give a presence/absence polymorphism.An in-sertion/deletion event within the SSR region or the amplified region would result in the absence of a product or length polymorphism,depending on the amplifiability of the resulting fragment size.Variab-ility in number of nucleotides within a microsatellite repeat would result in length polymorphisms when using a5’-anchored primer.(b)Nature of primer usedThe extent of polymorphism also varies with the nature(unachored,3’-anchored,or5’-anchored)and sequence of the repeats(motif)in the primer em-ployed.When unanchored i.e only the SSRs are used as primers,the primer tends to slip within the repeat units during amplification leading to smears instead of clear bands(Figure1a).Extending the primer(an-choring)with1to4degenerate nucleotides at the3’end(Figure1b)or5’end(Figure1c)assures anneal-ing only to the ends of a microsatellite in template DNA thus obviating internal priming and smear form-ation.Secondly,the anchor allows only a subset of the microsatellites to serve as priming sites.When 5’anchored primers are used,the amplified products include the microsatellite sequences and their length variations across a genome and therefore give more number of bands and a higher degree of ually di-nucleotide repeats,anchored either at3’or5’end reveal high polymorphism(Blair et al.,1999;Joshi et al.,2000;Nagaoka&Ogihara, 1997).The primers anchored at3’end(Figure1b) give clearer banding pattern as compared to those anchored at5’end(Tsumura et al.,1996;Blair et al., 1999;Nagaoka&Ogihara,1997).Since the primer is a SSR motif the frequency and distribution of the microsatellite repeat motifs in different species also influence the generation of bands.There is a difference of abundance of SSRs between nuclear and organ-elle DNA sequences.Taking di-and tri-nucleotides together,one SSR was found every33Kb in nuclear DNA compared to every423-Kb of organelle DNA sequence(Wang et al.,1994).In general,primers with(AG),(GA),(CT),(TC),(AC),(CA)repeats show higher polymorphism than primers with other di-,tri-or tetra-nucleotide repeats.(AT)repeats are the most abundant di-nucleotides in plants but the primers based on(AT)would self-anneal and not amplify.Tri-and tetra-nucleotides are less frequent and their use in ISSRs is lesser than the di-nucleotides.The(AG)and (GA)based primers have been shown to amplify clear bands in rice(Blair et al.,1999;Joshi et al.,2000; Reddy et al.,2000;Sarla et al.,2000),trifoliate orange (Fang et al.,1997),Douglasfir and sugi(Tsumura et al.,1996)and chickpea(Ratnaparkhe et al.,1998), whereas primers based on(AC)di-nucleotide repeats were found more useful in wheat(Nagaoka&Ogihara, 1997;Kojima et al.,1998)and potato(McGregor et al.,2000).Resolving power Rp is an index developed to compare the value of different primers in terms12of the informative bands obtained in a given set of germplasm(Prevost&Wilkinson,1999).(c)Detection methodThe level of polymorphism detected has been shown to vary with the detection method used.Polyacrylamide gel electrophoresis(PAGE)in combination with ra-dioactivity(labelled nucleotide in PCR reaction)was shown to be most sensitive,followed by PAGE with silver staining and then agarose-ethidium bromide sys-tem of detection.Markedly higher number of bands were resolved per primer when polyacrylamide was used compared to agarose(Moreno et al.,1998).In a study on trifoliate orange germplasm,silver stain-ing using high quality chemicals could detect all the bands detected by autoradiography(Fang et al.,1997). However,high levels of polymorphism have been detected even when products of ISSR amplification are resolved on agarose gels without radiolabelling (Tsumura et al.,1996;Arcade et al.,2000;Kojima et al.,1998;Wolff&Morgan-Richards,1998;Sankar &Moore,2001)Thus,the need for radioactivity can be avoided when many samples have to be screened as in germplasm characterization.ISSR-PCR is a simple,quick,and efficient tech-nique.It has high reproducibility.The use of radio-activity is not essential.The primers are not proprietary (as in SSR-PCR)and can be synthesized by any-one.Variations in primer length,motif and anchor are possible.The primers are long(16–25bp)resulting in higher stringency.The amplified products(ISSR markers)are usually200–2000bp long and amenable to detection by both agarose and polyacrylamide gel electrophoresis.In the literature this technique and its variations have been referred to by different names (Table1).ApplicationThe potential for integrating ISSR-PCR into programs of plant improvement is enormous(Table2).The ma-jor areas of the application of ISSR-PCR in different crops are discussed below.GenomicfingerprintingDNAfingerprinting is an important tool for character-ization of germplasm and establishment of the iden-tity of varieties/hybrids/parental sources etc.in plant breeding and germplasm management.Di-nucleotide based ISSR primers anchored at5’or3’end have been used infingerprinting studies with high reprodu-cibility for maintenance of cocoa collection(Charters &Wilkinson,2000).ISSRs showed sufficient poly-morphism to distinguish between various cultivars of chrysanthemum(Wolff et al.,1995).Microspore de-rived plants could be distinguished from those derived from somatic tissues in anther culture offlax at an early seedling stage(Chen et al.,1998).Genetic diversity and phylogenetic analysisISSRs have been successfully used to estimate the extent of genetic diversity at inter-and intra-specific level in a wide range of crop species which include rice (Joshi et al.,2000),wheat(Nagaoka&Ogihara,1997),fingermillet(Salimath et al.,1995),Vigna(Ajibade et al.,2000),sweet potato(Huang&Sun,2000)and Plantago(Wolff&Morgan-Richards,1998).Superi-ority of ISSR-PCR over other marker techniques has been brought out in such investigations by various workers.Anchored SSR primers for instance,have been found to be more useful and reproducible than isozymes,RFLPs and RAPDs in the diversity analysis of trifoliate orange germplasm(Fang et al.,1997). ISSRs were more useful for the analysis of diversity in the genus Eleusine in terms of quality and quant-ity of data output as compared to RFLP and RAPD (Salimath et al.,1995).Significantly,the efficiency of the technique was evident in characterization even at the varietal level of a species.For instance,three5’anchored primers together could distinguish20cul-tivars of Brassica napus(Charters et al.,1996).ISSR is the marker of choice for assessment of genetic diversity in cocoa(Charters&Wilkinson,2000),gym-nosperms such as Douglasfir and sugi(Tsumura et al., 1996)and even fungi(Hantula et al.,1996).In a study on white lupin it has been demonstrated that among 10primers used any two were sufficient to distinguish all the37accessions studied(Gilbert et al.,1999). Similarly,4primers were sufficient to distinguish34 cultivars of potato(Prevost&Wilkinson,1999)and3 primers could distinguish16genotypes of redcurrant (Lanham&Brennan,1998).The use of such highly informative primers lowers the cost,time and labour for diversity analysis.Various marker techniques have been used in phylogenetic investigations based on relative simil-arity.Inspite of their higher efficiency and repro-ducibility ISSR markers have as yet not been used extensively.It has however been found effective in13 Table1.Synonyms of the ISSR-PCR technique and its variantsS.No Terms used Reference1MP-PCR,Microsatellite primed PCR(refers to unanchored primer)Meyer et al.(1993)2SSR-anchored PCR,Inter-SSR amplification Zietkiewicz et al.(1994)3SPAR(single primer amplification reaction)Gupta et al.(1994)4RAMPs(random amplified microsatellite polymorphisms)Wu et al.(1994)5RAMs(randomly amplified microsatellites)Hantula et al.(1996)6AMP-PCR(anchored microsatellite primed PCR)Weising et al.(1998)7ASSR(anchored simple sequence repeats)Wang et al.(1998)resolving problems relating to the phylogeny of Asian cultivated rice Oryza sativa(Joshi et al.,2000),wheat (Nagaoka&Ogihara,1997),finger millet(Salimath et al.,1995),Vigna(Ajibade et al.,2000)and Dip-lotaxis species(Martin&Sanchez-Yelamo,2000). There is immense scope to use this powerful tech-nique in resolving species/inter-species status in many a genus and in deciding the distinctness of different genera within a family.Significantly,genome/species specific ISSR markers have been reported in four gen-era Oryza(Joshi et al.,2000),Lolium and Festuca (Pasakinskiene et al.,2000)and Diplotaxis(Mar-tin&Sanchez-Yelamo,2000)which are useful in delineating species.Genome mappingISSR markers are unmapped but can be used to sat-urate RFLP and SSR linkage maps.The RFLP map of barley was saturated with60ISSRs(referred as RAMPs in the study)which mapped to all chromo-somes(Becker&Heun,1995).Many of these markers are mapped in between clustered RFLPs,flanking RFLP clusters,at the tips of chromosomes and more importantly in areas of low RFLP marker density. In Einkorn wheats,however,the nine ISSR mark-ers mapped at or close to the RFLP marker positions (Kojima et al.,1998).ISSRs have also been used along with AFLP and RAPD markers in the mapping of Ja-panese and European larch genomes(Arcade et al., 2000).The genetic linkage map of Citrus was fur-ther saturated using75ISSR markers,which were dispersed among all the linkage groups(Sankar& Moore,2001).Also it was shown that the level of segregation distortion of ISSRs is lower compared to RAPDs.In soybean,58ISSR markers were mapped onto18RAPD/RFLP linkage groups(Wang et al.,1998).CA polymorphisms had a biased distribution and GA polymorphisms were randomly dispersed. Gene tagging and marker assisted selectionDNA markers closely linked to important agronomic traits greatly contribute to practical crop improvement programs.In rice,an ISSR marker generated by primer (AG)8YC was converted to a sequence tagged site (STS)marker to identify the fertility restoration gene, Rf-1(Akagi et al.,1996).This co-dominant marker can be used in management of genetic purity of hy-brid seed.In chickpea,ISSR markers UBC855500 generated by primer(AG)8YT and UBC8251200us-ing primer(AG)8T were linked to the gene conferring resistance to race4of Fusarium wilt(Ratnaparkhe et al.,1998).Markers closer to a given gene are gener-ated by altering5’or3’anchors.Recently,ISSR-PCR was used in identifying two allelic dominant DNA markers,one linked in coupling and the other in re-pulsion phase to a major locus Fgr,which modulates fructose to glucose ratio in tomatoes(Levin et al., 2000).These PCR products were obtained from two ISSR-PCR reactions using(TC)8CC and(TC)8CG as primers.Another trait of value in hybrid seed produc-tion viz.,temperature-sensitive genic male sterility has been tagged with an ISSR marker UBC8551060in rice (Hussain et al.,2000).ISSRs have also been used to generate species spe-cific,gene specific and trait specific markers.While delineating the phylogenetic relationship among dif-ferent species of the genus Oryza,87putative gen-ome/species specific markers were identified(Joshi et al.,2000).The582bp inter-SSR Festuca specific sequence and1350bp F.arundinacea specific se-quence have potential as markers to confirm presence of closely linked Festuca genes(Pasakinskiene et al., 2000).Likewise,race specific markers have been de-14Table2.Applications of ISSR-PCR techniqueS.No Application Reference1GenomicfingerprintingCocoa germplasm Charters&Wilkinson,2000Potato cultivars Prevost&Wilkinson,1999Chrysanthemum cultivars Wolff et al.,1995 2Genetic diversity and phylogenetic analysisRice cultivars Virk et al.,2000Oryza granulata Qian et al.,2001Wheat(Triticum sp.)Nagaoka&Ogihara,1997Barley(Hordeum vulgare)Sanchez et al.,1996Maize inbred lines(Zea mays)Kantety et al.,1995Fingermillet(Eleusine sp)Salimath et al.,1995Sorghum(Chinese)(Sorghum bicolor)Yang et al.,1996White lupin germplasm(Lupinus albus)Gilbert et al.,1999Vigna sp Ajibade et al.,2000Pea germplasm(Pisum sativum)Lu et al.,1996Soybean(Glycine max)Wang et al.,1998Oilseed rape cultivars(Brassica napus)Charters et al.,1996Sweet potato,wild relatives(Ipomoea sp)Huang&Sun,2000Potato cultivars(Solanum tuberosum)McGregor et al.,2000Redcurrant germplasm(Ribes sp)Lanham&Brennan,1998Grapevine germplasm(Vitis vinifera)Moreno et al.,1998Citrus cultivars(Citrus sp)Fang&Roose,1997Trifoliate orange germplasm(Poncirus trifoliata)Fang et al.,1997Plantago major subspecies Wolff&Morgan-Richards,1998Gymnosperms,Douglasfir and sugi Tsumura et al.,1996 3Genome mappingSaturating RFLP linkage map in barley Becker&Heun,1995Construction of a genetic linkage map in Einkorn wheat Kojima et al.,1998Genetic mapping of Japanese and European types of larch Arcade et al.,2000Saturating genetic linkage map in citrus Sankar&Moore,2001Saturating RFLP/RAPD linkage map in soybean Wang et al.,1998 4Determining SSR motif frequencyRecovery of microsatellite sequences in the mustard genome Varghese et al.,2000Distribution pattern of microsatellites across eukaryotic genomes Gupta et al.,1994Analysis of microsatellite frequency in rice cultivars Blair et al.,19995Gene tagging and use in marker assisted selectionRf-1gene for fertility restoration in rice Akagi et al.,1996Gene for resistance to Fusarium wilt Race4in chickpea Ratnaparkhe et al.,1998Temperature sensitive genic male sterility in rice Hussain et al.,2000Fgr gene for modulating fructose to glucose ratio in tomato Levin et al.,2000Genome/species specific markers in Lolium and Festuca Pasakinskiene et al.,2000Putative genome/species specific markers in Oryza.Joshi et al.,2000Race specific markers in fungi Hantula et al.,1996 6Evolutionary biologyDiplotaxis species Martin&Sanchez-Yelamo,2000Diploid hybrid speciation in Penstemon Wolfe et al.,199815veloped in various fungi groups using ISSRs(Hantula et al.,1996).Determining SSR motif frequencyISSR analysis provides insights into the organization (clustered or not),frequency and levels of polymorph-ism of different simple sequence repeats in a genome. In rice and wheat,di-nucleotide simple sequence re-peats used as primers gave the maximum number of bands and are,therefore,more common than any SSRs with larger units(Blair et al.,1999;Nagaoka&Ogi-hara,1997).Poly(GA)based3’-anchored primers pro-duced5times as many bands as those with poly(GT) motif indicating low frequency or lack of cluster-ing of(GT)motif(Blair et al.,1999).Using ISSRs it has been shown that tetra-nucleotide repeats were abundant across eukaryotic genomes(Gupta et al., 1994)and that tetramers of tetra-nucleotides AGAC and GACA are scattered within the genome of grasses (Pasakinskiene et al.,2000).It has been demonstrated in Brassica that enhanced recovery of microsatellite markers is possible using ISSR primers(Varghese et al.,2000).Studies on natural populations/speciationThe hypervariable nuclear ISSR markers have proved useful in testing hypotheses of speciation,introgres-sion and systematics(Wolfe et al.,1998).The hybrid origin of Penstemon clevelandi was clearly brought out by the use of just8ISSR markers.Population of P. clevelandi has been found to have an additive profile of bands of the two proposed progenitor species viz.P. centranthifolius and P.spectabilis.On the other hand the population of P.spectabilis lacked the additive profile of bands of its proposed putative parents.The hybrid origin of P.spectabilis was thus negated and its origin was attributed instead to introgression of genes and not the genome of a related species.The util-ity of the technique has been demonstrated in a wide range of applications in molecular ecology in plant families which include Asteraceae,Brassicaceae, Hippocastanaceae,Orchidaceae,Poaceae,Scro-phulariaceae and Violaceae(/∼awolfe/issri.issr.html).Variation within and between populations can be compared using dis-persed multilocus markers such as ISSR.It was shown that the amount of variation between O.granulata populations from different regions(49.2%)was higher than that between populations within a region(38%)or within a population(12%)using ISSR markers (Qian et al.,2001).PerspectivesAs the need to protect proprietary germplasm is likely to increase in the future,ISSRs will have an import-ant role in securing plant variety rights by virtue of its unique efficiency in distinguishing even closely re-lated germplasm.To date,more polymorphism has been detected with the use of ISSRs than with any other assay procedure(Gupta et al.,1994;Salimath et al.,1995;Virk et al.,2000).In many of the studies for determining the extent of polymorphism or com-paring marker systems only one family of SSRs,eg. tri-nucleotides or tetra-nucleotides had been used as primers.Such repeats are infrequent as compared to di-nucleotides and their use may not help arrive at precise classification.As more data on the occurrence and distribution of SSR motifs becomes available,it should be possible to use primers that give more ac-curate span of the whole genome.Also,different com-binations of the motif,anchor and length of primers can be used.Strategies to detect additonal polymorph-ism could include use of ISSRs in combination with RAPD(Joshi et al.,2000;Becker&Heun,1995;Wu et al.,1994)or AFLP primers in the same reaction or restriction digestion of ISSR products(Becker& Heun,1995).Unlimited combinations of motif and length of both primers and use of different restriction enzymes are thus possible.Well chosen primers can provide reasonably accuratefingerprinting and thereby quick estimate of genetic diversity especially in large sized accessions to identify core sets and the pattern of geographical distribution.The technique is not without limitations.For in-stance,there is the possibility as in RAPD,that fragments with the same mobility originate from non-homologous regions,which can contribute to some distortion in the estimates of genetic similarities (Sanchez et al.,1996).The molecular nature of the polymorphisms can be known only if the fragments extracted from the gel are sequenced.ISSR mark-ers linked to the traits of agronomic importance have been sequenced and used as STS markers in marker aided selection.An attractive possibility is thus the use of ISSRs as probes for in-situ hybridization for physical mapping of homologous chromosome sites (Pasakinskiene et al.,2000).Another advantage in the use of ISSR markers lies in their linkage to SSR loci.16Although microsatellites themselves are probably non-functional and selectively neutral,they are known to be linked to coding regions,so that ISSRs are likely to mark gene rich regions(Kojima et al.,1998). ReferencesAjibade,S.R.,N.F.Weeden&S.M.Chite,2000.Inter-simple sequence repeat analysis of genetic relationships in the genus Vigna.Euphytica111:47–55.Akagi,H.,Y.Yokozeki,A.Inagaki,A.Nakamura&T.Fujimura, 1996.A co-dominant DNA marker closely linked to the rice nuc-lear restorer gene,Rf-1,identified with inter-SSRfingerprinting.Genome39:1205–1209.Arcade,A.,F.Anselin,P.F.Rampant,M.C.Lesage,L.E.Paques&D.Prat,2000.Application of AFLP,RAPD and ISSR markersto genetic mapping of European and Japanese larch.Theor Appl Genet100:299–307.Becker,J.&M.Heun,1995.Mapping of digested and undiges-ted random amplified microsatellite polymorphisms in barley.Genome38:991–998.Blair,M.W.,O.Panaud&S.R.McCouch,1999.Inter-simple se-quence repeat(ISSR)amplification for analysis of microsatellite motif frequency andfingerprinting in rice(Oryza sativa L).Theor Appl Genet98:780–792.Charters,Y.M., A.Robertson,M.J.Wilkinson&G.Ramsay, 1996.PCR analysis of oilseed rape cultivars(Brassica napus L.ssp.oleifera)using5’-anchored simple sequence repeat(SSR) primers.Theor Appl Genet92:442–447.Charters,Y.M.&M.J.Wilkinson,2000.The use of self-pollinated progenies as‘in-groups’for the genetic characterization of cocoa germplasm.Theor Appl Genet100:160–166.Chen,Y.,G.Hausner,E.Kenaschuk,D.Procunier,P.Dribnenki&G.Penner,1998.Identification of microspore-derived plants inanther culture offlax(Linum usitatissimum L.)using molecular markers.Plant Cell Reports18:44–48.Fang, D.Q.,M.L.Roose,R.R.Krueger&C.T.Federici,1997.Fingerprinting trifoliate orange germplasm accessions with isozymes,RFLPs and inter-simple sequence repeat markers.Theor Appl Genet95:211–219.Fang,D.Q&M.L.Roose,1997.Identification of closely related citrus cultivars with inter-simple sequence repeat markers.Theor Appl Genet95:408–417.Gilbert,J.E.,R.V.Lewis,M.J.Wilkinson&P.D.S.Caligari,1999.Developing an appropriate strategy to assess genetic variab-ility in plant germplasm collections.Theor Appl Genet98: 1125–1131.Godwin,I.D.,E.A.B.Aitken&L.W.Smith,1997.Application of inter-simple sequence repeat(ISSR)markers to plant genetics.Electrophoresis18:1524–1528.Gupta,M.,Y-S.Chyi,J.Romero-Severson&J.L.Owen,1994.Amplification of DNA markers from evolutionarily diverse gen-omes using single primers of simple-sequence repeats.Theor Appl Genet89:998–1006.Gupta,P.K.&R.K.Varshney,2000.The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat.Euphytica113:163–185. Hantula,J.,M.Dusabenyagasani&R.C.Hamelin,1996.Random amplified microsatellites(RAMS)-a novel method for char-acterizing genetic variation within fungi.Eur J for Path26: 159–166.Huang,J.&S.M.Sun,2000.Genetic diversity and relationships of sweet potato and its wild relatives in Ipomoea series Batatas (Convolvulaceae)as revealed by inter-simple sequence repeat (ISSR)and restriction analysis of chloroplast DNA.Theor Appl Genet100:1050–1060.Hussain,A.J.,V.Gupta,J.Ali,P.K.Ranjekar&E.A.Siddiq,2000.Physiological characterization,genetics and molecular mapping of a new source of temperature sensitive genetic male sterility in rice.Fourth International Rice Genetics Symposium,22–27 October2000,IRRI,Philippines,Abstracts p.95.Joshi,S.P.,V.S.Gupta,R.K.Aggarwal,P.K.Ranjekar&D.S.Brar, 2000.Genetic diversity and phylogenetic relationship as revealed by inter-simple sequence repeat(ISSR)polymorphism in the genus Oryza.Theor Appl Genet100:1311–1320.Kantety,R.V.,X.P.Zeng,J.L.Bennetzen&B.E.Zehr,1995.As-sessment of genetic diversity in dent and popcorn(Zea mays L.) inbred lines using inter-simple sequence repeat(ISSR)amplific-ation.Molecular Breeding1:365–373.Kojima,T.,T.Nagaoka,K.Noda&Y.Ogihara,1998.Genetic link-age map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers.Theor Appl Genet96:37–45. Lanham,P.G.&R.M.Brennan,1998.Characterization of the ge-netic resources of redcurrant(Ribes rubrum:subg.Ribesia)using anchored microsatellite markers.Theor Appl Genet96:917–921. Levin,I.N.,E.Gilboa,S.Yeselson,Shen&A..A.Schaffer,2000.Fgr,a major locus that modulates the fructose to glucose ratio in mature tomato fruits.Theor Appl Genet100:256–262. 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Meyer,W.,T.G.Mitchell, E.Z.Freedman&R.Vilgays,1993.Hybridization probes for conventional DNAfingerprinting used as single primers in the polymerase chain reaction to distin-guish strains of Cryptococcus neoformans.J Clin Microbiol31: 2274–2280.Moreno,S.,J.P.Martin&J.M.Ortiz,1998.Inter-simple sequence repeats PCR for characterization of closely related grapevine germplasm.Euphytica101:117–125.Nagaoka,T.&Y.Ogihara,1997.Applicability of inter-simple se-quence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers.Theor Apppl Genet 94:597–602.Pasakinskiene,I.,C.M.Griffiths,A.J.E.Bettany,V.Paplauskiene, M.W.Humphreys,2000.Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses.Theor Appl Genet100:384–390.Prevost,A.&M.J.Wilkinson,1999.A new system of comparing PCR primers applied to ISSRfingerprinting of potato cultivars.Theor Appl Genet98:107–112.Qian,W.,S.Ge&D.Y.Hong,2001.Genetic variation within and among populations of a wild rice Oryza granulata from China。
三角梅ISSR反应体系的建立和优化
三角梅ISSR反应体系的建立和优化李房英;黄彦晶;吴少华【摘要】对影响三角梅ISSR-PCR扩增反应的各个参数进行优化,建立适合三角梅的ISSR反应体系:PCR反应体积为20μL,其中10×buffer(含Mg2+)2.0μL,dNTP 250 μmol/L,Taq酶1.0U,引物0.3μmol/L,模板DNA20ng.扩增程序为:94℃预变性5min;94℃变性1min,51.6℃退火1min,72℃延伸2min,34个循环;最后72℃延伸7min.该反应体系标记点位清晰、稳定、重复性好,适宜三角梅ISSR分析,为应用ISSR技术鉴定三角梅种质资源、分子标记辅助选择育种及其遗传多样性研究奠定了基础.【期刊名称】《海峡科学》【年(卷),期】2010(000)010【总页数】4页(P216-219)【关键词】三角梅;ISSR;体系建立;优化【作者】李房英;黄彦晶;吴少华【作者单位】福建农林大学园林学院;福建农林大学园艺学院;福建农林大学园艺学院【正文语种】中文三角梅(Bougainvillea spectabilis Willd)又名九重葛、毛宝巾、勒杜鹃，在我国已有100多年的栽培历史，品种丰富。
我国部分研究者在同工酶水平上对三角梅品种进行亲缘关系分析但存在难以解释之处[1]，至今仍有分类标准不统一、分类手段有一定的局限性，所得分类结果不一致以及同名异物、同物异名等问题，从而给生产、园林应用、交流等方面带来诸多困难。
而三角梅有关分子标记方面的研究仍是空白，因此利用分子标记技术进行三角梅的分类、品种鉴定等研究显得尤为重要。
ISSR（简单重复序列间隔区，Inter-Simple Sequence Repeat）是由Zietkiewicz等于1994年创建的一种简单序列重复区间扩增多态性的分子标记[2]。
ISSR具有操作简单，标记重复性好，稳定程度高，多态性丰富，DNA用量少，成本低等优点。
麻栗坡兜兰ISSR引物筛选及反应体系的优化
麻栗坡兜兰ISSR引物筛选及反应体系的优化高丽霞【摘要】[目的]对麻栗坡兜兰ISSR引物进行筛选,并建立一个稳定性高、重现性好、适合麻栗坡兜兰ISSR反应体系.[方法]以麻栗坡兜兰DNA为模板,分别对Mg2+、dNTP、引物、Taq DNA聚合酶、DNA等PCR反应成分进行优化,并用优化的体系对25条兰科中报道的ISSR引物进行筛选.[结果]确立了麻栗坡兜兰最适ISSR-PCR反应体系:在25μl反应体系中,Mg2+2 mmol/L、引物0.4 mmoL/L、dNTP 0.20 mmol/L、DNA 1.5μl、Taq酶1.4U.利用该体系,共筛选得到10条ISSR引物用于麻栗坡兜兰分析,并对21份麻栗坡兜兰材料进行扩增,获得了较好的扩增效果.[结论]该体系稳定可靠,为今后ISSR标记在兜兰属植物的种质鉴定、遗传多样性等方面的广泛应用奠定了重要基础.【期刊名称】《安徽农业科学》【年(卷),期】2014(000)020【总页数】3页(P6553-6555)【关键词】麻栗坡兜兰;ISSR-PCR;体系优化【作者】高丽霞【作者单位】河池学院化学与生物工程学院,广西宜州546300【正文语种】中文【中图分类】S188麻栗坡兜兰(Paphiopedilum malipoense)属兰科(Orchidaceae)兜兰属(Paphiopedilum)地生兰或半附生兰。
麻栗坡兜兰是兜兰属现存种类中最原始的类型，代表了由杓兰属向兜兰属过渡的种类[1]，主要分布于云南东南部、广西西部、贵州西北部，以及越南北部。
一方面由于近年来的过渡采摘，另一方面由于其自身的生物学原因，它没有像硬叶兜兰和杏黄兜兰那样延长的地下根状茎[2]，是当前最需要保护的濒临物种之一。
ISSR分子标记技术已在兰科中得以广泛应用。
赵谦等[3]用14条ISSR引物分析了14个蝴蝶兰品种间的遗传关系，其多态百分比为82%，表明蝴蝶兰品种间存在丰富的遗传多样性，并构建了ISSR遗传图谱；吴振兴等[4]用15条ISSR引物对兰属植物进行遗传多样性分析，其多态百分比为27.2%，并构建了ISSR遗传图谱；孙小琴等[5]用12条ISSR引物对江西省寒兰进行了遗传多样性分析，其多态百分比为78.9%；马佳梅等[6]用12条ISSR引物对西双版纳地区流苏石斛进行遗传多样性分析，其多态百分比为89.74%，表明流苏石斛品种间存在丰富的遗传多样性；严华等[7]采用ISSR分子标记技术对38种国兰亲缘关系进行分析；沈颖等[8]用ISSR分子标记技术对9种石斛属植物进行品种鉴定分析。
虉草ISSR-PCR反应体系优化与引物筛选
虉草ISSR-PCR反应体系优化与引物筛选张永亮;刘鹏;骆秀梅;刘杨【摘要】22个虉草基因组DNA为ISSR-PCR扩增模板,采用单因素试验方法,对影响PCR扩增体系中dNTP、引物浓度、Taq酶和模板DNA用量4个因素及引物退火温度进行梯度试验,优化得到最佳的ISSR-PCR反应体系,即20μL反应体系中分别加入0.3μL Taq DNA聚合酶(5 U/μL),2μL 10×PCRBuffer(mg2+ plus),1.5 μL dNTP (2.5 mmol/L),1.5 μL引物(10 pmol/μL),50 ng模板DNA,ddH2O补足体积.以此体系对24条引物进行筛选,最终获得了多态性高,重复性好的引物12条.引物UBC808、809、811、815、818、820、826的适宜退火温度为55℃,引物835,841和842的适宜退火温度为56℃,而引物810和834的适且退火温度分别为52℃和54℃.12条引物共扩增总条带数192条,其中,多态性条带数173条,多态位点百分率89.81％.【期刊名称】《草原与草坪》【年(卷),期】2016(036)003【总页数】7页(P1-6,11)【关键词】虉草;ISSR-PCR反应体系;引物筛选【作者】张永亮;刘鹏;骆秀梅;刘杨【作者单位】内蒙古民族大学,内蒙古通辽028042;内蒙古民族大学,内蒙古通辽028042;内蒙古民族大学,内蒙古通辽028042;内蒙古民族大学,内蒙古通辽028042【正文语种】中文【中图分类】S543;Q786虉草(Phalaris arundinacea)，属于禾本科(Gramineae)、虉草属植物，别名草芦、园草芦。
主要分布于欧洲、北美和亚洲，在我国主要分布于东北、华北、华中、华东等地区。
虉草耐盐碱、耐湿涝，又耐旱，生长快，营养繁殖能力强，产草量高、叶量丰富，蛋白质含量高，被广泛用于饲草、人工湿地植物、生物能源材料或造纸原料等[1-2]。
李种质资源ISSR反应体系引物筛选
安佰义，王 迦，王 ?，等．李种质资源ＩＳＳＲ反应体系引物筛选［Ｊ］．江苏农业科学，２０１９，４７（１０）：６３－６５．ｄｏｉ：１０．１５８８９／ｊ．ｉｓｓｎ．１００２－１３０２．２０１９．１０．０１４李种质资源ＩＳＳＲ反应体系引物筛选安佰义１，王 迦１，王 ?１，于慧颖１，范爱淇１，张艳波２，张 艳３，李 锋２（１．吉林农业大学园艺学院，吉林长春１３０１１８；２．吉林省农业科学院果树研究所，吉林长春１３００３３；３．吉林省农业科学院，吉林长春１３０１２４） 摘要：以李１８个品种种质资源为试验材料，对其开展ＩＳＳＲ反应体系引物筛选，结果表明，从４１个随机引物中筛选出２５个多态性引物用于ＰＣＲ扩增，每个多态性引物扩增出的条带数在８～２３条之间，扩增出的ＤＮＡ片段长度大多在１５０～２４００ｂｐ之间；共扩增出条带数为３８５条，其中，样品间相同的条带数有５３条；所选引物的多态位点百分率为８６．２３％。
关键词：李；种质资源；ＩＳＳＲ；引物；多态位点 中图分类号：Ｓ６６２．３０２．４ 文献标志码：Ａ 文章编号：１００２－１３０２（２０１９）１０－００６３－０３收稿日期：２０１８－０１－３１基金项目：吉林省重点科技攻关项目（编号：２０１６０２０４０３１ＮＹ）。
作者简介：安佰义（１９７８—），男，山东日照人，博士，副教授，从事园林植物种质资源及栽培生理研究。
Ｅ－ｍａｉｌ：ｌｌｉ１０４３７＠１２６．ｃｏｍ。
区间简单重复序列标记（ｉｎｔｅｒ－ｓｉｍｐｌｅｓｅｑｕｅｎｃｅｒｅｐｅａｔ，ＩＳＳＲ）是一种微卫星基础上的第２代分子标记技术，其基本原理是用锚定的微卫星ＤＮＡ为引物，在简单重复序列（ＳＳＲ）的３′端或５′端各加２～４个非重复随机核苷酸序列作为引物，在聚合酶链式反应（ＰＣＲ）中锚定引物可引起特定位点退火，并对重复序列间的ＤＮＡ片段进行ＰＣＲ扩增［１－３］。
ＩＳＳＲ标记结合了随机扩增多态性ＤＮＡ标记（ＲＡＰＤ）、ＳＳＲ技术的优点，具有模板ＤＮＡ用量少、引物设计简单、成本低、ＰＣＲ扩增产物多态性丰富、产物重复性和稳定性好、试验安全性较高及技术难度较低等特点［４］，现已在植物遗传作图、基因定位、品种鉴定、遗传多样性及亲缘关系分析等方面被广泛应用［５－９］。