Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Relationship among acidophilic bacteria from diverse environments as determined by randomly amplified polymorphic DNA analysis (RAPD)

Summary Random amplification of polymorphic DNA (RAPD), a PCR-based technique was applied to evaluate genomic diversity among three strains of Acidithiobacillus thiooxidans, five strains of Acidithiobacillus ferrooxidans and one acidophilic moderate thermophile strain, using 45 random primers of five different series. More than 2200 bands were observed, with an average of 45 bands per primer. Primer OPC-3 produced the maximum number of fragments whereas minimum numbers of fragments were produced with primer OPA-5. A dendrogram was generated using cluster analysis by the unweighted pair group method of arithmetic means (UPGMA). The dendrogram showed three groups with similarity ranging from 29 to 85%. The maximum similarity (85%) was observed between the strains T.t₁ and T.t₂ of Acidithiobacillus thiooxidans.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Phylogenetic relatedness among Spirulina and related cyanobacterial genera

Molecular polymorphisms in a selected set of Spirulina and related genera using random primers based on repetitive sequences along with biochemical parameters, led to the unambiguous differentiation of the strains and understanding of their phylogenetic relationships. A combination of 10 sets of dual primers generated 100% distinct polymorphic bands ranging from 150 to 5,000 bp. Total number of fragments ranged from 68 to 159 whereas polymorphic bands ranged from 13 to 32 for different Random Amplified Polymorphic DNA (RAPD) reactions. Spirulina platensis strains, Sp-2 and Sp-3, possessed quite comparable chlorophyll and protein content besides having maximum similarity coefficient (0.88) between them on the basis of RAPD reactions, thus proved to be closely related. Sp-8 (Spirulina from Loktak Lake) having the highest protein content and protein: chlorophyll ratio, showed close similarity with the mutant of Spirulina platensis (Sp-7) on the basis of RAPD analysis. Duncan’s Multiple Range Test (DMRT) ranking for the biochemical parameters were quite closer for the strains of Spirulina and Arthrospira. This is also supported by the cluster analysis based on RAPD data, as the strains of Spirulina and Arthrospira are placed together in the same subcluster in the dendrogram. The comparative closeness among the strains of Lyngbya, Oscillatoria and Phormidium is reflected by the low content of protein and protein: chlorophyll ratio, which is also supported by the dendrogram based upon RAPD; thus, exhibiting the usefulness of multiplex RAPD along with biochemical parameters for the phylogenetic analysis of Spirulina and related genera.     

A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite blackgram genotypes. A total of 25 random and 16 ISSR primers were used. Amplification of genomic DNA of the 18 genotypes, using RAPD analysis, yielded 104 fragments that could be scored, of which 44 were polymorphic, with an average of 1.8 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from two (OPA-13) to nine (OPK-4) and varied in size from 200 bp to 2,500 bp. Percentage polymorphism ranged from 16.6% (OPK-7) to a maximum of 66.6% (OPE-5, OPH-2, and OPK-8), with an average of 42.7%. The 16 ISSR primers used in the study produced 101 bands across 18 genotypes, of which 55 were polymorphic. The number of amplified bands varied from two (ISSR 858) to ten (ISSR 810), with a size range of 200–2,200 bp. The average numbers of bands per primer and polymorphic bands per primer were 6.3 and 3.4, respectively. Percentage polymorphism ranged from 25% (ISSR 885) to 100% (ISSR 858), with an average percentage polymorphism of 57.5% across all the genotypes. The 3-anchored primers based on poly(GA) and poly(AG) motifs produced high average polymorphisms of 54.98% and 58.32%, respectively. ISSR markers were more efficient than the RAPD assay, as they detected 57.4% polymorphic DNA markers in Vigna mungo as compared to 42.7% for RAPD markers. The Mantel test between the two Jaccards similarity matrices gave r =0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR.     

曲霉菌的RAPD分析及其在酿造工业中的应用

以米曲霉沪酿3.042(AS3.951)、黄曲霉GIM3.18、酱油曲霉AS3.495为参照,利用RAPD分子标记技术对16株曲霉菌进行系统发育分析。通过改进提取方法,获得了质量较好的模板DNA,凝胶电泳结果和分光光度法检测结果表明其适合用于进一步的RAPD-PCR试验。从9个待选引物中筛选到3个扩增产物谱带多、特征好、覆盖面广的引物:Primer1、Primer2、Primer5,重复实验证明其RAPD-PCR扩增图谱具有较好的稳定性,扩增产物谱带一般8~14条,各试验菌株主带4~9条,次带丰富。由此构建的系统进化树较好地吻合了传统的形态分类学,证实了RAPD分子标记技术在此类微生物系统发育分析中应用的可行性,也为酿造工业中检出产黄曲霉毒素的污染菌株提供了理论基础。     

Correspondence of ISSR and RAPD markers for comparative analysis of genetic diversity among different apricot genotypes from cold arid deserts of trans-Himalayas

The phylogenetic relationships of 36 locally grown Prunus armeniaca genotypes which are collected from nine sampling sites from two valleys viz. Nubra (9,600 ft) and Leh (11,500 ft) of trans-Himalayan region were analyzed using 31 PCR markers (20 RAPDs and 11 ISSRs). This is the first report of molecular genetic diversity studies in apricot from this region of the world. RAPD analysis yielded 139 fragments, of which 136 were polymorphic, with an average of 6.8 polymorphic fragments per primer. ISSR analysis produced 58 bands, of which 56 were polymorphic, with an average of 5.09 polymorphic fragments per primer. The primers based on (CT)n produced maximum number of bands (nine) while, (AT)n and many other motifs gave no amplification. RAPD markers were found more efficient with regards to polymorphism detection, as they detected 97.84 % as compared to 96.5 % for ISSR markers. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR. The results of PCA analysis were comparable to the cluster analysis. These analyses, allowed us to identify the groups corresponding to the two apricot collection sites.Key words: Prunus armeniaca, Apricot, Genetic Diversity, RAPD, ISSR, AMOVA     

Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 μg, respectively.     

Detection of Intra-Clonal Genetic Variability in Vegetatively Propagated Tea Using RAPD Markers

The role of random amplified polymorphic DNA (RAPD) markers in detecting intra-clonal genetic variability in vegetatively propagated UPASI-9 clone of tea (Camellia sinensis) was studied. Twenty five decamer primers were used, of which three did not amplify, three gave single bands and the rest of nineteen primers generated upto twelve bands (an average of 6.3 bands per primer). Twenty one primers exhibiting amplified products gave monomorphic banding patterns. Only one primer (OPE-17) gave a unique extra band of similar size in four plants.     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method

The genetic relationship between twenty-six strains of Agaricus bisporus were analysed by the RAPD (random amplified polymorphic DNA) method. DNA amplification was performed with the use of twelve arbitrary 10-mer primers. Four primers, which gave polymorphic band patterns were chosen for RAPD analysis. In total, they gave 24 distinguishable bands, of which nine were polymorphic. The conducted research showed that there is a great genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a limited genetic pool used in the cultivation of those strains.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Genetic diversity analysis and development of SCAR marker for detection of Indian populations of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis> causing chickpea wilt

Genetic diversity of the isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from 12 states representing different agro-ecological regions of India was determined through randomly amplified polymorphic DNA (RAPD) markers. The UPGMA cluster analysis grouped the isolates into eight categories showing high magnitude of genetic diversity. Each group had the isolates from different states present in various agro-ecological regions of India. Therefore, the groups generated through the RAPD analysis were not corresponding to area of the origin of the isolates. The RAPD primers, namely, OPA 7 and OPA 11 produced Foc specific fragment of ≈1.3 kb and ≈1.4 kb, respectively in all the isolates. These fragments were eluted, purified, cloned in pGEM-T Easy vector and sequenced. Primers were designed with sequence information of these two fragments using primer.3 software. Two sets of sequence characterized amplified region markers (SC-FOC 1 and SC-FOC 2) developed from the sequences of these fragments were found to be specific to Foc and produced an amplicon of 1.3 and 1.4 kb, respectively. These set of markers were validated against the isolates of the pathogen collected from different locations of India representing various races of the pathogen. They are non-specific to the other Fusarium species, Rhizoctonia solani and R. bataticola.     

满天星试管苗与其玻璃化苗的RAPD指纹图谱分析

采用分离群体分组分析法(BSA),用100个随机引物对满天星的正常苗和玻璃化苗进行RAPD分析的结果表明,7个随机引物扩增出多态性差异条带。再用上述7个引物分别对试管苗及其玻璃化苗个体进行DNA的PCR扩增的结果显示,引物J20在2种苗中出现差异条带。     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵（Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较。26个RAPD引物产生了总计192条DNA条带,大小分布 于0.26kb-1.98kb之间,其中165条（86．12％）具有多态性,每条引物产生DNA条带的平均数为7．38。8对AFLP引物组合共产生了576条带,分布于100bp-500bp之间,其中的341条具有多态性,多态百分率为76．00％,每对引物组合产生DNA条带的平均数为72。RAPD方法检测的每位点有效等位基因数（1．76）大于AFLP（1．65）,AFLP标记位点的平均多态性信息量（PIC）（0．38）低于RAPD标记位点PIC（0．41）,但AFLP标记具有很高的多态性检测效率（Ai=38．52）。用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47．84％－82．06％,平均相似性系数为0．6495,而采用AFLP的Nei氏相似性系数分布在54．15％－83．52％,平均相似性系数为0．6884。RAPD数据的标准差为0．13,而AFLP数据的标准差为0．08。因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反。源于两种不同标记的遗传相似矩阵的相关系数为0．51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性,无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群。     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Assessment of genetic fidelity of encapsulated microshoots of <Emphasis Type="Italic">Picrorhiza kurrooa</Emphasis>

In vitro grown microshoots of Picrrhiza kurrooa were encapsulated in the alginate beads. Regrowth of encapsulated microshoots, using alginate encapsulation, of P. kurrooa reached 89.33% following 3 months of storage. Amongst developing plantlets, 42.66% exhibited formation of multiple shoots at the onset of regrowth and 21.43% demonstrated simultaneous formation of shoots and roots. Healthy root formation was observed in plantlets following 2 weeks of their transfer to half-strength Murashige and Skoog medium containing 1 μM α-naphthalene acetic acid. Plants were transplanted to the greenhouse in three batches with 95% frequency of survival. The genetic fidelity of P. kurrooa plants growing out after storage in encapsulated form was ascertained by random amplified polymorphic DNA (RAPD) analysis. Molecular analysis of randomly selected plants from each batch was conducted using 45 random decamer primers. Of 45 primes tested, 14 produced scorable amplified products. Total 68 bands were observed amongst them 7.35% bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient of 0.966 thus confirming genetic stability of plants derived from encapsulated microshoots following 3 months of storage.