Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance

RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.     

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.     

Random amplified polymorphic DNA (RAPD) markers in hop, Humulus lupulus: level of genetic variability and segregation in F1 progeny

The level of genetic variation in 24 hop genotypes was studied using the recently developed technique for producing random amplified polymorphic DNAs (RAPDs). Of the 60 primers screened, eight produced polymorphic RAPD bands, 38 produced bands that were monomorphic for all genotypes and 19 did not produce any amplification product. It appeared that the level of polymorphism among the genotypes was generally low. Three of the primers, A11, A17 and C9, were used to determine the stability and segregation of RAPD markers in five families with a total of 182 F₁ progeny. The segregation ratios of these markers in the f₁ progeny suggested that they were inherited in a Mendelian manner. RAPD markers were stable and may be useful for the construction of linkage maps in hop.     

Evaluation of the genetic fidelity of in vitro-propagated gerbera (<Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus) using DNA-based markers

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard’s coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones.     

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。     

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．     

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.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

RAPD标记在山葡萄种质鉴定中的应用

采用修改后的CTAB 法获得了高质量的基因组DNA 。利用RAPD 标记技术对山葡萄7 份种质进行鉴定, 用4 个引物(从30 个引物中筛选)对试材进行PCR 扩增, 共扩增出30 条谱带, 平均每条引物产生7.5 条谱带, 其中21 条谱带为多态性谱带, 占总谱带数的70%。不同引物扩增的谱带数不同, 范围在6~9 条之间。利用4 个引物扩增出的多态性谱带可以将7 份山葡萄种质区分。     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

Analysis of genetic diversity through AFLP,SAMPL, ISSR and RAPD markers in <Emphasis Type="Italic">Tribulus terrestris</Emphasis>, a medicinal herb

Tribulus terrestris is well known for its medicinal importance in curing urino-genital disorders. Amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers were used for the first time for the detection of genetic polymorphism in this medicinal herb from samples collected from various geographical regions of India. Six assays each of AFLP and SAMPL markers and 21 each of ISSR and RAPD markers were utilized. AFLP yielded 500 scorable amplified products, of which 82.9% were polymorphic. SAMPL primers amplified 488 bands, 462 being polymorphic (94.7%). The range of amplified bands was 66 [(TC)₈G + M-CAG] to 98 [(CA)₆AG + M-CAC] and the percentage polymorphism, 89.9 [from (CT)₄C (AC)₄A + M-CTG] to 100 [from (GACA)₄ + M-CTA]. The ISSR primers amplified 239 bands of 0.4–2.5 kb, 73.6% showed polymorphism. The amplified products ranged from 5 to 16 and the percentage polymorphism 40–100. RAPD assays produced 276 bands, of which 163 were polymorphic (59%). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.9 for all the four marker systems. The dendrograms and PCA plots derived from the binary data matrices of the four marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. The relative efficiency of the four molecular marker systems calculated on the basis of multiplex ratio, marker index and average heterozygosity revealed SAMPL to be the best. Distinct DNA fingerprinting profile, unique to every geographical region could be obtained with all the four molecular marker systems. Clustering can be a good indicator for clear separation of genotypes from different regions in well-defined groups that are supported by high bootstrap values.     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Comparison of RAPD and ISSR markers for assessment of genetic diversity among endangered rare Dalbergia oliveri (Fabaceae) genotypes in Vietnam

Dalbergia oliveri is a leguminous tree of the Fabaceae family. This species is popular and valuable in Vietnam and is currently listed on the Vietnam Red List and on the IUCN Red List as endangered. Two PCR techniques using RAPD and inter-simple sequence repeat (ISSR) markers were used to make a comparative analysis of genetic diversity in this species. Fifty-six polymorphic primers (29 RAPD and 27 ISSR) were used. The RAPD primers produced 63 bands across 35 genotypes, of which 24 were polymorphic. The number of amplified bands varied from one to four, with a size range from 250 to 1400 bp. The percentage polymorphism ranged from 0 to 75. Amplification of genomic DNA of the 35 genotypes, using ISSR analysis, yielded 104 fragments, of which 63 were polymorphic. The number of amplified fragments using ISSR primers ranged from one to nine and varied in size from 250 to 1500 bp. The percentage polymorphism ranged from 0 to 100. ISSR markers were relatively more efficient than RAPDs. The mental test between two Jaccard's similarity matrices gave r ≥0.802, showing good fit correlation between ISSRs and RAPDs. Clustering of isolates remained more or less the same for RAPDs compared to combined RAPD and ISSR data. The similarity coefficient ranged from 0.785 to 1.000, 0.698 to 0.956 and 0.752 to 0.964 with RAPD, ISSR, and the combined RAPD-ISSR dendrogram, respectively.     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

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     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Analysis of genetic diversity among garden- and field-pea genotypes of higher Indian Himalayas

Genetic diversity among 31 genotypes of field and garden pea including primitive cultivated forms and widely cultivated varieties of India was studied using 40 random decamer and 9 ISSR primers. A total of 274 amplicons were detected using both types of markers, which amplified 192 RAPD and 82 ISSR amplicons. Average number of bands amplified per primer was higher in case of ISSR (9.1) as compared to RAPDs (4.8). ISSR primers also exhibited higher average polymorphism (89.0%) and resolving power (4.50) than RAPDs (72.4%, 1.87, respectively). Genetic similarity estimates based on the pooled data of both types of markers using Jaccard??s coefficient ranged from 0.58 to 0.85 delineating considerable diversity among the pea genotypes studied. The 31 genotypes clustered in two major groups based on pooled data. Popular cultivars of garden and field pea of the region exhibited high similarities among themselves. However, primitive cultivated forms collected from the higher Indian Himalayas were diverse from the current varieties and hold potential in pea breeding programmes.     

Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.