Detection of polymorphic loci in Arachis germplasm using random amplified polymorphic DNAs.

The development of easily scoreable genetic markers in Arachis will facilitate the introgression of desirable traits from wild species into adapted germplasm. We have used random amplified polymorphic DNAs (RAPDs) to identify polymorphic molecular markers in a range of wild and cultivated Arachis species. From a total of sixty 10-mer oligonucleotide primers, 49 polymorphic loci were identified between cultivated A. hypogaea type (TMV-2) and a synthetic amphidiploid (B x C)2 created from a A. batizocoi and A. chacoense cross. The inheritance of polymorphic markers, both in the amphidiploid and in the F1 progeny in a TMV-2 x (B x C)2 cross, has also been demonstrated. The potential exploitation of RAPD markers in groundnut improvement programs is discussed.     

Identification and analysis of genetic variation among rose cultivars using random amplified polymorphic DNA

Identified germplasm is an important component for efficient and effective management of plant genetic resources. Traditionally, cultivars or species identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and analysis of genetic variation within 34 rose cultivars through random amplified polymorphic DNA (RAPD) markers. Analysis was made by using twenty five decamer primers. Out of twenty five, ten primers were selected and used for identification and analysis of genetic relationships among 34 rose cultivars. A total of 162 distinct DNA fragments ranging from 0.1 to 3.4 kb was amplified by using 10 selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 34 rose cultivars form 9 clusters. The first cluster consists of eight hybrid cultivars, three clusters having five cultivars each, one cluster having four cultivars, two clusters having three cultivars each and two clusters having one cultivar each. The genetic distance was very close within the cultivars. Thus, these RAPD markers have the potential for identification of clusters and characterization of genetic variation within the cultivars. This is also helpful in rose breeding programs and provides a major input into conservation biology.     

Optimizing the generation of random amplified polymorphic DNAs in chrysanthemum

Many conditions of the RAPD reaction procedure may influence the result. This paper presents rapid detection of influential factors with a fractional factorial experiment. A more extensive study of these factors is also presented. Polymerase brand, thermal cycler brand, annealing temperature, and primer, are important factors in obtaining good DNA yields and optimal fragment patterns. Each primer has its optimal annealing temperature, and this is not correlated with the GC content of the primer. Optimal species-primer combinations have to be found by trial and error.     

Genetic diversity among elite Sorghum lines revealed by restriction fragment length polymorphisms and random amplified polymorphic DNAs

The genetic diversity of sorghum, as compared to corn, is less well characterized at the genetic and molecular levels despite its worldwide economic importance. The objectives of this study were to: (1) investigate genetic diversity for restriction fragment length polymorphism (RFLPs) and random amplified polymorphic DNAs (RAPDs) in elite sorghum lines, (2) compare similarities based on molecular markers with pedigree relationships, and (3) examine the potential of RFLPs and RAPDs for assigning sorghum lines to the A/B (sterile) and R (restorer) groups. Using four restriction enzymes, polymorphism was detected with 61% of the RFLP probes used, compared to 77% of the random primers. One hundred and sixteen (64%) probe-enzyme combinations yielded multiple-band profiles compared to 98% of the random primers. RFLP profiles generated 290 polymorphic bands compared to 177 polymorphic RAPDs. Pair-wise comparisons of polymorphic RFLPs and RAPDs were used to calculate Nei and Jaccard coefficients. These were employed to generate phenograms using UPGMA and neighborjoining clustering methods. Analysis of RFLP data with Jaccard's coefficient and neighbor-joining clustering produced the phenogram with the closest topology to the known pedigree.Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-365     

Identification of DNA polymorphism in cultivated groundnut using random amplified polymorphic DNA (RAPD) assay.

Construction of a genetic linkage map is necessary to apply marker-assisted selection tools in a crop improvement program. Except for the recent studies from two laboratories, most of the previous studies have shown little or no DNA polymorphism in cultivated groundnut (Arachis hypogaea L.). In the present study, 70 selected genotypes, representing variability for several morphological, physiological, and other characters, were studied for polymorphism employing random amplified polymorphic DNA (RAPD) assay with 48 oligonucleotide primers. Of the 48 oligonucleotide primers only 7 (14.6%) yielded polymorphic amplification products. The total number of bands from the 7 primers was 408, of which 27 were polymorphic. Detection of polymorphism in cultivated groundnut opens up the possibility of development of its molecular map by judicious selection of genotypes that show DNA polymorphism. This approach will be useful for developing marker-assisted selection tools for genetic enhancement of groundnut for desirable traits.     

Isolation and random amplified polymorphic DNA analysis of genomic DNA from soybean embryos

Summary An isolation procedure was developed for the extraction of genomic DNA and Random Amplified Polymorphic DNA (RAPD) analysis using individual soybean embryos. This procedure can be used to quickly and efficiently isolate DNA from a large number of individuals. DNA isolations were analyzed for total yield, integrity, and usefulness as a template in RAPD analysis.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA

Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly ifferentiated from T. mentagrophytes by using primer 5-GAAGGCTCCC-3 (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.This revised version was published online in October 2005 with corrections to the Cover Date.     

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.     

Estimating nucleotide diversity from random amplified polymorphic DNA and amplified fragment length polymorphism data

A way to estimate the index of nucleotide diversity (pi) from band match frequencies in random amplified polymorphic DNA and amplified fragment length polymorphism data is described. pi is shown to be a simple function of the proportion of mismatched bands between two individuals drawn at random from a population (phi) and the number of discriminating sites in the amplification system. The method is computationally and conceptually simple and avoids some of the assumptions inherent in other approaches: the relationship is independent of the base composition of the target DNA and avoids the bias inherent in estimations of allelic frequencies in dominant systems. Only two individuals from a population are needed to estimate pi. This economy of material suggests utility of this approach in conservation genetics or other fields where obtaining large samples is impractical or undesirable.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.     

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD)

Aim: Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Methods and Results: Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. Conclusion: A high level of genetic diversity was observed, which indicates that the Kolli Hills’C. violaceum isolates would fall into at least three new clusters. Significance and Impact of the Study: Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area.     

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs)

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

Detection of somaclonal variation in cultured rice cells using digoxigenin-based random amplified polymorphic DNA

A procedure for the non-radioactive detection of random amplified polymorphic DNA (RAPD) was developed and designated as digoxigenin (DIG)-based RAPD. Using this procedure, we analyzed somaclonal variation in cultured cells of rice. Somaclonal variation was found to increase with culture age. More than 50 polymorphic fragments were identified with the four primers tested. Random sequencing of 10 clones generated one intron, one 5′-noncoding, and eight non-redundant expressed sequences. A database search for homology showed that the eight exon sequences displayed a significant similarity to sequences already stored in EMBL, GenBank and DDBJ. The sources of the known genes ranged from microorganism to human, including three rice genes. The results showed that somaclonal variation might have occurred in transfer RNA, ribosomal protein, and other genes during cell culture. Received: 14 November 1997 / Revision received: 21 August 1998 / Accepted: 31 August 1998