R-ISSR marker as a useful tool for detection of new genomic loci in <Emphasis Type="Italic">Arthrocnemum macrostachyum</Emphasis>

Arthrocnemum macrostachyum, is a perennial halophytic shrub typical of Mediterranean salt marshes. The present study aims to investigate some combinations of inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) primers applied in real PCR. Thereby, the potential of R-ISSR markers to detect new genomic loci in 3 genotypes of A. macrostachyum grown in the Western coast of Syria was examined. Different combinations of RAPD and ISSR primers produced bands that were absent when single ISSR or RAPD primers were used. The results have demonstrated that ISSR primer (AG)₈TC gave more informative pattern when combined with different RAPD primers comparing to other tested primers. In contrast, the tested ISSR primer (GACA)₄ gave less informative pattern when used alone. These combinations were successfully applied in real PCR to detect new genomic variability in A. macrostachyum genotypes.     

Evaluation of RAPD, ISSR and AFLP Markers for Characterization of the Loose Smut Fungus Ustilago tritici

Random Amplified Polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) profiling were evaluated for assessing the extent of genetic variation among the isolates of Ustilago tritici (Pers.) Rostr., which causes the loose smut disease of wheat.Thirty random decamer primers, six random primer pairs, four SSR primers such as (GACA)₄, (GATA)₄, (CAA)₅ and (GTG)₅ and nine combinations of AFLP selective primers were used to characterize nine isolates of the fungus. These isolates were collected from infected earheads of seven commercial wheat cultivars grown at eight different locations in Haryana, which is a major wheat growing state in the North-West Plain Zone of India. The RAPD and ISSR primers generated 21 0 scorable amplified fragments, all of which were monomorphic among the isolates.The AFLP primer combinations generated 239 fragments out of which 193 were polymorphic. All the isolates could be precisely differentiated from each other employing AFLP and grouped into two distinct clusters.The molecular classification partly corresponded with geographic distribution and host origin of the isolates. AFLP profiling was found superior to RAPD and ISSR and can be effectively utilized for further characterization of loose smut pathogen.     

Analysis of VNTR loci in fish genomes using synthetic oligodeoxyribonucleotide probes

A set of synthetic oligodeoxyribonucleotide (oligo) probes, OAT18, OMS1 and OAT24 carrying the (TGG)₆, (GGAT)₄ and (GACA)₆ repeat motifs, respectively, was used to analyze the variable number tandem repeat (VNTR) loci in the genomes of Oncorhyncus mykiss (rainbow trout; family Salmonidae), Oreochromis mossambicus and Oreochromis niloticus (both tilapia belonging to family Cichlidae). Of all the oligos and enzymes (AluI, MboI, HaeIII and HinfI) used, the OAT18/HaeIII combination was found to be most informative for detecting DNA fingerprinting in rainbow trout, while the OMS1/MboI combination gave the most informative pattern for the Or. niloticus genome. In the rainbow trout genome, all three repeat loci were hypervariable, revealing varying degrees of polymorphism as compared to tilapia genomes. Startlingly, the OAT24 probe did not cross-hybridize with Or. mossambicus and lamprey salmon (Lampetra japonica) although GACA repeats have been reported to be evolutionarily conserved in all eukaryotes studied thus far. Cluster analysis with respect to GGAT repeat loci revealed that Or. niloticus diverged from Or. mossambicus before the separation of On. mykiss, suggesting the relatively recent evolution of these loci in rainbow trout, compared to the tilapia genomes. These highly informative probes will find application in various genetic studies of fishes.     

A new method for RAPD primers selection based on primer bias in nucleotide sequence data

Sequence analysis has proved that decamer nucleotides, used as primers of RAPD (random amplified polymorphic DNA), differ with each other greatly in number of annealing sites in the Arabidopsis thaliana genome. It is called the 'primer bias' by the authors. The biased primers produce a highly variable number of amplicons by polymerase chain reaction (PCR). The number of amplicons is proved to correlate with the number of annealing sites. Therefore, a statistical method is proposed for selecting efficient primers based on the primer bias in the genomic sequence. The method was tested by experiment in A. thaliana genome, and the results demonstrate that the method outperforms routine methods and can substantially increase the efficiency of RAPD methodologies. We also proved that the expressed sequence tags (ESTs) show a highly coincident bias pattern with that of the whole genomic sequence, and can therefore be used to assess efficiencies of primers for species whose genomic sequence data are currently unknown.     

Increased detectability of somatic changes in the DNA from human tumours after probing with “synthetic” and “genome-derived” hypervariable multilocus probes

Summary DNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)₄, (CAC)₄/ (GTG)₅] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensitities were observed. Together the probes 33.15 and (CAC)₅/(GTG)₅ detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)₄ and (CAC)₅/(GTG)₅.     

Conserved PCR primer set designing for closely-related species to complete mitochondrial genome sequencing using a sliding window-based PSO algorithm

Background

Complete mitochondrial (mt) genome sequencing is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. For long template sequencing, i.e., like the entire mtDNA, it is essential to design primers for Polymerase Chain Reaction (PCR) amplicons which are partly overlapping each other. The presented chromosome walking strategy provides the overlapping design to solve the problem for unreliable sequencing data at the 5′ end and provides the effective sequencing. However, current algorithms and tools are mostly focused on the primer design for a local region in the genomic sequence. Accordingly, it is still challenging to provide the primer sets for the entire mtDNA.

Methodology/Principal Findings

The purpose of this study is to develop an integrated primer design algorithm for entire mt genome in general, and for the common primer sets for closely-related species in particular. We introduce ClustalW to generate the multiple sequence alignment needed to find the conserved sequences in closely-related species. These conserved sequences are suitable for designing the common primers for the entire mtDNA. Using a heuristic algorithm particle swarm optimization (PSO), all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation. The overlap requirement for PCR amplicons in the entire mtDNA is satisfied by defining the overlapping region with the sliding window technology. Finally, primer sets were designed within the overlapping region. The primer sets for the entire mtDNA sequences were successfully demonstrated in the example of two closely-related fish species. The pseudo code for the primer design algorithm is provided.

Conclusions/Significance

In conclusion, it can be said that our proposed sliding window-based PSO algorithm provides the necessary primer sets for the entire mt genome amplification and sequencing.     

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.     

Genome and species relationships in genus<Emphasis Type="Italic"> Avena</Emphasis> based on RAPD and AFLP molecular markers

Species and genome relationships among 11 diploid (A and C genomes), five tetraploid (AB and AC genomes) and two hexaploid (ACD genome) Avena taxa were investigated using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers. The two primer pairs used for the AFLP reactions produced a total of 354 polymorphic bands, while 187 reproducible bands were generated using ten RAPD primers. Genetic similarities amongst the entries were estimated using the Jaccard and Dice algorithms, and cluster analyses were performed using UPGMA and neighbor joining methods. Principle coordinate analysis was also applied. The highest cophenetic correlation coefficient was obtained for the Jaccard algorithm and UPGMA clustering method (r=0.99 for AFLP and r=0.94 for RAPD). No major clustering differences were present between phenograms produced with AFLPs and RAPDs. Furthermore, data produced with AFLPs and RAPDs were highly correlated (r=0.92), indicating the reliability of our results. All A genome diploid taxa are clustered together according to their karyotype. The AB genome tetraploids were found to form a subcluster within the A_s genome diploids (AFLPs), indicating their near-autoploid origin. The AC genome tetraploids are clustered to the ACD genome hexaploids. Finally, the C genome diploids form an outer branch, indicating the major genomic divergence between the A and C genomes in Avena.Communicated by J.S. Heslop-Harrison     

Phylogenetic analysis of cyanobacterial strains of genus-Calothrix by single and multiplex randomly amplified polymorphic DNA-PCR

Analyses of molecular polymorphisms in a selected set of Calothrix strains, using primers based on repetitive sequences in the genome, led to the unambiguous differentiation of the strains as well as understanding of their genetic relationships. Seventeen 10 mer random primers were used singly and twelve dual primer combinations were used to examine the phylogenetic relatedness amongst the strains using RAPD- PCR. A total of nine hundred distinct polymorphic DNA fragments (bands), ranging from 0.18 kb to 5.00 kb were produced in PCR reaction with single oligos. A combination of twelve sets of primers generated nine hundred three distinct polymorphic DNA fragments (bands), ranging from 0.13 kb to 6.22 kb, which revealed a wide range of variability amongst the strains. The combined analysis of single and multiplex primer combination showed a maximum correlation coefficient of 0.821 amongst two strains (Ca28 and Ca29) with chlorophyll contents of 4.08 μg/ml and 3.57 μg/ml. These two isolates belonged to same geographical location. The study undertaken has revealed extensive evidence for the applicability of RAPD in cyanobacterial taxonomy, and furthermore, clearly demonstrated the superior discriminative power of RAPD towards the differentiation of geographically unrelated Calothrix strains.     

Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

Chloroplast and mitochondrial SSR help to distinguish allo-cytoplasmic male sterile types in cabbage (Brassica oleracea L. var. capitata)

The profiles of single sequence repeat (SSR) in six distinct allo-cytoplasmic male sterile (CMS) types of cabbage (Brassica oleracea L. var. capitata) were generated using 32 SSR primer pairs derived from the Arabidopsis thaliana chloroplast (cp) genome and another 21 SSR primers from the B. napus mitochondrial (mt) genome sequences. In total, 11 cpSSR and 4 mtSSR primers revealed polymorphism among the six cabbage CMS types, namely NigCMS, OguCMSR₁, OguCMSR₂, OguCMSR₃, OguCMSHY and PolCMS. Through cluster analysis, six cabbage CMS types could be unambiguously differentiated with just three sets of primers (ACP43, ACP47, mtSSR2). Analysis of the selected amplicon sequences showed high identity to that of the corresponding sequences in A. thaliana, B. rapa and B. napus. The aligned cluster analysis revealed that the polymorphism mainly included SSR number variation, single nucleotide polymorphism (SNP), and sequence insertion or deletion (InDel). Our results demonstrated that specific mitochondrial or chloroplast SSR analysis could be a feasible alternative means for cabbage CMS type identification.     

The chromosomal organization of simple sequence repeats in wheat and rye genomes

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)₁₂, (CAT)₅, (AAG)₅, (GCC)₅ and, in particular, (GACA)₄ hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)₄ at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae). Received: 13 February 1998; in revised form: 18 August 1998 / Accepted: 18 August 1998     

Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses

Simple-sequence repeats (SSRs) comprising three tetranucleotide repeat sequences with two-base ’anchors’, namely 5′-(AGAC)₄GC, 5′-AC(GACA)₄ and 5′-(GACA)₄GT, were used in PCR reactions as primers to develop inter-SSR DNA fingerprints of the outbreeding grass species Lolium multiflorum, L. perenne, Festuca pratensis and F. arundinacea. Each species was represented by DNA samples from 3 to 6 varieties. In all four species distinctive species-specific DNA profiles were produced that were common across a number of varieties despite their diverse origin. While the fingerprints of the two ryegrasses, L. multiflorum and L. perenne, were the most similar, a number of inter-SSR DNA markers were generated that enabled them to be distinguished from each other. Some slight variations were found between varieties, which provided putative variety-specific markers for cultivar identification. In addition, variations in the DNA profiles of the genotypes of L. multiflorum and F. pratensis were examined, and the results showed that variety-specific fingerprints are integrated patterns made up from the profiles of individual genotypes. Amongst the primers used, AC(GACA)₄ generated the best distinction between Lolium and Festuca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequences, ranging in size between 550 bp and 1,600 bp, were cloned: three clones for F. pratensis, one clone for L. multiflorum and one clone for F. arundinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridization confirmed the presence of this fragment in F. arundinacea (which contains one genome of F. pratensis), but no homology was found with L. multiflorum. However, a F. arundinacea clone amplified with (GACA)₄GT, pFa 104H1350, was found to be unique to the F. arundinacea genome. Received: 23 June 1999 / Accepted: 27 August 1999     

Efficiency of different PCR-based marker systems for assessment of Iris pumila genetic diversity

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccard? genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Nei? gene diversity and the Shannon? index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.     

(C-A) and (G-A) anchored simple sequence repeats (ASSRs) generated polymorphism in soybean, Glycine max (L.) Merr.

 We used thirty simple sequence repeats (SSRs) with a variable 2–4 base ‘anchor’ at their 5′ ends (ASSRs) independently or with arbitrary primers in analysis of soybean germplasm and the intercross of ‘Essex’ and PI 437654. (AG)₆, (GA)₆ or (CT)₆ and (GT)₆, (TG)₆ or (CA)₆ were efficient in the detection of (G-A) and (C-A) ASSR-generated polymorphisms, respectively. DNA sequence analysis of the ASSR-amplified fragments confirmed the presence of SSR sequences. (A-T) ASSRs failed to give amplification or generated fewer number of fragments. Only one of the four tested decamer primers altered ASSR banding patterns in the soybean. All the six long primers (18–20 mer) tested changed ASSR banding profiles. On average, seven polymorphic fragments per ASSR primer were produced in soybean germplasm and four in the intraspecific cross of ‘Essex’ and PI 437654. The grouping of 48 genotypes in UPGMA analysis using four (C-A) and four (G-A) ASSR primers was consistent with the classification obtained with RFLP markers. Seventy-seven (91%) ASSR markers were dominant, while the remaining 8 (9%) showed codominant segregation. Fifty-eight ASSR markers were mapped onto 18 RAPD/RFLP linkage groups, which covered approximately 50% of the soybean genome. Of the (G-A) ASSR-derived markers 49% remained unlinked compared with 17% of (C-A) ASSR markers at LOD 3.0. Map linkage information showed that the assigned (C-A) polymorphisms had a biased distribution, whereas (G-A) polymorphisms were randomly dispersed. Received: 24 July 1997 / Accepted: 17 November 1997     

R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.)

The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Ot1-3), hybrid F₁ and two bulks (tolerant and susceptible) formed from recombinant inbred lines—RILs (F₉) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons—R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low T _m were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer—repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to r _AB.C = 0.870.     

Evidence for a low level of genomic specificity of sequence-tagged-sites in Stylosanthes

Genome-specific DNA markers are of great value in many applications. Recent work on different plants and animal species indicated that PCR- (polymerase chain reaction) based genetic marker systems using specific primers are highly genome-specific. To test the genome specificity of sequence-tagged-sites (STSs) as genetic markers in Stylosanthes, 20 pairs of primers were generated. Fifteen were from randomly selected single-copy Pstl genomic clones, and the other five were from two known gene sequences. These primer pairs were analysed against a set of 24 genotypes representing 12 different Stylosanthes species. Thirteen of these primer pairs amplified successfully. Overall, there was a low level of genome specificity, suggesting a low degree of genomic divergence within this group of Stylosanthes species. Of the 312 entries (24 genotypes by 13 primer pairs), PCR amplifications were unsuccessful (little or no products) in only 16 cases. The number of banding patterns detected by each of these primer pairs varied from 2 to 12 with an average pair-wise polymorphism of 44.3%. The level of intraspecific variation detected on normal agarose gels was only 3.8%. Further evidence that diploid S. hamata and diploid S. humilis are progenitors of tetraploid S. hamata and that S. viscosa is a progenitor of S. scabra, was obtained.     

Differentiation ofFusarium sambucinum Fuckel sensu lato and related species by RAPD PCR

DNA polymorphisms generated by the random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) were used to analyse 41 isolates investigated in the EuropeanFusarium sambucinum Project (EFSP). Employing ten arbitrary (10-mer) oligonucleotides and simple repeat sequences (M13, (GACA)₄) as single primers, informative banding patterns typical for identifying European populations ofFusarium sambucinum Fuckel s. str.,F. torulosum (Berk. & Curt.) Nirenberg andF. venenatum Nirenberg were obtained.     

Identification,cloning and sequence analysis of a dwarf genome-specific RAPD marker in rubber tree [<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Muell.) Arg.]

High-yielding dwarf clones of Hevea brasiliensis are tolerant to wind damage and therefore useful for high-density planting. The identification of molecular markers for the dwarf character is very important for isolating true-to-type high-yielding dwarf hybrid lines in the early stage of plant breeding programs. We have identified a dwarf genome-specific random amplified polymorphic DNA (RAPD) marker in rubber tree. A total of 115 random oligonucleotide 10-mer primers were used to amplify genomic DNA by PCR, of which 19 primers produced clear and detectable bands. The primer OPB-12 generated a 1.4-kb DNA marker from both natural and controlled F₁ hybrid progenies (dwarf stature) derived from a cross between a dwarf parent and a normal cultivated clone as well as from the dwarf parent; it was absent in other parent (RRII 118). To validate this DNA marker, we analyzed 22 F₁ hybrids (13 with a dwarf stature and nine with a normal stature); the dwarf genome-specific 1.4-kb RAPD marker was present in all dwarf-stature hybrids and absent in all normal-stature hybrids. This DNA marker was cloned and characterized. DNA marker locus specificity was further confirmed by Southern blot hybridization. Our results indicate that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern based on the presence/absence of specific DNA markers than dye-stained gels or Southern blot analysis of RAPD blots using probes made from purified PCR products. Detection of RAPD markers in the hybrid progenies indicates that RAPD is a powerful tool for identifying inherited genome segments following different hybridization methods in perennial tree crops.