Sequence-specific Amplification Polymorphism Research Articles

Fine mapping of a cdt locus mutation that leads to increased cadmium tolerance

A pea mutant SGECdt (cdt), which has an increased cadmium tolerance and an increased cadmium accumulation, as compared to the initial line, was recently obtained. Earlier, a SSAP (sequence specific amplified polymorphism) analysis revealed localization of the cdt locus in VI linkage group. For fine mapping of the cdt locus a set of PCR based markers was developed. PCR markers were based on known sequences of pea genes, which were determined using analysis of genome microsynteny between pea and model legume Medicago truncatula. The close linkage of the cdt locus and markers based on the Pentatricopeptide repeat and Exosome complex exonuclease RRP 45 genes was revealed. Thus, prerequisites for cdt positional cloning were developed.

Genetic assessment of the genus Pisum L. based on sequence specific amplification polymorphism data

The present study was conducted to analyze genetic diversity among 56 accessions of Pisum sativum, Pisum elatius, Pisum pumilio, Pisum fulvum and Podandrogyne brevipedunculata from Syria (42) and a set of 16 reference accessions from Israel (3), Ethiopia (2), Greece (2), Italy (2), Turkey (3), Tunisia (1) and Georgia (1). The study was based on the analysis of multiple data sets generated using sequence specific amplification polymorphism (SSAP) and associated morphological and ecogeographic data. The diversity analysis based on SSAP markers resulted in 83 scorable fragments in P. sativum, P. elatius (63), P. fulvum (62), P. pumilio (53), P. brevipedunculata (31) and P. abyssinicum (29). Genetic variability was measured as Nei’s gene diversity and maximum polymorphism was found in P. elatius (33.87%) and P. fulvum (32.74%) whereas minimum diversity was observed in P. brevipedunculata (12.48%). Pair wise band sharing among Pisum species was also calculated, significant band sharing was observed between P. fulvum and P. elatius, P. elatius and P. sativum and P. pumilio and P. elatius. The relationship among species as revealed by SSAP data could not significantly be correlated with those based on the agro-morphologic characters, suggesting that the two inferences are independent and estimates of genetic relations differently among Pisum taxa. The ecogeographic data associated with Pisum species (latitude, longitude) were also plotted using Arc View GIS 3.2 in order to show the distribution of species in Syria.

Development of Ty1-copia retrotransposon-based S-SAP markers in strawberry (Fragaria × ananassa Duch.)

The most popular retrotransposon-based molecular marker system at the present time is the sequence-specific amplification polymorphism (S-SAP) system, which is widely used for various applications including phylogenetic analysis. In this study, we successfully developed a new S-SAP marker system based on FaRE1 retrotransposon in strawberry genome. Among 34 primer combinations used to develop the S-SAP marker system, seven primer combinations produced high quality and reliability banding patterns. A total of 288 polymorphic bands from 722 amplified bands were detected in 13 strawberry accessions, with a polymorphic ratio of 39.8%. Phylogenetic analysis showed that genetic coefficient ranged from 0.67 to 1.00 for 13 strawberry accessions. An 187bp S-SAP band was detected from 50 strawberry plantlets regenerated from leaves which were cultured on MS media supplemented with 1.0mg/l thidiazuron (TDZ) and 0.1mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The available evidences proved S-SAP marker had high level of polymorphism in strawberry accessions.

Genetic diversity of faba bean (Vicia faba L.) populations revealed by sequence specific amplified polymorphism (SSAP) markers

This study used sequence specifc amplifcation polymorphism (SSAP) markers to investigate the genetic diversity and population genetic structure of nine Tunisian Vicia faba populations belonging to the minor and major faba bean’s sub-species. Three primers were used (PDR1, Tps19 and Tvf4 ) in this study. These primers gave good SSAP marker profiles, high number of bands obtained per gel and a high percentage of polymorphic bands as confirmed in a previous study. Indeed, these primers provided a total of 173 amplified bands, with 123 of them being polymorphic. Shannon indexes ranges from 0.166 to 0.248 with an average of 0.207. The genetic diversity within population of 0.743 was clearly higher than that of among population genetic diversity (Dst = 0.138), indicating an out-crossing predominance in the studied populations. The Dst value showed that 15.6% of the total genetic variation resided among populations, a little lower than that of out-crossing species. The dendrogram grouping the populations by unweighted pair-group method with arithmetic averages (UPGMA) method revealed three main clusters. The local major faba bean ‘Batata’ was the most divergent population and was separated from other population.

English

Apple (malus domestica Borkh.), which is a widely cultivated, important and economic fruit crop with nutritive and medicinal importance, has emerged as a model horticultural crop in this post-genomic era. Wild apple resources are important and they develop gradually in apple industry and genetic diversity. In this study, two molecular markers (inter-simple sequence repeats, ISSR and sequence-specific amplification polymorphism, S-SAP) were evaluated for genetic analysis of 31 wild apple resources in Shandong. A total of 20 ISSR primers were used and 110 polymorphic bands were amplified. Five S-SAP primer sets yielded a total of 496 bands, of which 201 (40.52%) were polymorphic. The similarity coefficient between resources ranged from 0.70 to 0.94 for ISSR analysis and from 0.66 to 0.95 using the S-SAP methodology. This study indicates that the results obtained based on the dendrograms constructed using unweighted pair-group using arithmetic average (UPGMA) cluster analysis were significantly correlated. The ISSR and S-SAP markers were found to be useful for wild apple resources identification and assessment of phonetic relationships.   Key words: Apple, wild resource, inter-simple sequence repeats (ISSR), sequence-specific amplification polymorphism (S-SAP).

Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP, SSAP, SAMPL and SSR markers

In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel's test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae)

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.

Identification and Discrimination of Wheat Flour by DNA Analyses

Wheat flour is a common white powder material and sometimes encountered in criminal investigations. This report describes the application of DNA analysis to wheat flour samples for the identification and discrimination of white powder materials. Species identification of commercially available wheat flour samples was carried out by the partial sequence of rbcL, which is used as markers for phylogenetic and taxonomic studies. These wheat flour samples were discriminated by short tandem repeat (STR) analysis and sequence-specific amplification polymorphism (S-SAP) analysis. The partial sequences of rbcL were identical among all flour samples. And they were also identical to the rbcL sequences of wheat (Triticum aestivum L.) submitted to DNA data base. Furthermore, flour samples had characteristic patterns in both the STR and S-SAP analyses. These results suggested that identification of the wheat flour samples was possible by rbcL sequences and both the STR and S-SAP analyses were useful for the discrimination among wheat flour samples. It is considered that DNA analysis of wheat flour can contribute to criminal investigation.

Habitat differentiation between estuarine and inland Hibiscus tiliaceus L. (Malvaceae) as revealed by retrotransposon-based SSAP marker

Hibiscus tiliaceus L. (Malvaceae) is a pantropical coastal tree that extends to the tidal zone. In this study, the retrotransposon sequence-specific amplified polymorphism (SSAP) technique was used in order to understand the genetic variation between four population pairs of H. tiliaceus from repeated estuarine and inland habitat contrasts in China. The estuarine populations were consistently more genetic variable compared with the inland ones, which may be attributed to extensive gene flow via water-drifted seeds and/or retrotransposon activation in stressful estuarine environments. An AMOVA revealed that 8.9% of the genetic variance could be explained by the habitat divergence within site, as compared with only 4.9% to geographical isolation between sites, which indicates significant habitat differentiation between the estuarine and inland populations. The estuarine populations were less differentiated (ΦST = 0.115) than the inland (ΦST = 0.152) implying frequent gene interchange in the former. Accordingly, the principal coordinate analysis of genetic distance between individuals revealed that genetic relationships are not fully consistent with the geographic association. These results suggest that despite substantial gene flow via sea-drifted seeds, habitat-related divergent selection could be one of the primary mechanisms that drive habitat differentiation in H. tiliaceus at a local ecological scale.

GENETIC VARIATION ANALYSIS OF THE NEWLY SYNTHESIZED ALLOTETRAPLOIDS CUCUMIS HYTIVUS USING RETROTRANSPOSON-BASED SSAP MARKERS

Ty1-copia-like retrotransposons have been found to comprise the most common class of transposable elements in plant and occur in high copy number in plant genomes. The dispersion, ubiquity and prevalence of Ty1-copia retrotransposons in plant genomes potentially provide an excellent system for developing a multiplex DNA-based marker system. Sequence-specific amplification polymorphism (SSAP) system is the most popular and displayed a higher level of polymorphism than that of AFLPs in cucumber. In this study, we studied the genetic variation of the early generations of the synthetic allotetraploids Cucumis hytivus using DNA-SSAP marker systems in Cucumis. The results showed that allopolyploid formation was associated with rapid and extensive genomic changes. Most genomic changes involved loss of parental restriction fragments and appearance of novel fragments. In addition, the variation of the allotetraploids early generations was a rapid process, the loss of , parental fragments and the gaining of novel fragments were mainly occurred in F 1 hybrid, the S 1 and S 2 generations, and then stabilized.

Genetic variation in a sorghum line with multiple genetic instability induced with ethidium bromide in an in vitro culture

Ethidium bromide treatment (15 mg/l, 26 degrees C, 18 h) of a sorghum Zheltozernoe 10 callus culture yielded line Zh10-brl displaying multiple genetic instability. The line was characterized by a broad variety of mutations, which were identified in consecutive generations obtained from one initial regenerant via self-pollination. The mutation caused male sterility (male sterility, generation R1), a low plant height (dwarfness, R2), a reduced awn length (awnless, R3), yellow leaves in seedlings (xantha, R6), leaf variegation (leaf variegation, R6), leaf bleaching (virescence, R6), etc. In some cases, segregation in families suggested a monogenic recessive inheritance for the induced mutations. Male sterility was due to a range of defects that affected microsporogenesis and microgametogenesis and were probably caused by mutations of several genes. Leaf variegation was due to the appearance of green areas in originally albino seedling leaves; the reversion occurred only in somatic tissues without affecting male and female gametes. In male-sterile and variegated mutants, sequence-specific amplified polymorphism (SSAP) analysis with primers to the Isaak transposon revealed new DNA fragments, which were absent from the original line. The results supported the hypothesis that the mutations isolated in line Zh10-brl result from transposon mobilization induced by ethidium bromide and/or in vitro culture conditions.

WHAT IS THE IMPACT OF ALLOPOLYPLOIDY ON TRANSPOSABLE ELEMENTS? A STRUCTURAL APPROACH ON NEWLY SYNTHESIZED BRASSICA NAPUS ALLOTETRAPLOIDS

Transposable elements (TEs) represent a major component of plant genomes and may provide the host genome with the necessary plasticity for its adaptative response. Polyploidy has played a major role in the evolution of plants (Wendel, 2000) and its success can be explained by the fact that a polyploid genome is not only the strict addition of the progenitor genomes, but that polyploidisation induces structural and functional modifications which represent important sources of novelty (Adams and Wendel, 2005). Our study aims at evaluating the implication of TEs in the genomic modifications that occur during the formation of a polyploid genome. Our plant model is oilseed rape (Brassica napus, AACC) originating from interspecific hybridization between B. rapa (AA) and B. oleracea (CC), and we dispose of newly synthesized B. napus allotetraploids resulting from independent AA x CC crosses. We developed sequence-specific amplification polymorphism (S-SAP) markers anchored in two different families of Brassica-specific TEs, an Athila-like retrotransposon and a MITE, which are contrasting TEs regarding their genomic organisation, dynamics of evolution, and mode of replication. Comparison of the S-SAP profiles and characterization of the polymorphism identified in the newly synthesized allotetraploids will provide new insights into the putative role of TEs during the creation of an allopolyploid species.

Molecular markers based on LTR retrotransposons BARE-1 and Jeli uncover different strata of evolutionary relationships in diploid wheats

Molecular markers based on retrotransposon insertions are widely used for various applications including phylogenetic analysis. Multiple cases were described where retrotransposon-based markers, namely sequence-specific amplification polymorphism (SSAP), were superior to other marker types in resolving the phylogenetic relationships due to their higher variability and informativeness. However, the patterns of evolutionary relationships revealed by SSAP may be dependent on the underlying retrotransposon activity in different periods of time. Hence, the proper choice of retrotransposon family is essential for obtaining significant results. We compared the phylogenetic trees for a diverse set of diploid A-genome wheat species (Triticum boeoticum, T. urartu and T. monococcum) based on two unrelated retrotransposon families, BARE-1 and Jeli. BARE-1 belongs to Copia class and has a uniform distribution between common wheat (T. aestivum) genomes of different origin (A, B and D), indicating similar activity in the respective diploid genome donors. Gypsy-class family Jeli was found by us to be an A-genome retrotransposon with >70% copies residing in A genome of hexaploid common wheat, suggesting a burst of transposition in the history of A-genome progenitors. The results indicate that a higher Jeli transpositional activity was associated with T. urartu versus T. boeoticum speciation, while BARE-1 produced more polymorphic insertions during subsequent intraspecific diversification; as an outcome, each retrotransposon provides more informative markers at the corresponding level of phylogenetic relationships. We conclude that multiple retroelement families should be analyzed for an image of evolutionary relationships to be solid and comprehensive.

Mobilization of retrotransposons in synthetic allotetraploid tobacco

Allopolyploidy is a major driving force in plant evolution and can induce rapid structural changes in the hybrid genome. As major components of plant genomes, transposable elements are involved in these changes. In a previous work, we observed turnover of retrotransposon insertions in natural allotretraploid tobacco (Nicotiana tabacum). Here, we studied the early stages of allopolyploid formation by monitoring changes at retrotransposon insertion sites in the Th37 synthetic tobacco. We used sequence-specific amplification polymorphism (SSAP) to study insertion patterns of two populations of the Tnt1 retrotransposon in Th37 S4 generation plants, and characterized the nature of polymorphic insertion sites. We observed significant amplification of young Tnt1 populations. Newly transposed copies were amplified from maternal elements and were highly similar to Tnt1A tobacco copies amplified in response to microbial factors. A high proportion of paternal SSAP bands were not transmitted to the hybrid, corresponding to various rearrangements at paternal insertion sites, including indels or the complete loss of the Tnt1/flanking junction. These data indicate that major changes, such as retrotransposon amplification and molecular restructuring in or around insertion sites, occur rapidly in response to allopolyploidy.

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.

Characterisation of RNaseH-LTR sections of Ty1-copia retrotransposons in apple and fingerprinting of four apple clones by S-SAP analysis

SummaryRetrotransposons are the most widespread class of eukaryotic transposable elements and play an important role in genome evolution. Several of these elements have been sequenced and were found to contain different cis-regulatory elements involved in the response to different stress conditions in their long terminal repeat sequences (LTRs). Retrotransposon-based marker systems have proved useful because of their distribution, ubiquity, and prevalence in plant genomes. Here we report on the isolation and characterisation of new RNaseH-LTR sections of the Ty1-copia-like retro-elements in apple DNA. Twenty clones were identified with a variable degree of homology to the Ty1-copia RNaseH gene. Nineteen had the characteristic RNaseH gene, stop codon, polypurine tract (PPT), and putative 3’-LTR sequence elements. The RNaseH sections were aligned using ClustalV to seek potential relationships between sequences. This analysis revealed six distinct Clusters. Conserved motifs, corresponding to cis-regulatory elements, were discovered in the LTRs using plantCARE software and four were characterised. From one putative LTR fragment, we then designed a primer, which, through sequence-specific amplification polymorphism (S-SAP), could distinguish between ‘Wellspur Delicious’, ‘Hardispur Delicious’ and ‘Starkrimson’, and ‘Starking’ apple, by fingerprinting. The first three varieties originated from ‘Starking’ through bud mutations. Our results suggest that these bud mutations appeared to have been derived from retrotransposon insertions.

Isolation of two novel complete Ty1-copia retrotransposons from apple and demonstration of use of derived S-SAP markers for distinguishing bud sports of Malus domestica cv. Fuji

Retrotransposon-based molecular markers are a powerful tool for mapping and diversity studies. The scarcity of retrotransposon long terminal repeat (LTR) sequences limits the application of retrotransposon-based molecular marker systems. Here, we isolated two novel complete Ty1-copia retrotransposons (CTcrm1 and CTcrm2) in apple using a genome walking strategy. The CTcrm retrotransposons are nearly 5 kb long, and they have all the features of Ty1-copia retrotransposons. The differences in gene organization and nucleotide sequence length between the CTcrm retrotransposons and other reported complete retrotransposons in apple showed that CTcrm1 and CTcrm2 are the first two distinct complete Ty1-copia retrotransposons in the apple genome. To investigate the potential utility of the two retrotransposons as molecular markers, primers complementary to the CTcrm LTRs were designed to develop sequence-specific amplification polymorphism markers for discriminating bud sports of Fuji apple. Multiple polymorphisms corresponding to CTcrm1 and CTcrm2 were detected and could easily be used to discriminate bud sports from their Fuji progenitor, as well as from each other.

Markers and mapping revisited: finding your gene

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of this highly dynamic arena of plant science.

Comparison of four molecular markers for genetic analysis in Diospyros L. (Ebenaceae)

Four molecular markers, including inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplified polymorphism (SSAP), and amplified fragment length polymorphism (AFLP), were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 28 genotypes in the genus Diospyros. The results were as follows: (1) the highest level of detected polymorphism were observed for IRAP; (2) AFLP was the most efficient marker system due to the simultaneous detection of abundant polymorphism markers per single reaction; (3) the marker index (MI) value was lower for SSAP than for AFLP, but SSAP had a higher level of detected polymorphism than AFLP; (4) the correlation coefficients of similarity were statistically significant for all four marker systems; (5) the four molecular markers yielded similar phylogenetic trees. To our knowledge, this was the first detailed report of a comparison of performance among three retrotransposon-based molecular markers (IRAP, REMAP, SSAP) and the AFLP technique (DNA-based molecular marker) on a set of samples of Diospyros. The results provide guidance for future efficient use of these molecular methods in the genetic analysis of Diospyros.

Highly variable AFLP and S-SAP markers for the identification of 'Malbec' and 'Syrah' clones

The retrotransposon-based sequence-specific amplification polymorphism (S-SAP) and the amplified fragment length polymorphism (AFLP) marker systems were used to assess the clonal variation of 14 ‘Syrah’ and 22 ‘Malbec’ ( Vitis vinifera L.) clones. The utility of S-SAP markers was compared to that of AFLP markers. On the basis of our results, S-SAP is more informative marker system and showed higher average number of polymorphic bands per cultivar group than AFLP. Relationships among clones were analyzed by cluster analysis using unweighted pair-groups using arithmetic averages and in both cases revealed well defined groups of clones, in which ‘Malbec’ clones were separated from ‘Syrah’ clones. High variability of some clones could also be seen within these clusters. The different levels of polymorphism for ‘Malbec’ and ‘Syrah’ obtained in this study suggest that ‘Malbec’ exhibits a higher mutation frequency than ‘Syrah’. Our results indicate that higher proportion of polymorphic bands in S-SAP makes it a less labour-intensive and more efficient approach for developing markers for clonal identification.