Simple‐sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl.) are highly polymorphic and transferable to peach and almond

Thirty‐five polymorphic microsatellites were developed using a CT/AG enriched genomic library of Japanese plum cv. Santa Rosa. Twenty‐seven of them detected a single locus and eight two or more loci. A high level of variability was observed in a set of eight cultivars for the 27 single‐locus microsatellites: 5.7 average number of alleles per locus; 73% mean observed heterozygosity and 74% discrimination power. Most SSRs were transferable to peach (85%) and almond (78%).     

Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region.

Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.     

Microsatellites isolated in almond from an AC‐repeat enriched library

We have isolated 44 SSRs from an AC‐enriched genomic library from almond (Prunus amygdalus Batsch.). Twenty SSRs were screened for their polymorphism in 16 cultivars and for their transportability in seven different Prunus species (peach, nectarine, apricot, European plum, Japanese plum, sweet cherry, sour cherry) and in apple. The expected heterozygosity ranged from 0.62 to 0.89. About 30% of primers gave successful amplification in seven different Prunus species; in two cases amplifications were obtained also in apple.     

Assessment of genetic diversity and relatedness among Tunisian almond germplasm using SSR markers

Genetic diversity of 50 Tunisian almond (Prunus dulcis Mill.) genotypes and their relationships to European and American cultivars were studied. In total 82 genotypes were analyzed using ten genomic SSRs. A total of 159 alleles were scored and their sizes ranged from 116 to 227 bp. The number of alleles per locus varied from 12 to 23 with an average of 15.9 alleles per locus. Mean expected and observed heterozygosities were 0.86 and 0.68, respectively. The total value for the probability of identity was 4 × 10(-13) . All SSRs were polymorphic and they were able all together to distinguish unambiguously the 82 genotypes. The Dice similarity coefficient was calculated for all pair wise and was used to construct an UPGMA dendrogram. The results demonstrated that the genetic diversity within local almond cultivars was important, with clear geographic divergence between the northern and the southern Tunisian cultivars. The usefulness of SSR markers for almond fingerprinting, detection of synonyms and homonyms and evaluation of the genetic diversity in the Tunisian almond germplasm was also discussed. The results confirm the potential value of genetic diversity preservation for future breeding programs.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

New set of microsatellite loci isolated in apricot

We report 99 simple sequence repeats (SSRs) newly isolated from an apricot (Prunus armeniaca L.) genomic library enriched for AG/CT repeats. Twenty SSRs were screened for their polymorphism in 16 apricot cultivars. The number of alleles ranged from two to nine, whereas the expected heterozygosity (H_E) ranged from 0.26 to 0.82. The same SSRs showed also an appreciable transportability across different Prunus species, such as peach, nectarine, almond, European plum, Japanese plum, sweet cherry and sour cherry, with 20% of primers giving successful amplifications in all Prunus species assayed. None gave amplification in apple.     

Molecular markers for kernel bitterness in almond

Upon crushing, amygdalin present in bitter almonds is hydrolysed to benzaldehyde, which gives a bitter flavour, and to cyanide, which is toxic. Bitterness is attributable to the recessive allele of the Sweet kernel (Sk/sk) gene and is selected against in breeding programmes. Almond has a long intergeneration period due to its long juvenile phase, so breeders must wait 3 or 4 years to evaluate fruit traits in the field. For this reason, it is important to develop molecular markers to distinguish between sweet and bitter genotypes. The Sk gene is known to map to linkage group five (G5) of the almond genome, but its function is still undefined. Candidate genes involved in the amygdalin pathway have been mapped, but none of them were located to G5. We have saturated G5 with additional Simple Sequence Repeats (SSRs) using the progeny from the cross “R1000” × “Desmayo Largueta” and found six SSRs (UDA-045, EPDCU2584, CPDCT028, BPPCT037, PceGA025, and CPDCT016) closely linked to the Sk locus. The genotypes of four of these SSRs flanking the Sk locus, in a number of parents and a few seedlings of the CEBAS-CSIC almond breeding programme, allowed us to estimate the haplotypes of the parents, identifying the marker alleles adequate for an early and highly efficient selection against bitter genotypes. This analysis has established the usefulness of SSRs for screening populations of fruit trees such as almond by an easy, polymerase chain reaction-based method.     

Frequency, type, distribution and annotation of simple sequence repeats in Rosaceae ESTs

Genomic resources for peach, a model species for Rosaceae, are being developed to accelerate gene discovery in other Rosaceae species by comparative mapping. Simple sequence repeats (SSRs) are an important tool for comparative mapping because of their high polymorphism and transportability. To accelerate the development of SSR markers, we analyzed publicly available Rosaceae expressed sequence tags (ESTs) for SSRs. A total of 17,284 ESTs from almond, peach and rose were assembled into putatively non-redundant EST sets. For comparison, 179,099 ESTs from Arabidopsis were also used in the analysis. About 4% of the assembled ESTs contained SSRs in Rosaceae, which was higher than the 2.4% found in Arabidopsis. About half of the SSRs were found in the putative UTR, and the estimated average distance between SSRs in the UTR was 5.5 kb in rose, 5.1 kb in almond, 7 kb in peach and 13 kb in Arabidopsis. In the putative coding region, the estimated average distance was two to four times longer than in the UTR. Rosaceae ESTs containing SSRs were functionally annotated using the GenBank nr database and further classified using the gene ontology terms associated with the matching sequences in the SwissProt database. The detailed data including the sequences and annotation results are available from .     

Development,characterization and variability analysis of microsatellites in lychee (<Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn., Sapindaceae)

We report 12 microsatellites enriched in CT repeats obtained from a genomic library of the lychee (Litchi chinensis Sonn.) cultivar Mauritius. The polymorphisms revealed by these microsatellites were evaluated in a collection of 21 lychee cultivars. A total of 59 fragments were detected with these 12 SSRs, with an average of 4.9 bands/SSR. Three primer pairs seem to amplify more than a single locus. The mean expected and observed heterozygosities over the 9 single-locus SSRs averaged 0.571 (range: 0.137–0.864) and 0.558 (range: 0.169–0.779) respectively. The total value for the probability of identity was 7.53×10^-5. In addition, the selected SSRs were used to amplify DNA from four longan cultivars. Eleven of the 12 SSRs produced amplification fragments in longan, and eight of these fragments were polymorphic. All except two of the products amplified from longan were the same size as those amplified from lychee, suggesting a close genetic proximity between the two species. The SSRs studied produced 22 different patterns, allowing the unambiguous identification of 16 lychee and the 4 longan cultivars studied. Discrimination was possible with just four selected microsatellites. Two groups with two and three undistinguishable cultivars were obtained, reflecting probable synonymies. Unweighted pair-group method of artimetic averages (UPGMA) cluster analysis divided the lychee cultivars studied into two main groups, one consisting of ancient cultivars and the other with more diverse recent cultivars. This is the first report of microsatellite development in the Sapindaceae, and the results demonstrate the usefulness of microsatellites for identification, similarity studies and germplasm conservation in lychee and related species.Communicated by H.F. Linskens     

Genomic microsatellite adaptive divergence of wild barley by microclimatic stress in 'Evolution Canyon', Israel

We examined diversity levels and patterns of 19 nuclear microsatellites and four chloroplast microsatellites in 275 genotypes of wild barley Hordeum spontaneum, in seven stations at the ‘Evolution Canyon’ (EC) microsite, Lower Nahal Oren, Mt. Carmel, Israel. EC is sharply subdivided ecologically into a tropical savannoid, ‘African’, xeric, south‐facing slope (SFS) abutting the temperate, dense, liveoak, brushwood, ‘European’, mesic, north‐facing slope (NFS). We found the following. (i) 17 of 19 (89.5%) nuDNA simple sequence repeats (SSRs) were polymorphic across all seven subpopulations and three chDNA SSRs were polymorphic. (ii) A total of 216 nuDNA SSR alleles, with a maximum of 23 alleles in a nuclear locus, and ten chDNA SSRs, with a maximum of four alleles in a locus, were registered. (iii) There were striking and significant inter‐ and intraslope diversities, based on the 19 nuDNA SSRs, climaxing with a remarkable genetic distance between the mid‐slope stations on opposite slopes (D_A = 0.481), across a distance of 200 m. This genetic distance is as large as that between the H. spontaneum populations of Jerusalem and Sede Boqer, which are separated by 100 km (× 500 larger in transect length). (iv) Slope‐unique alleles (103 = 45.6%) were higher on the ‘European’ than on the ‘African’ slope. Slope‐specific (predominant) alleles (17) were equal on opposite slopes. (v) nuDNA SSR gene diversity was higher on the ‘European’ slope and the opposite was found for the chDNA SSR. (vi) nuDNA SSR genic differentiation was very high between opposite slopes, with Gst = 0.187; for chDNA SSR this value was 0.127. Our results are inexplicable by stochastic processes and suggest that: (i) microclimatic diversifying selection is the major evolutionary, fast‐acting, interslope force, overriding migration and drift, and (ii) ecological stress can generate local, regional and global adaptive patterns, suggesting that natural selection is a major differentiating force of both coding and noncoding SSRs linking micro‐ and macroevolutionary processes. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 205–224.     

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species

Genic microsatellites or simple sequence repeat markers derived from expressed sequence tags (ESTs), referred to as EST–SSRs, are inexpensive to develop, represent transcribed genes, and often have assigned putative function. The large apple (Malus × domestica) EST database (over 300,000 sequences) provides a valuable resource for developing well-characterized DNA molecular markers. In this study, we have investigated the level of transferability of 68 apple EST–SSRs in 50 individual members of the Rosaceae family, representing three genera and 14 species. These representatives included pear (Pyrus communis), apricot (Prunus armeniaca), European plum (P. domestica), Japanese plum (P. salicina), almond (P. dulcis), peach (P. persica), sour cherry (P. cerasus), sweet cherry (P. avium), strawberry (Fragaria vesca, F. moschata, F. virginiana, F. nipponica, and F. pentaphylla), and rose (Rosa hybrida). All 68 primer pairs gave an amplification product when tested on eight apple cultivars, and for most, the genomic DNA-derived amplification product matched the expected size based on EST (in silico) data. When tested across members of the Rosaceae, 75% of these primer pairs produced amplification products. Transferability of apple EST–SSRs across the Rosaceae ranged from 25% in apricot to 59% in the closely related pear. Besides pear, the highest transferability of these apple EST–SSRs, at the genus level, was observed for strawberry and peach/almond, 49 and 38%, respectively. Three markers amplified in at least one genotype within all tested species, while eight additional markers amplified in all species, except for cherry. These 11 markers are deemed good candidates for a widely transferable Rosaceae marker set provided their level of polymorphism is adequate. Overall, these findings suggest that transferability of apple EST–SSRs across Rosaceae is varied, yet valuable, thereby providing additional markers for comparative mapping and for carrying out evolutionary studies.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Microsatellite markers for Vaccinium from EST and genomic libraries

We present 30 microsatellite loci isolated from expressed sequence tag (EST) and genomic libraries in Vaccinium corymbosum L. Allele number per locus in 11 tetraploid and one diploid V. corymbosum accessions ranged from two to 15 (mean = 8.16) in 24 single‐locus simple sequence repeats (SSRs). Cross‐species amplification in a panel of 12 species representing nine sections ranged from 30 to 100% (mean = 83%).     

Exploration of presence/absence variation and corresponding polymorphic markers in soybean genome

This study was designed to reveal the genome‐wide distribution of presence/absence variation (PAV) and to establish a database of polymorphic PAV markers in soybean. The 33 soybean whole‐genome sequences were compared to each other with that of Williams 82 as a reference genome. A total of 33,127 PAVs were detected and 28,912 PAV markers with their primer sequences were designed as the database NJAUSoyPAV_1.0. The PAVs scattered on whole genome while only 518 (1.8%) overlapped with simple sequence repeats (SSRs) in BARCSOYSSR_1.0 database. In a random sample of 800 PAVs, 713 (89.13%) showed polymorphism among the 12 differential genotypes. Using 126 PAVs and 108 SSRs to test a Chinese soybean germplasm collection composed of 828 Glycine soja Sieb. et Zucc. and Glycine max (L.) Merr. accessions, the per locus allele number and its variation appeared less in PAVs than in SSRs. The distinctness among alleles/bands of PCR (polymerase chain reaction) products showed better in PAVs than in SSRs, potential in accurate marker‐assisted allele selection. The association mapping results showed SSR + PAV was more powerful than any single marker systems. The NJAUSoyPAV_1.0 database has enriched the source of PCR markers, and may fit the materials with a range of per locus allele numbers, if jointly used with SSR markers.     

Development of a second generation linkage map for almond using RAPD and SSR markers.

Fifty-four RAPD (random amplified polymorphic DNA) markers and 6 SSRs (simple sequence repeats) were included in a molecular marker map with 120 RFLPs (restriction fragment length polymorphisms) and 7 isozyme genes previously constructed using the offspring of a cross between the almond (Prunus amygdalus) cultivars 'Ferragnès' and 'Tuono'. Only highly reproducible RAPDs segregating 1:1 were used. To identify these markers, a total of 325 primers were screened, from which 41 produced RAPDs useful for mapping. Polymorphism was detected in six of the eight Prunus SSRs (simple sequence repeats) studied, thus enabling these to be mapped. All markers were placed on the 8 linkage groups previously identified. The number of new markers included in the map of 'Ferragnès' was 33 for a total of 126, and 30 in the map of 'Tuono' for a total of 99. The sizes of the maps of 'Ferragnès' (415 cM) and 'Tuono' (416 cM) were similar, representing a 5% increase over the maps constructed solely with isozymes and RFLPs. The estimated total size of the almond map was of 457 cM. Some markers were placed in zones with low density of markers and others in the extreme of linkage groups. The use of RAPD markers to complete genetic maps constructed with transferable markers is discussed.     

Evolutionary history of almond tree domestication in the Mediterranean basin

Genetic diversity of contemporary domesticated species is shaped by both natural and human‐driven processes. However, until now, little is known about how domestication has imprinted the variation of fruit tree species. In this study, we reconstruct the recent evolutionary history of the domesticated almond tree, Prunus dulcis, around the Mediterranean basin, using a combination of nuclear and chloroplast microsatellites [i.e. simple sequence repeat (SSRs)] to investigate patterns of genetic diversity. Whereas conservative chloroplast SSRs show a widespread haplotype and rare locally distributed variants, nuclear SSRs show a pattern of isolation by distance with clines of diversity from the East to the West of the Mediterranean basin, while Bayesian genetic clustering reveals a substantial longitudinal genetic structure. Both kinds of markers thus support a single domestication event, in the eastern side of the Mediterranean basin. In addition, model‐based estimation of the timing of genetic divergence among those clusters is estimated sometime during the Holocene, a result that is compatible with human‐mediated dispersal of almond tree out of its centre of origin. Still, the detection of region‐specific alleles suggests that gene flow from relictual wild preglacial populations (in North Africa) or from wild counterparts (in the Near East) could account for a fraction of the diversity observed.