Integration of dinucleotide microsatellites from hexaploid bread wheat into a genetic linkage map of durum wheat

 Seventy nine microsatellite markers from hexaploid bread wheat (T. aestivum L.) were integrated into a genetic linkage map of durum wheat (T. turgidum ssp. durum (Desf.) Huns.) created by RFLP segregation data from a population of 65 recombinant inbred lines. The results indicate a relatively even distribution of microsatellite loci and demonstrate that microsatellite markers from hexaploid wheat provide an excellent source of molecular markers for use in the genetics and breeding of durum wheat. Received: 16 July 1998 / Accepted: 13 October 1998     

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

 The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids. Received: 4 January 1999 / Accepted: 4 January 1999     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998     

Low levels of DNA sequence variation among adapted genotypes of hexaploid wheat

 PCR products from regions corresponding to sequences hybridising to wheat RFLP probes were sequenced in order to establish the level of DNA sequence variation among adapted wheat genotypes. Hexaploid bread wheat shows a very low rate of nucleotide polymorphism, approximately 1 polymorphic nucleotide per 1000 basepairs. Differences in PCR product length can be exploited to design genome-specific amplicons, which may have use in gene tagging or in diagnostic applications. Interpretation of results may be complicated by the simultaneous amplification of orthologous and paralogous sequences. These findings have significant implications for the use of STS markers in wheat and other polyploid species. Received: 3 October 1998 / Accepted: 28 November 1998     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.)

The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymorphism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of alleles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic library-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic diversity was seen in GA-containing microsatellites of genomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential location of specific motifs in coding versus non-coding regions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatellite motif and the standard deviation of the molecular-weight of amplified fragments. The reliability of molecular weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed. Received: 5 May 1999 / Accepted: 16 August 1999     

Genetic diversity among European cultivated spelt revealed by microsatellites

Spelt and common wheat constitute two of the six groups of the hexaploid wheats with an AABBDD genome. Spelt culture has been progressively replaced by that of common wheat which out-yields spelt under high-input conditions. In the last decades, spelt breeders intended to introduce the yield-potential and bread-making qualities of common wheat into spelt, by frequent crossings between accessions of these two different groups. The present study aims at determining the genetic basis of modern spelt cultivars in terms of intra-group variability and inter-group (spelt vs common wheat) distances, by using microsatellite markers developed for common wheat. The allelic composition of 30 spelt and nine common wheat accessions was determined at 17 microsatellite loci. The coefficient of co-ancestry (ƒ) and the genetic distances (1 - proportion of shared alleles) based upon allelic composition were calculated for all pairs of accessions. Two dendrograms were constructed using the UPGMA method. Amplification products were found for all loci on most accessions. A total of 113 alleles was identified, of which 60.2% were specific to spelt or common wheat. The correlation between (1 –ƒ ) and the genetic distance was high (0.701***). The mean pairwise genetic distance was 0.656 ± 0.181 over the 39 accessions, 0.706 ± 0.14 among common wheat and 0.573 ± 0.172 among spelt. The mean genetic distance between spelt and wheat was 0.782 ± 0.113. The two dendrograms were in accordance with each other and clearly separated the spelt from the common wheat accessions. It is concluded that microsatellites developed for common wheat and distances based on the proportion of shared alleles are powerful tools for reconstructing phylogenies in spelt, and that the genetic basis of modern spelt cultivars is narrow despite frequent crosses made with bread wheat. Received: 26 January 2000 / Accepted: 26 May 2000     

Genetic analysis of water-extractable arabinoxylans in bread wheat endosperm

 Two mapping populations were used for the analysis of the water-extractable arabinoxylans. One originated from a cross between the hexaploid cultivars ‘Courtot’ and ‘Chinese Spring’ and the other from a cross between an amphiploid (Synthetic) and cv ‘Opata’. Arabinose (Ara), and xylose (Xyl) contents were quantified for the 91 and 76 lines obtained from the two crosses, respectively. Relative viscosity (η_rel) of the wheat flour aqueous extract was evaluated by capillary viscometry. Both crosses gave similar correlation coefficients between sugar contents and relative viscosity. There were strong positive relationships between arabinose, xylose and arabinoxylan contents. The relative viscosity was strongly and positively related to the arabinoxylan content and strongly and negatively related to the Ara/Xyl ratio (arabinose content to xylose content). For one of the two crosses two measurements of relative viscosity were generated from 2 years of consecutive harvesting. As a strong correlation was observed between these two measurements, an important genotypic effect can be deduced for the relative viscosity of water-extractable arabinoxylans. QTL (quantitative trait locus) research did not reveal any chromosomal segments that were strongly implicated in variations in sugar content. However, a QTL was found for relative viscosity values and the Ara/Xyl ratio on the long arm of the 1B chromosome for the two crosses considered. This QTL explained 32–37% of the variations in relative viscosity and 35–42% of the variations in the Ara/Xyl ratio. Genes located at this QTL controlled relative viscosity through modifying the Ara/Xyl ratio. Variations in the Ara/Xyl ratio were supposedly related to differences in the molecular structure of water-extractable arabinoxylans. Minor QTLs were also obtained for relative viscosity and Ara/Xyl ratio, but the chromosomes concerned were different for the two populations evaluated. Received: 5 January 1998 / Accepted: 15 May 1998     

Fifty new microsatellite loci for the wheat genetic map

 Hexaploid bread wheat (Triticum aestivum) has low levels of RFLP. Simple sequence repeats, however, show high levels of polymorphism and are therefore especially useful in intervarietal breeding applications. We present 53 newly mapped microsatellite loci for the wheat genetic map, 41 primary loci and 12 additional loci from these same primer pairs. Markers have been accredited with a quality score on a scale of 1–5 which describes the complexity of the amplification product profile from each primer pair. Received: 29 June 1997 / Accepted: 4 February 1998     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Microsatellites confirm the authenticity of inter-varietal chromosome substitution lines of wheat (Triticum aestivum L.)

Ninety-five wheat microsatellite markers (WMS) were used to verify the authenticity of the set of Saratovskaya 29/Yanetzkis Probat inter-varietal wheat chromosome substitution lines developed using Saratovskaya 29 as the recipient variety. Polymorphic markers were available for all chromosome arms except 4DS, 6DS and 7DS. Each chromosome substitution line was tested by 2–8 microsatellite markers. The results demonstrate that most of the lines are correct. Out of 21 lines tested 17 showed the expected microsatellite pattern of the donor variety. Two entire chromosomes, 1B and 7A, and two chromosome arms, 3AL and 6DS, were not substituted with Yanetzkis Probat in their respective lines. Three microsatellite markers located in the distal regions of chromosome arms 4AL, 3BS and 5BL in the corresponding substitution lines did not reveal the expected microsatellite pattern of the recipient variety. The possible causes of the incorrect substitution line development and the appearance of incorrect distal microsatellite markers are discussed. The data confirm the idea that microsatellite markers provide ideal tools for testing the authenticity of genetic stocks of wheat. Received: 27 August 1999 / Accepted: 8 October 1999     

Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer ”stutter bands” as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar ’Chinese Spring’ were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) _n , (GGA/CCT) _n , (TAA/ATT) _n , (CAA/GTT) _n , (GGT/CCA) _n , (CAT/GTA) _n , (CGA/GCT) _n , (CTA/GAT) _n , and (CGT/GCA) _n repeats were estimated to be 5.4×10⁴, 3.5×10⁴, 3.2×10⁴, 1.2×10⁴, 6.3×10³, 4.9×10³, 4.5×10³, 4.5×10³ and 3.6×10³, i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) _n , 30 (43%) (CTT/GAA) _n , 16 (59%) (CAA/GTT) _n , 3 (27%) (CAT/GTA) _n and 2 (4%) (GGA/CCT) _n clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) _n , (CTT/GAA) _n and (CAA/GTT) _n microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) _n , four (14.8%) to (CTT/GAA) _n , and two (12.5%) to (CAA/GTT) _n resulted in polymorphic markers. The results indicated that (TAA/ATT) _n microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat. Received: 17 February 2001 / Accepted: 31 May 2001     

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.     

Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla

 We report on the development, genetic characterization and linkage mapping of a battery of SSR (simple sequence repeat) loci in Eucalyptus grandis and E. urophylla. This study reveals the abundance of SSRs in Eucalyptus, the very high information content of these markers for mapping and individual identification, and demonstrates the feasibility of constructing a comprehensive microsatellite-based linkage map for Eucalyptus. Primer sequence for a set of 20 highly informative EMBRA (Eucalyptus microsatellites from Brazil) loci are made available together with their map position and estimates of the expected heterozygosity and allele size range in these two species. Using genomic library enrichment and anchored-PCR screening prior to sequencing, the efficiency of SSR marker locus development was 63% from sequencing data to operationally useful SSR loci. Absolute transportability between the two species and very high levels of allelic variability and expected heterozygosity (H) were seen at all SSR loci surveyed. The number of alleles per locus ranged from 9 to 26 with an average of 16.3±4.8. The average H of 15 loci was 0.86±0.04, 0.83±0.08 and 0.89±0.04, respectively, for E. urophylla, E. grandis and the combined two-species estimate. In the mapping analysis 16 out of 20 marker loci segregated in a fully informative configuration, allowing the determination of synteny of six homologous linkage groups between the two species. The availability of transportable, multiallelic, PCR-based co-dominant SSR loci represents a dramatic improvement in our ability to carry out detailed population genetic analysis and to search, understand, and manipulate allelic variation at QTLs (quantitative trait loci) in species of Eucalyptus. Received: 16 March 1998 / Accepted: 22 March 1998