Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.)

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’. Received: 3 February 2000 / Accepted: 21 March 2000     

Development, characterization and inheritance of new microsatellites in olive (Olea europaea L.) and evaluation of their usefulness in cultivar identification and genetic relationship studies

Twelve new microsatellites have been developed in olive. For that purpose, a genomic library of the olive cultivar ‘Arbequina’ was enriched for GA, GT and ACT repeats. Two methods of screening yielded 27 sequences containing microsatellites out of the 119 clones sequenced. The GA repeat seems to be the most abundant motif. Among sequences containing microsatellites, 4 (14.8%) were redundant, 1 (3.7%) was previously described in the literature and 12 (44.4%) could not be used for primers design because the repeat motifs were incomplete. Suitable primer pairs were obtained for the remaining 10 (37.0%) sequences plus an additional 14 recovered from a formerly developed library. For the 24 primer pairs designed, 4 failed to amplify, 8 produced a complex bands pattern and 12 succeeded in giving amplification products. Considering these 12 primer pairs, 10 showed single locus amplification, whereas the other 2 revealed two loci each. This was demonstrated by studying allele segregation in two olive progenies. Sixty-eight alleles were detected for the 12 microsatellites when 51 olive cultivars were analysed. The number of alleles per locus ranged from 1 to 13. The expected heterozygosity varied between 0 and 0.83. All pairs of cultivars could be distinguished using only three microsatellites due to their great discrimination power value. The data coming from genotyping the 51 olive cultivars for 7 out of the 12 new microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice similarity coefficient. Cultivar association according to their geographical origin was observed.     

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     

Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species

 We have isolated and sequenced 263 microsatellite-containing clones from two small insert libraries of Actinidia chinensis enriched for (AC/GT) and (AG/CT) repeats, respectively. Primer pairs were designed for 203 microsatellite loci and successfully amplified from both plasmid and A. chinensis genomic DNA. In this paper we report the sequences of 40 primer pairs for which we have demonstrated Mendelian segregation in the progeny from controlled crosses. The polymorphism of ten microsatellites of each type was evaluated in four diploid and six tetraploid genotypes of A. chinensis. All microsatellites proved to be polymorphic, the number of alleles per locus detected in polyacrylamide sequencing gels ranging from 9 to 17. The high degree of polymorphism in Actinidia renders these markers useful either for mapping in A. chinensis or for fingerprinting cultivars of both domesticated kiwifruit species (A. chinensis and A. deliciosa). While most primer pairs produced single amplification products, about 20% generated banding patterns consistent with the amplification of two different loci. This supports the hypothesis that diploid species of Actinidia (2n=2x=58) are polyploid in origin with a basic chromosome number x=14/15 and that chromosome duplication may have occurred during the evolution of the genus. Finally, we have assayed the cross-species transportability of primer pairs designed from A. chinensis sequences and have found extensive cross-species amplification within the genus Actinidia; 75% of primer pairs gave successful amplification in the eight species assayed (A. arguta, A. rufa, A. polygama, A. chrysantha, A. callosa, A. hemsleyana, A. eriantha, and A. deliciosa), which are representative of the four sections into which the genus is currently split. Received: 14 February 1998 / Accepted: 26 May 1998     

Development of 1,030 genomic SSR markers in switchgrass

Switchgrass, Panicum virgatum L., a native to the tall grass prairies in North America, has been grown for soil conservation and herbage production in the USA and recently widely recognized as a promising dedicated cellulosic bioenergy crop. A large amount of codominant molecular markers including simple sequence repeats (SSRs) are required for the construction of linkage maps and implementation of molecular breeding strategies to develop superior switchgrass cultivars. The objectives of this study were (1) to identify SSR-containing clones and to design PCR primer pairs (PPs) in SSR-enriched genomic libraries, and (2) to validate and characterize the designed SSR PPs. Five genomic SSR enriched libraries were constructed using genomic DNA of ‘SL93 7 × 15’, a switchgrass genotype selected in an Oklahoma State University (OSU) southern lowland breeding population. A total of 3,046 clones from four libraries enriched in (CA/TG)n, (GA/TC)n, (CAG/CTG)n and (AAG/CTT)n SSR repeats were sequenced at the OSU Core Facility. From the sequences, we isolated 1,300 unique SSR-containing clones, from which we designed 1,398 PPs using SSR Locator V.1 software. Among the designed PPs, 1,030 (73.7%) amplified reproducible and strong bands with expected fragment size, and 802 detected polymorphic alleles, in SL93 7 × 15 and ‘NL94 16 × 13’, two parents of one mapping population. All of the four libraries contained a high rate of perfect SSR repeat types, ranging from 62.7 to 76.2%. Polymorphism of the effective SSR markers was also tested in two lowland and two upland switchgrass cultivars, encompassing ‘Alamo’ and ‘Kanlow’, and ‘Blackwell’ and ‘Dacotah’, respectively. The developed SSR markers should be useful in genetic and breeding research in switchgrass.     

<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Fingerprinting,embryo type and geographic differentiation in mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L., Anacardiaceae) with microsatellites

We report the sequence and variability parameters of 16 microsatellite primer pairs obtained from two mango (Mangifera indica L.) genomic libraries after digestion of DNA of the cultivar Tommy Atkins with HaeIII and RsaI and enrichment in CT repeats. Although no significant differences were recorded between the two libraries in the informativeness of the markers obtained, the RsaI library was shown to be more useful than the HaeIII taking into account the efficiency of the library and the feasibility of clone sequencing. The polymorphism revealed by those microsatellites was evaluated in a collection of 28 mango cultivars of different origins. A total of 88 fragments were detected with the 16 simple sequence repeats (SSRs) with an average of 5.5 bands/SSR. Two primer pairs amplified more than a single locus. The mean expected and observed heterozygosities over the 14 single-locus SSRs averaged 0.65 and 0.69 respectively. The total value for the probability of identity was 2.74 × 10⁻⁹. The SSRs studied allowed the unambiguous identification of all the mango genotypes studied and this discrimination can be carried out with just three selected microsatellites. UPGMA cluster analysis and Principal coordinates analysis group the genotypes according to their origin and their classification as monoembryonic or polyembryonic types reflecting the pedigree of the cultivars and the movement of mango germplasm. The results demonstrate the usefulness of microsatellites for studies on identification, variability, germplasm conservation, domestication and movement of germplasm in mango.     

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     

Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Identifying Novel Polymorphic Microsatellites from Cultivated Flax (Linum usitatissimum L.) Following Data Mining

One of the major concerns in genetic characterization and breeding of cultivated flax is the lack of informative microsatellite markers (SSRs). In this regard, the development of SSRs using molecular methods might be time-consuming, laborious, and expensive. On the other hand, using bioinformatics to mine sequences in public databases enables a cost-effective discovery of SSRs. A total of 3,242 Linum usitatissimum genomic sequences were surveyed for the identification of SSRs. Among them, 118 non-redundant sequences containing repeats were selected for designing primers. The most abundant motifs were tri- (72.4%) and dinudeotide (16.6%), within which AGG/CCT and AG/CT were predominant. Primers were tested for polymorphism in 60 L. usitatissimum cultivars/accessions including 57 linseed and three fiber flax. Eighty-eight pairs gave amplifications within the expected size range while 60 pairs were found to be polymorphic. The mean number of alleles amplified per primer was 3.0 (range, 2–8; 180 total alleles). The mean polymorphism information content (PIC) value was 0.39 (range, 0.06–0.87), and the highest average PIC was observed in dinucleotide SSRs (0.41). The SSR data mining presented here demonstrates the usefulness of in silico development of microsatellites. These novel genomic SSR markers could be used in genetic diversity studies, the development of genetic linkage maps, quantitative trait loci mapping, association mapping, and marker-assisted selection.     

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of microsatellites from cacao–<Emphasis Type="Italic">Moniliophthora perniciosa</Emphasis> interaction expressed sequence tags

Theobroma cacao L.–Moniliophthora perniciosa expressed sequence tags (ESTs) were converted into useful satellite markers for population analysis and genetic mapping. Forty-nine flanking primer pairs from TSH1188 (a resistant genotype) and Catongo (a susceptible genotype) ESTs were designed and screened for polymorphism analysis. Eleven were polymorphic, with an average of 3.81 alleles per locus and a total of 42 alleles. The satellite markers were tested on 21 cacao accessions and two bulked DNAs generated from 6 resistant and 6 susceptible plants from a segregating F₂ (SCA6 × ICS1) population for witches’ broom resistance. These results show that EST-derived microsatellites (short sequence repeats, SSRs) in Theobroma cacao have many potential applications in linkage mapping and the planning of crosses.     

AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus

 We report the sequences of 17 primer pairs of microsatellite loci, which we have cloned and sequenced from two genomic libraries of peach [Prunus persica (L) Batsch] ‘Redhaven’, enriched for AC/GT and AG/CT repeats respectively. For ten of these microsatellite loci we were able to demonstrate Mendelian inheritance in a segregating back-cross population; the remainder did not segregate. The polymorphism of the microsatellites was evaluated in a panel of ten peach genotypes, including true-to-type peaches, nectarines and one canning-peach. Fifteen microsatellites (88%) were polymorphic showing 2–4 alleles each. The mean heterozygosity, averaged over all loci, was 0.32 and significantly higher than that reported in the literature for isozymes and molecular markers, such as RFLPs and RAPDs. We have also assayed the cross-species transportability and found that ten microsatellite (59%) gave apparently correct amplification in all Prunus species surveyed, namely P. domestica (European plum), P. salicina (Japanese plum), P. armeniaca (apricot), P. dulcis (almond), P. persica var. vulgaris (peach), P. persica var. laevis (nectarine), P. avium (sweet cherry) and P. cerasus (sour cherry), with three of them also being amplified in Malus (apple). The remaining microsatellites gave less-extensive amplification. Because of their appreciable polymorphism and wide cross-species transportability, most of these new markers can be integrated into the linkage maps which are currently being constructed in peach, as well as in other stone fruit crops, such as almond, apricot, cherry and plum. Received: 3 September 1998 / Accepted: 28 November 1998     

Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

High Frequency and Large Number of Polymorphic Microsatellites in Cultured Shrimp, <Emphasis Type="Italic">Penaeus (Litopenaeus) vannamei</Emphasis> [Crustacea:Decapoda]

A total of 1479 recombinant clones were obtained from a Sau3A-digested genomic library of Penaeus (Litopenaeus) vannamei and used for probe hybridization. Of the 251 clones that tested positive to one or more of the probes and were sequenced, 173 (69%) contained 573 simple sequence repeats, or microsatellites, with 3 or more repeats. The frequency of microsatellites with 3, 5, and 10 or more repeats was 1 in 0.94 kb, 1 in 2.78 kb, and 1 in 5.94 kb, respectively. To increase the number of polymorphic markers for mapping, 136 primer sets that flanked microsatellites containing single or multiple motifs with 3 or more repeats were designed and tested. Of the 136 primers, 93 (68.0%) were polymorphic in cultured shrimp, with polymorphism information content (PIC) values ranging from 0.195 to 0.873, and observed heterozygosities ranging from 10% to 100%. These markers are being used along with other markers to construct a linkage map for P. vannamei.