Chloroplast microsatellite markers for the mangrove tree species Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa, and cross-amplification in other mangrove species

Chloroplast microsatellite (cpSSR) markers were developed for three ecologically and economically important tree species in the mangrove family, Rhizophoraceae: Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa. Noncoding regions of chloroplast DNA (cpDNA) from each species were separately amplified using universal chloroplast primers. Six, two, and three polymorphic cpSSR loci in B. gymnorrhiza, K. candel, and R. stylosa, respectively, were developed from amplified noncoding cpDNA regions. Characterization of 216, 156, and 253 individuals of B. gymnorrhiza, K. candel, and R. stylosa, respectively, collected from different natural mangrove populations (B. gymnorrhiza, 9; K. candel, 7; R. stylosa, 9) on Iriomote Island in Japan showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.027 and 0.480. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of the three species. Several of these markers may also be useful in similar studies of other mangrove species.     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Isolation and characterization of microsatellite markers in Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an important threatened medicinal plant (TMP) species. Here we isolated nine polymorphic microsatellite loci from D. versipellis using a modified biotin-capture method. Our isolated loci provided SSR markers with polymorphism of 2–7 alleles per locus. The expected and observed heterozygosities ranged from 0.507 to 0.864 and from 0.360 to 0.720, respectively. These markers would be the useful tools for analyzing questions concerning population genetic structure and mating system of D. versipellis.     

An improved technique for isolating codominant compound microsatellite markers

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)₆(AG)₅ or (TC)₆(AC)₅ and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.     

A kiwifruit (Actinidia spp.) linkage map based on microsatellites and integrated with AFLP markers

A genetic map of kiwifruit (Actinidia spp.) was constructed using microsatellite and AFLP markers and the pseudo-testcross mapping strategy. (AC)_n and (AG)_n microsatellite repeats were first isolated from Actinidia chinensis (2n = 2x = 58) enriched genomic libraries and tested for segregation in the interspecific cross between the diploid distantly related species A. chinensis and A. callosa. Some 105 microsatellite loci of the 251 initially tested segregated in the progeny in a 1:1 ratio as in a classical backcross, or in a ratio which could mimic the backcross, and were mapped using 94 individuals. AFLP markers were then produced using MseI and EcoRI restriction enzymes and 15 primer combinations. Nearly 10% of loci showed a distorted segregation at α = 0.05, and only 4% at α = 0.01, irrespectively to the marker class. Two linkage maps were produced, one for each parent. The female map had 203 loci, of which 160 (71 SSR and 89 AFLP) constituted the framework map at a LOD score ≥ 2.0. The map was 1,758.5 cM(K) long, covering 46% of the estimated genome length. The male map had only 143 loci, of which 116 (28 SSR, 87 AFLP and the sex determinant) constituted the framework map. The map length was only 1,104.1 cM(K), covering 34% of the estimate genome length. Only 35 SSR loci were mapped in the male parent because 18% of SSR loci that were characterised did not amplify in A. callosa, and 48% were homozygous. The choice of parents in the pseudo-testcross is critically discussed. The sex determinant was mapped in A. callosa. Received: 27 July 2000 / Accepted: 31 October 2000     

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     

Isolation, characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)

The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

Rapid Evolution of Simple Sequence Repeat Induced by Allopolyploidization

Microsatellite evolution normally occurs in diploids. Until now, there has been a lack of direct experimental evidence for microsatellite evolution following allopolyploidization. In the present study, F₁ hybrids and newly synthesized allopolyploids were derived from Triticum aestivum Chinese Spring × Secale cereale Jinzhou-heimai. One hundred and sixty-three wheat simple sequence repeat (SSR) markers were used to investigate the variation of wheat microsatellites after allopolyploidization and variation of the PCR products of 29 of the SSR markers was observed. Of these 29 SSR markers, 15 were unable to produce products from amphiploids. The other 14 SSR markers did produce products from parental wheat, F₁ hybrids and amphiploids. However, the length of the products amplified from amphiploids was different from the length of the products amplified from parental wheat and F₁ hybrids. Sequencing indicated that the length variation of the 14 microsatellites stemmed mainly from variation in the number of repeat units. The alteration of repeat units occurred in both perfect and compound repeats. In some compound SSR loci, one motif was observed to expand whereas another to contract. Almost all the microsatellite evolution observed in this study could be explained by the slipped-strand mispairing model. The results of this study seem to indicate that stress caused by allopolyploidization might be one of the factors that induce microsatellite evolution. In addition, the findings of present study provided an instance of how simple sequence repeats evolved after allopolyploidization.     

Accumulation,distribution and toxicological effects induced by cadmium on the development of mangrove plant <Emphasis Type="Italic">Kandelia candel</Emphasis> (L.)

Accumulation and distribution pattern of cadmium (Cd) and its toxic effect on growth of the mangrove plant of young Kandelia candel seedlings have been examined. This study demonstrated that under high concentration of Cd stress, the total biomass of K. candel decreased 41.57% compare to control (CK). At the end of 90 days exposure to 25 mg/l Cd, the average seedlings stem height and leaf number of the K. candel decreased by 30.54 and 42.68%, respectively. The results showed that K. candel seedlings, under the experimental condition, accumulated higher concentration of Cd in their roots (411.29 ± 3.60 mg/kg) when compared to hypocotyls, stems and leaves. More than 95% of Cd was accumulated mainly in roots. The distribution pattern of Cd concentration in K. candel seedlings was found in the following order: roots > hypocotyls > stems > leaves. Based on the leaf symptoms and morphological change of K. candel seedlings under heavy metal stress, this study showed that Cd is phytotoxic to K. candel.     

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.     

Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species,Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

Avicennia marina is an ecologically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from this species using an improved technique. Our isolated loci provided compound microsatellite markers with polymorphism of two to six alleles per locus. The observed and expected heterozygosities ranged from 0.025 to 0.625 and from 0.096 to 0.767, respectively. These markers would be the useful tools for researching on the genetic diversity and population genetic structure of A. marina.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Prunus verecunda</Emphasis> and <Emphasis Type="Italic">Prunus grayana</Emphasis> (Rosaceae)

Simple sequence repeat (SSR) markers were developed for Prunus verecunda and Prunus grayana to help understand the seed dispersal pattern of each species. We isolated and characterized nine microsatellite loci (four from P. verecunda and five from P. grayana). In P. verecunda, the number of alleles detected and the expected heterozygosity of five loci ranged from 11 to 24 and 0.59 to 0.92, respectively. In P. grayana, the number of alleles detected and the expected heterozygosity of five loci ranged from 4 to 14 and 0.62 to 0.86, respectively. These results show that the markers described here will be useful in studying the seed dispersal pattern of Prunus species.     

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.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

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     

Development of SSR markers for typing cultivars in the mushroom Auricularia auricula-judae

Auricularia auricula-judae, also known as black wood ear, is one of the most popular edible mushrooms in China. But the confusion about cultivars has limited the development of A. auricula-judae production. In this article, 17 polymorphic SSR markers were cloned and used to differentiate the cultivars of A. auricula-judae. The polymorphism information content (PIC) of these SSR ranged from 0.10 to 0.84, while the average was 0.47. The number of alleles detected for each locus was 2–11, with an average of 4.7 alleles per locus. The dendrogram, based on 17 SSR markers by UPGMA clustering, could differentiate the 16 A. auricula-judae cultivars in this study. In fact, the 16 cultivars analyzed in this study could be efficiently differentiated using a combination of three polymorphic SSR loci with high PIC. The total of 17 polymorphic SSR loci could also be amplified correctly in the A. polytricha strains surveyed. This is the first report on the development of SSR markers in the genus Auricularia.     

A panel of polymorphic EST-derived microsatellite loci for the small yellow croaker (Larimichthys polyactis)

Small yellow croaker (Larimichthys polyactis) is an important economic species of marine fishery. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Pseudosciaena crocea, Paralichthys olivaceus and Psetta maxima. Characteristics of nine EST–SSR loci were investigated using 46 L. polyactis individuals. The number of alleles per locus ranged from 2 to 6. The observed heterozygosity ranged from 0.0652 to 0.7391, while the expected heterozygosity ranged from 0.0638 to 0.7754. Seven loci departed from Hardy–Weinberg equilibrium (P < 0.01) significantly. These loci and markers will be useful for population genetics and systemic evolution among species of small yellow croaker.     

Simple sequence repeat (SSR) markers in the ectomycorrhizal fungus Laccaria bicolor for environmental monitoring of introduced strains and molecular ecology applications

Simple sequence repeat (SSR) markers were developed from SSR-enriched genome libraries for the ectomycorrhizal basidiomycete Laccaria bicolor. Seven markers were single-locus and amplified unambiguously in L. bicolor. The seven SSR markers were further characterized using an array of 15 L. bicolor strains representative of diverse origins worldwide. The observed number of alleles per locus varied from 5–9 and the values of observed heterozygosity from 0.167 to 0.667. The seven SSR loci could be amplified from DNA extracted from root tips of L. bicolor inoculated pine seedlings. All the L. bicolor ectomycorrhizas analysed exhibited the same SSR multi-locus profile as that detected for the UAMH8232 inoculant strain. The set of markers described represents a potent tool for the monitoring of introduced strains of L. bicolor and for molecular ecology applications.     

Analysis of microsatellite locus polymorphism in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) cultivars of Russian breeding

Polymorphism of microsatellite loci of the nuclear genome was examined in 29 cultivars and accessions of wild potato (S. tuberosum, S. stoloniferum, S. demissum, and S. phureja). Nine SSR markers, most informative (PIC = 0.61–0.92) for genotyping of the cultivars of Russian breeding were selected. Polymorphism of the selected SSR loci was characterized, and prevailing, as well as unique SSR allele phenotypes were described. A total of 87 allele phenotypes were identified. The highest number of allele phenotypes was detected for the SSR1 (17), ST83/84 (12), and STRBCS1b (12) loci. The least numbers of allele phenotypes were typical of the ST47/48 (5) and STWIN12G (6) loci. Based on the microsatellite loci analyzed, for each of the cultivars examined, its allele formula was established. The latter can be uses as the cultivar molecular genetic passport. Diagnostic sets of most informative loci (SSR markers), enabling identification of the genotypes of all potato cultivars of Russian breeding examined, were determined