Development of microsatellite markers in a mangrove tree species Aegiceras corniculatum (Myrsinaceae)

Aegiceras corniculatum is an ecologically important mangrove tree species. We isolated 18 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with polymorphism of two to eight alleles per locus. The observed and expected heterozygosities ranged from 0.050 to 0.550, and from 0.097 to 0.736, respectively. These markers will contribute to research on the conservation, genetic diversity and mating patterns of A. corniculatum.     

Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Isolation of microsatellite loci and cross‐species amplifications in three gobiid fish of the genus Pomatoschistus

Gobiids of the genus Pomatoschistus are increasingly being investigated as models for adaptation to coastal environments and for mating system studies. Among the dozen currently analysed species, microsatellite primers have been characterized only for Pomatoschistus minutus. This paper describes seven new polymorphic loci isolated from Pomatoschistus marmoratus and Pomatoschistus microps, two species that hybridize. Cross‐species amplification was tested for these new loci, together with seven already published P. minutus loci. Systematic amplification of samples of each of the three species provided a first indication of their polymorphism.     

Development and characterization of SSR markers in Ribes species

Eleven microsatellite loci were identified and characterized in blackcurrant (Ribes nigrum L.) and related species. An enriched library was constructed and screened with simple sequence repeat (SSR) oligonucleotides. Positive clones were sequenced and primers were designed flanking the repeat motifs. These 11 microsatellites produce amplification products polymorphic across a range of Ribes germplasm, predominantly from the Eucoreosma section of the genus. The number of alleles varied from 2 to 18 with levels of diversity ranging from 0.18 to 0.91.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species. Received: 6 October 2000 / Accepted: 13 March 2001     

Development of microsatellite markers in ginkgo (Ginkgo biloba L.)

Ginkgo biloba L. is one of the oldest unevolved tree species on Earth. We isolated five polymorphic microsatellite loci from G. biloba using a dual‐suppression polymerase chain reaction technique. These loci provided microsatellite markers with high polymorphism ranging from three to 13 alleles per locus. The observed and expected heterozygosities ranged from 0.667 to 0.952 and from 0.640 to 0.897, respectively. The markers will contribute to research on the conservation, genetic diversity and mating patterns of G. biloba.     

Isolation and characterisation of microsatellite markers for the Australian monitor lizard,Varanus acanthurus (Squamata: Varanidae) and their utility in other selected varanid species

There have been no molecular genetic examinations of mating systems in the Australian varanid lizards (genus Varanus) despite their high species diversity, the abundance of some species and difficulties with direct observation of behaviourally cryptic species in the field. We developed 10 polymorphic microsatellite loci and assessed their utility in a range of varanid species. Observed heterozygosities in the three species assessed ranged from 30% to 100%. These loci should be useful for investigation of population structure, gene flow and mating systems in Varanus acanthurus, V. baritji and V. tristis and may also be of use in other varanid species.     

Isolation and characterization of microsatellite loci in Schizolobium parahyba (Leguminosae)

Primer sequences for 16 microsatellite loci were developed from Schizolobium parahyba, a tropical tree species. Twelve loci were found to be polymorphic after screening diversity in individuals from Belize. A total of 39 alleles were found at nine loci. The markers are invaluable tools for studying the population genetics and mating system of the species.     

Characterization of tetranucleotide microsatellite markers in guppy (Poecilia reticulata)

Ten tetranucleotide microsatellite loci are characterized for guppy, Poecilia reticulata, an important model species in the study of adaptation and mating systems. Loci were isolated following a microsatellite enrichment procedure using probe‐labelled magnetic beads. These microsatellites were designed for use in examining gene flow, reproductive isolation, and parentage within natural guppy populations.     

Conservation of microsatellite loci within the genus Vitis

Eleven microsatellites isolated from grapevine (Vitis vinifera) were used to study the degree of conservation of these sequences across different Vitis species. Nine microsatellites were newly isolated, the remaining two (VVS2 and VVS5) came from the literature. A preliminary assay on the conservation of priming sites was carried out on 14 non-V. vinifera species, including relevant taxa for breeding. Parthenocissus quinquefolia was added as representative of a related genus. Cross-species amplification was obtained in 94% of the 176 genotype×locus tested combinations. Three microsatellite loci were then cloned and sequenced in ten species. The microsatellite repeat was found present in all cases. The repeat region was often longer in V. vinifera than in the other species. Furthermore the non-source species showed interruptions in the repeat. In spite of these constraints, which could reduce the polymorphism of microsatellites in non-source species, the results demonstrate the possibility of extending the use of microsatellite markers to wild germplasm and inter-specific hybrids. Point mutations have been found in microsatellite flanking regions and these variations have been used to investigate the genetic relationship among taxa. The Neighbor-joining tree that was obtained on the basis of ten nucleotide variations, showed that there is not a clear cut difference between American, Asian and European species and that the actual taxonomy which reflects the geographical distribution of species must most likely be revised. Moreover, in general, nucleotide variations which occur in microsatellite flanking regions provide new molecular tools for investigating the evolution of species. Received: 24 October 1999 / Accepted: 11 November 1999     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

Polymorphism of microsatellite loci of European chickpea cultivars

The polymorphism of 13 microsatellite loci in 61 chickpea varieties from the National Center of Genetic Plant Resources of Ukraine was studied. Forty-seven alleles were detected. Three loci were monomorphic; the rest showed polymorphism. The genetic diversity of chickpea varieties in all loci was 0.41 according to the Nei polymorphism index (D). It was concluded that chickpea varieties from Europe had insignificant microsatellite loci diversity. The most polymorphic group included chickpea varieties from Russia (D = 0.38); the least polymorphic group, Spanish varieties (D = 0.25). A consensus tree representing the most credible divergence of Europe chickpea varieties was constructed. The possible reasons for clustering chickpea varieties in a common cluster are discussed.     

Development of 10 microsatellite loci for <Emphasis Type="Italic">Rheum tanguticum</Emphasis> (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource with R. tanguticum. These microsatellite markers could provide a useful tool for genetic and conservation studies of the rhubarb species.     

Isolation and characterization of microsatellites in Echinochloa (L.) Beauv. spp.

Five primer pairs were developed that amplify microsatellite loci in three agronomically important Echinochloa (L.) Beauv. species: E. colona (L.) Link, E. crus‐galli (L.) Beauv. and E. crus‐pavonis (Kunth) Schultes. The microsatellites were tested on 24 individuals representing three species collected in rice fields from different geographical regions and revealed 3–7 alleles per microsatellite. Gene diversity [1 ? Σp_ij²] for four polymorphic loci within E. crus‐galli ranged from 0.12 to 0.61. Alleles at a fifth locus were useful in discriminating the species. The microsatellites should provide useful markers for intraspecific diversity studies and aid classification of species within this complex genus.     

Novel Polymorphic Microsatellite Markers for Panulirus ornatus and their Cross-species Primer Amplification in Panulirus homarus

Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species.     

Development and characterization of microsatellite markers in pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L.)

The pomegranate (Punica granatum L.) is a temperate climate species requiring high temperatures for proper and complete ripening. The species is consumed as a fresh fruit, but also can be used to obtain transformed products such as juice, jam, or preserve. It is a fruit tree species with a high degree of diversity, but the identification of cultivars by morphological traits is very difficult. Thus, the characterization of genotypes through molecular markers is of great value for germplasm preservation, genetic studies, and plant breeding. The number of simple sequence repeat (SSR or microsatellite) markers developed for this genus is too low, so in this work we report the development of 117 microsatellite loci from a CT/AG-enriched pomegranate genomic library. In order to check their utility, eleven accessions were analyzed. The polymorphism information content (PIC) value across all loci ranged between 0.09 and 0.71, with an average of 0.37. These markers will facilitate genetic diversity studies, mapping, and genotyping of pomegranate.     

Isolation of microsatellite loci from the scleractinian corals,Montastraea cavernosa and Porites astreoides

The ability to assess genetic variation is critical for determining genetic diversity and population structure. In corals, slow evolutionary rates in mitochondrial genomes have left allozymes as the only markers presently available to investigate patterns of intraspecific genetic variation. Characteristics of microsatellites render them more informative than allozymes for such analyses; however, few coral microsatellites are available. This study describes polymorphic microsatellite loci isolated from two scleractinian coral species. Most loci exhibit significant heterozygote deficiencies, likely due to nonrandom mating or Wahlund effects. These markers are being used to investigate gene flow among populations, providing insight into reef connectivity.     

Isolation and characterization of microsatellite loci from Eidothea hardeniana,an early Proteaceae

Eidothea hardeniana is a recently discovered rainforest species representing a basal Proteoid genus. It is known from a single location in New South Wales (Australia) with the only other member of the genus, Eidothea zoexylocarya, found in northern Queensland. We developed six microsatellite loci (five cross‐transferable) from an (AG) enriched library. The loci produced relatively high levels of diversity within 30 individuals of such a rare species. These microsatellites are now being used to provide new and useful information on the management of the species and on the processes shaping the evolution and persistence of ancient rainforest taxa.     

Two highly validated SSR multiplexes (8‐plex) for Euphrates' poplar,Populus euphratica (Salicaceae)

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight‐Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight‐plex of newly designed EST‐SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.