Eighteen microsatellite loci in Salix arbutifolia (Salicaceae) and cross-species amplification in Salix and Populus species

Salix arbutifolia is a riparian dioecious tree species that is of conservation concern in Japan because of its highly restricted distribution. Eighteen polymorphic loci of dinucleotide microsatellites were isolated and characterized. Among these, estimates of the expected heterozygosity ranged from 0.350 to 0.879. Cross-species amplification was successful at 9-13 loci among six Salix species and at three loci in one Populus species.     

Development of microsatellite markers for the endangered Neotropical tree species Tibouchina papyrus (Melastomataceae)

We isolated and characterized 12 microsatellite loci for Tibouchina papyrus (Melastomataceae), an endangered species with narrow and disjunct range, endemics to a few localities in "cerrado rupestre" from Central Brazil. These microsatellites were obtained by sequencing of a genomic shotgun library for primer design. Leaves from 96 individuals collected in the three known local populations were genotyped using the 12 primers designed to analyze the polymorphisms at each locus. The number of alleles per locus ranged from one to six; two loci were monomorphic. Among the polymorphic loci, expected heterozygosities ranged from 0.161 to 0.714. Combined paternity exclusion probability was 0.957 and combined genetic identity (0.051) was high for studies on parentage. Tibouchina papyrus is a rare and endemic tree species of outcrop quartzite and sandstone soils, with highly isolated populations, which may have lead to the low degree of polymorphism that we detected. Also, motifs of most loci are larger than dinucleotide, which typically display lower levels of polymorphism.     

Eight microsatellite markers for the neotropical tree Luehea seemannii (Tiliaceae)

We isolated eight polymorphic microsatellites from the neotropical tree Luehea seemannii for gene flow and genetic structure studies. We used a streptavidin subtractive enrichment technique to develop a library of CA/GT repeats. Eight loci were screened for diversity from 96 individuals from Barro Colorado Island (BCI) and neighbouring Gigante peninsula in Panama. Luehea seemannii shows moderate levels of genetic diversity within these two populations. Allelic richness ranged from four to nine alleles and averaged 6.44 alleles per locus. Average expected heterozygosity was 0.65 on BCI and 0.60 on Gigante. Results are compared to microsatellite data from another wind‐dispersed gap colonizing species common in Panama.     

Characterization and cross‐species amplification of microsatellite markers in cherimoya (Annona cherimola Mill., Annonaceae)

Fifteen polymorphic microsatellites were developed using a CT/AG‐enriched genomic library of cherimoya cv. Fino de Jete and screened on 23 genotypes from different geographical areas. This is the first set of microsatellites developed in Annonaceae. Fourteen of the microsatellites detected a single locus and only one detected two loci. The number of alleles in the single locus microsatellites ranged from two to six and the observed and expected heterozygosity ranged from 0.17 to 0.61 and from 0.12 to 0.76, respectively. Most of the simple sequence repeats were transferable to other species in the family.     

Development of microsatellite markers in peach [ Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry ( Prunus avium L.)

We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F(2) progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus x domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.     

Development and characterization of nine highly polymorphic microsatellite primers for Platycladus orientalis (Cupressaceae)

? Premise of the study: Microsatellite primers were developed and characterized for the long-lived tree species Platycladus orientalis for further investigation of its population characteristics. ? Methods and Results: Nine polymorphic microsatellites were identified for P. orientalis. The number of alleles per locus ranged from four to 22 (average = 11) for 94 individual specimens examined from four populations. The observed and expected heterozygosities ranged from 0.208 to 1.000 and 0.555 to 0.931, respectively. ? Conclusions: These nine markers can be used in future studies examining population genetics and the reproductive biology of P. orientalis, thereby revealing the importance of old-growth populations for the retention of genetic diversity, and providing useful insights into the maintenance of the potential genetic resource of this species.     

Development and characterization of microsatellites of an important medicinal plant Orthosiphon stamineus (misai kucing)

Orthosiphon stamineus (Lamiaceae) is an important medicinal plant in Malaysia that has great potential in the herbal medicine industry. In this study, we developed and characterized ten microsatellites for O. stamineus. The microsatellite loci were checked for variability using 28 individuals of O. stamineus sampled from 10 nurseries. The number of alleles ranged from two to four, and the observed heterozygosity ranged from 0.143 to 0.963. Neighbor-joining analysis revealed two main genetic clusters corresponding to the two varieties (white and purple). The application of these microsatellites will facilitate the cultivar/variety identification and germplasm management in the species.     

Development of polymorphic microsatellite markers for Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

Dalbergia nigra is an endangered tree restricted to the Brazilian Atlantic Forest. Ten polymorphic microsatellite loci were isolated and characterized in 47 trees from two populations. The total number of alleles per locus ranged from three to 12 alleles. The levels of observed and expected heterozygosities ranged from 0.304 to 0.740 and from 0.278 to 0.872, respectively. Significant deviations from Hardy-Weinberg were detected for only three loci in each population. No pair of loci exhibited significant linkage disequilibrium. These microsatellites provide an efficient tool to investigate genetic structure in forest remnants with the purpose of conservation of this species.     

Microsatellites for the net‐spinning caddisfly Plectrocnemia conspersa (Polycentropodidae)

Six polymorphic microsatellites were developed for the caddisfly Plectrocnemia conspersa from two enriched partial genomic libraries. These represent the first microsatellites published for the order Trichoptera. We show that, whilst it is possible to develop a highly enriched library, isolation of polymorphic microsatellites is difficult for this species, as has been found in some other of invertebrate groups. The genotypes of 160 individuals were determined using these new loci. Observed heterozygosities ranged from 0.013 to 0.788. Despite their problematic isolation, polymorphic microsatellite loci show greater levels of variation than previously studied allozyme markers.     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel microsatellite loci in the grass snake (Natrix natrix) and cross-amplification in the dice snake (Natrix tessellata)

Six novel polymorphic microsatellite loci are presented for the grass snake (Natrix natrix), a species with declining populations in many regions. The number of alleles per locus ranged from two to seven. Four dice snake (Natrix tessellata) microsatellites were polymorphic in the grass snake with three to four alleles. At two loci, the expected heterozygosity differed significantly from observed heterozygosity. Cross-amplification of the grass snake markers in the dice snake showed two polymorphic microsatellites with two and four alleles.     

Molecular data reveal isolation by distance and past population expansion for the shea tree (Vitellaria paradoxa C.F. Gaertn) in West Africa

While the genetic structure of many tree species in temperate, American and Asian regions is largely explained by climatic oscillations and subsequent habitat contractions and expansions, little is known about Africa. We investigated the genetic diversity and structure of shea tree (Vitellaria paradoxa,) in Western Africa, an economically important tree species in the Sudano-Sahelian zone. Eleven nuclear microsatellites (nuc) were used to genotype 673 trees selected in 38 populations. They revealed moderate to high within-population diversity: allelic richness ranged from R(nuc) = 3.99 to 5.63. This diversity was evenly distributed across West Africa. Populations were weakly differentiated (F(STnuc) = 0.085; P < 0.0001) and a pattern of isolation by distance was noted. No phylogeographic signal could be detected across the studied sample. Additionally, two chloroplast microsatellite loci, leading to 11 chlorotypes, were used to analyse a sub-set of 370 individuals. Some variation in chloroplast allelic richness among populations could be detected (R(cp) = 0.00 to 4.36), but these differences were not significant. No trend with latitude and longitude were observed. Differentiation was marked (G(STcp) = 0.553; P < 0.0001), but without a significant phylogeographical signal. Population expansion was detected considering the total population using approximate Bayesian computation (nuclear microsatellites) and mismatch distribution (chloroplast microsatellites) methods. This expansion signal and the isolation by distance pattern could be linked to the past climatic conditions in West Africa during the Pleistocene and Holocene which should have been favourable to shea tree development. In addition, human activities through agroforestry and domestication (started 10,000 bp) have probably enhanced gene flow and population expansion.     

Isolation and cross-amplification of microsatellites in pink abalone (Haliotis corrugata)

Ten novel microsatellite loci were isolated in pink abalone, Haliotis corrugata, using (GT)₁₅ and (CT)₁₅ enriched genomic libraries. Two previously reported Haliotis kamtschatkana microsatellites cross‐amplified in H. corrugata. A set of 12 polymorphic microsatellites were evaluated in a wild population sample (N = 49). The number of alleles ranged from two to 55, and the observed and expected heterozygosities ranged from 0.104 to 0.939 and from 0.213 to 0.982, respectively. Significant deviations from Hardy–Weinberg equilibrium at three loci and no linkage disequilibrium were observed. Haliotis corrugata microsatellites cross‐amplified in other abalone species, two in H. fulgens, and seven in H. rufescens.     

Isolation and characterization of novel microsatellite markers in commercial selected golden Malaysian arowana fish, Scleropages formosus (Osteoglossidae)

Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Isolation and characterization of microsatellite DNA primers in Juniperus przewalskii Kom (Cupressaceae)

Juniperus przewalskii (Cupressaceae) is a dominant tree species endemic to the northeast Qinghai-Tibetan Plateau. This species plays an important role in maintaining the arid ecosystem in this region. However, natural distributions of this species have been declined. In order to develop effective conservation methods, it is important to know the distribution of the genetic diversity within and among populations. In this study, we developed nine new microsatellite loci for this species. We used the combining biotin capture method to enrich AG/CT/AC/GGT microsatellites. The polymorphisms of each locus were further assessed in 12 individuals from four geographically distant populations. The number of alleles per locus varied from three to six and expected heterozygosity ranged from 0.58 to 0.70. These loci together provide a useful tool to investigate the genetic diversity of this species. In addition, all markers have been crossly checked in the other four congeneric species.     

Microsatellites from kousa dogwood (Cornus kousa)

Microsatellite loci were identified from Cornus kousa'National'. Primer pairs for 86 loci were developed and of these, eight were optimized and screened using genomic DNA from 22 kousa cultivars. All optimized loci were polymorphic and the number of alleles per locus ranged from three to 17. Observed heterozygosity ranged from 0 to 0.3 and expected heterozygosity ranged from 0.38 to 0.91. These microsatellites will be useful in population studies, and a breeding programme for cultivar development of Cornus species.     

Development of microsatellite markers for the wood cricket, Nemobius sylvestris (Orthoptera: Gryllidae)

Thirty-four novel microsatellite markers developed for wood cricket (Nemobius sylvestris) were tested and optimized. Twenty-five microsatellite loci were polymorphic, exhibiting between two and nine alleles. Observed heterozygosities ranged from 0.038 to 0.925. The microsatellites were also tested in a species belonging to another genus of the Gryllidae family (Gryllus bimaculatus). Two markers produced clear banding patterns with the expected product size. These markers will be used to study the effects of forest fragmentation on genetic connectivity using wood cricket as a model species.     

Two highly informative dinucleotide SSR multiplexes for the conifer Larix decidua (European larch)

We have designed two highly polymorphic microsatellite multiplexes for Larix decidua Mill (European larch), a coniferous tree species with a fragmented distribution across Europe. The multiplexes combine microsatellites previously designed for the sister species L. kaempferi and newly identified microsatellites obtained by pyrosequencing of an enriched microsatellite library and subsequent marker candidate selection. As we wanted to target highly polymorphic markers, only microsatellite motifs with a high number of repeats (≥ 12) were selected. An important proportion of the marker candidates presented multiple bands, bad amplification or insufficient polymorphism. Such difficulties were expected owing to the large genome size of the studied species. Our strategy for marker validation followed most recent recommendations for microsatellite development, for example verifying marker quality in terms of polymorphism and accurate allele binning before multiplexing. The most promising loci were combined in two multiplexes, a 7-plex and a 6-plex. These were tested on a sample of 413 individuals from 18 populations distributed across the natural range. The 13 loci had from 9 to 36 alleles. Markers were successfully tested in another laboratory, confirming robustness of the marker protocols. We also tested transferability on six other larch species from Asia and North America. Overall, this study shows that, even in species with large genome size and relatively low overall polymorphism, microsatellites can be successfully developed using next-generation sequencing technologies, provided that some additional precautions are taken compared to species lacking these characteristics.     

Development of microsatellite markers in the noxious red tide‐causing dinoflagellate Heterocapsa circularisquama (Dinophyceae)

We isolated 15 polymorphic microsatellites from one of the most noxious red tide‐causing dinoflagellate species, Heterocapsa circularisquama. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from two to six, and the estimate of gene diversity from 0.205 to 0.684 across the 15 microsatellites. These loci have the potential to reveal genetic structure and gene flow among H. circularisquama populations.