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.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.     

Characterization and cross-species amplification of microsatellites from the endangered Hawksbill turtle (Eretmochelys imbricate)

Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3–13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species.     

Characterization of microsatellites located within the genes of goldfish (Carassius auratus auratus)

The goldfish (Carassius auratus auratus; Cyprinidae) is not only an important ornamental fish species, but also a useful model for biological studies. The sequence of goldfish genes present in the public database was searched for short tandem repeats, and 11 polymorphic microsatellites were detected within eight important genes. Two microsatellites were located in coding regions of the c‐myc and GAP‐43 genes, respectively, whereas the others were mainly located in 5′ and 3′ untranscribed regions of other genes, such as gonadotropin and activin. The average allele number of these microsatellites was 5.5 per locus with a range between 3 and 9. These microsatellites will be useful for ecological and population genetic studies of this species, as well as for the ornamental fish industry.     

Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representative species of the genus Ficus

We developed microsatellites in fig (Ficus carica L.). A TC and TG‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Eight primer pairs produced amplification products that were both interpretable and polymorphic in 14 fig cultivars and two French wild‐growing populations of F. carica (n₁ = 9 and n₂ = 10). Number of alleles per locus ranged from three to six. Except for one microsatellite locus, the observed heterozygosity was higher than the expected value. The F. carica microsatellites gave amplification products in 17 other Ficus species in 86% of the cases.     

Isolation and multiplex analysis of six polymorphic microsatellites in the Antarctic notothenioid fish,Trematomus newnesi

Six polymorphic microsatellites were isolated from the dusky notothen, Trematomus newnesi (Boulenger 1902), using a microsatellite enrichment protocol and selective hybridization with di‐ tetra‐ and penta‐repeat probes. The loci were screened in 48 individuals captured in the Southern Ocean (coastal zone of Terre‐Adélie), revealing eight to 22 alleles per locus and an observed heterozygosity ranging from 0.70 to 0.92. These microsatellite markers provide a tool to study the relationship between the various morphs observed in this species and can be used for population genetic analysis and biodiversity studies.     

Atlantic capelin (Mallotus villosus) tetranucleotide microsatellites

Twelve microsatellite loci developed for Atlantic capelin (Mallotus villosus) using magnetic bead hybridization enrichment for tetranucleotide microsatellites revealed five loci composed of single repeat elements and six composed of complex repeats. Forty‐four beach‐spawning females from three different northwestern Atlantic Newfoundland beach‐spawning populations were screened at each locus. Loci were polymorphic (two to 59 alleles per locus) and all but two exhibited high heterozygosity (0.86–1). The loci are considered suitable for addressing questions related to fine‐scale population structure, spawning fidelity and survivorship/kinship issues.     

Isolation and characterization of microsatellite loci for Florida largemouth bass, Micropterus salmoides floridanus, and other micropterids

We isolated and characterized 52 novel microsatellite markers from Florida largemouth bass, Micropterus salmoides floridanus, for use in conservation, management and population genetic studies. Markers were assessed in M. s. floridanus from peninsular Florida (n = 30) and averaged eight alleles per locus with observed heterozygosity of 0.57 (range 0–0.97). Cross‐taxa amplification was successful among 88% of tested congeners. These polymorphic and potentially taxon‐diagnostic markers contribute to the limited number of microsatellites currently available for micropterids and specifically M. s. floridanus.     

Identification and characterization of nuclear microsatellites in Mediterranean cedars (Cedrus sp.)

We developed primers for the amplification of six polymorphic nuclear microsatellites in Mediterranean Cedrus taxa. Microsatellites originated from two Cedrus atlantica genomic libraries enriched for TC (four markers) and TG (two markers) motifs. No distortion from Mendelian segregation was observed. There were up to eight alleles per locus and mean expected heterozygosity was 0.75. Four microsatellites also amplified in the Himalayan cedar. These markers should be helpful for species identification, diversity studies, parentage analysis and genome mapping and to monitor biodiversity in endangered Mediterranean Cedrus forests.     

Isolation and characterization of microsatellite loci for the red panda,Ailurus fulgens

We report on the isolation and characterization of 10 microsatellites in the red panda Ailurus fulgens from genomic DNA‐enriched libraries. Twenty‐one microsatellites were screened from the libraries, and 10 of the screened microsatellites were polymorphic. The number of observed alleles for each locus in 24 individuals ranged from three to 12, and the expected and observed heterozygosity was 0.60–0.90 and 0.50–1.00, respectively. These markers should prove a useful tool for the study of genetic variation in red panda in the future.     

Somatic mutations at microsatellite loci in western Redcedar (Thuja plicata: Cupressaceae)

A per-generation somatic mutation rate for microsatellites was estimated in western redcedar (Thuja plicata, Donn ex D. Don.: Cupressaceae). A total of 80 trees representative of the average size and age of reproductive trees were sampled in four natural populations in southwestern British Columbia. Samples of bulked haploid megagametophytes were collected from two or three positions on each tree, assuming that the collections were far enough apart that the same mutant sector was not sampled twice. All samples were genotyped at eight microsatellite loci. A single mutation corresponding to a stepwise increase in one dinucleotide repeat was detected. The estimated mutation rate for microsatellites was 6.3 x 10(-4) mutations per locus per generation (or 3.1 x 10(-4) per allele per generation), with a 95% confidence interval of 3.0 x 10(-5) to 4.0 x 10(-3) mutations per locus. Somatic mutations can contribute to a greater mutational load in trees, as compared to shorter lived plants, and genotypic mosaics within an individual have important implications for plant defense strategies and plant evolution.     

Dinucleotide microsatellite loci isolated from flowering dogwood (Cornus florida L.)

We report eight variable dinucleotide microsatellite loci cloned from flowering dogwood (Cornus florida L.) using a biotin enrichment protocol. Degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR) was used to generate a population of DNA fragments, from which adenine‐cytosine dinucleotide (AC) and adenine‐guanine dinucleotide (AG) repeats were captured using biotinylated probes and streptavidin coated magnetic particles. The captured fragments were cloned into plasmids, and the plasmid library was screened for microsatellites using a simple PCR technique. Selected plasmids were sequenced, and PCR primers were designed and optimized using a thermal‐gradient thermocycler. The loci reported are highly variable with an average of 9.25 allele per locus and an average heterozygosity of 0.84.     

High somatic instability of a microsatellite locus in a clonal tree,<Emphasis Type="Italic"> Robinia pseudoacacia</Emphasis>

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13–71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.Communicated by H.F. Linskens     

Isolation and characterization of microsatellite loci in Bemisia tabaci

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo‐diploid species with a global distribution demonstrating strong geographical structure with eight recognizable genetic groups. Fifteen microsatellite loci (335 alleles, 6–44 alleles per locus) were derived from four of the eight groups and were then screened across 33 populations. These loci clearly differentiate the populations. The microsatellites amplified best in individuals from genetic groups rep‐resenting the Mediterranean, Middle East, Asia (three groups) and Australasia/Oceania and amplified less well with populations from sub‐Saharan Africa and the New World. This differential amplification pattern is a direct result of the relatedness to the microsatellite source material.     

PERMANENT GENETIC RESOURCES: PCR primers for 100 microsatellites in red drum (Sciaenops ocellatus)

One hundred nuclear‐encoded microsatellites from a genomic library of red drum (Sciaenops ocellatus) were isolated and characterized. Eight microsatellites had tetranucleotide motifs; 92 had dinucleotide motifs. The average number of alleles per microsatellite (sample of 22–24 fish) was 17.7 (range = 2–30); gene diversity averaged 0.796 (range = 0.227–1.000). Following Bonferroni correction, genotype frequencies at 90 microsatellites did not deviate significantly from Hardy–Weinberg equilibrium expectations. Occurrence of null alleles was inferred at 15 microsatellites; alleles differing by only a single base were observed at 11 microsatellites. The microsatellites developed should prove useful for population‐genetic studies of ‘wild’ red drum and in construction of a genetic map.     

Development of a genome-wide anchored microsatellite map for common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A total of 150 microsatellite markers developed for common bean (Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican × Andean population, DOR364 × G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.Communicated by H.F. Linskens     

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.     

PERMANENT GENETIC RESOURCES: Development of 52 new polymorphic SSR markers from cherimoya (Annona cherimola Mill.): transferability to related taxa and selection of a reduced set for DNA fingerprinting and diversity studies

Fifty-two single locus polymorphic microsatellites were developed using two genomic libraries digested with HaeIII and RsaI of cherimoya cv. Fino de Jete enriched in CT/AG repeats. A total of 222 alleles were detected with the selected simple sequence repeats (SSRs). The observed and expected heterozygosities ranged from 0.08 to 0.73 and from 0.20 to 0.84, respectively. Most of the SSRs were transferable to other species in the Annonaceae. A set of 20 microsatellites was selected to facilitate the exchange of data among laboratories.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Development and characterization of novel microsatellite markers from the olive ridley sea turtle (Lepidochelys olivacea)

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery‐related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat‐enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross‐species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species.