Microsatellite markers for paternity testing in fork‐marked lemurs (Phaner furcifer)

We report the development of three new microsatellites and four transferred across‐species for fork‐marked lemurs (Phaner furcifer). Two markers were isolated from Cheirogaleus medius and one from Microcebus murinus. The transferred markers also originate from Cheirogaleus medius and Microcebus murinus. The seven markers were tested on 30 individuals of Phaner furcifer and have proven to be useful for inclusion and exclusion of potential parents. The markers presented here are the first published for application on Phaner furcifer.     

Novel microsatellite markers for Dalechampia scandens (Euphorbiaceae) and closely related taxa: application to studying a species complex

We developed novel microsatellite markers for D alechampia scandens L. (Euphorbiaceae). The target plants belong to a distinct, but undescribed, species in the D . scandens species complex, characterized by small resin‐producing glands. In total, 110 alleles over 36 novel markers were identified across 39 individuals from three populations. The number of alleles varied from one to seven, with an average of 3.06 ± 0.26 alleles per locus. The developed markers, along with previously developed ones for a large‐glanded D . scandens species, were tested for amplification in 11 additional species of the genus D alechampia. Four markers did not produce any detectable allele in 37 individuals from two populations of the large‐glanded species. Average expected heterozygosity across all small‐ and large‐glanded specific loci was 0.36 and 0.15, for the small and large glanded populations, respectively. Cross‐species amplification showed that 89% of all markers were successfully amplified in at least one of the 11 other D alechampia species. These microsatellite markers may be useful for detecting undescribed species in the D . scandens species complex, and can be used for comparative analyses of genetic structure, mating system and phylogeography of other D alechampia species.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Characterization of Fusarium poae Microsatellite Markers on Strains from Switzerland and Other Countries

Fusarium poae is a pathogen of increasing importance within the disease complex Fusarium head blight (FHB). Eleven microsatellite markers were developed, and 72 F. poae strains from Switzerland and other countries were used to assess the level of marker polymorphism. The number of alleles for each of the markers ranged from 4 to 15, and the average gene diversity was 0.62, ranging from 0.25 to 0.84. Using these novel markers, 44 genotypes could be differentiated among all F. poae strains. Two genotypes were represented by nine and ten strains, respectively, deriving from distinct geographic areas within Switzerland and indicating a potential selection advantage. Four markers were F. poae‐specific, whereas seven markers also yielded amplification products in one to four strains of five other Fusarium species. Of the latter, five markers revealed F. poae‐specific allele size ranges. Hence, these microsatellite markers could be used both for FHB species differentiation and for intra‐specific distinction of F. poae strains.     

Isolation and characterization of microsatellite loci for the Potentilla core group (Rosaceae) using 454 sequencing

Microsatellites are valuable markers for the analysis of genetic diversity, linkage mapping or genotyping. The limited availability of microsatellites for the genus Potentilla (Rosaceae) stipulated the isolation of markers from a representative (Potentilla pusilla Host) of the Potentilla core group that constitutes the most species‐rich evolutionary lineage within the genus. Thousand four hundred and seventy‐six simple sequence repeat (SSR) containing candidate sequences were isolated from a single‐type line using 454 sequencing. Seventy‐four functional microsatellite markers were developed from 200 sequences selected for suitable priming sites flanking microsatellite repeats referring to a 37% primer‐to‐marker conversion ratio. Seventy‐two markers were polymorphic. These numbers confirm the increased efficiency of pyrosequencing over traditional isolation techniques in the development of microsatellites. Amplification primer sequences and the sequences of corresponding target fragments are provided for all functional markers, and molecular polymorphisms estimated for four accessions of P. pusilla and among seven core group species represented by 14 individuals are reported. Cross‐species transferability ranged between 86.4% and 97.3% among the studied taxa, and 57, 11 and six of the selected primer pairs amplified fragments of expected size and number in seven, six and five of the species, respectively. Reproducibility of the molecular phenotypes was 97.0%, which was inferred using a replicate sample of P. pusilla.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross‐species amplification

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)‐SSR markers from Brassica juncea and their cross‐amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST‐SSRs for genetic analysis of brassicas.     

Isolation of polymorphic tetranucleotide microsatellite markers for the silky short‐tailed bat Carollia brevicauda

We isolated 10 polymorphic microsatellite markers for the silky short‐tailed bat, Carollia brevicauda, from genomic libraries enriched for (AAGG)_n repetitive elements. The number of alleles ranged from six to 25 per locus with the observed heterozygosity ranging from 0.29 to 0.95. These markers will be useful for analysis of questions concerning population genetic structure and models of speciation. Results of cross‐species amplification in Carollia castanea and Carollia perspicillata are also reported.     

Genotype‐by‐sequencing of the plant‐pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology

Genetic and genomics tools to characterize host–pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non‐resource‐prohibitive methods that overcome the limitation of genotyping are now available through genotype‐by‐sequencing (GBS). The use of a two‐enzyme restriction‐associated DNA (RAD)‐GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high‐density genotyping of several fungal species. A total of 5783 and 2373 unique loci, ‘sequence tags’, containing 16 441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating‐type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re‐analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high‐density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies.     

Sequence characterized amplified polymorphic markers for the pitch canker pathogen,Fusarium circinatum

Fusarium circinatum is the causal agent of pitch canker disease of pines. This pathogen is thought to have originated in Central America and currently poses a serious threat to commercial pine plantations in many areas of the world. In this study, polymorphic molecular markers were developed for F. circinatum using the internal short sequence repeats‐polymerase chain reaction (ISSR‐PCR) technique. Nine sequence characterized amplified polymorphic markers were developed. Thirty‐two putative alleles were observed among 103 F. circinatum isolates from different geographical areas using the nine polymorphic markers. These sequence characterized amplified polymorphic markers can be used as genetic tools in populations studies of F. circinatum.     

Polymorphic dinucleotide microsatellites for the Mediterranean angler species (Lophiidae)

A microsatellite‐enriched genomic library was obtained from black‐bellied angler Lophius budegassa and six polymorphic dinucleotide markers were successfully optimized. These markers showed levels of polymorphism ranging from 0.363 to 0.662 and allele numbers ranging from three to seven. These markers were also found to successfully amplify in the closely related Lophius piscatorius (angler) and thus appear to be useful for genetic stock structure analysis in both species.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Identification and characterization of expressed sequence tags‐derived simple sequence repeats markers from robusta coffee variety ‘CxR’ (an interspecific hybrid of Coffea canephora × Coffea congensis)

SSR (simple sequence repeats) markers derived from ESTs (expressed sequence tags), commonly called EST‐SSRs or genic SSRs provide useful genetic markers for crop improvement. These are easy and economical to develop as by‐products of large‐scale EST resources that have become available as part of the functional genomic studies in many plant species. Here, we describe for the first time, nine genic‐SSRs of coffee that are developed from the microsatellite containing ESTs from a cDNA library of moisture‐stressed leaves of coffee variety, ‘CxR’ (a commercial interspecific hybrid between Coffea congensis and Coffea canephora). The markers show considerable allelic diversity with PIC values up to 0.70 and 0.75 for Coffea arabica and Coffea canephora, respectively, and robust cross‐species amplification in 16 other related taxa of coffee. The validation studies thus demonstrate the potential utility of the EST‐SSRs for genetic analysis of coffee germplasm.     

Fifty‐two polymorphic microsatellite loci in the rust fungus,Cronartium quercuum f.sp. fusiforme

We report on 52 microsatellite markers for use in Cronartium quercuum f.sp. fusiforme. The markers were developed from di‐, tri‐, and tetranucleotide repeat‐enriched genomic libraries. In 46 isolates collected from two natural populations in the southeastern USA, the number of alleles per locus ranged from two to 20 (mean 6.94) with gene diversity values ranging from 0.043 to 0.933 (mean 0.537). The markers should prove highly useful for genetic ‘fingerprinting’ of single‐spore isolates commonly used in host–pathogen gene interaction studies, as marker loci for linkage mapping studies, and for examining fine‐scale population genetic structure in natural populations of the fungus.     

Volatile Compounds of Viola odorata Absolutes: Identification of Odorant Active Markers to Distinguish Plants Originating from France and Egypt

Absolutes isolated from Viola odorata leaves, valuable materials for the flavor and fragrance industry, were studied. Violets are mainly cultivated in France and Egypt and extracted locally. The absolutes of the two origins showed different olfactory profiles both in top and heart notes, as evidenced by sensory analysis. The aims of this study were i) to characterize the volatile compounds, ii) to determine the odorant‐active ones, and iii) to identify some markers of the plant origin. Two complementary analytical methods were used for these purposes, i.e., headspace solid‐phase microextraction (HS‐SPME) using different fiber coatings followed by GC/MS analysis and gas chromatography – olfactometry/mass spectrometry (GC‐O/MS) applied to violet leaf extracts. From a total of 70 identified compounds, 61 have never been reported so far for this species, 17 compounds were characterized by both techniques (with seven among them known from the literature), 23 compounds were solely identified by HS‐SPME GC/MS (among them only two being already mentioned as components of violet absolutes in the literature), and, finally, 30 compounds were only identified by GC‐O/MS. According to the HS‐SPME GC/MS analyses, ethyl hexanoate and (2E,6Z)‐nona‐2,6‐dienol were specific volatile compounds of the sample with French origin, while (E,E)‐hepta‐2,4‐dienal, hexanoic acid, limonene, tridecane, and eugenol were specific of the samples with Egyptian origin. Additional compounds that were not detected by HS‐SPME GC/MS analysis were revealed by GC‐O analyses, some of them being markers of origin. Pent‐1‐en‐3‐ol, 3‐methylbut‐2‐enal, 2‐methoxy‐3‐(1‐methylethyl)pyrazine, 4‐ethylbenzaldehyde, β‐phenethyl formate, and 2‐methoxy‐3‐(2‐methylpropyl)pyrazine revealed to be odorant markers of the French sample, whereas cis‐rose oxide, trans‐rose oxide, and 3,5,5‐trimethylcyclohex‐2‐enone were odorant markers of the Egyptian samples.     

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.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.