Cross-species testing of nuclear markers in Pelophylax water frogs in Greece and examination of their power to detect genetic admixture

The genus Pelophylax has been considered a model organism for understanding hybridization. Despite being the most diverse within the Western Palearctic group of Pelophylax, the ridibundus/bedriagae lineage that includes six species, remains largely understudied, revealing many knowledge gaps in regards to their evolution and conservation. Using genetic data from populations among species inhabiting contact zones could prove vital in filling these gaps. We tested 17 microsatellite markers for cross-species amplification in mainland Pelophylax species distributed in southern Balkans and evaluated their power to successfully detect population/species structure. Importantly, we examined their potential for identifying hybrids and backcrosses between known hybridized species. We detected 12 highly polymorphic loci that cross-amplified all species that showed no significant Linkage Disequilibrium and were able to discriminate among species and between parental and hybrids. We suggest their future use in genetic studies for the genus Pelophylax in Greece, including the identification of contact zones.

A multilocus sequence typing scheme of Pseudomonas putida for clinical and environmental isolates

Pseudomonas putida is a bacterium commonly found in soils, water and plants. Although P. putida group strains are considered to have low virulence, several nosocomial isolates with carbapenem- or multidrug-resistance have recently been reported. In the present study, we developed a multilocus sequence typing (MLST) scheme for P. putida. MLST loci and primers were selected and designed using the genomic information of 86 clinical isolates sequenced in this study as well as the sequences of 20 isolates previously reported. The genomes were categorised into 68 sequence types (STs). Significant linkage disequilibrium was detected for the 68 STs, indicating that the P. putida isolates are clonal. The MLST tree was similar to the haplotype network tree based on single nucleotide morphisms, demonstrating that our MLST scheme reflects the genetic diversity of P. putida group isolated from both clinical and environmental sites.

Development and characterisation of 16 novel microsatellite markers for the brown alga Sargassum fusiforme (Harvey) Setchell

<p><em>Sargassum fusiforme</em> (Harvey) Setchell (Sargassaceae, Phaeophyta) is an ecologically and economically important kelp species in East Asia. Restoration and responsible utilisation of <em>S. fusiforme</em> would be facilitated by the availability of an appropriate set of molecular markers. In the present study, we developed 16 microsatellite markers for <em>S. fusiforme</em>. A total of 99 different alleles were observed at the 16 microsatellite loci across 50 individual samples. The number of alleles per locus ranged from 4 to 10, with an average of 6.2 per locus. The observed and xpected heterozygosities ranged from 0.483 to 0.833 and from 0.513 to 0.840, respectively. Only three of the loci deviated significantly from Hardy-Weinberg equilibrium after Bonferroni correction. No significant linkage disequilibrium was detected among the microsatellite loci. The obtained microsatellite markers will facilitate related research on <em>S. fusiforme</em>, such as ecological studies and genetic diversity assessments.</p>

Characterization of 17 novel microsatellite loci for the brown spiny lobster Panulirus echinatus Smith, 1869 using MiSeq

Using high-throughput sequencing technology 17 polymorphic microsatellites with perfect tetra-nucleotide repeats were identified for the brown spiny lobsterPanulirus echinatusSmith, 1869. Two to ten alleles were detected per locus across 30 samples analysed from the Canary Archipelago. Observed and expected heterozygosities per locus ranged between 0.100 and 0.867 and 0.095 and 0.799, respectively. No significant linkage disequilibrium was found between pairs of loci, and only one locus (Pe-L43) deviated from Hardy-Weinberg equilibrium, likely due to the presence of null alleles. Despite its economic importance and the threat of overfishing, population genetics studies of this species are lacking. Therefore, these 17 novel microsatellites markers will be a useful genetic resource for future conservation studies ofP. echinatus.

A new set of microsatellite loci for Cattleya walkeriana Gardner, an endangered tropical orchid species and its transferability to Cattleya loddigesii Lindl. and Cattleya nobilior Reichenbach

In this study, we isolate and analyse a new set of microsatellite loci for Cattleya walkeriana. Twenty-two primer pairs were screened for C. walkeriana (n = 32) and assessed for their transferability to Cattleya loddigesii (n = 12) and Cattleya nobilior (n = 06). All loci amplified for C. walkeriana; however, for C. loddigesii and C. nobilior, four and five primers, respectively, did not present amplification. The polymorphic loci presented between 2 and 13 alleles per locus for both C. walkeriana and C. loddigesii, with respective averages of 5.1 and 4.2. For C. nobilior, we found between two and five alleles per locus, with an average of 2.6. For C. walkeriana, observed heterozygosity varied from 0.100 to 0.966, whereas expected heterozygosity ranged from 0.097 to 0.900. The observed and expected heterozygosity for C. loddigesii and C. nobilior were also estimated. We found no significant linkage disequilibrium between any pair of loci, and evidence of null alleles at four loci (Cw16, Cw24, Cw30 and Cw31) for C. walkeriana. The combined power to exclude the first parent and combined non-exclusion probability of identity were 0.999 and 2.3 × 10−20, respectively. These new loci can be used in studies of germplasm resources, and assessments of genotypic and genetic diversity and population structure, thus improving the accuracy of such analyses and their applicability in the conservation and protection of these endangered species.

Development and characterisation of polymorphic microsatellite markers in the pearl oyster Pteria penguin (Roding, 1798)

We developed and characterised 12 polymorphic microsatellite loci for Pteria penguin (Roding, 1798), an important pearl oyster species for pearl production in China. The number of observed alleles per locus ranged from 5 to 26 within 46 individuals. The observed and expected heterozygosities per locus ranged from 0.2 to 0.925 and 0.707 to 0.945, respectively. The polymorphism information content values per locus ranged from 0.656 to 0.929. Ten loci were in accordance with Hardy-Weinberg equilibrium and no significant linkage disequilibrium was detected between loci. These new microsatellite markers will be useful for population and conservation genetic studies of P. penguin.

Microsatellite markers for Cattleya walkeriana Gardner, an endangered tropical orchid species

Cattleya walkeriana, one of the most improved Brazilian Cattleyas, is a popular tropical orchid endemic from Brazil and currently endangered. In the present study, for the first time microsatellite markers were developed for C. walkeriana and their transferability was tested for the species C. loddigesii and C. nobilior. The markers were used for genotyping 26 C. walkeriana specimens from different growers and from different levels of improvement. The transferability was successful, with five polymorphic loci transferred to C. loddigesii and six polymorphic loci to C. nobilior. Eight loci were polymorphic, revealing a maximum of two to ten alleles per locus in C. walkeriana and two to four and two to five in C. loddigesii and C. nobilior, respectively. There was no significant linkage disequilibrium in the studied loci. For C. walkeriana, the observed and expected heterozygosities ranged from 0 to 0.963 and from 0.138 to 0.841, respectively. These markers identified polymorphisms and may be used to study the genetic diversity, gene flow or hybridization of these species.

Rapid Changes in the Genetic Composition of Stagonosporopsis tanaceti Population in Australian Pyrethrum Fields

A novel set of microsatellite markers were developed and employed for geographical and temporal population analyses of Stagonosporopsis tanaceti, the cause of ray blight of pyrethrum in Australia. Genotyping of 407 isolates, using 13 markers, suggested an asexual mode of reproduction with significant linkage disequilibrium and high levels of clonality. Low geographical differentiation and widespread distribution of a few multilocus genotypes (MLGs), in the absence of airborne ascospores, suggested the role of human-mediated movement of seed as a major means of long-distance pathogen dispersal. The genetic composition of S. tanaceti was stable for a decade then changed rapidly in only 2 years. Bayesian clustering analyses and minimum spanning networks determined only two major clonal lineages in and prior to 2010. However, in 2012, a previously unobserved cluster of MLGs was detected, which significantly increased in frequency and displaced the historically dominant MLGs by 2013. This rapid change in the genetic composition of S. tanaceti could indicate a second introduction then a selective sweep, or strong selection pressures from recently introduced fungicides or pyrethrum varieties. These results may have serious implications for durability of management strategies for this disease.