New microsatellite markers for Ellipse, Venustaconcha ellipsiformis (Bivalvia: Unionidae), with notes on optimal sample size and cross-species amplification.

Venustaconcha ellipsiformis (Unionidae) is a freshwater mussel species inhabiting small to medium streams of the Midwestern United States; however, its occurrence is rather sporadic and populations are often isolated. Due to anthropogenic habitat degradation and water pollution, this species is designated as some sort of conservation status in many states. To prioritize conservation strategies, highly variable genetic markers are necessary to assess population genetic structure and potential genetic erosion of V. ellipsiformis. Using whole genome sequence data, we developed and characterized microsatellite markers for V. ellipsiformis. Among 23 tetranucleotide loci tested, 14 loci were consistently amplified and showed polymorphism. Analyses performed on three populations in the upper Mississippi River basin showed that the number of alleles per locus ranged from 2 to 11 and the observed heterozygosity varied from 0.15 to 0.75. Based on genotypic and allelic rarefaction curves, these loci had adequate statistical power to genetically discriminate between individuals and the sample size was large enough to capture most alleles available in the populations at most loci. Finally, cross-species screening of the loci successfully amplified and showed polymorphism in six species in the tribe Lampsilini. The microsatellite loci developed in this study provide a valuable addition to extant genetic markers for freshwater mussels and can be useful to provide high-level resolution of population genetic parameters for V. ellipsiformis. Such information will be of great value for resource managers developing and prioritizing conservation strategies for imperiled mussel species.

Isolation and characterization of 14 tetranucleotide microsatellite loci for the cannonball jellyfish (Stomolophus sp.) by next generation sequencing.

The Cannonball jellyfish (Stomolophus sp.) is a species of jellyfish with high relevance in artisanal fishing. Studies of their populations do not extend beyond the morphological descriptions knowing that presents a great morphological variability. However, there are no genetic studies to determine the number of independent populations, so microsatellite markers become a suitable option. Since there are no species-specific microsatellite loci, in this paper, 14 new microsatellite loci are characterized. Microsatellite loci were isolated de novo through next generation sequencing, by two runs on Illumina MiSeq. A total of 506,771,269 base pair were obtained, from which 142,616 were microsatellite loci, and 1546 of them could design primers. We tested 14 primer pairs on 32 individuals from Bahía de La Paz, Gulf of California. We observed low genetic variation among loci (mean number of alleles per locus = 4.33, mean observed heterozygosity 0.381, mean expected heterozygosity 0.501). These loci are the first ones described for the species and will be helpful to carry out genetic diversity and population genetics studies.

Heterologous microsatellite primers are informative for paca (Cuniculus paca), a large rodent with economic and ecological importance.

OBJECTIVE: This study was designed to facilitate genetic studies that would allow information on population structure and genetic diversity of natural or captive stocks of paca (Cuniculus paca), a species of ecological and socioeconomic importance, by testing cross-amplification of 20 heterologous microsatellite primer pairs developed for guinea pigs (Cavia porcellus) and capybara (Hydrochoerus hydrochaeris). RESULTS: Those primers that showed the best amplification profile in blood samples were subsequently applied to scats and saliva samples, to evaluate their efficiency. Of the 13 microsatellite pairs that amplified in blood, one-third (32%) were successfully amplified in saliva and scat samples. This initial work demonstrates successful cross-amplification in paca providing a solid and promising foundation for future genetic studies with this species. The ability to quantify genetic diversity using noninvasive samples from free-ranging paca is essential to developing applied management strategies for this large neotropical rodent that is not only a prey favored by wide-ranging carnivores [e.g., jaguar (Panthera onca), puma (Puma concolor)], but is also a species targeted by illegal hunting and wildlife trade.

Development of polymorphic simple sequence repeat markers in Huperzia serrata (Lycopodiaceae).

PREMISE: The natural population size of Huperzia serrata (Lycopodiaceae) has dramatically decreased and the species has become endangered due to overexploitation. Here, we developed simple sequence repeat (SSR) markers for H. serrata to survey both its genetic diversity and population structure. METHODS AND RESULTS: Based on 177 individuals, 120 SSR primer pairs were developed and optimized from five regions of the H. serrata transcriptomic data. Of these primer pairs, 20 were successfully amplified and 10 showed obvious polymorphism. These polymorphic loci were investigated to study the genetic diversity of H. serrata. Two to 11 alleles per locus were identified, the level of observed heterozygosity ranged from 0.00 to 1.00, and the level of expected heterozygosity ranged from 0.19 to 0.79. All loci were successfully amplified in H. crispata, H. sutchueniana, and H. selago. CONCLUSIONS: The 10 polymorphic primer pairs developed here will be valuable for studies of the endangered H. serrata and other related species.

Isolation, characterization, and cross-amplification of 20 microsatellite markers for Conospermum undulatum (Proteaceae).

PREMISE: Recent habitat fragmentation is posing a risk to the wavy-leaved smokebush, Conospermum undulatum (Proteaceae), a rare plant species endemic to southwestern Western Australia. Microsatellite markers are required to characterize the genetic diversity and structure of the species for conservation purposes and to facilitate ecological studies. METHODS AND RESULTS: Illumina MiSeq high-throughput sequencing was used to develop 20 novel microsatellite markers for C. undulatum. Polymorphism at each locus was assessed using 72 individuals from three natural populations. Nineteen markers were polymorphic, with the number of alleles per locus ranging from two to 21, and observed and expected heterozygosity ranging from 0.000 to 1.000 and 0.117 to 0.919, respectively. All markers successfully amplified in three congeneric species (C. stoechadis, C. canaliculatum and C. triplinervium). CONCLUSIONS: The microsatellite markers will be useful for revealing patterns of genetic diversity, dispersal dynamics, and hybridization events for C. undulatum to inform future conservation efforts.

Characterization and transferability of microsatellites for Gentiana lawrencei var. farreri (Gentianaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for a medicinal herb, Gentiana lawrencei var. farreri (Gentianaceae), for the future assessment of population genetic structure and potential hybridization events with related taxa. METHODS AND RESULTS: Using the 454 FLX+ sequencing platform, we obtained 81,717 clean reads with an average length of 291 bp. A total of 3031 primer pairs were designed, and 96 were selected for validation. A set of 20 fluorescently labeled primer pairs was further selected and screened for polymorphisms in three G. lawrencei var. farreri populations and one G. veitchiorum population. Among the four populations, the average number of alleles per locus was 15.2. Finally, a set of 17 unlinked loci were determined to be in Hardy-Weinberg equilibrium after two linked loci were removed. CONCLUSIONS: The identified simple sequence repeat markers will be useful for genetic diversity and evolution studies in G. lawrencei var. farreri and related taxa.

Characterization and transferability of microsatellites for the Kangaroo Paw, Anigozanthos manglesii (Haemodoraceae).

PREMISE OF THE STUDY: Microsatellites were developed for the future assessment of population genetic structure, mating system, and dispersal of the perennial kangaroo paw, Anigozanthos manglesii (Haemodoraceae), and related species. METHODS AND RESULTS: Using a Personal Genome Machine (PGM) semiconductor sequencer, ca. 4.03 million sequence reads were generated. QDD pipeline software was used to identify 190,000 microsatellite-containing regions and priming sites. From these, 90 were chosen and screened using PCR, and 15 polymorphic markers identified. These sites amplified di-, tri-, and pentanucleotide repeats with one to 20 alleles per locus. Primers were also amplified across congeners A. bicolor, A. flavidus, A. gabrielae, A. humilis, A. preissii, A. pulcherrimus, A. rufus, and A. viridis to assess cross-species transferability. CONCLUSIONS: These markers provide a resource for population genetic studies in A. manglesii and other species within the genus.

Isolation and characterization of microsatellite primers for the critically endangered shrub Styphelia longissima (Ericaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for population genetic analysis in the rare shrub Styphelia longissima (Ericaceae). METHODS AND RESULTS: We generated ca. 2.5 million sequence reads using a Personal Genome Machine semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80-480 bp. From these, 30 primer pairs were selected and screened using PCR; of these, 16 loci were found to be polymorphic, four loci were monomorphic, and 10 loci did not amplify reliably for S. longissima. For a sample of 57 plants from the only known population, the number of alleles observed for these 16 loci ranged from two to 21 and expected heterozygosity ranged from 0.49 to 0.91. These markers were also amplified in Astroloma xerophyllum, a closely related species. CONCLUSIONS: These markers will be used to characterize population genetic variation, spatial genetic structure, mating system parameters, and dispersal to aid in the management and conservation of the rare shrub S. longissima.

Characterization of microsatellite primers in the endangered orchid Phaius australis and cross-amplification to P. bernaysii (Orchidaceae).

PREMISE OF THE STUDY: The swamp orchid, Phaius australis (Orchidaceae), is nationally endangered due to illegal collection and habitat loss and fragmentation, resulting in a disjunct distribution in spring and coastal wetland ecotones along Australia's east coast. Polymorphic microsatellite markers were developed to study genetic diversity and population structure for conservation and restoration purposes. METHODS AND RESULTS: Illumina HiSeq high-throughput sequencing was used to develop 15 nuclear microsatellite markers, including 10 polymorphic markers for P. australis. Polymorphism at each marker was evaluated using 90 individuals from four natural populations. The number of alleles per locus ranged from one to three, and the observed and expected heterozygosity varied from 0.036 to 0.944 and from 0.035 to 0.611, respectively. These markers transferred successfully to congener P. bernaysii. CONCLUSIONS: The microsatellite markers will be useful for revealing levels of genetic diversity and gene flow for P. australis and may inform future conservation efforts.

Isolation and characterization of microsatellite loci for Smilax sieboldii (Smilacaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed for Smilax sieboldii (Smilacaceae), a member of the S. hispida group with a biogeographic disjunction between eastern Asia and North America, to study the phylogeography and incipient speciation of this species and its close relatives. METHODS AND RESULTS: Transcriptome sequencing produced 47,628 unigenes. Seventeen loci were developed from 122 randomly selected primer pairs. Polymorphism and genetic variation were evaluated for 68 accessions representing five populations of S. sieboldii. The number of alleles per locus ranged from four to 18; the expected heterozygosity ranged from 0.59 to 0.92. Twelve loci were successfully amplified in five related species: S. scobinicaulis, S. californica, S. hispida, S. moranensis, and S. jalapensis. CONCLUSIONS: These novel expressed sequence tag-derived microsatellite markers will facilitate further population genetic research of S. sieboldii and its close allies of the S. hispida group.

Isolation and characterization of 13 microsatellites for the rare endemic shrub Tetratheca erubescens (Elaeocarpaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the rare Tetratheca erubescens (Elaeocarpaceae) to assess genetic diversity and spatial structuring. METHODS AND RESULTS: We generated ca. 2.7 million sequence reads using a Personal Genome Machine (PGM) semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80-480 bp. From these, 30 primer pairs were selected and screened using PCR, from which 11 loci were found to be polymorphic and amplified reliably. For a sample of 95 plants from three populations, the number of alleles observed for these 11 loci ranged from two to seven and expected heterozygosity ranged from 0.06 to 0.72. No consistent evidence for null alleles or departure from Hardy-Weinberg equilibrium was found for any of the 11 loci. CONCLUSIONS: These markers will enable the quantification of genetic impact of proposed mining activities on the narrow endemic T. erubescens.

Development and characterization of 15 microsatellite markers for Cephalotaxus fortunei (Cephalotaxaceae).

PREMISE OF THE STUDY: To survey population variation and the adaptive evolution of Cephalotaxus fortunei (Cephalotaxaceae), an endemic and endangered conifer in China, microsatellite markers were developed and characterized for this species. METHODS AND RESULTS: Based on the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 15 microsatellite markers were developed for C. fortunei, 13 of which were polymorphic within a sample of 75 individuals representing five natural populations. The number of alleles per locus ranged from one to seven. The expected and observed heterozygosities were 0.108-0.738 and 0.000-1.000, respectively. Ten polymorphic loci were also successfully amplified in C. oliveri. CONCLUSIONS: These polymorphic loci provide a valuable tool for population genetic analysis of C. fortunei, which will contribute to its management and conservation.

Microsatellite primers for the rare sedge Lepidosperma bungalbin (Cyperaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the rare sedge Lepidosperma bungalbin (Cyperaceae) to assess genetic variation and its spatial structuring. METHODS AND RESULTS: We conducted shotgun sequencing on an Illumina MiSeq and produced 6,215,872 sequence reads. The QDD pipeline was used to design 60 primer pairs that were screened using PCR. We developed 17 loci, of which 12 loci were identified that were polymorphic, amplified reliably, and could be consistently scored. We then screened these loci for variation in individuals from three populations. The number of alleles observed for these 12 loci across the three populations ranged from nine to 19 and expected heterozygosity ranged from 0.41 to 0.89. CONCLUSIONS: These markers will enable the quantification of the potential impact of mining on genetic variation within L. bungalbin and establish a baseline for future management of genetic variation of the rare sedge.

Microsatellite primers for the rare shrub Acacia adinophylla (Fabaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the rare shrub Acacia adinophylla (Fabaceae) to assess genetic diversity and its spatial structuring. METHODS AND RESULTS: Shotgun sequencing on an Illumina MiSeq produced 6,372,575 reads. Using the QDD pipeline, we designed 60 primer pairs, which were screened using PCR. Seventeen loci were developed, of which 12 loci were identified that were polymorphic, amplified reliably, and could be consistently scored. These loci were then screened for variation in individuals from three populations. The number of alleles observed for these 12 loci ranged from three to 18 and expected heterozygosity ranged from 0.13 to 0.85. CONCLUSIONS: These markers will enable the quantification of genetic impact of proposed mining activities on the short-range endemic Acacia adinophylla.

Genetic diversity of Poa pratensis L. depending on geographical origin and compared with genetic markers.

BACKGROUND: Poa pratensis is one of the most common species of meadow grass in Europe. Most cultivars of the species found in Poland were originally derived from its ecotypes. We compared the effectiveness of the RAPD and ISSR methods in assessing the genetic diversity of the selected populations of P. pratensis. We examined whether these methods could be useful for detecting a possible link between the geographical origin of a given population and its assessed genetic variation. METHODS: The molecular markers RAPD and ISSR were used and their efficiency compared using, inter alia, statistical multivariate methods (UPGMA and PCA). RESULTS: The low value of Dice's coefficient (0.369) along with the significantly high percentage of polymorphic products indicates a substantial degree of genetic diversity among the studied populations. Our results found a correlation between the geographical origin of the studied populations and their genetic variations. For ISSR, which proved to be the more effective method in that respect, we selected primers with the greatest differentiating powers correlating to geographical origin. DISCUSSION: The populations evaluated in this study were characterized by a high genetic diversity. This seems to confirm the hypothesis that ecotypes of P. pratensis originating from different regions of Central Europe with different terrain structures and habitat conditions can be a source of great genetic variability.

Development and characterization of microsatellite markers for Central American Begonia sect. Gireoudia (Begoniaceae).

PREMISE OF THE STUDY: Transcriptome sequence data were used to design microsatellite primers for two widespread Central American Begonia species, B. heracleifolia and B. nelumbiifolia, to investigate population structure and hybridization. • METHODS AND RESULTS: The transcriptome from vegetative meristem tissue from the related B. plebeja was mined for microsatellite loci, and 31 primer pairs amplified in the target species. Fifteen primer pairs were combined in two multiplex PCR reactions, which amplified an average of four alleles per locus. • CONCLUSIONS: The markers developed will be a valuable genetic resource for medium-throughput genotyping of Central American species of Begonia sect. Gireoudia. A subset of these markers have perfect sequence matches to Asian B. venusta, and are promising for studies in other Begonia sections.

Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for Eucalyptus victrix (Myrtaceae) to evaluate the population and spatial genetic structure of this widespread northwestern Australian riparian tree species, which may be impacted by hydrological changes associated with mining activity. • METHODS AND RESULTS: 454 GS-FLX shotgun sequencing was used to obtain 1895 sequences containing putative microsatellite motifs. Ten polymorphic microsatellite loci were identified and screened for variation in individuals from two populations in the Pilbara region. Observed heterozygosities ranged from 0.44 to 0.91 (mean: 0.66) and the number of alleles per locus ranged from five to 25 (average: 11). • CONCLUSIONS: These microsatellite loci will be useful in future studies of population and spatial genetic structure in E. victrix, and inform the development of seed sourcing strategies for the species.