Development and characterization of microsatellite primers in Geranium carolinianum (Geraniaceae) with 454 sequencing

Abstract

• Premise of the study: Microsatellite primers were developed for Geranium carolinanum, a North American winter annual herb, for use in population genetic analyses.• Methods and Results: Genomic DNA enriched for repeat-containing fragments was sequenced on the Roche 454 Titanium platform, resulting in 470 primer pairs developed from 1115 microsatellite-containing sequences. A subset of 37 primer pairs was screened for polymorphism across three native and three invasive populations. We identified four monomorphic and eight polymorphic loci. Polymorphic loci contained between two and seven alleles per locus, and mean within-population expected heterozygosity ranged from 0.100 to 0.290. Within populations, observed heterozygosity for individual loci ranged from zero to 0.857, and expected heterozygosity ranged from 0.046 to 0.559.• Conclusions: These microsatellite markers will be useful for future studies of genetic diversity, structure, and mating systems across the geographic range of G. carolinianum, and may be transferable to other closely related species.

Genetic Characterization of the Fish Piaractus brachypomus by Microsatellites Derived from Transcriptome Sequencing

“…Microsatellites that showed polymorphism in 6% polyacrylamide gels were analyzed in a 3130xl sequencer (Life Technologies) to get better accuracy of allele determination. The sequencing strategy adopted in this study was according to protocols described by Schuelke ( 2000 ), using the CAGtag primer (5′-CAGTCGGGCGTCATCA-3′; Shirk et al, 2013 ) labeled with the fluorochromes HEX or FAM. The genotyping PCR was performed with the following reagents: 100 μM of each dNTP, 1.5 mM MgCl 2 , 1X Taq DNA buffer, 0.1 μM of each primer (F and R), 0.01 μM of the CAGtag primer, 0.5 units of Taq Polymerase (Invitrogen), and 10–50 ng of genomic DNA.…”

Patterns of genetic diversity reveal multiple introductions and recurrent founder effects during range expansion in invasive populations of Geranium carolinianum (Geraniaceae)

“…A total of 30 populations were sampled for genotyping at 6 microsatellite loci (Shirk et al, 2013) for comparison with the allozyme data set. We selected 15 invasive populations that spanned the sampled range in China (that is, only one population per city) and 15 native populations that included the 5 populations with leaf tissue collections that we were unable to use for allozyme genotyping (NC-2, TN-1, TX-1, VA-1 and VA-2) and 10 others that spanned the sampled range in the United States (Supplementary Table S1).…”

“…Total genomic DNA was extracted from 24 individuals per population using either field-collected leaf tissue or seedling tissue germinated from field-collected seeds with a modified CTAB (cetyltrimethylammonium bromide) protocol (Doyle and Doyle, 1990) and amplified using a 3-primer touchdown PCR for G. carolinianum primers GC10, GC38, GC29, GC31, GC39 and GC47. For PCR reaction conditions, see Shirk et al (2013). PCR products were analyzed on a 3730xl capillary sequencer (Applied Biosystems, Carlsbad, CA, USA) using a ROX-labeled internal size standard (GGF500R, Georgia Genomics Facility, Athens, GA, USA).…”

RBFOX1 and RBFOX2 are dispensable in iPSCs and iPSC-derived neurons and do not contribute to neural-specific paternal UBE3A silencing

“…2A ). As a control for lentiviral transduction and CRISPR/Cas9 integration and expression, we designed a scrambled sgRNA that has no match in the human genome 20 . For RBFOX2 knockouts (KOs), we amplified and sequenced the genomic DNA near the predicted CRISPR/Cas9 cut site to identify small indels that shifted the reading frame, leading to a premature stop codon.…”