Isolation and Characterization of Microsatellite Markers for Cotinus coggygria Scop. (Anacardiaceae) by 454 Pyrosequencing

Abstract

Abstract:Cotinus coggygria Scop. (Anacardiaceae) is a deciduous shrub or small tree that is native to a large area covering from southern Europe, east across central Asia, and the Himalayas in northern China. Shotgun 454 pyrosequencing was used to develop microsatellite markers from the genome of C. coggygria. In this study, 349 microsatellite loci were identified from 40,074 individual sequence reads produced by one-sixteenth run, and primer pairs were designed for these loci. To test the primer amplification efficiency, 50 microsatellite primer pairs were tested across 12 individuals from two C. coggygria populations (Wuzhi Mountain: 36°30'N, 113°39'E; Tianlong Mountain: 37°42'N, 112°26'E). Among the 50 tested primer pairs, eight were found to be polymorphic. The average allele number of the microsatellites was 3.5 per locus, with a range from two to five. The inbreeding coefficient ranged from −0.478 to 0.222. The observed and expected heterozygosities varied from 0.167 to 0.750 and from 0.163 to 0.743, respectively. This set of markers is potentially useful for assessing the genetic diversity, as well as for understanding the population structure and phylogeographical and landscape genetic patterns, of C. coggygria.

Adaptive genetic variation in the smoke tree (Cotinus coggygria Scop.) is driven by precipitation

“…Eight microsatellite primer pairs were selected for polymerase chain reaction (PCR) as suggested by Wang et al (2014a). Primers Cc 004F, Cc 012F, Cc 024F, and Cc 032F were 5 0 fluorescent labeled by FAM (Applied Biosystems, Foster City, CA, USA).…”

“…Twenty nanograms of template DNA was used in each 20 mL of PCR containing 0.2 mmol/L dNTP, 0.3 mmol/L primer, 2 mL of 10 Â polymerization buffer, and 0.6 unit of Taq polymerase (Takara, Dalian, Liaoning, China). PCRs were carried out in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) under the following conditions: initial denaturation for 5 min at 95 C, followed by 35 cycles of denaturation for 40 s at 94 C, primer annealing for 45 s at a locus-specific annealing temperature (Wang et al, 2014a), and extension for 50 s at 72 C with a subsequent extension step of 8 min at 72 C, and terminated by a final hold at 4 C. Fluorescently labeled PCR products were separated using the ABI 3730 DNA Sequence Analyzer at the Beijing Genomics Institute (Beijing, China). The size of the labeled PCR products was estimated using ABI proprietary software Genescan™ with ROX 500 (Applied Biosystems, Foster City, CA, USA).…”

“…This tree is native to a large area from eastern Asia across central Asia and the Himalayas to northern China. It is widely distributed in China's warm-temperate zone, which has elevations ranging from 330 to 2400 m above sea level (Wang et al, 2014a). The numerous small flowers of C. coggygria blossom from May to June.…”

Isolation and characterization of microsatellite markers for Axonopus compressus (Sw.) Beauv. (Poaceae) using 454 sequencing technology

“…Fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) has been proven to be the most effective magnetic bead enrichment method for isolating SSR markers (Sun et al, 2008;Yang et al, 2009;Hou et al, 2011;Wang et al, 2014a). However, this method is laborious and difficult to use, and cannot obtain a large number of SSRs in one experiment (Wang et al, 2014b). Next-generation sequencing (NGS) has become a more efficient approach to generate superior resources for the development of SSR markers (Kumar et al, 2014;Huang et al, 2014).…”

Isolation and characterization of novel microsatellites for Abies koreana and A. nephrolepis (Pinaceae)

“…To generate microsatellite primers for A. koreana, we used next-generation sequencing (NGS) with genomic DNA of A. koreana and then used a bioinformatics approach to obtain fragments encompassing repeat motifs from low-depth genomic sequences (Yu et al, 2011;Wang et al, 2014). To use the microsatellite markers for further comparison between A. koreana and A. nephrolepis, amplification of sequences in both the species and the evaluation of polymorphisms were performed.…”