Development of microsatellite markers in Robinsonia (Asteraceae) an endemic genus of the Juan Fernández Archipelago, Chile

Ten microsatellite markers were developed for Robinsonia (Asteraceae), a genus endemic to the Juan Fernández Archipelago, Chile. Polymorphisms of these markers were tested using one population each of R. evenia, R. gayana, and R. gracilis. The number of alleles for these markers ranged from 2 to 17 per locus, and expected heterozygosity ranged from 0 to 0.847 by population. A significant deviation from Hardy–Weinberg equilibrium was observed in zero to two markers in each population, and no significant linkage disequilibrium between markers was detected. The markers reported here would be useful for evolutionary studies and conservation strategies in Robinsonia.

Oceanic islands are significant ecosystems for the conservation of global plant diversity due to their small areas and high levels of endemism in comparison with continental regions (Kier et al. 2009). Tiny population sizes and unique characteristics of insular endemic species make them particularly sensitive to anthropogenic disturbances (Frankham 1997).
The Juan Fernández Archipelago is located in the Pacific Ocean 667 km west of continental Chile, and it consists of two major islands of different geological ages, Robinson Crusoe Island (4 million years old) and Alejandro Selkirk Island (1-2 million years old) (Stuessy et al. 1984). The flora of the Archipelago contains 132 endemic vascular plants (Marticorena et al. 1998), 74 % of which is regarded as ''threatened'' based on IUCN criteria (Ricci 2006). Biodiversity assessement in this archipelago, including population genetic study, is a pressing need as is the case with other oceanic islands (Caujape-Castells et al. 2010).
The genus Robinsonia DC. (Asteraceae) is endemic to the archipelago, and consists of eight species (Sanders et al. 1987;Danton and Perrier 2006). It should be pointed out that Pelser et al. (2007Pelser et al. ( , 2010 suggested submergence of all species of Robinsonia into the large genus Senecio in order to maintain strict holophyly of the latter, which would obviate endemic generic status for Robinsonia. We do not follow this suggestion, however, as we prefer to recognize Robinsonia as generically distinct based on its striking divergence in morphological features, such as a dioecious breeding system and a rosette tree habit. Morphological and ecological divergences occur among Robinsonia species, and genetic divergence among them has also been demonstrated with isozyme and ITS markers (Crawford et al. 1992;Sang et al. 1995). In view of the estimated ages of the islands, species of Robinsonia have diversified cladogenetically within the past 4 million years (Stuessy et al. 1990). Most of the species in Robinsonia are considered to be highly threatened (Ricci 2006). In this present study, we develop microsatellite markers for investigating evolutionary processes within the genus.
We used one individual of R. masafuerae Skottsb. for isolation of microsatellites, plus one individual each of R. evenia Phil., R. gayana Decne., R. gracilis Decne., Table 1 Characteristics of 24 microsatellite markers developed in Robinsonia masafuerae Length of fragments is shown by a single number (homozygote) or by two numbers separated by a slash (heterozygote) R. saxatilis Danton, and R. thurifera Decne. for crossspecies amplification tests. Polymorphism of microsatellite markers was evaluated in one population each of R. evenia, R. gayana, and R. gracilis, the most common species on Robinson Crusoe Island.
Total genomic DNA was extracted from leaf tissue by the cetyltrimethylammonium bromide method (Doyle and Doyle 1987) or Qiagen DNeasy 96 Plant Kit (Qiagen, Hilden, Germany). The extracted DNA of R. masafuerae was sequenced with one-fourth of the run on 1/8 of a 70 9 75 PicoTiterPlate using a multiplex identifier in 454 Genome Sequencer FLX System (Roche Applied Science, Penzberg, Germany) of LGC Genomics (Berlin, Germany). A total of 32,468 reads with an average length of 425 bp was generated.
The design for primer pairs was conducted with QDD 2.1 (Meglécz et al. 2010) using default settings. A total of 201 unique microsatellite regions contained pure/compound dinucleotide (140 regions) and trinucleotide microsatellite sequences (61 regions) with more than five repetitions, and primer designable flanking regions were found. First, a cross-species amplification test was performed in 24 selected primer pairs (Table 1) using the Qiagen Multiplex PCR plus Kit (Qiagen, Hilden, Germany) with the 5 0 -tailed primer method (Boutin-Ganache et al. 2001) with CAG-tailing (5 0 -CAGTCGGGCGTCATCA-3 0 ) and the PIG tailing (5 0 -GTTT-3 0 ) method (Brownstein et al. 1996) following Takayama et al. (2011). We applied single-plex PCR in touchdown thermal cycling programs as follows: initial denaturation at 95°C for 5 min, followed by first 15 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 90 s (decreased 0.5°C per cycle), and extension at 72°C for 60 s; and by second 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 90 s, and extension at 72°C for 60 s; a final extension step was performed at 60°C for 30 min. An automated sequencer (ABI 3130xl, Applied Biosystems, CA, USA) and Gene-Marker (SoftGenetics LLC, PA, USA) were used for scoring of the PCR products.
Next, we selected the ten best of the 24 markers, and confirmed the polymorphism using multiple individuals of three populations, one from each of the three species (Table 2). We applied multi-plex PCR as follows: RM-HD5GH, RM-G3THW, RM-HCT78 with 6-FAM, RM-HIFPY, RM-HBJ9H, RM-HJIWZ with VIC, RM-HMDCO, RM-HBZZR with NED, RM-HGNFW, RM-HGC8M with PET. Two to 17 alleles per locus were detected in the three populations, and expected heterozygosity ranged from 0.000 to 0.847 (Table 2). A significant heterozygote deficiency was detected for zero to two markers in each population, and no significant linkage disequilibrium between markers was observed in both populations (P \ 0.05, after Bonferroni correction) using GENEPOP 4.0 (Raymond and Rousset 1995). Ten microsatellite markers present easily scorable polymorphic peaks in the six species of Robinsonia, rendering these markers useful for populational genetic studies.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Table 2 Results of ten microsatellite markers in R. evenia, R. gayana, and R. gracilis