Twelve nuclear and 14 chloroplast microsatellites for Araucaria rulei, an endangered species
endemic to New Caledonia
MARKUS RUHSAM, ADRIEN S. WULFF, BRUNO FOGLIANI, PETER M. HOLLINGSWORTH
Microsatellite sequences were developed by Ecogenics GmbH (Switzerland) using an enrichment
protocol. Briefly, size selected fragments from genomic DNA were enriched for SSR content by using
magnetic streptavidin beads and biotin-labelled CT and GT repeat oligonucleotides. The SSR enriched
library was analyzed on a Roche 454 platform using the GS FLX titanium reagents. The total 42,914
reads had an average length of 144 base pairs. Of these, 742 contained a microsatellite insert with a
tetra- or a trinucleotide of at least 6 repeat units or a dinucleotide of at least 10 repeat units.
Suitable primer design was possible in 125 reads, of which 27 were tested for polymorphism. 12 loci
which were polymorphic showed clear amplification profiles and reliable amplification.
PCR amplifications were performed in volumes of 10 μL in two multiplex reactions with five (MP1,
Table 1) and seven microsatellites (MP2, Table 1) using the following protocol: 1x HotStar Taq Master
Mix (QIAGEN), 0.2 µM of each labelled forward primer (6-FAM, PET, VIC or NED), 0.2 µM of each
reverse primer and 1 µL of unquantified DNA. The mixture was then cycled through the profile: 15
min at 95°C, 35 cycles of 30 sec at 94°C, 90 sec at 56°C and 1 min at 72°C, ending with 30 min at 72°C
to complete extension and subsequent storage at 4°C. LIZ-500 labelled internal size standard (PE
Applied Biosystems) was added to each sample to size microsatellites. 29 samples were screened
from one Araucaria rulei population (Bogota, 21.54583 S, 166.05388 E).
Cross-species amplification was tested on five samples each from the other 12 Araucaria species
endemic to New Caledonia. All 12 nSSR loci could be amplified and were polymorphic in each
species.
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The aligned complete plastid genome of all 13 New Caledonian Araucaria species (Ruhsam et al.
2015, Genbank accessions KM678404-KM678430) was surveyed manually for microsatellites. For 18
regions which contained more than eight repeat motifs and had conserved primer sites in all species,
primers were designed using Primer3 (Untergasser et al. 2012). To reduce scoring errors for
mononucleotide repeats of ≥10 repeats, parts of the repeat motif were included in the reverse
primer where possible (Flores-Renteria & Whipple 2011). 14 out of the 18 microsatellite loci which
could be amplified reliably and produced unambiguous peaks, were grouped into four multiplex
reactions using the software MultiPLX (Kaplinski & Remm 2007) (Table 2). All forward primers were
tagged at the 5’ end of their sequence with an M13 tail (5’-CACGACGTTGTAAAACGAC-3’). PCR
reactions for each multiplex group (MP) were performed in volumes of 10 μL using the following
protocol: 1x QIAGEN Multiplex PCR master mix, 0.1 µM of each forward primer, 0.2 µM of each
reverse primer, 0.2 µM labelled (6-FAM, PET, VIC or NED) M13 primer (5’-CACGACGTTGTAAAACGAC3’) and 1 µL of unquantified DNA. The mixture was then cycled through the profile: 15 min at 95°C,
35 cycles of 30 sec at 94°C, 90 sec at 57°C and 1 min at 72°C, ending with 30 min at 60°C to complete
extension and subsequent storage at 4°C. LIZ-500 labelled internal size standard (PE Applied
Biosystems) was added to each sample to size microsatellites. 28 samples were screened from one
population (Bogota, 21.54583 S, 166.05388 E). Cross-species amplification was tested on five
samples each from the other 12 Araucaria species endemic to New Caledonia. All 14 cpSSR loci could
be amplified and were polymorphic in each species.
References
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Kaplinski L, Remm M (2007) MultiPLX: Automatic Grouping and Evaluation of PCR Primers. In:Yuryev
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