hide
Free keywords:
amphibians; Calotriton arnoldi; crested newt;
cross-amplification success; genome size; PAL_FINDER;
Pyrenean mountain newt
Abstract:
The development of microsatellite loci has become more efficient using nextgeneration
sequencing (NGS) approaches, and many studies imply that the
amount of applicable loci is large. However, few studies have sought to quantify
the number of loci that are retained for use out of the thousands of sequence
reads initially obtained. We analyzed the success rate of microsatellite loci
development for three amphibian species using a 454 NGS approach on tetranucleotide
motif-enriched species-specific libraries. The number of sequence
reads obtained differed strongly between species and ranged from 19,562 for
Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained
for Calotriton asper. PHOBOS was used to identify sequences with tetra-nucleotide
repeat motifs with a minimum repeat number of ten and high quality
primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319
for L. helveticus, we tested the amplification success, polymorphism, and degree
of heterozygosity for 41 primer combinations each for C. asper and T. cristatus,
and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci
for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially
amplifiable loci (PALs) resulted in estimated species-specific success rates
of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared
with representative Illumina NGS approaches, our applied 454-sequencing
approach on specifically enriched sublibraries proved to be quite competitive in
terms of success rates and number of finally applicable loci.
