Abstract
Nuclear microsatellite markers for Pungtungia herzi were developed using a combination of next-generation sequencing and Sanger sequencing. One hundred primer sets in the flanking region of dinucleotide and trinucleotide repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Of these primer sets, 16 new markers (16%) were successfully amplified with unambiguous polymorphic alleles in 16 individuals of Pungtungia herzi. Cross-species amplification with these markers was then examined in two related species, Pseudopungtungia nigra and Pseudopungtungia tenuicorpa. Fifteen and 11 primer pairs resulted in successful amplification in Pseudopungtungia nigra and Pseudopungtungia tenuicorpa, respectively, with various polymorphisms, ranging from one allele (monomorphic) to 11 alleles per marker. These results indicated that developing microsatellite markers for cross-amplification from a species that is abundant and phylogenetically close to the species of interest is a good alternative when tissue samples of an endangered species are insufficient to develop microsatellites.
Highlights
The Korean endemic genus Pseudopungtungia includes two endangered species, the black shinner (Pseudopungtungia nigra) and the slender shinner (Pseudopungtungia tenuicorpa), that have suffered drastic population decreases [1,2]
We developed a set of microsatellite markers from Pungtungia herzi using a Next-generation sequencing (NGS) protocol [22]
We examined the polymorphic markers from Pungtungia herzi in the two related endemic species, Pseudopungtungia nigra and Pseudopungtungia tenuicorpa
Summary
The Korean endemic genus Pseudopungtungia includes two endangered species, the black shinner (Pseudopungtungia nigra) and the slender shinner (Pseudopungtungia tenuicorpa), that have suffered drastic population decreases [1,2]. Insufficient genomic DNA and the lack of an appropriate number of individuals to screen polymorphic markers hinder further research progress. To overcome this difficulty, we used transferable microsatellite markers from a phylogenetically close species. The striped shiner (Pungtungia herzi) is widely distributed in most rivers and lakes in South Korea, northern China, and southern Japan [1,15]. It reaches lengths of 10–15 cm and feeds on attached algae and aquatic insect larvae. We examined the polymorphic markers from Pungtungia herzi in the two related endemic species, Pseudopungtungia nigra and Pseudopungtungia tenuicorpa
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