Isolation and characterization of novel microsatellite loci for the endangered orchid Cypripedium japonicum (Orchidaceae)

Abstract

Premise of the study:Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan.Methods and Results:Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively.Conclusions:The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.

Japanese “nameko” mushrooms (<i>Pholiota microspora</i>) produced via sawdust-based cultivation exhibit severe genetic bottleneck associated with a single founder

“…Additionally, while developing the markers, we achieved high rates of success in amplifying the primary candidate markers (21/24 = 0.85) as well as the final set of selected markers (14/24 = 0.58). This result was in contrast to the usual process of our microsatellite marker development, where the ratios of the final selected markers to the primary candidate ones were several to dozens in percentages: 18/45 = 0.40 in Gastrodia takeshimensis (Kishikawa et al, 2019); 13/102 = 0.13 in Phraortes elongatus (Nozaki et al, 2021); 15/293 = 0.05 in Pyrola japonica (Shutoh, Izuno, Isagi, Kurosawa, & Kaneko, 2017); and 26/238 = 0.11 in Cypripedium japonicum (Yamashita, Izuno, Isagi, Kurosawa, & Kaneko, 2016). Our results imply that the Chinese sample used for genome assembly is genetically similar to Japanese samples.…”

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

“…We defined ramets with identical multilocus genotypes at the 21 SSR loci as belonging to a genet. The combined non‐exclusion probabilities of a genet, using these 21 loci, were 0.000018 in YKD and 0.00012 in HKD (Yamashita et al, 2016) suggesting sufficient variation to determine genet identities. A tag sequence for fluorescence labeling was added to each forward primer (Boutin‐Ganache et al, 2001).…”

“…Genomic DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN, Germantown, MD) following the manufacturer's protocols. We used 21 SSR primer pairs (provided in Supporting Information S1) selected from the markers developed by Yamashita et al (2016). We defined ramets with identical multilocus genotypes at the 21 SSR loci as belonging to a genet.…”

“…We developed 26 simple sequence repeat (SSR) markers with a co-dominant mode of inheritance and high divergence (Yamashita et al, 2016). These markers also have sufficient resolution to investigate genetic variation among genets and differences among remnant populations.…”

Development of Microsatellite Markers for Cypripedium tibeticum (Orchidaceae) and their Applicability to Two Related Species

“…Microsatellites (simple sequence repeats [SSRs]) are widely used molecular markers that are applied in population genetics to investigate genetic diversity, population structure, and evolutionary history ( Hodel et al, 2016 ; Vieira et al, 2016 ). To date, microsatellite markers have been developed for several Cypripedium L. species; however, scarcely any markers are applicable to C. tibeticum ( Fay and Cowan, 2001 ; Pandey and Sharma, 2013 ; Yamashita et al, 2016 ). In this study, we developed and characterized 20 polymorphic SSR markers in C. tibeticum and tested their applicability in two related species: C. flavum P. F. Hunt & Summerh.…”