Characterization of 15 novel microsatellite loci for Cypripedium calceolus (Orchidaceae) using MiSeq sequencing

Abstract

Lady's slipper orchid (Cypripedium calceolus) serves as a flagship species for plant conservation in many European countries. Its populations are threatened by overcollecting and loss of suitable habitat. Information on local and regional genetic structure can help to develop appropriate conservation strategies. A total of fifteen novel microsatellite markers were developed using MiSeq sequencing. All loci found to be polymorphic, with the number of alleles per locus ranging from 2 to 8. Observed heterozygosity ranged from 0.19 to 0.89. The developed microsatellite markers will be useful to analyze genetic diversity and genetic structure of C. calceolus populations.Keywords Genetic diversity Á Genetic structure Á Microsatellites Á Orchidaceae Lady's slipper orchid (Cypripedium calceolus L.) is one of the largest and the most spectacular elements of European flora. This Euro-Asiatic species has suffered a marked decrease of localities and area occupied (Terschuren 1999). It is legally protected in all European countries, and listed in various national conventions and directives (Terschuren 1999). Studies of genetic diversity and fine-scale spatial genetic structure in relation to habitat management will improve management strategies of this vulnerable species.Two, out of four recently published microsatellite loci for C. calceolus, may pose scoring problems (Pedersen et al. 2012). Therefore a higher number of variable loci is needed to estimate genetic structure and distinguish close relatives. Here we report the isolation and characterization of polymorphic loci that will be useful in future population genetic studies.Seeds of C. calceolus were collected from population Bukówki (Poland) and asymbiotically germinated in in vitro culture. Total genomic DNA was isolated from three seedlings using procedure described by Bekesiova et al. (1999). Extracted DNA was used for a library preparation with a NEBNext Ò DNA Library Prep Master Mix Set for Illumina. The sequencing was performed on the MiSeq Benchtop Sequencer (Illumina) using the 2 9 250 bp read mode. The obtained data (10,19 Mega reads) was assembled using CLCGenomicWorkbench (CLCBio) into 513,225 contigs and the microsatellites were then detected using QDD 2.1 Beta (Meglecz et al. 2010). A total of 22,162 contigs contained at last one microsatellite of which 53 loci were selected for initial screening. We screened 32 plants from two Polish populations (Bukówki and Prokowo) for polymorphisms at these loci. All forward primers were tagged with M13(-21) (5 0 -TGTAAAACGACGGCCAGT-3 0 ) at the 5 0 end. The 10 lL PCR volume contained: 4.5 lL MyTaq TM HS Mix (Bioline), 0.4 lM of both forward and reverse primers, 0.2 lM dye labelled primer, 0.3 % DMSO, 1.2 lL water and 1 lL DNA template (*50 ng). The following PCR conditions were used: 2 min initial denaturation at 96°C, followed by 35 cycles of 95°C for 30 s/primer specific annealing temperature for 30 s/72°C for 45 s.

Combining current knowledge of Cypripedium calceolus with a new analysis of genetic variation in Italian populations to provide guidelines for conservation actions

“…Plant DNA Kit (Omega Bio‐tek, Norcross, Georgia, US). In addition to the previously employed 11 nuclear microsatellite loci (Gargiulo et al, 2019), we analyzed variation for the locus Ccal_50 (Minasiewicz & Znaniecka, 2014). We performed allele calling in GeneMapper v5.0 (Applied Biosystems) and used Micro‐Checker v2.2.3 (van Oosterhout, Hutchinson, Wills, & Shipley, 2004) with 1,000 randomizations to inspect the data set for scoring errors.…”

Spatial genetic structure of an endangered orchid Cypripedium calceolus (Orchidaceae) at a regional scale: limited gene flow in a fragmented landscape

“…Samples of yellow lady's slipper were also analyzed for diversity in species-specific 13 microsatellite loci (Online Resources S1; Minasiewicz and Znaniecka 2014). All polymerase chain reactions were conducted on an Eppendorf NEXUS GX1 (Eppendorf) following the protocol of Minasiewicz and Znaniecka (2014). Microsatellite genotyping was performed using an ABI 3130 sequencer (Applied Biosystem) and LIZ-500 size standard (Applied Biosystem).…”

Cypripedium calceolus (Orchidaceae) in Central Russia: a case study for its populations in two protected areas in the Republic of Mordovia (Russia)

“…In different regions of Eurasia, C. calceolus has been estimated as Near Threatened (Bilz 2011, Turis et al 2014, Vulnerable (Rassi et al 2001), Endangered (Witkowski et al 2003), and Critically Endangered (Blinova & Uotila 2011, Khapugin et al 2017a, Khapugin et al 2017c. International studies on C. calceolus related to its genetic diversity (Brzosko et al 2009, Brzosko et al 2011, Fay et al 2009, Kļaviņa et al 2014, Minasiewicz & Znaniecka 2014, population ecology and biology (Blinova 2002, Brzosko 2002, Davison et al 2013, Fardeeva et al 2010, Gajewski & Marcisz 2014, Khapugin et al 2014, Korczyński & Krasicka-Korczyńska 2014, García et al 2002, García et al 2010, Gorchakovskii & Igosheva 2003, Kirillova 2015, Kull 1998, Nicolè et al 2005, Puchnina 2017, Stetsuk 2013, Zheleznaya 2015, impacts of environment conditions and stress-factors on the species (Blinova 2002, Czerepko et al 2014, Kirillova 2016, Kirillova et al 2012, Puchnina 2017, Shefferson et al 2012, pollination (Antonelli et al 2009, Tremblay 1994, as well as the list of publications with new records of this threatened species is continuously enlarging ...…”