Development of microsatellite markers for the apomictic triploid fern Myriopteris lindheimeri (Pteridaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Myriopteris Fée is an early diverging lineage of cheilanthoid ferns (Pteridaceae) that contains approximately 47 species encompassed within three major clades—all of which, until recently , were circumscribed in the large, polyphyletic genus, Cheilanthes Sw. (Grusz and Windham, 2013 ; Grusz et al., 2014). The covillei clade is the largest subclade in the recently resurrected genus Myriopteris , within which M. lindheimeri (Hook.) J. Sm. resides (Grusz et al., 2014). Myriopteris lindheimeri itself comprises a number of relatively widespread, apomictic triploid lineages (n = 2 n = 90 chromosomes; Windham and Yatskievych, 2003) derived from a comparatively rare, sexual diploid cytotype through intraspecifi c whole genome duplica-Here, we use 454 next-generation sequencing to develop mi-crosatellite markers for M. lindheimeri. Like M. lindheimeri , many members of the covillei clade are also apomictic poly-ploids or, alternatively, sexual diploids that are involved in the formation of downstream polyploid taxa of hybrid origin (Grusz et al., 2009). For this reason, we tested our newly developed markers for cross-amplifi cation in diploid and polyploid taxa spanning the covillei clade, including: M. Genomic DNA of a single individual of diploid M. lindheimeri (voucher: Schuettpelz 450 [DUKE], collected from the Tonto National Forest, Pinal Co., Arizona, USA) was extracted from silica gel–dried material using the DNeasy Plant Mini Kit following the manufacturer's protocol (QIAGEN, Valencia, California, USA). Genomic DNA was run on two lanes (1/4 plate = 24 wells) using the Roche 454 GS-FLX Titanium sequencing platform (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) at the Duke University Center for Genomic and Computational Biology sequencing facility. The 454 run generated 234,428 sequence reads with a median length of 403 bp. Raw data were scanned for di-, tri-, tetra-, penta-, and hexanucleotide perfect micro-satellite repeats using MSATCOMMANDER version 0.8.2 (Faircloth, 2008). Of the 234,428 sequence reads searched, 25,295 sequences contained a total of 33,955 repeats. Given the surplus of repeat regions, we focused our efforts on a subset of nonplastid regions (determined by BLASTN against the M. lind-heimeri chloroplast genome; Wolf et al., 2011) containing di-, tri-, and tetra-nucleotide repeats with suffi cient fl anking sequence in which to develop primers (Chakraborty et al., 1997). A total of 159 unlabeled primer pairs were designed in Primer3, using default settings, implemented within MSATCOMMANDER Each microsatellite region (159 in total) was amplifi ed by PCR …