Development and characterization of 30 microsatellite loci for Plagiorhegma dubium (Berberidaceae)

Premise of the Study Plagiorhegma dubium (Berberidaceae) has been listed as an endangered species in Korea due to extensive collection and destruction of natural habitats. In this study, 30 microsatellite loci, including 25 polymorphic loci, were developed for P. dubium for use in population‐level genetic analyses. Methods and Results We carried out transcriptome sequencing and isolated a total of 30 expressed sequence tag–simple sequence repeat markers from P. dubium using Illumina HiSeq high‐throughput sequencing. To test utility of the developed markers, we genotyped 60 individuals from three populations and estimated the number of alleles and levels of observed and expected heterozygosity. Expected heterozygosity levels ranged from 0.000 to 0.594, 0.000 to 1.000, and 0.000 to 0.744 in the three populations, respectively. Conclusions These transcriptome‐derived simple sequence repeat markers are highly polymorphic and can be widely used in characterization of the endangered P. dubium. Population genetic studies with these markers will provide valuable insights for conservation by unraveling evolutionary patterns of P. dubium.

microsatellite markers is an expensive and time-consuming processes (Squirrell et al., 2003). Expressed sequence tag (EST)-derived SSRs can overcome some of the drawbacks of older methods, for example by decreasing processing time (Zhou et al., 2016). Here, we developed and characterized 30 EST-SSR markers for the rare and threatened P. dubium using transcriptome sequencing with the Illumina paired-end sequencing platform. We evaluated the performance of these markers using 60 individuals representing three populations of P. dubium.

Transcriptome sequencing
To prepare a cDNA library, total RNA of P. dubium was extracted from a fresh leaf of a single sample from Korea (voucher no. NIBRVP0000556155; Appendix 1). RNA was extracted using the RNeasy Kit version 2.2 (Illumina, San Diego, California, USA) following the manufacturer's instructions, and was used for TruSeq cDNA library preparation. The RNA libraries were sequenced on the Illumina HiSeq 2000 platform, producing 150-bp paired-end reads. All raw reads were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (Bioproject ID PRJNA472226). Trimmomatic 0.32 (Bolger et al., 2014) was used to remove adapters and low-quality reads with the following parameters: seed mismatch of 2, palindrome clip threshold of 30, simple clip threshold of 10, a minimum adapter length of 2, headcrop of 7, leading and trailing quality of 3, sliding window size of 4, with an average quality of 20 and a minimum sequence length of 50 bases. After trimming, reads were assembled into 94,785 transcripts using the short-read assembly program Trinity (Haas et al., 2013) with default settings; these were then clustered into 76,725 unigenes.

Development of microsatellite markers based on ESTs of P. dubium
Microsatellites from the unigenes were detected using MISA version 1.0.0 (Thiel et al., 2003) with the default parameters. The criteria for identifying SSRs were as follows: the minimum number of nucleotide repeats was six for mono-, di-, tri-, tetra-penta-, and hexanucleotides. MISA identified 11,458 SSRs, of which 96 primer pairs were selected for further testing based on (1) region containing at least five repetitions of di-or trinucleotide motifs; (2) PCR product size of 140-500 bp; (3) length ranging from 12 to 24 nucleotides; (4) annealing temperature 55-60°C; and (5) minimum GC content 50%. Primer pairs were designed using Primer3 (Rozen and Skaletsky, 1999) to flank the microsatellite-rich region with a minimum of six repeats. The utility of the selected 96 microsatellite markers was evaluated by PCR on three individuals from each population of P. dubium. Reactions were carried out in a total volume of 25 μL containing 2.5 μL of 10× Ex Taq buffer (TaKaRa Bio Inc., Shiga, Japan), 2 μL of 2.5 mM dNTPs, 0.1 μM of forward and reverse primer, 0.1 μL of TaKaRa Ex Taq, and 5-10 ng of template DNA. All PCRs were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Carlsbad, California, USA) using the following protocol: initial denaturation at 98°C for 5 min; followed by 30 cycles of denaturation at 95°C for 1 min, annealing at locus-specific annealing temperature (Table 1) for 1 min, and extension at 72°C for 1.5 min; and a final extension step at 72°C for 10 min. Of the 96 candidate markers, 34 markers amplified and were suitable for further testing. Finally, 30 markers were selected and included in the following analysis after excluding four markers with a low amplification rate of 80% or less. The PCR products were labeled with fluorescent dyes (HEX and FAM) and run on an ABI 3730XL automated sequencer (Applied Biosystems) using the GeneScan 500 LIZ Size Standard (Applied Biosystems). Genotyping was manually determined with GeneMapper 3.7 (Applied Biosystems). Identified microsatellite markers (Table 1) producing clear and polymorphic bands were subsequently used for genetic diversity assessments.

Genetic parameters
Fresh leaves from 60 P. dubium individuals were sampled from three populations from Korea, Japan, and China. The voucher specimens were deposited in the National Institute of Biological Resources Herbarium (KB), Incheon, Republic of Korea (Appendix 1). The precise locations of the sites have been withheld to prevent illegal collection. The level of polymorphism at each locus was assessed by calculating the number of alleles per marker (A), observed heterozygosity, and expected heterozygosity using GenAlEx 6.5 (Peakall and Smouse, 2012). Deviation from Hardy-Weinberg equilibrium was estimated with Arlequin 3.5 (Excoffier and Lischer, 2010).
Functional annotations for these 30 markers were compared against the NCBI nonredundant (NR) protein database with BLASTX (E-value 1 × 10 −5 ). A total of 30 markers were successfully amplified, of which polymorphism was detected in 25 (Table 1).