Development and Characterization of Microsatellite Markers for Three Pollination Morphs of Cimicifuga simplex (Ranunculaceae)

Cimicifuga simplex Wormsk. (Ranunculaceae) is a perennial herb distributed in eastern and northeastern Asia for which at least three different pollination morphs have been reported. It is classified as endangered or near threatened in some Japanese regions, and its rhizome is commercially used as a crude drug. To examine genetic differentiation and gene flow among the three morphs, we developed eight microsatellite markers by using next-generation sequencing and estimated the genetic structure of C. simplex. We tested eight primer pairs on 93 individuals from six populations of C. simplex in Nagano, central Japan, and found that heterozygosity in morphs I and III was low compared to expected heterozygosity. Bayesian clustering performed with the STRUCTURE program clearly distinguished the three morphs of C. simplex, and only a little gene flow was detected among the morphs. These eight microsatellite markers are expected to be useful in conservation genetic studies of this species and for future conservation planning.


Introduction
Cimicifuga simplex Wormsk. (Ranunculaceae) is a perennial herb distributed in eastern and northeastern Asia [1] [2] that is classified as endangered or near threatened in three of the 47 Prefecture Red Data Book lists of Japan [3]. Its rhi-How to cite this paper: Toji, T., Kameyama, Y., Hirao, A.S. and Itino, T. zome is commercially used as a crude drug [4]. Accordingly, for the conservation and sustainable pharmacological use of this species, it is important to evaluate its genetic diversity and genetic structure. C. simplex comprises at least three different pollination morphs [5] (henceforth morphs I, II, and III). Morphs I and III are pollinated mainly by bumblebees, and morph II is pollinated mainly by butterflies [5]. The three morphs differ in their altitudinal distribution, habitats, flowering season, and nuclear internal transcribed spacer gene sequences [6], although the actual state of gene flow among the different morphs is unknown. Therefore, we used next-generation sequencing to develop eight nuclear microsatellite markers for C. simplex and estimated its genetic structure for a preliminary assessment of genetic differentiation and gene flow among the three morphs.

DNA Extraction and Library Construction
Each sample was a fresh leaf collected from an individual C. simplex plant growing in Nagano Prefecture, Japan. Genomic DNA was extracted from the leaf with the DNeasy Plant Mini Kit (QIAGEN, Germantown, Maryland, USA), and the extracted DNA was used for library preparation. The library was constructed from morph II individual. A 5' and 3' adapter was ligated for random fragmentation DNA. The adapter-ligated fragments were then amplified by polymerase chain reaction (PCR) and gel purified. Each fragment was amplified into distinct clonal clusters by bridge amplification.

DNA Sequencing and Microsatellite Isolation
We performed DNA sequencing on a Miseq sequencer (Illumina, San Diego, CA) using approximately 10% of a plate. The filtered reads were assembled de novo into contigs by using Newbler software (Roche, Basel, Switzerland). The QDD version 3.1 [7] bioinformatics pipeline was used to identify contigs possessing microsatellite motifs as well as to design primer pairs. We searched for primers with the following specifications: melting temperature (T m ) of 57˚C -63˚C, PCR product between 100 and 300 bp long, and primer length between 18 and 27 nucleotides. We identified 96 candidate primer pairs meeting these specifications and tested them using DNA samples from eight C. simplex individuals.
For all tested microsatellite loci, the forward primers were synthesized with one of four different universal fluorescent sequences added, and a 5'-GTTTCTT-3' PIG-tail [9] was added to all reverse primers.

Microsatellite Genotyping
The PCR amplification was performed in a 6 µL reaction volume containing 10

Genetic Analysis
We used the eight primer pairs identified by the screening to analyze 93 individuals from six populations of C. simplex in Nagano Prefecture, central Japan   The eight microsatellite markers described in this study were successfully used to detect genetic variation within populations and genetically distinguish the three pollination morphs. Thus, they will be useful in conservation genetic studies of C. simplex. In addition, the finding that pollination morphs I and III have relatively low genetic diversity is useful information for future conservation planning for C. simplex.  Office (Nagano Prefectural Government) for permission to work in the area.

Results and Discussion
This study was supported by the Japan Ministry of Education, Culture, Sports, Science and Technology (15H02641 to TI) and Nagano Society for the Promotion of Science (to TT).