Development and characterization of EST‐SSR markers for Carex angustisquama (Cyperaceae), an extremophyte in solfatara fields

Premise of the Study Expressed sequence tag–simple sequence repeat (EST‐SSR) markers were developed for Carex angustisquama (Cyperaceae) to investigate the evolutionary history of this plant that is endemic to solfatara fields in northern Japan. Methods and Results Using RNA‐Seq data generated by the Illumina HiSeq 2000, 20 EST‐SSR markers were developed. Polymorphisms were assessed in C. angustisquama and the closely related species C. doenitzii and C. podogyna. In C. angustisquama, many loci were monomorphic within populations; the average number of alleles ranged from one to five, and levels of expected heterozygosity ranged from 0.000 to 0.580, while all markers were polymorphic in a population of C. doenitzii. This indicates that low genetic polymorphism of C. angustisquama is likely due to the species’ population dynamics, rather than to null alleles at the developed markers. Conclusions These markers will be used to assess genetic diversity and structure and to investigate evolutionary history in future studies of C. angustisquama and related species.

they are shown to be more reliable because they have lower frequencies of null alleles than anonymous genomic SSR markers (Ellis and Burke, 2007). Therefore, we developed EST-SSR markers and examined their polymorphisms and transferability to closely related taxa.
For the initial PCR amplification trial, we used two individuals from population CA09 (Appendix 1). For the 32 primer pairs that showed clear peaks, two individuals from each population (CA09, CA13, CA14, and CA15; Appendix 1) were then used to check polymorphisms among populations. Using 20 primers that were polymorphic over the eight samples (details for 12 monomorphic markers are provided in Appendix 2), 24 individuals from each population (CA09, CA14, and CA15) were evaluated for withinpopulation polymorphisms. However, because few polymorphisms were detected within each population, we examined the transferability and evaluated polymorphisms in two closely related species (C. doenitzii Boeckeler and C. podogyna Franch. & Sav.; Appendix 1) to test whether low genetic variation of C. angustisquama was the result of null alleles at the markers or of the species' genetic nature. GenAlEx 6.5 software (Peakall and Smouse, 2012) (Goudet, 1995) was used to test significance of Hardy-Weinberg equilibrium (HWE) by 1000 randomizations; the significance of the associated P values was adjusted by applying sequential Bonferroni correction. The test for the presence of null alleles was performed using MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004).
For C. angustisquama, all primer pairs (Table 1) were polymorphic when all populations were combined; A ranged from two to seven, and levels of H e and H o ranged from 0.100 to 0.703 and 0.000 to 0.286, respectively (Table 2). For each population, A ranged from one to five, and levels of H e and H o ranged from 0.000 to 0.580 and 0.000 to 0.524, respectively (Table 2). For cross-species amplification, 20 and nine primer pairs were polymorphic in C. doenitzii and C. podogyna, respectively (Table 3). Significant departures (P < 0.01) from HWE were detected in three loci (Cang4398, Cang7240, and Cang48335) in C. doenitzii, although no significant departures were detected for any of the populations or loci in both C. angustisquama and C. podogyna. Analysis with MICRO-CHECKER (at the 99% confidence level) highlighted the existence of null alleles at some loci in C. angustisquama and C. doenitzii (Tables 2, 3). EST-SSR markers were shown to have a disadvantage of less polymorphism than genomic SSR markers (Bouck and Vision, 2007;Ellis and Burke, 2007), and we found low genetic variation in all populations of C. angustisquama. This may be caused by presence of null alleles. However, substantial polymorphisms were detected in C. doenitzii, which is the most closely related species to C. angustisquama (K. Nagasawa, H. Setoguchi, M. Maki, H. Goto, K. Fukushima, Y. Isagi, S. Sakaguchi, Y. Suyama, and Y. Tsunamoto, unpublished data). Moreover, in C. angustisquama, although most loci were homozygous within populations, these loci were fixed with different alleles for each population, which likely reflects evolutionary history rather than null alleles. Thus, we conclude that low genetic variation of C. angustisquama is probably caused by the species' demographic history.

CONCLUSIONS
The 20 EST-SSR markers developed for C. angustisquama are less polymorphic within populations. However, in intraspecific and cross-species amplification, substantial polymorphisms were detected, indicating that low genetic variation in C. angustisquama results from the species' demographic history, and not from the markers' characteristics. Thus these markers will be useful for investigating intraspecific relationships among C. angustisquama populations occurring in disjunct solfatara fields. These markers are also useful in other Carex species, providing novel population genetic tools in this speciose genus.

ACKNOWLEDGMENTS
The authors thank Mr. K. Sawa (Yamagata Prefecture), Mr. S. Kurata (University of Tokyo), Dr. Y. Suyama (Tohoku University), Dr. Y. Tsunamoto (Forest Research and Management Organization), and Dr. K. Yonekura (Tohoku University) for providing plant materials. We are also grateful to Dr. J. R. P. Worth for his comments on an earlier version of the manuscript. This research was supported by the Japan Society for the Promotion of Science (Bilateral program "The spatial and temporal dimensions and underlying mechanisms of lineage divergence and plant speciation of keystone species in Sino-Japanese Forest subkingdom" and Grant-in-Aid for Young Scientists B [no. 17K15286]), the SICORP Program of the Japan Science and Technology Agency ("Spatial-temporal dimensions and underlying mechanisms of lineage diversification and patterns of genetic variation of keystone plant taxa in warm-temperate forests of Sino-Japanese Floristic Region"; grant no. 4-1403), the TAKARA Harmonist Fund, the Nissei Fund, and the Pro Natura Foundation Japan for the Ashiu Biological Conservation project.

DATA ACCESSIBILITY
Cleaned reads from the cDNA library have been deposited to the DNA Data Bank of Japan (DDBJ; Bioproject PRJDB6849). Sequence information for the developed primers has been deposited to the National Center for Biotechnology Information (NCBI); GenBank accession numbers are provided in Table 1.