ISSN 1346-7565 Acta Phytotax. Geobot. 71 (1): 73–76 (2020) doi: 10.18942/apg.201907 Primer Note Development and Characterization of EST-SSR Markers for Tricyrtis sect. Tricyrtis ()

* Riku Nakamasu, Shota Sakaguchi and Hiroaki Setoguchi

Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Nihonmatsu-cho, Sakyo-ku, Kyoto 606-8501, . *[email protected] (author for correspondence)

A set of 8 polymorphic EST-SSR markers has been developed to evaluate the genetic diversity and ge- netic structure of the populations of Tricyrtis sect. Tricyrtis (Liliaceae) in Kyushu, which reproduces sexually and asexually by bulbils. The total number of alleles for each locus ranged from 2 to 11 with an average of 5.5, while the values of observed and expected heterozygosity ranged from 0.043 to 0.875 and 0.043 to 0.849, respectively. The combined probabilities of identity (PI = 6.0E-07 and PI-Sib = 2.7E-03) of these markers suggest that they are useful for not only evaluating genetic diversity but also for estimat- ing clonal diversity within populations. Cross-species amplification was evaluated for three phylogenet- ically related species, T. macropoda, T. setouchiensis, and T. affinis, to show that all the EST-SSR mark- ers are transferrable among these species.

Key words: EST-SSR, genetic diversity, microsatellite loci, next generation sequencing, population genetics, Tricyrtis

Tricyrtis Wall. (Liliaceae) comprises approxi- cable to sect. Tricyrtis. To determine the genetic mately 20 species of perennial herbs in temperate structure and the spatial extent of genets within to subtropical mesic forests of eastern . The populations of the Tricyrtis, additional simple se- species are distributed from the Himalaya to east- quence repeat (SSR) markers that can be stably ern Asia (13 species in Japan) and include many amplified by polymerase chain reaction (PCR) local endemics and endangered species. Tricyrtis were needed. In this study, we developed EST- is divided into 4 sections, Flavae Masam., SSR (expressed sequence tag SSR) markers using Brachycyrtis (Koidz.) Kitag. & T. Koyama, Hir- transcriptome sequencing to evaluate the poly- tae Masam., and Tricyrtis (Takahashi 1980). The morphisms in the Tricyrtis populations produc- largest section Tricyrtis occurs mainly in Japan ing bulbils and in the closely related T. macropo- and (Takahashi 1987) and includes approx- da, T. setouchiensis Hir. Takah. bis and T. affinis imately 8 species. Recently, populations of Tri- Makino. cyrtis sect. Tricyrtis producing bulbils were dis- covered in northern Kyushu, Japan, while no oth- er species of Tricyrtis that reproduces both sexu- ally and vegetatively via bulbils has previously Materials and Methods been reported. Genetic markers can provide insights into the Fresh leaf tissues of Trichrtis setouchiensis relative contribution of sexual vs. clonal repro- (collected in Wakayama Prefecture, Japan; duction within populations. Although there are voucher accession number OSA60766, represen- genetic markers available for sect. Brachycyrtis tative specimen deposited in Osaka Museum of (Ohki & Setoguchi 2013), none have been appli- Natural History) were frozen in liquid nitrogen 74 Acta Phytotax. Geobot. Vol. 71 and total RNA was extracted by using RNeasy condition was as follows; primer size of 18–22 (Qiagen, Hilden, Germany) according to the bp, annealing temperature of 58–62 ºC, GC con- manufacturer’s protocol. The cDNA library was tent of 30–70%, and an expected product size of prepared following the Illumina protocol (Illumi- 100–450 bp. One of three different M13 universal na, San Diego, California, USA) and 100bp sequence (5’-CACGACGTTGTAAAACGAC-3’, paired-end short-read sequencing was conducted 5’-TGTGGAATTGTGAGCGG-3’, or using the Illumina Hiseq 2000 (Illumina; se- 5’-CTATAGGGCACGCGTGGT-3’) was added quencing performed by BGI, Shenzhen, Guang- to all forward primers and the reverse primers dong Province, China). A total of 34,033,518 raw were tagged with PIG-tail sequences reads were generated. After quality assessment (5’-GTTTCTT-3’). In total, 384 primer pairs were and data filtering (ambiguous limit = 2, quality randomly selected to evaluate amplification and limit = 0.03, eighteen numbers from the begin- polymorphism in the Tricyrtis. ning of the 5’ terminal nucleotides were re- Twenty-four individuals of Tricyrtis from moved), 34,004,071 clean reads were obtained three populations were used to screen the primers and assembled using CLC Genomics Workbench (Pop. A = 32.978ºN, 130.107ºE; Pop. B = v.7.5.1 software (DLC bio, Aarhus, Denmark) 33.430ºN, 130.249ºE; Pop. C = 32.751ºN, (DRA accession No.; DRA006602), resulting in 130.284ºE). Sixteen individuals of T. macropoda, 44,618 contigs (word size 23, bubble size 50, min- T. setouchiensis and T. affinis other taxa in sect. imum contig length = 200 bp, average length was Tricyrtis were used to check the versatility of the 669). We screened microsatellite regions for ≥ 10 primers. Silica gel-dried leaf material stored in dinucleotide and ≥ 8 trinucleotide repeats. In to- deep freezer (Panasonic, Japan) at -80 ºC were tal, 1,445 SSR motifs were found. Primers for pulverized with a TissueLyser (Qiagen). After the each SSR were designed using MSATCOM- polysaccharides were removed from this powder MANDER (Faircloth 2008). The designed primer with HEPES buffer (pH 8.0) (Setoguchi & Ohba

Table 1. Characteristics of the 8 microsatellite markers developed for sect. Tricyrtis. Both pigtail and M13 sequences are un- derlined. Only proteins whose E-values were lower than E-05 are shown. Repeat Allele size BLASTX top GenBank Locus name Primer sequence (5'-3') E-value accession motif range (bp) hit description no. F : CACGACGTTGTAAAACGACGAGGATGAGACCCGGATCTG No Tri_885 (AAG)9 276-297 - LC375740 R : GTTTCTTACCGCATCTGTTATCCTCCC significant hit

F : TGTGGAATTGTGAGCGGCATCGCCATTGTTGAGGGTC No Tri_3252 (CTT)12 105-135 - LC375741 R : GTTTCTTCGGCGATACTGTTTCGATCG significant hit

F : CACGACGTTGTAAAACGACAGCACCCAAAGCAAGACAAC No Tri_4011 (AAG)11 204-222 - LC375742 R : GTTTCTTGTGCACTTCATCTCCGTGTC significant hit PREDICTED: mediator F : CACGACGTTGTAAAACGACAATCAGCAACCCTCTTTGGC of RNA Tri_10106 (AAC)10 285-324 polymerase II 5.00E-05 LC375743 transcription R : GTTTCTTACTTCCTGGAGCCCTCTTTG subunit 15a [Vitis vinifera] uncharacterized F : CTATAGGGCACGCGTGGTAGAGTAAGCGGCGGACTATG protein Tri_4485 (TTC)10 289-328 LOC8071232 2.00E-13 LC375744 R : GTTTCTTAGATAGCTACTCGTGCAGGC [Sorghum bicolor] F : TGTGGAATTGTGAGCGGAGGTTCATGCTCCAAGGAAC Tri_13531 (AT)12 294-344 No significant hit - LC375745 R : GTTTCTTTTCAGAGGTGGTGGTTCAGG F : CACGACGTTGTAAAACGACACCCTCACCGACAATACCAC Tri_32962 (ACC)8 103-130 No significant hit - LC375746 R : GTTTCTTTGAGGGATTTGAGGGCTGTG F : TGTGGAATTGTGAGCGGACAATCACGAGAACAGTGCG Tri_37933 (AG)10 260-308 No significant hit - LC375747 R : GTTTCTTTATCGCCCTTTCCCACCTTC February 2020 Nakamasu & al. — Microsatellite Markers for sect. Tricyrtis (Liliaceae) 75

Table 2. Genetic diversity statistics for the three populations of Tricyrtis producing bulbils based on newly developed EST-

SSR primers. NA, number of alleles; HO, observed heterozygosity [significant deviation from Hardy-Weinberg equilibri-

um (P < 0.05) is indicated by *]; HE, expected heterozygosity; PI, probability of identity for two unrelated individuals; PI-Sibs, probability of identity for two siblings; CPI, combined PI; CPI-Sibs, combined PI-Sibs. Geographic coordinates for the populations are; Pop. A = 32.978ºN, 130.107ºE; Pop. B = 33.430ºN, 130.249ºE; Pop. C = 32.751ºN, 130.284ºE. Pop. A (n = 24) Pop. B (n = 24) Pop. C (n = 24)

Locus name NA HO HE PI PI-Sibs NA HO HE PI PI-Sibs NA HO HE PI PI-Sibs Tri_885 4 0.609 0.612 2.3E-01 5.0E-01 6 0.750 0.707 1.2E-01 4.3E-01 4 0.708 0.651 1.9E-01 4.7E-01 Tri_3252 2 0.043 0.043 9.2E-01 9.6E-01 3 0.250* 0.497 3.4E-01 5.9E-01 3 0.375 0.378 4.2E-01 6.7E-01 Tri_4011 4 0.522 0.615 2.2E-01 5.0E-01 4 0.667 0.619 2.2E-01 5.0E-01 4 0.333 0.322 4.8E-01 7.1E-01 Tri_10106 6 0.818 0.823 5.6E-02 3.5E-01 4 0.522 0.556 2.4E-01 5.3E-01 7 0.875 0.704 1.2E-01 4.3E-01 Tri_4485 6 0.364* 0.810 6.3E-02 3.6E-01 8 0.609 0.830 5.1E-02 3.5E-01 6 0.625 0.799 7.0E-02 3.7E-01 Tri_13531 3 0.435 0.552 2.8E-01 5.5E-01 5 0.583* 0.613 2.2E-01 5.0E-01 6 0.261* 0.376 4.0E-01 6.6E-01 Tri_32962 6 0.739 0.718 1.2E-01 4.2E-01 6 0.458 0.597 2.1E-01 5.0E-01 5 0.875 0.720 1.2E-01 4.2E-01 Tri_37933 10 0.870 0.849 4.0E-02 3.4E-01 11 0.792 0.839 4.3E-02 3.4E-01 9 0.750* 0.760 8.7E-02 3.9E-01 Average 5.125 0.550 0.628 5.875 0.579 0.657 5.500 0.600 0.589 CPI 2.2E-07 2.2E-07 1.4E-06 CPI-Sibs 2.3E-03 2.0E-03 3.8E-03

Table 3. Cross-amplification and genetic diversity statistics of EST-SSR primers in related taxa of Tricyrtis. NA, number of

alleles, HO, observed heterozygosity [significant deviation from Hardy-Weinberg equilibrium (P < 0.05) is indicated by *];

HE, expected heterozygosity. Geographic coordinates for the populations are; T. macropoda: 32.530ºN, 131.623ºE; T. setouchiensis: 34.734ºN, 135.117ºE; T. affinis: 35.343ºN, 135.758ºE. T. macropoda (n = 16) T. setouchiensis (n = 16) T. affinis (n = 16)

Locus name NA HO HE NA HO HE NA HO HE Tri_885 8 0.625 0.770 4 0.733 0.656 5 0.625 0.711 Tri_3252 4 0.563 0.455 7 0.563* 0.693 2 0.625 0.469 Tri_4011 2 0.063 0.061 4 0.750 0.615 3 0.563 0.486 Tri_10106 3 0.750 0.615 7 0.813 0.770 8 0.938 0.787 Tri_4485 8 0.813 0.789 7 0.938 0.789 5 0.500* 0.693 Tri_13531 4 0.625 0.633 5 0.333* 0.684 2 0.125 0.117 Tri_32962 5 0.750 0.629 5 0.625 0.678 4 0.375 0.363 Tri_37933 8 0.688 0.676 10 0.867* 0.858 7 0.688* 0.641 Average 5.25 0.609 0.578 6.13 0.703 0.718 4.50 0.555 0.533

1995), total DNA was extracted using the CTAB fornia, USA) using the GeneScan LIZ-600 size method (Doyle & Doyle 1987). The extracted standard (Applied Biosystems), POP7 polymer DNA was dissolved in 30 μL of TE buffer and (Applied Biosystems) and 36-cm capillary array. used for PCR. The total PCR reaction volume Fragment size was determined using GeneMap- was 5 µL, which contained approximately 0.5 ng per (Applied Biosystems). DNA, 2.5 µL of 2×Multiplex PCR Master Mix To evaluate the polymorphisms of the mark- (Qiagen), 0.01 µM of forward primer, 0.2 µM of ers and the genetic diversity, genetic diversity in- reverse primer and 0.1 µM of fluorescently la- dices (number of alleles, allelic richness, ob- beled M13 primer. PCR amplification was per- served heterozygosity and expected heterozygos- formed with an initial denaturation at 94 ºC for 3 ity) were calculated by using GenAlex v.6.5.2 min followed by 35 cycles at 94 ºC for 30 sec, 60 (Peakall & Smouse 2006). The significance of the ºC for 3 min, and 68 ºC for 1 min followed by a Hardy-Weinberg equilibrium was tested with final extension at 68 ºC for 20 min. Amplified 1,000 randomizations using FSTAT 2.9.3 (Gou- products were loaded onto an ABI 3130 auto-se- det 1995). The probability identity of two unre- quencer (Applied Biosystems, Foster City, Cali- lated individuals (PI) and two siblings (PI-Sib) of 76 Acta Phytotax. Geobot. Vol. 71

Tricyrtis sp. was also calculated by using GenAl- nese Society for the Promotion of Science (Nos. ex v.6.5.2. JP24247013 and JP26304013), the Environmental Re- search and Technology Development Fund of the Minis- try of the Environment (grant no. 4-1702), and SICORP Program of the Japan Science and Technology Agency (grant no. 4-1403; Spatial-temporal dimensions and un- Results and Discussion derlying mechanisms of lineage diversification and pat- terns of genetic variation of keystone taxa in warm- By testing 384 primer pairs, 8 loci showed temperate forests of Sino-Japanese Floristic Region). polymorphism in all Tricyrtis taxa examined in this study. The corresponding sequences of these regions have been deposited in GenBank (Table 1). For the populations of the Tricyrtis producing bulbils, the number of alleles (NA), the mean val- References ues of HO and HE and probability of identity (PI) are shown in Table 2. NA ranged from 2 to 11 Doyle, J. & J. L. Doyle. 1987. Genomic plant DNA prepa- ration from fresh tissue-CTAB method. Phytochem. with an average of 5.5; the mean values of HO and Bull. 19: 11–15. HE were 0.576 and 0.625; and the average of com- Faircloth, B. C. 2008. msatcommander: detection of mic- bined probabilities of identity (PI and PI-Sib) rosatellite repeat arrays and automated, locus-specif- were 6.0E-07 and 2.7E-03, respectively. The re- ic primer design. Molec. Ecol. Resour. 8: 92–94. sults of cross-species transferability confirmed Goudet, J. 1995. FSTAT (version 1.2): a computer pro- that all 8 loci were successfully amplified and gram to calculate F-statistics. J. Hered. 86: 485–486. Ohki, N. & H. Setoguchi. 2013. New microsatellite mark- polymorphic in the other taxa of Tricyrtis (Table ers for Tricyrtis macrantha (Convallariaceae) and 3). cross-amplification in closely related species. Appl. In conclusion, all 8 polymorphic EST-SSR Plant Sci. 1: 1200247. loci were reliably scorable and transferable Peakall, R. O. D. & P. E. Smouse. 2006. GENALEX 6: among the three taxa of Tricyrtis, indicating that genetic analysis in Excel. Population genetic software for teaching and research. Molec. Ecol. Resour. 6: these markers can be used with confidence in fu- 288–295. ture genetic studies of populations of Tricyrtis. Setoguchi, H. & H. Ohba. 1995. Phylogenetic relation- Additionally, the markers with low combined ships in Crossostylis (Rhizophoraceae) inferred from probability of identity will be useful for identify- restriction site variation of chloroplast DNA. J. Plant ing the spatial distribution of clonal ramets in Res. 108: 87–92. Takahashi, H. 1980. A taxonomic study on the genus Tri- populations of Tricyrtis. cyrtis. Sci. Rep. Fac. Educ., Gifu Univ. (Nat. Sci.). 6: 583–635. Takahashi, H. 1987. Distribution of Tricyrtis and its phy- We are grateful to Mr. Yasuhiko Inoue for his efforts in togeographical problems. Acta Phytotax. Geobot. 38: collecting the plant materials. This work was supported 123–132. by Grants-in-Aid for Scientific Research from the Japa-

Received April 18, 2019; accepted April 24, 2019