Development of 16 microsatellite markers for the Korean endemic Vicia hirticalycina (Fabaceae).

PREMISE OF THE STUDY: Vicia hirticalycina (Fabaceae) is a narrowly endemic species restricted to mountain valleys of southern Korea. To investigate its fine-scale genetic diversity and differentiation in Korea, we developed polymorphic microsatellite markers. METHODS AND
RESULTS: Sixteen polymorphic microsatellite markers were developed from Illumina MiSeq data. In 74 individual plants from four populations, one to seven alleles were expressed for each locus. The levels of observed and expected heterozygosity ranged from 0.000 to 0.778 and from 0.000 to 0.738, respectively. Cross-amplification was conducted with three related species and seven to 11 markers were successfully amplified.
CONCLUSIONS: These new microsatellite markers will be useful in future studies on the population genetics of V. hirticalycina.

The genus Vicia L. (Fabaceae, Papilionoideae, Fabeae) includes approximately 160 species distributed across temperate regions of the Northern Hemisphere and South America (Lock and Maxted, 2005). Of these, 15 to 16 species exist naturally in Korea and two to three are deemed endemic (Lee, 1996, 2003; Choi, 2007). Uncertainty in the number of endemic species may have resulted from the taxonomic ambiguity of V. hirticalycina Nakai and V. anguste‐pinnata Nakai, which are endemic to the southern region of the Korean Peninsula. Plants of V. hirticalycina are diploid (Nam et al., 2012) perennial herbs that prefer shady, humid sites in Korean mountain valleys (National Institute of Biological Resources, 2013). Vicia anguste‐pinnata was first separated from V. hirticalycina by Nakai (1914) based mainly on leaflet size, plant height, and number of flower characteristics. However, morphological analysis (Kwon and Choi, 2001) has revealed no serious distinction between the two species. Therefore, V. anguste‐pinnata has been synonymized with V. hirticalycina (Kwon and Choi, 2001). Nevertheless, the taxonomic dispute about these species is ongoing (e.g., Kim et al., 2009).

The molecular analysis by Kwon and Choi (2001) detected moderate geographical differentiations between western and eastern populations in Korea. However, because that investigation included only one individual as the representative from each of 14 locations, the reported results are limited in terms of resolution and applicability to population and genetic dynamics. Furthermore, that research utilized a random‐amplified polymorphic DNA (RAPD) technique. Whereas RAPDs are dominant markers, microsatellite markers are co‐dominant markers with a high level of polymorphism and thus allow for easy estimation of allele frequencies in populations (Ouborg et al., 1999); therefore, microsatellite markers would be especially useful to understand the genetic diversity of V. hirticalycina. To this end, we developed 16 polymorphic microsatellite markers, which will uncover genetic diversity in V. hirticalycina and potentially help resolve the taxonomic dispute concerning populations of this Korean endemic plant.

METHODS AND RESULTS

We sampled four populations (74 individuals) of V. hirticalycina in Korea: Jogye Mountain (n = 30), Ibam Mountain (n = 12), the Byeonsan Peninsula (n = 18), and Gaya Mountain (n = 14) (Appendix 1). Voucher specimens were deposited in the herbaria of Inha University (IUI) and Korea National Arboretum (KH) (Appendix 1). To obtain the microsatellite sequences of V. hirticalycina, we employed Illumina sequencing technology as described by Jin et al. (2016). Briefly, genomic DNA was extracted from the leaves of one individual from Jogye Mountain (voucher no. JW1605003) with a DNeasy Plant Mini Kit (QIAGEN, Seoul, Korea). Using a Covaris S220 Ultrasonicator (Covaris, Woburn, Massachusetts, USA), we fragmented the genomic DNA (300 ng) to 500 bp and then prepared a library with a TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, California, USA). Sequencing was performed on the Illumina MiSeq platform at Life is Art of Science (Gimpo, Korea). In all, 23,484,184 paired reads (300 × 300 bp) were produced. Using the SSR_pipeline v.0951 (Miller et al., 2013), which cleans and aligns Illumina reads, we searched candidate sequences to design primers containing di‐, tri‐, or tetrameric microsatellites with at least 10, six, or five repeats, respectively. Candidates with flanking regions smaller than 100 bp and microsatellite motifs of more than 25 repeats were eliminated. The remaining candidates were rechecked by reference‐mapping of raw reads using Geneious R 7.1.9 (Biomatters Ltd., Auckland, New Zealand). For this procedure, we selected candidates based on the following criteria: (1) few variations at the primer sites and (2) no other single‐nucleotide polymorphisms in the flanking regions.

From the sequences meeting those criteria, primer pairs were designed for 129 loci with Primer3 (Rozen and Skaletsky, 1999). The M13(–21) sequence (5′‐TGTAAAACGACGGCCAGT‐3′) was affixed to the 5′ end of all forward primers intended for fluorescence labeling. To investigate the amplification of 129 loci, we conducted a preliminary PCR analysis with eight individuals from the Jogye Mountain population. After successful amplification, we selected 25 of those loci and tested them in the remaining 66 individuals from four populations using a GeneAmp PCR System 2700 Thermal Cycler (Applied Biosystems, Foster City, California, USA). Each reaction mixture (total volume 20 μL) contained 5 ng of DNA, plus 10 μL of Dr. Taq Master Mix without dye (2×) (MG‐med, Seoul, Korea) that consisted of 0.4 mM dNTPs, 2× PCR buffer with 4 mM MgSO4, and 0.4 U·μL−1 of Taq DNA polymerase. These mixtures also included the 0.08 μM forward M13(–21)‐tagged primer, a 0.3 μM reverse primer, and a 0.3 μM M13(–21) fluorescence label (NED, PET, VIC, 6‐FAM). Amplification conditions consisted of initial denaturation at 94°C for 2 min, followed by 30 cycles at 94°C for 30 s, 55°C for 25 s, and 72°C for 30 s; no final extension was applied. The PCR products were checked by electrophoresis with 2% agarose gels and resolved to genotype on an ABI 3730xl Sequencer with GeneScan 500 LIZ Size Standard (Applied Biosystems). Allele sizes were scored with GeneMapper 3.7 (Applied Biosystems). Using GenAlEx 6.5 (Peakall and Smouse, 2006), we calculated the number of alleles, observed heterozygosity, expected heterozygosity, and deviation from Hardy–Weinberg equilibrium for each population. The occurrence of null allele frequencies was estimated by MICRO‐CHECKER version 2.2 (van Oosterhout et al., 2004), and 18 loci showed no evidence of null alleles in all individuals. In addition, we tested cross‐amplification with other congeners, i.e., tetraploid V. unijuga A. Braun, tetraploid V. venosa (Willd. ex Link) Maxim. subsp. cuspidata (Maxim.) Y. Endo & H. Ohashi, and diploid V. chosenensis Ohwi, in successfully amplified primers. Sequences of the raw reads are available in the Sequence Read Archive database of the National Center for Biotechnology Information (BioProject no. PRJNA431503).

Of the 25 loci, 23 showed successful amplification for all individuals. Among those, seven loci showed genotyping errors. In total, we developed 16 primer pairs, all of which were polymorphic (Table 1). The values calculated for genetic diversity for each population of V. hirticalycina were as follows (Table 2): number of alleles varied from one to seven; levels of observed and expected heterozygosity per locus ranged from 0.000 to 0.778 and from 0.000 to 0.749, respectively; and loci VMS14 (Jogye Mountain), VMS127 (Ibam Mountain), VMS08 (Gaya Mountain), and VMS125, VMS141, and VMS154 (Byeonsan Peninsula) showed significant deviation from Hardy–Weinberg equilibrium. In all, 11, 10, and seven primers for these loci were successfully amplified in each plant representing V. unijuga, V. venosa subsp. cuspidata, and V. chosenensis, respectively (Table 3).

Table 1

Characteristics of 16 microsatellite loci developed for Vicia hirticalycina

Locusa	Primer sequences (5′–3′)	Repeat motif	Allele size range (bp)	Fluorescent label	GenBank accession no.
VMS8	F: AATAACCACCCACCAAGTTT	(CT)10	200–226	VIC	MG841044
	R: GAGATTCACTGCATAGCTCA
VMS14	F: TGTTGTCACTTCAATTTCGG	(TAA)9	291–309	PET	MG841045
	R: CTTTGCAGCCTGGAAGATCT
VMS53	F: CGGGAAAGTTTTCAAGTTGC	(TTC)7	281–305	NED	MG841046
	R: ACCAGAATCCCTCATTTCAC
VMS77	F: AGCAGAGAAATGAACCATAACC	(CCAA)6	215–227	6‐FAM	MG841047
	R: TCACTGAAGCTACCAATAAAGG
VMS94	F: AGATGGTGTCCAAAGCCAC	(CTC)7	197–218	VIC	MG841048
	R: AGATTAGAATGCTCCTTGACTG
VMS125	F: GCTTATACCTGATGCTGGTG	(ATC)6	227–239	6‐FAM	MG841049
	R: TCACATGATCGTTCTTCCAC
VMS127	F: GTCTCGACGCCGTAGATG	(CAG)6	256–262	VIC	MG841050
	R: CACACTGGGAATTGATGAGTG
VMS128	F: AAGGCAATGATCCCTCCGG	(CAC)6	164–173	NED	MG841051
	R: GCGAAAGTTACCGAAGAGG
VMS134	F: CTAGTTGCCTCACCATGC	(CAA)6	282–300	PET	MG841052
	R: GGAATTTGCATCTTCGCC
VMS137	F: CTGACCCAACCTGATGCC	(CCA)6	215–227	6‐FAM	MG841053
	R: GAGGTCCGAGTGGCAAAG
VMS141	F: GAAGCCAGCCTCTATGTGC	(GAT)7	196–208	VIC	MG841054
	R: GATGAAGAACAAGACGTCGGAG
VMS144	F: GCTCAACATCTGTGCTTG	(GTG)6	292–304	NED	MG841055
	R: GGACCATATCATGGCTGAAG
VMS148	F: GGAGTCAATTCTGACATACG	(GAT)6	201–204	6‐FAM	MG841056
	R: CACTACACTCATATTGGACC
VMS154	F: CTCGTGGACATGTAAAGCC	(GTG)7	294–306	NED	MG841057
	R: TCTCAGCCTACTGTTGGTC
VMS156	F: AGCAGAGAAAGGGGTTGC	(TGT)6	286–301	PET	MG841058
	R: GAGTGACAAAATGGCTTCACC
VMS161	F: GGCAGTATTATGAAGGAGTGAG	(GCT)6	247–268	VIC	MG841059
	R: CTATATGGTTTGCCGTTGGC

Annealing temperature was 55°C for all loci.

Table 2

Values for genetic diversity among 74 individuals of Vicia hirticalycina across 16 microsatellite loci.a

Locus	Jogye Mountain (n = 30)			Ibam Mountain (n = 12)			Byeonsan Peninsula (n = 18)			Gaya Mountain (n = 14)
	A	H o	H e	A	H o	H e	A	H o	H e	A	H o	H e
VMS8	7	0.500	0.609	3	0.667	0.538	6	0.667	0.677	5	0.666	0.690*
VMS14	6	0.567	0.687*	6	0.750	0.736	4	0.500	0.659	5	0.643	0.667
VMS53	6	0.700	0.656	6	0.667	0.684	5	0.778	0.688	6	0.712	0.738
VMS77	3	0.500	0.655	3	0.500	0.601	2	0.278	0.424	3	0.605	0.627
VMS94	7	0.500	0.628	5	0.667	0.663	4	0.333	0.295	6	0.722	0.749
VMS125	4	0.200	0.186	1	0.000	0.000	2	0.000	0.105*	1	0.000	0.000
VMS127	4	0.533	0.502	3	0.167	0.292*	3	0.333	0.290	3	0.518	0.537
VMS128	2	0.033	0.033	2	0.083	0.080	2	0.222	0.198	1	0.000	0.000
VMS134	4	0.500	0.517	5	0.417	0.628	4	0.722	0.616	3	0.472	0.489
VMS137	4	0.100	0.097	3	0.250	0.288	1	0.000	0.000	2	0.069	0.071
VMS141	4	0.300	0.266	4	0.250	0.229	5	0.500	0.588*	5	0.367	0.381
VMS144	4	0.400	0.446	3	0.250	0.344	2	0.111	0.105	2	0.436	0.452
VMS148	1	0.000	0.000	2	0.083	0.080	1	0.000	0.000	1	0.000	0.000
VMS154	3	0.400	0.376	3	0.167	0.156	3	0.056	0.156*	2	0.069	0.071
VMS156	4	0.433	0.531	4	0.667	0.632	4	0.778	0.647	5	0.704	0.730
VMS161	6	0.633	0.612	4	0.417	0.413	3	0.556	0.579	7	0.638	0.661

A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals.

Voucher and locality information are provided in Appendix 1

Significant departure from Hardy–Weinberg equilibrium at P < 0.05.

Table 3

Cross‐amplification of 16 microsatellite loci developed for Vicia hirticalycina in three related species.a

Locus	Vicia unijuga (n = 6)			Vicia venosa subsp. cuspidata (n = 6)			Vicia chosenensis (n = 7)
	Amplification	A	Allele size (bp)	Amplification	A	Allele size (bp)	Amplification	A	Allele size (bp)
VMS8	++	5	200–222	++	5	203–215	++	5	207–219
VMS14	+	5	282–300	+	4	285–297	—	NA	NA
VMS53	+	6	281–299	+	6	281–296	+	1	284
VMS77	+	3	222–230	++	3	214–226	+	2	222–226
VMS94	—	NA	NA	—	NA	NA	—	NA	NA
VMS125	++	4	230–245	++	2	233–236	+	2	241–250
VMS127	++	3	247–259	++	4	253–265	++	1	258
VMS128	++	2	164–173	++	1	173	++	1	173
VMS134	++	2	291–294	+	2	294–297	++	1	294
VMS137	++	2	222–228	+	2	222–225	+	1	222
VMS141	++	4	196–205	++	5	193–205	++	2	196–202
VMS144	++	3	295–304	++	2	292–298	++	2	286–298
VMS148	+	1	198	++	2	195–198	—	NA	NA
VMS154	++	2	297–303	+	2	294–297	+	1	297
VMS156	++	4	289–298	++	5	289–304	+	4	290–308
VMS161	++	4	250–259	++	3	247–256	++	2	253–256

++ = successful amplification; + = successful amplification for more than one individual but not for all; — = failed amplification; NA = not applicable.

Voucher and locality information are provided in Appendix 1.

CONCLUSIONS

We have developed 16 polymorphic microsatellite markers in V. hirticalycina. Cross‐amplification was also tested for the related species V. unijuga, V. venosa subsp. cuspidata, and V. chosenensis, and allele sizes were determined. These new markers will be useful as tools to reveal the population genetics of V. hirticalycina and congeners.

Species	Voucher specimen accession no.	Collection locality	Geographic coordinates	n
Vicia hirticalycina Nakai	JW1605003a	Jogye Mountain, Suncheon‐si, Jeollanam‐do	35.002483°N, 127.313599°E	30
V. hirticalycina	JW1605101b	Ibam Mountain, Jangseong‐gun, Jeollanam‐do	35.484356°N, 126.827151°E	12
V. hirticalycina	JW1605201b	Byeonsan Peninsula, Buan‐gun, Jeollabuk‐do	35.626333°N, 126.569711°E	18
V. hirticalycina	JW1709101b	Gaya Mountain, Hapcheon‐gun, Gyeongsangnam‐do	35.821682°N, 128.098094°E	14
V. unijuga A. Braun	DP1605004b	Okseok Mountain, Bonghwa‐gun, Gyeongsangbuk‐do	37.021419°N, 128.782077°E	1
V. unijuga	DP1609057b	Hyangsan‐ri, Danyang‐gun, Chungcheongbuk‐do	37.059290°N, 128.451105°E	1
V. unijuga	DP1709151b	Palgong Mountain, Gunwi‐gun, Gyeongsangbuk‐do	36.016582°N, 128.695000°E	1
V. unijuga	DP1709013b	Samseong Mountain, Chungdo‐gun, Gyeongsangbuk‐do	35.715614°N, 128.685199°E	1
V. unijuga	JW1709324b	Ibam Mountain, Jangseong‐gun, Jeollanam‐do	35.484356°N, 126.827151°E	1
V. unijuga	JW1709223b	Biseul Mountain, Daegu	35.715163°N, 128.523369°E	1
V. venosa (Willd. ex Link) Maxim. subsp. cuspidata (Maxim.) Y. Endo & H. Ohashi	DP1606223b	Chiak Mountain, Wonju‐si, Gangwon‐do	37.371277°N, 128.050546°E	1
V. venosa subsp. cuspidata	JW1709006b	Jeoksang Mountain, Muju‐gun, Jeollabuk‐do	35.947298°N, 127.689751°E	5
V. chosenensis Ohwi	JH1307001b	Bawi Mountain, Chuncheon‐si, Gangwon‐do	36.175598°N, 127.802492°E	3
V. chosenensis	DP1605001b	Okseok Mountain, Bonghwa‐gun, Gyeongsangbuk‐do	37.021419°N, 128.782077°E	4

DP = collector Dong‐Pil Jin; JH = collector Jung‐Hyun Lee; JW = collector Jong‐Won Park; n = number of individuals sampled.

Voucher deposited in Korea National Arboretum (KH), Pocheon, Gyeonggi‐do, Korea.

Voucher deposited in Herbarium of Inha University (IUI), Incheon, Korea.