Development of chloroplast microsatellite markers for the endangered Maianthemum bicolor (Asparagaceae s.l.).

PREMISE OF THE STUDY: Ten polymorphic chloroplast microsatellite (cpSSR) markers were developed and characterized in an endemic and endangered herb, Maianthemum bicolor (Asparagaceae s.l.), for use in conservation genetics. METHODS AND
RESULTS: Primer sets flanking each of the 10 cpSSR loci in noncoding regions of the chloroplast genome of M. bicolor were designed. These cpSSR markers were tested on a total of 33 adult individuals from three natural populations in South Korea. The number of alleles per locus ranged from two to three. The unbiased haplotype diversity per locus ranged from 0.061 to 0.682. All markers were successfully transferred to the congeneric species M. japonicum, M. bifolium, and M. dilatatum with polymorphisms among the species.
CONCLUSIONS: The developed cpSSR markers will be useful in assessing the genetic diversity and population structure of M. bicolor and will help to infer its molecular identification, thereby providing a basis for conservation.

Maianthemum bicolor (Nakai) Cubey (Asparagaceae s.l.) is an endemic perennial herb of South Korea (Oh, 2007). This species is characterized by dioecious flowers and tepal color that is green at the early stage of the flowering period and changes to dark purple as it grows (Yang, 2007). Therefore, it is visually possible to distinguish the species after the flower and fruit appear. Natural populations of M. bicolor are facing extinction due to habitat deterioration, low germination rates, and overexploitation by humans due to the species’ ornamental value (Chang et al., 2005; Lee et al., 2007). In addition, all local populations of M. bicolor growing on the ridges of mountains at altitudes higher than 1300 m above sea level are highly fragmented, and those habitats are isolated with discontinuous distribution (Lee et al., 2007; Oh, 2007). Both in situ and ex situ conservation programs are being undertaken to secure the wild populations (Lee et al., 2007), but neither the extent of genetic diversity nor the population structure of M. bicolor has been clearly analyzed. Thus, there is an urgent need for development of useful molecular markers for conservation genetics.

Although nuclear markers are excellent for use in most population genetic analyses (Ding et al., 2008; Kim et al., 2008; Tang et al., 2008; Guichoux et al., 2011), they are not suitable for phylogenetic and phylogeographic studies above species level due to high substitution rates (Powell et al., 1996). Chloroplast simple sequence repeat (cpSSR) markers are mainly distributed throughout noncoding regions, which show higher sequence variation than coding regions (Powell et al., 1995). Moreover, the cpSSR markers developed from a species are frequently applicable to amplify homologous regions across related taxa (Diekmann et al., 2012). Therefore, cpSSR markers can be applied for conservation genetics of endangered plant species and can be used to determine molecular identification and genetic relationships among closely related species (Clark et al., 2000; Huang et al., 2015) as complementary tools of nuclear markers.

Here, we provide 10 cpSSR markers for M. bicolor to facilitate conservation and molecular identification. These markers were developed from the complete chloroplast genome sequences of M. bicolor and tested on three natural populations. We also examined the transferability of the markers to three congeneric species, M. japonicum (A. Gray) LaFrankie, M. bifolium (L.) F. W. Schmidt, and M. dilatatum (Alph. Wood) A. Nelson & J. F. Macbr.; the former is closely related to M. bicolor and the latter two are more distantly related within the genus (Kim and Kim, unpublished).

METHODS AND RESULTS

To determine the complete chloroplast genome sequences of M. bicolor, we sampled fresh leaf material from Mt. Deogyu, South Korea. Genomic DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). The chloroplast genome of M. bicolor was sequenced using a genome skimming approach on an Illumina MiSeq sequencer (Illumina, San Diego, California, USA) and assembled with Geneious version 7.13 (Biomatters, Auckland, New Zealand). The chloroplast genome of M. bicolor is 157,176 bp in length. It displays a typical quadripartite structure, with the large single-copy region (85,698 bp, 54.5% of the total genome) separated from the small single-copy region (18,394 bp, 11.7%) by two inverted repeat regions (26,542 bp, 16.9% each). The GC content is 37.6% (Park et al., unpublished).

The cpSSR regions of M. bicolor were searched through the complete chloroplast genome sequence using a tandem repeat search tool in Geneious with the parameters set to ≥7 mononucleotide repeats, ≥4 di- and trinucleotide repeats, and ≥3 tetra-, penta-, and hexanucleotide repeats. A total of 169 repeat motifs were identified in the chloroplast genome, among which the most frequent types were mononucleotide (124 [73.4%]) and dinucleotide (32 [18.9%]), while tri- (3 [1.8%]), tetra- (8 [4.7%]), and pentanucleotides (2 [1.2%]) were rare. Forty-one loci were randomly selected for screening. We designed 41 primer pairs using Primer3 (Rozen and Skaletsky, 1999) under the following criteria: (1) guanine-cytosine content 30–70%, (2) annealing temperature (Ta) 55–65°C, (3) primer size 18–25 bp in length, and (4) amplicon size 150–500 bp in length. To check the amplification success and variability, two individuals from each of three populations of M. bicolor (n = 6; Mt. Deogyu, Mt. Seorak, and Mt. Daeam; Appendix 1) were selected (Appendix 2).

PCR amplification was carried out in a total volume of 25 μL, containing 2.5 μL of 10× PCR buffer (10 mM Tris-HCl [pH 8.3], 1.5 mM MgCl2, and 50 mM KCl), 0.25 mM of each dNTP, 5 μM of each 6-FAM fluorescently labeled forward and unlabeled reverse primer, 1 unit of Taq DNA polymerase, and 100 ng of DNA template. Amplification of genomic DNA was performed on a Veriti thermal cycler (Applied Biosystems, Carlsbad, California, USA). Fragment length polymorphism was detected on an ABI 3730 DNA Analyzer with GeneScan 500 ROX Size Standard (Applied Biosystems). Peak data were analyzed using Peak Scanner version 1.0 (Applied Biosystems). The authenticity of the loci amplified in M. bicolor populations was confirmed by sequencing the representative PCR products on an ABI 3730 automated sequencer (Applied Biosystems) using the amplification primers. Consensus DNA sequences were assembled for each individual using DNA Baser version 3 (Heracle BioSoft SRL, Pitești, Romania). Of the 41 primer pairs screened, 10 primer sets showed length variation in cpSSR regions and were developed further (Table 1); 31 were monomorphic and not developed any further (Appendix 2).

Table 1.

Characteristics of 10 polymorphic chloroplast microsatellite markers developed for Maianthemum bicolor.

Locus	Primer sequences (5′–3′)	Repeat motif	Ta (°C)	Position	Allele size rangea (bp)	GenBank accession no.
MA2	F: GAATTGGGAGAGATGGCTGA	(AT)3(A)12	58	psbI–trnS_GCU	326–327	KU845594
	R: TGCTTACTCTCAAACTCTTCGTTT
MA15	F: TCGATTTATTGAGGCATACTATGAT	(T)10	58	ndhF–rpl32	265–266	KU845595
	R: TTCCAATTCAAATGATATGACCA
MA30	F: TTGTTTCGTTCCATTCGATCT	(T)10	59	rpoC1 intron	228–229	KU845596
	R: ATGGTCGATCCCGTAACAGA
MA32	F: TCACGTTGTCCCAAGTGAAA	(A)11	60	rpoB–trnC-GCA	325–326	KU845597
	R: ATGATCCGATGGGGTACAAA
MA33	F: GGGTTCGAATCCCTCTCTCT	(A)8	59	psbC–trnS-UGA	189–190	KU845598
	R: TCCATGACTCCCCTTAGCTG
MA421	F: GGACCCATTCAGGAACAAGA	(T)11	60	clpP intron	273–274	KU845599
	R: TTTGGATGCATACGGTTCAA
MA422	F: GAGATAGGATTGGAATCGACACA	(T)7	59	clpP intron	200–201	KU845600
	R: CTGAGATATACCATGAGACCAACAA
MA481	F: GCTTTTATTGCAGCTAAATATCCTTT	(T)7	59	ndhF–rpl32	396–397	KU845601
	R: CACGGCAATATGAGCTTATTCA
MA482	F: AACAAGACATTCTATTACCTAAGAA	(T)9	57	ndhF–rpl32	148–150	KU845602
	R: TACCTAACCCATTGCGGAAC
MA483	F: TTGAGTCGATTTATTGAGGCATAC	(T)2(T/G)(T)7	59	ndhF–rpl32	270–271	KU845603
	R: TTCCAATTCAAATGATATGACCA

Note: Ta = annealing temperature.

Allele sizes are based on 33 individuals of three populations of M. bicolor; see Appendix 1 for population information.

Thirty-three adult individuals from three natural populations of M. bicolor were used to characterize 10 polymorphic cpSSR loci (Table 2). All polymorphic loci are located in the noncoding regions in which the mutations are usually neutral (Table 1). Polymorphism parameters were calculated with POPGENE version 1.31 (Yeh et al., 1997). The allelic variation (A) in 10 cpSSR loci ranged from two to three, while the unbiased haplotype diversity (H) ranged from 0.061 to 0.682, with an average value of 0.409 (Table 2). Ten cpSSR markers amplified a total 21 alleles. Of these, 10 alleles specifically occurred in one or more individuals of each population; four were specific for the Mt. Deogyu population, three for the Mt. Seorak population, and three for the Mt. Daeam population (Table 2). All polymorphic primers developed for M. bicolor were able to successfully amplify cpSSR regions in three congeneric species (M. japonicum, M. bifolium, and M. dilatatum) sampled from two populations in South Korea and showed polymorphisms among the species (Appendix 3).

Table 2.

Number of alleles, haplotype diversity, and allele distribution of 10 polymorphic chloroplast microsatellite markers developed for Maianthemum bicolor.

			Allele distributiona
			Mt. Deogyu (n = 10)		Mt. Seorak (n = 13)		Mt. Daeam (n = 10)
Locus	A	H	Repeat motif	n	Repeat motif	n	Repeat motif	n
MA2	2	0.492	(AT)3(A)12	10	(AT)3(A)11*	13	(TA)3(A)12	10
MA15	2	0.436	(T)10	10	(T)10	13	(T)9*	10
MA30	2	0.511	(T)9	2	(T)9	13	(T)9	10
			(T)10*	8
MA32	2	0.458	(A)11	10	(A)10	12	(A)10	10
					(A)11	1
MA33	2	0.436	(A)8*	10	(A)9	13	(A)9	10
MA421	2	0.515	(T)11	8	(T)12	13	(T)11	9
			(T)12	2			(T)12	1
MA422	2	0.061	(T)7	9	(T)7	13	(T)7	10
			(T)8*	1
MA481	2	0.061	(T)7	10	(T)7	12	(T)7	10
					(T)8*	1
MA482	3	0.682	(T)9*	10	(T)7*	13	(T)8*	10
MA483	2	0.436	(T)2(T/G)(T)7	10	(T)10	13	T(T/G)(T)7*	10
Mean	2.1	0.409	—		—		—
Total	21	0.775	—		—		—

Note: A = number of alleles; H = unbiased haplotype diversity; n = number of individuals.

Asterisk denotes specific alleles occurred in one or more individuals of each population.

CONCLUSIONS

The cpSSR markers developed here are useful for formulating an in situ conservation strategy of the endangered M. bicolor populations by estimating the level of genetic diversity and population structure. The cpSSR markers will also be useful to determine the minimum size of the ex situ core collection that captures the genetic diversity of wild populations of M. bicolor. All of the cpSSR markers designed here for amplification of M. bicolor are applicable to other congeneric species and are a means for species verification in combination with morphological identification.

Appendix 1.

Collection data for three populations of Maianthemum bicolor and three congeneric species.

Species	Locationa	N	Voucher specimensb
M. bicolor (Nakai) Cubey	South Korea: Mt. Deogyu, Seolcheon-myeon, Muju-gun, Jeonbuk	10	C.Kim 2015-2
	South Korea: Mt. Seorak, Buk-myeon, Inje-gun, Gangwon	13	C.Kim 2015-42
	South Korea: Mt. Daeam, Buk-myeon, Inje-gun, Gangwon	10	C.Kim 2015-101
M. bifolium (L.) F. W. Schmidt	South Korea: Mt. Deogyu, Seolcheon-myeon, Muju-gun, Jeonbuk	2	C.Kim 2015-69
M. dilatatum (Alph. Wood) A. Nelson & J. F. Macbr.	South Korea: Mt. Seorak, Buk-myeon, Inje-gun, Gangwon	3	C.Kim 2015-28
M. japonicum (A. Gray) LaFrankie	South Korea: Mt. Deogyu, Seolcheon-myeon, Muju-gun, Jeonbuk	1	C.Kim 2015-3
	South Korea: Mt. Seorak, Buk-myeon, Inje-gun, Gangwon	2	C.Kim 2015-35

Note: N = number of samples.

GPS coordinates are not included because this is an endangered species in South Korea.

All voucher specimens are deposited at Gachon University herbarium (GCU), Seongnam, South Korea.

Appendix 2.

Characteristics of 31 monomorphic chloroplast microsatellite primers designed in Maianthemum bicolor.

Locus	Primer sequences (5′–3′)	Repeat motif	Ta (°C)	Position	Allele size (bp)a
MA1	F: CCCCGGAAACAGAAAGTCCA	(A)11	61	matK-trnK_UUU	403
	R: TCGATCCTTACCAGATCATTCCA
MA8	F: GAGAGGGATTCGAACCCTCG	(AAT)3	63	trnS_GCU-trnG_UCC	367
	R: CCAGTACTTAACCAGGCCGG
MA10	F: TGGCAGTTGAAGGGGAAGTT	(AT)8	58	trnS_GCU-trnG_UCC	440
	R: ACGAATCGCACTTTTACCACT
MA13	F: TCGGTGGAAACCAAATTCTT	(AT)4	55	trnG-UCC-trnR-UCU	452
	R: TCTAATGGATAGGACAGAGGT
MA14	F: TCGGTGGAAACCAAATTCTT	(A)9	55	trnG-UCC-trnR-UCU	452
	R: TCTAATGGATAGGACAGAGGT
MA19	F: TCATAACGTAAACCACCCGCA	(AT)5	59	atpH-atpI	344
	R: TCCTATACCTGTTATGTTTCTTGGA
MA21	F: AACCTTTTCCCGCATCAGGC	(A)10, (AAT)3	61	matK-trnK_UUU	393
	R: CGAGTACTCTACCGTTGAG
MA29	F: GACTACTAGAAAAGTTTTGATGCTT	(AAT)5	60	petN-psbM	416
	R: CATCTATGTAAAAACAGTCAGCCA
MA31	F: TCCTAGTTCGGGACTGACGG	(A)9	63	trnD_GUC-trnY_GUA	411
	R: CGGTTAATGGGGACGGACTG
MA34	F: AGGATTCTTCCTATTTGTGG	(AG)4	55	trnS-UGA	296
	R: TTTTCAAGACCGGAGCTATC
MA46	F: TTGCTGACCAACAGTATCTC	(A)10	56	atpB-rbcL	385
	R: ACGCACTTCACTTACTCACAA
MA50	F: GGTGGTTCAATTCGATGTTG	(A)8	56	accD	430
	R: GTTATCTTCGTTTTGAAGTCAGT
MA51	F: GAGAAACCCCCGGATCCTTT	(AAT)3	61	psbE-petL	405
	R: TTTCAGATACGACGGTAAGAAAAA
MA53	F: GTCAATAAGGAATCGCCAGGC	(AT)7	61	rpl33-rps18	377
	R: TCAATTCGATCCCCCGATTCA
MA56	F: GTTCGACGTCTCCGAGCTAT	(AAAT)3	61	rpl20-rps12	449
	R: ACGGACTAACAAGGTCAGCT
MA57	F: AGTCGCACATACACCCTAGT	(AT)4	58	rps12-clpP	407
	R: TAGTTCTTCCGCTTCCAGGA
MA61	F: AACGAGTCACACACTAAGCA	(A)9	56	rpl16-rps3	286
	R: TCCATTCGACACGTGCAATTT
MA64	F: TGGTCTACAGGGTTCATAAC	(A)8	56	rpl2intron	430
	R: ATCCAAATCTGCCGAATCAC
MA69	F: TCAACCATTTCATTTTACCA	(A)8	55	ycf1-ndhF	200
	R: CACGATTATATGACCAATTGTAT
MA70	F: ATCATTATTGACGGTCCAAGACC	(A)9	61	ndhF-rpl32	400
	R: CACTTTTATCTAGCTATTTGGTCA
MA73	F: AGTTCCAATTCAAATGATATGACCA	(AAGT)3	59	ndhF-rpl32	310
	R: TCTGATACTTTCTTATTGGATTGGA
MA76	F: CTTCTTAGGTAATAGAATGTCTTGT	(AT)4	62	ndhF-rpl32	435
	R: TGACTTCGCTAAAGAGAAAGCT
MA77	F: TTTGTGCGACAAACAAAAACAAA	(A)10	56	rpl32-trnL_UAG	332
	R: CAACTTGGTCTTGAGTTAGGCG
MA86	F: GTTGACGTTTTCCCAGAATT	(AAT)4	55	rps15-ycf1	456
	R: CAAATCGTTTCTTTGGCCCA
MA88	F: TTCAATAGCTTTGCGTCCGC	(AAACG)3	58	trnR_ACG-rrn5	381
	R: AAAATAGCTCGACGCCAGAA
MA89	F: AGGCAAAGGGTCGAGAAACT	(A)8	58	trnV_GAC-rps12	389
	R: TCGAAAAATGAAATGGCGTGG
MA90	F: ACTGGTAGGAGAAGAACCCGA	(A)9	61	trnV_GAC-rps12	372
	R: TGGACCCTCATGTTGATCCG
MA91	F: CGCAACTCAATCATGTATGATGGA	(AAG)3	62	ycf2	394
	R: ATGGTTCCTTACTTCGACAGGGT
MA92	F: CTCAACATGAGGGAGGAGAAAGA	(A)8	63	rps19-psbA	389
	R: ATCAACCGTGCTAACCTTGG
MA115	F: AGAGCAATGCCTATTGTACC	(A)10	56	atpA-atpF	358
	R: TAAGGCTTGTTGGAAAACTC
MA132	F: CCTCCTTGAAAGAGAGATGT	(C)8	56	trnE_UUC-trnT_GGU	354
	R: TGGCTTAGTGCGAGATAGTGA

Note: Ta = annealing temperature.

Allele sizes are based on six individuals from three M. bicolor populations; see Appendix 1 for population information.

Appendix 3.

Comparison of allele distributions of 10 polymorphic cpSSR between Maianthemum bicolor and its congeneric species M. bifolium, M. dilatatum, and M. japonicum.

Locus	M. bicolora (n = 33)	M. bifolium (n = 2)	M. dilatatum (n = 3)	M. japonicum (n = 3)
MA2	(AT)3(A)12, (AT)3(A)11*	(AT)7(A)4	(AT)7(A)4	(AT)3(A)8
MA15	(T)9, (T)10	(T)15, (T)16	(T)14	(T)9, (T)10
MA30	(T)9*, (T)10	(T)11	(T)10	(T)11
MA32	(A)10, (A)11*	(A)9	(A)10	(A)9, (A)10
MA33	(A)8*, (A)9	(A)11	(A)9	(A)10
MA421	(T)11, (T)12*	(T)10, (T)11	(T)10	(T)10
MA422	(T)7, (T)8*	(T)7	(T)7	(T)7
MA481	(T)7, (T)8*	(T)6, (T)7	(T)7	(T)7
MA482	(T)7, (T)8*, (T)9	(T)9	(T)9	(T)7
MA483	(T)2(T/G)(T)7, T(T/G)(T)7	(T)15	(T)14	(T)9, (T)10

Note: n = number of individuals.

Asterisk denotes specific alleles for M. bicolor.