A new set of microsatellite primers for Coelogyne fimbriata (Orchidaceae) and cross-amplification in C. ovalis.

PREMISE OF THE STUDY: Declining orchid populations have made it necessary to prioritize the study of population structure and genetic diversity for species including Coelogyne fimbriata (Orchidaceae). METHODS AND
RESULTS: A biotin-streptavidin capture method was used to construct a microsatellite library for C. fimbriata. A total of 15 polymorphic nuclear microsatellite loci were isolated and characterized using 47 C. fimbriata individuals from two natural populations in China. The number of alleles per locus for the two populations ranged from two to 17. The observed and expected heterozygosities ranged from 0.000 to 1.000 and from 0.000 to 0.867, respectively. Among these polymorphic primers, 11 loci were also successfully amplified in C. ovalis, and 10 loci showed moderate to high-level polymorphism. Cross-amplification of the 15 polymorphic loci was tested in five related species: C. cumingii, C. eberhardtii, C. mayeriana, C. peltastes, and C. velutina.
CONCLUSIONS: Fifteen microsatellites in C. fimbriata and 10 in C. ovalis have moderate to high-level genetic variation, indicating their utility in population genetic studies, thus contributing to orchid conservation.

Coelogyne fimbriata Lindl. (Orchidaceae), a medicinal orchid, is mainly distributed in southern China, Cambodia, northeastern Indonesia, Laos, Malaysia, Thailand, and Vietnam (Clayton and Beaman, 2002). Because southern China is the northernmost edge of its distribution region, Chinese C. fimbriata populations are of particular concern because populations on distribution margins are most vulnerable to disturbance (Channell and Lomolino, 2000). Furthermore, in consideration of global climate change and habitat fragmentation, it is urgent to design effective conservation strategies for endangered natural orchid populations (Swarts and Dixon, 2009). Coelogyne fimbriata is an epiphytic or lithophytic orchid, which requires a dormancy period in winter. This species grows on its substrate with creeping and slender rhizomes. It can reproduce both sexually via seed and vegetatively by rhizomatic growth. Usually blooming in late summer, it produces one or two flowers on a scape. The flowers exhibit a type of pollinator deception in which the flower odor mimics food for foraging female wasps (Cheng et al., 2009).

Many studies have focused on the pollination syndromes of orchids (Tang et al., 2014); however, there is a lack of genetic information documented for this species. Because genetic information is important for the conservation and sustainable utilization of orchids (Gijbels et al., 2015), we developed microsatellite markers to allow studies of the genetic diversity, genetic structure, and mating system of C. fimbriata. In total, 15 polymorphic microsatellite loci were isolated and characterized to study genetic variation within this species clade. These highly polymorphic loci displayed high genetic variation and extensive usability in congeneric species, and may serve as a universal tool for orchid genetic studies.

METHODS AND RESULTS

A biotin-streptavidin capture method was employed to construct a microsatellite-enriched DNA library (Jiang et al., 2011). First, we extracted genomic DNA from silica gel–dried leaves of one C. fimbriata individual using a Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China). The enzyme MseI (New England Biolabs, Beverly, Massachusetts, USA) was used to digest approximately 300 ng of genomic DNA in a 25-μL reaction volume for 2 h at 37°C. Fragments 200–1000 bp in length were then ligated to an MseI-adapter pair (F: 5′-TACTCAGGACTCAT-3′ and R: 5′-GACGATGAGTCCTGAG-3′). The ligation-digestion mixture was diluted with ultrapure water (1:4), and the diluted fragments were amplified using MseI-N primer (5′-GATGAGTCCTGAGTAAN-3′) in a 25-μL PCR reaction volume at 95°C for 5 min, followed by 23 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min. Next, to obtain microsatellite-enriched DNA fragments, the PCR products were hybridized with 5′-biotinylated (AC)15 probes. We used streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA) to capture single-stranded DNA fragments containing microsatellites. The enriched products were amplified using MseI-N primers for 28 cycles. After the PCR products were purified using a multifunctional DNA Extraction Kit (Bioteke Corporation, Beijing, China), they were ligated into Escherichia coli strain DH5α with the pMD19-T vector (TaKaRa Biotechnology Co., Dalian, Liaoning, China).

We randomly selected and sequenced 249 positive clones using M13+/M13− primers on an ABI 3730 DNA Sequence Analyzer (Applied Biosystems, Foster City, California, USA). Of the 249 sequenced clones, 136 contained microsatellites. Twenty-four sequences were discarded because of short flanking regions for primer design. Finally, we designed 112 primer pairs using Premier 5.0 (PREMIER Biosoft International, Palo Alto, California, USA). We selected 28 individuals from Dawei Mountain, Yunnan Province, and 19 from Diaoluo Mountain, Hainan Province, China (Appendix 1), for PCR using these 112 primers. Of the 112 primers, 47 produced an expected band on 1% agarose gel, 40 failed to obtain amplification products, and 25 others produced multiple bands that were difficult to discriminate. To test for polymorphism, we used the M13(–21)-tailed primer method to fluorescently label alleles and PCR products, which were electrophoretically resolved using an ABI 3730 DNA Sequence Analyzer (Applied Biosystems) with an internal lane standard (GeneScan 500[−250] LIZ) (Schuelke, 2000). Microsatellite loci were amplified under the following conditions: 5 min of denaturation at 94°C; 30 cycles of 30 s at 94°C, 30 s at 53–65°C, and 30 s at 72°C; eight cycles of 30 s at 94°C, 30 s at 53°C, and 30 s at 72°C; and a final 10-min extension at 72°C. Allele binning and calling were conducted using GeneMapper 4.0 (Applied Biosystems), revealing 15 polymorphic loci.

Characteristics of the 15 polymorphic microsatellite loci developed for C. fimbriata are shown in Table 1. An additional 32 monomorphic loci are described in Appendix 2. Each polymorphic locus had two to 17 alleles, with a mean of 5.1. At the population level, the observed and expected heterozygosities were calculated with GenAlEx 6.5 (Peakall and Smouse, 2012) and ranged from 0.000 to 1.000 and from 0.000 to 0.867, respectively (Table 2). FSTAT 2.9.2.3 (Goudet, 1995) was used to analyze linkage disequilibrium and Hardy–Weinberg equilibrium (HWE). No significant linkage disequilibrium at any locus was detected for either population. However, significant deviation from HWE was found for most loci in the two populations (Table 2), which may be indicative of strong clonality. Signs of null alleles were detected in the loci CF1-11, CF1-51, and CF2-126 with MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004).

Table 1.

Characteristics of 15 polymorphic microsatellite markers developed for Coelogyne fimbriata.

Locus	Primer sequences (5′–3′)a	Ta (°C)	Repeat motif	A	Allele size range (bp)	GenBank accession no.
CF1-11	F: <6-FAM>CAACATCCTCTCGGCATAT	60	(GT)9	7	429–443	KP676048
	R: GACCACACTACACCTACAC
CF1-26	F: <6-FAM>ATAACTCACGCCCGATTC	60	(CT)5	3	210–218	KP676049
	R: CCTGTTGTTGCCTGCTGT
CF1-30	F: <6-FAM>CACCTCTCCTCAATTACATCA	58	(ATC)6	2	103–109	KP676050
	R: AGTTGGCGTAAGGCTAATG
CF1-51	F: <HEX>TGAGAATGTCCGTAGGTT	58	(AG)14	7	336–362	KP676052
	R: GGGATTGGAGTAAAGGGT
CF1-60	F: <ROX>AAACCTTCGTTCGCTCCT	60	(TC)5…(CT)5…(TC)5	2	344–346	KP676053
	R: GTGCCTGCTAGGGTTCCA
CF1-120	F: <6-FAM>GGAATCACTCTCAACTTCAC	60	(GT)6	5	362–372	KP676054
	R: ATCATAGGATGGACTCTGTAG
CF1-167	F: <ROX>CAAGCAAGCACTGAGCAA	58	(AG)6	8	259–293	KP676055
	R: GAGACCATCACCGCATTC
CF1-229	F: <6-FAM>AGGCTTACTCGCATACTCT	52	(CT)7…(TC)7	2	181–187	KP676056
	R: ATCTCGCTTCTGGCTTCA
CF1-231	F: <HEX>GGTGCTATGTATGTGAA	52	(AG)33…(GA)18	2	285–289	KP676057
	R: CAGACCATCAAGAAGCATA
CF2-26	F: <6-FAM>CTCCCATACCCACCATTT	55	(AG)16…(AG)6	4	153–171	KX237659
	R: ATAGCCTACCTCAAGACG
CF2-29	F: <ROX>TTGTAGTCTTCATCCTTT	52	(TG)5	5	270–308	KX237660
	R: TCTAGTCTACCCATACTT
CF2-57	F: <HEX>TGACTTAGGACCAGGAA	55	(CT)27	17	173–213	KX237661
	R: GTCGCAAGCACAGATA
CF2-126	F: <6-FAM>CTCCCGTGCCTATGTTTC	52	(CT)20	4	253–259	KX237663
	R: ATTCCGCTCTGATTTCCA
CF2-147	F: <HEX>GGAGGTCTTTGATTAGAT	52	(CT)6CA(CT)5	4	254–260	KX237664
	R: ATGGAGGATTATCAGTAT
CF2-172	F: <ROX>CTTGTATTTCCCTTTCTTG	52	(CT)9(CA)6	4	257–271	KX237666
	R: TGAGATAACTAAACCCAGA

Note: A = number of alleles; Ta = annealing temperature.

Fluorescent dyes (i.e., HEX, ROX, and 6-FAM) are presented with the forward primers.

Table 2.

Characteristics of 15 polymorphic microsatellite loci in Coelogyne fimbriata and C. ovalis populations, respectively.

	Coelogyne fimbriata						Coelogyne ovalis
	DWS population (n = 28)			DLS population (n = 19)			MHX population (n = 21)			JGX population (n = 16)
Locus	A	Ho	He	A	Ho	He	A	Ho	He	A	Ho	He
CF1-11	7	0.259*	0.740	4	0.500	0.475	—	—	—	—	—	—
CF1-26	1	0.000	0.000	3	0.053*	0.101	9	0.333*	0.859	5	0.188*	0.756
CF1-30	2	1.000*	0.500	2	0.158	0.229	1	0.000	0.000	2	0.000*	0.469
CF1-51	7	0.286*	0.665	4	0.250	0.736	—	—	—	—	—	—
CF1-60	2	0.643	0.436	2	0.688	0.451	—	—	—	—	—	—
CF1-120	4	0.889*	0.658	3	0.316	0.277	4	0.095	0.255	5	0.500*	0.585
CF1-167	7	0.964*	0.766	5	0.579*	0.672	1	0.000	0.000	4	0.000	0.516
CF1-229	2	0.179	0.316	2	0.063	0.061	—	—	—	—	—	—
CF1-231	2	0.680	0.449	1	0.000	0.000	1	0.000	0.000	5	0.200*	0.391
CF2-26	2	0.750	0.469	3	0.368	0.597	6	0.667*	0.604	10	0.625*	0.813
CF2-29	3	0.769	0.500	4	0.333	0.474	4	0.900	0.546	3	0.929	0.554
CF2-57	5	0.926*	0.598	14	0.789*	0.867	4	0.952*	0.585	6	0.875*	0.809
CF2-126	2	0.111*	0.500	4	0.474*	0.669	2	0.000	0.472*	3	0.333*	0.611
CF2-147	1	0.000	0.000	3	0.842	0.554	1	0.000	0.000	1	0.000	0.000
CF2-172	2	0.036	0.035	3	0.421	0.639	10	0.529	0.877	7	0.063*	0.510

Note: — = failed to amplify; A = number of alleles; He = expected heterozygosity based on Hardy–Weinberg equilibrium; Ho = observed heterozygosity; n = number of individuals genotyped.

Voucher and locality information for the populations are shown in Appendix 1.

*Indicates significant deviation from Hardy–Weinberg equilibrium (P < 0.001).

Two wild C. ovalis Lindl. populations (Appendix 1) were used to test the cross-compatibility and polymorphism of the 15 microsatellite loci. This testing was performed because C. ovalis and C. fimbriata have been visually confused as the same species, with no clear differences shown in studies of pollinaria or other morphological characters (Pelser et al., 2000). However, many researchers do recognize C. fimbriata and C. ovalis as separate species (Govaerts, 1999; Wu and Hong, 2009; George and George, 2011). In our results, 11 of 15 loci could obtain clear PCR products and 10 showed moderate to high levels of polymorphism, with the exception of one locus (CF2-147). No significant linkage disequilibrium was detected in C. ovalis populations; however, significant deviation from HWE was found in these populations at most loci (Table 2). Signs of null alleles were detected in the CF1-26, CF1-120, CF2-126, and CF2-172 loci. In addition, cross-amplification of 15 polymorphic loci was conducted on another five related species (n = 5 for each species): C. cumingii Lindl., C. eberhardtii Gagnep., C. mayeriana Rchb. f., C. peltastes Rchb. f., and C. velutina de Vogel; these samples were collected from living plants at Shanghai Chenshan Botanical Garden (Appendix 1). Four to eight loci were successfully amplified in all five Coelogyne species (Table 3).

Table 3.

Amplification of 15 microsatellite loci developed for Coelogyne fimbriata in five other Coelogyne species.

Locus	C. cumingii (n = 5)	C. mayeriana (n = 5)	C. eberhardtii (n = 5)	C. peltastes (n = 5)	C. velutina (n = 5)
CF1-11	—	—	—	—	—
CF1-26	+	+	+	+	+
CF1-30	+	+	+	+	+
CF1-51	—	—	—	—	—
CF1-60	—	—	—	—	—
CF1-120	—	+	+	+	—
CF1-167	+	+	+	+	+
CF1-229	—	—	—	—	—
CF1-231	—	—	—	—	+
CF2-26	—	+	—	+	+
CF2-29	—	—	+	—	—
CF2-57	—	—	+	+	+
CF2-126	—	—	—	—	—
CF2-147	—	—	+	—	—
CF2-172	+	+	+	+	+

Note: + = primer successfully amplified; — = primer failed to amplify; n = number of individuals.

CONCLUSIONS

In the current study, although a majority of the developed loci showed monomorphism (68.1%), 15 polymorphic loci were identified in C. fimbriata. These polymorphic loci are valuable for orchid population genetic studies. For example, these markers can be used to characterize the clonal structure of C. fimbriata to estimate seed and pollen flow at a fine scale. Furthermore, these polymorphic loci can provide more information, such as genetic diversity indices, which are important for the conservation and management of the species.

Appendix 1.

Locality information for the Coelogyne fimbriata and C. ovalis samples used in this study.

Species	Locality ID	Collection locality	Geographic coordinates	Collector	Collection no.	n
Coelogyne fimbriata Lindl.	DWS	Yunnan, China	22.931°N, 103.685°E	Wei-Chang Huang	CS-HWC201606-2	28
Coelogyne fimbriata	DLS	Hainan, China	18.659°N, 109.916°E	Ming-Zhong Huang	CS-HMZ201610-6	19
Coelogyne ovalis Lindl.	MHX	Yunnan, China	23.051°N, 103.356°E	Wei-Chang Huang	CS-HWC201606-5	21
Coelogyne ovalis	JGX	Yunnan, China	23.523°N, 100.646°E	Wei-Chang Huang	CS-HWC201509-8	16
Coelogyne cumingii Lindl.b	—	Taiwan	—	Wei-Chang Huang	—	5
Coelogyne eberhardtii Gagnep.b	—	Thailand	—	Wei-Chang Huang	—	5
Coelogyne mayeriana Rchb. f.b	—	Taiwan	—	Wei-Chang Huang	—	5
Coelogyne peltastes Rchb. f.b	—	Taiwan	—	Wei-Chang Huang	—	5
Coelogyne velutina de Vogelb	—	Taiwan	—	Wei-Chang Huang	—	5

Note: — = no detailed information available; n = number of individuals sampled.

All voucher specimens were deposited in Shanghai Chenshan Herbarium (CSH), Shanghai, China.

Samples of Coelogyne cumingii, C. eberhardtii, C. mayeriana, C. peltastes, and C. velutina were collected from living plants at Shanghai Chenshan Botanical Garden (introduced from Taiwan and Thailand according to the record).

Appendix 2.

Characteristics of 32 monomorphic microsatellite markers developed for Coelogyne fimbriata.

Locus	Primer sequences (5′–3′)	Ta (°C)	Repeat motif	Allele size (bp)	GenBank accession no.
CF1-33	F: TAAGTAATTCAGCCTCCC	55	(CT)8	197	KP676050
	R: CAGACCATCAAGAAGCATA
CF2-2	F: CAAGTCCAAATCAGCGAAGG	56	(AG)15	152	KY744706
	R: TCCAGAATACATCCAGGCAC
CF2-3	F: GAAGAAATCAAGGACCAATG	64	(TCT)5	198	KX237656
	R: TCTGAGAACGAAGGAGGC
CF2-8	F: TAGGAGGTGAGGAGGAA	56	(AG)15	402	KX237657
	R: CCAGATGCCAAGATAAA
CF2-15	F: CGACTTTCTCCGGTATCTC	56	(AG)14	248	KY744707
	R: CACTCACTCAGCCTCTTCC
CF2-20	F: GGAAAATAGTAAAAGCCAT	56	(TC)18	181	KY744708
	R: TCCCAAACTTCAAACC
CF2-23	F: CTCCCCGTTGTAATCCAATCAT	64	(CT)12	178	KY744709
	R: GTTCCTCCTTCGGCTACGTTAG
CF2-24	F: ACCCTTCCTATCGCTGTATT	62	(CT)7	186	KX237658
	R: CTCTTCCCACCAAGTCTTTT
CF2-27	F: GAGAGTGGAGGTAGGAGAA	62	(AG)6	111	KY766112
	R: GGAGGAGGCTATGGAGAA
CF2-59	F: GAAGCAGAAAATAACATA	56	(TC)22	90	KY744710
	R: TCTCACTCCACTCTATCT
CF2-101	F: TGGTCAGTCGGAGGAG	64	(TC)27	355	KY744711
	R: ATGGAGGTGGTAGTGTTGG
CF2-112	F: GGGATTCGGACTGAGATT	64	(GA)39	228	KX237663
	R: TTAGTAGGGATGCGAGGAG
CF2-127	F: TCAAGTCCCATCAATC	53	(CT)23	153	KY744712
	R: TTTAGTGCTCCACATT
CF2-129	F: TTGGCATTTCGCTTCT	59	(CT)12…(TC)16	235	KY744713
	R: CGTGTCTTTGTCGGTTT
CF2-136	F: TCGACCCGTAGTACGCAACA	64	(TC)8	285	KY744714
	R: ATGGACACCAGGGCAAGG
CF2-137	F: GGAAGGCTATGGAGAAAT	64	(TC)10…(CT)7	126	KY744715
	R: ATGGGATGACCAGAGGAT
CF2-140	F: GAAGATGGGAAGAAAGAA	62	(GA)10	108	KY744716
	R: TGAAAGGAGGAGTAGGAG
CF2-146	F: TATGCAAATGATATGA	53	(TC)15…(AC)10	347	KY744717
	R: GAGAATGTGAGAAAGT
CF2-148	F: TGAATAAGATATTCGGATCA	56	(AG)20	227	KY744718
	R: AAATCGGTGTATGGAGAC
CF2-149	F: GTCAAACAGAAAGCCAAG	56	(CT)18	350	KX237665
	R: AAAGATCCGCTCCACTAT
CF2-155	F: TCTCGTCTTTTCCTCTTACC	56	(CT)20	104	KY744719
	R: CCATTACCTCCTCACCATAC
CF2-160	F: GAATCCTCGCTCCCATTT	53	(TG)11…(GA)15	109	KY744720
	R: GTTGGTTTAGAGTTTGCAGGTA
CF2-171	F: TCCTTGTTCGCGTGAAAC	59	(GA)43	232	KY744721
	R: GAGATCCCTCGACCATAC
CF2-177	F: AAGAGTTAGAAGTGGGGAGG	59	(AG)19AA(AG)15	405	KY744722
	R: GGGGAAGTGCCTTATGAT
CF2-192	F: ACGGGTGAGTATCTTGGC	64	(CA)16	274	KY744723
	R: GAGGTGGTGAACTCCATTTA
CF2-213	F: ACCAATAGGAAGTGAGGAGGAA	64	(CT)16	151	KY744724
	R: ATGGCGGAGCAAGAAAGG
CF2-217	F: CTTGTTTCATAAAGCGAAGT	64	(CT)23…(CA)13	181	KX237667
	R: CTTTTATCACAGTCACCCAT
CF2-222	F: TAGGGGAAACTATGGACAAA	63	(CT)46	181	KY744725
	R: CGAGTTAGGGATTAGAGGGT
CF2-232	F: AATAAGATAATGGAAGGA	62	(GA)9	108	KY744726
	R: ACTCCAGTTTGTCTTTTA
CF2-234	F: ATCAAAGCCTATTATTCCC	64	(TC)8	292	KY744727
	R: AGATTTACCGTCGTCAGC
CF2-238	F: AACGCCCACCACCAAGTA	64	(GA)11	379	KY744728
	R: CGGCCCTATTCCCTCACA
CF2-240	F: TACACGCCCTAATACCCA	60	(TC)13	488	KY744729
	R: CGAGGAGGAGGTTGAGAT

Note: Ta = annealing temperature.