Development of Microsatellite Markers for<i>Cypripedium tibeticum</i>(Orchidaceae) and their Applicability to Two Related Species

Keywords: Cypripedium tibeticum, genetic diversity, microsatellites, Orchidaceae

Cypripedium tibeticum King ex Rolfe (Orchidaceae), known as Chinese lady slipper, is a unique orchid species with high ornamental and medicinal value. It is endemic to southwestern China, including Tibet, Gansu, Yunnan, Guizhou, and Sichuan provinces. Cypripedium tibeticum mainly grows in alpine meadows, scrub forests, and forest margins at high altitudes (2800-4200 m a.s.l.).  Bronstein et al., 2014). . 
. . In this study, we developed and characterized 20 polymorphic SSR markers in C. tibeticum and tested their applicability in two related species: C. flavum P. F. Hunt & Summerh. and C. bardolphianum W. W. Sm. & Farrer. These microsatellites will be valuable tools for ecological, phylogeographic, and conservation studies of C. tibeticum and other related Cypripedium species. 

METHODS AND RESULTS

Plant sample collection and DNA extraction-Leaf samples of C. tibeticum were collected from three locations in Sichuan Province, including Huanglong, Kangding, and Xiaojinxian in China (population codes: HL, KD, and XJX, respectively [Appendix 1]). Genomic DNA was extracted from fresh leaf tissue of each individual using the DNeasy Plant Mini Kit according to manufacturer instructions (QIAGEN, Valencia, California, USA). 

Development and screening of microsatellite markers-Total genomic

DNA was digested with the restriction enzyme HaeIII, and products were 5′ phosphorylated and a single A was added at the 3′ end. DNA was ligated to a Solexa adapter and sequenced on a single HiSeq 2500 flow cell (Illumina, 

were obtained, and raw reads with quality scores less than 25 and lengths shorter than 25 bp after stripping the adapters were filtered using SeqPrep (https://github.com/jstjohn/seqprep). De novo assembly was performed using CLC Genomics Workbench (QIAGEN) and produced 1,920,476 contigs, where parameters were set as: average coverage value of 25× and minimum contig length of 299 bp. MSATCOMMANDER 1.0.8 software was used to identify contigs carrying di-, tri-, and tetranucleotide repeats with a minimum of five repeats and a minimum tract length of 100 bp (other parameters were set to default settings) (Faircloth, 2008). Ninety-four primer pairs were designed for microsatellite loci candidates using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, California, USA) and tested with 15 C. tibeticum samples (five individuals from each population) for amplification efficiency and polymorphism. Each 15-μL PCR reaction contained ~15 ng of genomic DNA, 1.5 μL of 10× PCR buffer, 0.8 μL of fluorescently labeled TP-M13 (5 mM), 1.0 μL of each primer (10 mM), 1.5 μL of dNTP (10 mM), and 0.1 μL of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, California, USA). All candidate primer pairs were tested by a touchdown PCR protocol as follows: 94°C for 2 min; five cycles of 94°C for 30 s, 60-56°C (Δ1°C touchdown per cycle) for 30 s, 72°C for 30 s; followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s; and a final extension at 60°C for 30 min. 
Results from testing primer pairs showed that 26 pairs produced bands within the expected size range on agarose gels in all the samples tested. The size of amplified fragments from these loci was further analyzed on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA), and allele sizes were scored by GeneMapper version 3.2 software (Applied Biosystems). Twenty loci were polymorphic and showed clear peaks. The authenticity of these amplified loci was confirmed by Sanger sequencing of representative PCR products, and sequences have been deposited in GenBank (Table 1). Sequence library data of this study were deposited to the Sequence Read Archive of the National Center for Biotechnology Information (NCBI; BioProject ID: PRJNA393499). 

Data analysis and results

-These 20 microsatellite loci were PCRamplified in an additional 74 individuals from three C. tibeticum populations collected in Sichuan Province. For each locus, the observed number of alleles, effective number of alleles, observed heterozygosity, and expected heterozygosity were calculated using PopGene32 version 1.32 (Yeh et al., 1999). Tests for Hardy-Weinberg equilibrium and linkage disequilibrium were performed by GENEPOP Web version 4.2 (Rousset, 2008). The total number of alleles per locus ranged from two to 21 (mean ± SD: 6.350 ± 4.320). The observed heterozygosity and expected heterozygosity ranged from 0.261 to 0.967 (0.664 ± 0.143) and from 0.441 to 0.960 (0.745 ± 0.119), respectively (Table 2). Of the 20 polymorphic loci, nine loci in the Huanglong population, six loci in the Kangding population, and five loci in the Xiaojinxian population deviated significantly from Hardy-Weinberg equilibrium, respectively (P < 0.05; 3). 

CONCLUSIONS

In this study, we developed and validated 20 polymorphic microsatellite markers for the orchid C. tibeticum, most of which showed applicability in two related Cypripedium species: 

Table 2

 2 

). Linkage disequilibrium was not detected at any locus.The utility of these 20 microsatellite loci developed for C. tibeticum was also detected in two other Cypripedium species: C. flavum and C. bardolphianum. 
tabLe 1 .

 1 Characteristics of 20 microsatellite loci developed in Cypripedium tibeticum. 


	Locus	Primer sequences (5′-3′)	Repeat motif	Allele size range (bp)	Fluorescent label	T a (°C)	GenBank accession no.
	M150 M164 M172 M182 M209 M289 M294 M372 M401 M576 M112 M130 M136 M139 M142 M233 M370 M681 M880 M886	F: GGATGAGGAAACCCTAGCGT R: GATTCTCCTTGATGGCAATGT F: GTTAGGTGAAGGCTAGAAGGT R: AGCTGAGAGTGGAAGATCATAT F: CACCTCTATTCCACTTTGCCT R: GTAAAGGGATAGCACATGCAC F: GTGGTGGACCGATCATACCT R: GGAGAATTTAGTGGAAGAGAGAT F: GGGTCCTTTTGTTGACCTACT R: AACCCTAATTGGTCAATTCGAT F: ATCACCTTTCCACAATCATGT R: TGCTGTCACCATTGAACTCT F: ATACAGAGCCAGCCTACACT R: AGTAACTAGTGGAGCACAGCT F: TCCTAGTTCGAAGATGATGGT R: TAGCTACAGCCAAATTCGGT F: TCAGGATTACAGCCCTAAACT R: GGGAGATTCAGTTACTAGAGCT F: AATCATCAAAGGGAGAGCAAT R: TTTGTCACCACCATGATAGAGT F: AAGACTGGAAGTAGCGGAGAT R: GAAGTCTACATCCACTGTTCCT F: TTCAGCAGATTCAGATTTAGCT R: CTTCTGACAGATCTTCTCCAAT F: TCCAACCTCTCTATCCTCGAT R: GATCTGAGGAGGATGAAGGT F: GAGGGAGGATTCTGACAACT R: GTGGTTCGACTTCGTGAAAT F: TTGTGCATCGTCATCAAGGT R: GATCACAAACATGCTGGGAT F: ACACGCTACAGACCGAGCT R: CTTGTTTGCAAGATGAATGCT F: GGAGATTCAGTTATCAGAGCTG R: AGTCTACATCCACTATTCCTTGT F: ATGCCAGACGGCAGAGGT R: GGCTTTGAGCTAATGGCTCT F: GGAGAACAGGAAAACTGTTCAT R: AGCCGATTACAAAGTCACTGT F: CGAAATTCACTGGGATTCAGT R: TCTCAGGATTACAACAGTAGCT	(GTA) 5 (AT) 7 (AAG) 8 (AG) 8 (TTC) 16 (CT) 8 (AAG) 8 (AT) 9 (TTC) 5 (AG) 8 (AAG) 9 (CT) 10 (CAT) 5 (CTT) 3 (TTC) 9 (TC) 11 (TC) 7 (CAT) 6 (TC) 24 (TG) 10 (TC) 10 (CT) 9	153-164 137-188 153-161 148-163 170-182 177-201 161-169 154-160 161-175 301-310 232-304 182-218 185-191 213-231 153-155 169-184 271-339 150-160 137-147 156-164	HEX FAM HEX FAM HEX HEX FAM HEX FAM FAM FAM HEX HEX FAM HEX FAM FAM HEX FAM HEX	60 60 62 62 57 58 63 60 59 62 53 51 53 53 53 51 52 55 52 52	KY490005 KY490006 KY490007 KY490008 KY490009 KY490010 KY490011 KY490012 KY490013 KY490014 MF398589 MF398590 MF398591 MF398592 MF398593 MF398594 MF398595 MF398596 MF398597 MF398598
	Note: T a = annealing temperature.					

tabLe 2 .

 2 Genetic variation of the 20 polymorphic microsatellite loci in three populations of Cypripedium tibeticum. a Note: A = number of alleles; A e = effective number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals sampled for each population. * Significant deviation from Hardy-Weinberg equilibrium (P < 0.05). a Voucher and locality information are provided in Appendix 1. 


			Huanglong (n = 30)			Kangding (n = 23)			Xiaojinxian (n = 21)	
	Locus	A	A e	H o	H e	A	A e	H o	H e	A	A e	H o	H e
	M150	5	3.141	0.533*	0.693	5	3.752	0.696*	0.750	5	4.027	0.619	0.770
	M164	9	4.369	0.667	0.784	9	3.586	0.522*	0.737	10	7.230	0.620*	0.883
	M172	4	3.352	0.700	0.714	4	3.765	0.826	0.751	4	3.303	0.667	0.714
	M182	4	3.834	0.607	0.753	6	5.042	0.636	0.820	5	4.376	0.737	0.792
	M209	5	4.380	0.700*	0.785	6	4.038	0.609	0.769	5	3.920	0.619	0.763
	M289	5	4.478	0.967*	0.790	5	4.640	0.957	0.802	6	4.624	0.800	0.804
	M294	5	3.939	0.633	0.759	4	3.174	0.636	0.701	5	4.302	0.714*	0.786
	M372	3	2.410	0.833*	0.595	3	2.489	0.870	0.612	3	2.513	0.619	0.617
	M401	5	3.516	0.933*	0.728	5	2.315	0.696	0.581	7	4.432	0.667	0.793
	M576	3	2.925	0.379*	0.670	4	3.010	0.619	0.684	3	2.882	0.714	0.669
	M112	20	10.852	0.724*	0.924	21	16.133	0.636*	0.960	20	14.700	0.714*	0.955
	M130	7	5.202	0.700*	0.822	7	5.426	0.696	0.834	6	5.313	0.667*	0.832
	M136	3	2.062	0.467	0.524	3	1.796	0.435	0.453	3	1.757	0.286	0.441
	M139	9	4.485	0.690	0.791	9	5.728	0.682	0.845	7	5.714	0.700	0.846
	M142	2	2.000	0.310	0.509	2	1.910	0.261	0.487	2	1.930	0.429	0.494
	M233	5	3.666	0.667*	0.740	6	4.172	0.591	0.778	5	3.571	0.667	0.738
	M370	17	8.476	0.786	0.898	15	10.377	0.810*	0.926	12	8.112	0.632*	0.900
	M681	5	3.303	0.733	0.709	5	4.445	0.739*	0.792	6	3.571	0.762	0.738
	M880	5	4.000	0.767	0.763	6	4.944	0.783*	0.816	6	4.846	0.619	0.813
	M886	5	4.854	0.700	0.753	5	4.165	0.739	0.777	5	4.027	0.667	0.770
	Mean	6.300	4.212	0.675	0.735	6.500	4.745	0.672	0.744	6.250	4.758	0.646	0.756

tabLe 3 .

 3 Characteristics and polymorphism of 20 microsatellite loci developed for Cypripedium tibeticum in C. flavum and C. bardolphianum. a Eighteen loci were successfully PCR-amplified in C. flavum, and 17 loci were amplified in C. bardolphianum (locus M294 did not amplify in C. bardolphianum, M370 and M886 did not amplify in either species). Genotyping results showed locus M372 was monomorphic in both species, and M136 was polymorphic in C. bardolphianum but monomorphic in C. flavum. All the other markers were polymorphic in both species (Table 


			C. flavum (n = 16)			C. bardolphianum (n = 15)	
	Locus	Allele size range (bp)	A	H o	H e	Allele size range (bp)	A	H o	H e
	M150	155-217	6	0.750	0.807	157-217	4	0.733	0.687
	M164	143-188	6	0.625	0.786	140-185	6	0.667	0.752
	M172	155-159	3	0.625*	0.647	147-169	5	0.733	0.736
	M182	136-163	4	0.688	0.659	142-163	4	0.667	0.720
	M209	156-173	6	0.813	0.730	151-181	6	0.800	0.786
	M289	180-197	4	0.875	0.724	183-192	3	0.867	0.674
	M294	161-163	2	0.563	0.466	-	-	-	-
	M372	157	1	0.000	0.000	157	1	0.000	0.000
	M401	161-167	4	0.813	0.684	151-173	7	0.867	0.777
	M576	301-307	3	0.750	0.679	301-304	2	0.400	0.497
	M112	234-286	13	0.733*	0.906	238-280	11	0.769	0.905
	M130	189-198	4	0.667*	0.687	174-186	4	0.750	0.736
	M136	188	1	0.000	0.000	182-185	2	0.308	0.492
	M139	215-221	3	0.600	0.662	215-225	5	0.643	0.743
	M142	147-151	2	0.154*	0.492	155-159	2	0.333	0.391
	M233	173-182	3	0.500	0.623	171-177	2	0.200	0.186
	M370	-	-	-	-	-	-	-	-
	M681	146-152	3	0.563	0.684	150-152	2	0.400	0.515
	M880	140-146	3	0.571	0.659	144-150	3	0.539	0.655
	M886	-	-	-	-	-	-	-	-
	Mean		3.944	0.572	0.605		4.059	0.569	0.603

Note: -= unsuccessful amplification; A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals sampled for each population. * Significant deviation from Hardy-Weinberg equilibrium (P < 0.05). a Voucher and locality information are provided in Appendix 1. 
http://www.bioone.org/loi/apps 

The authors thank Dejun An for help with sample collection and Dr. Brad S. Coates (Research Geneticist, U.S. Department of Agriculture-Agricultural Research Service) for assistance in editing this manuscript. This study was supported by the National Natural Science Foundation of China (NSFC 31300366), the Natural Science Foundation of Shaanxi Province (2015JQ3091), the Natural Science Foundation of Xi'an Science and Technology Bureau (CXY1531WL28), and the Scientific Research Foundation of the Education Department of Shaanxi (15JK2146).
Jing Li, Yibo Luo, Lingling Xu
Development of Microsatellite Markers for<i>Cypripedium tibeticum</i>(Orchidaceae) and their Applicability to Two Related Species