Development of chloroplast microsatellite markers for Glyptostrobus pensilis (Cupressaceae).

PREMISE: Glyptostrobus pensilis (Cupressaceae) is a critically endangered conifer native to China, Laos, and Vietnam, with only a few populations remaining in the wild. METHODS AND
RESULTS: Using a complete chloroplast genome sequence, we designed 70 cpSSR loci and tested them for amplification success and polymorphism in 16 samples. Ten loci were found to be polymorphic and their genetic diversity was characterized using a total of 83 individuals from three populations in China. A total of 43 haplotypes were present, the effective number of haplotypes varied from 4.55 to 13.36, and the haplotypic richness ranged from 8.04 to 16.00. Gene diversity ranged from 0.81 to 0.97 (average 0.89). The number of alleles per locus and population ranged from one to eight, and the effective number of alleles ranged from 1.00 to 3.90. All polymorphic loci were successfully amplified in the related species Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata.
CONCLUSIONS: These newly developed chloroplast microsatellites will be useful for population genetic and phylogeographic analyses of G. pensilis and related species.

Glyptostrobus pensilis (Staunton ex D. Don) K. Koch, the only extant species in the genus Glyptostrobus Endl., is a relict conifer in the family Cupressaceae (Hao et al., 2016). In China, it is mainly distributed in the Pearl River delta region of Guangdong Province, the central region of Fujian Province, the lower reaches of the Minjiang River, and northeastern Jiangxi Province (Li and Xia, 2004). A few wild populations have recently been found in Laos and Vietnam, extending its latitudinal distribution from 28°N to 13°N (Averyanov et al., 2009; Thomas and LePage, 2011). The species preferred habitat of riverbanks and flood plains have been severely degraded by human activities (e.g., agriculture and rice cultivation) in many locations, which has led to a rapid decline of most G. pensilis populations (Li and Xia, 2004, 2005; Nguyen et al., 2013). Currently, the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species has evaluated G. pensilis as Critically Endangered (CR) (Thomas et al., 2011).

Chloroplast microsatellites have been widely used to investigate the population genetic structure and phylogeographic history of a range of tree species (Ruhsam et al., 2016; Gryta et al., 2017). Previous molecular studies of G. pensilis have only used nuclear markers such as inter‐simple sequence repeats (Li and Xia, 2005; Wu, 2011), and recently Wang et al. (2019) developed 10 polymorphic nuclear microsatellite markers for this species. Compared with nuclear simple sequence repeats (SSRs), chloroplast SSRs (cpSSRs) are more likely to detect historical bottlenecks or genetic drift due to their uniparental inheritance, slower mutation rate, and lack of recombination (Ennos et al., 1999; Pleines et al., 2009; Li and Liu, 2012). Nguyen et al. (2013) analyzed G. pensilis populations from Vietnam using six cpSSRs; however, these loci were developed from Pinus thunbergii Parl. and designed for use in Pinaceae species (Vendramin et al., 1996). In this study, we developed new species‐specific chloroplast microsatellite loci using the complete chloroplast genome of G. pensilis (Hao et al., 2016). Additionally, we tested the transferability of these loci in four related species: Cryptomeria japonica (Thunb. ex L. f.) D. Don var. sinensis Miq., Taxodium distichum (L.) Rich., T. ascendens Brongn., and Cunninghamia lanceolata (Lamb.) Hook.

METHODS AND RESULTS

We searched the complete chloroplast genome of G. pensilis (Hao et al., 2016; GenBank accession number KU_302768) for microsatellite loci exhibiting a minimum of eight repeats as these loci are likely to exhibit a higher level of polymorphism (Ueno et al., 2012). For loci with a minimum of eight repeats, primers were designed using the online software Primer3Plus (Untergasser et al., 2007) using default parameters. In total, 70 cpSSR loci were selected and evaluated for their amplification efficiency and level of polymorphism using 16 G. pensilis DNA samples from different populations (Appendix 1). DNA was extracted from G. pensilis leaves using a modification of the cetyltrimethylammonium bromide (CTAB) method (Tsumura et al., 1995).

PCR amplification was carried out in volumes of 15 μL using the following protocol: 7.5 μL of 2× Taq PCR Master Mix (Tiangen, Beijing, China), 0.75 μL of forward primer (10 μM), 0.75 μL of reverse primer (10 μM), 3 μL of 20–50 ng/μL DNA template, and 3 μL of ddH2O. The mixture was then cycled using the following profile: 94°C for 4 min; 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; with a final extension at 72°C for 30 min. PCR products were visualized on a 1.6% agarose gel. All loci that could be amplified successfully were tested individually using 16 G. pensilis samples to establish their polymorphism. These amplifications were carried out using fluorescently labeled primers (FAM, HEX, TAMRA, and ROX; Applied Biosystems, Foster City, California, USA) and the same PCR protocol as detailed above. PCR products were run on an ABI 3730xL DNA Analyzer adding a GeneScan 500 LIZ internal size standard (Applied Biosystems) to size fragments. The software GeneMarker version 1.9 was used to score the electropherograms of all samples (Hulce et al., 2011). Sixty‐five of the 70 cpSSR loci amplified successfully across the 16 test individuals, but 55 loci were monomorphic, and only 10 loci were polymorphic (Table 1, Appendix 2). These polymorphic loci were used to investigate the genetic diversity of 83 individuals across three Chinese G. pensilis populations (Appendix 1).

Table 1

Characterization of 10 polymorphic chloroplast microsatellite loci developed in Glyptostrobus pensilis.a

Locus	Primer sequences (5′–3′)	Location	Repeat motif	Allele size range (bp)	GenBank accession no.
Gp_cp_1	F:(ROX)TGACACACGGGTCTGTATCA	ycf4 to psaI	(AT)10	261–271	MK386658
	R: GCCTTTGGTGGGCTTGTTTT
Gp_cp_6	F:(FAM)GCTGTTCCCCTGTGCATCAT	trnL to trnF	(AT)11	195–203	MK386659
	R: GATCAATTTGTGTCTGCTTCTGT
Gp_cp_7	F:(HEX)ACCTGTCTCAAATCGACTTCCC	ycf3 to psaA	(T)11	149–167	MK386660
	R: CTCCTCTTTCCAGACGAGACA
Gp_cp_8	F:(HEX)TGAACCGATGACTTACGCCT	psb to trnE	(TA)9	267–279	MK386661
	R: AAATCGAATCCCCGTTGCCT
Gp_cp_11	F:(TAMRA)AATCCTGAAAGTCGACTAGAATTAAGT	chlB to rps16	(AT)23	363–414	MK386662
	R: GCTAAGAGCATCTTCGAATAAAAATAG
Gp_cp_12	F:(TAMRA)TTAAGTCGAGTGAGTCAGATGG	accD to clpP	(T)11	379–437	MK386663
	R: TGCCCATAGGATGCCAAGTG
Gp_cp_13	F:(TAMRA)TGGGGGATCAAAATAACACAGA	rbcL to accD	(AT)16	208–334	MK386664
	R: GTTTTCCAATGTGAATTTGAAAATCGA
Gp_cp_14	F:(FAM)TCCCCGCAGAACTATCGTTT	ccsA to petA	(T)12	208–214	MK386665
	R: AGGAAAGAATTTGGTAATCTTGGCT
Gp_cp_17	F:(FAM)ACCTACCCAGAATTAGCAAGCC	trnD to psbM	(T)12	116–119	MK386666
	R: AGAATTGGCGGTTGCTTCCT
Gp_cp_35	F:(HEX)TTTTCCTCTACCGCGAACCC	psaJ to rpl33	(A)10	115–117	MK386667
	R: ACTTCACCCCTCCTTGAATTCT

Optimal annealing temperature was 55°C for all loci.

The software Haplotype Analysis version 1.05 (Eliades and Eliades, 2009) was used to calculate the following statistics: number of haplotypes (A), number of private haplotypes (P), effective number of haplotypes (N e), haplotypic richness (R h), and gene diversity (H e). The software GenAlEx6.5 (Peakall and Smouse, 2012) was used to calculate the following parameters: number of alleles (N a), effective number of alleles (N e), Shannon's information index (I), and diversity (H).

A total of 43 haplotypes were detected in the three assayed populations. The number of haplotypes per population ranged from 11 to 18, the number of private haplotypes ranged from nine to 16, the effective number of haplotypes ranged from 4.55 to 13.36, the haplotypic richness ranged from 8.04 to 16.00, and the gene diversity ranged from 0.81 to 0.97 (Table 2). The number of alleles per locus ranged from one to eight per population, the effective number of alleles ranged from 1.00 to 3.90, Shannon's information index ranged from 0.00 to 1.52, and the diversity ranged from 0.00 to 0.74 (Table 3). The 10 polymorphic loci could also be successfully amplified in five individuals in each of the following four related species: Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata (Table 4, Appendix 1).

Table 2

Haplotype diversity in three Chinese Glyptostrobus pensilis populations based on 10 polymorphic chloroplast microsatellite markers.a

Population	A	P	N e	R h	H e	D 2 sh
DM (N = 33)	18	16	7.026	11.857	0.884	76.6
GZHN (N = 21)	17	15	13.364	16.000	0.971	470.5
PNSL (N = 29)	11	9	4.546	8.035	0.808	1.4
Mean	15.333	13.333	8.312	11.964	0.888	182.8

A = number of haplotypes; P = number of private haplotypes; N e = effective number of haplotypes; R h = haplotypic richness; H e = genetic diversity; D 2 sh = mean genetic distance between individuals; N = number of individuals sampled.

Locality and voucher information are provided in Appendix 1.

Table 3

Characteristics of 10 polymorphic chloroplast microsatellite markers in 83 individuals of three Chinese Glyptostrobus pensilis populations.a

Locus	DM (N = 33)				GZHN (N = 21)				PNSL (N = 29)
	N a	N e	I	H	N a	N e	I	H	N a	N e	I	H
Gp_cp_1	3	1.824	0.765	0.452	2	1.960	0.683	0.490	2	1.890	0.664	0.471
Gp_cp_6	2	1.198	0.305	0.165	3	2.194	0.852	0.544	3	1.324	0.479	0.245
Gp_cp_7	3	2.139	0.883	0.533	3	2.845	1.071	0.649	3	1.979	0.779	0.495
Gp_cp_8	3	1.280	0.437	0.219	3	2.110	0.832	0.526	1	1.000	0.000	0.000
Gp_cp_11	5	1.806	0.917	0.446	5	3.084	1.301	0.676	1	1.000	0.000	0.000
Gp_cp_12	3	1.280	0.437	0.219	2	1.893	0.665	0.472	1	1.000	0.000	0.000
Gp_cp_13	8	2.515	1.371	0.602	6	3.903	1.524	0.744	3	1.235	0.398	0.190
Gp_cp_14	3	1.203	0.363	0.169	3	2.110	0.832	0.526	1	1.000	0.000	0.000
Gp_cp_17	2	1.424	0.474	0.298	3	2.384	0.940	0.580	2	1.071	0.150	0.067
Gp_cp_35	2	1.271	0.369	0.213	2	1.995	0.692	0.499	1	1.000	0.000	0.000
Mean	3.400	1.594	0.632	0.331	3.200	2.448	0.939	0.571	1.800	1.250	0.247	0.147

N

= number of individuals sampled; N a = number of alleles; N e = effective number of alleles; I = Shannon's information index; H = diversity.

Locality and voucher information are provided in Appendix 1.

Table 4

Results of cross‐amplification of 10 polymorphic chloroplast microsatellite markers developed for Glyptostrobus pensilis in four closely related species.a , b

Locus	Taxodium distichum (N = 5)	Taxodium ascendens (N = 5)	Cryptomeria japonica var. sinensis (N = 5)	Cunninghamia lanceolata (N = 5)
Gp_cp_1	254–256	250–254	258–262	254–256
Gp_cp_6	179	179	197	171
Gp_cp_7	149	149	133	177
Gp_cp_8	276	276	284	286
Gp_cp_11	379–383	381–383	375	371
Gp_cp_12	378	378	384	432
Gp_cp_13	303–305	303	299	299
Gp_cp_14	205	205	201–213	213
Gp_cp_17	117	115–117	117	117
Gp_cp_35	116–117	114–117	115–116	116–117

N = number of individuals sampled.

Locality and voucher information are provided in Appendix 1.

Numbers shown represent the size in base pairs of the amplified fragments.

CONCLUSIONS

In this study, we developed 10 polymorphic cpSSRs (as well as 55 pairs of monomorphic primers) that can be used to assess the population genetic and phylogeographic structure of G. pensilis populations. The high number of private haplotypes in the three assayed populations suggests geographically isolated populations. Additionally, the 10 loci can be successfully amplified in four related species of G. pensilis.

Species	Population code	Voucher no.	Collection locality	Geographic coordinates	Elevation (m)	N
Glyptostrobus pensilis (Staunton ex D. Don) K. Koch	PNSL	Lin170804	Pingnan, Fujian, China	27°0′27.87″N, 118°51′59.75″E	1260	29
	GZHN	Lin170411	Guangzhou, Guangdong, China	23°11′24.6″N, 113°21′38.13″E	40	21
	DM	Lin170729	Doumen, Guangdong, China	22°23′42.5″N, 113°15′14.65″E	20	33
Taxodium distichum (L.) Rich.		Li180522	Changsha, Hunan, China	28°8′16.48″N, 112°59′28.36″E	90	5
Taxodium ascendens Brongn.		Li180522	Changsha, Hunan, China	28°8′16.48″N, 112°59′28.36″E	90	5
Cryptomeria japonica (Thunb. ex L. f.) D. Don var. sinensis Miq.		Wang180720	Jiujiang, Jiangxi, China	29°32′59.77″N, 115°58′03.32″E	911	5
Cunninghamia lanceolata (Lamb.) Hook.		Li180522	Changsha, Hunan, China	28°8′16.48″N, 112°59′28.36″E	90	5

N = number of individuals sampled.

Locus	Primer sequences (5′–3′)	Repeat motif	Product size (bp)
Gp_cp_2	F: ACATTGATTTCTAAAAGAGAGGAGTCA	(A)11	211
	R: TCAGTGTCAGAAATTTGGCTGA
Gp_cp_3	F: TGATGAGCTACTCTACGTGCT	(T)13	369
	R: ATCTGCCATTGTACCCGCAA
Gp_cp_4	F: ATAGATTCCGAGCGGCTGTG	(T)18	292
	R: ACCGCTGAGTTATATCCCTTTCC
Gp_cp_5	F: GCGATCGTACCTTCATCGGA	(T)20	230
	R: TCCTTTTTCAATATCGTTCCCTGG
Gp_cp_9	F: ATTTCTCGCCAAGCTGTCCA	(AT)10	332
	R: CGAGCAATGCCATCTCCTACT
Gp_cp_10	F: CGAACCCGCATCGTTAGCTT	(A)15	280
	R: GGTTGTTCACCTGAAATTAAGAGGA
Gp_cp_15	F: TCAAGCAAAGGTAGATGGTGAG	(A)12	257
	R: TCTCAACCTTCATGTGGGAG
Gp_cp_16	F: ATGCTCTTTCGCAACGTTCG	(A)12	201
	R: TGAACACAAAGAAAGGTAAGGTCT
Gp_cp_18	F: TCCGCTCAATTCCGTTACTC	(T)12	145
	R: TCCATGATTGATTTTCCCTTCGT
Gp_cp_19	F: TCTTGCAAAATCCGGACCG	(A)11	201
	R: TGAACCAAGTCAGTTCGCTTG
Gp_cp_20	F: CGAAAACCGTCGGGAAACAT	(A)11	260
	R: GCTTCTTCCTTCCCGCCAT
Gp_cp_21	F: GGCTCGCGGGTATGTTAACT	(A)11	190
	R: TCGGGCAATTTTGTCATGTACC
Gp_cp_22	F: AGGGGCAGAATCTAGGGTT	(A)11	194
	R: CCGCTATTTTCCACGTTGAGC
Gp_cp_23	F: ATCCGCCTTGATTCCCGTTT	(A)11	263
	R: ACAGGCGCTGTGGAAAGAT
Gp_cp_24	F: TCTCTTTTGCGTCCTTCCCC	(T)11	231
	R: AAGAATTAGTTCGCCATGGGT
Gp_cp_25	F: TCCTTCGGGATTAATTCTTCATTCT	(T)11	264
	R: AATCCTGAGCAGCCAAACC
Gp_cp_26	F: TTGTAGCTCTACGTGGCAC	(AT)11	263
	R: AGGCATAAACAAAAACAGGGCT
Gp_cp_27	F: CGGGGGAATGATACCTGTCG	(T)10	138
	R: ACGGAGACTTGATATTGATGCTC
Gp_cp_28	F: TCGTGAATTCGTTGGACAG	(TA)10	213
	R: TCCATCTGACTCACTCGACT
Gp_cp_29	F: GAGCTTACTTGGGTACTGAGC	(A)10	126
	R: CATCCGGCTCGAGCAATAGT
Gp_cp_30	F: TGAGTATCCGTTTCCTTTCTTTTGC	(A)10	201
	R: TAAGTTTTCCCTTACTATAGTGTGTGT
Gp_cp_31	F: CGGGAAGAGTAGTATGAAGCTC	(A)10	233
	R: GCATATGTGCGATGAATAGACTCC
Gp_cp_32	F: CCGAGAACGAACCGAATGGA	(A)10	157
	R: GGGATTGACTGTTGGATTGGC
Gp_cp_33	F: ATTAGCGGGGAGTTCCATCC	(A)10	226
	R: CGGACTTGTGATTCGTTTGATCT
Gp_cp_34	F: ACGCGGCGATCAATTGGATA	(A)10	143
	R: CCTACAGAGCGTGATCCTGC
Gp_cp_36	F: TCATTTTTACCCAGGAATAGAAACAT	(A)10	156
	R: GATGGCTTCATTTTATTCATAGTTTGT
Gp_cp_37	F: ACCCAAAAAGAGGAGACAAGC	(A)10	165
	R: GAATGACTTCGGGGTGGGAG
Gp_cp_38	F: ACTTGGACGAACTCCCTATTGA	(AT)9	221
	R: CAGCCGGGATAGCTCAGTTG
Gp_cp_39	F: TCTTATGTTCTTAGTAACACGCCT	(A)9	125
	R: TGGAGTAGGAGGAAAATCCGT
Gp_cp_40	F: ATGTCTCGTTATCGCGGACC	(A)9	248
	R: TGACCTGTTGATCCCTTGGC
Gp_cp_41	F: CTGCACATCTGTCCCTCTGT	(T)9	162
	R: TGCTTTCATCCTCCCGCAAT
Gp_cp_42	F: TGCGATCGTAAGGAAATCCA	(A)9	211
	R: TTCTCCCCTGAAGCCATTGG
Gp_cp_43	F: GGTTGATGGCTCTGGTCTTGA	(A)9	186
	R: TGAATCCTTGTTGCTCGGCT
Gp_cp_44	F: CCATTCGATCCCTATCCGGTC	(T)9	224
	R: CATCAACCACTCGGCCATCT
Gp_cp_45	F: AGTGAGGTAGATTACGCCTAATCT	(A)9	222
	R: AGCCCAGTGTTCATTTTGAATATT
Gp_cp_46	F: GCGAGTCAAGCCGAAGTACA	(A)9	132
	R: AATTTTTCGTTTCCTTCGTACTACT
Gp_cp_47	F: GAAGCAACCGCCAATTCTTCA	(T)9	190
	R: TGTTCGGGTGAGAAAGGTGT
Gp_cp_48	F: TCTCTTACATATCTCTGGAAAAAGGA	(T)9	228
	R: TGCTGCTCTGTCCCAACTAT
Gp_cp_49	F: AGCGAAGAATCCCTTGTCCTG	(A)9	171
	R: ATCTGGGCCCTCCGTCTAAT
Gp_cp_50	F: CAGATACTGGCCGGGCTAGA	(A)9	140
	R: CGCTCAGCCATCTCTCCTAG
Gp_cp_51	F: CGCCATCTTGGATGGAATGG	(T)9	233
	R: TGTGGCGGGTATAGTTTAGTGG
Gp_cp_52	F: CGGCTTTTAAGTGCGACTATGG	(A)9	298
	R: TGACTTAATCACCCGCACTC
Gp_cp_53	F: GGCACGAGAACTTGAAGATCG	(A)9	164
	R: ATTGATTCATCGACCCGCGG
Gp_cp_54	F: TGCATAAGAATGAGCCAACTTGA	(T)9	187
	R: TCATACGGCTTAAACAAGAACAC
Gp_cp_55	F: CAGGCATTTACTTTTTGTTTTGGAGT	(T)9	134
	R: TTTGGGTGGAATGGGGATTG
Gp_cp_56	F: ATATTCCGCAAGAATTTTGGGTT	(T)9	202
	R: TGCATTTGTCAACTTGTTTATCGAGA
Gp_cp_57	F: CGCACGGCTCCTAAGTGAT	(T)9	257
	R: ACCCTAAGATGAGCATCGC
Gp_cp_58	F: TGTGTATTTGGCTTTGAAACGA	(T)9	176
	R: TGTCTTTGTTTGCTCAATTTTGC
Gp_cp_59	F: TATTGGACCAGCGGTAGTGG	(T)9	144
	R: ATAAGCAGTCCAAGGGGAGC
Gp_cp_60	F: ACGATTATTCAGATTGAGCTCCGA	(T)9	201
	R: CCCCATTTACCTGTATGCTATACT
Gp_cp_61	F: GTTCAGCCAATAGGGGAGGG	(T)9	159
	R: TAAGTCCCAGGTCCCGCAT
Gp_cp_62	F: TGTCTACGTGCATAAACTCTTTTC	(T)9	209
	R: ACCACGCTCATCTCATGTAC
Gp_cp_63	F: CCACCTATGCCCATACGGTC	(T)9	127
	R: TCGATTGACCTGAGGACCTT
Gp_cp_64	F: GGGTACCGGGTTCTATTGAAT	(A)8	149
	R: TCGATCTATGCCGCCTTACT
Gp_cp_70	F: TCGAGCCGTATGAAGATAAACCT	(G)11	137
	R: GCTCTTCCTTCGCTTCGAG

Optimal annealing temperature was 55°C for all loci.