Development and characterization of microsatellite loci for the endemic Thalictrum smithii (Ranunculaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the gynodioecious Chinese endemic Thalictrum smithii (Ranunculaceae). METHODS AND
RESULTS: Thirty-nine microsatellite primers were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. Thirteen microsatellite loci were found to be highly polymorphic after screening 114 specimens (60 hermaphrodite and 54 female) from three T. smithii populations. The number of alleles per locus ranged from three to 13, and the levels of observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.204 to 0.834, respectively. Twenty-six of these primers were polymorphic in T. petaloideum and T. finetii.
CONCLUSIONS: These markers will be useful for examining genetic diversity, polyploidy, and mating system in populations of T. smithii and for guiding study on the evolution of speciation in Thalictrum.

Evolutionary transitions of sexual systems from hermaphrodite to unisexual have repeatedly occurred in different lineages of flowering plants. Transitions between sexual systems are often associated with changes in pollination modes. For example, many wind‐pollinated species have unisexual flowers (Delph et al., 2007; Friedman and Barrett, 2008). The genus Thalictrum L. (meadow‐rues; Ranunculaceae) has been considered an ideal group to explore the evolutionary transitions in sexual systems and pollination modes. There are numerous hermaphrodite and dioecious species, which are insect‐ or wind‐pollinated (Boivin, 1944; Soza et al., 2012, 2013). The evolutionary transition from hermaphrodite to dioecy has been repeatedly observed in diverse lineages of flowering plants via an intermediate stage of gynodioecy, a dimorphic sexual system in which female and hermaphrodite individuals co‐exist within the populations (e.g., Delph et al., 2007). Of the approximately 70 Thalictrum species found in China, our recent investigations in the Hengduan Mountains revealed that T. smithii B. Boivin was the only known gynodioecious species in this genus.

Microsatellite or simple sequence repeat (SSR) markers are co‐dominant with high levels of polymorphism and have been widely used in studies of genetic diversity, genome mapping, mating systems, and genetic differentiation (Jarne and Lagoda, 1996; Zhang and Hewitt, 2003; Li et al., 2015a). We developed and characterized 13 polymorphic microsatellite loci for T. smithii and tested the applicability of these SSR loci in three wild populations with different sex ratios to estimate genetic diversity of T. smithii.

Methods and results

Three T. smithii populations were sampled in western Sichuan Province, China (Songgangcun [SGC], Xianggelilazhen [XGLL], and Yajiangxian [YJX]; Appendix 1). Fresh leaves were collected from up to 20 randomly chosen individuals per sex type from each population; individuals were at least 10 m apart from each other. Furthermore, two monoecious Thalictrum species were collected in the Hengduan Mountains: T. petaloideum L. and T. finetii B. Boivin. Location, sample sizes, and sex ratio of these sampled populations are listed in Appendix 1. All flowering individuals were counted in each population to estimate the sex ratio.

Microsatellite loci were developed according to the method described by Li et al. (2015b). Using the QIAGEN DNA Extraction Kit (QIAGEN, Hilden, Germany) and the cetyltrimethylammonium bromide (CTAB) method (Porebski et al., 1997), total genomic DNA was extracted from the leaves of T. smithii sampled from the SGC population.

The Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) procedure was used for isolating microsatellite loci from an enriched (AG)n library (Zane et al., 2002). After being digested with MseI (Fermentas, Burlington, Ontario, Canada), genomic DNA was ligated to an MseI‐adapter pair (F: 5′‐TACTCAGGACTCAT‐3′, R: 5′‐GACGATGAGTCCTGAG‐3′) with T4 ligase (Promega Corporation, Madison, Wisconsin, USA). The diluted (1 : 10) digestion‐ligation mixture was directly amplified with MseI‐N primers (5′‐GATGAGTCCTGAGTAAN‐3′; 25 μM) in a total volume of 20 μL using the following thermocycler conditions: initial denaturation at 95°C for 3 min; followed by 26 cycles at 94°C for 30 s, annealing at 53°C for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 5 min.

Microsatellite repeats were selected using magnetic beads with 5′‐biotinylated (AC)15 and (AG)15 probes, which enriched amplified DNA fragments in the range of 200–800 bp. Nonspecific DNA fragments were removed by three non‐stringency washes with TEN1000 (10 mM Tris‐HCl, 1 mM EDTA, 1 M NaCl [pH 7.5]) followed by three stringency washes using 0.2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS). The enriched DNA fragments were then amplified via PCR using the primers MseI‐N for 26 cycles. After purification with a Gel Extraction Kit (TaKaRa Biotechnology Co., Dalian, Liaoning, China), the PCR products were cloned into the pGEM‐T vector (Promega Corporation) and transformed into DH5α cells (TransGen Biotech, Beijing, China). Recombinant clones were screened by blue/white selection, and the positive clones were tested by PCR using primers T7 and Sp6 separately to screen (AC)10‐rich or (AG)10‐rich clones.

The total of 96 clones with positive inserts were sequenced on an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, California, USA). Using the software SSR Hunter (Li and Wang, 2005), microsatellite repeat motif regions were identified. Among the 96 sequences analyzed, 77 contained microsatellite motifs. Thirty‐nine high‐quality sequences were selected after exclusion of redundant sequences, and the software OLIGO 7.0 (Rychlik, 2007) was used to design microsatellite primers with the following criteria: product length 80–300 bp, primer length 21 bp, melting temperature 48–68°C, GC content 27–60%. Fluorescent dyes 6‐FAM, TAMRA, or HEX (Invitrogen, Carlsbad, California, USA) were used to label the 39 pairs of primers (Table 1). Among three T. smithii populations (Appendix 1), 114 individuals were randomly selected to assess polymorphism and performance for the 39 SSR loci. Amplifications were performed in a 25‐μL reaction system containing 30–50 ng of genomic DNA, 0.6 μM of each primer, and 7.5 μL of 2× Taq PCR MasterMix (0.1 unit Taq polymerase/μL, 0.5 mM dNTP each, 20 mM Tris‐HCl [pH 8.3], 100 mM KCl, 3 mM MgCl2; Tiangen, Beijing, China). The thermocycling conditions were: 94°C for 5 min; 32 cycles at 94°C for 30 s, at an annealing temperature optimized for each specific primer for 30 s (Table 1), and at 72°C for 60 s; and a final extension at 72°C for 5 min. The PCR products were separated using an ABI PRISM 3730xl DNA sequencer with GeneScan 500 LIZ Size Standard (Applied Biosystems) and were genotyped using GeneMapper version 4.0 (Applied Biosystems).

Table 1

Characteristics of 39 microsatellite loci developed for Thalictrum smithii

Locus	Primer sequences (5′–3′)	Repeat motif	Allele size range (bp)	T a (°C)	Fluorescent dye	GenBank accession no.
Tsp01	F: CAGGTATATTGCTTCCTTTAC	(AG)10	100–140	56	6‐FAM	KY243242
	R: ATGGCTGGCCTCCTTGTTTGG
Tsp02	F: ATCAATGGCTATGAACATCTA	(AG)14	172	53	HEX	KY243241
	R: CATCCTAAACACCTATCTCTT
Tsp03	F: TTGAGAAAAACAGAGATGAAA	(AG)4(TG)5(AG)9	145–170	54	HEX	KY243240
	R: CAATAGCCTAAAACGGACACC
Tsp04	F: GATGGAAAAAATTCGAGCAGA	(CT)11	92–122	54	6‐FAM	KY243239
	R: ATTCGGGAGAGGAGAGCAAAA
Tsp05	F: AAAACGAAATTACACCAACCG	(TC)16	123–158	51	6‐FAM	KY243238
	R: TGCCCTGAAAGAGAACCACAT
Tsp06	F: CCCAAGATAGTTTACTCCGAG	(CT)12	125–137	53	6‐FAM	KY243257
	R: CAGTTGAAAGTGTTGACCTGA
Tsp07	F: AAGAGAATAGCGTGAGAGATA	(AG)8	185–240	51	HEX	KY243256
	R: AAAACAGAGAAGAAACAAAAA
Tsp08	F: GCTGCACATCACTACTCACT	(GA)13	279–295	50	6‐FAM	KY243255
	R: CAACACTTCATTCCCTTTTT
Tsp09	F: AACAGATAATTCTCACCTTTG	(AG)13	92–116	56	HEX	KY243254
	R: CCTTACACCTTTTGCTCCCGC
Tsp10	F: ATTCCCGATTCCAACTATAAA	(TC)9	98–125	55	6‐FAM	KY243253
	R: CGATCAAGAATTTGGGATTTT
Tsp11	F: AATGTAGCAAGCTATGGTACA	(AG)10	122–132	54	6‐FAM	KY243252
	R: CTCCACTAAATGCCTTTCAGA
Tsp12	F: CACTCTTGTCACTCACTCTCT	(GA)9	230–270	52	HEX	KY243251
	R: TTACTAAACCCAAATCATCTA
Tsp13	F: TTAATCAAATTGAAACCTAGA	(AG)12	179–201	50	6‐FAM	KY243250
	R: CAAGAAAAGAAAGAAGAGAAT
Tsp14	F: GCCTTGTTCAACCCTCCATAC	(CT)10	100–110	51	HEX	KY243249
	R: TACTACTTCTACCTTTCTTTC
Tsp15	F: TCACACCTCTTCCACCACATT	(TC)10	92–108	50	TAMRA	KY243248
	R: AATTCTGGTTTTCTGGCCGGA
Tsp16	F: TTTTGATTTTCTCTCTCTTCTA	(CT)11	116–140	52	HEX	KY243247
	R: CTTGAGATAGGTTAGTGTACCA
Tsp17	F: TCCTACAGCCAGCGAATCAATG	(TC)11	128–164	50	6‐FAM	KY243246
	R: ACACCACTTCGTCAGACGGTCA
Tsp18	F: TCTACTCTGTTCTTCTGCTGG	(TC)12	203–241	52	HEX	KY243245
	R: TAAAAAATGGGGTACCTTGCG
Tsp19	F: CGGAAGGAGATTGAAGAAGGA	(CT)10	212–216	58	TAMRA	KY243244
	R: TGCCCAAGGGGGTTGGGTATG
Tsp20	F: ACTTCTCTCTTTCTCTTCTCC	(CT)12	115–150	54	6‐FAM	KY243243
	R: GATTGATGCTTTTTTTTTATT
Tsp21	F: ATTCAAAGCCTGTGTCAGATC	(AG)10	120–140	55	6‐FAM	KY243265
	R: GCAGCCATTCTTATTTCATTT
Tsp22	F: TATCTTTCATTCACTCCACAC	(TC)9	86–105	58	6‐FAM	KY243264
	R: ACCTAATTAGCCGCATGAAGC
Tsp23	F: TTAAGAACAGACATCGATCCA	(TC)9	120–132	56	6‐FAM	KY243263
	R: TCAGAAACAGTAAACACAAAG
Tsp24	F: TTAAGGAAAACCGAGAAGCAT	(AG)9	96	52	TAMRA	KY243262
	R: TTCCAAGATGAAACGAGTAAA
Tsp25	F: CACCAGTAACAGAAACAAAAA	(GT)8	160–180	50	6‐FAM	KY243261
	R: GCCACAAGTCAATTCCAAGAT
Tsp27	F: ATCTCAGTTTCAGTACTTTCT	(CA)8	123–138	52	6‐FAM	KY243259
	R: TATATTCTCTTTCAATTTTTC
Tsp28	F: ATCAGAACTGTCTCAGGCTTT	(CA)7	140–158	54	6‐FAM	KY243258
	R: TAATTGGTTGGCATTATGTCT
Tsp29	F: GTTATGGCTAGAATGTTTTGTT	(TA)8	142–162	55	6‐FAM	KY243277
	R: TGTTGGTCTCAGCTTGAGAGGG
Tsp30	F: GAATTCGATATAGACTACTTC	(CA)9	118–128	50	6‐FAM	KY243276
	R: AATAATGTAGACAATCTAGTC
Tsp31	F: TCATTCTTCTGCTTCCTTTGC	(TG)9	169–202	56	HEX	KY243275
	R: CCACTACCTCCATCATCACCT
Tsp32	F: ACTTTACTACCACTTCCTCAA	(GT)9	200–224	52	6‐FAM	KY243274
	R: AGCCACAAACATGCCACCTTC
Tsp33	F: AAGGTAGCTGCTACAAGATCA	(AC)6	100–120	54	6‐FAM	KY243273
	R: TTCTGAGTACCAACCACTTCA
Tsp34	F: ATTGATGTTTTGATTTTTTGG	(CA)7	105–122	50	6‐FAM	KY243272
	R: AATTTAGCTGGATTTGCTTAG
Tsp35	F: GCGACTGTCAAACTTTAGAGA	(TG)8	80–110	52	6‐FAM	KY243271
	R: TTAGCGGCAAATGTAATTATC
Tsp36	F: TGCCTTTTATGGTCAGATTCC	(TG)6	200–330	53	HEX	KY243270
	R: TATTTTTGTTGCTCACGTTGG
Tsp37	F: TCACCAAAATAATAACGGCAC	(AC)9	97–121	53	6‐FAM	KY243269
	R: ATCCAAAATGGACACCAGACC
Tsp38	F: TTGTCACAAGTTTACAAGACA	(CA)8	183–200	52	TAMRA	KY243268
	R: GAAGCATAGGTAGAGGAAGGA
Tsp39	F: ACCTGGAATGTTCAGAAAGAT	(CT)6NNN(AC)11	128–164	52	6‐FAM	KY243267
	R: AACGACTTAGAAAATGGCAGA
Tsp40	F: AATTTACAGTACATACTTGGC	(AC)7	125–160	51	TAMRA	KY243266
	R: TTAACTCTAGATAGGTCTTGG

T a = annealing temperature.

Of the 39 primer pairs, 13 displayed polymorphism among individuals from the three T. smithii populations (Table 2). CERVUS version 3.0.7 (Kalinowski et al., 2007) was used to analyze the number of alleles per locus (A), observed and expected heterozygosity (H o and H e), and deviation from Hardy–Weinberg equilibrium. Across the three T. smithii populations, A ranged from three to 13, with a mean of 5.308 in the SGC population, 6.308 in the XGLL population, and 6.846 in the YJX population. Levels of H o and H e per locus ranged from 0.000 to 1.000 and from 0.204 to 0.834, respectively. A relatively high level of genetic diversity was found in the SGC population (H o = 0.489, A = 5.308) compared with the XGLL population (H o = 0.346, A = 6.308) and the YJX population (H o = 0.437, A = 6.846). Some loci showed significant deviation from Hardy–Weinberg equilibrium (Table 2).

Table 2

Results of initial primer screening of 13 polymorphic microsatellite loci in three populations of Thalictrum smithii.a

Locus	SGC (N = 34)			XGLL (N = 40)			YJX (N = 40)
	A	H o	H e b	A	H o	H e b	A	H o	H e b
Tsp04	9	0.618	0.722	7	0.450	0.597	8	0.450	0.768**
Tsp08	6	0.500	0.702	3	0.075	0.420	9	0.750	0.834
Tsp09	4	0.853	0.617*	6	0.625	0.516	5	0.750	0.602
Tsp13	4	0.382	0.673**	7	0.125	0.774**	8	0.325	0.802
Tsp15	5	0.235	0.445	3	0.025	0.204	5	0.200	0.640**
Tsp16	4	0.382	0.559*	4	0.150	0.670**	5	0.300	0.692**
Tsp17	4	0.500	0.712**	11	0.450	0.769**	9	0.675	0.829
Tsp18	6	0.794	0.767	12	0.500	0.803**	13	0.700	0.791
Tsp19	3	0.000	0.381	3	0.000	0.466**	3	0.050	0.507**
Tsp31	5	0.235	0.495**	6	0.300	0.574**	7	0.225	0.708**
Tsp32	5	1.000	0.569**	4	0.875	0.664**	7	0.575	0.492
Tsp37	9	0.500	0.757**	6	0.525	0.628	7	0.625	0.781
Tsp39	5	0.353	0.614**	10	0.400	0.828**	3	0.050	0.224
Average	5.308	0.489	0.616	6.308	0.346	0.609	6.846	0.437	0.667

A = number of alleles; H e = expected heterozygosity; H o = observed heterozygosity; N = sampled individuals from each population.

aSee Appendix 1 for locality and voucher information.

bDeviations from Hardy–Weinberg equilibrium: *P < 0.05, **P < 0.01.

The 39 primer pairs were also tested in T. petaloideum and T. finetii, and 26 of the developed primer pairs were found to be polymorphic (Table 3).

Table 3

Cross‐amplification and polymorphism analysis of 39 microsatellite markers developed for Thalictrum smithii in two related monoecious Thalictrum species.a , b

Locus	T. petaloideum					T. finetii
	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5	Sample 1	Sample 2	Sample 3	Sample 4	Sample 5
Tsp04c	—	—	—	—	—	94/110	94/110	110	110/112/120	120
Tsp08c	—	—	161	161	161	—	—	—	—	—
Tsp09c	92/98	98	98	98	98	—	98	84/98	—	98
Tsp13c	—	—	—	—	—	—	—	169	—	—
Tsp15c	92/96	92/96	92/96	92/96	92/96	96	96	96	98	96
Tsp16c	—	—	—	—	—	102/106/116	102	102/106/116	102	—
Tsp17c	146	146	146/158	146	146/152/158	158	158	158	—	—
Tsp18c	225	213/217/225	203/213/217	203/213	213/217	215/217	213/217	213/215/219	—	219
Tsp19c	—	—	—	—	—	—	—	—	210	—
Tsp31c	—	202	202/204/208	202/204	—	202/204	202	204/208/212	212	212
Tsp32c	216	216	216/220	216	—	206	206	206	206	206
Tsp37c	101/103	99/101/103	97/99	97/101	97/99	95/99	99	95/99	101	107
Tsp39c	118/134/136	134/136/138	118/134/138	118/136	118/136	110	110	110	110	110
Tsp01	—	—	135	135/139	135/139	105/119	119/137	105/119	—	—
Tsp02	—	—	—	—	—	—	—	—	—	—
Tsp03	—	—	—	147	147	—	—	—	—	—
Tsp05	135/137/141	137/141	125	125/151	125/151	121/125/129	125/137	125	121/125/151	125/129/137
Tsp06	—	—	—	136	—	—	—	—	—	—
Tsp07	188	188	188	186	188	198	198/216	198/216	212	212
Tsp10	—	—	102/104	102	102/104	—	—	—	—	—
Tsp11	—	—	—	—	—	—	—	—	—	—
Tsp12	—	—	—	—	—	251	251	251	263	263
Tsp14	—	—	—	—	—	—	—	—	—	—
Tsp20	—	—	—	—	—	—	—	—	—	—
Tsp21	—	—	—	—	—	—	—	—	—	—
Tsp22	—	—	—	—	—	—	—	—	—	—
Tsp23	—	—	—	—	126	—	—	—	—	—
Tsp24	—	—	—	—	—	—	—	—	—	—
Tsp25	169	—	169	169	169	165/169	165/169	165	169/167	165
Tsp27	—	—	—	—	—	—	—	—	—	—
Tsp28	—	—	—	—	—	—	—	—	—	—
Tsp29	152/154	152/156	154/156	152	154/156	154	154	154	152/154	152/154
Tsp30	—	—	—	—	—	—	—	—	—	—
Tsp33	103	103	103	103	103	103/119	103/119	103/119	103/105	103/105
Tsp34	—	—	116	116	116	—	—	—	116	—
Tsp35	80/82	82	82	80	82	84	84	84	82	84
Tsp36	—	—	—	—	—	—	—	—	—	—
Tsp38	—	—	—	—	—	—	—	—	—	—
Tsp40	148/154	154	138/144	138/144	138/144	144	144	144	154	154

— = no amplification product.

See Appendix 1 for locality and voucher information.

Values separated by a slash (/) represent band sizes, which vary as a result of either duplication(s) in other parts of the genome or nonspecific amplification.

Loci marked with an asterisk have high levels of polymorphism and have been grouped at the beginning of the table to facilitate review of these loci and their cross‐amplification and polymorphism information.

Conclusions

The SSR markers developed here can be used to estimate the levels of genetic diversity and gene flow in populations of T. smithii and to explore the evolutionary transitions in sexual systems in this genus from hermaphrodite to dioecy. Detailed information on the genetic consequences of sexual differentiation on T. smithii could also be obtained by using these SSR markers at larger scales.

Data accessibility

Sequence information for the developed primers has been deposited to the National Center for Biotechnology Information (NCBI); GenBank accession numbers are provided in Table 1.

Species	Population code	Population locality	Geographic coordinates	Sex ratioa	N	Voucher no.
T. smithii B. Boivin	SGC	Songgangcun, Sichuan Province, China	31.925939N, 102.108678E	0.87	F: 14, H: 20	CSUC605069
	XGLL	Xianggelilazhen, Sichuan Province, China	28.564108N, 100.351135E	0.59	F: 20, H: 20	CSUC607152
	YJX	Yajiangxian, Sichuan Province, China	30.033902N, 101.022107E	0.62	F: 20, H: 20	CSUC607145
T. petaloideum L.	LZP	Muli, Sichuan Province, China	28.026361N, 101.195139E	—	5	CSUC508033
T. finetii B. Boivin	KW	Muli, Sichuan Province, China	28.123889N, 101.213333E	—	5	CSUC508041

F = female; H = hermaphrodite; N = number of individuals sampled.

Sex ratio represents the frequency of hermaphrodites in each population.