Characterization of 30 microsatellite markers in distylous Primula sinolisteri (Primulaceae) using HiSeq sequencing.

PREMISE OF THE STUDY: Microsatellite markers were developed for Primula sinolisteri, a perennial distylous herb belonging to section Obconicolisteri (Primulaceae), to facilitate future investigations of the population genetics and mating patterns of populations in this species. METHODS AND
RESULTS: We developed 30 microsatellite markers for P. sinolisteri using HiSeq X-Ten sequencing and measured polymorphism and genetic diversity in a sample of 36 individuals from three natural populations. The markers displayed relatively high polymorphism, with the number of observed alleles per locus ranging from one to 19 (mean = 4.42). The observed and expected heterozygosity ranged from 0-1.000 and 0.083-0.882, respectively. Twenty-nine of the loci were also successfully amplified in homostylous P. sinolisteri var. aspera.
CONCLUSIONS: The microsatellite markers we have identified in P. sinolisteri provide powerful tools for investigating patterns of population genetic diversity and the evolutionary relationships between heterostyly and homostyly in this species.

Primula sinolisteri Balf. f. var. sinolisteri (Primulaceae) is an animal‐pollinated, perennial, herbaceous species belonging to Primula L. sect. Obconicolisteri. It is restricted to northwestern Yunnan, from the Dali range to the Tibetan border (Hu and Kelso, 1996; Richards, 2003), and commonly occurs in dry rocky pastures at elevations between 2300 and 3000 m. Primula sinolisteri var. sinolisteri exhibits distyly, with populations comprising long‐styled and short‐styled floral morphs. However, P. sinolisteri var. aspera W. W. Sm. & H. R. Fletcher possesses a different floral phenotype in which stigmas and anthers occur at a similar height within flowers, a condition known as homostyly. Distyly and homostyly are widely reported in Primula (Richards, 2003). Phylogenetic reconstructions clearly indicate that the most recent common ancestor of Primula was distylous, and that homostyly has evolved repeatedly in the genus as a result of the evolutionary breakdown of heterostyly and transitions from outcrossing to selfing (Mast et al., 2006; Zhou et al., 2017).

Recent investigations of the molecular genetic architecture of the heterostyly linkage group in Primula provide an opportunity for comparative genetic analysis of the evolutionary events associated with the origin and breakdown of heterostyly (Huu et al., 2016; Li et al., 2016; Burrows and McCubbin, 2017). Therefore, because both intraspecific and interspecific variation in floral conditions occur in P. sinolisteri var. sinolisteri and closely related taxa within section Obconicolisteri, this group provides an outstanding opportunity for investigating the evolutionary relationships between distyly and homostyly and the ecological causes and population genetic consequences of mating system transitions.

Next‐generation sequencing technology is now widely used in many areas of evolutionary biology, including the development of microsatellite markers for population genetic studies. Highly polymorphic microsatellite markers are useful tools for measuring the genetic diversity and structure of plant populations as well as patterns of mating (e.g., Matheny et al., 2013; Zhou et al., 2015, 2017; Yuan et al., 2017). Here, we used next‐generation sequencing to develop a set of variable microsatellite markers in P. sinolisteri var. sinolisteri.

METHODS AND RESULTS

We isolated total genomic DNA from leaf tissue of one P. sinolisteri var. sinolisteri individual from the population SIN_QBX (Appendix 1) following a modified version of the cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). We prepared a library using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA). We performed sequencing on a HiSeq X‐Ten sequencer (Illumina, San Diego, California, USA) using 2 × 150 bp read length. Raw reads were obtained and deposited in the National Center for Biotechnology Information Sequence Read Archive (BioProject ID PRJNA485859; accession no. SRP157868). Using Geneious version 6.0 (Biomatters, Auckland, New Zealand), the resulting 10,687,169 raw reads were quality filtered by trimming adapter sequences and by removing reads with quality scores <10. Using the built‐in Geneious assembler, the cleaned reads were then assembled into 55,878 contigs with high sensitivity/medium for the sensitivity setting. Plastome contigs were identified using BLASTX against GenBank and were excluded.

We used the MIcroSAtellite identification tool (MISA; Thiel et al., 2003) to identify unique reads containing microsatellites based on the following criteria: more than five repeats for dinucleotides to hexanucleotides and 100 bp for the maximal number of bases between two adjacent microsatellites. Minimum product size was set to 100 bp. A total of 3264 contigs contained at least one microsatellite. Two hundred simple sequence repeat (SSR) loci with di‐ or trinucleotide repeats were randomly selected for further characterization. Primers were designed for these loci using PRIMER version 5.0 (Clarke and Gorley, 2001) using the automatic search model to detect paired PCR primers of 24 bp in length. We used a Veriti 96‐well Thermal Cycler Gradient PCR Machine (Applied Biosystems, Foster City, California, USA) to test and optimize these primers initially.

Preliminary amplification tests were carried out with four individuals of P. sinolisteri var. sinolisteri from the SIN_QBX population (Appendix 1). We performed PCR amplification using the following protocol: 20‐μL total reaction volume containing 10 μL of Master Mix (Tiangen Biotech, Beijing, China; including 3 mmol∙L−1 MgCl2, 100 mmol∙L−1 KCl, 0.5 mmol∙L−1 of each dNTP, 20 mmol∙L−1 Tris‐HCl [pH 8.3], and 0.1 units Taq polymerase), 0.6 μmol∙L−1 of each primer, 8.4 μL of deionized water, and 30–50 ng of genomic DNA. We conducted PCR amplification under the following conditions: 95°C for 3 min followed by 30 to 35 cycles at 95°C for 30 s, at the annealing temperature for each specific primer (optimized for each locus; Table 1) for 30 s, 72°C for 30 s for extension, and a final extension step at 72°C for 5 min. We separated and visualized PCR products using a QIAxcel capillary gel electrophoresis system (QIAGEN, Valencia, California, USA) with an internal 10–300‐bp size standard. Out of the 200 primer pairs that we tested, 30 microsatellite loci amplified successfully with suitable fragment lengths and showed polymorphism (Table 1).

Table 1

Characteristics of 30 microsatellite loci isolated from Primula sinolisteri var. sinolisteri

Locus	Primer sequences (5′–3′)	Repeat motif	Fragment size range (bp)	T a (°C)	GenBank accession no.
PROB15	F: ATTGCCAGACAGAAAAAGGC	(AT)7	295–307	53.9	MH180228
	R: CACAGTAAATTCATCACAGCAACA
PROB29	F: GCTTCCCAATCAAAACAATACC	(AC)11	151–209	54.7	MH180229
	R: GACTCGTCGGATTTGTCCAT
PROB46	F: AGGCCATTACCCCCATAAAC	(CT)11	152–166	54.4	MH180230
	R: TGGGCAAAGGAAGAAGAAGA
PROB48	F: CATTTGAATTTTGGACGCCT	(AC)7	256–304	52.3	MH180231
	R: TACGGGTGAATTCGTCATTG
PROB54	F: CGACCAGGATTGATGTTGTG	(TG)7	146–156	55.4	MH180232
	R: TGGTTCCGGAAACTACCATC
PROB55	F: TGTCTATCGTGGTGGGTTCA	(GA)11	105–121	54.4	MH180233
	R: ATTCCGGGGTTTAATATCGG
PROB63	F: CCGCACCCAATCATATATCC	(AG)15	140–154	55.4	MH180234
	R: GCTCAAAGATCTGCGAAACC
PROB70	F: TGAGGAAATTGATGGTGCAA	(AG)14	111–125	54.4	MH180235
	R: GAAAGGTCAAGTGGAGCAGC
PROB72	F: TTTGGCCTTGCTTATTCACC	(AG)12	168–190	54.3	MH180236
	R: AAATTTAGGGTGGTGGGGG
PROB73	F: ACCGATTTGACCTCTCATGC	(TC)6	95–111	55.4	MH180237
	R: CATGCCTCTGTCATCCATTG
PROB83	F: TGCCAATTGCCATCCTTAAT	(TG)8	189–207	52.3	MH180238
	R: TAAGTGGCAATGGTGGTGAA
PROB100	F: GCTTTTGTTGTTCCAGCCAT	(TCT)7	106–176	55.4	MH180239
	R: AGCCCAGCAGTTCTGGAGTA
PB01	F: TCGTCATCATCCATTCACAA	(AG)11	144–172	50.9	MH180240
	R: GATGAGATTGGGTTTGTGGC
PB02	F: AGCATGCTGAAGTAAGGCTTC	(AT)6	202–254	52.0	MH180241
	R: GGATCGGTTTGAATGGAATG
PB18	F: GGGGAAATTGAGGACACAAA	(AG)10	228–238	50.9	MH180242
	R: TGGATCGGTATCAGCATTGA
PB31	F: GCCATAAAGCAGGGTCCATA	(CT)10	152–174	55.0	MH180243
	R: CTGTCGCTTGAGTAGCCGGT
PB34	F: TTTTTCTCCTGTGTGGGGAC	(GT)14	193–205	51.9	MH180244
	R: AATCGTGCATTCGTTCCTTC
PB35	F: TCACCCTCTCAACAAAACCC	(GT)10	182–208	51.9	MH180245
	R: GCTTTGATAAGCGGCATCAT
PB49	F: AAAGGGGAATGGATTGAACC	(TC)7	167–205	51.9	MH180246
	R: ACCAGTGTTGGCGTTAGCTT
PB51	F: GAACTTCAAGGTGAGCTGCC	(GA)7	225–251	54.9	MH180247
	R: GGTGGTGTTGGGTTCGTATC
PB56	F: GCACGAACGAGGAGTAGGAG	(GA)10	234–262	55.0	MH180248
	R: AAAGCAACCAACTCCCCTCT
PB59	F: GGCCCCATGACAAACATATC	(TA)8	224–248	53.9	MH180249
	R: GGTGAGAACCGTACTCCGAA
PB60	F: ATGTTTGGGAACCCATTGAA	(CA)15	221–233	49.9	MH180250
	R: TCATTGAGACATGGCGAGTT
PB61	F: GAGACACCTGCTCACAACGA	(CT)15	211–221	53.9	MH180251
	R: TCTTGCAGGCAGCTACAGAA
PB64	F: TATTGGATCGGAGTTGGAGC	(AC)8	151–171	51.9	MH180252
	R: AGGCTTAAAAGATGCAGCCA
PB66	F: GAAAAGCAAAATGGAACGGA	(GA)7	162–174	50.9	MH180253
	R: GCTGCCTTTCAGGTGTGTTT
PB72	F: CAAAGTCGATGACCGGAACT	(TC)8	201–209	53.9	MH180254
	R: CCAGATCCCACGGTTAGTGT
PB84	F: CACTTTGGTGGGCTATGGAA	(CT)14	141–171	51.9	MH180255
	R: AGCCAAGATTTGTGCAATCC
PB85	F: GGGCCAAAGCGAATAGACAT	(TG)17	164–174	52.9	MH180256
	R: ATATACGCCGGTCTCCCTTT
PB95	F: TGGAGGTGAAACTGGAGGAG	(GTG)6	129–135	52.9	MH180257
	R: TTTGTTAATGAGAGCGCGTG

T a = annealing temperature.

For these 30 successful loci, we measured polymorphism in 36 individuals obtained from three natural populations of distylous P. sinolisteri var. sinolisteri and six individuals from one population of homostylous P. sinolisteri var. aspera (Appendix 1). We calculated basic population genetic parameters of diversity, including the number of alleles and observed and unbiased expected heterozygosity, using GenAlEx version 6.5 (Peakall and Smouse, 2012). We tested for deviations from Hardy–Weinberg equilibrium at each locus using GENEPOP version 4.0.7 (Rousset, 2008). Null alleles were detected by MICRO‐CHECKER (van Oosterhout et al., 2004).

The number of alleles per locus ranged from one to 19, with a mean (±SD) = 4.42 ± 1.977 (Table 2). Among polymorphic loci, the observed heterozygosity and expected heterozygosity ranged from 0–1.000 (mean ± SD = 0.564 ± 0.260) and 0.083–0.882 (0.626 ± 0.180), respectively. The inbreeding coefficient ranged from −0.599 to 1.000. Some loci deviated significantly from Hardy–Weinberg equilibrium in each population (Table 2), as a result of heterozygote deficiency. This can likely be attributed to the presence of null alleles as detected by MICRO‐CHECKER (Table 2). Among the 30 SSR markers, 29 loci were successfully amplified in P. sinolisteri var. aspera (Table 2).

Table 2

Population genetic parameters in three populations of Primula sinolisteri var. sinolisteri and amplification tests in P. sinolisteri var. aspera.a

Primula sinolisteri var. sinolisteri													P. sinolisteri var. aspera
	QBX (n = 12)				JZ (n = 12)				MRS (n = 12)				TCA (n = 6)
Locus	A	H o	H e	F IS	A	H o	H e	F IS	A	H o	H e	F IS	Total A	Size range (bp)
PROB15	3	0.250	0.351c	0.287	2	0.545	0.496	−0.100	3	0.250	0.601c	0.584	5	305
PROB29	7	0.900	0.790	−0.139	7	0.818	0.661	−0.238	11	0.917	0.882	−0.040	19	203
PROB46	3	0.583	0.469	−0.244	5	0.833	0.712c	−0.171	5b	0.417	0.601c	0.306	6	154
PROB48	3	0.667	0.497	−0.343	1	NA	NA	NA	1	NA	NA	NA	3	256
PROB54	3	0.500	0.403	−0.241	2	0.545	0.397	−0.375	3	0.500	0.663c	0.246	4	154–158
PROB55	7b	0.500	0.799c	0.374	5	0.750	0.781c	0.040	7	0.750	0.851	0.118	9	105–107
PROB63	4	0.545	0.550c	0.008	5	0.750	0.726	−0.033	5b	0.333	0.590c	0.435	7	168
PROB70	7b	0.500	0.802c	0.377	6	0.833	0.767	−0.086	6b	0.500	0.788	0.366	8	117
PROB72	8	0.909	0.826	−0.100	6b	0.417	0.715	0.417	5	0.667	0.750	0.111	9	186
PROB73	3	0.455	0.483	0.060	3	0.333	0.538c	0.381	3	0.333	0.611c	0.455	5	97–101
PROB83	6	0.667	0.740	0.099	5	0.833	0.736c	−0.132	6b	0.455	0.727	0.375	8	205
PROB100	3	0.583	0.594	0.019	1	NA	NA	NA	7	0.917	0.813	−0.128	9	110–112
PB01	6b	0.455	0.769	0.408	4b	0.417	0.719c	0.420	4	0.500	0.705	0.291	10	146
PB02	5b	0.167	0.632c	0.736	6	0.917	0.767c	−0.196	5	0.667	0.681	0.020	8	204
PB18	5	0.750	0.712	−0.054	4	0.750	0.656	−0.143	1	NA	NA	NA	6	232
PB31	b	0.750	0.833c	0.100	7	1.000	0.844	−0.185	7	0.667	0.819	0.186	9	162
PB34	1	NA	NA	NA	6	0.833	0.781c	−0.067	1	NA	NA	NA	6	—
PB35	7	0.917	0.795	−0.153	4b	0.083	0.358c	0.768	5	0.750	0.701	−0.069	10	196–198
PB49	6	0.750	0.767	0.022	5	1.000	0.722c	−0.385	5	0.583	0.642c	0.092	9	171–173
PB51	2	0.000	0.153c	1.000	2	0.080	0.083	0.036	2	0.250	0.330	0.242	3	227
PB56	4	0.417	0.413	−0.010	4	0.583	0.660	0.117	8b	0.417	0.674c	0.381	8	238
PB59	3b	0.250	0.455c	0.451	3	0.750	0.594	−0.263	3	0.167	0.156	−0.067	4	264–266
PB60	5	0.750	0.681	−0.101	4	0.667	0.694c	0.039	2	0.091	0.087	−0.048	7	223
PB61	4b	0.083	0.615c	0.865	1	NA	NA	NA	5b	0.364	0.702	0.482	5	235
PB64	6	0.583	0.771c	0.244	4	0.250	0.295c	0.153	6	0.750	0.792	0.053	9	151–153
PB66	3	0.333	0.288	−0.156	4	0.333	0.545	0.389	4b	0.167	0.573	0.709	6	168
PB72	5	0.917	0.788	−0.164	4	0.636	0.680	0.065	1	NA	NA	NA	5	205
PB84	5	0.667	0.767	0.130	3	0.083	0.226c	0.633	6b	0.500	0.743	0.327	6	161–163
PB85	5	0.583	0.757	0.239	2	0.512	0.417	−0.228	4	0.417	0.663c	0.372	6	172
PB95	2	0.750	0.469c	−0.599	2b	0.000	0.500c	1.000	2	0.333	0.278	−0.200	3	129–132
Mean	4.633	0.558	0.620	0.107	3.900	0.576	0.595	0.069	4.433	0.416	0.547	0.218	7.067	—

— = unsuccessful PCR amplification; A = number of alleles per locus; F IS = inbreeding coefficient; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals; NA = not applicable.

Voucher and locality information are provided in Appendix 1.

Significant frequency of null alleles (P < 0.05).

Significant deviation from Hardy–Weinberg equilibrium (P < 0.05).

CONCLUSIONS

The microsatellite markers that we have isolated in P. sinolisteri var. sinolisteri will provide a valuable resource for investigating mating systems, population genetic structure, and phylogeography in P. sinolisteri and its varieties. It will be of particular interest to investigate the evolutionary relationships between distylous and homostylous populations and determine the number of transitions from outcrossing to selfing and their genetic consequences. The high discriminatory power of the microsatellite markers that we have identified will also be useful for parentage analysis and measures of disassortative mating in populations and should provide opportunities to evaluate the potential influence of ecological, demographic, and reproductive factors on mating patterns.

DATA ACCESSIBILITY

Raw reads were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (BioProject ID PRJNA485859; accession no. SRP157868). Sequence information for the developed primers has been deposited to NCBI; GenBank accession numbers are provided in Table 1.

Species	Population code	Floral morph structure	Voucher no.	Location	Geographic coordinates	Elevation (m)	n
Primula sinolisteri Balf. f. var. sinolisteri	SIN_QBX	Distyly	Z. Wei 123	Dali, China	25°64.386′N, 100°13.554′E	2743	12
Primula sinolisteri var. sinolisteri	SIN_JZ	Distyly	Z. Wei 112	Jinzhan, China	25°80.183′N, 99°99.014′E	2736	12
Primula sinolisteri var. sinolisteri	SIN_MRS	Distyly	Z. Wei 118	Heqing, China	25°25.829′N, 100°128.13′E	2934	12
Primula sinolisteri var. aspera W. W. Sm. & H. R. Fletcher	ASP_TCA	Homostyly	Z. Wei 203	Gaoligong Mountain, China	25°17.27′N, 98°43.50′E	2963	6