Development of polymorphic microsatellite markers for distylous–homostylous Primula secundiflora (Primulaceae) using HiSeq sequencing

ABSTRACT

Primula secundiflora is an insect-pollinated, perennial herb belonging to the section Proliferae (Primulaceae) that exhibits considerable variation in its mating system, with predominantly outcrossing populations comprising long-styled and short-styled floral morphs and selfing populations comprising only homostyles. To facilitate future investigations of the population genetics and mating patterns of this species, we developed 25 microsatellite markers from P. secundiflora using next-generation sequencing and measured polymorphism and genetic diversity in a sample of 30 individuals from three natural populations. The markers displayed high polymorphism, with the number of observed alleles per locus ranging from three to 16 (mean = 8.36). The observed and expected heterozygosities ranged from 0.100 to 1.000 and 0.145 to 0.843, respectively. Twenty-one of the loci were also successfully amplified in P. denticulata. These microsatellite markers should provide powerful tools for investigating patterns of population genetic diversity and the evolutionary relationships between distyly and homostyly in P. secundiflora.

MAIN

Primula (Primulaceae) is an iconic taxon for the investigation of pollination and plant mating system evolution. Beginning with Darwin’s classic work on heterostyly in Primula (Darwin, 1877), the genus has received over a century of sustained interest and today is the most intensively studied heterostylous taxon (Mast and Conti, 2006). The vast majority of the ~400–500 Primula species are distylous, with the remaining species monomorphic for stylar condition and homostylous (Richards, 2003; de Vos et al., 2014). Homostyly is reliably reported from nearly half of the 38 sections of the genus, with phylogenetic analyses and ancestral state reconstructions indicating a single origin of distyly in Primula followed by numerous independent transitions to homostyly (Mast et al., 2006; de Vos et al., 2014; Zhong et al., 2019).

Primula secundiflora is an insect-pollinated, perennial herb belonging to the section Proliferae (Primulaceae). The species is widely distributed throughout southwest China and eastern Tibet and Qinghai, commonly occurring in moist meadows, on open slopes, and among shrubs (Hu and Kelso, 1996). As recorded, P. secundiflora exhibits distyly (Richards, 2003), with populations comprising long-styled and short-styled floral morphs enabling outcrossing mediated by pollinators. However, our recent field investigation found that the species also possesses monomorphic populations with a single floral phenotype, a condition known as homostyly. Therefore, because of the intraspecific variation in population floral morph structure (as shown in Yuan et al., 2017; Zhou et al., 2017; Shao et al., 2019; Zhong et al., 2019; Wang et al., 2021; Zhang et al., 2021; Zeng et al., 2024), this species provides an outstanding opportunity for investigating the evolutionary relationships between distyly (outcrossing) and homostyly (selfing) and the ecological causes and genetic consequences of mating system transitions.

Of the different types of PCR-based molecular markers, microsatellites, also known as simple sequence repeats or short tandem repeats, have been widely used in molecular population genetics studies of non-model species because the large amount of allelic variation at each locus can be readily amplified by PCR. The markers are commonly abundant, distributed in both nuclear and plastome genomes, and are highly polymorphic compared with other genetic markers, as well as being co-dominant and cross-species-transferable. With technical developments such as next-generation sequencing and multiplex PCR, the costs and labor of identifying and genotyping large numbers of microsatellite loci in non-model species have been greatly reduced. Marker identification therefore remains a popular approach for addressing certain types of questions in evolution and ecology. Here, we employed next-generation sequencing to develop a set of variable microsatellite markers in P. secundiflora for our further molecular population genetic analysis of this distylous–homostylous species.

We isolated total genomic DNA from silica-dried leaf tissue of one P. secundiflora individual sampled from population CD (see Supplementary Table S1) following a modification of the cetyl trimethyl ammonium bromide protocol (Doyle and Doyle, 1987). The purified DNA was sheared into ~700-bp fragments to construct a library according to the preparation procedures of NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). We then generated paired-end reads (2 × 150 bp) using a HiSeq X-Ten sequencer (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (Shenzhen, Guangdong, China). Approximately 2 Gb raw reads were obtained and deposited in the National Genomics Data Center of the China National Center for Bioinformation (BioProject: PRJCA021303; Genome Sequence Archive: CRA013486). We performed sequence quality screening using NGS QC TOOL KIT (Patel and Jain, 2012) with default parameters. Clean reads were generated from the raw sequences by removing adapter sequences, ambiguous reads (‘N’ > 10%) and low-quality reads (Phred score < 30) using Trimmomatic v.0.39 (Bolger et al., 2014). Using the built-in Geneious assembler (Biomatters, Auckland, New Zealand), the clean reads were then assembled into 63,752 contigs with high/medium for the sensitivity setting. We identified and excluded plastome contigs using BLASTX against GenBank.

We used QDD v.3.1.2 (Meglécz et al., 2014) to identify unique reads containing microsatellites from the scaffolds generated by de novo assembly, and these satisfied the following criteria: more than four repeats for dinucleotides to hexanucleotides, and 100 bp for the maximal number of bases between two contiguous microsatellites. The minimum product size was set to 120 bp. In total, we identified 4,762 contigs containing at least one microsatellite. One hundred simple sequence repeat loci with di- or trinucleotide repeats were randomly selected for further characterization. We designed primers for these loci using the automatic search model of PRIMER v.5.0 (Clarke and Gorley, 2001), taking into consideration the optimal PCR product size (100–300 bp), primer annealing temperature (57–61 ℃) and primer length (18–25 bp). We used a Veriti 96-well Thermal Cycler PCR Machine (Applied Biosystems, Foster City, CA, USA) to optimize these primers initially.

Preliminary amplification tests were carried out with four individuals of P. secundiflora from the CD population (Supplementary Table S1). 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 mM MgCl₂, 100 mM KCl, 0.5 mM each dNTP, 20 mM Tris-HCl [pH 8.3] and 0.1 U Taq polymerase), 0.5 μM each primer, 8.5 μl of deionized water and 30–50 ng of genomic DNA. We conducted PCR amplification under the following conditions: 95 ℃ for 3 min followed by 30–35 cycles at 95 ℃ for 30 s, at the annealing temperature for each specific primer (optimized for each locus; Table 1) for 30 s, 72 ℃ for 30 s for extension, and a final extension step at 72 ℃ for 10 min. We separated and visualized PCR products using a QIAxcel capillary gel electrophoresis system (QIAGEN, Valencia, CA, USA) with an internal 10–300-bp size standard. We first determined allele sizes using GENEMAPPER v.3.7 software with GeneScan-500 ROX as an internal-lane size standard (Applied Biosystems) and then rechecked the data manually to reduce scoring errors. Out of the 100 primer pairs that we tested, 25 microsatellite loci amplified successfully with suitable fragment lengths and showed polymorphism (Table 1). This number of microsatellite loci with high polymorphism is sufficient for addressing most questions on population genetics and mating systems of Primula populations, as evidenced by recent studies of congeneric species (Zhou et al., 2017; Zhong et al., 2019).

Table 1. Characteristics of 25 microsatellite loci isolated from Primula secundiflora

Locus	Primer sequence (5'–3')	Repeat motif	Fragment size (bp)	Ta (°C)	GenBase accession no.
PS647	F: GAGTAAATGCTGCTCCAGCTC R: CAAAACCCTCGTATGGTGCT	(AAT)10	262	59.7	C_AA051754
PS216	F: AGCAACCTAACGGATCAACTG R: CCGACAACGTTAAGGTAGGG	(AAT)5	197	59.5	C_AA051743
PS020	F: CATACATCAGCCATCGCTTC R: GCCTATGACGCCTTCTACCA	(CGC)6	214	60.2	C_AA051738
PS620	F: CGGTGTCTTCTATACTTTACGCC R: AGGTATAGAAAAGGGCCAACA	(TTA)5	286	57.6	C_AA051753
PS117	F: TGGGTTTCTGTTCTTGTTTCAG R: CCACCTCCACTACCACAACC	(AGC)8	256	60.1	C_AA051740
PS375	F: CAAAGCCTAAATCAGCACCA R: GGGGTTTGTGGTATTGGATG	(CT)7	116	58.8	C_AA051747
PS554	F: TGTTCGCCAAAGAACATAGC R: AATCAACTCAAAAGGGGGCT	(AG)6	227	58.4	C_AA051750
PS013	F: CCTTAAACACGGAGCTCACA R: GGTCGGTGTCTATGCATGTTT	(AC)7	150	59.8	C_AA051737
PS765	F: CTTTTCCTCAACATCAGAGGC R: CCTGACGTGTACAGAGGGGT	(AT)10	126	60.0	C_AA051756
PS897	F: AGATGAAGGCTTTTGTGAGGA R: TTTCGCGAATTGCCTTAAAC	(CT)8	234	59.8	C_AA051758
PS982	F: GACAACCCTAGGGAGGGAA R: TCCAAAATCGATAAGCGTCC	(AG)8	284	60.4	C_AA051761
PS223	F: TTTTCCCTTACATGCAAATCC R: CCTCTTTCTTTCGGCCTCTC	(AG)10	125	59.7	C_AA051744
PS413	F: AGGAGTGACGCCTCCTTTTA R: AATGAAGCGGTGGTGCTTAC	(TA)7	229	60.5	C_AA051748
PS433	F: TGCGTTACATCAACGCTACA R: CGTTGGAGATACAAAACGACG	(AG)10	145	57.0	C_AA051749
PS560	F: TGAAAGAGAAAGGACGGAAAA R: GCATTCCTCTTTTGGGTTCA	(AG)6	269	59.4	C_AA051751
PS768	F: CTTTCAACTGACATGAAACTTCG R: GGTGGGTGAAAAAGAAATGC	(TG)12	161	59.5	C_AA051757
PS282	F: TTCTTGCGAGTTTTTGTTGG R: ACTGGAATGCAATTGACCGT	(TA)6	165	60.0	C_AA051746
PS684	F: TTGGGGACATGTGTCACTCT R: AATAGCCAACCCGTTCTCCT	(AG)8	276	58.8	C_AA051755
PS082	F: TGATGCACGTGTGATTATGG R: CTCTACAACGAAAAACACTGGG	(TA)6	199	60.3	C_AA051739
PS940	F: CCGTTGGATTAAATCAAGATGA R: TTGGAACAGATCCACAGGGT	(TA)6	265	59.5	C_AA051759
PS957	F: CTACCGTACCCTTTTCCCAA R: TGCTGCAAGAAATCAAGCAT	(TC)6	168	60.1	C_AA051760
PS247	F: GAATTAAGGTTTCATCTCCATTTAC R: GGAGGAGAGAGAAAGTGGGG	(CT)6	266	59.6	C_AA051745
PS607	F: CGTTGTCGGGGAGTAATTTT R: CGGATCTCAAGAATCAGATGG	(TG)7	314	59.9	C_AA051752
PS212	F: GCACCAATGACAATTTAACAAAA R: TATGTGTCGATGACCGGAAA	(TA)6	132	60.1	C_AA051742
PS136	F: ACCAGAGGATGGACACTTCC R: TTGCTTCCATGATCCCTTTC	(AT)6	260	58.4	C_AA051741

For these 25 successful loci, we measured polymorphism in 30 individuals obtained from three natural distylous populations of P. secundiflora in Tibet and four individuals from one distylous population of P. denticulata in Tibet (Supplementary Table S1). We calculated basic population genetic parameters of diversity, including the number of alleles (N_A), observed (H_O) and expected heterozygosity (H_E) and inbreeding coefficient (F_IS), using GenAlEx v.6.5 (Peakall and Smouse, 2012). We tested for deviations from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium between microsatellite loci using GENEPOP v.4.7.2 (Rousset, 2008). Frequencies of null alleles were detected by MICRO-CHECKER (van Oosterhout et al., 2004).

The number of alleles per locus ranged from 2 to 16, with the mean ± SD = 8.36 ± 3.627 (Table 2). Among all the polymorphic loci, the H_O and H_E ranged from 0.100 to 1.000 (0.580 ± 0.269) and 0.145 to 0.843 (0.543 ± 0.205), respectively. The locus deviation from HWE in each population is indicated in Table 2, and there were no loci showing deviation from HWE in all three populations. Among the 25 microsatellite markers, 21 loci were successfully amplified in a population of P. denticulata sampled from Tibet, with allele numbers ranging from 1 to 5.

Table 2. Genetic diversity statistics of 25 polymorphic microsatellite loci in three populations of Primula secundiflora

Locus	Population												Total (n = 30)
	CD (n = 10)				MK (n = 10)				XLS (n = 10)
	NA	HO	HE	FIS	NA	HO	HE	FIS	NA	HO	HE	FIS	NA	Size range (bp)	HO	HE	FIS
PS647	4	1.000	0.715	-0.399	9	0.900	0.810	-0.111	5	1.000	0.735	-0.361	13	247–287	0.967	0.753	-0.290
PS216	5	0.800	0.630	-0.270	3ab	0.200	0.340	0.412	4	0.600	0.510	-0.176	9	175–199	0.533	0.493	-0.011
PS020	3abc	0.100	0.515	0.806	7b	0.800	0.760	-0.053	4ac	0.111	0.735	0.849	11	192–222	0.337	0.670	0.534
PS620	3a	1.000	0.605	-0.653	2a	1.000	0.500	-1.000	7	1.000	0.780	-0.282	9	264–292	1.000	0.628	-0.645
PS117	5b	0.400	0.485	0.175	2a	1.000	0.500	-1.000	1	0.000	0.000	0.000	7	200–252	0.467	0.328	-0.275
PS375	3	0.300	0.265	-0.132	3b	0.300	0.265	-0.132	2	0.300	0.255	-0.176	5	106–124	0.300	0.262	-0.147
PS554	6b	0.800	0.650	-0.231	5b	1.000	0.715	-0.399	3b	0.300	0.535	0.439	11	184–250	0.700	0.633	-0.064
PS013	3a	1.000	0.545	-0.835	2b	0.700	0.455	-0.538	3b	0.600	0.535	-0.121	5	156–180	0.767	0.512	-0.498
PS765	4ab	0.400	0.510	0.216	2b	0.500	0.495	-0.010	1	0.000	0.000	0.000	6	142–160	0.300	0.335	0.069
PS897	8	0.700	0.750	0.067	2b	0.200	0.480	0.583	4abc	0.300	0.565	0.469	12	227–284	0.400	0.598	0.373
PS982	3b	0.700	0.535	-0.308	4abc	0.200	0.480	0.583	2a	0.000	0.180	1.000	5	270–286	0.300	0.398	0.425
PS223	7	0.800	0.795	-0.006	9	1.000	0.850	-0.176	10	1.000	0.885	-0.130	15	141–171	0.933	0.843	-0.104
PS413	7b	0.700	0.775	0.097	10a	0.900	0.845	-0.065	9	0.800	0.840	0.048	16	204–246	0.800	0.820	0.027
PS433	2	0.100	0.095	-0.053	4b	0.500	0.625	0.200	1	0.000	0.000	0.000	6	147–165	0.200	0.240	0.049
PS560	1	0.000	0.000	0.000	2b	0.600	0.480	-0.250	1	0.000	0.000	0.000	2	264–267	0.200	0.160	-0.125
PS768	4	0.500	0.615	0.187	2a	1.000	0.500	-1.000	3	0.900	0.580	-0.552	6	146–173	0.800	0.565	-0.455
PS282	7b	0.800	0.790	-0.013	8	0.900	0.785	-0.146	10	0.900	0.845	-0.065	14	150–180	0.867	0.807	-0.075
PS684	7	0.700	0.765	0.085	5ac	0.400	0.760	0.474	6	0.500	0.730	0.315	10	260–298	0.533	0.752	0.291
PS082	5a	0.900	0.685	-0.314	4	0.600	0.565	-0.062	4	0.700	0.525	-0.333	8	187–220	0.733	0.592	-0.236
PS940	4b	0.200	0.345	0.420	4	0.500	0.575	0.130	4	0.600	0.575	-0.043	5	227–294	0.433	0.498	0.169
PS957	7b	1.000	0.760	-0.316	3b	0.800	0.640	-0.250	4	1.000	0.715	-0.399	7	156–204	0.933	0.705	-0.322
PS247	6b	0.700	0.600	-0.167	4	0.900	0.635	-0.417	3	0.600	0.620	0.032	7	262–286	0.733	0.618	-0.184
PS607	1	0.000	0.000	0.000	2	0.100	0.095	-0.053	3a	0.200	0.340	0.412	4	309–321	0.100	0.145	0.180
PS212	5	0.300	0.420	0.286	3b	0.700	0.565	-0.239	4	0.600	0.525	-0.143	7	139–160	0.533	0.503	-0.032
PS136	4	0.600	0.685	0.124	6b	0.500	0.700	0.286	7b	0.800	0.730	-0.096	9	264–294	0.633	0.705	0.105
Mean	4.56	0.580	0.541	-0.054	4.28	0.648	0.577	-0.129	4.20	0.512	0.510	0.033	8.36	–	0.580	0.543	-0.050

Note: Bold loci were successfully amplified in Primula denticulata; N_A, number of alleles per locus; H_O, observed heterozygosity; H_E, expected heterozygosity; F_IS, inbreeding coefficient; n, number of individuals.

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

^b Significant linkage disequilibrium (P < 0.05).

^c Null allele present.

The microsatellite markers that we have isolated in P. secundiflora will provide a valuable resource for investigating mating system, population genetic structure and phylogeography in P. secundiflora and its closely related species. It will be of particular interest to investigate the evolutionary relationships between distylous and homostylous populations and determine the direction of transitions between outcrossing and selfing and their genetic consequences. The high polymorphism of the microsatellite markers that we have identified will also be useful for parentage analysis and measures of mating patterns within the natural populations and should provide opportunities to assess the potential influence of demographic, ecological and reproductive factors on mating system evolution.

ACKNOWLEDGMENTS

This research was funded by the Top-notch Young Talents Project of Yunnan Provincial ‘Ten Thousand Talents Program’ (YNWR-QNBJ-2019-203), the Key Basic Research Program of Yunnan Province (202201AS070057, 202101BC070003 and 202103AC100003) and Yunnan Provincial Basic Research Special Project (202301AT070254). We are grateful to Z. R. Zhang for technical assistance. Laboratory work was performed at the Laboratory of Molecular Biology at the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences.

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