Characterization of 30 microsatellite markers for distylous Primula denticulata (Primulaceae) using HiSeq sequencing

Primula denticulata exhibits considerable variation in ﬂoral morphology and ﬂowering phenology along elevational gradients in SW China. We isolated 30 microsatellite markers from P. denticulata to facilitate further investigation of population genetics and ﬂoral evolution in this species. We used the HiSeq X-Ten sequencing system to develop a set of markers, and measured polymorphism and genetic diversity in a sample of 72 individuals from three natural populations of P. denticulata subsp. denticulata . The markers displayed relatively high polymorphism, with the number of alleles ranging from two to seven (mean = 3.567). The observed and expected heterozygosity ranged from 0 to 1.000 and 0.041 to 0.702, respectively. Twenty-eight of the loci were also successfully ampliﬁed in P. denticulata subsp. sinodenticulata . The microsatellite markers we have identiﬁed will provide valuable tools for investigations of the population genetic structure, mating systems and phylogeography of the P. denticulata complex, and will help to address questions concerning the ecological and genetic mechanisms responsible for the evolution of reproductive traits in the species.

Primula denticulata Smith (Primulaceae) is an insectpollinated, perennial herb belonging to Primula section Denticulata. The species is mainly restricted to China and is distributed from the southern margin of the Tibetan plateau to the Hengduan mountain region, commonly occurring in moist meadows, on open slopes, along roadsides and among shrubs (Hu and Kelso, 1996;Richards, 2003). Populations are typically distylous, comprising long-styled and short-styled floral morphs (Hu and Kelso, 1996). Primula denticulata is composed of two subspecies: subsp. denticulata is restricted to the Tibetan plateau at elevations between 2,800 and 4,600 m and subsp. sinodenticulata occurs in Yunnan, Sichuan and north-west Guizhou at lower elevations between 1,500 and 3,000 m. Primula denticulata subsp. sinodenticulata flowers from February to April whereas P. denticulata subsp. denticulata flowers from the middle of April to July. The flowering time of populations varies clinally along elevational gradients, along with a range of other reproductive characters including scape height, flower number and size, and herkogamy (stigma-anther separation). It is not yet known whether clinal variation is adaptive and maintained by natural selection, and evaluating this hypothesis is a major goal of our ongoing research on the ecological genetics of the species.
Distylous Primula species are well-known model systems for investigations of the pollination biology and mating systems of populations (Richards, 2003;de Vos et al., 2014de Vos et al., , 2018, including several Chinese species (Yuan et al., 2017;Zhou et al., 2017;Zhong et al., 2019). In addition, Primula species have been the recent focus of detailed studies of the molecular genetic architecture of the distylous linkage group (Nowak et al., 2015;Huu et al., 2016Huu et al., , 2020Li et al., 2016). The occurrence of conspicuous population differentiation of ecologically relevant traits along elevational gradients in P. denticulata provides opportunities for investigating a range of questions associated with the mechanisms that may be responsible for clinal variation in the species. The development of genetic markers to enable studies of the patterns of genetic diversity and the phylogeographic relationships of populations across elevational gradients is a necessary first step for any future microevolutionary studies of the P. denticulata complex.
Of the different types of PCR-based molecular markers, microsatellites [or simple sequence repeats (SSRs)] have been used most extensively in molecular population genetics because they can be readily amplified by PCR and, importantly, because of the large amount of allelic variation that is usually evident at each locus (Merritt et al., 2015). These markers are abundant, distributed throughout the genome, and are highly polymorphic compared with other genetic markers, as well as being species-specific and co-dominant (Vieira et al., 2016). For these reasons, they are widely used as genetic markers in population genetic studies in most non-model species. Although direct genotyping-by-sequencing methods, such as restriction site-associated DNA sequencing (Baird et al., 2008), are useful for addressing certain types of questions in evolution and ecology, SSR markers are still an efficient, less labor-intensive and relatively cheap approach for isolating genetic markers in non-model species for studies of population genetic structure and mating systems (e.g., Barrett, 2007, 2008;Zhou et al., 2012Zhou et al., , 2015Yakimowski and Barrett, 2014). Here, we use next-generation sequencing to develop a set of variable microsatellite markers in P. denticulata for our further molecular population genetic analysis of the complex.
We chose a single individual of P. denticulata subsp. denticulata from population LX to design markers (see Supplementary Table S1), and isolated total genomic DNA from the leaf tissue using a modification of the CTAB (cetyltrimethylammonium bromide) protocol (Doyle and Doyle, 1987). Library preparation was carried out using using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). We performed paired-end sequencing on a HiSeq X-Ten sequencer (Illumina, San Diego, CA, USA) with 2 × 150 bp read length. We obtained approximately 2 Gb of sequence data and deposited it in the National Center for Biotechnology Information Sequence Read Archive. The BioProject ID is PRJNA588183, and the accession number is SRR10419389. We performed a preliminary quality check of 3,122,591 raw reads with FastQC version 0.11.2. The clean reads were filtered from raw reads after trimming adapter sequences and removing both ambiguous ('N' > 10%) and low-quality reads (Phred score < 30) to ensure overall quality. We used the built-in Geneious assembler tool version 6.0 (Biomatters, Auckland, New Zealand) to assemble the cleaned reads into 16,109 contigs with high sensitivity/medium for the sensitivity setting. We identified and excluded plastome contigs using BLASTX against GenBank.
We used the MIcroSAtellite identification tool (Thiel et al., 2003) to identify unique reads containing microsatellites, and these satisfied the following criteria: more than five repeats for dinucleotides to hexanucleotides, and 100 bp for the maximal number of bases between two contiguous microsatellites. Minimal product size was set to 100 bp. In total, we isolated 2,939 contigs containing at least one microsatellite site. We randomly selected 150 SSR loci with di-or trinucleotide repeats for further characterization. We designed primers for these loci using PRIMER version 5.0 (Clarke and Gorley, 2001), in which we used the automatic search model to detect paired PCR primers. We used a Veriti 96-well Thermal Cycler gradient PCR machine (Applied Biosystems, Foster City, CA, USA) to initially test and optimize these primers.
We carried out the preliminary amplification tests with five individuals of P. denticulata subsp. denticulata from the LX 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 MgCl 2 , 100 mM KCl, 0.5 mM of each dNTP, 20 mM Tris-HCl, pH 8.3 and 0.1 units Taq polymerase), 0.5 μl of each primer, 8.5 μl of deionized water and 30-40 ng of genome DNA. The PCR amplification was implemented under the following conditions: 95 °C for 3 min; 30 cycles at 95 °C for 30 s, at the annealing temperature (optimized for each locus in Table 1) for each specific primer for 30 s, and 72 °C for 30 s for extension; and a final extension step at 72 °C for 10 min. We separated PCR products on 8% polyacrylamide denaturing gels using a 100 bp ladder molecular size standard (Thermo Fisher Scientific; Fermentas, Shenzhen, China) and visualized them by silver staining. Out of the 150 primer pairs that we tested, 30 microsatellite loci amplified successfully with suitable fragment lengths and all exhibited polymorphism ( Table  1). As evident from earlier studies of congeneric species of Primula (four loci in Yuan et al., 2017;10 loci in Zhou et al., 2017;15 loci in Zhong et al., 2019), 30 microsatellite loci with high polymorphism is sufficient for addressing most questions on the population genetic structure and mating systems of Primula populations.
We measured polymorphism for the above-mentioned 30 loci in 72 individuals sampled from three natural populations of P. denticulata subsp. denticulata located in Tibet and five individuals from one population of P. denticulata subsp. sinodenticulata from Yunnan (Supplementary Table S1). We calculated basic population  Table 2. Population genetic parameters in three populations of P. denticulata subsp. denticulata and amplification tests in P. denticulata subsp. sinodenticulata a Locus Primula denticulata subsp. denticulata Primula denticulata subsp. sinodenticulata  genetic parameters of diversity, including the number of alleles and observed/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), and detected null alleles using MICRO-CHECKER (van Oosterhout et al., 2004). The number of alleles of each locus ranged from 2 to 7, with the mean ± SD = 3.567 ± 1.278 (Table 2). Among all the polymorphic loci, the observed and expected het-erozygosity ranged from 0 to 1.000 (0.394 ± 0.267) and 0.041 to 0.702 (0.436 ± 0.165), respectively. The inbreeding coefficients ranged from − 1.000 to 1.000. The locus deviation from Hardy-Weinberg equilibrium in each population is indicated in Table 2. Among the 30 SSR markers, 28 loci were successfully amplified in a population of P. denticulata subsp. sinodenticulata sampled from Yunnan (Table 2).
Our results for P. denticulata are consistent with the distylous and outbreeding mating system of populations, and are broadly similar to those we have previously reported in distylous populations of P. sinolisteri . All loci were polymorphic across three populations of P. denticulata subsp. denticulata sampled from Tibet. In addition, we confirmed cross-amplification of these microsatellite loci in P. denticulata subsp. sinodenticulata from Yunnan. The markers will be useful tools for future genotyping and studies of population genetic structure in the P. denticulata complex. The high discriminatory power of the markers will also be useful for parentage analysis in dimorphic populations and should provide opportunities to investigate the potential influence of ecological and demographic factors on mating patterns.