Characterization of 24 microsatellite markers in Primula chungensis (Primulaceae), a distylous-homostylous species, using MiSeq sequencing.

Primula chungensis is a species with considerable floral and mating-system variation, including distylous (outcrossing), homostylous (selfing) and mixed populations that contain both outcrossing and selfing forms. We isolated 24 microsatellite markers from P. chungensis using Illumina MiSeq sequencing. Polymorphism and genetic diversity were then measured based on a sample of 24 individuals from a natural population in southern Tibet. All loci were polymorphic with the number of alleles per locus ranging from 2 to 4. The observed and expected heterozygosity ranged from 0 to 1 and 0.219 to 0.708, respectively. The microsatellite markers we have identified will serve as valuable tools for the investigation of the population genetic structure and phylogeography of P. chungensis and will inform models of the evolutionary history of mating systems in the species.

Introduction

Primula chungensis I. B. Balfour & Kingdon-Ward is a herbaceous, insect-pollinated, perennial species belonging to sect. Prolifera (Primulaceae) and restricted to the mountainous regions of Yunnan, Sichuan and Tibet of China (Hu and Kelso, 1996, Richards, 2002). It commonly occurs in wet meadows, forest edges, open slopes, and roadsides at altitudes between 2900 and 3200 m. The species flowers from May to June and produces one to three inflorescences composed of pale orange flowers (about 20 flowers per inflorescence) that last for up to 6 days. Our field investigations indicate that P. chungensis exhibits considerable variation in floral biology across its geographical range. Most populations are composed of a single self-pollinating floral phenotype with anthers and stigmas at equivalent height (homostyly). In contrast, other populations contain outcrossing long-styled and short-styled floral morphs typical of the floral polymorphism distyly. Populations containing both homostylous and heterostylous morphs also occur, and finally some populations are monomorphic for heterostylous morphs, especially the long-styled morph.

Primula is a well-known model system for studies of the evolution, function and breakdown of heterostyly (Crosby, 1949, Ornduff, 1979, Piper et al., 1984, Richards, 2002, Mast and Conti, 2006, Mast et al., 2006, de Vos et al., 2014, Keller et al., 2014), and more recently for investigations of the molecular genetics of the heterostyly linkage group (McCubbin et al., 2006, Li et al., 2011, Nowak et al., 2015). The occurrence of polymorphism for floral morphology and mating system in P. chungensis provides outstanding opportunities for investigating a range of questions associated with the evolutionary maintenance and breakdown of heterostyly. Development of genetic markers to investigate the patterns of genetic diversity in populations of contrasting mating systems (outcrossing vs. selfing), and to determine the evolutionary relationships between populations containing different floral phenotypes is a necessary first step for evolutionary studies of P. chungensis. Here, we report the isolation and characterization of 24 polymorphic microsatellite markers from P. chungensis, which will be valuable for furthering our understanding of the evolutionary genetics of this species.

Materials and methods

We isolated total genomic DNA from leaf tissue of one P. chungensis individual from the Muli population (28°54.604′ N, 100°47.268′ E; 3489 m a.s.l.) in Sichuan using DNeasy Plant Mini Kit (QIAGEN, Irvine, USA) following the manufacturer's protocol. Extracted DNA was used for a library preparation with a Nextera XT Library Prep Kit for Illumina. We performed sequencing on the MiSeq Benchtop sequencer (Illumina, Inc., San Diego, California, USA) using the 2 × 250 bp read length. Raw Illumina reads (679,708 reads) were analyzed and assembled using Geneious version 6.0 (Biomatters, Auckland, New Zealand) into 56,092 contigs. The contigs were BLASTed against NCBI GenBank using BLASTx to identify and exclude contigs with chloroplast genome hits. Microsatellites with at least 5 repeats were then detected using QDD version 2.1 Beta (Meglécz et al., 2010). A total of 2341 contigs contained at least one microsatellite, of which 127 loci were selected for primer design using the software PRIMER version 5.0 (Clarke and Gorley, 2001). These primers were initially tested and optimized using a Veriti 96-well Thermal Cycler Gradient PCR Machine (Applied Biosystems, Foster City, California, USA). A total of 24 primer pairs amplified consistently, and were used for further screening (Table 1). We assessed polymorphism at these loci on 24 individuals obtained from a distylous population located in southern Tibet (29°46.616′N, 94°44.545′E; 3305 m a.s.l.).

Table 1

Primer sequences and characterization of 24 microsatellite loci isolated from Primula chungensis.

Locus name	Primer sequence (5′–3′)	Repeat motif	Size (bp)	Ta (°C)	GenBank accession no.
PC109	F:ACGGGTCATTGCGTTTAAGTCG	(CGA)5	190	54	KT033877
	R:TTGTCGTTTGTCGGAGTGTTCG
PC4007	F:ATGGTGCTAAACGCCTTGTG	(CT)10	308	54	KT033878
	R:TTTGAAGTAGGGGTGAGGTG
PC4198	F:CTACACCTCTCCTCCTCTTC	(TC)9	153	56	KT033879
	R:CCCTAGCCGTCCAACTTA
PC9123	F:CAAAAGAGCGTTAGGAGTGA	(TG)7	338	48	KT033880
	R:GATGGTTGTCGTTGTCGGT
PC11592	F:TACCCTAACCTATTTTTCCC	(AG)15	165	56	KT033881
	R:CGCTACCTTACCTCTCTTCT
PC14297	F:ACTTACTGGTCTTTCGGTGA	(AG)16	246	52	KT033882
	R:CTCTCGCTTTATGTGCCTG
PC15519	F:ACTCGTGTTCCGTTTGTTCTAA	(GTTTT)8	240	50	KT033883
	R:AGTAACTGTTTGTTTGGCTATTGA
PC15877	F:CGGTTTATTTGGTTGAAGTC	(CT)6	228	50	KT033884
	R:CTCCTTGTTCTTTGTTTGAC
PC20540	F:CTCATCGTCTTTCCTATTTC	(TTTTTG)5	213	48	KT033885
	R:AAGGCAATCTGTTCTGAC
PC21731	F:ATGTGTCCTTGTTTTATGTG	(GA)6	173	48	KT033886
	R:ATGGTGTTTATCAATGTAGG
PC29976	F:CAATGATTTCTCTCTCGTCC	(TC)6	184	50	KT033887
	R:AGATGGTTGAAGATGGTGAT
PC30591	F:GGAAATCGCAGCCTCATAAC	(TG)7	220	52	KT033888
	R:GAGAGCGGAGTGTTCACCTA
PC30882	F:GCACAAACCCTAACTAAAGT	(AG)8	245	48	KT033889
	R:GCACAATCAAGCCAATG
PC32026	F:CTTTTCACCAGACGCTACCC	(CT)6	221	56	KT033890
	R:GCGGAACATCATTGAACCA
PC33802	F:ACTAAACATAACAAGCATCG	(AG)9	134	52	KT033891
	R:CAACTGTAATCTGAGCCAAT
PC33837	F:GTAGACACCTTGAATCATAACAAGTAAC	(GAT)5	266	52	KT033892
	R:TTGAAACCAAATTGCCACCT
PC34870	F:AATCAGCATAGGGTTCTCAA	(AG)8	113	52	KT033893
	R:CCTCTCTTCTCCTCCCTTTC
PC37139	F:GATGGCTCTCGTTTTTTTAC	(TC)6	126	50	KT033894
	R:TCAGAGTAGAACCACATCAG
PC39361	F:GCATTCCACGTCTTTTGTTG	(TG)7	134	52	KT033895
	R:GGTAATCCCCTCGCATGACT
PC39450	F:CGTGGCAATTTCTCGTAG	(AAGA)9	177	52	KT033896
	R:ATGGCATTTGTCCTGTCTT
PC46911	F:GCAAAAGAAACTCCCGCAAT	(AG)8	255	52	KT033897
	R:CTAAGCACAATGTTGAGGCA
PC47381	F:GAGGGAGACCGAAAGAGGA	(CTC)7	189	54	KT033898
	R:GCGGTAGGCACAGGAGAGTA
PC50689	F:GGGTTTTTGTTCATCTTAGC	(AAC)10	183	48	KT033899
	R:TCTTCGGTATGTGTCTCGTG
PC52377	F:TCTCTCTCTTTTCTTTGTTC	(TC)9	111	48	KT033900
	R:TAGAGGAAATGGAGGAAG

Note: Ta = annealing temperature.

We performed PCR amplification using the following protocol: 20 μL reaction volume containing 25–50 ng of genomic DNA, 0.6 μM of each primer, 10 μL 2 × Taq PCR MasterMix [Tiangen (Tiangen Biotech, Beijing, China); 3 mM MgCl2, 100 mM KCl, 0.5 mM of each dNTP, 20 mM Tris-HCl (PH 8.3), 0.1 U Taq polymerase] and 10 × PCR buffer. We conducted PCR amplifications under the following conditions: 95 °C for 4 min followed by 30–35 cycles at 94 °C for 45 s, at the annealing temperature for each specific primer (optimized for each locus; Table 1) for 45 s, 72 °C for 1.5 min for extension, and a final extension step at 72 °C for 10 min. PCR products were separated and visualized using a QIAxcel capillary gel electrophoresis system (QIAGEN, Irvine, USA) with an internal 10–300 bp size standard.

Results and discussion

Of 127 primer pairs tested, 24 primer pairs amplified microsatellite loci displaying polymorphism, whereas the remaining 103 pairs were monomorphic. All sequences were deposited in GenBank (Table 1). We calculated basic population genetic parameters of diversity, including the number of alleles (Na), observed and expected heterozygosities (HO, HE), Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between pairs of loci using the package GENEPOP version 4.0 (Raymond and Rousset, 1995). The number of alleles per locus (Na) ranged from 2 to 4, with a mean of 2.458. In the population investigated, the observed (HO) and expected (HE) heterozygosities ranged from 0 to 1.000 and from 0.219 to 0.708, with averages of 0.516 and 0.469, respectively (Table 2). Nine loci (PC9123, PC11592, PC20540, PC30882, PC33837, PC37139, PC39361, PC47381, PC50689; see Table 2) deviated significantly from Hardy–Weinberg equilibrium indicating the possibility of null alleles, the Wahlund effect and disassortative mating in this distylous population. After Bonferroni correction, no significant pairwise linkage disequilibrium was observed for any pair of loci.

Table 2

Genetic diversity parameters of the 24 microsatellite loci developed for Primula chungensis based on 24 individuals sampled from a distylous population located in southern Tibet.

Locus	Na	HO	HE
PC109	2	0.667	0.444
PC4007	2	0.250	0.219
PC4198	2	0.333	0.278
PC9123a	2	0.917	0.497
PC11592a	2	0.750	0.469
PC14297	2	0.250	0.219
PC15519	4	1.000	0.653
PC15877	3	1.000	0.611
PC20540a	2	0.000	0.500
PC21731	3	0.792	0.598
PC29976	3	1.000	0.625
PC30591	3	0.778	0.512
PC30882a	2	0.111	0.494
PC32026	2	0.250	0.219
PC33802	2	0.500	0.375
PC33837a	2	0.000	0.444
PC34870	3	0.875	0.539
PC37139a	2	0.000	0.500
PC39361a	3	0.833	0.559
PC39450	4	0.833	0.708
PC46911	3	0.750	0.531
PC47381a	2	0.000	0.375
PC50689a	2	0.000	0.500
PC52377	2	0.500	0.375

Note: Na = the number of allele; HO = observed heterozygosity; HE = expected heterozygosity.

Geographic coordinate of the population: 29°46.616′N, 94°44.545′E.

Indicates significant departure from Hardy–Weinberg equilibrium (P < 0.01).

In this study, we isolated 2341 microsatellite loci from P. chungensis. Primer pairs were designed to test 127 of these loci for polymorphism in 24 individual plants from a distylous population. Among the loci we tested, 24 microsatellite markers were polymorphic. These 24 polymorphic microsatellite markers will be powerful molecular tools for analyzing population structure and mating systems of P. chungensis, particularly the evolutionary relationship between distylous and homostylous populations. The high discriminatory power of these microsatellite loci will also be useful for parentage analysis in floral polymorphic populations of this species, which may provide an opportunity to evaluate the potential influence of ecological and reproductive factors on mating patterns (Zhou et al., 2015).