Luculia gratissima (Wall.) Sweet (Rubiaceae) is an evergreen perennial shrub distributed in the southeast margin of the Tibetan Plateau in southwest China and adjacent region of Nepal and Myanmar at altitudes between 800 and 2400 m [1,2]. The species flowers from August to December, and produces several compact pink to white tubular flowers on each tight inflorescence that are sweetly fragrant. It is very interesting that the species is distylous with complementary positioning of stigmas and anthers in the two floral forms [3]. Previous studies suggest that the floral syndrome of distylous species would facilitate disassortative mating during pollinator visitation [4,5]. However, the potential functions of distyly are not well understood, and DNA microsatellites would provide a precise molecular marker to trace the characteristic of pollination patterns by parentage analysis. In this study, we have developed and characterized 19 microsatellite markers for L. gratissima using the Fast Isolation by Amplified Fragment Length Polymorphism (AFLP) of sequences containing repeats (FIASCO) [6], which will be used for further studies of breeding system, gene flow patterns, and population structure.

A total of 275 positive clones were captured, among these 102 clones (37%) were found to contain simple sequence repeats (SSR). Finally, 33 sequences contained SSR loci were selected for primer design. Nineteen microsatellite loci successfully amplified in L. gratissima for 33 microsatellite loci and 10 of them were polymorphic amplification, the remaining nine microsatellite loci were monomorphic as a result of capillary gel (Table 1).

The number of alleles ranged from two to five in 24 individuals of the species sampled from the two natural populations. Values for H_O and H_E ranged from 0.000 to 1.000 and from 0.289 to 0.760, with averages of 0.303 (SD = 0.285) and 0.555 (SD = 0.133), respectively (Table 2). For all 10 microsatellite loci, except LG2 and LG25, the genotypic frequencies showed significant deviation from Hardy-Weinberg equilibrium (HWE) (P < 0.01) indicating the possibility of null alleles and the disassortative mating of this distylous species.

These microsatellite markers developed in our study will be a useful tool for further studies of population genetics, and will be used to assign parentage to seeds which will help us understand the characteristic of pollination patterns for this distylous plant.

Genomic DNA samples of L. gratissima were extracted from silica-gel-dried leaves of three different individuals using a modified hexadecyltrimethylammonium bromide (CTAB) method [7]. The extracted DNA was dissolved in 30 μL TE buffer. The fast isolation by AFLP of sequences containing repeats (FIASCO) [6] was performed in this study. Total genomic DNA (approximate 250–500 ng) was completely digested with 2.5 U of Mse I restriction enzyme (New England Biolabs, Beverly, MA, USA), and then ligated to an Mse I AFLP adaptor (5′-TAC TCA GGA CTC AT-3′/5′-GAC GAT GAG TCC TGA G-3′) using T4 DNA ligase (Fermentas, Burlington, ON, Canada). The digested-ligated fragments were diluted in a ratio of 1:10, and 5 μL of them were used amplification reaction with adaptor-specific primers (5′-GAT GAG TCC TGA GTA AN-3′/5′-TTA CTC AGG ACT CAT CN-3′). The amplified DNA fragments (200–800 bp) were enriched by magnetic bead selection with a 5-biotinylated (AG)₁₅, (AAG)₁₀ and (AC)₁₅ probe, respectively [6]. The Recovered DNA fragments were reamplified with Mse I-N primers. The purified PCR products using EZNA Gel Extraction Kit (Omega Bio-Tek, Guangzhou, China), were ligated into pBS-T II vector (Tiangen, Beijing, China), and then transformed into E. coli strain DH5α competent cells (TaKaRa, Dalian, Liaoning, China). The positive clones were picked out and tested using vector primers T3/T7 and primer (AC)₁₀/(AG)₁₀/(AAG)₇ respectively to select appropriate fragments which contained SSR. In other words, a set of tested PCR included three reactions was performed using T3 and T7, T3 and (AC)₁₀, (AC)₁₀ and T7 as primers, respectively. The second set of tested PCRs was done using T3 and T7, T3 and (AG)₁₀, (AG)₁₀ and T7 as primers, respectively. The last set of tested PCRs was done using T3 and T7, T3 and (AAG)₇, (AAG)₇ and T7 as primers, respectively. All these PCR reactions had the same conditions: 95 °C for 3 min followed by 30 cycles at 94 °C for 45 s, 52 °C for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 7 min. The positive clones were captured for sequencing with an ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences contained simple sequence repeat and enough flanking regions were selected for primer design using Primer Premier 5.0 program [8].

The designed Primer pairs were assessed in 24 individuals of L. gratissima pooled from two natural populations collected in Nepal: NMD (Daman, Makwanpur, Narayani: 27°40′N, 85°05′E, 1970 m a.s.l.) and BLG (Godawari, Lalitpur, Bagmati: 27°40′N, 85°19′E, 1385 m a.s.l.). Herbarium voucher deposited in Kunming Institute of Botany, Chinese Academy of Science (code ZW0153-0176). The PCR reactions were performed in 20 μL of reaction volume containing 10–50 ng genomic DNA, 0.5 μM of each primer, 10 μL 2× Taq PCR MasterMix (Tiangen; 0.1 U Taq Polymerase/μL, 0.5 mM dNTP each, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl₂). PCR amplifications were conducted under the following conditions: 95 °C for 3 min followed by 32–35 cycles at 94 °C for 30 s, at the annealing temperature for each specific primer (optimized for each locus, Table 1) for 45 s, 72 °C for 1 min, and a final extension step at 72 °C for 7 min. The amplification products were separated and visualized using QIAxcel of capillary gel electrophoresis system (QIAGEN, Irvine, CA, USA).

The data was analyzed by GENEPOP 4.0 [9], which included test of observed heterozygosity (H_O), expected heterozygosity (H_E), and departure from Hardy-Weinberg equilibrium (HWE) for the 10 polymorphic microsatellite loci.

In summary, 19 microsatellite markers have been specifically developed for L. gratissima in this study. The high discriminatory power of 10 polymorphic loci suggests that they should be suitable for survey of population structure and parentage analysis in this distylous species. These developed and characterized SSR markers for L. gratissima would also be useful for exploring genetic diversity and genetic structure of other species in Luculia.