Syzygium paniculatum Gaertn. is a rare rainforest tree of the Myrtaceae family, endemic to a narrow coastal strip of New South Wales, Australia. S. paniculatum produces polyembryonic seeds, however, little else is known about its reproductive biology. Seeds of S. paniculatum are likely to be apomictic, originating clonally from maternal tissues, as this is evident in other polyembryonic species of Syzygium (Nic Lughadha and Proença 1996; Sahai and Roy 1962; Roy 1961 as Eugenia). If so, the population genetic structure of the species is likely to be affected.

We sought to develop molecular microsatellite markers that could be used to understand the reproductive and population biology within S. paniculatum. Genomic DNA was extracted and purified from leaf material from one individual from The Entrance Peninsula (New South Wales, S 33° 18′ 29.4″, E 151° 31′ 18.3″) using QIAGEN DNeasy Plant mini kit. A microsatellite AG-enriched library was constructed using the method of Glenn and Schable (2005). Polymerase chain reactions (PCRs) were performed on 96 clones and sequencing using M13 universal primers revealed 65 containing microsatellites. BioEdit v5.0.6 (Hall 1999) was used to edit sequences. Primer 3 (Rozen and Skalesky 2000) and NetPrimer (PREMIER Biosoft International) were principally used for primer design. Fourteen primer pairs were designed (Table 1) and to facilitate fluorescent labelling of PCR products an M13 tag (5′-TGTAAAACGACGGCCAGT-3′) was attached to the 5′ end of all forward primers (Schuelke 2000). These were tested for amplification and polymorphism in S. paniculatum, six other Syzygium species as well as three related taxa (Table 2).

Table 1 Fourteen microsatellite loci characterized for Syzygium paniculatum across 40 individuals representing 11 populations
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Table 2 Cross species amplification of microsatellite loci developed for S. paniculatum
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Microsatellite loci were amplified simultaneously using the Qiagen multiplex PCR Kit in 10 μL volumes each containing 5 μL 2× QIAGEN Multiplex PCR Master Mix, 0.05 μM each forward primer, 0.2 μM each reverse primer, 0.3–0.4 μM of M13 forward primer (with fluorescent dye attached) and approximately 5 ng of DNA. Reaction conditions: one cycle of 95°C for 15 min; 30 cycles of 94°C for 30 s, 60°C for 90 s, 72°C for 60 s; eight cycles of 94°C for 30 s, 53°C for 90 s, 72°C for 60 s; one cycle of 60°C for 30 min and 25°C for 1 min. Genotyping was performed on an ABI 3730 Capillary Sequencer (Applied Biosystems). GeneMapper version 3.7 (Applied Biosystems) was used to investigate and score alleles for each locus.

After optimisation for amplification and genotyping, markers were characterized (Table 1) using 40 individuals in total, 20 from The Entrance Peninsula and four each from five other S. paniculatum populations representing the entire range of S. paniculatum along the NSW coast. The number of alleles amplified per locus in S. paniculatum ranged from one to seven, with a mean of three. All 14 loci were genotyped in all 40 individuals; however, six loci either did not amplify across all populations or produced no heterozygosity in populations tested. The remaining loci showed heterozygosity and most showed variation across populations of S. paniculatum (Table 1).

One to four alleles were observed per locus in each individual indicating polyploidy which precludes the calculation of a range of standard genetic statistics, including deviations from Hardy–Weinberg and linkage equilibria. ATETRA (Van Puyvelde et al. 2010) was used to estimate expected heterozygosity (H E), which ranged from 0.41 to 0.68 (mean H E = 0.54), and Shannon Weiner diversity index (H′) which ranged from 0.59 to 1.27 (mean H′ = 0.85). Low allelic variation observed may be explained by the polyembryonic and potentially clonal nature of S. paniculatum seeds. Most loci were successfully amplified across the other species and were found to be polymorphic when compared to S. paniculatum (Table 2).

We aim to use these markers to conduct a fine scale study of the spatial distribution of genotypes across the geographic range of S. paniculatum. The markers can also be used to investigate the reproductive biology of the species to determine whether polyembryonic seeds of S. paniculatum are formed asexually by apomixis and whether apomixis is likely to occur in all individuals. This will be helpful in developing a long term conservation plan for this rare species. These loci can also be used to investigate the population and reproductive biology of other Syzygium species, some of which are of economic significance in Asia, as well as for closely related Myrtaceous species.