Development and characterization of microsatellite loci for Ficus hirta (Moraceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Figs (Ficus L., Moraceae) and their pollinating wasps (Hymenoptera: Agaonidae) are a textbook example of pollination mutualism and one of the best cases of coevolution known (Jousselin et al., 2003). With more than 750 species, fi gs are a common component of tropical and subtropical habitats and are completely dependent on their pollinator wasps for pollen dispersal and the production of viable seeds. In turn, the pol-linating wasps gall a subset of the developing fi g seeds and so are completely dependent on the fi g for the completion of their life cycle. Ficus hirta Vahl is a dioecious shrub or small tree with a broad distribution at low elevations in Southeast Asia, where it plays an important role in forest succession and renewal in human-disturbed mountainous landscapes. Consequently , understanding the genetic diversity of this species is relevant to forest restoration and conservation, as well as co-evolutionary studies. Microsatellite loci have become a powerful tool for analyzing population genetic structure, gene fl ow, and levels of inbreeding. Many microsatellite primers have been developed for genetic studies of fi gs (Zavodna et al. Moreover, mic-rosatellite primers for F. hirta 's pollinator have been developed (Tian et al., 2011). Six of the microsatellite primers developed for other fi g species are polymorphic in F. hirta ; however, two of these loci exhibit signifi cant excesses of homozygotes within populations, suggestive of null alleles (Yu et al., 2010). Additional marker loci are needed for population genetic studies of F. hirta ; therefore, we have used a next-generation transcriptome sequencing approach (RNA-Seq) to develop microsatellites specifi cally for this species. Plant material and RNA/DNA extraction — Fresh, female fl owers from pre-receptive and from receptive male syconia of F. hirta growing at the South China Botanical Garden (IBSC; Guangdong Province) were excised with twee-zers and then immediately frozen in liquid nitrogen for RNA extraction. A voucher specimen (Huiyu 368) has been preserved in the IBSC herbarium. Total mRNA was extracted from each of the two samples using a modifi ed cetyl-trimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). For each sample, an RNA-Seq library was constructed using the Illumina TruSeq following the manufacturer's recommendations, and then sequenced on an Illumina HiSeq 2000 platform. Both cDNA library construction and Illumina sequencing were carried out by Novogene (Beijing, China). In total, 56,047,406 (pre-receptive) …