Molecular phylogeny and population genetics of the Grevillea Thelemanniana group (Proteaceae)

A set of 12 polymorphic microsatellite markers were developed for Grevillea thelemanniana subsp. thelemanniana, a rare and threatened shrub, endemic to southwest Western Australia. Genomic sequences were obtained from next generation (454) sequencing in the target species. Primer pairs for a total of 30 microsatellite loci were designed from these of which 13 were successfully amplified in 25 individuals from Kenwick Nature Reserve, Perth. Twelve loci were polymorphic with observed heterozygosity ranging from 0.12 to 0.6 and the number of alleles per locus ranged from 2 to 8. These markers were trialed in all species within the Grevillea thelemanniana species complex. Amplification success and level of polymorphism varied among loci and taxa, but most (96%) were successful. These loci will be useful in understanding the genetic variation, molecular ecology and phylogenetic relationships in the Grevillea thelemanniana complex. 156 Grevillea thelemanniana Endl. subsp. thelemanniana is a priority listed taxon in Western Australia (Priority 4 Rare, near threatened and in need of monitoring) (Smith 2012). It is found only in a fragmented habitat southeast of Perth, Western Australia. The species is part of a larger complex comprised of 16 species and 4 subspecies, endemic to Western Australia and found primarily in the coastal sand plains from as far south as Busselton and north to Exmouth. The Grevillea thelemanniana complex has been recognized as a group with high conservation importance and poorly known taxonomic relationships (Makinson 2000). Here we develop 12 microsatellite markers for Grevillea thelemanniana subsp. thelemanniana that show amplification and polymorphism among other members of the complex. Relatively few microsatellite panels exist for Grevillea, and of these all are for eastern Australian species, making this the first library for a western Australian Grevillea (England et al 1999,Hoebee et al 2011, Mantello et al 2011, James et al 2012). Genomic DNA was extracted from one individual of G. thelemanniana subsp. thelemanniana collected at Kenwick Nature Reserve. Extraction from freeze-dried, ground leaf material followed the modified CTAB extraction method (Doyle and Doyle 1987). The DNA solution (4μg) was sent to the Ramaciotti Centre for Gene Function Analysis (http://www.ramaciotti.unsw.edu.au) in Sydney for 454 shotgun sequencing on a Roche GS-FLX Titanium machine (454 Life Sciences, Branford, CT, USA) following the protocol of Gardner et al (2011). Sequences have been deposited in the Dryad Digital Repository (see Meglecz et al. 2012; http://dx.doi.org/10.5061/dryad.jd183 ) The resulting raw sequences (total length 101,949,487 bases) were searched for unique microsatellite motifs two to six base pairs long with C8 repeats (Gardner et al 2011) in QDD 1.3 software (Meglecz et al 2010). The program Primer3 1.1. 1 (Rozen and Skaletsky 2000) within QDD 1.3 was used to design primers, using the basic search defaults. The resulting 501 unique loci were sorted based on PCR product size, repeat class, repeat length, GC content and multiplexing potential. The best 30 (15 dinucleotide and 15 trinucleotide) were chosen for evaluation of amplification and polymorphism. Reactions were performed with 10-50 ng of G. thelemanniana subsp. thelemanniana DNA in 10 μl reaction volumes containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 0.2 mM each dNTP, 0.08 μM forward primer and reverse primer, 0.01 μl Taq DNA polymerase and 4 mM MgCl2. Amplification was conducted in EppendorfTM