Styphelia longissima Hislop & Puente-Lel. (Ericaceae) is a newly described species (Hislop and Puente-Lelièvre, 2017) and found only in a single population on sand within remnant kwongan vegetation near Eneabba, in the South West Australian Floristic Region (SWAFR), an international biodiversity hotspot (Hopper and Gioia, 2004). Until very recently, this taxon was assigned to the genus Leucopogon R. Br. (Ericaceae) with the temporary name of Leucopogon sp. ciliate Eneabba (F. Obbens & C. Godden s.n. 3/7/2003). Recent taxonomic revision has placed this and some other Leucopogon species within the Styphelieae, with sister taxa Leucopogon sp. Ongerup and Astroloma sp. sessile leaf (Puente-Lelièvre et al., 2015; Hislop and Puente-Lelièvre, 2017). In 2007, the population consisted of just 1993 individuals (Woodman Environmental Consulting Pty. Ltd., 2008). Since then, however, mortality has substantially exceeded recruitment (Harris, 2013), and the species is currently listed as Rare Flora under the Wildlife Conservation Act 1950 (Western Australian Minister for the Environment, 2015). Styphelia longissima is a spindly to dense shrub, to 0.5(−0.8) m high, with cream-white colored flowers in July that are most likely insect pollinated, and seed dispersal is myrmecochorous (Harris, 2013). Microsatellite markers were developed for S. longissima to enable an assessment of population genetic variation, spatial genetic structure, mating system parameters, and dispersal for management and conservation. Astroloma xerophyllum (DC.) Sond., a sister taxon, was chosen for cross-amplification based on molecular phylogeny of the Styphelieae (Puente-Lelièvre et al., 2015).
METHODS AND RESULTS
Genomic DNA was extracted from a single tissue-cultured plant, sampled from the only known population (Appendix 1), using a Carlsons method (Carlson et al., 1991) with modifications outlined in Anthony et al. (2016). Next-generation sequencing was performed on a Personal Genome Machine (PGM) semiconductor sequencer (Life Technologies, Carlsbad, California, USA) at the Lotterywest State Biomedical Facility Genomics Node in Perth, Western Australia, as described previously (Anthony et al., 2016). After sequencing, signal processing, base-calling, and quality trimming were performed using the default settings of TorrentSuite 4.0 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and library-specific FASTQ files were also generated. Sequencing resulted in >2.5 million reads, with a modal read length of 344 bp and a total data output of 766 Mb (National Center for Biotechnology Information [NCBI] Sequence Read Archive Bioproject no. PRJNA397350).
The raw sequences were screened using QDD version 3.1 pipeline (Meglécz et al., 2014) to remove redundant sequences and design primers for >12,000 sequences with PCR product lengths of 80–480 bp. The default parameters of the program were used both for the screening steps and for primer design. The resultant sequences were filtered to ensure that the primer was not overlapping the repeat sequence, there were no poly- ‘A’ or poly- ‘T’ runs for more than seven base pairs within the sequence, and there was only one repeat motif between the primers. Subsequently, 30 primer pairs were selected based on the suggestions of Meglécz (2014).
Initial screening was performed with CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, California, USA) using 5 µL of SsOAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), 0.2 µM each of forward and reverse primers, and 5–10 ng of genomic DNA in a 10-µL reaction volume. Initially, screening included reliable amplification of a single sample across a range of temperatures to determine the most appropriate annealing temperature, followed by evidence of polymorphism among eight individuals. Consequently, 16 polymorphic loci (including three loci that were fixed heterozygotes) and four monomorphic loci were selected to complete the study (Table 1), and the remaining 10 loci did not amplify reliably for S. longissima. Amplification of 57 individuals using 16 polymorphic loci was performed using a Veriti thermocycler (Life Technologies) within four multiplex mixes containing 6.25 µL of 2× Multimix and 2.25 µL of 5× Q-Solution (Type-It Microsatellite PCR kit; QIAGEN, Hilden, Germany), 1.25 µL of primer mix, and 2.25 µL of 5–100 ng DNA in a 12-µL reaction. Primer Mix (PM) 1 contained the primers Sl36, Sl65, Sl53; PM 2 contained Sl17, Sl18, Sl57, Sl60; PM 3 contained Sl6, Sl26; and PM 4 contained Sl01 Sl47, Sl67, Sl71 using the following PCR conditions: an initial 1-min denaturation at 95°C; 35 cycles of 94°C for 10 s, 62°C (PM 1, 2, 3) or 56°C (PM 4) for 30 s, and 72°C for 45 s; followed by a final extension of 15 min at 72°C. Electrophoresis was performed using the ABI 3500 sequencer (Life Technologies), and allele sizes were determined using Geneious version 7.1 (Biomatters Ltd., Auckland, New Zealand). Multiple replicate runs were performed to ensure the accuracy of the final data set. Genetic diversity parameters were calculated using GenAlEx version 6.4 (Peakall and Smouse, 2006). Departure from Hardy-Weinberg equilibrium (HWE) was assessed for each locus and population by X2 tests, and the possibility of null alleles was checked using MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004).
Characteristics of microsatellite loci developed for Styphelia longissima and cross-amplified in Astroloma xerophyllum.a
Results of primer screening with 16 polymorphic primers for Styphelia longissima.
The number of alleles observed for the 16 polymorphic loci ranged from two to 21, and the observed and expected heterozygosities ranged from 0.25 to 1.00 and 0.49 to 0.91, respectively (Table 2). There were significant departures from HWE for 12 of the 16 loci, with four loci showing no evidence of stuttering, large allele dropout, or null alleles (Table 2). Two loci showed evidence of stuttering indicated as a deficit of heterozygote genotypes with alleles of one base pair repeat difference. These loci have been thoroughly checked by repeat PCR and analysis scoring, and the one base pair difference shown to be real. These departures from HWE are most likely due to inbreeding within a small isolated population.
Using the same extraction method, five individuals were cross-amplified for the sister taxon A. xerophyllum. Amplification was performed using the same initial screening method outlined above and resulted in 12 polymorphic and four monomorphic loci (Table 1).
These 16 polymorphic microsatellites will be used for conservation genetic studies in the rare S. longissima to underpin management and conservation. These microsatellites are likely to be useful for genetic studies in other related species given the initial success in cross-amplification for A. xerophyllum.