Janet M. Anthony

Somatic Embryogenesis for Mass Propagation of Ericaceae – A Case Study with Leucopogon verticillatus

An efficient three-phase culture has been developed for plant regeneration of Leucopogon verticillatus (R. Br.) (Ericaceae formerly Epacridaceae [Ann. Missouri Bot. Gard. 85 (1998): 531–553]) via somatic embryogenesis as indicative of likely culture scenarios for other Ericaceae. The Ericaceae, particularly many Australian species, are often difficult to propagate by conventional forms of nursery propagation. Initiation of somatic embryos was best achieved using Gamborg’s B5 medium, pH 6, 4% maltose, 0.7% agar with the plant growth regulators 10 µM TDZ and 5 µM IAA. Somatic embryos were removed from the parent tissue and transferred to half strength basal GB5 medium for elongation. Root development did not occur unless specific treatments were used, a 2–5 day pulse treatment of 100 µM IBA significantly increased root production. All roots produced in agar-medium were fine and easily damaged when removed from culture. The most successful rooting medium (>60%) was sand on oat medium, which facilitated easy removal from the substrate and improved the survival of plants when transferred to soil.

Microsatellite markers from the Ion Torrent: a multi-species contrast to 454 shotgun sequencing

The development and screening of microsatellite markers have been accelerated by next‐generation sequencing (NGS) technology and in particular GS‐FLX pyro‐sequencing (454). More recent platforms such as the PGM semiconductor sequencer (Ion Torrent) offer potential benefits such as dramatic reductions in cost, but to date have not been well utilized. Here, we critically compare the advantages and disadvantages of microsatellite development using PGM semiconductor sequencing and GS‐FLX pyro‐sequencing for two gymnosperm (a conifer and a cycad) and one angiosperm species. We show that these NGS platforms differ in the quantity of returned sequence data, unique microsatellite data and primer design opportunities, mostly consistent with the differences in read length. The strength of the PGM lies in the large amount of data generated at a comparatively lower cost and time. The strength of GS‐FLX lies in the return of longer average length sequences and therefore greater flexibility in producing markers with variable product length, due to longer flanking regions, which is ideal for capillary multiplexing. These differences need to be considered when choosing a NGS method for microsatellite discovery. However, the ongoing improvement in read lengths of the NGS platforms will reduce the disadvantage of the current short read lengths, particularly for the PGM platform, allowing greater flexibility in primer design coupled with the power of a larger number of sequences.

Characterization and cross application of novel microsatellite markers for a rare sedge, Lepidosperma gibsonii (Cyperaceae).

PREMISE OF THE STUDY Ten polymorphic microsatellite loci for the rare sword sedge Lepidosperma gibsonii (Cyperaceae) were characterized for the future study of population structure, hybridization, and clonality. METHODS AND RESULTS Twenty samples from each of three populations were screened with the markers to assess genetic variation. Observed population heterozygosities ranged from 0.35 to 1.00, and number of alleles observed per locus ranged from eight to 23. No departures from Hardy-Weinberg equilibrium were detected for any locus in any population. Single samples from 14 species were screened to examine the transferability of the microsatellites to other species of Lepidosperma. At least eight out of 10 loci amplified in all species tested. CONCLUSIONS These loci will be useful for studying genetic variation, hybridization, dispersal, and breeding systems in Lepidosperma, a ubiquitous element of the flora of southern Australia.

Isolation and characterization of microsatellite markers for the banded ironstone endemic Acacia karina (Leguminosae: Mimosaceae) and cross-species amplification with A. stanleyi and A. jibberdingensis

Microsatellite markers were developed for the banded ironstone endemic shrub Acacia karina to examine genetic diversity, range-wide differentiation and mating system parameters. Nine loci were developed and in a sample of 20 individuals from one population the number of alleles ranged from 4 to 12 per locus and observed heterozygosities from 0.556 to 0.824. All loci were tested for cross-species amplification in two other south-west Australian Acacia species thought to be closely related to A. karina. Of these nine loci, eight were polymorphic in A. jibberdingensis and three in A. stanleyi.

Characterisation of polymorphic microsatellite markers isolated from Drakaea glyptodon Fitz. (Orchidaceae)

Drakaea glyptodon is a sexually deceptive terrestrial orchid endemic to Western Australia. We developed fourteen polymorphic microsatellite markers to assess genetic variability within and among populations. Moderate to high levels of variation were detected at most loci. There was evidence for inbreeding with high inbreeding coefficients for some loci. This suite of markers will be used to assess population genetics and investigate the causes of rarity and speciation of Drakaea.

Characterization and Transferability of Microsatellites for the Kangaroo Paw, Anigozanthos manglesii (Haemodoraceae)

Premise of the study: Microsatellites were developed for the future assessment of population genetic structure, mating system, and dispersal of the perennial kangaroo paw, Anigozanthos manglesii (Haemodoraceae), and related species. Methods and Results: Using a Personal Genome Machine (PGM) semiconductor sequencer, ca. 4.03 million sequence reads were generated. QDD pipeline software was used to identify 190,000 microsatellite-containing regions and priming sites. From these, 90 were chosen and screened using PCR, and 15 polymorphic markers identified. These sites amplified di-, tri-, and pentanucleotide repeats with one to 20 alleles per locus. Primers were also amplified across congeners A. bicolor, A. flavidus, A. gabrielae, A. humilis, A. preissii, A. pulcherrimus, A. rufus, and A. viridis to assess cross-species transferability. Conclusions: These markers provide a resource for population genetic studies in A. manglesii and other species within the genus.

Isolation and characterization of 13 microsatellites for the rare endemic shrub Tetratheca erubescens (Elaeocarpaceae).

Premise of the study: Microsatellite markers were developed for the rare Tetratheca erubescens (Elaeocarpaceae) to assess genetic diversity and spatial structuring. Methods and Results: We generated ca. 2.7 million sequence reads using a Personal Genome Machine (PGM) semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80–480 bp. From these, 30 primer pairs were selected and screened using PCR, from which 11 loci were found to be polymorphic and amplified reliably. For a sample of 95 plants from three populations, the number of alleles observed for these 11 loci ranged from two to seven and expected heterozygosity ranged from 0.06 to 0.72. No consistent evidence for null alleles or departure from Hardy– Weinberg equilibrium was found for any of the 11 loci. Conclusions: These markers will enable the quantification of genetic impact of proposed mining activities on the narrow endemic T. erubescens.

Isolation and Characterization of Microsatellite Primers for the Critically Endangered Shrub Styphelia longissima (Ericaceae)

Premise of the study: Microsatellite markers were developed for population genetic analysis in the rare shrub Styphelia longissima (Ericaceae). Methods and Results: We generated ca. 2.5 million sequence reads using a Personal Genome Machine semiconductor sequencer. Using the QDD pipeline, we designed primers for >12,000 sequences with PCR product lengths of 80–480 bp. From these, 30 primer pairs were selected and screened using PCR; of these, 16 loci were found to be polymorphic, four loci were monomorphic, and 10 loci did not amplify reliably for S. longissima. For a sample of 57 plants from the only known population, the number of alleles observed for these 16 loci ranged from two to 21 and expected heterozygosity ranged from 0.49 to 0.91. These markers were also amplified in Astroloma xerophyllum, a closely related species. Conclusions: These markers will be used to characterize population genetic variation, spatial genetic structure, mating system parameters, and dispersal to aid in the management and conservation of the rare shrub S. longissima.