Development and characterization of microsatellite markers for Osyris lanceolata Hochst. & Steud., an endangered African sandalwood tree species

Abstract

Osyris lanceolata Hochst. & Steud. is a multipurpose tree species widely spread in many of the sub-Saharan countries ranging from Algeria to Ethiopia all the way to South Africa. In Kenya, the species is endemic to the Arid and Semi-Arid Lands (ASALs). It is highly valued for its essential oils used in the cosmetic and pharmaceutical industries. Despite its endangered status and economic importance, little is known about its genetic diversity status and only few conservation strategies exist for the species. Overexploitation of the species has resulted in the decline of its population and reduced availability of its products. The mode of harvesting of sandalwood is destructive and unsustainable. This is because the whole tree is usually uprooted to get the heartwood from the stem, stump and roots. The exploitation of African sandalwood could soon drive the species to extinction unless proper control measures are put in place through regulation of its trade and development of conservation strategies. Despite its endangered status and economic importance, no genetic study has been carried out on the species to provide information vital for conservation strategies. This paper reports the development and characterization of a set of 12 polymorphic and five (5) monomorphic microsatellite markers isolated and characterized of O. lanceolata. One plant leaf sample was used as the source of DNA for genomic library construction. Total genomic DNA was extracted from silica gel dried leaf using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The DNA sample was then sent to The Gene Pool Institute of Evolutionary Biology, University of Edinburgh for sequencing. Simple sequence repeats (SSRs) were extracted through PAL Finder software version 0.02.04 (Castoe et al. 2012) and primer pairs developed. Identified microsatellites and designed primers were assembled using QDD (Meglécz et al. 2010) with parameters given in set_qdd_default.ini.file. The gaps emerging during the scaffolding process were closed using GapCloser (vs. 1.12). The contigs >1000 bp of the draft assembly were analyzed and functionally annotated using Blast2GO (Conesa et al. 2005). Based on this information, 48 primer pairs consisting of either di- or trinucleotide repeats were selected. After testing, 17 primer pairs were identified and used to characterize 84 samples of O. lanceolata from three natural populations, namely Mt. Elgon (28), Gachuthi (27) and Kitui (29). The PCR analysis was performed using Multiplex PCR Mater Mix (QIAGEN) and 10 ng of DNA as described by (Omondi et al. 2015). The PCR mix contained a fluorescently labelled M13 primer, M13-tailed forward primer and a reverse primer in the concentration ratio of 0.15:0.01:0.15 μM. For all loci, a touchdown thermal cycling program was used with annealing temperature ranging between 57–55°C. The cycling profile consisted of initial denaturation of 95°C for 15 min followed by 10 cycles at 94°C for 30 s, 57°C for 90 s and 72°C for 60 s (annealing temperature decreasing by 1°C per cycle); and 22 cycles at 94°C for 30 s, 55°C for 90 s and 72°C for 60 s and a single final cycle at 60°C for 30 min using Verity 96 well thermocycler (Applied Biosystems).