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14 June 2016 Development of Microsatellite Loci of Pod Mahogany, Afzelia quanzensis (Fabaceae), by Illumina Shotgun Sequencing, and Cross-Amplification in A. africana
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Afzelia quanzensis Welw. (Fabaceae) is a deciduous, medium to large tree that naturally occurs in eastern and southern Africa. It is a lowland species that grows well in hot temperatures and sandy soils. Its wood possesses an ornamental grain, which is very strong and flexible. It glues firmly and takes a good varnish, properties that make it eagerly sought after by woodcarvers. Apart from woodcarving, A. quanzensis is also used for railway sleeper and door construction, and as timber for roofing and fencing. As a result, it has been heavily logged in its native range (Gerhardt and Todd, 2009). The International Union for Conservation of Nature (IUCN) has regionally listed A. quanzensis as vulnerable in Malawi (Golding, 2002), while in South Africa, it is now a protected species. No microsatellite loci have been developed specifically for the species. Here, we describe the development of microsatellite loci that will be used in genetic studies.

METHODS AND RESULTS

Genomic DNA was extracted from a leaf of one A. quanzensis individual (population geographic coordinates: 19°36.056′S, 32°30.084′E; representative voucher deposited at the National Herbarium and Botanic Garden, Harare, Zimbabwe [SRGH], voucher number 1) using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA) following the manufacturer's instructions. The DNA was used to prepare a sequencing library using the KAPA DNA Library Preparation Kit for Illumina Sequencing (Kapa Biosystems, Wilmington, Massachusetts, USA) following the manufacturer's instructions. The final library was quantified using the KAPA Library Quantification Kit for Illumina. The DNA library was sequenced by an Illumina MiSeq Benchtop Sequencer (Illumina, San Diego, California, USA).

The resulting raw Illumina paired-end sequencing reads were analyzed with a Perl script, PAL_FINDER_v0.02.04 (available at  http://sourceforge.net/projects/palfinder), which identifies microsatellite loci without the need for prior sequence trimming and assembly (Castoe et al., 2012). The Perl script was run with Primer3 version 2.0.0 (Rozen and Skaletsky, 1999) for simultaneous primer design. Default settings were used except for the following adjustments: primer minimum annealing temperature (Ta) 50°C, primer maximum Ta 60°C, and primer optimum Ta 55°C. A total of 961,804 potential loci were identified, of which 7789 had primer pairs. We tested 70 potentially amplifiable loci with amplifiable primer pairs that occurred only once.

Of the 70 loci tested, 39 amplified successfully and these were checked for polymorphisms in 40 individuals randomly collected from a population near Chaseyama, southeastern Zimbabwe. Forward primers were tagged with a labeled M13 primer tail (TGTAAAACGACGGCCAGT). All PCR reactions were performed in a total volume of 10 µL, with 10 ng of template DNA, 0.6 µM of the reverse primer, 0.15 µM of the forward primer, 0.25 mM each dNTP, 0.6 µL bovine serum albumin (BSA; 10% w/v), 1 µL 10× reaction buffer with 15 mM MgCl2, and 0.25 units of Taq DNA polymerase (Bulldog Bio, Rochester, New York, USA). Loci Afq45, Afq51, Afq62, Afq68, and Afq69 had an additional 0.1 mM MgCl2. The thermocycling profile consisted of an initial denaturation at 94°C for 5 min; then 35 cycles of 94°C for 30 s, 55.0°C or 59.4°C (Table 1) for 30 s, 72°C for 30 s; and a final extension at 72°C for 7 min. The PCR amplicons were electrophoresed on an ABI 3730 DNA analyzer with GeneScan 500 LIZ (Applied Biosystems, Foster City, California, USA) as the size standard. The genotypes were scored using GeneMapper version 3.7 (Applied Biosystems).

Table 1 shows the 39 loci that amplified, their repeat motifs, number of alleles per locus, allele size range, and Ta. Twenty-seven loci were monomorphic while 12 were polymorphic. For the 12 polymorphic loci, number of alleles per locus (A), observed heterozygosity (Ho), and expected heterozygosity (He) were calculated using GenAlEx version 6.5 (Peakall and Smouse, 2006, 2012), and are shown in Table 2. The program Arlequin version 3.5 (Excoffier and Lischer, 2010) was used to perform an exact test (Guo and Thompson, 1992) with a Markov chain for Hardy–Weinberg equilibrium (HWE), while the program MICRO-CHECKER version 2.2.3 (van Oosterhout et al., 2004) was used to estimate null allele frequencies (FNULL) with Bonferroni correction.

Table 1.

Characteristics of 39 microsatellite loci developed for Afzelia quanzensis.

t01a_01.gif

Continued.

t01b_01.gif

The number of alleles for polymorphic loci ranged from three to 10 with an average of 5.583. Ho ranged from 0.138 to 0.737, while He ranged from 0.313 to 0.832. Three loci, Afq12, Afq51, and Afq67, showed evidence of null alleles. Loci Afq35, Afq51, and Afq67 showed departure from HWE (Table 2). It is suspected that a relatively small sample size may have contributed to the departure from HWE, and the result does not invalidate the utility of the markers. The alleles that show potential null alleles can be used with adjusted genotypes. Cross-amplification of the 12 polymorphic loci was tested in 24 individuals of a congeneric species, A. africana Sm., another African tree species prized for its high-quality wood. Eight loci amplified in A. africana, with the number of alleles ranging from one to five, as shown in Table 3.

CONCLUSIONS

Thirty-nine microsatellite loci were developed, of which 27 were monomorphic and 12 showed polymorphisms. The microsatellite loci are the first developed specifically for A. quanzensis. The loci provide a set of markers to study the genetic diversity, gene flow patterns, and population structure of the species. The species is being increasingly harvested for its wood, so the effects of overharvesting on long-term genetic viability need to be understood for conservation purposes. Eight loci can also be used for genetic studies of A. africana, another species that is being logged for its valuable wood.

Table 2.

Polymorphic microsatellite locus-specific measures of genetic diversity of a population of 40 individuals of Afzelia quanzensis.a

t02_01.gif

Table 3.

Genetic properties of eight Afzelia quanzensis microsatellite loci tested in 24 A. africana individuals.a

t03_01.gif

ACKNOWLEDGMENTS

The authors thank K. Feldheim for assistance with the sequencing run and K. Ramanauskas for providing the platform to run the Perl script. Appreciation also goes to A. S. L. Donkpegan for providing Afzelia africana samples. Funding was provided by the University of Illinois at Chicago (UIC) Biological Sciences Department's Hadley Award, the UIC Office of International Affairs' Chicago Consular Corps Award, and the Institute of International Education's Dr. Benjamin L. Van Duuren Grant.

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Percy Jinga, Jason Palagi, and Mary V. Ashley "Development of Microsatellite Loci of Pod Mahogany, Afzelia quanzensis (Fabaceae), by Illumina Shotgun Sequencing, and Cross-Amplification in A. africana," Applications in Plant Sciences 4(6), (14 June 2016). https://doi.org/10.3732/apps.1600010
Received: 26 January 2016; Accepted: 1 February 2016; Published: 14 June 2016
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