Isolation and characterization of microsatellite markers for the threatened African endemic tree species Pterocarpus erinaceus Poir.

Abstract To study the genetic diversity and structure of the forest species Pterocarpus erinaceus Poir., seventeen polymorphic nuclear microsatellite markers were isolated and characterized, using next‐generation sequencing. Three hundred and sixty‐five (365) individuals were analyzed within fifteen (15) West African populations. The number of alleles for these loci varied from 4 to 30, and the heterozygosity varied from 0.23 to 0.82. The seventeen (17) primers designed here will allow characterizing the genetic diversity of this threaten species on its natural stands and to better understand the population differentiation mechanisms shaping it.


| MATERIAL AND ME THODS
We sampled nine to thirty adult trees in 15 populations (Table 1) with a total of 365 trees in four countries of West Africa which are Benin, Burkina Faso, Niger, and Togo ( Figure 1).
Freshly collected leaves were dried in a coffee filter containing 10 g Silica gel. Each filter containing sample was put in a plastic zip bag for transport to the laboratory. Our genomic library was constructed using DNA of P. erinaceus samples from twelve randomly selected individuals among populations (Table 1).

| DNA extraction
Total genomic DNA extraction was performed with a solution of alkyltrimethylammonium bromide (MATAB) using twenty milligrams of dried leaves from each tree sample. Extraction protocol used derived from Bousquet et al. (1990) methodology.
The quality of the genomic extracted DNA was controlled on a 1% agarose gel, and quantification was done by Hoechst assay using fluoroskan (Fluoroskan™ Microplate Fluorometer). Albumine; Qiagen ® ), and 0.06 U/µL of Taq DNA polymerase. PCR running conditions were as follows: initial denaturation at 94°C for 4 min followed by 36 cycles each at 92°C in 30 s, 1 min at 52°C, 45 s at 72°C, and with a final extension step at 72°C for 5 min.

| Cross-amplification test of P. officinalis microsatellites markers on P. erinaceus
Electropherograms were analyzed, and allele sizes were determined using GeneMapper ® software version 4.1 using GeneScan 600 LIZ as a size standard (Applied Biosystems). Four out of the eight primers failed to amplify the target loci (mPoCIRE01, mPoCIRE04, mPoCIRH02, and mPoCIRE09), and two showed little polymorphism (Table 2).
These eight loci were considered noninformative and showed insufficient variability to be used for genetic studies of P. erinaceus.

| Construction of the DNA library and validation
The Westburg NGS DNA Library PrepKit was used to prepare the DNA library with a pooled DNA extract from twelve samples. The library was built following the manufacturer instructions. Using this kit, enzymatic fragmentation allows for obtaining DNA fragment sizes from 200 to 600 bp (suggested by the manufacturer) depending on the reaction time and the amount of DNA input. DNA was fragmented in an Eppendorf Mastercycler ® nexus using 35 µl volume of pooled DNA (1µg) to which 5 µl of ER/A-tailing buffer (10X) and 10 µl of ER/A-tailing enzyme mix (5X) were added. The fragmentation program used in the thermocycler included a first step of precooling at 4°C for 5 min followed by the second step of fragmentation with three fragmentation times, 1 min at 4°C, 10 min at 32°C, and 30 min at 65°C. Purification on magnetic beads (Agencourt AMPure XP beads -A63881-, Beckman Coulter) was performed before and after PCR.
The amplification conditions included initial denaturation at 98°C for 2 min, followed by 7 cycles of 98°C for 20 s, annealing at 60°C for 35 s, elongation at 72°C for 30 s, and a final 1 min elongation step at 72°C.
The quality of DNA library was controlled using an Agilent 4,200 TapeStation with a screen tape D5000, and the fragments sized between 100 and 600 pb mainly, with an average of 260 bp. The DNA library (fragments) was quantified using the Takara kit (638,324) on a qPCR machine (LightCycler ® 480 Real-Time PCR System, Roche Life Science).

| Sequencing
MiSeq system Illumina sequencer DNA was used to perform DNA sequencing on the genotyping platform at CIRAD-Montpellier. A 500 cycles NANO V2 cartridge Illumina (2 x 250 pb) was used to sequence the library.

| Design and choice of primers
A total of 800,000 reads were generated for P. erinaceus DNA library.  (Thiel, 2003) and primer modeling software Primer3 (Whitehead Institute) were used for the identification and design of microsatellites primers in the generated draft assembly. A data matrix containing all the microsatellite primers was obtained as output.
Thirty microsatellites were identified and selected for initial screening on the basis of the type and size of the repeat motif, as well as the annealing temperature as previously described Muller et al. (2006). Therefore, dinucleotide and trinucleotide SSRs with a repeat motif of 15 to 30 bp were randomly selected from those generated. The selected primers amplified SSR motifs with a minimum of five repetitions. The annealing temperature varied from 54 to 56°C, including that used by Muller et al. (2006) for P. officinalis (54°C). This first test was performed on an ABI 3500XL sequencer (Life technologies) using genomic DNA extract from eight individuals selected from different countries (Table 1).
An M13 tailed primer (5′-CACGACGTTGTAAAACGAC-3′), allowing detection of fluorescence, was added to the forward primers. Each PCR amplification was performed in a 96-well plate using The analysis of electrophoregrams with GeneMapper ® software version 4.1 using GeneScan 600 LIZ as a size standard (Applied Biosystems) allowed determining allele sizes. Among the 30 primer pairs tested, 17 were selected. Indeed, we eliminated primers with profiles that were difficult to read on GeneMapper®, or with no or little polymorphism. The 17 selected primers are shown in Table 4 and were used for screening the remaining individuals in order to calculate genetic parameters.
Genetic parameters including alleles' number per locus, number of private alleles, observed heterozygosity (Ho), and expected heterozygosity (He) were computed using GenAIEx software version 6.0 (Peakall & Smouse, 2012      = no amplification equilibrium (HWE) was measured for each locus by chi-squared tests and p-value significance assessed in the context of multiple testing with a Bonferroni correction procedure (Rice, 1989). Significant linkage disequilibrium was rated among these loci by using GENETIX software version 4.05 (Belkhir et al., 1996)

| RE SULTS AND D ISCUSS I ON
Contrary to Hong et al. (2020)