** = Geographic origin of individuals selected for the first
screening
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 ttwenty 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 done by Hoescht assay using fluoroskan
(Fluoroskan™ Microplate Fluorometer).
Construction of the DNA library and validation
The Westburg NGS DNA Library PrepKit was used to prepare the DNA bank
with extract mixed DNA of twelve individuals. We started with 1µg ofPterocarpus mixed DNA. This DNA was fragmented, and ligated with
Illumina adapters. Purification on magnetic beads (Agencourt AMPure XP
beads -A63881-, Beckman Coulter) was performed before and after PCR. PCR
amplification was run with 7 cycles.
The quality of DNA library was controlled using an Agilent 4200
TapeStation with a screen tape D5000. The size of the fragments were
expected between 100 and 600 pb. The library was quantified using the
Takara kit (638324) on a qPCR machine (LightCycler® 480 Real-Time PCR
System, Roche Life Science).
Sequencing the DNA library
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 was generated for P. erinaceus samples.
Development of optimised and streamlined microsatellites was based on
the bioinformatics Galaxy pipeline and its following tools: FASTQ
Groomer tool, Filter FASTQ tool and ABySS parallel assembler (Simpson et
al., 2009). The MISA MIcroSAtellite identification tool (Thiel, 2003)
and primer modelling software Primer3 (Whitehead Institute) were used
for the identification and design microsatellites primers in nucleotide
sequences generated. A data matrix containing all the microsatellite
primers was obtained as output.
Among the 38,715 single sequence repeats identified, primers were
designed for 11,718 sequence repeats of which 3,530 were dinucleotide
repeats, 2,970 trinucleotide repeats, 2,847 tetranucleotide repeats,
1,001 pentanucleotide repeats, 525 hexanucleotide repeats, and 844
contained complex SSR motifs.
Dinucleotide and trinucleotide microsatellites motifs were selected for
SSR-PCR amplification screening. These primers exhibited a minimum of
five repetitions of the repeated motif and amplified fragments between
100 and 400 bp in length. Thirty suitable microsatellite were identified
and selected for initial screening. This first test was performed on an
ABI 3500XL sequencer (Life technologies, Carlsbad, California, USA)
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 10-μL volume reaction containing
20 ng of DNA, 1X PCR buffer (without MgCl2), 0.08 μM of
the M13-labeled primer, 0.1 μM of each forward fluorescent primer (FAM,
NED, PET, and VIC) and the reverse primer, 0.1 μM of M13 fluorescent
primer, 2 mM of MgCl2, 200 μM dNTPs, 0.4X Solution “S”
(additive solution that facilitates amplification of difficult
templates) and 0.05 U/µL of Taq DNA polymerase. PCR running
conditions were: 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.
Electropherograms were analysed and allele sizes were determined with
GeneMapper® software version 4.1 using GeneScan 600 LIZ as a size
standard (Applied Biosystems). 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 2 and they were used for
screening of remaining individuals in order to calculate genetic
parameters.
Genetic parameters including alleles’ number per locus, observed (Ho)
and expected (He) heterozygosity were computed using GenAIEx software
version 6.0 (Peakall and Smouse, 2012). Deviation from the
Hardy–Weinberg equilibrium (HWE) was measured for each locus by χ2
tests, and with a Bonferroni correction procedure (Rice, 1989), p-value
significance was assessed in the context of multiple testing.
Significant linkage disequilibrium was rated among these loci by using
GENETIX software version 4.05 (Belkhir et al., 1996). MICRO-CHECKER
software version 2.2.3 (Van Oosterhout et al., 2004) was used to check
for the null alleles in microsatellite data.
Results and Discussion
A total of 237 alleles were identified for the 17 locus on the 365
characterized trees, with each locus having from 4 to 30 alleles (mean
of 13.9 alleles per locus). Mean values for the expected heterozygosity
(He) varied from 0.307 to 0.781 (0.571 ± 0.176) and from 0.234 to 0.821
(0.551 ± 0.190) for observed heterozygosity (Ho) for the individuals
screened. Evidence of significant linkage disequilibrium, was found for
12 for 136 possible SSR pairwise combinations after Bonferroni
corrections. Significant departures from Hardy–Weinberg equilibrium for
14 of the 17 loci was recorded after Bonferroni corrections and presence
of null alleles was suggested for all loci excepted for mPeCIR_D2 and
mPeCIR_T3. This set of 17 pairs of specific primers ofPterocarpus erinaceus would serve to study the genetic diversity
of this species in West Africa.
Table 2 : Characteristics of 17 microsatellite primers designed
for Pterocarpus erinaceus Poir.