2.1. Genotyping and genetic clustering
For the present work, a total of 361 accessions of KG collected from
Benin, Burkina Faso, Ghana and Togo were used. The DNA of each sample
was extracted from young leaves of each accession, using the protocol of
the Integrated genotyping service and support (IGSS) at the Biosciences
Eastern and Central Africa (BecA:
http://hub.africabiosciences.org/activities/services) located in
Nairobi, Kenya. The quality of the DNA was confirmed by electrophoresis
in 0.8% agarose, and the quantification carried out using UVP
BioDoc-It2 Imaging System. All of the samples were diluted to 50 ng/µl
for the DArT genotyping platform. Genotyping was performed using the
DArT-Seq™ platform at Diversity Arrays Technology
(Kilian et al. 2012). The quality
analysis of the genotypic data was performed using Illumina HiSeq 2500
(Illumina 2015). The SilicoDArt calling
algorithms (DArTsoft14) was used to score DArTseq markers into a binary
format (presence = 1 and absence = 0) for each sample genomic
representation. A total of 2844 SNP markers were obtained and processed
in TASSEL v5 (Buckler et al.2014), for quality check. SNPs were filtered with TASSEL v5 for further
analysis using the parameters Minor Allele Frequency (MAF)
>0.05 and proportion of missing data < 30%. A
total of 142 SNPs were retained and Nipals model in kdcompute
(https://kdcompute.igss-africa.org)
was used for data imputation.
The program Structure 2.3.4 (Pritchardet al. 2010) was used to assign individuals to different genetic
clusters based on admixture model. The population structure was
evaluated based on Bayesian clustering approach using the following
settings: correlated allelic frequencies, burn-in period of 10,000 and
10,000 Markov Chain Monte Carlo (MCMC) interactions; and grouping (K)
ranging from 1 to 5 in 10 independent runs. The results generated were
used as input to the POPHELPER, an R package
(Francis 2016), to predict the best K
value based on Evanno method (Evannoet al. 2005) and to generate the bar graph and delta K graph.
The pairwise Fixation index (Fst) and molecular variation (AMOVA) were
performed using R package adegenet
(Jombart & Ahmed 2011) in order to
reveal genetic differentiation among the two distinct groups occupying
the three agroclimatic zones of Burkina Faso, Benin, Ghana and Togo.
General patterns of genetic diversity were also evaluated by calculating
observed (Ho) versus expected (He) heterozygosity and gene diversity
(Hs) within each population.