DNA extraction and genotyping by sequencing

Kersting’s groundnut plants were grown at the University of Abomey-Calavi (Benin) under field conditions. Three-week old leaves were collected into 96 deep well samples collection plates and sent to the Integrated Genotyping Service and Support (IGSS) platform (https://ordering.igssafrica.org/cgibin/order/login.pl) located at Biosciences Eastern and Central Africa (BecA-ILRI) Hub in Nairobi for Genotyping. DNA extraction was done using Nucleomag Plant Genomic DNA extraction kit. The genomic DNA extracted was in the range of 50–100 ng/ul. DNA quality and quantity were checked on 0.8% agarose. Libraries were constructed according to Kilian et al. [42] Diversity Arrays Technology and Sequencing (DArTSeq) complexity reduction method through the digestion of genomic DNA and ligation of barcoded adapters followed by Polymorphic Chain Reactions (PCR) amplification of adapter-ligated fragments. Libraries were sequenced using Single Read sequencing runs for 77 bases. Next generation sequencing was carried out using Hiseq2500.

DArTseq markers scoring was achieved using the DArt Proprietary Limited (PL’S) proprietary SNP and SilicoDArt calling algorithms (DArTsoft14). SNP markers were scored as binary fashion for presence/absence (1 and 0, respectively) of the restriction fragment with the marker sequence in genomic representation of the sample. SNP markers were aligned to the reference genomes of mung bean [Vigna radiata (L.) R.Wilczek] and adzuki bean [Vigna angularis (Willd.) Ohwi & Ohashi] [43, 44], two related species of Kersting’s groundnut, in order to identify chromosome positions.