Taxon sampling and total genomic DNA isolation
To investigate the geographical distribution of watermelon haplotypes,
we included in the study the four most economically importantCitrullus species: 1) C. lanatus , widely cultivated
throughout the world (78 accessions from four continents out of which
only 14 were from West Africa); 2) C. mucosospermus , restricted
to West Africa and the closest sister species of cultivated watermelon
(13 accessions); 3) C. amarus , a wild species from
Southern Africa that has spread to Europe and the closest relative toC. ecirrhosus (22 accessions); and C. colocynthis , a wild
species found in northern Africa and East-Asia (22 accessions). In
total, 135 accessions were assessed, including 53 from Africa, 41 from
Asia, 25 from Europe, and 16 from North America (Table 1). Voucher
specimens of all accessions were deposited in the herbarium of the
Institute of Plant Genetics (Achigan-Dako
et al., 2015) (IPK-Gatersleben).
As indicated in Table 1, a total of 53 accessions were received from the
USDA National Plant Germplasm System, 66 were received from
IPK-Gatersleben, and 16 were collected throughout West Africa as part of
this study. Seeds of all accessions were germinated in a greenhouse at
IPK-Gatersleben, and approximately 100 mg of leaf tissue was collected
from one seedling per accession and dried with silica gel. Total genomic
DNA was extracted from the dried leaf tissues using the QIAGEN DNAeasy
Plant Kit, and one washing step was added according to the
manufacturer’s instructions to increase the quality of the DNA.
Concentrations were estimated on 1% agarose gels stained with ethidium
bromide. Samples exhibiting sub-optimal PCR amplification were purified
via the QIAquick PCR Purification Kit (QIAGEN) and resuspended in 50 ml
1x TE buffer.