Woad (Isatis tinctoria L.) was the most important source of natural blue indigo, a pigment used mainly for dyestuff until the beginning of the 20th century, when the increased use of synthetic dyes by the industry lead to a decrease in the interest of natural dyes and to the abandonment of dye crops. The aim of this study was to characterize, according to morphological and agronomical traits, 11 woad accessions from different countries and to quantify the indigo precursor content by high-performance liquid chromatograph-diode array detection (HPLC-DAD). Qualitative traits revealed a low variability and a great variation was observed in quantitative traits. Principal component analysis (PCA) diagrams divided the accessions into four groups, primarily according to their geographic origin. The first three components of the PCA accounted for 76.8% of the total variation. Reciprocal interactions between indigo precursors and fresh leaf weight were compared through genetic diversity, with significant differences in isatan B (0.5–5.1 g/kg), indoxyl (0.3–2.0 g/kg) and residual indigo (0.3–0.5 g/kg). This information, together with genetic data, can be used to assist local farmers to re-introduce Isatis species in the European agricultural system, not only indicating the higher indigo yielding genotypes, but also the most suitable harvest time.

Woad (Isatis tinctoria L.) was introduced in Europe in ancient times to produce indigo, a natural blue pigment used mainly for dyestuff. This species was cultivated in Portugal until the beginning of the 20th century, especially in the inner North and South. A set of nine inter-simple sequence repeat (ISSR) markers generated 177 reproducible fragments, of which 171 were polymorphic. The mean number of fragments/accession was 111, ranging between 100 (Portugal-Coimbra) and 124 (Poland). The total polymorphism observed was 0.3272, the average polymorphism was 0.1784 and the gene differentiation between accessions was 0.4546. Polymorphism ranged between 53.8% (Austria) and 73.1% (Belgium). The genetic relationship among woad accessions was obtained with unweighted pair group method with arithmetic mean dendrogram based on a molecular marker, clearly clustering the woad accessions according to their geographic origin. The genetic diversity observed in this collection shows that there is a considerable potential for its improvement and that ISSR could be used to evaluate intra- and inter-accession similarities in I. tinctoria species.