Introduction
Painting and colour used along human history relied on natural dyes, giving rise to the textile industry in Europe, an industry that decreased with the development of synthetic dyes in the 20th century. Today there is an increasing interest in natural products from renewable sources, such as natural dyes, mainly from dye plants, due to the fact that they are friendly to the environment. Woad or glastum (Isatis tinctoria L.) is a dye plant that belongs to the Cruciferae family (Brassicaceae), tetraploide (2n = 4x = 28), outbreeding and biennial (Darlington and Wyle, Reference Darlington and Wyle1955). The centre of origin of this plant appears to be Central Asia (Spataro et al., Reference Spataro, Taviani and Negri2007). It produces indigo, a natural blue pigment used mainly for dyestuff. Woad is also used for medical purposes and in cosmetic industries manufacturing soaps and body creams (Spataro and Negri, Reference Spataro and Negri2008). The three main indigo precursors, isatan A, isatan B and indican, are present in the leaves (Gilbert and Cooke, Reference Gilbert and Cooke2001; Oberthur et al., Reference Oberthur, Graf and Hamburger2004). Various landraces of woad present differ in their phenotypes and amounts of indigo precursors (Stoker et al., Reference Stoker, Cooke and Hill1998). The evaluation of genetic diversity could promote the efficient use of genetic variations in breeding programmes (Paterson et al., Reference Paterson, Tanksley and Sorrells1991). Inter-simple sequence repeat (ISSR) markers have been used, in Brassicaceae, to study genetic relationships among species of the genus Diplotaxis (Martín and Sánchez-Yélamo, Reference Martín and Sánchez-Yélamo2000); distinction of cultivated and wild on B. rapa (Andersen et al., Reference Andersen, Poulsen, Andersen, Kiær, D'Hertefeldt, Wilkinson and Jørgensen2009); cultivar identification and genetic diversity analysis of B. oleracea (Lu et al., Reference Lu, Liu, Gong, Zhao, Song and Zhu2009); and to detect genotoxic effect of heavy metals on Eruca sativa (L.) (Al-Qurainy, Reference Al-Qurainy2010). They are based on amplification of DNA sequence present at an amplifiable distance between two identical microsatellite adjacent regions oriented in opposite direction, using a single microsatellite primer (Zietkiewicz et al., Reference Zietkiewicz, Rafalski and Labuda1994). This type of marker has advantages similar to random amplified polymorphic DNAs (RAPDs) and simple sequence repeats, since they are polymorphic, have good reproducibility, cheap in cost, require no prior knowledge regarding the sequences of the genome, require only a small amount of DNA and are fast in reacting (Zietkiewicz et al., Reference Zietkiewicz, Rafalski and Labuda1994; Rakoczy-Trojanowska and Bolibok, Reference Rakoczy-Trojanowska and Bolibok2004). However, ISSRs are more reproducible than the RAPD; they use longer primers and hybridize at higher temperatures (Pasqualone et al., Reference Pasqualone, Caponio and Blanco2001).
The aim of this work was to evaluate the genetic diversity and similarity levels among and within accessions of woad from Europe and Central Asia by using ISSR markers. This information, combined with data from agronomic and phytochemical content, can contribute to the prediction of the potential of this crop in subsistence agriculture and as an alternative to the production of synthetic dyes.
Material and methods
Plant material
Eleven accessions of woad from nine different European countries – UK, Spain (SP), Belgium (BL), Germany (GR), Austria (AU), Italy (IT), Poland (PL), Portugal (Portugal-Madeira (PT-M) and Portugal-Coimbra (PT-C) and one from Central Asia – Kazakhstan (KZ) – were studied. The seeds were sown in jiffy pots in August and transplanted to the field in September 2008.
DNA extraction and ISSR analyses
Young woad leaves were collected from nine individual plants, frozen in liquid nitrogen and stored at − 80°C. DNA was extracted using the DNeasyTM Plant Mini Kit (Qiagen) and its concentration and purity were evaluated by Nanodrop ND-1000.
Initially, 28 primers of the University of British Columbia (UBC) were tested, of which nine primers (UBC 809, UBC 811, UBC 817, UBC 826, UBC 847, UBC 850, UBC 880, UBC 888 and UBC 889) were chosen, due to the polymorphism and higher efficiency amplification. Each PCR reaction contained 60 ng of DNA, 10 μl Taq-PCR Master mix (Quiagen) and 0.5 μM of each primer. Amplification was performed in an Biometra One II thermocycle with an initial denaturation at 94°C for 5 min and 44 cycles at 94°C for 30 s, 52°C for 45 s, 72°C for 2 min, followed by 10 min at 72°C. Amplified products were separated in 1.8% agarose gel in 1 × tris-borate-EDTA buffer at 120 V. The gels were stained with 0.5 μg/ml ethidium bromide solution and visualized by illumination under an ultra violet light.
The ISSR fragments were scored manually as present (1) or absent (0). Cluster analyses, using simple matching (SM) coefficient and unweighted pair group method with arithmetic mean (UPGMA), were performed with the NTSYSpc-2.0 g software.
Result and discussion
The nine ISSR primers selected yielded very clear identifiable bands and informative patterns in the eleven accessions. This set of primers produced fragments with sizes between 250 and 2500 bp and generated a total of 177 reliable fragments, of which 171 (96.6%) were polymorphic (Table 1), with an average of 19.7 polymorphic fragments/primer. Spataro et al. (Reference Spataro, Taviani and Negri2007), in a I. tinctoria Eurasian collection, using amplified fragment length polymorphism (AFLP) and selective amplified microsatellite polymorphic locus, found similar results, 95.63 and 96.44% of polymorphic fragments, respectively. However, Gilbert et al. (Reference Gilbert, Garton, Karam, Arnold, Karp, Edwards, Cooke and Barker2002) found 86.9% of polymorphic fragments in three species of Isatis (I. glauca, I. tinctoria and I. indigotica) with AFLP, results that could be attributed to the molecular marker (ISSR) and to the different species used.
Total number of bands and percentage of polymorphism detected in each accession and primer
The 11 accessions showed an average of 111 fragments, ranging from 100 in the accession from PT-C, to 124 in the accession from PL. The accession from AU revealed the lowest percentage of polymorphism (53.8%), while the accession from BL showed the highest percentage of polymorphism (73.1%). For the assembly of accessions, the average of polymorphism was 65.4% (Table 1). Gilbert et al. (Reference Gilbert, Garton, Karam, Arnold, Karp, Edwards, Cooke and Barker2002) found a higher polymorphism (86.8%) in a collection of five landraces in the same species. The primer UBC 8882000 generated a specific fragment for the Italian accession. Seven plants, of the nine studied, from the UK accession, also presented one specific allele with primer UBC 817600. Such specific bands could be useful for identifying accessions. Two unique alleles were found in accessions from PT-M, BL and UK with primers UBC 8172000, UBC 8501800 and UBC 888600, respectively.
The dendrogram based on UPGMA analysis grouped the 11 accessions into three major clusters with SM similarity coefficient, ranging from 0.63 to 0.94 (Fig. 1). The accessions from PL, BL and Hungary (HU) are grouped in Cluster I. Cluster II comprises seven accessions, and within this cluster, we can find two subclusters. Subcluster IIa comprises the accessions from UK, SP, PT-M and PT-C, while subcluster IIb comprises the accessions from AU, GR and KZ. Cluster III is formed only by the accession from IT, which shows less similarity with the other accessions.
Dendrogram of genetic similarity relationship based on total 99 plants data using UPGMA.
The analysis of molecular variance showed that the percentage of variation within accessions (58%) was higher than the variation among accessions (42%). The first three components in the principal coordinate analysis (PCoA) that resulted from the Nei genetic distance matrix accounted for 61.5% of the total variations (22.4, 20.9 and 18.3% for the first, second and third components respectively). The PCoA grouped the accessions into three clusters, which are in accordance with the clusters obtained with UPGMA. The total variation in PCoA (61.5%) was lower than the principal component analysis observed with the morpho-physiological traits (79%, not published), and the graphic projection of the accessions was not similar.
The detection of high levels of polymorphism makes ISSR a powerful tool for assessing genetic diversity in woad, as it is fast, simple and efficient. None of the individual plants were genetically identical according to the ISSR analysis, indicating that the level of resolution in our study was sufficient to distinguish all genotypes.
This study, together with the morphological and chemical characterization, could contribute to the selection of woad accessions for breeding programmes, in order to have plants with higher number of leaves and/or bigger leaves and higher content of indigo precursors, becoming an economically viable crop in the coming years.
Acknowledgements
The authors thank the Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; the Royal Botanic Garden of Madrid; the Botanic Garden of Coimbra; the National Botanic Garden of Belgium; Germplasm Bank of Hungary and Dr. David Hill from University of Bristol for providing the woad material. This work was supported by FCT project POCTI/AGR/56087/2004.
