Wild Seakale (Crambe Maritima L.) Diversity As Investigated by Morphological and RAPD Markers M

Scientia Horticulturae 95 (2002) 1–12

Wild seakale (Crambe maritima L.) diversity as investigated by morphological and RAPD markers M. Briarda,*, A. Horvaisb, J.Y. Pe´rona aInstitut National d’Horticulture, 2 rue le Noˆtre, 49045 Angers Cedex 01, France bDgenos S.A., Parc Technologique des Capucins, CHRU Bat Monte´clair, 49033 Angers 01, France Accepted 29 January 2002

Abstract

Seakale is a Brassicaceae, native to the coastal sands of Northwestern Europe. To bring this species closer to commercialisation and thereby enhance the diversification of vegetable crops, a breeding program was initiated in 1992. A systematic search for wild populations was undertaken in France, from Quiberon (south Brittany) to Dunkerque (north France near Belgium) to enlarge its genetic basis. Many sites previously described in the literature have disappeared, while five large sites, not previously described, were found. Morphological descriptors and molecular markers (RAPD) were used to study the phenotypic and phenetic variability of the collected plants. A great variability for leaf and leaf-stalk colour, limb, flowers and siliques sizes, was observed. Among the wild collected plants, molecular similarity varied from 25 to 85%. The mean distance from all the wild genotypes to the breeding material already in collection was large (50%). Even if no clear correlation was found between morphological assessment and molecular data except for the leaf-stalk descriptor, the collecting trip was a success. A real enlargement of the variability was obtained. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Seakale; Crambe maritima L.; Germplasm diversity; Molecular markers; RAPD

1. Introduction

To cope with the decrease in the number of vegetables available in the market for consumption since the 1950s, the National Institute of Horticulture (INH) of Angers, France, has been working on vegetable crop diversiﬁcation since 1975 (Pe´ron, 1986). Two different approaches for enlarging the number of vegetables have been undertaken namely resurgence of currently unused crops and wild plant domestication. These two approaches

* Corresponding author. Tel.: þ33-241-225463; fax: þ33-241-225515. E-mail address: [email protected] (M. Briard).

0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0304-4238(02)00022-5 2 M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12 are being employed for the development of seakale, Crambe maritima L. as a commercial vegetable. Seakale is a member of the Brassicaceae. It is native to Northwestern Europe, found in coastal sands, shingles, rocks and cliffs. It is distributed along the Atlantic coast of Europe from the Oslo fjord to north Spain, along the Baltic coast and around the Black and Azov Sea, but not in Mediterranean coast (Claham et al., 1962). In France, several botanists (Antoine, 1953; des Abbayes et al., 1971; Gehu, 1960) have described large sites where seakale was found. It is a perennial herb with branched fleshy rootstock sprouting readily after burial in the shingles. Its leaves are ovate, long-stalked, glaucous, and glabrous with irregularly toothed and wavy margins (Claham et al., 1962). Its blanched leaf petioles were collected and consumed as a wild vegetable by indigenous people when the sand had, by chance, covered enough the overwintered crowns before the reappearance of new leaves in the early spring (Bois, 1927). It could also be consumed as a forced winter vegetable. For this purpose, it was grown in the 19th and early 20th centuries in many private gardens of the UK and also commercially by market gardeners (Evans, 1982). It is also cited as a vegetable crop in almost all the French specialised agricultural encyclopaedias of the last century (Nichol- son, 1893; Vilmorin-Andrieux, 1883). Its asparagus-like shoots were regarded as a highly prized gourmet vegetable. Because of a strong root system with a very high capacity to reproduce, the crop was managed from root cuttings. Nowadays the crop is non-existent. In France, as well as in the UK, there is no commercial seakale production and amateur gardeners also ignore it. Its decline has been caused by several factors such as the deterioration of seakale stocks due to its vegetative multiplication resulting in viruses infestation (Brown, 1937), the high labour input for its production (Evans, 1982) and its difficult reproduction mainly due to the low percentage of germinating seeds and long germination time (Scott and Randall, 1976). A nutritional composition study (Pe´ron et al., 1991) revealed a low caloric value, a low nitrate content, high protein and fibre content, an interesting distribution among mineral (high quantity of potassium/low sodium; excellent calcium/phosphorus ratio), and a good content of some vitamins such as thiamine (Vitamin B1). This very valuable nutritional composition together with an attractive taste encouraged us to proceed with this project. To rehabilitate seakale as a crop, Evans (1982) recommended forcing seakale crowns, obtained from root cuttings or seeds, in growing rooms as is done for chicory. At the same time, our work in rehabilitating seakale as a crop included applying new technologies for production: the in vitro micropropagation of cuttings, the use of the chicory-forcing chambers with specific growing conditions for seakale (Pe´ron, 1986) and the development of a new clone called CCo (Pe´ron, 1985; Pe´ron and Re´gnier, 1987). Moreover, field studies were done both from cuttings as well as from tissue culture; in the latter case proper acclimatisation was done before field setting. These experiments resulted in the development of a cultural practice for growing and forcing the plants (Pe´ron, 1989, 1990). We focused on two aspects in order to enhance the economics of the crop. The first aspect was sexual reproduction to replace rooted cuttings. Growth of the rooted cuttings was highly variable, production of successive generations of cuttings was difficult to maintain, and in vitro plantlet propagation was too expensive. The second objective was final product quality especially in terms of uniformity and homogeneity (Fig. 1). Therefore, M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12 3

Fig. 1. Ideal shape of shoots from forced crowns of the CCo seakale clone. physiological studies were begun to overcome seed dormancy (Lan et al., 1998) and to improve the crown quality before forcing (Lan and Pe´ron, 1998). However, this physiological selection was not enough because the plant material used was still close to the wild type and CCo, our cultivated clone, is auto-incompatible. Thus, a breeding project was initiated in 1992. The ﬁrst step was to search for and collect new plant material for a collection of genotypes to enlarge the genetic basis. The second step was to analyse this collection and to evaluate its variability. Morphological characters and molecular markers, random ampliﬁed polymorphic DNA (RAPDs; Williams et al., 1990) were involved to characterise its phenotypic and phenetic variability.

2. Materials and methods

A systematic approach was undertaken to determine the variability of seakale. Following identiﬁcation of seakale sites from the scientiﬁc literature, a comprehensive germplasm collection was initiated in France, along the Channel and Atlantic coasts (Fig. 2). Each 4 M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12

Fig. 2. The five important collection sites in France. C: Cayeux, G: Gatteville, L: Locquemeau, K: Kerlouan and T: Tre´vignon; IH: limits of the prospected area. seakale site was described including the number of native plants, area of the site, position with respect to the sea, the ecosystem (sand, shingle, other plants). Seeds were collected from several plants at different places of each site to give as diverse a morphological population as possible. CCo, the clone selected in the laboratory, was the reference genotype already studied in all previously cited experiments. Twenty CCo cuttings were grown. For the wild populations, depending on the germination rate or transplanting success, 8–30 plants were grown. Therefore, the ﬁeld experiment consisted of 20 CCo plants and 76 wild plants, six from Kerlouan, 13 from Gatteville, 30 from Cayeux, nine from Trevignon and eight from Locquemeau. Seed germination and growing conditions were as previously described (Lan et al., 1998; Pe´ron, 1989). Morphological characteristics were evaluated through foliage descriptors including presence or absence of anthocyanin, general colour, habit, leaf type, leaf indentation, leaf-stalk form and vigour of the plant. For the molecular analysis, a subset of 12 plants was chosen. There were two plants of each origin. Their morphological characteristics were shown in the Table 1. From each plant, fully expanded young leaves were collected. This leaf material was frozen (À20 8C) and DNA extraction was performed following the procedure described by Edwards et al. (1991). The ﬁnal solution was diluted to 25 ng/ml, 1 ml of which was mixed in a 25 ml reaction mixture that contained 1X PCR buffer (75 mM Tris–HCl, pH 9; 20 mM Table 1 1 (2002) 95 Horticulturae Scientia / al. et Briard M. Foliage characteristics of the genotypes studied with RAPD markers

Anthocyanic colourationa General colour Habit Leaf dissection Leaf indentation Leaf-stalk Vigour

K3 À Pale green Prostrate Highly dissected Slighlty indented Very thick Intermediate K4 À Pale green Semi-erect Intermediate Slightly indented Thin Intermediate G1 À Pale green Semi-erect Poorly dissected Intermediate Short Intermediate G2 À Pale green Erect Intermediate Absent Medium size Vigorous C2 À Blue green Semi-erect Poorly dissected Slightly indented Long, thin Vigorous C9 À Pale green Semi-erect Intermediate Intermediate Short, thick Vigorous T2 À Blue green Erect Intermediate Intermediate Medium size Vigorous T10 À Blue green Semi-erect Intermediate Slightly indented Medium size Vigorous L2 À Blue green Semi-erect Poorly dissected Slightly indented Long, thin Intermediate L5 À Blue green Semi-erect Poorly dissected Slightly indented Long, thin Intermediate Cco7 þþ Pale green Erect Intermediate Highly indented Long, thin Vigorous Cco10 À Dark green Erect Intermediate Intermediate Long, thin Vigorous

a –

(À) absent, (þþ) present. 12 5 6 M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12

(NH4)2SO4; 0.01% (w/v) Tween 20); 2 mM MgCl2; dNTP 0.1 mM; 0.2 mM primer Eurogentec and Genosys or 0.4 mM primer Operon and BBV and 1 U Taq polymerase (Eurogentec). Amplification was carried out in a Trio Thermoblock Biometra using the following conditions: 45 cycles consisting of 1 min at 94 8C, 1 min at 32 8C, 2 min at 72 8C. Then, 10 ml of the PCR reactions was loaded on a 1% agarose gel in TBE buffer and separated by electrophoresis (100 V, 2 h). A Biomarker EXT from Bioventures was included as a size marker. Gels were stained with ethidium bromide and observed under UV light. Markers were scored as present (1) or absent (0). The Sokal and Michener (1958) distance index was computerised using a macro written for Microsoft Excel 1997. Twenty two primers were tested: they were either randomly chosen from Eurogentec, Genosys and Operon or given as a gift by BBV (Bretagne Biotechnologie Vegetale, Saint Pol de León, France) based on previous results on Brassica oleracea (Boury et al., 1992). For each of the 22 primers, a limited number of duplicate reactions were completed to confirm repeat- ability: with repeated PCR reactions on repeated extracts of several individuals. From the distance matrix, a radial UPGMA dendrogram was drawn using the DARwin 3.5 software developed by Perrier (1998).

3. Results and discussion

Except for Cayeux and Gatteville (Fig. 2), seakale populations had disappeared from most of the sites previously described in the literature. However our systematic search from Quiberon (south Brittany) to Dunkerque (north France near Belgium) resulted in finding new sites. Among them, three sites were particularly large: from north to south Locque- meau, Kerlouan (Fig. 3) and Tre´vignon (Fig. 4). Phenotype observation of the 96 plants grown in the field indicated new types compared to CCo. Indeed, all the 76 plants from the wild population collections were, at least for one character, different from the 20 plants belonging to CCo. Among the 2877 pairs feasible within the 76 wild plants, only six pairs were constituted with two exactly similar plants (data not shown). A great variability for leaf and leaf-stalk colour, limb, flowers and siliques sizes, was observed. Therefore, from these morphological results we could conclude that our collecting trip was a success in terms of variability enlargement. However, the greatest care must be taken, since within Brassica, maybe more so than within other species, environmental conditions may have greater effects on the phenotype that are not genotypic. Furthermore, in nature, the seakale seed is dispersed by seawater in which it will float for many days without loss of viability (Claham et al., 1962). Therefore, one can imagine that all along the Channel and Atlantic coasts only one population exists, spread from a place to another one by seawater. Moreover, within a site, some plants may have very deep and long caulinary ramifications, and the sand, shingles and dunes movements due to tides and storms could also be responsible for a vegetative plant multiplication. These two phenomena could result in apparently individual plants which could, in fact, belong to the same stock. Because developing 76 new inbred lines from the 76 wild plants that we were able to grown will be high costly, it is necessary to avoid duplicates. Even though we tried to collect seeds from distant plants of each site (see Section 2), a molecular characterisation of M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12 7

Fig. 3. Kerlouan site, seakale plants (indicated with an arrow) growing in the coastal sand at the upper limit of the high tide. a subset of plants was done in order to check if populations are as different from CCo and from each other as we could imagine from their morphological variability. Therefore, we choose two plants from each population and from CCo (Table 1). To check if plants issuing from the same site and looking highly similar were duplicates or not, due to natural vegetative multiplication, L2 and L5 from Locquemeau and T2 and T10 from Tre´vignon, were studied. To evaluate if plants issuing from two different sites but looking very similar could be sister lines, due to seawater seeds transportation, C2 from Cayeux, was compared with L2 and L5. The others were chosen in order to evaluate reliability of morphological descriptors for genome variability assessment. Finally, the morphological evaluation of the 20 CCo plants revealed one plant quite different from the others, with anthocyanin present in leaves and stalks. In order to check, how distant from the others this violet plant (CCo7) was, we compared it with a standard one (CCo10).

Fig. 4. Tre´vignon site, seakale plants (mentioned with arrows) growing on the cliff. 8 M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12

Table 2 RAPD markers produced by six primers

Primer Sequence Polymorphic Molecular Frequencya markers code weight (bp)

SB 1 GTTAGACCATTC a 1710 8 b 680 3 JW 2 CGGTCACTGT c 2240 5 SB 4 GTTAGACCATTCCA d 1120 6 e 890 7 f 680 4 OPB 1 GTTTCGCTCC g 2000 6 OPB 7 GGTGACGCAG h 2150 4 i 860 7 j 630 8 k 540 10 l 450 10 OPD 12 CACCGTATCC l0 2240 6 m 2150 8 n 1710 10 o 1360 8 p 1260 3 q 930 6 r 860 6 s 660 5 a Number of genotypes in which the band was present (among 12).

As a preliminary, all the RAPD primers were checked for their reproducibility and their ability to detect polymorphism. Among the 22 primers studied, six revealed polymorphism and generated 100% reproducible proﬁles. Markers were considered reliable if the presence or absence of the fragment could be visually determined without ambiguity. A total of 59 reliable, repeatable markers were obtained. As indicated in Table 2, 20 of them (34%) were polymorphic and were scored for the accessions in this study. The matrix of similarity percentages was calculated (Table 3) and the resulting dendrogram was drawn (Fig. 5). The two morphologically similar plants belonging to the Locquemeau population were also close to each other for molecular markers (80% similarity—same subgroup on the dendrogram). On the other hand, those from Tre´vignon were rather distant from each other (45% similarity) and were branching on two different subgroups of the dendrogram. C2 from Cayeux, in spite of a high morphological similarity with L2 and L5, reached only 75 and 65%, respectively. However, the three plants were clustered in the same subgroup. From this, we can conclude that even if some plants were phenetically close to each other, none, among all the plants analysed in the present study, were duplicates, even those looking very similar. It means that no evidence can be given for seawater seeds transportation from this preliminary study. It also means that vegetative multiplication within a site was not as important as we feared or our collecting method was efﬁcient enough to avoid plants belonging to the same stock. Our initial question was to know if, by collecting new plants, we really enlarged our genetic basis or not? Even without a more M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12 9

Table 3 Matrix of Sokal and Michener’s similarity index (%)a

K3 K4 G1 G2 C2 C9 T2 T10 L2 L5 CCo7 Cco10

K3 60 75 85 50 65 60 25 35 45 35 25 K4 65 55 40 35 60 45 55 45 35 45 G1 80 45 60 75 30 40 40 30 30 G2 45 70 65 30 30 30 30 20 C2 35 40 75 75 65 85 75 C9 65 30 40 40 30 30 T2 45 55 45 35 35 T10 80 70 80 80 L2 80 80 80 L5 70 70 CCo7 90 a Letters are sites with C: Cayeux, G: Gatteville, L: Locquemeau, K: Kerlouan, T: Tre´vignon and CCo: reference clone. Numbers after letters are the number of the plant analysed. deﬁnitive work with larger numbers of individuals analysed, the answer is deﬁnitively yes. Except for the Locquemeau site, we found genotypes distant from CCo in each wild population. The most distant individual is from Gatteville (G2/CCo10 similarity is only 20%). The mean distance from all the wild genotypes to the two CCo is large (50%). Finally, between the different origins we can also found very distant individuals (e.g. K3/

Fig. 5. UPGMA dendrogram obtained from the similarity analysis of 12 Crambe genotypes. 10 M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12

T10 similarity is only 25%). In short, among wild populations, molecular similarity varied from 25 to 85%. All this confirms the collecting trip success: a variability enlargement has been obtained. The molecular similarity (35%) between the two Cayeux plants was consistent with their morphological distinction. This was also confirmed on the dendrogram with their branching in two different subgroups. Regarding their phenotypes, Kerlouan 3 and Cayeux 2 were supposed to be very different. They were effectively distant (50%) but a bit less than the Tre´vignon 2 and 10 (45%) which were supposed to be closed. Finally, CCo7 and CC10, very different in terms of morphology, were the closest of all the genotypes studied (90%). From these results, no clear correlation between morphological and molecular data was shown. It is probably due to the neutral character of RAPD markers. Therefore, it appears that morphological characteristics have to be interpreted with caution, especially colours. This misinterpretation of phenotypes is probably because foliage colour and presence of anthocyanin are deeply influenced by environmental conditions as for other Brassica.For example, a small stone disturbing a Cauliflower root growth is enough to cause a dramatic change in anthocyanins. The only morphological character, which seems consistent with molecular results, was leaf-stalk thickness and length. CCo has long and thin leaf stalk as does Cayeux 2 (85 and 75% similar) or Locquemeau 2 and 5 (from 70 to 80%). They are clustering in the same subgroup. In contrast Cayeux 9, Gatteville 1 and Kerlouan 3 have very thick and/or short leaf stalk. Their marker similarity with the clone varies from 25 to 35%. For future collections, we will consider this descriptor with specific care. Even if they were the closest of all the genotypes studied, the two individuals belonging to the clone CCo (90% similarity) were not identical. Distance is not as dramatic as we could imagine from their morphological appearances, which is excluding a mix in plantlets during transplantation process. The 10% difference between the two plants could be partly explained by a certain degeneration of the cutting due to viruses infestation stock as mentioned by Brown (1937), or by somaclonal variation. It could also, more probably, be compared to the off-types recently described in other Brassica (Ruffio-Chaˆble et al., 2001). Indeed, the appearance of such off-type plants in CCo is not unusual: each time we multiplied the clone we got some of them, without finding their origin. Ruffio-Chaˆble et al. (2001) reported ‘‘aberrant plants’’ as well in hybrids as in inbred lines or populations varieties of B. oleracea. Studies have been undertaken to identify the underlying agronomic factors and the genetic mechanisms involved. The authors suggested the involvement of epigenetic phenomena causing modification in gene expression. Changes in chromatin structure and/or DNA methylation may be induced by environmental stresses. Reactivation of mobile elements will also be considered. The ‘‘aberrant’’ Crambe plants could be a complementary model to this Brassica study. In conclusion, the present results were very encouraging. We began with the breeding program trying to improve final product quality and yield as well as decreasing seed dormancy. After a few breeding cycles it will be possible to check the level of homogeneity of the lines with the RAPD primers developed in this study. We will also use them to characterise four new populations we collected in Eastern and Western England and a new one from north Brittany. Finally, it would be interesting to determine the genetic distance between our material and populations around the Black Sea. Schultz (1919) has described a botanical variety M. Briard et al. / Scientia Horticulturae 95 (2002) 1–12 11

Fig. 6. Plantlet of Crambe maritima var. pontica; cotyledons and first leaves are pubescent. native of the Black Sea coast, called C. maritima var. pontica. Thirteen years ago, we got seeds of plants belonging to this botanical variety which were preserved in the Museum d’Histoire Naturelle in Paris. Only one plant grew and it effectively looked different at the juvenile stage with pubescent cotyledons and ﬁrst leaves (Fig. 6).

Acknowledgements

We are grateful to Nathalie Blanchard and Richard Guyon for their participation in the collection and molecular analysis, respectively.

References

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