Evaluation of root yield traits and glucosinolate concentration of different Armoracia rusticana accessions in Basilicata region (southern Italy)

Scientia Horticulturae 170 (2014) 249–255

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Evaluation of root yield traits and glucosinolate concentration of different Armoracia rusticana accessions in Basilicata region (southern Italy) Rosa Agneta a , Christian Möllers b , Susanna De Maria c , Anna Rita Rivelli c,∗ a

Doctoral School of “Crop Systems, Forestry and Environmental Sciences”, University of Basilicata, Via dell’Ateneo Lucano, 85100 Potenza, PZ, Italy Department of Crop Sciences, Georg-August-Universität Göttingen, Von Siebold-Str. 8, D-37075 Göttingen, Germany c School of Agricultural, Forest, Food and Environmental Sciences, University of Basilicata, Via dell’Ateneo Lucano, 85100 Potenza, PZ, Italy b

a r t i c l e

i n f o

Article history: Received 21 January 2014 Received in revised form 6 March 2014 Accepted 11 March 2014 Available online 1 April 2014 Keywords: Horseradish Brassicaceae Phenotypical traits Glucosinolates AFLP markers

a b s t r a c t Armoracia rusticana (horseradish) is a Brassicaceae species cultivated for its roots. It is appreciated for its intense flavour due to the richness of secondary metabolites, such as glucosinolates (GLS) and their breakdown products. Roots of horseradish well suitable for agro-industrial and pharmaceutical sectors are selected based on yield, biometrical characteristics and qualitative composition. Six horseradish accessions from southern Italy were compared based on root traits, yield and GLS concentration. The genetic variability of the accessions was also investigated using the amplified fragment length polymorphism (AFLP) markers. Phenotypically, the six accessions showed significant differences for the sprout number on the crown as well as for the root diameters and length. Among the accessions, wide diversity in total and marketable root yield per plant was also found. The root GLS analysis revealed significant differences for the individual and total GLS concentration. Particularly, this latter ranged from 1.73 to 37.7 ␮mol g−1 of dry weight, with sinigrin as predominant GLS in all accessions, contributing from 53% to 87% of the total GLS concentration. By using AFLP markers, the percentage of polymorphisms detected was high. The similarity clusters developed based on root biometric characteristics, yield and total GLS concentration was different from the cluster based on AFLP markers. Wide variability among the horseradish accessions was found on the basis of the morphological and qualitative traits, and molecular markers. The genetic diversity of horseradish germplasm still available in the Basilicata region deserves to be preserved and protected against genetic erosion. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Horseradish (Armoracia rusticana P. Gaertner, B. Meyer & Scherbius), a member of the Brassicaceae family, is a neglected medical herb known since antiquity as folk medicinal plant, and which is currently used as dish condiment (Agneta et al., 2013; Wedelsbäck Bladh and Olsson, 2011). The plant is particularly appreciated for its thick, fleshy and white root characterized by a pungent flavour when cut, which is mainly grated fresh or processed into a sauce. The typical intense aroma and odour are due to the richness of secondary metabolites such as glucosinolates (GLS) and their volatile breakdown products, the latter ones released by enzymatic degradation of GLS (Li and Kushad, 2004). The wide anti-microbial (Tierens et al., 2001; Yano et al., 2006)

∗ Corresponding author. Tel.: +39 971 205382; fax: +39 971 205378. E-mail address: [email protected] (A.R. Rivelli). http://dx.doi.org/10.1016/j.scienta.2014.03.025 0304-4238/© 2014 Elsevier B.V. All rights reserved.

and anti-carcinogenic activities (Munday, 2002; Srivastava et al., 2003) shown by breakdown products of GLS and their potential utilization as natural substances in the agro-pharmaceutical sector are the main reasons for the increasing scientific and commercial interest to characterize these secondary metabolites in planta. Horseradish is considered an important source of these metabolites, since some studies report that its tissues contain a large number of GLS such as sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, gluconasturtin, 4-methoxyglucobrassicin, glucoiberin, gluconapin, glucocochlearin, glucoconringianin, glucosativin, glucoibarin, 5-hydroxyglucobrassicin, glucocapparilinearisin or glucobrassicanapin, glucotropaeolin, glucoarabishirsutain, 2methylsulfonyl-oxo-ethyl-GLS (Agneta et al., 2012; Li and Kushad, 2004; Redovnikovic´ et al., 2008; Wedelsbäck Bladh et al., 2013a). Interestingly, Li and Kushad (2004) and Wedelsbäck Bladh et al. (2013a) have shown a large variability of GLS concentration in roots and leaves of various horseradish accessions of different parts of the world, whose values ranged from 2 to 296 ␮mol g−1

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of dry weight. Despite the large variation in GLS content among accessions, Li and Kushad (2004) noted that the geographical origin of the accession did not seem to have an effect on total GLS level. Horseradish, native of the temperate regions of eastern Europe and western parts of Russia, has become naturalized in many parts of the world and can be found cultivated and growing wild, e.g., in fields, home gardens, weedy areas, farmland, roadsides and ditches. The main areas of commercial cultivation are located in Europe ´ and in North America (Łuczaj and Szymanski, 2007; Sampliner and Miller, 2009; Wedelsbäck Bladh and Olsson, 2011). Nowadays, in several European countries such as Bulgaria, Romania, Poland, Russia and Italy, horseradish is still used to prepare tradi´ tional dishes and as popular medical herb (Łuczaj and Szymanski, 2007; Sampliner and Miller, 2009; Sarli et al., 2012). Particularly, horseradish is largely diffused in the internal areas of the Basilicata region (southern Italy) where this plant is semi-cultivated. The plant is locally consumed, from ancient times as food and condiment, mainly utilized grated fresh or to prepare traditional gastronomic dishes in the period between January and April, and in some cases it is used as folk medicinal remedy (Pieroni and Quave, 2005; Pieroni et al., 2004; Sarli et al., 2012). Recently, a morphological characterization study reported large phenotypic differences among various accessions collected from different villages of the Basilicata region (Sarli et al., 2012). The different morphology of the horseradish plants belonging to various sites of southern Italy, suggests the existence of a large variability among them. Moreover, in this area, the local farmers that still cultivate horseradish mainly maintain the plants in their home gardens. Hence, these home gardens play a strategic role in the preservation of different horseradish landraces. Furthermore, the exploration of the phenotypical, chemical, and genetic variability of the available germplasm can be useful to prevent the risk of losing genotypes of high nutritional value and specific taste, especially in view of the fact that horseradish is vegetatively propagated by planting clones of root sections collected from the previous year’s crop (Walters and Wahle, 2010). However, despite the great interest on this species, most researches have focused mainly on the GLS profile and composition and only few studies are available on GLS content and even less are on the productivity and agronomic performances. Since the commercial value of the horseradish root depend on yield performance, root biometric characteristics (i.e., diameter, length, dry weight), and flavour due to the richness of GLS, the aim of this study was to assess the differences on root yield, morphological traits and GLS concentration among six accessions of A. rusticana from different geographical areas of the Basilicata region. Additionally, considering that the GLS content and composition in planta can be strongly influenced by genotype and environment (Rosa et al., 1997; Rosen et al., 2005; Sarikamis¸ et al., 2009; Kabouw et al., 2010), the purpose of this study was also to investigate the genetic diversity occurring among the different accessions, by using the amplified fragment length polymorphism (AFLP) markers, and to compare the genetic variation among the same accessions with their variability concerning the root yield, root morphological traits and GLS concentration. 2. Materials and methods 2.1. Plant material and sample preparation The investigations were carried out on A. rusticana plants grown in the field collection of the Institute of Plant Genetics, National Research Council, Thematic Centre for the Preservation of Mediterranean Biodiversity, located in Policoro (MT) (40◦ 17 30 N, 16◦ 65 16 E), where many accessions of horseradish, previously collected from various villages of the mountainous internal areas of

the Basilicata region (whose map is reported in Sarli et al., 2012), are vegetatively maintained and propagated. The germplasm consisted of root cuttings, transplanted in single rows (130 cm between rows and 70 cm in the row) by the end of April 2010 (detailed information is reported in Sarli et al., 2012). Irrigation, plant protection, and weed control were carried out according to local practices. The geographical origin and the alphabetic code of the six accessions characterized in this study are reported in Table 1. The accessions selected were the most locally consumed and were originally from Matera (i.e., A and F accessions from Accettura and Stigliano towns, respectively) and Potenza (i.e., B, C, D and E accessions were originally from Catelsaraceno, Guardia Perticara, Montemurro and San Severino Lucano towns, respectively) provinces, by municipalities located in mountainous areas from 723 to 960 m a.s.l. On the selected six accessions, morphological traits of leaves, petioles and stem (i.e., flowering stem) were observed and evaluated on four plants during two years (2010 and 2011, leaving the plants in field as a perennial crop), according to the modified UPOV guidelines for the conduct of tests for distinctness, uniformity and stability for horseradish (UPOV, 2001) as described by Sarli et al. (2012). According to such procedures, the observations on the leaves were carried out on the fourth fully developed leaf and on the first incised leaf. In February 2011, three plants of each accession were manually harvested, cleaned with tap water and divided in sprouts and roots. Then, root biometric characteristics (number of roots and sprouts on the crown, length, equatorial and polar diameters at the top and basal diameter of the marketable roots) and root yield (fresh weight of total and marketable roots) were recorded. Subsequently, samples of the roots were cut, cleaned with distilled water, dried with paper towels and quickly frozen at −80 ◦ C for the GLS analysis; the remaining roots were dried in a ventilated oven at 75 ◦ C until steady weight to determine the dry weight (dry wt). Later, during the vegetative re-growing stage (by the end of April 2011) for each accession on two plants left in the field, four youngest fully expanded leaves (approximately 8 cm in width and 20 cm in length) were collected and stored at −80 ◦ C until DNA extraction. 2.2. Glucosinolate analysis Before chemical analysis, root tissues were quickly frozen in liquid nitrogen to allow the crushing, then lyophilized and homogenized into fine powder using a laboratory mill. Afterwards, polypropylene tubes, containing 200 mg of dry material for each sample, were placed in a water bath heated to 75 ◦ C for 1 min. GLS were extracted following the protocol by Kräling et al. (1990). Briefly, for GLS extraction, 2 ml of 70% methanol and 200 ␮l of internal standard solution (6 mmol glucotropaeolin) were added to each sample and vortexed shortly, then the mixture was incubated for 10 min in a water bath at 75 ◦ C, mixed twice on a Vortex mixer, centrifuged (Heraeus Varifuge F) for 5 min at 2400g, and separated in a supernatant, that was decanted into a polypropylene tube and in a remaining pellet. This latter was once extracted again with 2 ml of 10% methanol and treated following the same procedure described before. Afterword, the supernatants were combined and vortexed; then 500 ␮l of the extract were transferred onto a small ion-exchange column (Pasteur pipette) containing 20 mg of Sephadex DEAE-A 25 in the formiate form. The column was washed twice with 1 ml of deionized water. The GLS were desulfated by adding 100 ␮l sulfatase type H-1 (Sigma S-9626) diluted 1:2.5 and incubated overnight at 39 ◦ C. The desulfated GLS were eluted with 3 × 500 ␮l water, collected in 70/12 PP tube and vortexed shortly. Finally, the solution from each sample was transferred into 1 ml sample vials. For each sample 30 ␮l were injected into the high-performance liquid chromatography (HPLC) analyzer equipped with a photodiode array detector (PDA). The GLS were

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Table 1 Alphabetic and identification code (ID) and geographical origin (province, altitude, and geographic coordinates) of the six accessions of horseradish analyzed. Code

ID

Town of provenance

Province

Altitude (m a.s.l.)

Latitude

Longitude

A B C D E F

ACC CASTS GP MON SANS STI

Accettura Castelsaraceno Guardia Perticara Montemurro San Severino Lucano Stigliano

MT PZ PZ PZ PZ MT

770 960 750 723 877 909

40◦ 40◦ 40◦ 40◦ 40◦ 40◦

16◦ 15◦ 16◦ 15◦ 16◦ 16◦

separated by using a C18 column (Nucleodur C18, 125 mm × 3 mm, 3 ␮m; Macherey-Nagel) and UV detection at 229 nm. Separation was carried out with linear solvent gradient from 1% to 20% acetonitrile in water over 20 min, followed by linear gradient to 20% acetonitrile for 5 min, then from 20% to 1% acetonitrile for 2 min. The flow rate was 0.6 ml/min at 35 ◦ C. The individual GLS were quantified by integration of their peak areas taking into account the specific response factors elaborated by Buchner (R. Buchner 1988, PhD Thesis, Georg-August-Universität Göttingen, Germany) and by comparing the peak area of the samples with the peak area of the internal standard. The GLS determined by HPLC–UV were sinigrin (SIN); gluconasturtiin (NAS); glucobrassicin (GBC); glucoiberin (IBE); 4-methoxyglucobrassicin (4ME); glucobarbarin (BAB); gluconapin (GNA); glucobrassicanapin (GBN). The identification of the GLS quantified was performed by comparison with retention time of single standards. Particular, desulfo glucotropaeolin, sinigrin (SIN) and glucobarbarin (BAB) were isolated at the Department of Crop Sciences, Georg-August-Universität Göttingen, from Lepidium sativum, Brassica nigra and Barbarea vulgaris, respectively; desulfo 4-methoxyglucobrassicin (4ME) standard was obtained from the Department for Plant Biochemistry Albrecht-von-HallerInstitute for Plant Sciences, Georg-August-Universität Göttingen; gluconapin (GNA), glucobrassicanapin (GBN), glucobrassicin (GBC) and gluconasturtiin (NAS) were obtained from Phytolab (Germany), whereas glucoiberin (IBE) was obtained from Carl Roth (Germany). 2.3. DNA isolation and AFLP marker analysis The leaf material previously stored at −80 ◦ C, was frozen in liquid nitrogen and rapidly ground to a fine powder. Then, subsamples of 100 mg were processed for DNA extraction using the DNeasy Plant kit (QIAGEN), following the protocol provided by the manufacturer. The DNA concentration was quantified using a Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies). For AFLP analysis 8 AFLP primer combinations were used, following the protocol of Vos et al. (1995) slightly modified as described in Radoev et al. (2008). The primer combinations used were chosen because they have given in previous experiments with Brassica napus a large number of polymorphisms. The EcoRI primers used in AFLP reactions were labelled with one of three different fluorescent dyes: FAM, HEX, and NED (Applied Biosystems, Darmstadt, Germany). The amplification products were separated on an ABI PRISM 3100 genetic analyzer (Applied Biosystems) using 36-cm capillary arrays and the GeneScan-500 ROX size standard (Applied Biosystems). 2.4. Data analysis Data on yield and root biometric characteristics (i.e., fresh weight of total and marketable roots, number of sprouts on the crown, length, diameters, number and dry weight of marketable roots) and GLS concentration were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s test to separate means. Statistical analysis was performed by M-STAT software (version 2.00). Before ANOVA, data expressed in percentage were normalized using arcsin transformation.

29 10 22 18 01 24

0 N 0 N 0 N 0 N 0 N 0 N

09 59 06 59 08 14

0 E 0 E 0 E 0 E 0 E 0 E

Cluster analysis of data obtained from chemical (i.e., total GLS) and root biometric characteristics and yield were constructed based on Euclidean distances and the unweighted pair group method of arithmetic averages (UPGMA) clustering methods was used to construct the phenetic dendrograms with the statistical software PAST (version 3.0). The means of each character were normalized before the cluster analysis using Z-scores, to prevent effects due to the differences of measurement scale utilized. The AFLP data obtained from ABI PRISM 3100 genetic analyzer (Applied Biosystems) were analyzed with Gene Mapper software (v4.0, Applied Biosystems). A binary data matrix was created by scoring a particular peak as present (1) or absent (0) for each sample. Data were entered in a computer file as a binary matrix, one for each kind of marker. The binary matrix was used to estimate Jaccard’s genetic similarity coefficients (Jaccard, 1908). The similarity matrix was subjected to UPGMA clustering methods to obtain the phenetic dendrograms with PAST software (version 3.0). 3. Results and discussion 3.1. Morphological traits, root yield and glucosinolate concentration Phenotypically, the six horseradish accessions analyzed could be clearly distinguished by observing the morphological traits of their above-ground organs, as they differed from each other in the size and shape of the leaves and petiole and height of the flowering stem (Table 2). The biometric characteristics measured, i.e., the number of the sprouts on the crown, length, equatorial and polar diameters at the top and basal diameter of the marketable roots, were significantly different among the accessions as well (Table 3). Horseradish roots exhibit distinct polarity with a proximal end (or point of attachment to the main root with a number of sprouts producing new vegetation) and distal end (Walters and Wahle, 2010). Above the ground, multiple sprouts on the crown can be formed also during the growing season. At the root harvest the number of sprouts ranged from 1 for accession B to 11 for accessions C and E. The highest number of sprouts was usually related to the largest diameters at the top (accessions C and E) and vice versa. Large differences were also found for the marketable root length, which varied from 13.8 cm in accession C to 24.0 cm in accession E. The biometric differences found among the accessions reflected on root yield traits. As shown in Table 4 the total root per plant significantly varied among the accessions, ranging from 121 g of accession B up to 1063 g of accession E. Regarding the marketable root yield (generally one for plant except for accessions A and E), the accession E showed the highest value (732 g), followed by accessions C, A, D and F and, finally B that had the lowest value (78 g). Perlaki and Djurovka (2009), studying over three years the horseradish yield found the highest value of root of 310 g/plant and an average value of 233 g/plant, that was considered as a fairly good yield. The dry weight of the roots ranged from 24.9% (accession D) to 36.4% (accession E). Sahasrabudhe and Mullin (1980) reported values of root dry weight in horseradish of around 28–30%, values very close to those found in the accessions A, B and F. The

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Table 2 Description of the morphological traits of leaf, petiole and flowering stem of the six horseradish accessions. Portion

Accessions

Trait

Shape Lengtha Widtha Twisting of tip Undulation of margin Serration Green colour of midrib Lengtha Widtha Time of appearance Incisions of the margin Heighta

Leaf

Petiole Incised (secondary) leaf Flowering stem

A

B

C

D

E

F

Elliptic Medium Medium Medium Medium Medium Light Medium Medium Early Weak Short

Elliptic Medium Medium Absent or very weak Medium Medium Medium Medium Medium Early Medium Short

Broad elliptic Long Broad Medium Strong Strong Medium Long Broad Medium Weak Tall

Elliptic Medium Medium Weak Medium Weak Light Medium Medium Medium Weak Short

Elliptic Long Medium Medium Strong Strong Medium Long Broad Medium Weak Tall

Broad elliptic Long Broad Medium Strong Strong Light Long Medium Late Weak Medium

a Values were separated for: leaf length as medium <55 cm, long ≥55 cm; leaf width as medium <20 cm, broad ≥20 cm; petiole length as medium <15 cm, long ≥15 cm; petiole width as medium <1 cm, broad ≥1 cm; stem height as short ≤55 cm, medium ranging from 55.1 to 70 cm, long ≥70 cm.

Table 3 Number of sprouts on the crown, root length and diameters of the six horseradish accessions. Accession (code)

Sprouts on the crown (n)

A B C D E F

Root diameter

Length (cm)

5.3b 1.0d 11.7a 6.0b 11.0a 3.5c

19.5b 17.8c 13.8d 20.5b 24.0a 17.0c

Equatorial at the top (cm)

Polar at the top (cm)

At the base (cm)

7.5c 2.6f 15.3a 5.9d 12.0b 4.5e

5.4b 2.6d 9.0a 5.5b 8.9a 4.1c

2.3b 1.8b 3.0a 3.1a 2.0b 3.4a

Values were analyzed by one-way ANOVA; the significance level of F ratio was P ≤ 0.001 for each parameter. In each column, values followed by the same letter do not significantly differ for P ≤ 0.05 according to Duncan’s test. Table 4 Yield of total and marketable roots of six horseradish accessions. Accession (code)

A B C D E F

Total roots

11 GLS were identified, respectively, in sprouts and roots. Referring instead to the GLS content, it is mostly documented in plants cultivated in vitro, embryoids, suspension cells and calli (Alnsour et al., 2012; Mevy et al., 1997; Redovnikovic´ et al., 2008), whereas little information is available in plants cultivated in open field. In our study, significant differences were found for individual and total GLS concentration among the accession tested (Table 5). The total GLS concentration ranged from 1.73 to 37.7 ␮mol g−1 of dry wt (Table 5), with accessions A and C showed the highest values. In all accessions, sinigrin (SIN) was the predominant GLS, therefore the variation in total GLS concentration was mainly due to the variation in SIN concentration, followed by gluconasturtiin (NAS) and glucobrassicin (GBC). Similar results were found by Redovnikovic´ et al. (2008) that found sinigrin accounted for more than 80% of total GLS, followed by lower amounts of NAS and GBC. Li and Kushad (2004) evaluating root GLS content in 27 horseradish accessions found that GLS concentration varied among genotypes from 2 to 296 ␮mol g−1 of dry wt, with SIN representing on average 83% of the total GLS, followed by NAS (11%) and GBC (1%). Recently, also Wedelsbäck Bladh et al. (2013a), on a total of 168 Nordic accessions

Marketable roots

(g/plant)

(g/plant)

(n/plant)

(% of dry wt)

376c 121f 939b 293d 1063a 202e

264c 78e 413b 169d 732a 147d

2a 1b 1b 1b 2a 1b

29.9b 30.6b 36.1a 24.9c 36.4a 30.1b

Values were analyzed by one-way ANOVA; the significance level of F ratio was P ≤ 0.001 for each parameter. In each column, values followed by the same letter do not significantly differ for P ≤ 0.05 according to Duncan’s test.

quantitative and qualitative characteristics of the marketable roots and the intensity of the flavour due to the composition and richness of glucosinolates are important traits for the industrial processing and fresh uses alike (Perlaki and Djurovka, 2009; Wedelsbäck Bladh et al., 2013a). Recently, Agneta et al. (2012) gave a complex profile of naturally occurring intact GLS in horseradish plant, in which 16 and

Table 5 Concentration of individual (SIN, sinigrin; NAS, gluconasturtiin; GBC, glucobrassicin; IBE, glucoiberin; 4ME, 4-methoxyglucobrassicin; BAB, glucobarbarin; GNA, gluconapin; GBN, glucobrassicanapin) and total glucosinolates in roots of six horseradish accessions. Accession (code)

SIN

NAS

GBC

IBE

4ME

BAB

GNA

GBN

Total

0.04b 0.15a 0.11a 0.12a 0.14a 0.04b

0.07bc 0.06c 0.12a 0.09b 0.06c 0.07bc

0.07a 0.06a 0.07a 0.07a 0.02b 0.05a

0.03ab 0.02bc 0.04a 0.02bc 0.01c 0.01c

35.70a 6.88b 37.67a 8.44b 1.73b 6.16b

**

***

**

**

***

(␮mol g−1 of dry wt) A B C D E F F probability

31.21a 5.25b 32.32a 6.75b 0.92b 4.88b

1.96a 0.62b 2.58a 0.42b 0.26c 0.37b

2.07a 0.51b 1.79a 0.72b 0.15b 0.31b

0.25c 0.18c 0.64a 0.26c 0.17c 0.42b

***

***

***

*** **

Values were analyzed by one-way ANOVA; the significance level of F ratio is given ( significantly differ for P ≤ 0.05 according to Duncan’s test.

P ≤ 0.01,

***

P ≤ 0.001). In each column, values followed by the same letter do not

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of horseradish found that sinigrin levels varied between 10 and 45 ␮mol/g of dry wt, gluconasturtiin between 1.3 and 7.4 ␮mol/g of dry wt and glucobrassicin between 0.1 and 2.6 ␮mol/g of dry wt, with accessions showing high levels of both sinigrin and gluconasturtiin. Considering our results, the percentage of the individual GLS on the total concentration of GLS varied in each accession. In particular SIN concentration represented from 53% to 87% of the total GLS (in E and F accessions, respectively), NAS accounted from 5% to 15% of the total GLS (in D and E accessions, respectively), whereas GBC was from 4.7% to 8.6% of the total GLS (in C and E accessions, respectively). All remaining GLS were in concentration below 4% of the total GSL concentration. In particular, gluconapin (GNA) and 4-methoxyglucobrassicin (4ME) as GLS in minor concentration have been already reported by Li and Kushad (2004). Recently, Tomsone et al. (2013) reported that the genotype has great influence on the content of volatile compounds of horseradish roots. In our study, among the six accessions compared, the accession C showed the highest values for all the individual GLS, including SIN, whereas the accession E, except for 4ME, showed always the lowest concentrations; however, the accessions showing the higher GLS concentration are not always associated with the best root yield traits. Nevertheless, as highlighted by Wedelsbäck Bladh et al. (2013a), the large variation in the concentration of GLS of the different accessions of horseradish should satisfy chefs and consumers, who look for specific flavour, when horseradish is used as a condiment in food. 3.2. AFLP marker and cluster analysis Using the 8 AFLP primers combinations 189 amplification products could be scored with the 6 different accessions (Table 6). The size of the amplification products varied from 50 bp (EcoAAG/MseCTA) to 498 bp (EcoACA/MseCAA), with an average number of 23.6 bands scored per reaction. The number of scorable bands ranged from 12 for the EcoATG/MseCTA primer combination to 33 for the EcoAAC/MseCAA primer combination. The number of polymorphic amplicons per reaction was only slightly lower and ranged from 11 (EcoATG/MseCTA) to 30 (EcoAAC/MseCAA) showing an average of 95.5% polymorphic bands. The percentage of polymorphisms detected ranged from 90.3% (for primer combination EcoAAG/MseCTA) to 100% (for both primer combination EcoACA/MseCAA and EcoAAT/MseCAA). The percentage of polymorphisms detected appears high. Using a biparental doubled haploid population derived from a cross of a resynthesized Brassica napus line and a winter oilseed rape cultivar Radoev et al. (2008) were able to score 144 polymorphic markers after using 23 AFLP primer combinations. By using 132 primer combinations Ecke et al. (2010) scored 1463 polymorphic markers in a set of 85 northern European canola quality winter rapeseed varieties and breeding lines, which equals 11 polymorphic markers per primer combination. Recently, Wedelsbäck Bladh et al. (2013b), by using the AFLP method with three primer combinations, that yielded 65 polymorphic bands, found a significant genetic diversity among 176 Nordic horseradish accessions, although a low genetic diversity was expected, since the plant is usually clonally propagated. Cluster analysis was performed on data for root biometric characteristics and yield, GLS concentration and AFLP marker analysis. On the basis of such parameters, a UPGMA dendrogram was constructed (Fig. 1), which showed a significant cophenetic correlation value of 0.93. At a Euclidean distance of 4.8 a clear separation of all accessions in two main groups was found, with a first cluster formed by accession E and C, and a second cluster formed by accessions B, D, F and A. The second group consisted of accessions that together were characterized by a lower root yield, dry matter, number of sprouts on the crown, equatorial and polar diameters at the top of the marketable roots, and GLS concentration. Although

253

Fig. 1. Dendrogram of Armoracia rusticana accessions basis on Euclidean distance from root biometric characteristics, yield and glucosinolate concentration data.

accession A showed similar root biometric characteristics and yield compared to the accessions of the second cluster, it was separated as single sub-cluster from those, on the basis of its high total GLS concentration (35.70 vs. 7.16 ␮mol g−1 of DW). Moreover, although accessions C and E showed similar high values related to marketable root yield, dry matter content (Table 4), number of sprouts on the crown and equatorial and polar diameters at the top (Table 3), a large difference in individual and total GLS concentration was found (i.e., 37.7 vs. 1.73 ␮mol g−1 of dry wt; Table 5). In general, root yield and quality, including the GLS content are the main characteristics to obtain a product well suitable for agro-industrial and pharmaceutical sectors. Considering together the chemical, yield and root morphological data, we confirm the existence of a great variability in horseradish accessions occurring in the Basilicata region, as previously showed by Sarli et al. (2012) on the basis of morphological traits. This means that the characterization of various genotypes available is extremely important to select the best individuals for industrial or fresh market destinations, especially in view of the fact that in some cases accessions with a high root yield may contain a low GLS concentration (e.g., genotype E). Recently, Tomsone et al. (2013) showed that the hierarchical cluster analysis can be performed to group horseradish genotypes according to the composition of volatile compounds. Additionally, the UPGMA cluster developed based on the Jaccard similarity matrix (Jaccard, 1908) using AFLP-marker information showed a significant cophenetic correlation value of 0.999 (Fig. 2). Accessions B and A and C and F reached high similarity values of 1.0, whereas lower values of 0.81 were obtained for accessions C, F, and E and of 0.38 for D, B and A. The AFLP markers revealed that based on the available polymorphisms, accessions B and A, and C and F coincided, whereas groups D, B, A and C, F, E were quite different from each other. However, the cluster obtained by using root traits and yield, and GLS concentration was quite different from the cluster obtained using molecular markers. This may be explained by the fact that only few root yield and GLS traits were available for cluster development. In contrast, a fairly large number of anonymous AFLP markers were available for cluster development and a more or less random distribution of the markers over the chromosomes can be anticipated. Hence, the clustering based on molecular markers appears much more reliable.

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Table 6 Polymorphism detected by individual primers: primer combination, range of amplicons, total number of amplicons, and number and percentage of polymorphic amplicons of 8 different AFLP primer combination used in accession characterization. Primer combination

Size of amplicons (bp)

EcoATG/MseCTA EcoATC/MseCTA EcoACC/MseCTA EcoAGA/MseCTA EcoACA/MseCAA EcoAAT/MseCAA EcoAAC/MseCAA EcoAAG/MseCTA

54–404 58–424 71–489 64–306 51–498 88–490 90–482 50–484

Total Average

– –

Amplicons (total n)

Polimorphism (n)

Polimorphism (%)

12 22 21 20 26 24 33 31

11 21 21 20 26 24 30 28

91.7 95.5 95.2 100 100 100 90.9 90.3

189 –

181 –

– 95.5

also Wedelsbäck Bladh et al. (2013b), suggested that, in Nordic countries, horseradish has probably been introduced at many occasions during a long period of time, which may explain the among-accession diversity and the differences between the countries, especially in view of the fact that as a medicinal plant it was early collected in different geographical regions (Wedelsbäck Bladh and Olsson, 2011; Wedelsbäck Bladh et al., 2013b). 4. Conclusion

Fig. 2. Dendrogram of Armoracia rusticana accessions obtained using the Jaccard’s genetic similarity distance from AFLP data.

Nevertheless, the different grouping of e.g., accessions E and C and the identity of accessions B and A, and C and F is surprising. Since there were large differences between the GLS profile, yield and root traits of those identical accessions, probably more polymorphic markers would be needed to detect genetic differences between them. However, on the base of AFLP marker, Wedelsbäck Bladh et al. (2013b), by using three primer combinations, have yielded 65 polymorphic bands and showed rather good separation of 176 accessions from four Nordic countries, grouped into 17 clusters, depending on their country of origin. While, similar to our study, Tomsone et al. (2012) have found no similarity among clusters of nine horseradish genotypes based on their contents of biologically active compounds (i.e., total phenol content and antioxidant properties) and on the basis of genetic data from random amplified polymorphic DNA (RAPD) analyses. The same authors (Tomsone et al., 2012) have assumed that the randomly amplified loci were not connected with the loci responsible for the level of the active compounds analyzed. Additionally, we cannot separate the genotypes on the basis of site of provenance as already reported by Sarli et al. (2012) who assumed a distribution of the species facilitated by territorial contiguity and similarity. Moreover, the existence of different genotypes could be related with the hypothesis that horseradish plant has been introduced in Basilicata from different European regions, such as Swabia or Albania, during different historical periods (Hammer et al., 2011; Pieroni et al., 2005). Interestingly,

Root biometric characteristics, yield, glucosinolate concentration and molecular marker analysis have been successfully applied to detect similarities and differences among the horseradish. Since horseradish is a clonally propagated crop there is a special interest to assess and maintain local landraces diversity. Among the accessions analyzed, the GLS concentration varied greatly, such as the yield and root traits. The results suggest that the GLS content and the yield performances of horseradish could be independent characteristics. In fact, not always the individuals of horseradish showing the best root yield are associated with the higher GLS concentration. Moreover, the cluster obtained by using root traits, yield and GLS concentration was quite different from the cluster obtained using molecular markers. According to what was observed by Tomsone et al. (2012), who used RAPD markers, it is possible that also in our study the loci sampled with AFLP markers are not related with the loci coding for the root traits, yield and GLS concentration. The chemical, agronomic and molecular characterization of available horseradish germplasm still occurring in the Basilicata region is an important task to preserve this species against genetic erosion and to identify the genotypes with high-value bioactive compounds and highest yield performances. Acknowledgements The authors are grateful to Uwe Ammermann and Rosi Clemens for their technical assistance in HPLC and AFLP analysis. References Agneta, R., Möllers, C., Rivelli, A.R., 2013. Horseradish (Armoracia rusticana), a neglected medical and condiment species with a relevant glucosinolate profile: a review. Genet. Resour. Crop Evol. 60, 1923–1943, http://dx.doi.org/10.1007/s10722-013-0010-4. Agneta, R., Rivelli, A.R., Ventrella, E., Lelario, F., Sarli, G., Bufo, S.A., 2012. Investigation of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (Armoracia rusticana) using LC-ESI−hybrid linear ion trap with Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation. J. Agric. Food Chem. 60, 7474–7482, http://dx.doi.org/10.1021/jf301294h. Alnsour, M., Kleinwächter, M., Böhme, J., Selmar, D., 2012. Sulfate determines the glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.). J. Sci. Food Agric. 93, 918–923, http://dx.doi.org/10.1002/jsfa.5825.

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