Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.     

Batch solvent extraction of flavanolignans from milk thistle (Silybum marianum L. Gaertner)

Seeds of milk thistle (Silybum marianum L. Gaertner) contain silymarins and ca. 25% (w/w) of oil. A pre-treatment step involving refluxing with petroleum ether is usually performed before extraction of the silymarins using organic solvents. This paper compares the extraction of whole and defatted milk thistle seeds in various solvents as a function of temperature. The extraction of whole seeds of milk thistle with water at 50, 70 and 85 degrees C was also examined: the yield of silymarin increased with increasing water temperature. In most cases, ethanol at 60 degrees C recovered the largest quantities of silymarins. However, boiling water proved to be an efficient extraction solvent for the more polar silymarins such as taxifolin and silychristin, even when using whole seeds. Extractions of defatted seed meal with boiling ethanol returned maximum yields of 0.62, 3.89, 4.04, and 6.86 mg/g defatted seed of taxifolin, silychristin, silybinin A and silybinin B, respectively. When extracting defatted seed meal with ethanol, yields of taxifolin, silybinin A and silybinin B were, respectively, 6.8-, 0.95-, 1.7- and 1.6-fold higher than when extracting whole seeds. When extracting with boiling water, the yields of silychristin, silybinin A, and silybinin B were 380, 47 and 50% higher for whole seeds compared with defatted seeds.     

Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry

A sensitive method for the simultaneous quantitation of six active constituents in commercial silymarin standardized extracts was developed based on liquid chromatography (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely separated and quantified by LC/MS. Silymarin obtained from Sigma-Aldrich Co. was evaluated and used as standard reference material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in commercial standardized silymarin extracts, respectively. Significant variation was found between different commercial silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in commercial milk thistle extracts. The calibration curves were over the range from 0.25 to 100 microg/mL for silychristin and silydianin, and from 0.10 to 100 microg/mL for silybin A, silybin B, isosilybin A and isosilybin B, respectively (r(2)> or =0.9958). For all six active constituents, the overall intra-day precision values, based on the relative standard deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodology could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.     

Study of different Trichoderma strains on growth characteristics and silymarin accumulation of milk thistle plant

Abstract

Biological control involves the use of beneficial organisms such as Trichoderma that promote positive responses by the plant. Trichoderma species produce and/or release a variety of compounds, including cell wall degrading enzymes and secondary metabolites, which enhance root development, crop productivity and resistance to biotic and abiotic stresses. This study examines the effects of the five Trichoderma strains (M7, KHB, G124-1, G46-3 and G46-7) on milk thistle including morphological characteristics and silymarin accumulation. This research indicated that treating milk thistle by M7 strain, significantly increased the highest branch length of plants, while strain KHB induced the production of silychristin, isosilybin and silymarin. Finally, strain G46-7 dramatically increased silydianin content in milk thistle plant. The findings suggested that certain Trichoderma strains are positive elicitors for promoting growth characteristics and silymarin accumulation in Silybum marianum (L.) Gaertn.     

Preparative method for isosilybin isolation based on enzymatic kinetic resolution of silymarin mixture

The flavonolignan isosilybin (1) is one of the silybin congeners contained in the silymarin complex, which is isolated from the seeds of the milk thistle (Silybum marianum). A number of recent studies have demonstrated that isosilybin is probably the most potent anticancer agent found in silymarin. It occurs as a mixture of two diastereoisomers, A and B. Lipase Novozym 435 was found to allow the preparative production of both optically pure isosilybin A and B in a diastereoisomeric purity of over 95%. A preparatory method based on the enzymatic resolution and other purification methods has been developed, enabling multigram amounts of both optically pure isosilybins A (1a) and B (1b) to be obtained.     

Molecular insights of genetic variation in milk thistle (Silybum marianum [L.] Gaertn.) populations collected from southwest Iran

Milk thistle (Silybum marianum) is among the world’s popular medicinal plants. Start Codon Targeted (SCoT) marker system was utilized to investigate the genetic variability of 80 S. marianum genotypes from eight populations in Iran. SCoT marker produced 255 amplicons and 84.03% polymorphism was generated. The SCoT marker system’s polymorphism information content value was 0.43. The primers’ resolving power values were between 4.18 and 7.84. The percentage of polymorphic bands was between 33.3 and 100%. The Nei’s gene diversity (h) was 0.19–1.30 with an average 0.72. The Shannon’s index (I) ranged from 0.29 to 1.38 with an average value of 0.83. The average gene flow (0.37) demonstrated a high genetic variation among the studied populations. The variation of 42% was displayed by the molecular variance analysis among the populations while a recorded variation of 58% was made within the populations. Current investigation suggested that SCoT marker system could effectively evaluate milk thistle genotypes genetic diversity.

     

Isolation and purification of silychristin,silydianin and taxifolin in the co-products of the silybin refined process from the silymarin by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of silychristin, silydianin and taxifolin in the co-products of the silybin refined process from the silymarin was successfully established by n-hexane–chloroform–methanol–water (0.5:11:10:6 (0.5 acetic acid), v/v/v/v) as the two-phase solvent system. 146 mg silydianin, 280 mg silychristin and 63 mg taxifolin from 1.463 g co-products sample in one separation were obtained with the purities of 95.1%, 99.3% and 98.2%, respectively, determined by HPLC. The structures of the compounds were identified by means of ESI-MS-MS, TOF-MS, ¹H NMR, ¹³C NMR and 2DNMR-HMBC. Silychristin, silydianin and taxifolin had been separated as standards by HSCCC for the first time. A comparative study between HSCCC and RPLC for separation and isolation of taxifolin, silychristin and silydianin was investigated. The differences between the two preparative chromatographic methods were all discussed. The results demonstrated that HSCCC was a powerful separation tool and could contribute to identifying and quantifying plant ingredients.     

Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense

Flavonolignans silybin and isosilybin are major components of silymarin complex isolated from seeds of the milk thistle (Silybum marianum) featuring strong antioxidant and hepatoprotective effects, and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activities. Natural silybin and isosilybin are mixtures of diastereoisomers: silybin/isosilybin A (1a, 1b) and silybin/isosilybin B (2a, 2b). The metabolism of these compounds is supposed to be strongly linked to Phase II of biotransformation and the respective conjugates are rapidly excreted in bile and urine. The aim of this study was to obtain optically pure sulfated metabolites of both silybins and isosilybins. Aryl-sulfate sulfotransferase (EC 2.8.2.22) from Desulfitobacterium hafniense was found to be a highly effective tool for the regiospecific enzymatic synthesis of silybin A-20-O-sulfate, silybin B-20-O-sulfate, isosilybin A-20-O-sulfate and isosilybin B-20-O-sulfate providing nearly quantitative yields and employing cheap p-nitrophenyl sulfate as sulfate donor. The isolated sulfated products will be used as authentic standards in metabolic studies of both silybins and isosilybins.     

Comparative assessment of genetic diversity in wild and primitive cultivated barley in a center of diversity

Wild barley (Hordeum spontaneum K.) and indigenous primitive varieties of cultivated barley (Hordeum vulgare L.), collected from 43 locations in four eastern Mediterranean countries, Jordan, Syria, Turkey and Greece, were electrophoretically assayed for genetic diversity at 16 isozyme loci. Contrary to a common impression, cultivated barley populations were found to maintain a level of diversity similar to that in its wild progenitor species. Apportionment of overall diversity in the region showed that in cultivated barley within-populations diversity was of higher magnitude than the between-populations component. Neighboring populations of wild and cultivated barleys showed high degree of genetic identity. Groups of 3 or 4 isozyme loci were analyzed to detect associations among loci. Multilocus associations of varying order were detected for all three groups chosen for the analysis. Some of the association terms differed between the two species in the region. Although there was no clear evidence for decrease in diversity attributable to the domestication of barley in the region, there was an indication of different multilocus organizations in the two closely related species.     

Solid-state fermentation of industrial solid wastes from the fruits of milk thistle Silybum marianum for feed quality improvement

The industrial solid wastes generated during the production of silymarin from the fruits of milk thistle Silybum marianum was used as the substrate. Preparation and evaluation of the feeds produced by solid-state fermentation (SSF) of the industrial solid wastes was carried out. The protein content of the fermented feed (FF) from a combination of Aspergillus niger and Candida tropicalis was the highest among the examined strains. The optimal process parameters for protein enrichment with SSF using A. niger and C. tropicalis included incubation temperature of 30.8 °C, fermentation time of 87.0 h, and initial moisture content of 59.7 %. Under these conditions, the value additions of FF occurred. The fiber of FF was decreased by 25.07 %, while the digestibility of protein, protein content, and the ratio of total essential amino acids to total amino acids were increased by 79.85, 16.22, and 8.21 %, respectively. The analysis indicated that FF contained 1.44 mg/kg flavonoids and 0.5 mg/kg silybin, which significantly increased by 2.42 and 1.63 times, respectively than those in unfermented substrates. FF recorded reduced molecular weight of proteins from 20.1 to 44.3 kDa to below 14.3 kDa. The results of feeding trial of FF replacement with soybean meal in broilers diets for 8 weeks showed that FF significantly improved carcass characteristics including abdominal fat rate, serum biochemical parameters including aspartate transaminase, blood urea nitrogen and high density lipoprotein cholesterol, and immune responses of broilers. A potential feed quality improvement was achieved through mixed strains SSF of industrial solid wastes of S. marianum fruits.     

水飞蓟宾及其类似物的化学修饰研究进展

以天然黄酮类化合物为活性先导物,研究其化学结构与生物活性的关系,进而进行化学修饰研究,是目前创新药物的一条重要思路。药用植物水飞蓟用来治疗肝胆疾病已有2000多年的历史,水飞蓟宾作为水飞蓟素的主要活性成分具有保肝、抗炎和抗癌等活性而备受人们关注,但由于水飞蓟宾极低的溶解性极大地限制了其生物利用度。为此,国内外学者通过化学和物理等方法对其进行修饰和改造,并取得了一定的成果。本文就近10年来水飞蓟宾及其类似物的化学修饰研究进行综述。     

Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver

The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damage. Silymarin, derived from the milk thistle plant, Silybium marianum, has been used in traditional medicine as a remedy for diseases of the liver and biliary tract. In the present study, the effect of hepatoprotective drug silymarin on body weight and biochemical parameters, particularly, antioxidant status of ethanol-exposed rats was studied and its efficacy was compared with the potent antioxidant, ascorbic acid as well as capacity of hepatic regeneration during abstention. Ethanol, at a dose of 1.6 g/kg body wt/day for 4 wks affected body weight in 16-18 week-old male albino rats (Wistar strain weighing 200-220 g). Thiobarbituric acid reactive substance (TBARS) level, superoxide dismutase (SOD), and glutathione-s-transferase (GST) activities were significantly increased, whereas GSH content, and catalase, glutathione reductase (GR) and GPx (glutathione peroxidase) activities significantly reduced, on ethanol exposure. These changes were reversed by silybin and ascorbic acid treatment. It was also observed that abstinence from ethanol might help in hepatic regeneration. Silybin showed a significant hepatoprotective activity, but activity was less than that of ascorbic acid. Furthermore, preventive measures were more effective than curative treatment.     

Landscape management and domestication of Stenocereus pruinosus (Cactaceae) in the Tehuacan Valley: human guided selection and gene flow

ABSTRACT: BACKGROUND: Use of plant resources and ecosystems practiced by indigenous peoples of Mesoamerica commonly involves domestication of plant populations and landscapes. Our study analyzed interactions of coexisting wild and managed populations of the pitaya Stenocereus pruinosus, a columnar cactus used for its edible fruit occurring in natural forests, silviculturally managed in milpa agroforestry systems, and agriculturally managed in homegardens of the Tehuacan Valley, Mexico. We aimed at analyzing criteria of artificial selection and their consequences on phenotypic diversity and differentiation, as well as documenting management of propagules at landscape level and their possible contribution to gene flow among populations. METHODS: Semi-structured interviews were conducted to 83 households of the region to document perception of variation, criteria of artificial selection, and patterns of moving propagules among wild and managed populations. Morphological variation of trees from nine wild, silviculturally and agriculturally managed populations was analyzed for 37 characters through univariate and multivariate statistical methods. In addition, indexes of morphological diversity (MD) per population and phenotypic differentiation (PD) among populations were calculated using character states and frequencies. RESULTS: People recognized 15 pitaya varieties based on their pulp color, fruit size, form, flavor, and thorniness. On average, in wild populations we recorded one variety per population, in silviculturally managed populations 1.58 +/- 0.77 varieties per parcel, and in agriculturally managed populations 2.19 +/- 1.12 varieties per homegarden. Farmers select in favor of sweet flavor (71% of households interviewed) and pulp color (46%) mainly red, orange and yellow. Artificial selection is practiced in homegardens and 65% of people interviewed also do it in agroforestry systems. People obtain fruit and branches from different population types and move propagules from one another. Multivariate analyses showed morphological differentiation of wild and agriculturally managed populations, mainly due to differences in reproductive characters; however, the phenotypic differentiation indexes were relatively low among all populations studied. Morphological diversity of S. pruinosus (average MD = 0.600) is higher than in other columnar cacti species previously analyzed. CONCLUSIONS: Artificial selection in favor of high quality fruit promotes morphological variation and divergence because of the continual replacement of plant material propagated and introduction of propagules from other villages and regions. This process is counteracted by high gene flow influenced by natural factors (pollinators and seed dispersers) but also by human management (movement of propagules among populations), all of which determines relatively low phenotypic differentiation among populations. Conservation of genetic resources of S. pruinosus should be based on the traditional forms of germplasm management by local people.     

Genetic structure of Pinus brutia stands exposed to wild fires

This study investigates the genetic structure of brutia pine (Pinus brutia Ten.) stands that were exposed to wild fires. A systematic investigation within the species distribution in Greece first identified areas of frequent wild forest fires and then located stands that had experienced ground fires about 20 years ago. In these stands it was possible to sample the pre-fire population that had survived the ground fire event (“mature” population) as well as the post-fire population that has reached reproductive capacity (“young” population). Gel electrophoresis was used in order to study isoenzyme variability in four such populations present in two sites (Kourteri, Lesvos island and Mytilineoi, Samos island). Results indicated absence of notable differences in genetic diversity among the remnant mature populations and the young populations after regeneration. No significant inbreeding was detected and genetic identity among young and mature stands was high. However, some differences were observed in the frequencies of rare alleles and in the presence of interspecific (P. brutia x halepensis) hybrids in the young populations. This paper discusses the above results in the context of the evolution of brutia pine and the maintenance of its genetic variation.     

Comparing the genetics of wild and captive populations of White‐headed Ducks Oxyura leucocephala: consequences for recovery programmes

The White‐headed Duck is a globally threatened species historically recorded from Spain in the west to China in the east. It has suffered major population declines, local extinctions and range fragmentation. Several projects have attempted to reintroduce captive‐bred birds into parts of the former range in Europe, but with little success. Two captive stocks currently exist, one originating from Pakistan in 1968 and the other originating from Spain in 1982. This study compares the suitability of these captive stocks for specific reintroduction projects by using 11 microsatellite markers and mtDNA control region sequences to assess genetic differences between captive populations and wild birds from Spain and Greece. No significant population structure was found and all microsatellite alleles recorded in captive birds originating from Pakistan were also observed in the wild Spanish population. A higher diversity of alleles was observed in wild birds from Greece than from Spain, probably due to the effects of a strong bottleneck experienced in Spain in the 1970s. Compared with wild populations, both captive stocks have suffered a significant loss of diversity in microsatellites and mitochondrial DNA owing to founder effects and/or genetic drift, and therefore may not be well suited for release programmes. We recommend the development of a more diverse captive breeding programme based on birds taken from different areas of the range, in particular by supplementing the Spanish population with birds from North Africa. Our study shows the value of molecular tools in developing conservation programmes for threatened bird species and has implications for the design of recovery programmes.     

A preliminary study on population genetic structure and phylogeography of the wild and cultivated Zizania latifolia (Poaceae) based on Adh1a sequences

Recent decades have witnessed growing interests in exploring the population genetics and phylogeography of crop plants and their wild relatives because of their important value as genetic resources. In this study, sequence variation of the nuclear Adh1a gene was used to investigate the genetic diversity and phylogeographic pattern of the wild and cultivated Zizania latifolia Turcz. Sequence data were obtained from 126 individuals representing 21 wild populations in China and 65 varieties of the cultivated Zizania latifolia. Low to medium level nucleotide diversity was found in the wild populations, with northeastern populations being the most variable. We detected significant population subdivision (F _ST = 0.481) but no significant phylogeogaphical structure, suggesting limited gene flow and dispersal among populations. The current pattern of genetic variation in the wild populations might be explained by a fragmentation of ancient populations due to habitat destruction and degradation during recent decades. The heterogeneous levels and spatial apportionment of genetic diversity among wild populations also suggested a history of gradual colonization of Zizania latifolia populations from the northeast to the south of China. Interestingly, all 65 varieties of the cultivated Zizania latifolia possessed a single identical genotype, implying a single domestication associated with very few initial individuals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Wildlife uses and hunting patterns in rural communities of the Yucatan Peninsula, Mexico

Background

Use of plant resources and ecosystems practiced by indigenous peoples of Mesoamerica commonly involves domestication of plant populations and landscapes. Our study analyzed interactions of coexisting wild and managed populations of the pitaya Stenocereus pruinosus, a columnar cactus used for its edible fruit occurring in natural forests, silviculturally managed in milpa agroforestry systems, and agriculturally managed in homegardens of the Tehuacán Valley, Mexico. We aimed at analyzing criteria of artificial selection and their consequences on phenotypic diversity and differentiation, as well as documenting management of propagules at landscape level and their possible contribution to gene flow among populations.

Methods

Semi-structured interviews were conducted to 83 households of the region to document perception of variation, criteria of artificial selection, and patterns of moving propagules among wild and managed populations. Morphological variation of trees from nine wild, silviculturally and agriculturally managed populations was analyzed for 37 characters through univariate and multivariate statistical methods. In addition, indexes of morphological diversity (MD) per population and phenotypic differentiation (PD) among populations were calculated using character states and frequencies.

Results

People recognized 15 pitaya varieties based on their pulp color, fruit size, form, flavor, and thorniness. On average, in wild populations we recorded one variety per population, in silviculturally managed populations 1.58 ± 0.77 varieties per parcel, and in agriculturally managed populations 2.19 ± 1.12 varieties per homegarden. Farmers select in favor of sweet flavor (71% of households interviewed) and pulp color (46%) mainly red, orange and yellow. Artificial selection is practiced in homegardens and 65% of people interviewed also do it in agroforestry systems. People obtain fruit and branches from different population types and move propagules from one another. Multivariate analyses showed morphological differentiation of wild and agriculturally managed populations, mainly due to differences in reproductive characters; however, the phenotypic differentiation indexes were relatively low among all populations studied. Morphological diversity of S. pruinosus (average MD = 0.600) is higher than in other columnar cacti species previously analyzed.

Conclusions

Artificial selection in favor of high quality fruit promotes morphological variation and divergence because of the continual replacement of plant material propagated and introduction of propagules from other villages and regions. This process is counteracted by high gene flow influenced by natural factors (pollinators and seed dispersers) but also by human management (movement of propagules among populations), all of which determines relatively low phenotypic differentiation among populations. Conservation of genetic resources of S. pruinosus should be based on the traditional forms of germplasm management by local people.     

Genome and population dynamics under selection and neutrality: an example of S-allele diversity in wild cherry (Prunus avium L.)

Self-incompatibility, a common attribute of plant development, forms a classical paradigm of balancing selection in natural populations, in particular negative frequency-dependent selection. Under negative frequency-dependent selection population genetics theory predicts that the S-locus, being in command of self-incompatibility, keeps numerous alleles in equal frequencies demonstrating a wide allelic range. Moreover, while natural populations exhibit a higher within population genetic diversity, a reduction of population differentiation and increase of effective migration rate is expected in comparison to neutral loci. Allelic frequencies were investigated in terms of distribution and genetic structure at the gametophytic self-incompatibility locus in five wild cherry (Prunus avium L.) populations. Comparisons were also made between the differentiation at the S-locus and at the SSR loci. Theoretical expectations under balancing selection were congruent to the results observed. The S-locus showed broad multiplicity (16 S-alleles), high genetic diversity, and allelic isoplethy. Genetic structure at the self-incompatibility locus was almost four times lower than at 11 nSSR loci. Analysis of molecular variance revealed that only 5?% of the total genetic variation concerns differentiation among populations. In conclusion, the wealth of S-allelic diversity found in natural wild cherry populations in Greece is useful not only in advancing basic population genetics research of self-incompatibility systems in wild cherry but also in the development of breeding programs.     

Metabolic effects of silibinin in the rat liver

The flavonolignan silibinin, which is a mixture of two diastereoisomers, silybin A and silybin B, is a component of the extract obtained from the fruit and seeds of the variegated milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin. Among the therapeutic properties credited to silibinin, its antihyperglycaemic action has been extensively explored. Silibinin is structurally related to the flavonoids quercetin and fisetin, which have been previously demonstrated to be very active on liver metabolic processes related to glycaemic regulation. The aim of the present work was to investigate the effects of silibinin on metabolic pathways responsible for the maintenance of glycaemia, particularly glycogenolysis and gluconeogenesis, in the perfused rat liver. The activities of some key enzymes in these pathways and on parameters of energy metabolism in isolated mitochondria were also examined. At a concentration range of 50-300μM, silibinin inhibited gluconeogenesis in the fasted condition and inhibited glycogenolysis and glycolysis in the fed condition. The mechanisms by which silibinin exerted these actions were multiple and complex. It inhibited the activity of glucose 6-phosphatase, inhibited the pyruvate carrier, and reduced the efficiency of mitochondrial energy transduction. It can also act by reducing the supply of NADH for gluconeogenesis and mitochondria through its pro-oxidative actions. In general, the effects and the potency of silibinin were similar to those of quercetin and fisetin. However, silibinin exerted some distinct effects such as the inhibitory effect on oxygen consumption in the fed condition and a change in the energy status of the perfused livers. It can be concluded that the effects of silibinin on liver glucose metabolism may explain its antihyperglycaemic property. However, this effect was, in part, secondary to impairment in cellular energy metabolism, a finding that should be considered in its therapeutic usage.     

Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm

Hazelnut (Corylus avellana L.) is a traditional nut crop in southern Europe. Germplasm exploration conducted on-farm in five countries (Portugal, Spain, Italy, Slovenia, and Greece) identified 77 landraces. The present work describes phenotypic variation in nut and husk traits and investigates genetic relationships using ten simple sequence repeat (SSR) markers among these landraces, 57 well-known references cultivars, and 19 wild accessions. Among the 77 landraces, 42 had unique fingerprints while 35 showed a SSR profile identical to a known cultivar. Among the 42 unique landraces, morphological observations revealed high phenotypic diversity, and some had characteristics appreciated by the market such as nut round and caliber. Analysis of genetic relationships and population structure allowed investigation of the origin and spread of the cultivated germplasm in southern Europe. Our results indicate the existence of three primary centers of diversity in the Mediterranean basin: northwestern Spain (Tarragona) and southern Italy (Campania) in the West and Black Sea (Turkey) in the East. Moreover, the data suggest the existence of secondary gene pools in the Iberian (Asturias) and Italian (Liguria and Latium) Peninsulas, where local varieties were recently domesticated from wild forms and/or from introduced ancient domesticated varieties.