- Email: [email protected]
Identification of zucchini varieties in commercial food products by DNA typing
Accepted Manuscript Identification of zucchini varieties in commercial food products by DNA typing Maria Verdone, Rosa Rao, Mariangela Coppola, Giandomenico Corrado PII:
S0956-7135(17)30391-2
DOI:
10.1016/j.foodcont.2017.07.039
Reference:
JFCO 5732
To appear in:
Food Control
Received Date: 22 May 2017 Revised Date:
28 July 2017
Accepted Date: 29 July 2017
Please cite this article as: Verdone M., Rao R., Coppola M. & Corrado G., Identification of zucchini varieties in commercial food products by DNA typing, Food Control (2017), doi: 10.1016/ j.foodcont.2017.07.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Identification of zucchini varieties in commercial food products by DNA typing
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Maria Verdone, Rosa Rao, Mariangela Coppola and Giandomenico Corrado*
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Dipartimento di Agraria, Università degli Studi di Napoli “Federico II”, via Università 100, 80055
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Portici (NA), Italy
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* Corresponding author. Tel.: +39 081 2539294; fax: +39 081 2539481. E-mail address:
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[email protected] (G. Corrado).
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Traceability, SSR, STR, molecular markers, Cucurbita pepo, DNA profile, random match probability
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Keywords
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ACCEPTED MANUSCRIPT ABSTRACT
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DNA typing has been long proposed as a component of food traceability especially for the ability
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to provide genetic information on processed and/or mixed commercial products. However, the
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use of DNA testing to deliver legal evidence has been very limited in the agro-food chain, also
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because in this field, a genetic concordance is rarely sustained with indications on the
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interpretation and statistics of a DNA profile.
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Zucchini is the most important and diverse horticultural group belonging to Cucurbita pepo, a
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plant species that yields a large variety of edible fruits such as squashes and pumpkins. Challenges
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in genetic discrimination of zucchini material exiting the agro-food chain typify the problems in
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DNA typing of horticultural food products, because of limited allelic diversity, presence of common
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alleles, highly processed food and genetic similarity with other edible species.
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The aim of this work was to identify zucchini varieties in commercial products by a means of
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Simple Sequence Repeat (SSR) analysis and to provide indications for the statistical interpretation
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of a match. We assessed the efficacy of SSRs analyzing some of the most diffused hybrids and
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open pollinated zucchini varieties. SSRs were also screened for transferability in DNA isolated from
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edible organs of different plant species that are frequently present in food products containing
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zucchini. Moreover, we demonstrate the possibility to identify the variety in food products of
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different origins. The statistical evaluation on the rarity of DNA profiles in zucchini indicated that
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the genetic match presented in this work cannot be attributed to chance alone. Based on this
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evidence, the advantages and pitfalls of food DNA typing for variety identification of a horticultural
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crop are discussed.
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ACCEPTED MANUSCRIPT 1. Introduction
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Cucurbita pepo L. is considered the most diverse species of its genus with respect to fruit size,
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shape and color (Paris, 1996). This plant species yields a large variety of fruits used for culinary
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purposes, such as pumpkin, gourd, marrow and winter and summer squash. These designations do
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not have a precise botanical meaning because they are not associated to one particular species
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(Paris, 1996). For instance, summer squash is a lay term to designate edible, tender fruits of C.
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pepo that are harvested and consumed before maturity, although not all fruits of this species are
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considered summer squashes (Whitaker & Robinson, 1986).
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Zucchini is currently the most diffused and economically important horticultural group of C. pepo
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(Paris, 2016b). The term zucchini is an Americanism originating from the Italian word (masculine,
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plural) for little pumpkin (“zucca” plus a diminutive suffix) (http://www.accademiadellacrusca.it).
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Zucchini designates a rather uniform, cylindrically-shaped fruit with length-to-broadest width ratio
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generally comprised from 3.5 to 4.5 (Paris, 1996). Zucchini fruits, also named courgettes in English
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speaking countries, typically have a green skin color, being harvested immature, and are eaten
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only after cooking. However, traditional varieties with more spherical fruits (e.g. “Tondo”), grown
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mainly in Italy and France, and more recently, varieties with yellow fruits are also included in the
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zucchini group by plant breeders and retailers.
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The zucchini cultivation has experienced a recent success in many countries mainly due to ease of
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cultivation in both open-field and protected agriculture, short crop cycle, adaptability to
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temperate and subtropical climates, suitability to low-calories diet regimes and cooking versatility.
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The diffusion of summer squash, mainly zucchini, has increased proportionally more than many
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other vegetables, especially in Western countries (Paris, 2016b). This success is also determined by
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the world wide increasing consumption of fresh and frozen fruits and vegetables, principally as
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ACCEPTED MANUSCRIPT alternative to canned products (Barbosa-Cánovas, Altunakar, & Mejía-Lorío, 2005; Harris &
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Shiptsova, 2007). For instance, mixed vegetables are the second most important frozen product
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after potatoes (Barbosa-Cánovas, et al., 2005).
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Traceability provides the ability to characterize and certify products throughout the food chain,
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from producers to consumers (Dabbene, Gay, & Tortia, 2014). Different technologies are required
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for effective traceability implementation and one important element is genetic traceability (Opara,
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2003). DNA markers are widely used molecular tools to trace or reveal the genetic identity of a
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biological component of food and feed. For genetic traceability, preference is given to markers
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amenable to the analysis of raw material and highly processed commercial products. Compared to
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other analytical methods, the DNA analysis offers a great level of precision and accuracy to
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identify the origin and composition of food in terms of botanical species or varieties (Barcaccia,
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Lucchin, & Cassandro, 2015; Corrado, 2016).
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Zucchini represents a challenge for DNA traceability. Because of its more recent origin, zucchini is
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characterized by a reduced diversity compared to other C. pepo horticultural groups (Formisano,
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et al., 2012). Moreover, genetic traceability needs to consider that zucchini is frequently present in
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complex, mixed products (i.e. those with different vegetable ingredients). Additionally, the
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botanical relationships between zucchini and other edible Cucurbitacae (e.g. pumpkin, gourd,
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cucumber, etc.) may pose a limitation in the discrimination power of some DNA markers (Li Gong,
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et al., 2012). Finally, many zucchini cultivars are hybrids, requiring co-dominant markers for an
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easier discrimination. Among DNA markers available, Simple Sequence Repeat (SSR), also known
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as Simple Tandem Repeats (STR), are the markers of choice in forensics (Schlötterer, 2004) and
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they are widely used also to trace the agro-food chain because of their high polymorphism,
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codominant nature, reproducibility and amenability to degraded DNA (Corrado, 2016).
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ACCEPTED MANUSCRIPT The aim of this work was to type the DNA of zucchini varieties in commercial food products by a
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means of SSR analysis. In order to evaluate the genetic differences among popular zucchini
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varieties, we assessed the efficacy of SSRs by analyzing some of the most diffused hybrids and
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open pollinated cultivars. SSRs were also screened for transferability in DNA isolated from edible
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organs of different plant species that are frequently present in commercial products containing
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zucchini. Finally, it was constructed a profiles database, which was used to support the statistical
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evaluation on the rarity of DNA profiles in zucchini. This evaluation allowed discussing the
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advantages and pitfalls of food DNA typing for variety identification of a horticultural crop.
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2.1. Plant material and DNA isolation
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We analysed 30 varieties of zucchini (Cucurbita pepo ssp. pepo L.) namely, ‘Altea’, ‘Anissa’, ‘Asso’,
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‘Aurore’, ‘Aymaran’, ‘Black Beauty’, ‘Cavili’, ‘Coucourzelle’, ‘De Nice à fruit rond’, ‘Genovese’,
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‘Genovese Antico’, ‘Giulia’, ‘Gulliver’, ‘Lanka’, ‘Mikonos’, ‘Milos’, ‘Ontano’, ‘Parthenon’,
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‘President’, ‘Quine’, ‘Rhodos’, ‘Rigas’, ‘Romanesco’, ‘San Pasquale’, ‘Sitos’, ‘Syros’, ‘Temprano de
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Argelia’, ‘Vertre noir maraîchère’, ‘Virginia’ and ‘Vitulia’. This set includes 20 hybrid cultivars and
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10 open-pollinated varieties. Fruit general shape and colour are reported in Supplementary Table
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1. Plants were grown from seed and the first true leaf was harvested and frozen in liquid nitrogen
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for subsequent analysis. We analysed three plants per variety.
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For the SSR transferability experiments, DNA was isolated from fresh edible organs of the
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following species: cabbage (Brassica oleracea L.), carrot (Daucus carotae L.), celery (Apium
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graveolens L.), cucumber (Cucumis sativus L), onion (Allium cepa L.), pea (Pisum sativum L.), potato
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(Solanum tuberosum L.), pumpkin (Cucurbita maxima Duchesne), tomato (Solanum lycopersicum
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L.). Cabbage, carrot, celery, onion, pea, potato and tomato are very frequently present in
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processed food products containing zucchini (e.g. vegetable soups, frozen mixed vegetables, etc.).
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Cucumber and pumpkin were included because they are two edible plants belonging to the
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Cucurbitacae family. For all plant material, DNA isolation and quantification was carried out as
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described (Scarano, Rao, Masi, & Corrado, 2015)
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2.2. Commercial products and DNA isolation
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We analysed five types of commercial products containing zucchini: fresh fruits (with cylindrical or
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round shape) from the shelf of a large-scale distribution; frozen zucchini (sliced and sold as a
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frozen product); mixed vegetables (different clean vegetables cut to prepare a soup, raw and not
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ACCEPTED MANUSCRIPT seasoned, sold as a frozen product); chargrilled zucchini (sliced zucchini, chargrilled and stored in a
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transparent glass jar with olive oil); and Caprese (aka Scapece) zucchini (canned zucchini, fried
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with vegetable oil, seasoned with vinegar, garlic and mint leaves, stored in olive oil). Frozen
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zucchini, mixed vegetables and chargrilled zucchini were purchased from the same retail store
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brand. For DNA isolation, fresh and frozen products were pulverised in liquid nitrogen with a
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mortar and pestle for subsequent manipulations. Zucchini stored in oil were first blotted on
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kitchen towels and then centrifuged for 30 second at 1000 g on kitchen paper to remove the
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residual liquid phase. Samples were then pulverised as described above. DNA isolation was carried
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out using the DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instruction.
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2.3. SSR analysis
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DNA amplification reactions were conducted in a 25 μl volume containing 20ng of genomic DNA,
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1X PCR buffer (Promega), 0.1 mM dNTPs, 0.2 μM labelled forward primer, 0.2 μM reverse primer
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and 0.5U of Taq polymerase (Promega). The amplification reactions were carried out in a
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Mastercycler Gradient (Eppendorf) thermocycler with the following conditions: a DNA
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denaturation step at 94 °C for 4'; 35 cycles comprising a denaturation step at 94 °C for 45'', an
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annealing phase at 56 °C for 45'' and elongation step at 72 °C for 90”. A final elongation phase at
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72 °C for 15' ended the reaction. The eight SSR loci employed comprised different core structures
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(L Gong, Stift, Kofler, Pachner, & Lelley, 2008). Primer and core sequences are reported in
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Supplementary Table 2. Amplification products were separated by agarose gel electrophoresis to
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verify the presence of amplified fragments. For allelic discrimination, the fluorescent fragments
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were resolved by capillary electrophoresis in an ABI PRISM 3130 (Applied Biosystems) system
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using the POP6 polymer (Applied Biosystems). Signal peak height and allele sizes were calculated
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using the ABI PRISM Genotyper (v. 4.0) software (Applied Biosystems) based on the GeneScan
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500Liz molecular weight standard (Applied Biosystems).
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ACCEPTED MANUSCRIPT 2.4. Estimation of the amplicon size range for processed products.
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DNA from processed products was amplified with primers targeting the same DNA regions. The
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primers and their main features are reported in Supplementary Table 3. Primers were designed on
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the "Cucurbita pepo Unigene PU020788" clone, retrieved at the International Cucurbit Genomics
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Initiative (ICuGI) database (http://www.icugi.org/) based on sequence similarity BLAST search.
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2.5. Data analysis
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For each SSR locus, we calculated the number of alleles, their frequency, the Observed
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Heterozygosity (Ho), the Polymorphic information content (PIC) (Powell, et al., 1996), the Fixation
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Index and the Probability of identity (Hartl, Clark, & Clark, 1997). Pairwise genetic distances were
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calculated as reported (Nei, 1972). These calculations were performed using the GenAlEx 6.5
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software (Peakall & Smouse, 2012). Hierarchical clustering using the UPGMA algorithm was carried
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out with the MEGA software (Tamura, Dudley, Nei, & Kumar, 2007). The calculation of the
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expected genotype frequency (i.e. genotype probability) based on allele frequency under Hardy–
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Weinberg expectation for random union of alleles, without taking population structure into
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account (National Research Council, 1996). DNA profile frequency estimates were calculated by
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considering the genotype frequency for each locus and then multiplying the frequencies across all
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loci, yielding the Random Match Probability. For the analysis of the cumulative product of
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genotype probability for an increasing number of SSR loci, we calculated the descriptive statistics
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considering the probability of each scored genotype using all the combinations without repetition
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(C) of different number of loci (Cn,k=n!/(k!(n-k)!), where n is the number of values to choose from,
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and k the element chosen). This analysis was performed in R (Team R Core, 2000).
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ACCEPTED MANUSCRIPT 3. Results
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3.1. Allelic diversity
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We analysed 30 cultivated varieties of C. pepo using eight SSRs representatives of various repeat
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classes. Main genetic parameters of the population under investigation are presented in Table 2.
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All the loci were polymorphic. We detected 29 alleles, whose length ranged from 65 to 236 bp, for
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an average of 3.6 allele per locus. The mean observed heterozygosity (Ho) was just above 25% yet,
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two loci (CMTp69 and CMTp176) had a Ho below 0.1%. The Ho did not significantly correlate with
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the number of alleles (p >0.05; Spearman’s Rho). The PIC values indicated that the degree of
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polymorphism in each locus was high (i.e. >0.5) for the majority (5 out of 8) of SSRs (Botstein,
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White, Skolnick, & Davis, 1980) and that PIC significantly correlated with the number of alleles (p
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>0.05; Spearman’s Rho). The Probability of Identity, an estimation of the probability that two
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unrelated individuals will have by chance the same genotype, inversely correlated with the
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number of alleles. The fixation index was substantial positive for all but two loci (CMTp98 and
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CUTC022867) and considering the outcrossing nature of C. pepo, it may indicate an effect of
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breeding. This hypothesis is also consistent with the presence in many loci of one allele with a
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predominant frequency, evident especially for the multiallelic SSRs (e.g. those with more than
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three alleles). The genetic divergence between varieties were calculated considering the SSR
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profiles of three plants per variety using the Nei’s genetic distance between populations.
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Hierarchical clustering illustrated that all the varieties could be discriminated (Figure 2).
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3.2. SSR transferability
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Although SSR markers are generally regarded as species-specific, different studies indicated that
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the sequence similarity across related species or genera is sufficient to obtain amplification
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products using primers designed for one plant species (Rai, Phulwaria, & Shekhawat, 2013; Saha,
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ACCEPTED MANUSCRIPT Cooper, Mian, Chekhovskiy, & May, 2006; Yamamoto, et al., 2001). For instance, it is known that
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the close botanical relationship allows the transferability of the SSR primers among cultivated
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Curbitaceae (Li Gong, et al., 2012). Considering that zucchini processed products can be present in
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foodstuff containing different plant species (e.g. mixed vegetables), we assessed the transferability
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of the employed SSR primers in cabbage, carrot, celery, onion, pea, potato and tomato. Moreover,
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we also analysed two edible Cucurbits, cucumber and pumpkin. For all these plant species, the
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DNA was isolated from edible organs (fruits, seeds, leaves, etc.) available in grocery stores. We
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tested the eight SSR loci in the same amplification conditions used for the analysis of the zucchini.
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Amplifications were then subjected to capillary electrophoresis. We classified the output in three
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categories: “no amplification” (when the fluorescent signal was similar to the background line);
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“nonspecific amplification” (presence of more than two peaks, usually in a wide size range) and
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“within range” (one or two well-defined peaks, whose size was within the detected allelic size
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range for zucchini; table 2). The results are summarised in Table 3 and an illustrative example of
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the capillary electrophoresis output is reported in Supplementary Figure 1. We did not obtain a
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detectable amplicon for the majority of the samples (39%), while the percentage of nonspecific
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and specific amplification were similar. The set of eight primers was not completely transferable in
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the plant species tested and, as expected, the maximum transferability was obtained in pumpkin
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(6 loci) and cucumber (4 loci). For the other species, the number of transferable SSR ranged from
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three to zero. The most transferable SSR was CMTp69 (in seven plant species). The most specific
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loci were CMTp132 and CMTp142 (transferable in one species) yet, the EST-SSR CUTC022867 was
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transferable only in the two Cucurbitaceae analysed and, differently for the other SSRs, did not
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yield nonspecific amplifications in the other plant species.
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3.3. Amplificability of the DNA from commercial products
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ACCEPTED MANUSCRIPT Food processing and storage can decrease the quality of the DNA that can be isolated from
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commercial products. We analysed five types of commercial products that differ in the
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transformation process, ranging from “no treatment” (fresh fruits) to a combination of mechanical
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and heat treatments (Table 1). Considering the allelic range observed, we tested if the DNA
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isolated from the food products containing zucchini can consistently yield PCR products within the
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desired interval (up to 280 bp). To this aim, we used primers designed to amplify the same target
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region of a single gene of the C. pepo genome (Figure 1A). By using different primer combinations,
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the result indicated that is possible to amplify fragments within the 280-89 bp range in the
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commercial products tested (Figure 1B, C and D).
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3.4. Analysis of commercial products
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Having tested the amplificability of the isolated DNA, we screened the commercial products
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containing zucchini (Table 1) with the eight SSR markers. All the SSRs loci were able to amplify
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consistently DNA (Figure 3). We observed, also for DNA isolated from complex food (e.g. those
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with additional ingredients) the presence of one or two allele, confirming the codominance of the
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markers employed. Moreover, to test for the possible presence of spurious alleles deriving from
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other plant species, we compared the profile of the DNA isolated form mixed vegetable with the
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one from the frozen zucchini manually separated from the commercial product, without finding
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differences.
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In the commercial products (including fruits), we detected in total 11 alleles, which were also
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present in our zucchini collection. The comparison of the food samples’ and the references’
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profiles revealed a match for the frozen zucchini and mixed vegetables. We therefore computed
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the match probability to weight this evidence. For this estimation, we considered the allele
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frequencies collected from all the plants analysed (90), mainly to take into account the variability
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ACCEPTED MANUSCRIPT in the genetic profiles in the open pollinated varieties. The random match probability calculations
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are reported in table 4. The expected frequency (RMP) of the genotype identified in some
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commercial product is 2.95 x 10-4 considering our population of zucchini varieties. To interpret and
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assess the weight of this DNA evidence, we built an accumulation curve of the possible random
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match probability for each of the genotypes under investigation for increasing combinations of
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loci. This analysis provided evidence on the number of loci required for reliable genetic tagging.
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Summary statistics are reported in Supplementary Table 4. To increase readability, Figure 4
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boxplots the log10 of the genotype frequencies according to an increasing number of SSRs. The
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curve based on raw RMPs started to flatten with five loci (not shown) and the average genotype
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frequency was always significantly smaller by increasing the number of SSR loci (p <0.001,
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Wilcoxon Signed-Rank test). Increasing the number of loci also reduced data dispersion and
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strongly limited the occurrence of outliers (i.e. values outside 1.5 times the interquartile range
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above the upper and below the lower quartile).
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ACCEPTED MANUSCRIPT 4. Discussion
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The zucchini market, as other horticultural crops, is characterized by an increasing standardization
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of production (i.e. uniformity in fruit shape and dimension as well as in qualitative and
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organoleptic aspects of commercial products), which has recently boosted an interest for more
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typical productions (Brugarolas, Martínez-Carrasco, Martínez-Poveda, & Ruiz-Martínez, 2009;
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Johns, Powell, Maundu, & Eyzaguirre, 2013). Zucchini represents the most important and
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cosmopolitan horticultural group of the Cucurbita genus (Paris, 1996, 2016a) and an ingredient of
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very popular frozen products (Barbosa-Cánovas, et al., 2005) and challenges in genetic
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characterization of zucchini food products are illustrative of the specific issues about DNA typing
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of horticultural products.
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This study demonstrated the possibility of using SSR markers for the characterization of different
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zucchini varieties and the identification of the variety in commercial processed products. The DNA
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analysis indicated that the germplasm under investigation has a number of alleles that is
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consistent with other published works in C. pepo as well other herbaceous annual crops
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(Bredemeijer, et al., 2002; Li Gong, et al., 2012; Shehata, Al-Ghethar, & Al-Homaidan, 2009; Singh,
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et al., 2013). The SSRs markers were all polymorphic and their informativeness mainly correlated
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with the number of alleles. Significant differences between SSRs were found with respect to the
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observed heterozygosity, which may be explained by fixation of some SSRs due to association of
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some favorable alleles. For each locus, the percentage of observed genotypes over the possible
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ones was high (78%), indicating that our zucchini population represent a suitable panel of different
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genotypes. The usefulness of a DNA marker is also reflected by frequencies of the most common
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genotypes (Edwards, Hammond, Jin, Caskey, & Chakraborty, 1992). For many loci, we observed
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the presence of common alleles, which can limit the value of the genotype’s combined frequency.
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Moreover, the presence of alleles with high frequency renders a locus less powerful in
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ACCEPTED MANUSCRIPT discriminating two unrelated individual. Based on these criteria, the CMTp257 yielded the most
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useful results among the employed SSRs.
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The eight loci could discriminate the varieties under investigation and proved to be suitable to
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amplify DNA from a range of commercial products. In spite of the thermal and/or mechanical
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treatment, we could reliably amplify PCR products up to 280 bp, a dimension that includes almost
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all SSR loci for Cucurbita (L Gong, et al., 2008). Compared to other processed vegetables such as
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tomato (Scarano, et al., 2015), it is likely that this dimension also reflects the fact that zucchini are
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not peeled and, if not frozen, they are stored in oil.
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SSR transferability may represent an advantage for plant forensics mainly because it allows
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reducing the cost and effort to develop SSRs for orphan plant species (Craft, Owens, & Ashley,
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2007). Our work indicated that caution should be applied when analyzing a complex food mixture
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because of the possibility to obtain within-range allelic profiles also from very different species.
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For mixed vegetables, the data suggest that the profile of the isolated product does not come
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from multiple sources. It is fair to add that zucchini was the only Cucurbit in this commercial
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product. Large differences were present in the cross-species transferability of the SSRs, implying
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that a preliminary screening of SSR markers is useful, if not necessary, when aiming at the genetic
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traceability of mixed products. Interestingly, the EST-SSR CUTC022867 did not yield nonspecific
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amplifications in non-Cucurbits. EST-SSRs are thought to be more transferable in closely related
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species (Varshney, Graner, & Sorrells, 2005), and the possibility that genic SSRs are less
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transferable in more distant taxonomic ranks (e.g. across genera) should be tested analyzing a
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larger panel of SSRs.
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The significance of non-human DNA typing for court use has advanced recently especially for
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animal species and products (Johnson, Wilson-Wilde, & Linacre, 2014; Linacre, et al., 2011). The
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ACCEPTED MANUSCRIPT relevance is much more limited for the plant sector (Iyengar, 2014), in spite of a wide range of
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applications (Barcaccia, et al., 2015; Corrado, 2016; Ferri, et al., 2015). While DNA typing cannot
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be considered anymore a novel approach, a limitation of its use in forensic plant genetics is
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represented by the strength of statistical interpretations (Sensabaugh & Kaye, 1998). A number of
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studies have identified the genetic makeup of plant-derived food products yet, hardly ever this
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information is accompanied by an estimate of a profile frequency (Craft, et al., 2007; Howard,
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Gilmore, Robertson, & Peakall, 2009). To this aim, the allele frequency information from our
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database was used to assess relative rarity of DNA profiles. This analysis underlined the
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importance of the number of loci under investigation. Although the SSR loci employed displayed
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large difference in their discriminating power, the accumulation curve gives reason to believe that
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the rarity of a full DNA profile frequency estimate in zucchini could be significantly lowered by
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adding more SSR loci. The shortage of statistical analysis for the interpretation of the rarity of
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genetic profile in horticultural crops does not allow an evaluation of the general probability values
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(Foreman & Evett, 2001). For cultivated plants, the random match probability represents the
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frequency of an SSR profile expected to occur in a population of cultivars, rather than individuals
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(Craft, et al., 2007). It is difficult to put forward an estimate of the cultivated zucchini varieties,
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mainly because varieties are registered as C. pepo (hence including marrow, pumpkin, winter
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squash as well as different horticultural types of summer squash). Based on the catalogues of main
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breeding companies, a sensible estimation of the number of zucchini varieties currently sold in
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Italy should be around 50-60, which suggests that the genetic match presented in this work cannot
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be attribute to chance alone. A main priority to strengthen the use of DNA testing of horticultural
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products in courts will be to establish international database of genetic profile. Further studies
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should also explore genetic differentiation and population substructure, which is likely to be
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present when comparing different horticultural groups of C. pepo.
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Acknowledgments
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The research activity was supported by the “Valorizzazione di produzioni ortive campane di
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eccellenza
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PON02_00395_3215002. MC was supported by a scholarship of the Regione Campania, POR
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Campania FSE 2014/2020, RIS: Salute, biotecnologie, agroalimentare.
strumenti
di
genomica
avanzata”
(GenHORT)
project,
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TABLES
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Table 1: Commercial products analysed and their main features. Product
Mechanical Treatment
Heat treatment
Fruits from the shelf (cylindrical and round) Frozen zucchini Mixed Vegetables (frozen) Chargrilled, in olive oil* Fried in oil (Caprese/Scapece)* *: pasteurised product
No
No
Yes (slices) Yes (dice) Yes (slices) Yes (slices)
No No Yes (grilled) Yes (fried)
No Yes No Yes
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Other vegetable ingredients No
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ACCEPTED MANUSCRIPT Table 2: Main genetic indices of the zucchini genotypes under investigations.
CMTp69
65-75
CMTp98
212-236
CMTp132
141-165
CMTp142
150-196
CMTp176
93-109
CMTp257
123-147
CMTp260
120-156
CUTC022867 124-139 Mean SE
PI Na 5
Ho PIC F 0.067 0.528 0.874 0.200
0.215
0.068
0.640
0.878
3
0.289
0.376
0.232
0.432
0.767
4
0.144
0.561
0.743
0.239
0.617
2
0.078
0.299
0.740
0.536
0.817
6
0.395
0.674
0.414
0.164
0.426
4
0.400
0.610
0.344
0.224
0.506
3
0.500
0.524
0.047
0.310
3.625 0.498
0.259 0.057
0.473 0.057
0.433 0.113
0.352 0.059
0.600
Legend: Na: number of alleles; Ho: Observed heterozygosity; He: Expected Herozygosity; F: Fixation index; PI: Probability of identity at a locus, MAF: major allele frequency.
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MAF 0.664
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431 432 433
0.271
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Size range (bp)
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CMTp98
CMTp132
P. sativum
WR
NONSP
NOAMP
NOAMP
WR
WR
NONSP
NOAMP
D. carotae
WR
WR
NOAMP
NONSP
NOAMP
WR
NONSP
NOAMP
C. sativus
WR
NONSP
NOAMP
NOAMP
WR
NONSP
WR
WR
C. maxima
WR
NONSP
WR
NONSP
WR
WR
WR
WR
S. tuberosum
WR
WR
NONSP
NOAMP
NOAMP
NONSP
NONSP
NOAMP
A. graveolens
WR
WR
NONSP
WR
NOAMP
NONSP
NONSP
NOAMP
A. cepa
NOAMP
NONSP
NONSP
NOAMP
NOAMP
NOAMP
NONSP
NOAMP
B. oleracea
NOAMP
NONSP
NONSP
NOAMP
NOAMP
NONSP
NOAMP
NOAMP
WR
NOAMP
NOAMP
NOAMP
NONSP
NOAMP
NONSP
NOAMP
CMTp176
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CMTp142
CMTp257
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Table 3: Transferability of the SSRs in other plant species.
CMTp260
CUTC022867
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Legend: NONSP: nonspecific PCR products; NOAMP: no PCR amplification; WR: within-range PCR amplicon (see Table 2 for the allelic size range considered).
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Population
Allele I
Allele II
Expected
frequency
frequency
Genotype
Alleles
Observed/Possible
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frequency 0.664
0.664
0.415
5
CMTp98
0.878
0.878
0.770
2
CMTp132
0.767
0.767
0.588
CMTP142
0.617
0.617
0.380
CMTp176
0.817
0.817
CMTp257
0.426
0.148
CMTp260
0.506
0.506
CUTC022867
0.600
0.333
6/15 3/3
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5/6
4
8/10
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2
3/3
0.126
6
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0.123
4
8/10
0.400
3
6/6
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Fruit colour (intensity)
Altea Anissa Asso Aurore Aymaran Black Beauty Cavili Coucourzelle De Nice à fruit rond Genovese Genovese Antico Giulia Gulliver Lanka Mikonos Milos Ontano Parthenon President Quine Rhodos Rigas Romanesco San Pasquale Sitos Syros Temprano de Argelia Vertre noir maraîchère Virginia Vitulia
cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical tapered cylindrical globular tapered cylindrical tapered cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical tapered cylindrical cylindrical tapered cylindrical cylindrical
grey green (dark) green (medium) green (dark) green (very light) green (dark) green (very light) grey green (very light) green (very light) green (very light) green (light) green (medium) green (light) green (medium) green (medium) grey green (dark) green (medium) green (dark) green (medium) grey green (very light) grey green (light) green (dark) green (very light) green (dark) green (very light) green (light)
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Legend: H: hybrid; OP: open-pollinated variety.
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Type H H H H H OP H OP OP OP OP H H H H H H H H H H H OP OP H H OP OP OP H
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Variety
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Primer label (forward)
GCAGAGGAGAAGTGGGTTTG CTTTATCCGACCAAGCGTTC
(AAG)9
6-FAM
CMTp142
TCAACCAAGTGCCAATCTCA ACTGATCCACCGACTGATACG
(TC)12+5
VIC
CMTp257
CACGAAGATTTGATGGCCTTA GGATTGGGATGGTGAAGATG
(CGT)11
PET
CMTp260
CCCTAGACCCATCATAGTCG ACATTTGGTTACTTCCCCATT
(CAT)7
NED
CMTp69
ATACTTGCTCCCCAAGTTTA AAATAAAAAGACAACGTAATGGT
(TATT)4
VIC
CMTp176
CCTGGACTTCCACATCAGTT ACTACGTGTCTCTGCAGGAAG
(TC)12
6-FAM
CMTp132
CCATTTCCATTTCCATTTCA AGGTTAGAAACAGGGGGAATC
CUTC022867
TTGGACAATCTGAGGAAGTTGG TTGGACAATCTGAGGAAGTTGG
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CMTp98
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Primer sequences (5' to 3')
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Locus SSR
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(GAT)12
NED
(AGC)8
PET
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Primer sequence (5’ to 3’)
GC-EF1pepo-RVa
CACGAACAGCAAAACGACCC
GC-EF1pepo-RVb
CTTGGTCACCTTTGGCTCCC
GC-EF1pepo-FW
GTATTGCCACACCTCCCCAC
EF1A-FW
ATTCGAGAAGGAAGCTGCTG
EF1A-RV
TTGGTGGTCTCAAACTTCCAC
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Primer
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ACCEPTED MANUSCRIPT Supplementary Table 4: Descriptive statistics of the random match probability (RMP) values for an increasing number of SSR loci. The RMP values for the population under investigation were obtained considering the combinations (without repetition) from 1 to 8 SSR loci. Descriptive statistics were then obtained using the summary() function in R. IQR: Interquartile Range. . Number of SSR loci 2
3
4
5
6
7
8
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1 1.37E-03
6.77E-06
1.20E-07
0.00E+00
7.30E-09
4.26E-09
0.00E+00
2.19E-09
1st Quartile
1.23E-01
1.98E-02
3.32E-03
5.76E-04
1.04E-04
1.93E-05
3.78E-06
5.24E-07
Median
3.60E-01
7.97E-02
1.86E-02
4.05E-03
8.71E-04
1.85E-04
4.30E-05
8.11E-06
Mean
3.60E-01
1.28E-01
4.51E-02
1.49E-02
5.75E-03
2.08E-03
7.49E-04
2.67E-04
3rd Quartile
5.88E-01
2.08E-01
5.74E-02
1.67E-02
5.32E-03
1.53E-03
4.05E-04
1.11E-04
Maximum
1.00E+00
7.70E-01
5.14E-01
3.02E-01
1.25E-01
5.02E-02
1.91E-02
4.88E-03
IQR
4.65E-01
1.88E-01
5.41E-02
4.01E-04
1.10E-04
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1.51E-03
ACCEPTED MANUSCRIPT Figure legends Figure 1 Hierarchical clustering (UPGMA algorithm) of the C. pepo varieties based on Nei’s genetic distance. The dendrogram shows that the 30 cultivars analyzed were discriminated.
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Figure 2 Amplificability of DNA isolated from commercial products. (A) Scheme of the annealing of the primers to the Elongation Factor 1-alpha gene (not to scale). (B) PCR with the EF1A-FW and EF1A-RW primers; (B) PCR with the GC-EF1A-pepo-FW GC-EF1Apepo-RVb; (C) PCR with the GC-
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EF1Apepo-FW and GC-EF1Apepo-Rva. 1: 1 kb + DNA ladder (ThermoFisher); 2: water negative control; 3: positive control (leaf DNA); 4: cylindrical fruit; 5: mixed vegetables; 6: frozen zucchini; 7:
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chargrilled zucchini; 8: fried zucchini. See table 1 for a description of the commercial products. Figure 3 Agarose gel electrophoresis of PCR reactions with the CMTp260 primers using the DNA isolated from different commercial food products. 1: 1 kb DNA ladder (Thermo Fisher); 2: water
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negative control; 3: positive control (leaf DNA); 4: cylindrical fruit; 5: rounded fruit; 6: mixed vegetables; 7: frozen zucchini; 8: chargrilled zucchini; 9: fried zucchini. See table 1 for a description of the commercial products
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Figure 4 Box and whisker plot of the random match probability (RMP) values of the genotypes
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Supplementary Figure 1 Examples of electrophoretic profiles obtained in the SSR transferability test. Panel A and B are an example electropherograms classified as «no amplification» (A: CMTp69 amplification of cabbage DNA; B: CMTp69 amplification of onion DNA). Panel C and D are an example of electropherograms classified as «nonspecific amplification» (C: CMTp98 amplification 31
ACCEPTED MANUSCRIPT of pea DNA; D: CMTp98 amplification of pumpkin DNA). Panel E and F are an example of electropherogram classified as “within range” (E: CMTp69 amplification of cucumber DNA; F: CMTP69 amplification of pumpkin DNA). Panel G and H are an example of electropherograms obtained with zucchini DNA using the CMTp69 primers. In each panel, the allelic bar is present at
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the top, while in the squares below the peaks it is reported the allelic dimensions (at the second decimal digit; sz) and peak height (Ht) in relative fluorescence units. The different colours are selected by the analysis software (GeneMapper v.4.0) according to the fluorochrome of the
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Highlights
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We used SSR markers for DNA typing of zucchini varieties in commercial products encompassing different levels of food processing We assessed SSR transferability in closely related Cucurbits and other horticultural species
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DNA typing identified the zucchini varieties in two commercial products
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We provide indications for the statistical interpretation of the observed genetic
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concordance
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• The identified matches cannot be attributed to chance alone
S0956-7135(17)30391-2
DOI:
10.1016/j.foodcont.2017.07.039
Reference:
JFCO 5732
To appear in:
Food Control
Received Date: 22 May 2017 Revised Date:
28 July 2017
Accepted Date: 29 July 2017
Please cite this article as: Verdone M., Rao R., Coppola M. & Corrado G., Identification of zucchini varieties in commercial food products by DNA typing, Food Control (2017), doi: 10.1016/ j.foodcont.2017.07.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Identification of zucchini varieties in commercial food products by DNA typing
2
Maria Verdone, Rosa Rao, Mariangela Coppola and Giandomenico Corrado*
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Dipartimento di Agraria, Università degli Studi di Napoli “Federico II”, via Università 100, 80055
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Portici (NA), Italy
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* Corresponding author. Tel.: +39 081 2539294; fax: +39 081 2539481. E-mail address:
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[email protected] (G. Corrado).
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Traceability, SSR, STR, molecular markers, Cucurbita pepo, DNA profile, random match probability
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Keywords
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ACCEPTED MANUSCRIPT ABSTRACT
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DNA typing has been long proposed as a component of food traceability especially for the ability
15
to provide genetic information on processed and/or mixed commercial products. However, the
16
use of DNA testing to deliver legal evidence has been very limited in the agro-food chain, also
17
because in this field, a genetic concordance is rarely sustained with indications on the
18
interpretation and statistics of a DNA profile.
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Zucchini is the most important and diverse horticultural group belonging to Cucurbita pepo, a
20
plant species that yields a large variety of edible fruits such as squashes and pumpkins. Challenges
21
in genetic discrimination of zucchini material exiting the agro-food chain typify the problems in
22
DNA typing of horticultural food products, because of limited allelic diversity, presence of common
23
alleles, highly processed food and genetic similarity with other edible species.
24
The aim of this work was to identify zucchini varieties in commercial products by a means of
25
Simple Sequence Repeat (SSR) analysis and to provide indications for the statistical interpretation
26
of a match. We assessed the efficacy of SSRs analyzing some of the most diffused hybrids and
27
open pollinated zucchini varieties. SSRs were also screened for transferability in DNA isolated from
28
edible organs of different plant species that are frequently present in food products containing
29
zucchini. Moreover, we demonstrate the possibility to identify the variety in food products of
30
different origins. The statistical evaluation on the rarity of DNA profiles in zucchini indicated that
31
the genetic match presented in this work cannot be attributed to chance alone. Based on this
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evidence, the advantages and pitfalls of food DNA typing for variety identification of a horticultural
33
crop are discussed.
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ACCEPTED MANUSCRIPT 1. Introduction
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Cucurbita pepo L. is considered the most diverse species of its genus with respect to fruit size,
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shape and color (Paris, 1996). This plant species yields a large variety of fruits used for culinary
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purposes, such as pumpkin, gourd, marrow and winter and summer squash. These designations do
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not have a precise botanical meaning because they are not associated to one particular species
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(Paris, 1996). For instance, summer squash is a lay term to designate edible, tender fruits of C.
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pepo that are harvested and consumed before maturity, although not all fruits of this species are
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considered summer squashes (Whitaker & Robinson, 1986).
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Zucchini is currently the most diffused and economically important horticultural group of C. pepo
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(Paris, 2016b). The term zucchini is an Americanism originating from the Italian word (masculine,
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plural) for little pumpkin (“zucca” plus a diminutive suffix) (http://www.accademiadellacrusca.it).
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Zucchini designates a rather uniform, cylindrically-shaped fruit with length-to-broadest width ratio
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generally comprised from 3.5 to 4.5 (Paris, 1996). Zucchini fruits, also named courgettes in English
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speaking countries, typically have a green skin color, being harvested immature, and are eaten
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only after cooking. However, traditional varieties with more spherical fruits (e.g. “Tondo”), grown
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mainly in Italy and France, and more recently, varieties with yellow fruits are also included in the
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zucchini group by plant breeders and retailers.
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The zucchini cultivation has experienced a recent success in many countries mainly due to ease of
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cultivation in both open-field and protected agriculture, short crop cycle, adaptability to
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temperate and subtropical climates, suitability to low-calories diet regimes and cooking versatility.
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The diffusion of summer squash, mainly zucchini, has increased proportionally more than many
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other vegetables, especially in Western countries (Paris, 2016b). This success is also determined by
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the world wide increasing consumption of fresh and frozen fruits and vegetables, principally as
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ACCEPTED MANUSCRIPT alternative to canned products (Barbosa-Cánovas, Altunakar, & Mejía-Lorío, 2005; Harris &
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Shiptsova, 2007). For instance, mixed vegetables are the second most important frozen product
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after potatoes (Barbosa-Cánovas, et al., 2005).
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Traceability provides the ability to characterize and certify products throughout the food chain,
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from producers to consumers (Dabbene, Gay, & Tortia, 2014). Different technologies are required
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for effective traceability implementation and one important element is genetic traceability (Opara,
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2003). DNA markers are widely used molecular tools to trace or reveal the genetic identity of a
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biological component of food and feed. For genetic traceability, preference is given to markers
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amenable to the analysis of raw material and highly processed commercial products. Compared to
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other analytical methods, the DNA analysis offers a great level of precision and accuracy to
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identify the origin and composition of food in terms of botanical species or varieties (Barcaccia,
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Lucchin, & Cassandro, 2015; Corrado, 2016).
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Zucchini represents a challenge for DNA traceability. Because of its more recent origin, zucchini is
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characterized by a reduced diversity compared to other C. pepo horticultural groups (Formisano,
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et al., 2012). Moreover, genetic traceability needs to consider that zucchini is frequently present in
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complex, mixed products (i.e. those with different vegetable ingredients). Additionally, the
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botanical relationships between zucchini and other edible Cucurbitacae (e.g. pumpkin, gourd,
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cucumber, etc.) may pose a limitation in the discrimination power of some DNA markers (Li Gong,
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et al., 2012). Finally, many zucchini cultivars are hybrids, requiring co-dominant markers for an
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easier discrimination. Among DNA markers available, Simple Sequence Repeat (SSR), also known
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as Simple Tandem Repeats (STR), are the markers of choice in forensics (Schlötterer, 2004) and
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they are widely used also to trace the agro-food chain because of their high polymorphism,
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codominant nature, reproducibility and amenability to degraded DNA (Corrado, 2016).
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ACCEPTED MANUSCRIPT The aim of this work was to type the DNA of zucchini varieties in commercial food products by a
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means of SSR analysis. In order to evaluate the genetic differences among popular zucchini
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varieties, we assessed the efficacy of SSRs by analyzing some of the most diffused hybrids and
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open pollinated cultivars. SSRs were also screened for transferability in DNA isolated from edible
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organs of different plant species that are frequently present in commercial products containing
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zucchini. Finally, it was constructed a profiles database, which was used to support the statistical
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evaluation on the rarity of DNA profiles in zucchini. This evaluation allowed discussing the
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advantages and pitfalls of food DNA typing for variety identification of a horticultural crop.
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ACCEPTED MANUSCRIPT 2. Material and Methods
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2.1. Plant material and DNA isolation
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We analysed 30 varieties of zucchini (Cucurbita pepo ssp. pepo L.) namely, ‘Altea’, ‘Anissa’, ‘Asso’,
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‘Aurore’, ‘Aymaran’, ‘Black Beauty’, ‘Cavili’, ‘Coucourzelle’, ‘De Nice à fruit rond’, ‘Genovese’,
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‘Genovese Antico’, ‘Giulia’, ‘Gulliver’, ‘Lanka’, ‘Mikonos’, ‘Milos’, ‘Ontano’, ‘Parthenon’,
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‘President’, ‘Quine’, ‘Rhodos’, ‘Rigas’, ‘Romanesco’, ‘San Pasquale’, ‘Sitos’, ‘Syros’, ‘Temprano de
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Argelia’, ‘Vertre noir maraîchère’, ‘Virginia’ and ‘Vitulia’. This set includes 20 hybrid cultivars and
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10 open-pollinated varieties. Fruit general shape and colour are reported in Supplementary Table
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1. Plants were grown from seed and the first true leaf was harvested and frozen in liquid nitrogen
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for subsequent analysis. We analysed three plants per variety.
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For the SSR transferability experiments, DNA was isolated from fresh edible organs of the
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following species: cabbage (Brassica oleracea L.), carrot (Daucus carotae L.), celery (Apium
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graveolens L.), cucumber (Cucumis sativus L), onion (Allium cepa L.), pea (Pisum sativum L.), potato
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(Solanum tuberosum L.), pumpkin (Cucurbita maxima Duchesne), tomato (Solanum lycopersicum
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L.). Cabbage, carrot, celery, onion, pea, potato and tomato are very frequently present in
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processed food products containing zucchini (e.g. vegetable soups, frozen mixed vegetables, etc.).
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Cucumber and pumpkin were included because they are two edible plants belonging to the
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Cucurbitacae family. For all plant material, DNA isolation and quantification was carried out as
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described (Scarano, Rao, Masi, & Corrado, 2015)
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2.2. Commercial products and DNA isolation
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We analysed five types of commercial products containing zucchini: fresh fruits (with cylindrical or
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round shape) from the shelf of a large-scale distribution; frozen zucchini (sliced and sold as a
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frozen product); mixed vegetables (different clean vegetables cut to prepare a soup, raw and not
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ACCEPTED MANUSCRIPT seasoned, sold as a frozen product); chargrilled zucchini (sliced zucchini, chargrilled and stored in a
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transparent glass jar with olive oil); and Caprese (aka Scapece) zucchini (canned zucchini, fried
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with vegetable oil, seasoned with vinegar, garlic and mint leaves, stored in olive oil). Frozen
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zucchini, mixed vegetables and chargrilled zucchini were purchased from the same retail store
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brand. For DNA isolation, fresh and frozen products were pulverised in liquid nitrogen with a
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mortar and pestle for subsequent manipulations. Zucchini stored in oil were first blotted on
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kitchen towels and then centrifuged for 30 second at 1000 g on kitchen paper to remove the
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residual liquid phase. Samples were then pulverised as described above. DNA isolation was carried
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out using the DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instruction.
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2.3. SSR analysis
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DNA amplification reactions were conducted in a 25 μl volume containing 20ng of genomic DNA,
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1X PCR buffer (Promega), 0.1 mM dNTPs, 0.2 μM labelled forward primer, 0.2 μM reverse primer
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and 0.5U of Taq polymerase (Promega). The amplification reactions were carried out in a
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Mastercycler Gradient (Eppendorf) thermocycler with the following conditions: a DNA
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denaturation step at 94 °C for 4'; 35 cycles comprising a denaturation step at 94 °C for 45'', an
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annealing phase at 56 °C for 45'' and elongation step at 72 °C for 90”. A final elongation phase at
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72 °C for 15' ended the reaction. The eight SSR loci employed comprised different core structures
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(L Gong, Stift, Kofler, Pachner, & Lelley, 2008). Primer and core sequences are reported in
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Supplementary Table 2. Amplification products were separated by agarose gel electrophoresis to
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verify the presence of amplified fragments. For allelic discrimination, the fluorescent fragments
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were resolved by capillary electrophoresis in an ABI PRISM 3130 (Applied Biosystems) system
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using the POP6 polymer (Applied Biosystems). Signal peak height and allele sizes were calculated
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using the ABI PRISM Genotyper (v. 4.0) software (Applied Biosystems) based on the GeneScan
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500Liz molecular weight standard (Applied Biosystems).
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ACCEPTED MANUSCRIPT 2.4. Estimation of the amplicon size range for processed products.
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DNA from processed products was amplified with primers targeting the same DNA regions. The
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primers and their main features are reported in Supplementary Table 3. Primers were designed on
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the "Cucurbita pepo Unigene PU020788" clone, retrieved at the International Cucurbit Genomics
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Initiative (ICuGI) database (http://www.icugi.org/) based on sequence similarity BLAST search.
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2.5. Data analysis
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For each SSR locus, we calculated the number of alleles, their frequency, the Observed
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Heterozygosity (Ho), the Polymorphic information content (PIC) (Powell, et al., 1996), the Fixation
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Index and the Probability of identity (Hartl, Clark, & Clark, 1997). Pairwise genetic distances were
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calculated as reported (Nei, 1972). These calculations were performed using the GenAlEx 6.5
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software (Peakall & Smouse, 2012). Hierarchical clustering using the UPGMA algorithm was carried
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out with the MEGA software (Tamura, Dudley, Nei, & Kumar, 2007). The calculation of the
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expected genotype frequency (i.e. genotype probability) based on allele frequency under Hardy–
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Weinberg expectation for random union of alleles, without taking population structure into
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account (National Research Council, 1996). DNA profile frequency estimates were calculated by
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considering the genotype frequency for each locus and then multiplying the frequencies across all
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loci, yielding the Random Match Probability. For the analysis of the cumulative product of
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genotype probability for an increasing number of SSR loci, we calculated the descriptive statistics
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considering the probability of each scored genotype using all the combinations without repetition
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(C) of different number of loci (Cn,k=n!/(k!(n-k)!), where n is the number of values to choose from,
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and k the element chosen). This analysis was performed in R (Team R Core, 2000).
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ACCEPTED MANUSCRIPT 3. Results
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3.1. Allelic diversity
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We analysed 30 cultivated varieties of C. pepo using eight SSRs representatives of various repeat
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classes. Main genetic parameters of the population under investigation are presented in Table 2.
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All the loci were polymorphic. We detected 29 alleles, whose length ranged from 65 to 236 bp, for
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an average of 3.6 allele per locus. The mean observed heterozygosity (Ho) was just above 25% yet,
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two loci (CMTp69 and CMTp176) had a Ho below 0.1%. The Ho did not significantly correlate with
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the number of alleles (p >0.05; Spearman’s Rho). The PIC values indicated that the degree of
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polymorphism in each locus was high (i.e. >0.5) for the majority (5 out of 8) of SSRs (Botstein,
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White, Skolnick, & Davis, 1980) and that PIC significantly correlated with the number of alleles (p
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>0.05; Spearman’s Rho). The Probability of Identity, an estimation of the probability that two
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unrelated individuals will have by chance the same genotype, inversely correlated with the
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number of alleles. The fixation index was substantial positive for all but two loci (CMTp98 and
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CUTC022867) and considering the outcrossing nature of C. pepo, it may indicate an effect of
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breeding. This hypothesis is also consistent with the presence in many loci of one allele with a
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predominant frequency, evident especially for the multiallelic SSRs (e.g. those with more than
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three alleles). The genetic divergence between varieties were calculated considering the SSR
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profiles of three plants per variety using the Nei’s genetic distance between populations.
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Hierarchical clustering illustrated that all the varieties could be discriminated (Figure 2).
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3.2. SSR transferability
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Although SSR markers are generally regarded as species-specific, different studies indicated that
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the sequence similarity across related species or genera is sufficient to obtain amplification
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products using primers designed for one plant species (Rai, Phulwaria, & Shekhawat, 2013; Saha,
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ACCEPTED MANUSCRIPT Cooper, Mian, Chekhovskiy, & May, 2006; Yamamoto, et al., 2001). For instance, it is known that
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the close botanical relationship allows the transferability of the SSR primers among cultivated
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Curbitaceae (Li Gong, et al., 2012). Considering that zucchini processed products can be present in
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foodstuff containing different plant species (e.g. mixed vegetables), we assessed the transferability
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of the employed SSR primers in cabbage, carrot, celery, onion, pea, potato and tomato. Moreover,
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we also analysed two edible Cucurbits, cucumber and pumpkin. For all these plant species, the
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DNA was isolated from edible organs (fruits, seeds, leaves, etc.) available in grocery stores. We
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tested the eight SSR loci in the same amplification conditions used for the analysis of the zucchini.
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Amplifications were then subjected to capillary electrophoresis. We classified the output in three
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categories: “no amplification” (when the fluorescent signal was similar to the background line);
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“nonspecific amplification” (presence of more than two peaks, usually in a wide size range) and
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“within range” (one or two well-defined peaks, whose size was within the detected allelic size
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range for zucchini; table 2). The results are summarised in Table 3 and an illustrative example of
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the capillary electrophoresis output is reported in Supplementary Figure 1. We did not obtain a
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detectable amplicon for the majority of the samples (39%), while the percentage of nonspecific
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and specific amplification were similar. The set of eight primers was not completely transferable in
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the plant species tested and, as expected, the maximum transferability was obtained in pumpkin
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(6 loci) and cucumber (4 loci). For the other species, the number of transferable SSR ranged from
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three to zero. The most transferable SSR was CMTp69 (in seven plant species). The most specific
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loci were CMTp132 and CMTp142 (transferable in one species) yet, the EST-SSR CUTC022867 was
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transferable only in the two Cucurbitaceae analysed and, differently for the other SSRs, did not
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yield nonspecific amplifications in the other plant species.
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3.3. Amplificability of the DNA from commercial products
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ACCEPTED MANUSCRIPT Food processing and storage can decrease the quality of the DNA that can be isolated from
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commercial products. We analysed five types of commercial products that differ in the
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transformation process, ranging from “no treatment” (fresh fruits) to a combination of mechanical
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and heat treatments (Table 1). Considering the allelic range observed, we tested if the DNA
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isolated from the food products containing zucchini can consistently yield PCR products within the
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desired interval (up to 280 bp). To this aim, we used primers designed to amplify the same target
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region of a single gene of the C. pepo genome (Figure 1A). By using different primer combinations,
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the result indicated that is possible to amplify fragments within the 280-89 bp range in the
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commercial products tested (Figure 1B, C and D).
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3.4. Analysis of commercial products
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Having tested the amplificability of the isolated DNA, we screened the commercial products
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containing zucchini (Table 1) with the eight SSR markers. All the SSRs loci were able to amplify
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consistently DNA (Figure 3). We observed, also for DNA isolated from complex food (e.g. those
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with additional ingredients) the presence of one or two allele, confirming the codominance of the
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markers employed. Moreover, to test for the possible presence of spurious alleles deriving from
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other plant species, we compared the profile of the DNA isolated form mixed vegetable with the
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one from the frozen zucchini manually separated from the commercial product, without finding
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differences.
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In the commercial products (including fruits), we detected in total 11 alleles, which were also
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present in our zucchini collection. The comparison of the food samples’ and the references’
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profiles revealed a match for the frozen zucchini and mixed vegetables. We therefore computed
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the match probability to weight this evidence. For this estimation, we considered the allele
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frequencies collected from all the plants analysed (90), mainly to take into account the variability
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ACCEPTED MANUSCRIPT in the genetic profiles in the open pollinated varieties. The random match probability calculations
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are reported in table 4. The expected frequency (RMP) of the genotype identified in some
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commercial product is 2.95 x 10-4 considering our population of zucchini varieties. To interpret and
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assess the weight of this DNA evidence, we built an accumulation curve of the possible random
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match probability for each of the genotypes under investigation for increasing combinations of
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loci. This analysis provided evidence on the number of loci required for reliable genetic tagging.
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Summary statistics are reported in Supplementary Table 4. To increase readability, Figure 4
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boxplots the log10 of the genotype frequencies according to an increasing number of SSRs. The
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curve based on raw RMPs started to flatten with five loci (not shown) and the average genotype
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frequency was always significantly smaller by increasing the number of SSR loci (p <0.001,
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Wilcoxon Signed-Rank test). Increasing the number of loci also reduced data dispersion and
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strongly limited the occurrence of outliers (i.e. values outside 1.5 times the interquartile range
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above the upper and below the lower quartile).
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ACCEPTED MANUSCRIPT 4. Discussion
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The zucchini market, as other horticultural crops, is characterized by an increasing standardization
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of production (i.e. uniformity in fruit shape and dimension as well as in qualitative and
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organoleptic aspects of commercial products), which has recently boosted an interest for more
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typical productions (Brugarolas, Martínez-Carrasco, Martínez-Poveda, & Ruiz-Martínez, 2009;
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Johns, Powell, Maundu, & Eyzaguirre, 2013). Zucchini represents the most important and
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cosmopolitan horticultural group of the Cucurbita genus (Paris, 1996, 2016a) and an ingredient of
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very popular frozen products (Barbosa-Cánovas, et al., 2005) and challenges in genetic
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characterization of zucchini food products are illustrative of the specific issues about DNA typing
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of horticultural products.
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This study demonstrated the possibility of using SSR markers for the characterization of different
253
zucchini varieties and the identification of the variety in commercial processed products. The DNA
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analysis indicated that the germplasm under investigation has a number of alleles that is
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consistent with other published works in C. pepo as well other herbaceous annual crops
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(Bredemeijer, et al., 2002; Li Gong, et al., 2012; Shehata, Al-Ghethar, & Al-Homaidan, 2009; Singh,
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et al., 2013). The SSRs markers were all polymorphic and their informativeness mainly correlated
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with the number of alleles. Significant differences between SSRs were found with respect to the
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observed heterozygosity, which may be explained by fixation of some SSRs due to association of
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some favorable alleles. For each locus, the percentage of observed genotypes over the possible
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ones was high (78%), indicating that our zucchini population represent a suitable panel of different
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genotypes. The usefulness of a DNA marker is also reflected by frequencies of the most common
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genotypes (Edwards, Hammond, Jin, Caskey, & Chakraborty, 1992). For many loci, we observed
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the presence of common alleles, which can limit the value of the genotype’s combined frequency.
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Moreover, the presence of alleles with high frequency renders a locus less powerful in
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ACCEPTED MANUSCRIPT discriminating two unrelated individual. Based on these criteria, the CMTp257 yielded the most
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useful results among the employed SSRs.
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The eight loci could discriminate the varieties under investigation and proved to be suitable to
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amplify DNA from a range of commercial products. In spite of the thermal and/or mechanical
270
treatment, we could reliably amplify PCR products up to 280 bp, a dimension that includes almost
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all SSR loci for Cucurbita (L Gong, et al., 2008). Compared to other processed vegetables such as
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tomato (Scarano, et al., 2015), it is likely that this dimension also reflects the fact that zucchini are
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not peeled and, if not frozen, they are stored in oil.
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SSR transferability may represent an advantage for plant forensics mainly because it allows
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reducing the cost and effort to develop SSRs for orphan plant species (Craft, Owens, & Ashley,
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2007). Our work indicated that caution should be applied when analyzing a complex food mixture
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because of the possibility to obtain within-range allelic profiles also from very different species.
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For mixed vegetables, the data suggest that the profile of the isolated product does not come
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from multiple sources. It is fair to add that zucchini was the only Cucurbit in this commercial
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product. Large differences were present in the cross-species transferability of the SSRs, implying
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that a preliminary screening of SSR markers is useful, if not necessary, when aiming at the genetic
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traceability of mixed products. Interestingly, the EST-SSR CUTC022867 did not yield nonspecific
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amplifications in non-Cucurbits. EST-SSRs are thought to be more transferable in closely related
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species (Varshney, Graner, & Sorrells, 2005), and the possibility that genic SSRs are less
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transferable in more distant taxonomic ranks (e.g. across genera) should be tested analyzing a
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larger panel of SSRs.
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The significance of non-human DNA typing for court use has advanced recently especially for
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animal species and products (Johnson, Wilson-Wilde, & Linacre, 2014; Linacre, et al., 2011). The
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ACCEPTED MANUSCRIPT relevance is much more limited for the plant sector (Iyengar, 2014), in spite of a wide range of
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applications (Barcaccia, et al., 2015; Corrado, 2016; Ferri, et al., 2015). While DNA typing cannot
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be considered anymore a novel approach, a limitation of its use in forensic plant genetics is
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represented by the strength of statistical interpretations (Sensabaugh & Kaye, 1998). A number of
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studies have identified the genetic makeup of plant-derived food products yet, hardly ever this
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information is accompanied by an estimate of a profile frequency (Craft, et al., 2007; Howard,
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Gilmore, Robertson, & Peakall, 2009). To this aim, the allele frequency information from our
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database was used to assess relative rarity of DNA profiles. This analysis underlined the
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importance of the number of loci under investigation. Although the SSR loci employed displayed
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large difference in their discriminating power, the accumulation curve gives reason to believe that
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the rarity of a full DNA profile frequency estimate in zucchini could be significantly lowered by
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adding more SSR loci. The shortage of statistical analysis for the interpretation of the rarity of
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genetic profile in horticultural crops does not allow an evaluation of the general probability values
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(Foreman & Evett, 2001). For cultivated plants, the random match probability represents the
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frequency of an SSR profile expected to occur in a population of cultivars, rather than individuals
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(Craft, et al., 2007). It is difficult to put forward an estimate of the cultivated zucchini varieties,
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mainly because varieties are registered as C. pepo (hence including marrow, pumpkin, winter
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squash as well as different horticultural types of summer squash). Based on the catalogues of main
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breeding companies, a sensible estimation of the number of zucchini varieties currently sold in
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Italy should be around 50-60, which suggests that the genetic match presented in this work cannot
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be attribute to chance alone. A main priority to strengthen the use of DNA testing of horticultural
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products in courts will be to establish international database of genetic profile. Further studies
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should also explore genetic differentiation and population substructure, which is likely to be
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present when comparing different horticultural groups of C. pepo.
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Acknowledgments
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The research activity was supported by the “Valorizzazione di produzioni ortive campane di
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eccellenza
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PON02_00395_3215002. MC was supported by a scholarship of the Regione Campania, POR
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Campania FSE 2014/2020, RIS: Salute, biotecnologie, agroalimentare.
strumenti
di
genomica
avanzata”
(GenHORT)
project,
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TABLES
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Table 1: Commercial products analysed and their main features. Product
Mechanical Treatment
Heat treatment
Fruits from the shelf (cylindrical and round) Frozen zucchini Mixed Vegetables (frozen) Chargrilled, in olive oil* Fried in oil (Caprese/Scapece)* *: pasteurised product
No
No
Yes (slices) Yes (dice) Yes (slices) Yes (slices)
No No Yes (grilled) Yes (fried)
No Yes No Yes
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Other vegetable ingredients No
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ACCEPTED MANUSCRIPT Table 2: Main genetic indices of the zucchini genotypes under investigations.
CMTp69
65-75
CMTp98
212-236
CMTp132
141-165
CMTp142
150-196
CMTp176
93-109
CMTp257
123-147
CMTp260
120-156
CUTC022867 124-139 Mean SE
PI Na 5
Ho PIC F 0.067 0.528 0.874 0.200
0.215
0.068
0.640
0.878
3
0.289
0.376
0.232
0.432
0.767
4
0.144
0.561
0.743
0.239
0.617
2
0.078
0.299
0.740
0.536
0.817
6
0.395
0.674
0.414
0.164
0.426
4
0.400
0.610
0.344
0.224
0.506
3
0.500
0.524
0.047
0.310
3.625 0.498
0.259 0.057
0.473 0.057
0.433 0.113
0.352 0.059
0.600
Legend: Na: number of alleles; Ho: Observed heterozygosity; He: Expected Herozygosity; F: Fixation index; PI: Probability of identity at a locus, MAF: major allele frequency.
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MAF 0.664
2
431 432 433
0.271
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Size range (bp)
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CMTp98
CMTp132
P. sativum
WR
NONSP
NOAMP
NOAMP
WR
WR
NONSP
NOAMP
D. carotae
WR
WR
NOAMP
NONSP
NOAMP
WR
NONSP
NOAMP
C. sativus
WR
NONSP
NOAMP
NOAMP
WR
NONSP
WR
WR
C. maxima
WR
NONSP
WR
NONSP
WR
WR
WR
WR
S. tuberosum
WR
WR
NONSP
NOAMP
NOAMP
NONSP
NONSP
NOAMP
A. graveolens
WR
WR
NONSP
WR
NOAMP
NONSP
NONSP
NOAMP
A. cepa
NOAMP
NONSP
NONSP
NOAMP
NOAMP
NOAMP
NONSP
NOAMP
B. oleracea
NOAMP
NONSP
NONSP
NOAMP
NOAMP
NONSP
NOAMP
NOAMP
WR
NOAMP
NOAMP
NOAMP
NONSP
NOAMP
NONSP
NOAMP
CMTp176
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CMTp142
CMTp257
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Table 3: Transferability of the SSRs in other plant species.
CMTp260
CUTC022867
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ACCEPTED MANUSCRIPT Table 4: Genotype frequency of the SSR profiles of the two identified processed food products. For each locus, the table reports the frequency of the alleles in the genotypes of the zucchini plant varieties under investigation (n=90) and the expected frequency of the genotype. For each locus, the table also reports the number of alleles and the observed genotype in our population over the theoretical number of genotypes possible [(n x (n+1))/2]. Identified Sample
Population
Allele I
Allele II
Expected
frequency
frequency
Genotype
Alleles
Observed/Possible
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genotypes
frequency 0.664
0.664
0.415
5
CMTp98
0.878
0.878
0.770
2
CMTp132
0.767
0.767
0.588
CMTP142
0.617
0.617
0.380
CMTp176
0.817
0.817
CMTp257
0.426
0.148
CMTp260
0.506
0.506
CUTC022867
0.600
0.333
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5/6
4
8/10
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2
3/3
0.126
6
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0.123
4
8/10
0.400
3
6/6
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ACCEPTED MANUSCRIPT Supplementary Table 1: Plant varieties under investigation and their main features Fruit general shape
Fruit colour (intensity)
Altea Anissa Asso Aurore Aymaran Black Beauty Cavili Coucourzelle De Nice à fruit rond Genovese Genovese Antico Giulia Gulliver Lanka Mikonos Milos Ontano Parthenon President Quine Rhodos Rigas Romanesco San Pasquale Sitos Syros Temprano de Argelia Vertre noir maraîchère Virginia Vitulia
cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical tapered cylindrical globular tapered cylindrical tapered cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical cylindrical tapered cylindrical cylindrical tapered cylindrical cylindrical
grey green (dark) green (medium) green (dark) green (very light) green (dark) green (very light) grey green (very light) green (very light) green (very light) green (light) green (medium) green (light) green (medium) green (medium) grey green (dark) green (medium) green (dark) green (medium) grey green (very light) grey green (light) green (dark) green (very light) green (dark) green (very light) green (light)
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Legend: H: hybrid; OP: open-pollinated variety.
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Type H H H H H OP H OP OP OP OP H H H H H H H H H H H OP OP H H OP OP OP H
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Variety
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Primer label (forward)
GCAGAGGAGAAGTGGGTTTG CTTTATCCGACCAAGCGTTC
(AAG)9
6-FAM
CMTp142
TCAACCAAGTGCCAATCTCA ACTGATCCACCGACTGATACG
(TC)12+5
VIC
CMTp257
CACGAAGATTTGATGGCCTTA GGATTGGGATGGTGAAGATG
(CGT)11
PET
CMTp260
CCCTAGACCCATCATAGTCG ACATTTGGTTACTTCCCCATT
(CAT)7
NED
CMTp69
ATACTTGCTCCCCAAGTTTA AAATAAAAAGACAACGTAATGGT
(TATT)4
VIC
CMTp176
CCTGGACTTCCACATCAGTT ACTACGTGTCTCTGCAGGAAG
(TC)12
6-FAM
CMTp132
CCATTTCCATTTCCATTTCA AGGTTAGAAACAGGGGGAATC
CUTC022867
TTGGACAATCTGAGGAAGTTGG TTGGACAATCTGAGGAAGTTGG
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CMTp98
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Primer sequences (5' to 3')
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core
Locus SSR
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(GAT)12
NED
(AGC)8
PET
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Primer sequence (5’ to 3’)
GC-EF1pepo-RVa
CACGAACAGCAAAACGACCC
GC-EF1pepo-RVb
CTTGGTCACCTTTGGCTCCC
GC-EF1pepo-FW
GTATTGCCACACCTCCCCAC
EF1A-FW
ATTCGAGAAGGAAGCTGCTG
EF1A-RV
TTGGTGGTCTCAAACTTCCAC
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Primer
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ACCEPTED MANUSCRIPT Supplementary Table 4: Descriptive statistics of the random match probability (RMP) values for an increasing number of SSR loci. The RMP values for the population under investigation were obtained considering the combinations (without repetition) from 1 to 8 SSR loci. Descriptive statistics were then obtained using the summary() function in R. IQR: Interquartile Range. . Number of SSR loci 2
3
4
5
6
7
8
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1 1.37E-03
6.77E-06
1.20E-07
0.00E+00
7.30E-09
4.26E-09
0.00E+00
2.19E-09
1st Quartile
1.23E-01
1.98E-02
3.32E-03
5.76E-04
1.04E-04
1.93E-05
3.78E-06
5.24E-07
Median
3.60E-01
7.97E-02
1.86E-02
4.05E-03
8.71E-04
1.85E-04
4.30E-05
8.11E-06
Mean
3.60E-01
1.28E-01
4.51E-02
1.49E-02
5.75E-03
2.08E-03
7.49E-04
2.67E-04
3rd Quartile
5.88E-01
2.08E-01
5.74E-02
1.67E-02
5.32E-03
1.53E-03
4.05E-04
1.11E-04
Maximum
1.00E+00
7.70E-01
5.14E-01
3.02E-01
1.25E-01
5.02E-02
1.91E-02
4.88E-03
IQR
4.65E-01
1.88E-01
5.41E-02
4.01E-04
1.10E-04
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1.51E-03
ACCEPTED MANUSCRIPT Figure legends Figure 1 Hierarchical clustering (UPGMA algorithm) of the C. pepo varieties based on Nei’s genetic distance. The dendrogram shows that the 30 cultivars analyzed were discriminated.
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Figure 2 Amplificability of DNA isolated from commercial products. (A) Scheme of the annealing of the primers to the Elongation Factor 1-alpha gene (not to scale). (B) PCR with the EF1A-FW and EF1A-RW primers; (B) PCR with the GC-EF1A-pepo-FW GC-EF1Apepo-RVb; (C) PCR with the GC-
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EF1Apepo-FW and GC-EF1Apepo-Rva. 1: 1 kb + DNA ladder (ThermoFisher); 2: water negative control; 3: positive control (leaf DNA); 4: cylindrical fruit; 5: mixed vegetables; 6: frozen zucchini; 7:
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chargrilled zucchini; 8: fried zucchini. See table 1 for a description of the commercial products. Figure 3 Agarose gel electrophoresis of PCR reactions with the CMTp260 primers using the DNA isolated from different commercial food products. 1: 1 kb DNA ladder (Thermo Fisher); 2: water
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negative control; 3: positive control (leaf DNA); 4: cylindrical fruit; 5: rounded fruit; 6: mixed vegetables; 7: frozen zucchini; 8: chargrilled zucchini; 9: fried zucchini. See table 1 for a description of the commercial products
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Figure 4 Box and whisker plot of the random match probability (RMP) values of the genotypes
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Supplementary Figure 1 Examples of electrophoretic profiles obtained in the SSR transferability test. Panel A and B are an example electropherograms classified as «no amplification» (A: CMTp69 amplification of cabbage DNA; B: CMTp69 amplification of onion DNA). Panel C and D are an example of electropherograms classified as «nonspecific amplification» (C: CMTp98 amplification 31
ACCEPTED MANUSCRIPT of pea DNA; D: CMTp98 amplification of pumpkin DNA). Panel E and F are an example of electropherogram classified as “within range” (E: CMTp69 amplification of cucumber DNA; F: CMTP69 amplification of pumpkin DNA). Panel G and H are an example of electropherograms obtained with zucchini DNA using the CMTp69 primers. In each panel, the allelic bar is present at
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Highlights
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We used SSR markers for DNA typing of zucchini varieties in commercial products encompassing different levels of food processing We assessed SSR transferability in closely related Cucurbits and other horticultural species
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DNA typing identified the zucchini varieties in two commercial products
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We provide indications for the statistical interpretation of the observed genetic
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• The identified matches cannot be attributed to chance alone