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A reliable assay for the detection of soft wheat adulteration in Italian pasta is
based on the use of new DNA molecular markers capable of discriminating
between Triticum aestivum and Triticum durum
Anna Paola Casazza a, *, Caterina Morcia b, Elena Ponzoni a, Floriana Gavazzi a, Stefano Benedettelli c,
Diego Breviario a
a
b
c
Istituto di Biologia e Biotecnologia Agraria e IBBA, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milan, Italy
Genomics Research Centre, CRA, Via S. Protaso 302, 29017 Fiorenzuola dArda (PC), Italy
Universit degli Studi di Firenze, Dipartimento di Scienze delle Produzioni Vegetali, del Suolo e dellAmbiente Agroforestale, Piazzale delle Cascine 18, 50144 Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2012
Received in revised form
24 August 2012
Accepted 27 August 2012
Italian pasta must be prepared using exclusively durum wheat. According to current Italian rules, only
a maximum of 3% Triticum aestivum is allowed to account for cross-contamination that may occur during
the agricultural process. Efcient methods for the detection of accidental or intentional contamination of
common wheat to durum wheat products are therefore required. This article describes a novel approach
for the detection and quantication of soft wheat adulteration in whole grain durum ours and dried
pasta. The assay relies on the presence of intron-specic DNA length polymorphisms in the plant
b-tubulin gene family, which can be highlighted through the PCR-based TBP (Tubulin-Based Polymorphism) method. In wheat, the TBP method produces species-specic amplication products, which
can be either directly used as new DNA molecular markers capable of discriminating between T. aestivum
and Triticum durum or analyzed at the sequence level for the design of species-specic probes. The latter
approach allowed the development of new sequence-specic targets that can be exploited in RT-PCR
assays for a rapid and accurate quantication of soft wheat adulteration in durum wheat pasta.
2012 Elsevier Ltd. All rights reserved.
Keywords:
b-Tubulin introns
DNA molecular markers
Soft and durum wheat
Pasta adulteration
1. Introduction
Pasta is the Italian national dish, not only because it is the staple
daily food for the majority of the population (in 2011 the annual per
capita consumption of dry pasta amounts to 26 kg) but also because
it is part of Italys identity and self-image. Italy is the world leader in
pasta production covering a market share of 26% with 3.25 million
tons produced in 2010, half of which sold in Italy and the remaining
half exported either to other European countries (70%) or outside
Europe, mainly to the USA and Japan (ISTAT, 2012). Italian pasta is
normally made from 100% durum wheat (Triticum durum Desf.,
tetraploid AABB) semolina and is considered a product of superior
quality with respect to those containing common wheat (Triticum
aestivum L., hexaploid AABBDD) or just made from soft wheat. The
734
Eureka [11], Frassineto [12], Gentil Bianco [13], Gentil Rosso aristato
[14], Gentil Rosso mutico [15], Inallettabile [16], Marzuolo dAqui 3
[17], Marzuolo Val Pusteria [18], Mieti [19], Palesio [20], Postarello
[21], San Francisco [22], Sieve [23], Terricchio [24], Verna [25]) and
seven durum wheat varieties (cv. Ancomarzio [26], Arcangelo [27],
Claudio [28], Iride [29], Triticum turanicum [30], Levante [31], Urria
[32]) were obtained from the University of Florence. Soft wheat cv.
Aubusson [A] and durum wheat cv. Claudio [C] kernels were
provided by CRA, Genomic Research Centre, Fiorenzuola dArda.
The selected wheat panel comprises modern varieties commonly
cultivated in Italy, as well as old ones grown in the early 20th
century, to have a representative sample of the wheat genome.
Besides, the old varieties, in the last period, are interesting for their
functional quality and for their yield capacity in organic growing
(Dinelli et al., 2009).
Fourteen commercial samples of Italian pasta [1/14] manufactured by eleven different brands (indicated with a lower-case
letter following the sample number) were purchased from
various Italian food shops: 100% durum wheat pasta dried at high
(Vermicellini [2b], Gnocchi [3b]) or low temperature (Mezze
Maniche Rigate [1a], Toffarelle [4c], Mezzi Rigatoni [5d], Farfalle
[6e], Gnocchi Sardi [7f], Linguine [8g]); 100% durum wheat egg
pasta (Maltagliati [9b], Lasagne [10d] and [11h], Tagliatelle [12i], all
containing 19-19, 36% fresh hen-eggs Category A); 100% soft wheat
pasta for infants (Stelline [13l]) and 100% emmer wheat (Triticum
dicoccum AABB) pasta (Ditalini [14m]).
Wheat kernels and pasta specimens were reduced respectively
to whole grain our or ne powder using the vibration mill TissueLyser (Qiagen) with stainless steel grinding jars (Retsch). For the
preparation of dened our blends, the durum wheat cultivar
Ancomarzio or Claudio and the common wheat cultivar Bilancia or
Aubusson were used as indicated. Whole grain durum our was
blended with different percentages of common wheat our and
genomic DNA (gDNA) was extracted using the GenElute Plant
Genomic DNA Miniprep kit (SigmaeAldrich). gDNA from pasta
specimens was extracted using the Speedtools Food DNA Extraction
kit (Biotools). DNA quality was assessed by agarose gel electrophoresis and EtBr staining, while DNA quantication was performed spectroscopically using the Qubit (1.0) Fluorometer
(Invitrogen). For each sample, gDNA extraction was repeated on the
same material at least three times.
2.2. DNA fragments isolation, cloning and sequencing
TBP amplication fragments, electrophoretically separated on
polyacrylamide gels and visualized by silver staining as described
previously (Breviario et al., 2007), were excised from the gel matrix
using a sharp blade and immediately rehydrated with deionized
water in order to collect the gel slide. DNA was directly extracted
from the gel matrix with the QIAEII Gel Extraction Kit (Qiagen)
and cloned using the TOPO TA-Cloning Kit (Invitrogen). Positive
transformants were selected by colony PCR (see Section 2.3) and
plasmid DNA isolated using the Nucleospin Plasmid Kit
(Macherey-Nagel).
Sequencing of cloned fragments was performed by the ABI
PRISM 310 Genetic Analyzer, using the BigDye Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems). Nucleotide sequences
have been bioinformatically analyzed by performing BLAST (Basic
Local Alignment Search Tool, Altschul et al., 1997) searches on NCBI
(http://www.ncbi.nlm.nih.gov/).
735
for the PCR reaction (10 ml total volume). Reaction mixtures contained: 0.5 mM each primer (M13-Reverse and T7-Promoter,
universal primers), 0.15 mM each dNTP, 2.5 mM Mg2, 0.6 U Taq
DNA Polymerase (Eppendorf) and corresponding buffer. PCR
program: 2 min initial denaturation at 94 C followed by 25
amplication cycles (94 C for 1 min, 55 C for 1 min, 72 C for
1 min) and nal extension step for 7 min at 72 C.
PCR products were separated by agarose gel electrophoresis and
visualized by EtBr staining. DNA molecular weight markers
(SHARPMASS50, pUC Mix and GeneRuler 1 kb Plus DNA
Ladders) were purchased from EuroClone and Fermentas,
respectively.
The method that leads from TBP to the isolation of plant specic
diagnostic probes is under pending patent protection (No.: PCT/
IB2011/000146 deposited on the 31st of January 2011).
2.4. Real-time PCR and data analysis
Reactions for the RT-PCR using SYBR Green detection consisted of
12.5 ml of SYBR Green PCR Master Mix (Applied Biosystems), 900 nM
forward and reverse primers, 100 ng of DNA template and water to
25 ml. All PCR samples and controls were prepared in duplicate. The
Fig. 1. TBP amplication proles (1st intron, hTBP and 2nd intron approach) of ve selected wheat varieties. T. aestivum cultivars: [7] Bilancia; [14] Gentil Rosso aristato and [17]
Marzuolo dAqui 3. T. durum cultivars: [26] Ancomarzio and [28] Claudio. Diagnostic bands and groups of bands (numbered in ascending order from low to high molecular weight
and labeled with A or B for 1st and 2nd intron amplication fragments, respectively) are highlighted by boxes. The insert on the left side shows a magnication of the boxed
portion of the 1st intron prole. m, molecular weight ladder (fragments lengths are indicated in base pair).
736
Fig. 2. TBP 1st and 2nd intron analysis of gDNA extracted from T. durum (cv. Ancomarzio [26]), T. aestivum (cv. Bilancia [7]) and blends of durum wheat whole grain our containing
different amounts of soft wheat (1, 3, 6, 12, 24 and 50% w/w). Diagnostic bands A1 and B4 are highlighted by arrows. Asterisks indicate samples where bands A1 and B4 are still
clearly detectable, i.e. the detection limit of the approach (see Section 3.2). m, molecular weight ladder (fragments lengths are indicated in base pair).
737
Fig. 3. TBP 1st and 2nd intron analysis of gDNA extracted from selected 100% durum
wheat pasta dried at low [1a] or high temperature [2b and 3b], egg pasta [10d] and
100% soft wheat pasta [13l] (more details on pasta specimens can be found in Section
2.1). For reference, the amplication patterns of T. aestivum (cv. Bilancia [7]) and
T. durum (cv. Ancomarzio [26]) are also shown. T. aestivum diagnostic bands A1 and B4
are highlighted by arrows.
738
Fig. 4. Sequence alignment of the b-tubulin rst introns corresponding to the isolated bands A7, A8 and A9. Positions of the T. aestivum specic primers are underlined (Tae_109F
and Tae_109R) or highlighted in gray (4FDf and Tae_109a-r1), respectively.
On the basis of these evidences, the bands B1, B2, B3, B4 and A1
(see Fig. 1) have been discarded from the pool of putatively useful
fragments and further investigation was restricted to the 1st intron
amplied fragments highlighted in the insert of Fig. 1. Bands A4, A5
and A6 all correspond to the rst introns of b-tubulin isoform 2
(GenBank ID: U76745). Band A4 is present in both T. aestivum and
T. durum, while A5 and A6 are specic for T. aestivum and T. durum,
respectively. Bands A7, A8 and A9 correspond to the rst introns of
b-tubulin isoform 5, with both A7 and A8 common to T. aestivum
and T. durum, while A9 was specic for T. aestivum. In the T. aestivum
varieties Bilancia [7] and Mieti [19], the band A8 has a short
insertion of 15 nucleotides and therefore migrates very close to A9
(see Fig. 1 and Fig. A.1, Supplementary data). After an extensive
work of sequence analysis and comparison, a few nucleotide
stretches which are highly specic for T. durum and for T. aestivum
could be identied. Fig. 4 shows the alignment obtained for the
intron sequences derived from bands A7, A8 and A9. The positions
where T. aestivum specic primers (see Section 2.3) have been
designed are underlined (Tae_109F and Tae_109R) or highlighted in
gray (4FDf and Tae_109a-r1), respectively. The same procedure has
been followed for bands A4, A5 and A6 (sequence alignment is
shown in Fig. A.4, Supplementary data) in order to design T. durum
specic primers (Tdu_106F and Tdu_106R, see Section 2.3).
739
Table 1
Mean Cts obtained from real-time PCR analysis of pasta specimens using the
T. aestivum 4FDf/Tae_109a-r1 specic probe and quantication of soft wheat content
(% w/w).
Samplea
Mean Ct
1a
2b
3b
4c
5d
6e
7f
8g
9b
10d
11h
12i
13l
14m
NTCb
32.07
28.05
28.24
32.49
30.66
28.01
30.60
29.21
32.87
29.89
32.30
30.41
23.73
28.07
0
0.4
5.9
5.2
0.3
1.0
6.1
1.0
2.7
0.2
1.7
0.3
1.2
100
5.9
0
a
b
740
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