Traceability of the local cultivar ‘Caaveiro’ in flour mixtures used to produce Galician bread by simple sequence repeats and droplet digital polymerase chain reaction technology

Original article Traceability of the local cultivar ‘Caaveiro’ in flour mixtures used to produce Galician bread by simple sequence repeats and droplet digital polymerase chain reaction technology Ana Mar ıa Ramos-Cabrer, 1 * † Nerea Fern andez-Canto, 1,2,3† Fernando Almeida-Garc ıa, 4 Ana Gorostidi, 5 Matilde Lombardero-Fern andez, 2 Mar ıa  Angeles Romero-Rodr ıguez 3 & Santiago Pereira-Lorenzo 1 1 Department of Crop Production and Engineering Projects, Universidade de Santiago de Compostela, Campus de Lugo, 27002, Lugo, Spain 2 Department of Anatomy, Animal Production and Clinical Veterinary Sciences, Universidade de Santiago de Compostela, Campus de Lugo, 27002, Lugo, Spain 3 Area of Food Technology, Department of Analytical Chemistry and Food Science, Faculty of Science, University of Santiago de Compostela, Campus de Lugo, 27002, Lugo, Spain 4 Sementares S.L. Illa de Tambo 5, 15005, Poio, Pontevedra, Spain 5 Biodonostia Health Research Institute, Genomic Platform, 20014, San Sebastian, Spain (Received 8 June 2022; Accepted in revised form 24 August 2022) Summary The analysis of simple sequence repeats (SSRs) and of bulk ground samples by droplet digital polymerase chain reaction (ddPCR) was investigated as an alternative to individual kernel testing for assessing the presence of local cultivars in common wheat (Triticum aestivum L.) varietal blends. The recent Protected Geographical Indication (PGI) of ‘Pan Galego’ (Galician bread) requires that the flour comprise a minimum 25% local wheat cultivars. As a test for compliance with this minimum level, wheat flours were prepared by mixing commercial flours with 0%, 5%, 20%, 25% and 30% ‘Caaveiro’ and 100% ‘Caaveiro’ and 100% commercial flours were used as controls. A second analysis was performed with a second set of wheat flours with 5% and 25% ‘Caaveiro’. These were mixed with two different commercial flours to assess the potential ability of five SSRs to identify the percentage of ‘Caaveiro’, constituting the first reference of the use of SSRs in the traceability of specific autochthonous cultivars in flour blends. ddPCR using the QX200 system platform was used to the targeted proportions across the simulated range with two out of five SSRs, indicating that they can be used in the traceability of ‘Caaveiro’ in mixed flours and breads. Keywords autochthonous wheat, ddPCR, flour blends, microsatellites, traceability. Introduction In Spain, six million ha of cereals are cultivated producing 25.4 million t of cereal grain, of which 7.1 t are wheat (MAPA, 2020). Commercial flours used in the Galician are currently produced by mixing commercial wheat cultivars, depending on the year, with the Galician cultivar ‘Caaveiro’ to produce Galician bread (C^ amara-Salim et al., 2020). The cultivar ‘Caaveiro’ is a soft winter wheat, which imparts a distinct flavour and taste to bread. The recently created Protected Geographical Indication (PGI) Pan Galego (UE, 2019a) commits to the inclusion of a minimum of 25% Galician wheat. Nowadays, there is no way to certify that this minimum is accomplished by flour mills or bakeries in the production of Galician bread under the auspices of the PGI. ‘Pan Galego’/‘Pan Gallego’ (PGI) (2019/C 243/03) (UE, 2019b) ‘is a crusty bread (the hardness of the crust varying depending on the format) with a soft and springy crumb dotted with a great many irregular air pockets. It is made by artisan bakeries using common wheat flour, some of which is made from native Galician wheat varieties and ecotypes (those commonly known as “trigo pa ıs” (autochthonous wheat) or “trigo gallego” (Galician wheat)) grown in the Autonomous Community itself. The bread is made using a sourdough starter (making up at least 15% of the total flour weight) and a large amount of water (at least 75 L per 100 kg of flour), with long rising (minimum 3 h) and baking times. Ovens floored with stone or other refractory materials are used for baking.’ *Correspondent: E-mail: [email protected] †Both co-authors equally contributed to the manuscript. International Journal of Food Science and Technology 2022 doi:10.1111/ijfs.16048 Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1 ‘Processes are slow as a result of using sourdough starters for fermentation, resulting in a soft and springy crumb, lots of air pockets, an intense flavour and a touch of acidity.’ (UE, 2019b) ‘The use of flour made from local wheat ecotypes, mixed with the expert eye of Galicia’s bakers –with flours from outside Galicia, is another typical feature of this bread that contributes to its specificity. The qualities conferred on ‘Pan Gallego’ by most of the local wheat ecotypes (native wheats) derive from their high protein content and extensibility, plus the fact that the gluten they contain is ideal for bread-making. However, as these are weak or medium-strong flours, local bakers have traditionally combined them in varying proportions with stronger, more elastic flours, usually imported from Castile. The use of these native flours also affects the colour of the crumb, making it darker than that found in other wheat breads, and, according to the artisan bakers, gives the bread more intense aromas and flavours. On this last point, it has been demonstrated that the bread’s aromas derive from certain components of the flour, which is why using these wheats would affect its organoleptic characteristics.’ (UE, 2019b). Droplet digital polymerase chain reaction (ddPCR) is considered the next generation of PCR (Hindson et al., 2011). It is performed by partitioning the reaction mixture into an emulsion of minuscule droplets and treating each one as an endpoint reaction. The proportion of PCR outcomes defines the absolute quantification of a specific sequence. ddPCR allows absolute quantitation of nucleic acids in a sample without the need for standards. It is less affected by inhibitors due to dilution by partitioning the reactions into thousands of nanodroplets and the insignificance of primer efficiencies. A single PCR mixture is divided into over 20 000 water-in-oil droplets as a base for PCR amplification of single template molecules. A DNA binding dye is included in the Super-Mix in order to differentiate positive droplets (amplification of the target molecule) from negative droplets (no target molecule present) by using automated droplet flow cytometry. Finally, the number of target molecules in the mixture is estimated from the positive and negative droplets by using Poisson statistics. Primer-probe systems for real-time PCR and droplet digital PCR (ddPCR) have been used previously for the detection of cereal species (Schulze et al., 2021). ddPCR was effective in detecting and quantifying species of rye and common wheat in mixtures, but the results depended on the DNA content, and the ploidy of cereal species and were also influenced by comminution. ddPCR has been used to perform quantitative analysis of bulk samples rather than individual seeds in varietal blends of wheat (Perry & Lee, 2017). Resistance to orange wheat blossom midge Sitodiplosis mosellana (G ehin), a relevant insect pest in wheat, has been identified under the control of the single gene Sm1, which has been incorporated into several wheat varieties, such as ‘Unity’. However, the breakdown of resistance of this single gene through evolution of virulence by the wheat midge was expected. Hence, an interspersed refuge strategy was implemented by mixing midge-tolerant wheat (MTW) seeds with a midgesusceptible cultivar in a 9:1 ratio. This blend was submitted to verification testing to ensure that the blend ratio remained within the acceptable range. Tests were performed on individual seeds, and up to 1000 seeds were tested per sample. ddPCR was used successfully to verify that the coefficients of variation had been achieved, as they were generally below 5%. In another study, a duplex chip digital PCR (cdPCR) assay was used to quantify the 3% of contamination by wheat in the pasta production chain, from raw materials to the final products based on durum wheat (Triticum durum) (Morcia et al., 2020). Although studies based on SSRs have been conducted to identify wheat cultivars (Ruiz et al., 2012) and on ddPCR to quantify the presence of a specific cultivar or species in mixtures of seeds (Perry & Lee, 2017) or flours (Morcia et al., 2020), there have been no previous studies on the use of SSRs to identify Galician cultivars or to quantify their presence in blends at the minimum 25% level required by the PGI ‘Galician bread’ and no studies on the use of ddPCR for the same purpose. The objective of this study was to test eighteen simple sequence repeats (SSRs) for their usefulness in identifying wheat cultivars in the traceability of the autochthonous cultivar ‘Caaveiro’ in the flours used for the production of ‘Galician bread’ under the auspices of the PGI ‘Pan Galego’/‘Pan Gallego’ and to test five out of the eighteen SSRs by ddPCR. This is the first study to identify and quantify the use of Galician wheats in the production of ‘Galician bread’ by using SSRs and ddPCR. Material and methods Materials We evaluated two Galician cultivars, ‘Caaveiro’ and ‘Callobre’ one Galician ecotype ‘Carral’; and fourteen commercial cultivars: ‘Enebro’, ‘Nogal’, ‘Rebelde”, ‘Ovalo’,’Valbuena’, ‘Montecarlo’, ‘Algoritmo’, ‘Basilio’, ‘Tocayo’, ‘Atomo’, ‘Sensos’, ‘Acorazado’, ‘Alhambra’, and ‘Radia’. Additionally, we evaluated three commercial flours for bread, named type A (‘Castilian strong force’ used by Da Cunha group), B (‘Carrefour’ brand) and C (‘Eroski bio’ brand), all of them blends of commercial wheat cultivars. Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al.2 First trial flour with 100% local cultivar ‘Caaveiro’ and commercial flour (type A) with 0%, 5%, 20%, 25% and 30% of ‘Caaveiro’ flour. Second trial flour with 100% local cultivar ‘Caaveiro’, 95% commercial flour (type B) with 5% ‘Caaveiro’ flour, 75% commercial flour (type B) with 25% ‘Caaveiro’ flour, and 75% commercial flour (type C) with 25% ‘Caaveiro’ flour. DNA extraction A 0.5 g sample of flour or grain was used for DNA extraction with the E.Z.N.A.ÒPlant DNA Kit (OMEGA Bio-Tek Inc., Norcross, GA, USA) and DNeasyÒPlant Mini Kit (Qiagen, Hilden, Germany). SSRs DNA integrity and quality were assessed by gel electrophoresis and visualisation of the previously stained samples. DNA samples were quantified using the Invitrogen Qubit 4 Fluorometer with the Qubit dsDNA HS (High Sensitivity) Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Five out of eighteen SSRs adapted from durum wheat (Triticum turgidum L.) (Ruiz et al., 2012) were selected from a previous analysis (unpublished) with soft wheat samples to identify specific markers for autochthonous wheat cultivars and for ddPCR (Table 1). A combination of high Polymorphism Information Content (PIC) values, a high number of alleles and location in different Linkage Group (LG) was sought. PCR Polymerase chain reaction amplifications were performed in a final volume of 20 lL containing 12 mM PCR buffer pH 8.3, 2.0 mM MgCl2, 0.31 mM of each nucleotide (dNTPs), 1 lM of the primer Forward, 1lM from the primer Reverse, 1 ld of Taq DNA polymerase and 50 ng of DNA, completed with distilled water up to 20 lL. With these microsatellite primers, the samples were amplified in a thermocycler (PTC-100 from M.J. Research, INC or GeneAmp PCR System 2700) based on the protocol of Reija (Reija Abelairas, 2017). Polymerase chain reaction amplification was performed following the thermocycling program described below, taking into account that the ‘annealing’ temperature selected was optimal for the SSRs in each of the multiplexes: 94 °C for 2 min, followed by thirty cycles of 94 °C for 35 s, the annealing temperature for 1 min 30 s, 72 °C for 1 min 30 s and a final extension of 5 min at 72 °C. Multiplexes, as expected after set-up, showed amplification products for all SSRs (Table 1). Multiplexes, as expected after set-up, showed amplification products for all SSRs (Table 1). ddPCR Previously designed primers were used to perform ddPCR experiments with EvaGreen Dye (Table 1). Each reaction contained 19mix (QX200 EvaGreen Digital PCR Supermix, Bio-Rad Laboratories, Hercules, CA, U.S.A.), 200 nM of each forward and reverse primer solution, and 2.03 lL of DNA template, resulting in a final volume of 20.3 lL. Droplets were generated with the QX200 droplet generator (Bio-Rad Laboratories), and PCR was performed in a C1000 touch thermal cycler. A thermal-gradient PCR experiment was conducted to establish the optimal annealing temperature for the primers. The temperatures tested were 55.0, 56.4, 58.5, 60.9, 62.9, 64.3, and 65 °C. The negative control (NDT), water, was run at 54.2 °C. PCR thermal cycling conditions were as follows: initial denaturation stage of 95 °C for 10 min, followed by forty cycles of denaturation at 94 °C for 30 s, optimal annealing temperature for 60 s, and a final incubation of 10 min at 98 °C. PCR plates were transferred into a QX200 droplet reader (Bio-Rad), and readings were analysed using QuantaSoft software (Bio-Rad). The annealing temperature for PCR amplification was set at 59 °C for the five assays (Fig. 1). First trial Serial dilutions (1:2; D1 and D2) (Table 2) of the stock DNA were prepared for quantification purposes by ddPCR for each of the six samples including 0, 5, 20, 25, 30, and 100% autochthonous ‘Caaveiro’ flour with an industrial flour used in Galicia for bread (Type A). A 2.03 lL volume of each dilution for each sample was added to each one of the five assays (Fig. 2). Second trial The same amplification conditions were employed as described above. Four new samples (S7, 100% local cultivar ‘Caaveiro’; S8, 95% commercial flour type B with 5% ‘Caaveiro’ flour; S9, 75% commercial flour type B with 25% ‘Caaveiro’ flour, and S10, 75% commercial flour type C with 25% ‘Caaveiro’ flour) were diluted to 20 ng/lL (D3 for Xgwm0312 and Xgwm0156), 10 ng/lL (D4 for Xgwm0099 and Xgwm0332) and 5 ng/lL (D5 for Xgwm0570) and the working concentration was chosen from previous trial criteria. Reactions were performed in duplicate for each of the five assays (Fig. 3). Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al. 3 Table 1 SSRs evaluated in this study in Galician flours and bread. Adapted from Ruiz et al. (2012) SSRs LG Multiplex Fluoroc hrome Number of alleles (unique alleles) PIC Primer forward Primer reverse Repetition motive Tªof annealing Opata wheat (bp) Synthetic wheat (bp) Xgwm0186 5A 1 VIC 28 (15) 0.77 GCA GAG CCT GGT TCA AAA AG CGC CTC TAG CGA GAG CTA TG 50(GA)26 60°132 106 Xgwm0234 5B 1 6-FAM 19 (9) 0.75 GAG TCC TGA TGT GAA GCT GTT G CTC ATT GGG GTG TGT ACG TG (CT)16(CA)20 55°250 229 Xgwm0312*2A 1 6-FAM 26 (4) 0.85 ATC GCA TGA TGC ACG TAG AG ACA TGC ATG CCT ACC TAA TGG (GA)37 60°216 219 Xgwm0577 7B 1 NED 41 (15) 0.77 ATG GCA TAA TTT GGT GAA ATT G TGT TTC AAG CCC AAC TTC TAT T (CA)14(TA)6 55°164 155 Xgwm0099*1A 2 6-FAM 11 (3) 0.94 AAG ATG GAC GTA TGC ATC ACA GCC ATA TTT GAT GAC GCA TA (CA)21 60°117 120 Xgwm0148 2B 2 NED 12 (4) 0.8 GTG AGG CAG CAA GAG AGA AA CAA AGC TTG ACT CAG ACC AAA (CA)22 60°165 167 Xgwm0155 3A 2 VIC 16 (3) 0.86 CAA TCA TTT CCC CCT CCC AAT CAT TGG AAA TCC ATA TGC C (CT)19 60°143 127 Xgwm0156*5A 2 VIC 20 (4) 0.91 CCA ACC GTG CTA TTA GTC ATT C CAA TGC AGG CCC TCC TAA C (GT)14 60°300 279 Xgwm0332*7A 2 6-FAM 20 (4) 0.86 AGC CAG CAA GTC ACC AAA AC AGT GCT GGA AAG AGT AGT GAA GC (GA)36 60°290 211 Xgwm0570*6A 2 PET 15 (3) 0.9 TCG CCT TTT ACA GTC GGC ATG GGT AGC TGA GAG CCA AA (CT)14(GT)18 60°149 143 Xgwm0060 7A 3 NED 19 (5) 0.8 TGT CCT ACA CGG ACC ACG T GCA TTG ACA GAT GCA CAC G (CA)30 60°190 224 Xgwm0088 6B 3 6-FAM 17 (4) 0.72 CAC TAC AAC TAT GCG CTC GC TCC ATT GGC TTC TCT CTC AA (GT)18TT(GA) 4 60°162 – Xgwm0389 3B 3 VIC 14 (3) 0.75 ATC ATG TCG ATC TCC TTG ACG TGC CAT GCA CAT TAG CAG AT (CT)14(GT)16 60°117 128 Xgwm0513 4B 3 PET 4 (0) 0.79 ATC CGT AGC ACC TAC TGG TCA GGT CTG TTC ATG CCA CAT TG (CA)12 60°152 146 BARC155 5A 4 6-FAM 11 (4) 0.67 GCG AGT ATT GAC GTC TTA TTT TTG AA GCG TCA TGA ATT CTA ACA ATG TGC ATA (TAA)10 50°182 BARC80 1B 4 NED 17 (6) 0.88 GC GAA TTA GCA TCT GCA TCT GTT TGA G CGG TCA ACC AAC TAC TGC ACA AC 51 112 103 WMC468 4A 4 VIC 5 (1) 0.5 AGC TGG GTT AAT AAC AGA GGA T CAC ATA ACT GTC CAC TCC TTT C 51° Xgwm0002 3A 4 PET 4 (0) 0.36 CTG CAA GCC TGT GAT CAA CT CAT TCT CAA ATG ATC GAA CA (CA)18 50°128 130 *SSRs selected from previous analysis (unpublished) with soft wheat samples to identify specific markers for autochthonous wheat cultivars for ddPCR. Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al.4 Results SSRs and alleles Capacity to distinguish of flours and their percentages Selected SSRs were able to distinguish the autochthonous wheat ‘Caaveiro’ from other cultivars, showing specific alleles for Xgwm0312, Xgwm0332, Xgwm0099, Xgwm0570, Xgwm0060 and WMC468 (Table 3). Moreover, commercial flours used for bread also showed specific profiles, a combination of the cultivars mixed every year (Table 3). Once the monovarietal flours (‘Caaveiro’) and their mixtures were subject to PCR amplification, it was found that they had not undergone changes in DNA quality due to milling (Figure S1), and the PCR products obtained were of very good quality; therefore, PCR is a very useful tool for traceability studies and the detection of fraud in flours. On the other hand, not all SSRs were useful for this purpose (Table 3), since some of them had common alleles in monovarietal flours and, therefore, flour mixtures contained matching alleles. An example of this was Xgwm0148, for which ‘Caaveiro’ flour presented allele 144, and type A flour presented alleles 144 and 160. Therefore, mixtures of both flours presented alleles 144 and 160, but this result does not discriminate a pure type A flour from one mixed with ‘Caaveiro’, since both would present the common allele 144 (Figure S2). Therefore, the microsatellites Xgwm0148, Xgwm0155, Xgwm0186, Xgwm0234, Xgwm0088, Xgwm0513, Xgwm0389, Xgwm0577, BARC155, BARC80 and Xgwm0002 were not useful for this purpose (Table 3). On the other hand, the SSRs Wgwm0156, Wgwm0312, Wgwm0332, Wgwm0099, Wgwm0570, Xgwm0060 and WMC468 were useful in this differentiation (Table 3). In the case of the microsatellites Wgwm0312, Wgwm0099 and Xgwm0060, the differentiation capacity was larger (indicated in Table 3as ‘++’), because the alleles are not repeated in monovarietal flours. For example, in the case of locus Xgwm0312 (Figure S3), ‘Caaveiro’ presented alleles 250 and 252, while type A flour showed alleles 192, 194, 208, 214, 222 and 230; hence, alleles 250 and 252 were not repeated. Thus, when the two mixtures are combined, it should be noted that the alleles of both varieties appear in proportion to the amount of each flour type and do not overlap. However, Wgwm0156, Wgwm0332, WMC468 and Wgwm0570 (Table 3, indicated with ‘+’, Figure S4) predicted the presence or absence of flours depending on their origin, but the degree of precision was lower, since there are one or more alleles in common between the monovarietal ‘Caaveiro’ and type A flours. This case is depicted in Figure S4, where the 190 allele is repeated in both the ‘Caaveiro’ and type A flours. Consequently, this allele has a low discrimination capacity, as it will always appear, and its peak height will not indicate the percentage of each variety. Once the percentage composition of all the mixtures had been analysed, it was possible to quantify the proportion of wheat in the blend with a minimum level of detection of 5% (Figure S5) by the comparison of the discriminant allelic profiles according to Figure 1 Temperature amplification gradient with the sample S1 and the five SSRs (from left to the right, Xgwm0312, Xgwm0099, Xgwm0156, Xgwm0332, Xgwm0570) from left to right: 65; 64.3; 62.9; 58.5; 56.4; 55 and 54.2 °C. Table 2 Serial dilutions (1:2; named D1 and D2) of the stock DNA for each of the six samples prepared for quantification purposes by ddPCR Sample % of ‘Caaveiro’ flour mixed with commercial flour (type A) Dilutions ng/lL S1 0 D1 37.50 0 D2 9.38 S2 5 D1 20.20 5 D2 5.05 S3 20 D1 24.30 20 D2 6.08 S4 25 D1 17.60 25 D2 4.40 S5 30 D1 27.10 30 D2 6.78 S6 100 D1 25.20 100 D2 6.30 Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al. 5 the proportion of ‘Caaveiro’ and type A flours. The maximum peak heights of the specific alleles in 100% type A flour (192, 194, 208, 214, 222 and 230) are presented in Figure S5a, and those in 100% ‘Caaveiro’ (250 and 252) in Figure S5b. The height of the allele peak decreased as the proportion of that allele in the mixture was reduced (Figure S5c, d, e, f, g, h, i and j). Because the amount of ‘Caaveiro’ in the mixture increased as that of type A flour decreased, the peaks corresponding to alleles specific to ‘Caaveiro’ increased in height, while those specific to type A flour gradually decreased (on the left side of the figure are the alleles representing type A flour, and on the right are those representing ‘Caaveiro’). The peak heights began to equalise when type A flour constituted 60% of the mixture (Figure S5k) and were fully balanced in the 50:50 mixture (Figure S5l). From this value, it is clearly seen how the peak heights of alleles specific to type A flour decreased, while those of alleles specific to ‘Caaveiro’ increased (Figure S5m, n, ~ n, o). When the percentage of ‘Caaveiro’ rose to 75% (Figure S5p), all the alleles of type A flour were present, but their peaks were very low. In the 85% ‘Caaveiro’ mixture, the alleles of type A flour were more weakly amplified, but all of them were detectable (Figure S5q, r, s, t). The quantification of ‘Caaveiro’ in flour blends containing 100%, 75%, 50% and 25% ‘Caaveiro’ and 100% type A flour could also be performed in the same amplification (Fig. 4), specifically using alleles 250 and 252. The quantification of ‘Caaveiro’ in flour blends can be also determined in the same amplification (Fig. 4) when we represent flours with 100% ‘Caaveiro’, 75% ‘Caaveiro’, 50% ‘Caaveiro’, 25% ‘Caaveiro’ and 100% type A flour, specifically for alleles 250 and 252. ddPCR Droplet digital polymerase chain reaction amplifications of Xgwm0312 and Xgwm0099 showed highly significant R-squared values in the blends tested (Table 4, Figs 5and 6). Xgwm0312 showed R 2 over 0.8 in both Figure 3 From left to right, amplification of samples S7, 100% local cultivar ‘Caaveiro’; S8, 95% commercial flour (type B) with 5% ‘Caaveiro’ flour; S9, 75% commercial flour (type B) with 25% ‘Caaveiro’ flour; S10, 75% commercial flour (type C) with 25% ‘Caaveiro’ flour; followed by their duplicates and a negative control, water, for each SSR (M1–M5). Purple lines show the manual threshold set for each assay. Figure 2 From left to the right, dilutions D1 and D2 for each sample (S1, 0% ‘Caaveiro’, to S6, 100% Caaveiro, plus a negative control, water) for each SSR (M1–M5). Red lines show the manually set threshold for each assay. Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al.6 Table 3 Number of alleles detected in different wheat varieties by using eighteen microsatellites, quality of the amplification, capacity to distinguish between seventeen varieties and ability to discriminate three types of commercial flours (A, B and C) and flour mixtures containing the autochthonous wheat ‘Caaveiro’ Marker No. of alleles detected in this study Number of alleles (unique alleles) (Ruiz et al., 2012) Allelic rank Alleles Amplification quality in agarose Amplification quality in the sequencer Ability to distinguish cultivars Ability to discriminate flours mixtures Xgwm0148 8 12 (4) 138–164 89, 138, 140 2 , 142, 144 1,2,3 , 146, 160 1,3 , 164 Good Good Yes No Xgwm0155 6 16 (3) 139–147 139, 141 1,2,3 , 143 1,2,3 , 145 1,2 , 147, 149 Good Good Yes No Xgwm0156 11 20 (4) 283–317 283 1,2,3 , 287 1,2,3 ,289 2,3 ,291, 293, 296, 300, 312 1,2 , 315 1 , 317 1 , 319 Good Good Yes Yes+ Xgwm0186 8 28 (15) 117–137 100, 117, 119 1 , 121 1,2,3 , 123, 127 1,2,3 , 129, 137 1,2,3 Good Good Yes No Xgwm0234 12 19 (9) 198–245 198, 201 1,2,3 , 224, 226 1 , 228, 230, 234, 236, 238 1,2,3 , 240 1,2,3 , 244 Good Good Yes No Xgwm0312 27 26 (4) 184–250 184, 192 1 , 194 1 , 196, 208 1,2 , 210, 212, 214 1,2 , 218 2 , 220, 222 1,2,3 , 224, 226, 228, 230 1,2 , 232, 235, 237, 239, 241, 243, 245, 248, 250, 252, 256, 258 Good Good Yes Yes++ Xgwm0332 22 20 (4) 190–245 190 1,2,3 , 193 1,2,3 ,195 3 , 200 1,2 , 204, 208 1,2,3 , 211 3 , 213, 217, 219, 221, 223, 225, 227, 229, 231, 233, 236, 241 1 , 245, 250, 256 Good Good Yes Yes+ Xgwm0099 21 11 (3) 105–155 105 1,2,3 , 107, 109, 111, 113, 115 1 , 122, 124, 127, 130, 132, 134, 136, 138, 140, 142, 144, 147, 149, 153, 155 Good Good Yes Yes++ Xgwm0570 13 15 (3) 96–159 96, 106, 136 1,2,3 , 138, 141, 143, 145 1 , 148 1,3 , 151 1,2,3 , 153, 155, 157, 159 Good Good Yes Yes+ Xgwm0577 24 41 (15) 128–219 128 1,2 , 130, 134 1 , 136, 138, 140, 142, 147, 150, 152 1 ,155, 157 1,2 , 159 1,2 , 162 1 , 176, 203, 205, 207, 209, 211, 213, 215, 217, 219 Good Bad Yes No Xgwm0060 10 19 (5) 116–138 116, 118 1,2,3 , 122, 126, 128, 130, 132, 134, 136, 138 Good Good Yes Yes++ Xgwm0088 6 17 (4) 141–151 141, 143 1 , 145 2,3 , 147 1,2,3 , 149, 151 1 Good Good Yes No Xgwm0513 17 4 (0) 148–213 148 1,2,3 ,150 1,2,3 ,154 1,2,3 , 160, 162, 174, 184, 188, 189, 194, 200, 203, 205, 207, 209, 211 1,2,3 , 213 Good Good Yes No Xgwm0389 15 14 (3) 114–151 114, 116 1,2 , 118, 120 3 , 122, 124, 126, 128, 131, 133 3 , 135, 137, 139 1 , 143 1,2,3 , 151 Good Good Yes No BARC155 9 11 (4) 180–206 180, 184 1,2,3 , 186, 188, 194, 198, 201, 203, 206 Good Good Yes No BARC80 6 17 (6) 101–133 101, 103, 106 1,2 ,109 1,2,3 , 114 1,3 , 133 Good Good Yes No WMC468 12 5 (1) 129–157 129 3 , 131, 133 1,2,3 , 135 1 , 140, 144, 146 1,2 , 148, 151, 153, 155, 157 1,2,3 Good Good Yes Yes+ Xgwm0002 11 4 (0) 116–277 116 1,2,3 ,118, 120 1,3 , 127, 220 1,2,3 ,222, 224 1,3 , 231, 255, 257 1,2,3 , 277 Good Good Yes No In bold, alleles of ‘Caaveiro’; in bold and underlined, specific alleles of ‘Caaveiro’. 1, Alleles of flour A; 2, alleles of flour B; 3, alleles of flour C. Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al. 7 trials (without flour type B in the second trial due to the lack of the blend with 5% of ‘Caaveiro’ flour). Xgwm0099 presented R 2 over 0.95 in both trials. Amplification of Xgwm0156 and Xgwm0570 showed adequate R-squared values in the first trial for copies per microlitre of sample but not for copies per nanogram of sample. Xgwm0332 only showed adequate results for trial 1 and copies per microlitre sample with D1. Xgwm0156, Xgwm0332, and Xgwm0570 did not show adequate results in the second trial for blends with two commercial flours (B and C). Xgwm0312 showed very reduced amplification in ‘Caaveiro’ wheat with respect to commercial cultivars, with a maximum of three copies per nanogram of sample for the two dilutions D1 and D2 in the first trial (Table 5) and for the D3 of the second trial (Table 6), meanwhile, in 25% ‘Caaveiro’ flour, there were thirteen copies per nanogram of sample (D3) to fourteen (D4) for commercial flour type A, four copies per nanogram of sample (D3) for type B and five copies per nanogram of sample (D3) for type C. A maximum of 1291 to 6020 copies of Xgwm0099 per microlitre and 138 to 161 copies per nanogram of sample were amplified in each reaction (Table 6). Our results indicated that the number of copies per nanogram of sample in 25% ‘Caaveiro’ flour was seventythree for samples D3 and D4 of commercial flour type A, eighty-nine D4 of type B and seventy-seven copies per ng sample D4 of type C. Discussion SSRs and genetic variability In this study fourteen varieties of wheat were identified, the autochthonous wheat ‘Caaveiro’ (identical to Figure 4 Comparison of the discriminant allelic profiles according to the proportion of ‘Caaveiro’ flour and type A flour by SSR Xgwm0312 (in green, alleles of type A flour; in orange, alleles of ‘Caaveiro’). Table 4 R-squared values (R 2 ) of the linear regressions for the copies per microlitre and nanogram of samples of five SSRs in two trials with different dilutions and different mixes of ‘Caaveiro’ flour with commercial flours Molecular marker Trial Dilution R 2 for copies per lL sample R 2 for copies per ng sample Xgwm0312 1 D1 0.777 0.8907 Xgwm0312 1 D2 0.7401 0.9456 Xgwm0312 2 D3 0.6774 0.6672 Xgwm0312 2 D3 0.813 a 0.8135 a Xgwm0099 1 D1 0.9882 0.9872 Xgwm0099 1 D2 0.9843 0.9639 Xgwm0099 2 D4 0.9515 0.9516 Xgwm0156 1 D1 0.8623 0.5934 Xgwm0156 1 D2 0.8935 0.1324 Xgwm0156 2 D3 0.4339 0.433 Xgwm0332 1 D1 0.8058 0.0031 Xgwm0332 1 D2 0.4781 0.4049 Xgwm0332 2 D4 0.6069 0.4781 Xgwm0570 1 D1 0.8395 0.0216 Xgwm0570 1 D2 0.9243 0.0025 Xgwm0570 2 D5 0.0049 0.0049 a Only for blends with flour type B. Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al.8 ‘Carral’), ‘Callobre’ and three commercial flours used for bread with eighteen markers that had previously been used in wheat studies in Spain (Ruiz et al., 2012). Pasqualone et al.(1999) and Ruiz et al.(2012) both reported on the use of the microsatellite locus Xgwm0155; the latter obtained two alleles (135 and 136), while the present work described six alleles, ranging from 139 to 149 bp. R€ oder et al.(2002) also used 426 373 236 309 363 114 91 94 54 85 81 31 167 87 97 51 y = -2,7527x + 386,08 R² = 0,777 y = -0,5916x + 90,415 R² = 0,7401 y = -0,9524x + 137,28 R² = 0,6774 0 50 100 150 200 250 300 350 400 450 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 elpmaSLµ_rePseipoC 0 - 100 % 'Caaveiro' flour Xgwm0312 D1 D2 D3 Lineal (D1) Lineal (D2) Lineal (D3) 21 15 13 13 13 3 18 15 12 14 12 3 8,4 4,3 4,8 2,6 y = -0,1511x + 17,534 R² = 0,8907 y = -0,1366x + 16,433 R² = 0,9456 R² = 0,6772 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 el pm aSgnrePseipoC 0 - 100 % 'Caaveiro' flour Xgwm0312 D1 D2 D3 Lineal (D1) Lineal (D2) Lineal (D3) Figure 5 Linear regressions for Xgwm0312 by ddPCR for ‘Caaveiro’ flour and 3 commercial flours (type A, blue and green, types B and C, red) and some blends for the first trial (dilutions D1, blue; D2, green) and the second trial (dilution D3, red). Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022 Traceability of ‘Caaveiro’ wheat by SSRs A. M. Ramos-Cabrer et al. 9