ASPECTOS METODOLÓGICOS GENERALES

DNA from each sample was extracted using Qiagen DNeasy blood and tissue DNA extraction kits following the manufacturers protocol. Small tissue samples (≤10mg) were placed in 1.5ml microcentrifuge tubes. 180uL ATL buffer was added to the samples followed by 20 ul of proteinase k (>600 mAU/ml). The proteinase k breaks down the tissue’s proteins and cells allowing suspension of the DNA into solution. The solution was vortexed throughout this procedure. The vial was incubated at 56° for at least 30 minutes. AL buffer (200uL) was added followed by 200 ul of 96-100% ethanol. The total mixture was then pipetted into a special designed flow-through column placed within a 2mL collection tube. The flow-through column has a silica membrane which retains the DNA while allowing other solution to be centrifuged through. The column and collection tube was centrifuged at 8000rpm for one minute. The collection tube was discarded, and another was replaced. 500 ul of AW1 buffer is added to the column. Centrifuging occured again at 8000pm for 1 minutes. The collection tube was discarded and replaced. 500uL of AW2 buffer was added. Centrifuging occurred again now at 14,000rpm for 3 minutes. The collection tube was discarded. The spin column was placed in a 1.5ml microcentrifuge tube. 200uL Buffer AE was added to the column. This was centrifuged at 8000rpm for 1 minute. Finally, the spin column was discarded and the extracted DNA was isolated in the microcentrifuge tube. This DNA was stored at -20°C until amplified through PCR (DNeasy Blood & Tissue Handbook).

A 25uL cocktail of ultrapure water, 10x buffer, MgCl2, dNTP, taq, extracted sample DNA, and forward and reverse loci-specific CO1 primers was created for

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with an annealing temperature between 42-52°C dependent on which temperature provided the highest DNA yields. Gel electrophoresis was then used to determine whether samples successfully amplified. The procedure follows: 100mL of TBE buffer was added to a flask followed by 1.5g of agarose. This flask was microwaved until the agarose had completely dissolved in the TBE. Then, 1uL of ethidium bromide was added to the solution. This solution was poured onto a plate for the solution to solidify. Well combs were placed across the plate. Once the gel solidified well combs were removed and the gel was placed in a horizontal gel electrophoresis system. 5uL of PCR product mixed with 2uL loading dye buffer was added to each well. 7uL of a standard ladder was added to one well in each row to ensure control. The machine was turned on to ~80V for 45 minutes. After, the gel was removed and observed under UV light. Observation of distinct fluorescent bands indicated successful amplification.

Fluorescent base pairs were then added to each successfully amplified product. This was done by adding a 2uL mixture of EXO-1, SAP, and UPW to 2uL to the PCR product. This was incubated at 37C for 30mintues and 95 for 5 minutes. 1.5uL of this solution was then added to a cocktail of BigDye, 5x Buffer, forward primer, and UPW. The samples were fully run through the PCR process again to adhere fluorescent base pairs to sequences that allow for detection by machine sequencing.

Samples were subsequently purified through ethanol precipitation. 1uL of sodium acetate and 40uL of 95% ethanol was added to the sample product. The product was centrifuged (1500xg) for 45mintues. The plate was inverted and centrifuged at 300G for 2 minutes. 40uL of 70% ethanol was added. The plate was centrifuged (1500xg) for 10m minutes again, and then inverted and spun at 300g for 2 minutes. The plate was kept at -

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20C until it was shipped to Functional Biosciences for sequencing by machine based fluorescent sequencing.

3.3 DNA Barcoding

DNA barcoding was used to identify individual tissue samples. This process used a universal PCR-based assay of the Cytochrome Oxidase-I (COI) locus; primer sequences and PCR conditions are standardized as part of the Fish Barcode of Life (FBOL)

initiative. Barcoding uses COI sequences compared to a large COI FBOL sequence database. Statistical analyses were used to assign individual samples to species with a probability assignment. Given genetic identification, individual samples could be confidently assigned to species, thus allowing the estimate of Atlantic cod mislabeling rates across Spain to be derived.

Before sequences were compared to barcoding databases however they were first edited using the program Sequencher. These sequences were then aligned using the program BioEdit. Of the total 418 CO1 sequences, 335 sequences were greater than 600 base pairs (bp) long (typically 636 bp). Due to degradation of DNA however numerous samples provided shorter sequences. Between 500-600bp there were 24 sequences, 39 between 400-500 bp, 16 between 300-400bp, and four less than 300bp long. After editing and aligning, the sequences were compared to both the Barcode of Life Database (BOLD) and GenBank databases. These programs both use different assignment

statistics and compare to different databases. This redundant analysis ensured that each sequence was not assigned to an erroneous species sequence that may have been

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species assignment was documented (Table 4.10). There is ~.02% intraspecific variation in Atlantic cod (G. morhua) CO1 and a ~2% interspecific variation in CO1 between Atlantic cod (G. morhua) and its next closest match of Alaskan pollock (G.

Chalcogrammus). Therefore, a threshold cutoff for Atlantic cod sequences at 98% was established. No species besides Gadus morhua were identified above this threshold for samples identified as Gadus morhua.

3.4 Retesting with 16s

Samples that did not amplify with the use of the CO1 locus, or were shown to be mislabeling by CO1 sequencing were retested using 16s. The first reason for this retesting is that collected samples had the potential to have been degraded. Many of the samples collected were highly processed food products, and all samples were shipped without ethanol for a short period to the United States. Thus, the failure to amplify a portion of samples was expected. 16S was shown to work in our lab in many cases where CO1 would not, and thus after initial CO1 sequencing, 16S was used to increase

sequencing yield and substantiate the final dataset to the highest degree. 16s is a much conserved, universal locus that has comprehensive presence in the GenBank barcoding database making it a good locus to use as an alternative or in conjunction with CO1.

The other use of 16S retesting was to validate mislabeling assignments. Samples initially matching mislabeled species were again barcoded using the 16s locus in order to reinforce the validity of the findings, and ensure no errors were made in process of CO1 barcoding. In four mislabeled samples however, only the 16s provided a sequence.

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3.5 Geographic Analysis

Geographic analysis of mislabeling rates between cities, and other geographic parameters (such as coastal and inland cities) will be tested by use of chi squared tests of independence and principle component analysis. This will statistically determine

whether differences in mislabeling rates are random or related. This chi squared test was also used to determine if differences in mislabeling rates between types of fish products, and if differences between where a product is purchased (markets, supermarket, and restaurant), are random or related. The principle component analysis will compare additional geographic trends with mislabeling such as the population of each city, the distance to a coastline, and distance to the Atlantic coast.

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CHAPTER 4

RESULTS

This data reveals a mislabeling rate for Atlantic cod (Gadus morhua) of 6.4% (35/546) throughout Spain. Of the samples collected there was a high yield rate for successful sequencing of the collected samples at 86.1%. The results presented in this chapter are partitioned into mislabeling by city, mislabeling by product type, mislabeling by location of purchased, relatedness of substituted species, and substituted species data. All data in the presented tables and figures is compiled from the Appendix Table A which contains data for all collected and sequenced samples including DNA barcoding species assignment statistics. Color scales in all tables correspond to increased values and are used for facilitated interpretation of the data.

4.1 Mislabeling by City

The lowest mislabeling was recorded in Madrid (4.0%) and the highest

mislabeling in Seville (11.1%) (Table 4.1). The other six cities maintained a confined range of mislabeling rates between 4.9% and 7.5%. Figure 4.1 represents this data in a visualized map view.

City # of Collected Samples % of Collected Samples # of Sequenced Samples % of Sequenced Samples # of Mislabeled Samples % Mislabeling Madrid 112 17.7% 90 16.5% 3 3.3% Salamanca 72 11.4% 65 11.9% 4 6.2% Santiago 72 11.4% 61 11.2% 3 4.9%

Table 4.1: City Summary Data

35 Bilbao 72 11.4% 66 12.1% 5 7.6% Barcelona 92 14.5% 81 14.8% 6 7.4% Valencia 72 11.4% 65 11.9% 4 6.2% Seville 71 11.2% 63 11.5% 7 11.1% Granada 71 11.2% 55 10.1% 3 5.5% Totals 634 100% 546 100% 35 6.4%

Table 4.1: Summary of collection, sequencing, and mislabeling data for each city. This data is compiled from table A of the appendix.