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. 2020 Aug 4;15(8):e0237111. doi: 10.1371/journal.pone.0237111

aTBP: A versatile tool for fish genotyping

Silvia Gianì 1,#, Silvia Silletti 1,#, Floriana Gavazzi 1, Laura Morello 1, Giacomo Spinsanti 2, Katia Parati 3, Diego Breviario 1,*
Editor: Tzen-Yuh Chiang4
PMCID: PMC7402489  PMID: 32750100

Abstract

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.

Introduction

With approximately 35.000 described species, fishes account for about 50% of all vertebrates. Fish exhibit a great level of diversity, reflecting processes of adaptation to very different aquatic environments. High species number, significant morphological and genetic diversity and environmental fitness, are at the basis of several important scientific issues. These may refer to taxonomy and correct species identification, evolutionary biology and assessment of variation and changes in allele frequencies, resilience and adaptability to extremely variable climate conditions, diversification and parental recognition, traceability of seafood. All these issues find in cellular DNA a common and effective target for investigation. In fact, cellular DNA can potentially be retrieved from any species and any kind of organic substrate, such as muscle, fin, or blood and DNA-based analyses can be applied to any of the issues just mentioned. Species identification is nowadays largely based on DNA barcoding, through the amplification and sequencing of some mitochondrial genes where a sufficient interspecies variation can be detected [1–3]. The fish section of the consortium for barcoding of life (http://www.boldsystems.org/ or https://ibol.org/) includes about 8.000 fish species and relies on the sequence of the 650 bp region of the mitochondrial gene cytochrome c oxidase I (COI). It represents an effective and comprehensive resource for the analysis of fishes and fish products [4, 5]. More recently, and for the purpose of tracing species in food matrixes that contain a low quality DNA, due to harsh food processing, the use of minibarcodes (shorter fragments of the full length DNA barcode approximately 200 bp long) has been applied with some success. Several minibarcode regions have been identified that allow for differentiation of a range of species, and these regions have also been tested in silico to differentiate commercially important salmon and trout species [6–8]. However, limits in the classical DNA barcoding approach may be encountered in the analysis of mixtures composed of multiple species, in the recognition of undeclared substitutions, especially with local varieties, in the availability of specific, known target sequences, and in the need for sequencing and related costs for data elaboration and instrument maintenance.

Genomic DNA data are also very important for conservation management of genetic resources and for assessment of variations occurring in natural populations. This data provides a novel opportunity to investigate how populations have responded to changes, to identify mechanisms underlying these changes, and to evaluate the adaptive potential and vulnerability of populations in the future. A recent and worrisome example has been reported concerning a 60% decline in the populations of salmon of North America and Europe, clearly associated to warmer winter temperatures. Using single nucleotide polymorphisms (SNPs) as molecular tools, declining and near to decline populations have been identified [9]. These declining fish numbers are not only problematic for biodiversity, but their loss also represents an impediment to improving our scientific understanding of key fundamental adaptation strategies revealing molecular responses to life in cold conditions. Cited in the line of our present contribution, this is reminiscent of a well known and early reported adaptation process that explained the occurrence of microtubule polymerization at cold temperatures as dependent on specific amino acid substitutions found in the α- and β-tubulin moieties [10, 11]. In more general terms, the availability of a key functional marker is of importance to monitor the effect of climate changes on population fitness. In this way genomic screening can effectively assess population vulnerability. This has been successfully applied for salmon in a Canadian alpine environment where the maintenance of an almost balanced population of red and white Chinook salmon (Onchorhynchus tshawytscha) has been associated to increased carotenoids synthesis and increased heterozygosity at the major histocompatibility complex loci [12, 13], respectively. In addition, the reproduction system can obviously affect variation in natural populations and thus the use of suitable molecular markers like polymorphic microsatellite loci and COI can help in assigning parentage, in identifying hybridization events and in recording the breeding system [14, 15].

As previously reported, different molecular markers may be utilized for different purposes. Thus, we want to direct the attention to a relatively new molecular marker, animal Tubulin Based Polymorphism (aTBP; [16]), sufficiently versatile to assist these many different purposes. Based on the natural occurrence of polymorphisms in the intron length and nucleotide composition of the β-tubulin genes, the approach may offer an attractive and workable alternative to the genetic identification of fish species, as well as subpopulations and local varieties, with no need for sequencing. Hereby, we present experimental evidence in favour of the use of aTBP for fish genotyping and discuss its possible applications.

Materials and methods

Experimental samples

Total DNA extracts made from the following fish species: Sparus aurata, Dicentrarchus labrax, Oncorhynchus mykiss, Acipenser naccarii, Thunnus thynnus, Salmo carpio and Salmo trutta f. fario were provided by the Spallanzani Institute (Rivolta d’Adda, Italy). These were obtained from different research projects in which the Institute has been involved: Competus—CRAFT-017633; Cobice—LIFE–04NAT/IT/000126; FP7-SME-2010-1-262523; FP4-FAIR989211; Salvacarpio–Regional project n. 1220; MIIPAF, Three-year plans for fishing and aquaculture—VI 2000–02. The DNA extracts were originally produced from fin-clipped samples by using the semi-automatic BioSprint 96 DNA system (QIAGEN) and the BioSprint 96 DNA Blood Kit (Qiagen) following the manufacturer’s protocols. Fish species identification of these samples was performed by the use of a panel of Single Sequence Repeats markers (SSRs), as reported [17–21], with the exception of T. thynnus and O. mykiss. 15–20 samples of each species were randomly chosen and used for the aTBP molecular analyses. The DNA samples identified by the prefix FT were instead provided by the Life Sciences Department of the University of Siena. These included 34 fish specimens, purchased frozen from local Tuscan markets, consisting of 6 specimens of Sparus aurata, 2 of Acipenser transmontanus, 6 of Thunnus albacares, 4 of Pangasisus hypophthalmus, 8 of Salmo salar, 4 of Oncorhynchus mykiss, and 4 of Dicentrarchus labrax. Total DNA extractions were performed by using the Wizard® SV Genomic DNA Purification System (Promega), following the manufacturer’s instruction for animal tissues. Fish species identification of the FT samples was obtained by DNA sequencing of the fragments amplified with the use of the following universal primers: 5’-TCAACYAATCAYAAAGATATYGGCAC- 3’ for the forward and 5’-ACTTCYGGGTGRCCRAARAATCA-3’ for the reverse, known to target a conserved portion of the COI gene [1–3]; DNA sequencing was performed on both strands and sequences matched to each other. Unaligned and aligned COI sequences are provided in the S1 Data.

aTBP amplification and capillary electrophoresis

30 ng of any total DNA sample, previously characterized either by SSRs or COI, were used as template for aTBP PCR amplification. PCR conditions and primer sequences for amplification of intron III (aFex3.2 and aRex3.2) have been recently reported [16]. The forward primer was labeled in 5’ position as described in [22]. Two negative controls (no template) were always included in each PCR reaction and all PCR amplifications were repeated at least twice to check the consistency of the amplification profile. 4 μl of each PCR reaction was preliminary loaded on a 2% agarose gel, stained by Atlas Clear Sight DNA Stain (1μg mL-1) (Bioatlas) and compared to gene Ruler™1 Kb plus ladder as reference, to verify the intensity of the amplification signal to proceed with the appropriate dilutions to be used for amplicon resolution analysis done by capillary electrophoresis. 2μL of each diluted sample was mixed with 0.2 μl of 1200 LIZ Size Standard and 17.8 μl Hi-Di formamide to a final volume of 20 μL. Samples were denaturated at 95°C for 5 min and, after cooling to -20°C, were loaded onto the ABI 3500 Genetic Analyzer (Thermo Fisher Scientific) for CE separation following the running protocol described by [23].

Data analysis

The amplicons resolution data were collected using the Data Collection Software v. 3.1 (Thermo Fisher Scientific) and then analyzed by the Gene Mapper Software v. 5.0 tool (Thermo Fisher Scientific). Data analysis was made by comparison of the numerical output of the ABI 3500 analyzer, converted in an excel spreadsheet which allows the association of each specific amplicon profile to each fish species. At least two different electrophoretic runs were performed for each amplified product in order to confirm the aTBP profile. The PCA analysis was carried out with Past3 software [24] based on a presence-absence matrix, obtained from the score of the aTBP markers.

Results

As shown in Fig 1, the ability of the aTBP method in discriminating among animal species is based on the variation of the length of intron III, commonly found in members of the animal β-tubulin gene family that may differ by number. Therefore, the same couple of primers conveniently located at the boundaries of the third intron amplifies, in a typical PCR reaction, a group of fragments that can vary in number and length in each analyzed species. If resolved in a capillary electrophoresis system, they eventually define a species-specific DNA code. The separation resolution is such that peaks/fragments differing from just 1–2 bp can be recognized. Each peak of the electropherogram is defined by sizes, expressed in bp, and by a height, expressed in Relative Fluorescence Units (RFU) values.

Fig 1. aTBP: An intron length polymorphism—Based method.

Fig 1

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The genomic organization of a generic vertebrate β-tubulin gene is shown. Intron III is used as the source of DNA polymorphism in a PCR reaction that is triggered by the use of the aTBP primer pair.

A good and paradigmatic example of the level of information that is retrievable by aTBP, when applied to individuals of the same species, is provided in Table 1 with reference to gilthead seabream (S. aurata). Sixteen different individuals coming from aquaculture, already characterized for their morphological traits and with a panel of SSRs primers, were analyzed with aTBP together with 6 individuals of the same species purchased in the market and classified by COI barcoding. The data reveal the presence of five amplified fragments that are commonly shared among all of the analyzed samples (grey columns in Table 1), In addition, a quite diffuse and interesting intra-species variation, characterized in both subgroups by either missing or supplementary amplicons, likely corresponding to allelic variations, was identified. It is of relevance to note that, with the exception of the 312 bp long fragment amplified from the DNA extracted from three individuals of the Spallanzani group of specimens, the DNA polymorphisms detected at intra-species level are present in both the analyzed groups, likely reflecting ongoing variations in the general gilthead seabream population.

Table 1. aTBP profile of 22 samples of gilthead seabram (S. aurata).

Sample CE peaks
A5 Size 210.4 214.4 243.9 284.6 316.6 348.8 356.9 562.1 645.4
Height 31306 22426 21955 9556 15357 1519 1239 4798 4774
B5 Size 209.0 210.0 211.1 214.6 244.0 284.8 312.6 315.8 349.0 562.4 645.9
Height 12144 13097 25174 14470 15301 4806 8911 7919 1262 6889 2472
C5 Size 208.9 209.8 210.9 214.4 244.0 284.6 316.8 348.9 357.0 562.3 646.4
Height 16768 16189 30909 19393 18316 8103 15216 900 980 5384 5089
D5 Size 209.7 214.4 244.0 284.6 297.3 316.8 348.8 357.1 562.1 646.4
Height 30384 30428 31122 11928 18227 18857 1707 1579 16842 10168
E5 Size 209.6 214.2 243.8 284.6 297.4 316.7 348.8 357.1 562.3 646.4
Height 29737 29886 32583 13433 20264 21847 1816 1779 19978 11679
F5 Size 211.0 214.6 244.1 284.8 316.8 349.0 357.2 562.6 645.8
Height 30631 12439 14485 4736 15886 675 556 6427 2255
G5 Size 210.9 214.5 244.0 284.5 297.4 317.0 348.8 562.0 646.3
Height 24018 9643 8789 3323 5381 5070 851 4471 1470
H5 Size 211.0 214.6 244.0 284.6 317.0 348.9 357.2 562.5 646.8
Height 15524 5955 6998 2243 6551 305 287 2542 1971
A6 Size 210.8 214.4 244.0 284.6 297.3 316.6 348.9 357.0 562.2 646.5
Height 28890 11578 11432 4860 4819 5642 710 548 3836 1560
B6 Size 210.8 214.4 244.0 284.6 312.5 348.9 357.0 562.2 646.4
Height 31948 7584 8826 3609 6286 359 372 1127 1619
C6 Size 210.8 214.4 243.9 284.5 316.7 348.8 357.1 562.2 646.3
Height 26365 9916 11818 4212 7978 581 400 1849 1049
D6 Size 210.8 214.4 243.9 284.6 316.7 348.8 356.9 562.0 645.4
Height 11943 4865 4567 2147 3466 289 234 1212 1334
E6 Size 210.8 214.4 244.0 256.3 284.6 316.6 348.7 357.1 562.5 646.3
Height 26742 10695 9726 6251 3716 5840 422 567 4371 1757
F6 Size 210.9 214.5 243.9 284.6 312.4 315.5 348.8 562.1 645.5
Height 24948 10458 9518 3975 5936 4762 1018 4322 1975
G6 Size 208.9 210.9 214.4 243.9 284.6 297.3 316.6 356.9 562.3 646.4
Height 8466 16142 5015 3607 1466 4713 4649 197 6864 2020
H6 Size 209.0 210.5 243.9 284.6 316.8 348.8 357.1 562.1 646.4
Height 14288 31750 17309 15993 7099 14240 995 784 5823 4895
FT49 Size 209.0 210.1 211.1 214.6 244.1 284.8 297.6 317.0 349.0 562.4 646.8
Height 16896 19169 28429 18824 17453 6065 9577 8942 1376 7652 3003
FT99 Size 208.9 209.9 211.0 214.6 244.0 256.5 284.7 297.6 348.7 357.1 562.5 646.8
Height 27505 21764 30946 20052 15411 12155 4287 10438 187 925 5081 2402
FT128 Size 208.8 209.9 210.9 214.6 244.1 284.8 297.6 317.1 349.1 357.1 562.5 646.7
Height 25141 30944 7263 30616 25256 7525 16964 15525 933 675 8415 3913
FT290 Size 209.0 209.6 210.5 214.4 244.0 284.6 297.3 356.9 562.2 646.6
Height 14445 28478 28478 20145 17842 4714 11055 773 2247 507
FT287 Size 209.8 210.4 214.4 243.9 284.5 297.3 316.7 348.8 357.0 562.2 646.3
Height 24362 31171 18013 17275 4347 9780 7490 567 589 6011 1498
FT310 Size 210.0 211.1 214.6 244.1 256.6 284.7 297.5 348.9 357.1 562.4 646.6
Height 19834 29679 19140 16886 11181 5112 8455 493 447 4689 2169
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Size refers to the length, in base pairs, of the amplified TBP fragment; Height refers to the signal intensity, expressed in RFU. A5-H6: samples provided by the Lazzaro Spallanzani Research Institute; FT49-FT310: samples characterized by COI sequencing at the University of Siena.

Grey columns show the peaks shared among all the analyzed samples.

A similar situation was found when analysing 16 samples of the Adriatic sturgeon (A. naccarrii). Once more, commonly shared amplicons were found together with intraspecific polymorphisms, as shown in the upper panel of Table 2. Quite remarkably, one of these samples (A10) showed a very different pattern of aTBP amplification, perfectly matching that found in two available samples of white sturgeon (A. transmontanus). This reassignment is fully consistent with data previously obtained on the same experimental group with the use of a panel of SSR markers [19].

Table 2. aTBP profile of sturgeon (Acipenser spp.).

Sample CE peaks
Adriatic sturgeon A9 Size 245.0 250.4 251.2 254.3 289.7 630.9 775.6 860.2 863.2 865.0 1013.9
Height 12175 9723 12043 5772 7682 249 2272 391 364 412 1549
B9 Size 245.1 250.5 251.4 254.5 290.0 631.1 775.8 853.6 860.2 863.7 865.4 1014.1
Height 21322 13189 21903 17113 18187 870 4125 851 1425 784 454 2563
C9 Size 245.1 250.4 251.3 254.3 289.8 630.9 775.6 853.8 860.1 865.1 1014.0
Height 11809 4626 7084 5467 7036 598 925 618 733 481 1135
D9 Size 245.0 250.3 251.2 254.3 289.7 630.8 775.5 852.6 860.2 865.2 1013.9
Height 9122 14802 5291 4354 4024 261 557 538 482 128 748
E9 Size 245.0 250.4 251.2 254.3 289.7 630.8 775.2 860.2 863.1 864.9 1013.9
Height 11901 9286 11547 5505 7441 212 2284 263 350 600 1479
F9 Size 244.6 250.0 251.2 254.3 289.6 775.4 860.2 863.0 865.0 1013.7
Height 29308 29415 11547 29563 32188 13497 2866 3175 2505 8511
G9 Size 245.1 250.5 251.4 254.4 290.0 631.1 775.9 853.2 860.5 863.8 865.6 1014.2
Height 28093 10014 17103 12533 18004 792 3808 472 2209 649 535 2412
H9 Size 245.1 250.5 251.4 254.5 290.0 631.2 775.9 863.7 865.5 1014.5
Height 21460 13604 16855 7033 14811 413 2481 966 773 1547
B10 Size 245.1 250.4 251.3 254.4 289.9 631.2 775.8 860.8 863.8 1014.7
Height 26052 13531 24414 15436 22283 1547 3387 1281 1231 2272
C10 Size 245.0 250.4 251.2 254.3 289.8 630.8 775.5 860.2 863.1 1013.8
Height 6318 5740 9844 6722 7374 173 1195 637 343 625
D10 Size 245.0 250.4 251.2 254.2 289.7 630.9 775.4 853.0 860.2 865.2 1014.0
Height 7802 2364 3730 2900 2078 92 248 404 281 367 211
E10 Size 245.1 250.5 251.3 254.4 290.0 631.1 776.0 860.6 864.8 1014.6
Height 7883 5876 8189 3158 6151 386 723 254 191 504
F10 Size 245.0 250.4 251.2 254.4 289.8 630.9 775.5 860.3 864.8 1014.1
Height 6602 6250 6427 2409 5185 388 1024 235 502 643
G10 Size 245.1 251.3 254.4 290.0 631.1 775.8 852.0 860.4 865.3 1014.2
Height 12875 11766 8299 9505 953 1264 790 1114 741 574
H10 Size 245.0 251.2 254.2 289.8 631.0 775.6 853.2 859.9 1014.4
Height 7699 5837 4089 4713 312 461 309 395 311
White sturgeon A10 Size 251.3 252.3 255.4 289.5 297.7 760.3 795.8 816.3 902.9
Height 7998 9431 2598 6235 1270 1356 1531 691 486
FT274 Size 251.3 252.3 254.4 255.3 289.4 297.7 760.3 795.8 816.1 902.2
Height 5841 2923 673 1440 4419 1454 1636 1026 1024 658
FT284 Size 251.3 252.3 255.4 289.4 297.7 760.2 795.7 816.0 902.7
Height 6761 10520 2375 6673 1156 1676 1141 390 493
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Analysis of 16 samples of Adriatic sturgeon provided by the Lazzaro Spallanzani Research Institute and 2 samples of white sturgeon purchased in the market. For each analysed sample, numerical values refer to the sizes of the amplified TBP fragment, in bp, and to the signal height in RFU, respectively.

Light grey columns: peaks commonly shared between the two species.

Dark grey columns with white numbers: species-specific diagnostic fragments.

Bold fonts: sample of the first set erroneously classified as A. naccarii.

Data reported in Table 3 more adequately underscore the application of the aTBP method for the discrimination of two different, important and largely commercialized tuna species: red tuna (Thunnus thynnus) and yellowfin tuna (Thunnus albacares). As can be easily appreciated, the two tuna species show commonly shared amplified aTBP fragments, referable to their genus, and species-specific fragments, two of 255 bp and 778 bp, and one of 282 bp in yellowfin and red tuna, respectively. Once more, both groups are further characterized by the presence of additional intraspecies polymorphic fragments that may be shared or not between the two species.

Table 3. aTBP profiles of tunafish (Thunnus spp.).

Sample Size values
Red tuna A11 219.3 220.7 230.5 253.2 282.9 387.0 519.5 796.3
B11 219.7 221.1 230.6 283.1 387.2 519.8 796.6
C11 219.1 220.5 230.3 282.8 387.0 519.5 796.3
D11 219.1 220.5 230.3 282.8 373.2 387.0 519.5 797.2
E11 219.7 221.2 230.7 283.2 387.2 520.0 796.7
F11 219.1 220.5 230.2 282.8 387.1 519.7 796.1
G11 219.7 221.0 230.6 283.2 519.8 797.4
H11 219.7 221.0 230.6 283.1 387.3 519.9 797.0
A12 219.7 221.1 230.7 283.2 373.5 387.2 519.9 796.9
B12 219.7 221.1 230.6 283.2 373.0 387.4 519.8 797.8 804.7
C12 218.9 220.4 230.2 282.7 386.9 519.5 796.3
D12 219.9 221.3 230.7 283.4 387.5 520.2 797.4
E12 219.9 221.3 230.8 283.3 387.5 520.1 797.4
F12 219.7 221.1 230.6 283.0 387.3 519.9 805.3
G12 219.7 221.1 230.6 283.1 373.5 519.9 804.6
H12 219.7 221.1 230.6 283.1 387.1 520.0 797.0 805.7
Yellowfin tuna FT95 221.1 230.7 255.4 387.5 519.9 778.3
FT201 220.6 230.7 255.4 373.7 519.9 777.9 796.7
FT189 219.5 221.1 230.4 253.4 255.4 373.6 387.4 519.8 778.1
FT267 219.9 221.1 230.6 255.4 373.6 387.4 519.8 778.4
FT308 220.6 230.6 255.4 387.5 519.8 778.4
FT357 220.2 221.4 224.8 230.8 255.6 387.6 520.0 778.5 797.2
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Analysis of 16 samples of red tuna provided by the Lazzaro Spallanzani Research Institute and 6 samples of yellowfin tuna previously characterized at the University of Siena. Only numerical values referring to the sizes, in bp, of the ampiflied TBP fragments are shown.

Light grey columns: commonly shared peaks.

Dark grey columns with white numbers: species-specific diagnostic fragments.

The ability of aTBP to easily discriminate among different fish species, revealed by the data just presented, motivated us to verify if the method could be used as a simple way to detect fraud and substitutions, frequently reported, and to a vast scale, in the fisheries market [25]. To this purpose, we analyzed and compared the aTBP profile of two fish species, pangasius (P. hypophthalmus) and European seabass (D. labrax), because the latter is often replaced by the former when commercialized as fillets or canned food.

Fig 2 readily shows how the two species look completely different from each other when their corresponding aTBP profiles are compared. Not a single amplified fragment is shared among those that are species- specific. As shown, in case of a suspected substitution, this difference can be conveniently revealed by a simple electrophoresis run of the amplified fragments in an agarose gel.

Fig 2. Comparison between seabass and pangasius aTBP.

Fig 2

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A) electropherograms obtained from pangasius (top panel) and European seabass (bottom panel) respectively. Reported numbers on top of the peaks refer to the sizes of the amplified fragments. A typical example is shown. Complete data are reported in S1 Table. B) Separation in agarose gel of the aTBP fragments amplified from pangasius or seabass samples. The numbers and letters above the agarose gel indicate the samples analyzed. The numbers next to the “1kb plus” marker indicate the molecular weights of each fragment.

The Salmonidae is a particularly relevant fish family often studied with reference to multiple important issues such as variations in response to climate changes, reproductive habits and parentage recognition, metabolic species-specific features, and, of course, market traceability. Table 4 shows the data obtained applying the aTBP method to individuals of four different salmonid species: carpione trout (Salmo carpio), an endemic species of the Garda lake in Italy, brown trout fario (Salmo trutta f. fario), Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), belonging to two genera of the family and present in natural environments as different as ocean or fresh water.

Table 4. aTBP analysis of the Salmonidae family.

Species Sample Size values
Carpione C3 214.7 219.9 220.8 223.8 228.9 230.9 234.0 236.3 259.3 289.1 303.8 330.6 338.4
D3 214.7 219.9 220.8 223.7 228.8 230.9 233.9 236.3 259.3 289.0 303.8 330.6 338.4
E3 213.7 214.7 219.9 220.8 223.8 228.9 230.8 234.0 236.3 259.4 289.0 303.8 330.6 338.4
F3 214.7 219.9 220.7 223.7 228.8 233.9 236.3 259.4 289.0 303.8 330.6 338.4
G3 214.7 219.9 220.7 223.7 228.8 233.9 236.2 259.4 289.1 303.8 330.6 338.4
A4 214.6 219.7 220.5 223.5 228.7 233.8 236.1 259.2 288.8 303.7 330.3 338.1
B4 214.7 219.9 220.7 223.6 228.8 230.9 236.2 259.3 289.0 303.8 330.5 338.3
C4 213.7 214.7 219.9 220.8 223.7 228.9 230.8 234.0 236.2 259.3 289.0 303.9 330.5 338.3
D4 214.8 219.9 220.8 223.7 228.9 230.9 236.2 259.3 289.0 303.8 330.5 338.3
E4 214.5 219.7 220.5 223.5 228.6 230.6 236.0 259.2 288.9 303.6 330.3 338.1
F4 213.5 214.6 219.7 220.6 223.6 228.6 230.7 236.1 259.1 288.8 303.6 330.4 338.2
G4 214.6 219.7 220.5 223.5 228.7 230.6 236.1 259.2 288.8 303.6 330.3 338.1
Brown trout fario 5 213.3 219.6 220.4 223.5 228.5 233.6 236.0 259.1 288.7 305.4 330.2 338.1
6 213.5 219.7 220.6 223.6 228.7 233.7 236.1 259.2 288.8 305.5 330.4 338.3
8 213.3 219.6 220.4 223.4 228.5 233.5 235.9 259.1 288.6 305.4 330.3 338.1
10 213.5 219.7 220.6 223.6 228.7 233.8 236.1 259.2 288.9 305.6 330.5 338.2
11 213.5 219.7 220.5 223.6 228.7 233.8 236.2 259.2 288.8 305.6 330.4 338.3
12 213.5 219.7 220.5 223.5 228.6 233.8 236.1 259.2 288.8 305.5 330.4 338.2
13 213.4 219.6 220.4 223.4 228.5 233.6 235.9 259.1 288.7 305.3 330.3 338.1
14 213.3 219.6 220.4 223.5 228.7 233.6 235.9 259.1 288.7 305.5 330.3 338.0
15 213.3 219.5 220.4 223.4 228.5 233.6 236.0 259.1 288.7 305.4 330.3 338.0
17 213.3 219.6 220.4 223.5 228.6 233.6 236.0 259.1 288.7 305.3 330.2 338.1
19 213.3 219.6 220.4 223.5 228.6 233.6 236.0 259.1 288.7 305.4 330.2 338.1
22 213.6 219.8 220.6 223.6 228.8 233.8 236.1 259.3 288.8 305.5 330.4 338.2
Salmon FT1 213.3 219.3 220.3 224.5 225.8 228.4 230.5 259.1 288.4 290.5 330.0 331.4
FT113 213.3 219.3 220.3 224.5 225.8 228.5 230.6 259.1 288.5 290.6 330.0 331.4
FT198 213.3 219.4 220.4 224.5 225.9 228.5 230.6 259.1 288.5 290.6 330.0 331.4
FT203 213.3 219.3 220.4 224.5 225.8 228.5 230.6 259.1 288.5 290.6 330.0 331.5
FT252 213.3 219.3 220.4 224.6 225.9 228.5 230.6 259.2 288.5 290.6 330.1 331.4
FT273 213.3 219.3 220.4 224.4 225.8 228.5 230.6 259.1 288.6 290.7 330.0 331.4
FT296 213.3 219.4 220.4 224.4 225.8 228.5 230.6 259.1 288.5 290.6 329.9 331.4
FT368 213.3 219.3 220.4 224.5 225.8 230.6 259.1 288.5 290.6 330.0 331.4
Rainbow trout A1 213.2 220.5 223.6 224.8 225.9 232.8 258.3 290.5 309.4 324.5 334.0
D1 212.9 220.5 223.5 224.6 226.0 258.1 290.7 309.1 324.2 333.9
E1 213.0 220.4 223.5 224.6 225.7 232.6 258.1 290.4 309.1 324.3 333.8
G1 212.4 220.3 223.4 224.5 225.7 258.2 290.4 309.0 324.3 333.7
E2 213.1 220.5 223.6 224.8 225.8 232.8 258.4 290.6 309.4 324.5 334.0
F2 213.3 220.5 223.6 224.9 225.9 232.8 258.3 290.7 309.3 324.5 333.9
G2 212.9 220.3 223.4 224.6 225.9 232.6 258.2 290.5 309.2 324.2 333.8
H2 213.0 220.4 223.4 224.6 225.7 232.7 258.2 290.4 309.1 324.3 333.9
FT119 212.6 220.1 223.1 224.6 225.9 232.7 257.8 290.3 309.2 324.4 333.7
FT22 213.0 220.4 223.5 224.7 225.7 232.7 258.2 290.6 309.1 324.2 333.7
FT262 213.1 220.4 223.5 224.6 226.0 232.6 258.2 290.5 309.1 324.2 333.8
FT334 212.5 220.4 223.5 224.7 225.7 258.2 290.6 309.2 324.2 333.9
Species Sample Size values
Carpione C3 358.6 374.5 388.9 412.5 414.3 747.0 790.1
D3 358.6 374.4 388.9 412.5 414.3 747.0 790.1
E3 358.7 374.5 388.9 412.6 414.3 746.9 790.2
F3 358.6 374.5 388.9 412.4 414.3 747.0 790.1
G3 358.6 374.5 388.9 412.5 414.3 747.0 790.1
A4 358.4 374.3 388.6 412.2 414.0 746.3 789.7
B4 358.6 363.4 374.5 388.8 412.5 414.3 746.9 789.9
C4 358.6 374.5 388.9 412.4 414.3 746.8 790.3
D4 358.5 374.5 388.8 412.5 414.3 746.8 790.1
E4 358.4 363.3 374.3 388.6 412.3 414.0 746.4 789.7
F4 358.4 363.3 374.2 388.7 412.3 414.1 746.3 789.5
G4 358.4 363.2 374.2 388.6 412.3 414.1 746.4 789.7
Brown trout fario 5 357.2 363.1 388.5 412.2 414.0 746.1 789.5
6 357.5 363.3 388.7 412.3 414.1 746.3 789.8
8 357.2 363.1 388.5 412.2 413.8 746.1 789.6
10 357.4 363.2 388.7 412.4 414.1 746.2 789.7
11 357.4 363.3 388.6 412.4 414.1 746.6 789.9
12 357.4 363.2 388.7 412.4 414.0 746.7 789.9
13 357.2 363.1 388.6 412.1 414.0 746.2 789.6
14 357.3 363.2 388.6 412.2 413.9 746.1 789.7
15 357.3 363.2 388.5 412.2 413.9 746.1 789.4
17 357.3 363.1 388.5 412.2 414.0 746.1 789.5
19 357.2 363.1 388.5 412.2 414.2 746.1 789.5
22 357.4 363.2 388.8 412.3 414.0 746.4 789.8
Salmon FT1 340.7 356.2 388.5 391.9 414.0 476.3 745.5 858.2
FT113 340.7 347.5 358.1 388.5 392.0 414.0 476.3 745.4
FT198 340.8 356.2 388.5 392.1 414.0 476.3 745.4 858.1
FT203 340.7 347.5 356.1 388.5 392.0 414.1 476.3 745.6 858.2
FT252 340.8 356.2 358.2 388.5 392.1 414.0 476.4 745.4 858.2
FT273 340.8 347.5 356.3 388.5 392.0 414.1 476.4 745.4 858.1
FT296 340.8 347.6 356.3 388.4 392.0 414.1 476.4 745.7 858.0
FT368 340.7 356.2 358.2 388.5 392.1 414.0 476.4 745.5 858.2
Rainbow trout A1 361.5 388.2 393.5 398.5 414.0 742.4 860.3 1013.6
D1 361.5 393.2 398.4 414.0 742.3 860.0 1013.4
E1 361.3 388.0 393.1 398.2 413.8 742.0 859.7 1013.2
G1 361.3 387.9 393.1 398.3 413.8 741.9 859.6 1013.1
E2 361.5 388.2 393.2 398.5 414.1 742.6 860.2 1013.9
F2 361.5 388.2 391.3 393.5 398.5 414.0 742.6 860.3 1013.7
G2 361.4 388.0 393.2 398.2 413.8 742.1 859.9 1013.2
H2 361.3 393.1 398.2 413.8 742.0 859.9 1013.5
FT119 361.3 388.0 393.2 398.3 414.0 742.2 860.0 1013.2
FT22 361.4 393.3 413.9 742.1 860.0 1013.2
FT262 361.3 388.0 393.2 398.3 413.9 742.1 859.8 1013.4
FT334 361.4 388.0 393.2 414.0 742.1 859.9 1013.4
Open in a new tab

With the exception of salmon, for which just 8 samples were available, a selection of 12 individuals out of the total for each of the other three salmonid species, is shown. The complete dataset is provided in S2 Table.

Intermediate grey columns: Salmonidae family; Light grey columns: Salmo genus; Dark grey columns with white numbers: species; boxed numbers: peaks/amplicons common to the Carpione trout and the brown trout fario.

With the premise that intraspecific polymorphisms, present also in these groups, have been reduced to a minimum to set up a consultable table of immediately appreciable results, Table 4 delivers several useful information. First, the amplified fragments can be individually assigned to different taxonomic ranks, starting with the 220 bp long amplicon that is attributable to the Salmonidae family since it is present in all the samples we have analyzed. The three species belonging to the Salmo genus also share five common aTBP amplified fragments (219, 228, 259, 289 and 330 bp) while each single species is characterized by the presence of a small yet variable number of clearly specific amplification products, shown in the dark grey columns of Table 4. Additional similarities, such as those between carpione trout and brown trout fario, are notable (boxed columns).

Similarity between these two species, indicating their more recent separation, was further confirmed by the Principal Component Analysis (PCA) of Fig 3, where the four salmonid species are distributed in three major directions for a cumulative contribution of the first two principal components that explains 76% of the total variance. The complete data set used for PCA is provided in S2 Table.

Fig 3. PCA of the Salmonidae diversity based on aTBP.

Fig 3

Open in a new tab

The presence-absence matrix obtained by scoring the TBP markers was used to explore the distribution of four fish species belonging the Salmonidae family. The first two principal components explain the 58 and 18% of the variance, respectively.

Overall, the data shown indicate that the aTBP method can be easily and conveniently used to monitor variations occurring at different taxonomic ranks, providing a useful and very versatile tool for different kind of investigations.

Discussion

This paper presents evidence in favour of the use of the aTBP method for the genetic characterization of fish at different taxonomic levels and for different purposes. We have demonstrated that using a single PCR-based reaction with the same pair of primers, the TBP method can amplify from the genome of any fish sample a number of fragments that delineate a specific DNA profile, or barcode. The aTBP amplification products of a single barcode can then be sequentially attributed to the family, genus, species and subspecies categories. In its essence, aTBP adds to the two fundamental features of an ideal DNA barcode: high taxonomic coverage and high interspecific resolution. Thus, with aTBP recognition of subspecies polymorphisms become simpler and more efficient providing immediate data, with no need for sequencing or necessary prior knowledge of the target sequences. The power of the discrimination of the aTBP genomic profiling method is also shown to be unaffected by ploidy since sturgeon and salmonid species, known polyploidys [26, 27], can be easily distinguished. In fact, the two sturgeon species we have analysed, A. naccarii and A. transmontanus, are natural octaploid with 240–264 chromosomes. Due to the high level of fragments resolution granted by CE (1–2 bp), aTBP is expected to perform well also in presence of higher ploidy and chromosome numbers. Problems may arise in the reading of the electropherogram output that can become complex for the presence of numerous peaks. A software that can help in the fast recognition of the output is presently under development. Finally, aTBP is a functional and nuclear-based molecular marker. All these features may offer new opportunities to studies that are performed in diverse fields of investigation. The exception is molecular taxonomy where a long term, well established, rapidly diffused and internationally supported method based on the sequencing of the mitochondrial COI gene has provided the deposition of more than 80.000 barcoding sequences corresponding to approximately 8.000 different fish species. Nevertheless, as also shown in this paper, since aTBP substantially confirm COI data, it may be useful when species assignment, based on COI, is uncertainly relying on minimal SNPs differences.

This stated, the use of aTBP for identification, authentication and detection of fish species in food samples is quite appropriate and particularly suitable for all those laboratories that are not equipped with demanding sequencing facilities. As a classical DNA barcoding, aTBP can be applied to a high number of species, characterized by a large spectrum of variation. Differently from a classical DNA barcoding, the aTBP primers are effective independently from the taxonomic rank while COI primers must be often optimized for the successful use at ranks higher than species. In addition, aTBP can be used for detecting subspecies populations and local varieties. Anyhow, both applications, aTBP and classical DNA barcoding, are particularly suitable for seafood traceability, especially when transformation processes make morphological inspection impossible for fillets, frozen and canned foods, fostering frauds and substitutions. These irregularities could be easily uncovered by the detection of the aTBP species-specific diagnostic peaks as well as the visualization, even in a very simple agarose gel, of very diverse patterns of amplification as here shown for pangasius versus seabass (Fig 2). aTBP can also be of help for assessing variation in a natural population, a major goal in the field of evolutionary biology. To this regard, it is of interest to highlight the finding of a hierarchical distribution that assigns specific aTBP amplification fragments to different taxonomic ranks, as observed in Thunnus, Acipenser and Salmonidae. It looks like evolution has left molecular traces of its action in the introns of tubulin, from family down to species, and the presence of intra specific subpopulations, characterized by the sharing of few polymorphisms, promise to be a renovated handle for monitoring future evolutions. Since these intraspecific changes in allele frequency can be easily scored, they provide useful information on the overall structure of populations with respect to vulnerability, or resilience, in response to environmental changes and in natural selection constraints. Unique responses often are associated with mutations in genomic regions related to metabolic, developmental, immunogenic and physiological processes. aTBP genomic profiling is based on a functional marker, that is tubulin, since long related to cold response because of the identification of cold-inducible promoters and aminoacid changes exclusively present in the α- and β-tubulin moieties of the Antarctic fishes. Thus it is reasonable to consider the aTBP genomic profiling as a useful tool that can further our understanding of changes in fish genotypes and variations in population fitness.

Another field of possible and useful application of the aTBP method is the potential contribution to our understanding of the role that natural or anthropogenic hybridization and sexual competition play in genetic diversity including breeding among native and introduced species. For example, aTBP could be used for identifying preferential occupation of spawning grounds by a given species as well as recognition of the breeding system and parental assignment. Since the aTBP is a nuclear-based codominant marker, its usage may favor the recognition of hybrids already present in the F1 generation, rather than the F2 populations as is commonly practiced by the use of the mitochondrial, maternally inherited COI gene. In summary, understanding the processes underlying diversification can aid in formulating appropriate conservation management plans that will help to maintain the evolutionary potential of taxa, particularly under human-induced activities and climate changes.

Under most practical terms, aTBP is a simple and quick technique, based on a single PCR reaction and the resolution of the amplified fragments by electrophoresis, that may take few hours for an easy recognition on an agarose gel. Several samples can be concomitantly analyzed, 24 a day in our experience, providing consistent and reproducible genomic profiles that assist in the characterization of the genetic variation of the investigated species. A possible further improvement could be obtained by combining aTBP amplification to High Resolution Melting, as recently done for a combination of different plant DNA barcodes [28]. Also, efforts are in place to establish a practical aTBP data base with the help of Institutions and fishery companies. In conclusion, aTBP should be considered as valuable new tool of genetic investigation in fish for its simplicity of use, good costs/effectiveness ratio, usefulness in different fields of application and wide taxonomic coverage.

Supporting information

S1 Table. Seabass and pangasius aTBP analysis, complete dataset.

(XLSX)

Click here for additional data file. (11.9KB, xlsx)
S2 Table. Salmonidae aTBP analysis, complete dataset used for PCA.

(XLSX)

Click here for additional data file. (25.3KB, xlsx)
S1 Data. COI sequences, aligned and unaligned, of different fish species.

(TXT)

Click here for additional data file. (48.6KB, txt)
S1 Fig

(TIF)

Click here for additional data file. (1.5MB, tif)

Acknowledgments

We wish to thank Dr. Luca Braglia for his contribution on PCA. We also want to acknowledge Prof. Khidir Hilu of Virginia Tech, USA and Prof. Sara Patterson, Emeritus at Wisconsin University USA for their critical reading of the manuscript.

This work was partially supported by the Future Home for Future Communities (FHfFC) project funded by Regione Lombardia. There was no additional external funding received for this study and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was partially supported by the FHfFC (Future Home for Future Communications) project funded by Regione Lombardia. DB was the recipient. GA: FHfFC 2016 There was no additional external funding received for this study and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Hebert PD, Ratnasingham S, deWaard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci. 2003;270 Suppl 1:S96–9. 10.1098/rsbl.2003.0025 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD. DNA barcoding Australia's fish species. Philos Trans R Soc Lond B Biol Sci. 2005;360(1462):1847–57. 10.1098/rstb.2005.1716 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN. Universal primer cocktails for fish DNA barcoding.: Molecular Ecology Notes; 2007. p. 544–8. [Google Scholar]
  • 4.Galal-Khallaf A, Ardura A, Mohammed-Geba K, Borrell Y, Garcia-Vazquez E. DNA barcoding reveals a high level of mislabeling in Egyptian fish fillets. Food Control 2014. p. 441–5. [Google Scholar]
  • 5.Jennings WB, Ruschi PA, Ferraro G, Quijada CC, Silva-Malanski ACG, Prosdocimi F, et al. Barcoding the Neotropical freshwater fish fauna using a new pair of universal COI primers with a discussion of primer dimers and M13 primer tails. Genome. 2019;62(2):77–83. Epub 2019/02/07. 10.1139/gen-2018-0145 . [DOI] [PubMed] [Google Scholar]
  • 6.Shokralla S, Hellberg RS, Handy SM, King I, Hajibabaei M. A DNA Mini-Barcoding System for Authentication of Processed Fish Products. Sci Rep. 2015;5:15894 Epub 2015/10/30. 10.1038/srep15894 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Sultana S, Ali ME, Hossain MAM, Asing, Naquiah N, Zaidul ISM. Universal mini COI barcode for the identification of fish species in processed products. Food Res Int. 2018;105:19–28. Epub 2017/11/03. 10.1016/j.foodres.2017.10.065 . [DOI] [PubMed] [Google Scholar]
  • 8.Mitchell JK, Hellberg RS. Use of the mitochondrial control region as a potential DNA mini-barcoding target for the identification of canned tuna species. Food Anal. Methods 2016. p. 2711–20. [Google Scholar]
  • 9.Lehnert SJ, Kess T, Bentzen P, Kent MP, Lien S, Gilbey J, et al. Genomic signatures and correlates of widespread population declines in salmon. Nat Commun. 2019;10(1):2996 Epub 2019/07/05. 10.1038/s41467-019-10972-w . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Detrich HW, Prasad V, Ludueña RF. Cold-stable microtubules from Antarctic fishes contain unique alpha tubulins. J Biol Chem. 1987;262(17):8360–6. . [PubMed] [Google Scholar]
  • 11.Detrich HW. Microtubule assembly in cold-adapted organisms: functional properties and structural adaptations of tubulins from antarctic fishes. Comp Biochem Physiol A Physiol. 1997;118(3):501–13. 10.1016/s0300-9629(97)00012-1 . [DOI] [PubMed] [Google Scholar]
  • 12.Evans ML, Neff BD. Major histocompatibility complex heterozygote advantage and widespread bacterial infections in populations of Chinook salmon (Oncorhynchus tshawytscha). Mol Ecol. 2009;18(22):4716–29. Epub 2009/10/08. 10.1111/j.1365-294X.2009.04374.x . [DOI] [PubMed] [Google Scholar]
  • 13.Lehnert SJ, Pitcher TE, Devlin RH, Heath DD. Red and white Chinook salmon: genetic divergence and mate choice. Mol Ecol. 2016;25(6):1259–74. Epub 2016/03/01. 10.1111/mec.13560 . [DOI] [PubMed] [Google Scholar]
  • 14.Haddeland PJ, Junge C, Serbezov D, Vøllestad LA. Genetic parentage analysis confirms a polygynandrous breeding system in the European grayling (Thymallus thymallus). PLoS One. 2015;10(3):e0122032 Epub 2015/03/20. 10.1371/journal.pone.0122032 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Faulks L, Östman Ö. Genetic Diversity and Hybridisation between Native and Introduced Salmonidae Fishes in a Swedish Alpine Lake. PLoS One. 2016;11(3):e0152732 Epub 2016/03/31. 10.1371/journal.pone.0152732 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Gianì S, Di Cesare V, Gavazzi F, Morello L, Breviario D. Tubulin-based polymorphism genome profiling: A novel method for animal species authentication in meat and poultry. Food Control 2020; 110, 107010, 10.1016/j.foodcont.2019.107010. [DOI] [Google Scholar]
  • 17.Parati K, Chavanne H, Pozzi A, Previtali C, Cenadelli S, Bongioni G. Isolation and characterization of novel microsatellite DNA markers in the gilthead seabream (Sparus aurata) Conservation Genetic Research 2011. p. 83–5. [Google Scholar]
  • 18.Vandeputte M, Dupont-Nivet M, Haffray P, Chavanne H, Cenadelli S, Parati K, et al. Response to Domestication and Selection for Growth in the European Sea Bass (Dicentrarchus labrax) in Separate and Mixed Tanks. Aquaculture; 2009. p. 20–7. [Google Scholar]
  • 19.Congiu L, Pujolar JM, Forlani A, Cenadelli S, Dupanloup I, Barbisan F, et al. Managing polyploidy in ex situ conservation genetics: the case of the critically endangered Adriatic sturgeon (Acipenser naccarii). PLoS One. 2011;6(3):e18249 Epub 2011/03/29. 10.1371/journal.pone.0018249 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Galli A, Greppi G, Boscari E, Bonacina C. Parental allocation in Acipenser naccarii–statistical aspects and software development. J. Appl. Ichthyol.; 2011. p. 651–4. [Google Scholar]
  • 21.Parati K, Gentili G, Alborali GL, Viglione M. IL CARPIONE DEL GARDA: TUTELA DI UNA SPECIE A RISCHIO DI ESTINZIONE.: Quaderni della Ricerca; 2013.
  • 22.Gavazzi F, Casazza A, Depedro C, Mastromauro F, Breviario D. Technical improvement of the TBP (tubulin-based polymorphism) method for plant species detection, based on capillary electrophoresis. Electrophoresis. 2012;33(18):2840–51. 10.1002/elps.201200144 [DOI] [PubMed] [Google Scholar]
  • 23.Braglia L, Manca A, Gianì S, Hatzopoulos P, Breviario D. A Simplified Approach for Olive (Olea europaea L.) Genotyping and Cultivars Traceability. American Journal of Plant Sciences; 2017. p. 3475–89. [Google Scholar]
  • 24.Hammer Ø, Harper D. Paleontological Data Analysis. Oxford Blackwell Publishing; 2006. p. 351 pp. [Google Scholar]
  • 25.FAO (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS). Overview of food fraud in the fisheries sector, by Alan Reilly. Fisheries and Aquaculture Circular No. 1165. 2018, Rome, Italy.: FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS. 2018 Rome, Italy.
  • 26.Zhou L, Gui J. Natural and artificial polyployds in aquaculture. Aquaculture and Fisheries; 2017; 2:103–11 [Google Scholar]
  • 27.Du K, Stock M, Kneith S. and others. The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization. Nature Ecology & Evolution. 2020; 10.1038/s41559-020-1166-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ballin NZ, Onaindia JO, Jawad H, Fernandez-Carazo R, Maquet A. High-resolution melting of multiple barcode amplicons for plant species authentication. Food Control. 2019;105:141–50. 10.1016/j.foodcont.2019.05.022 . [DOI] [PMC free article] [PubMed] [Google Scholar]

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Tzen-Yuh Chiang

22 Apr 2020

PONE-D-20-08308

aTBP: a versatile tool for fish genotyping

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Reviewer #1: The manuscript by Gianì et al. entitled “aTBP: a versatile tool for fish genotyping”, the author developed new genotyping method and applied to the identification of fish species. This article develops the new method to indicate the relationship of inter- and intra- species. But, I found there are some mistakes as well as experimental method in this manuscript. The author only uses seven fish species for this research and these samples come from the market. Farmed individuals may come from a single ancestor. The sample is too small and not representative. The author must use more species and evidence to prove that this method is useful. Teleost fishes represent a highly diverse group consisting of more than 20,000 species. The author cannot prove that this method can distinguish all species. The author must substantially modify the method and the description of the results. I consider the methodology of this article to be inappropriate in their current form, in my opinion, this manuscript does not meet criteria for publication and must therefore be reject.

Reviewer #2: The authors tried to prove that the popular aTBP method is a versatile tool for fish genotyping. In general, they provided solid data to support the main conclusions. However, minor revisions are required before acceptance for publication.

1. Extra editing is necessary.

2. The authors should discuss about how to deal with polyploid species, which would bring many more variants to improve the practical difficulty in genotyping. By the way, it would be much better if a practical database can be established. These issues should be mentioned in the discussion section.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 4;15(8):e0237111. doi: 10.1371/journal.pone.0237111.r002

Author response to Decision Letter 0

18 May 2020

REF 1

Q : The manuscript by Gianì et al. entitled “aTBP: a versatile tool for fish genotyping”, the author developed new genotyping method and applied to the identification of fish species. This article develops the new method to indicate the relationship of inter- and intra- species. But, I found there are some mistakes as well as experimental method in this manuscript. The author only uses seven fish species for this research and these samples come from the market. Farmed individuals may come from a single ancestor. The sample is too small and not representative. The author must use more species and evidence to prove that this method is useful. Teleost fishes represent a highly diverse group consisting of more than 20,000 species. The author cannot prove that this method can distinguish all species. The author must substantially modify the method and the description of the results. I consider the methodology of this article to be inappropriate in their current form, in my opinion, this manuscript does not meet criteria for publication and must therefore be reject.

A: We feel sorry to realise that our contribution has not found the appreciation of referee n.1. We are afraid that he/she could have possibly overlooked or missed some of the delivered information. With reference to the sampling, Ref.1 seems to have failed to appreciate that the vast majority of the analyzed fish samples came from aquaculture and were preliminary characterized at both morphological and at molecular levels with a panel of SSRs. We have clearly stated this in the M&M section and all over the manuscript, providing references to related projects and publications. This material was purposely and properly used to verify the reliability of our aTBP findings, acting as a gold standard. In addition we have collected samples from a completely different source, that is the fish market, authenticated with the COI marker so to build up a 3 markers crossed/referenced data : SSRs, COI and aTBP, an experimental strategy that serves the purpose of validating the aTBP method. The number of the analyzed species , seven, was limited by the availability of enough individuals and data that could corroborate the 3-markers approach and yet major species of commercial and scientific interests (tuna, sturgeon, gilthead seabream, European seabass, salmonids) have been included. Inclusion of additional fish species would have been redundant not adding more information, and would have made the paper too long and burdensome. With reference to the number of species distinguishable by aTBP, we certainly cannot exclude that out of 20.000 teleost species some aTBP profiles could overlap but that, in our opinion, would also be informative about their close genetic relationships very much similar to what we have shown for S. carpio, postulated as emerging from S. trutta fario as a recent speciation event. The common ancestor argument raised, is very much theoretical and in principle we cannot drop it but the experimental evidences we have collected from two different groups of sampling, fish market and aquaculture, where some intraspecific DNA polymorphisms are shared, tend to exclude it. Anyhow the aTBP tool applied to fish genotyping is also addressing the future, that is the monitoring of population changes that are in progress or will be in response to several adaptation events, thus providing a method that is very convenient , sustainable and affordable for many laboratories.

REF 2

Q: Extra editing is necessary.

A : It has been done in accordance to PLOS ONE style

Q. The authors should discuss about how to deal with polyploid species, which would bring many more variants to improve the practical difficulty in genotyping. By the way, it would be much better if a practical database can be established. These issues should be mentioned in the discussion section.

A: We thank the referee for his/her kind consideration of our work. With reference to ploidy, as it has been documented for sturgeon and the salmonids, known polyploid species with a prevalent 4N and 8N evolutionary/natural scale ploidy (4N functional scale) and very different chromosome numbers (from 54/58 to 240/264), aTBP is well performing and we think that its discrimination capacity could extend even further to a higher ploidy level since the major restriction of the technique is the limit of-resolution of the amplified fragments in CE, that is 1-2 bp. Clearly a higher number of target sequences would increase the number of peaks, not linearly because it would depend from the allelic variance, and this call for a faster reading, recognition and comparison of the profiles. This is the reason we are currently developing a software that could efficiently y compare the aTBP profile of the analyzed samples with authentic profiles of reference. This also calls for the establishment of a dedicated data base, as correctly suggested by the referee. We are doing this within the limit of our possibility because that requires a full collaboration with Institutions that can provide certified material . We are also in contact with some producers (caviar/ sturgeon) to expand our data. As requested by the referee we report these considerations in the discussion section together with a couple of new references.

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Decision Letter 1

Tzen-Yuh Chiang

15 Jun 2020

PONE-D-20-08308R1

aTBP: a versatile tool for fish genotyping

PLOS ONE

Dear Dr. Breviario,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study, the authors initially found size polymorphism in aTBP fragments, and examined this scenario further with evidence in other 7 fish species. The authors concluded the statement below, by the discovery of varying size fragments existing within and between species as well as at inter-population level,

“These data are discussed with respect to the application of the aTBP method to diverse fields of investigation that may include the characterization of a fish population and assessment of its variations in response to environmental changes, the recognition of genetic diversification resulting from hybridization events and studies on parental assignment as well as species traceability, authentication and detection in seafood.”

The above derivation, regarding to the function of aTBP, seems to be a courageous assumption while many unknows are yet, or left to be answered.

However, aTBP is a gene locus residing on the certain region of microsatellite, and the scoped and resolution in population genetic study would be constrained by analyzing various fragment sizes of homologous alleles of single gene, especially limited information revealed by fragment sizes only. Simply speaking, it is rarely, or nearly practical to study population genetic on targeted species through single genetic locus of one single microsatellite gene as found in this study.

Otherwise, fragment size is not recommended studying population genetic structure for its unknown mutation mode or mechanism affecting fragment length difference. For example, a set of homologous genes with similar genetic length may probably have difference nucleotide composition, in which a wrong conclusion might be easily obtained under this assumption.

By thoroughly consideration, despite the significant efforts from the authors, this paper of describing genotyping methodology to fish may present limited value and depth, as not claimed by the authors. Regretfully, I would suggest rejecting this paper as my final decision.

Reviewer #2: As mentioned in my previous comments, the authors should pay much attention to the overall writing of the manuscript. Extra editing from a professional company or a native English speaker is necessary. Other issues are Ok with good answers.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 4;15(8):e0237111. doi: 10.1371/journal.pone.0237111.r004

Author response to Decision Letter 1

24 Jun 2020

REF1 Remarks : This time ref1 objection, different from those made on the first version of the manuscript, seems to be motivated by his/her reluctancy to accept that aTBP can be a useful tool for studying popolulation genetics that is allele variance that may occur in a given popolutation across time and in response to different external changes. We say it seems because his/her consideration, as far as we can tell from his/her wording, starts from an ill-based assumption that is that aTBP is .. a gene locus residing on the certain region of microsatellite (cited) .. . As reported TBP is instead based on intron-length variation occurring in the numerous and different members of the beta-tubulin gene family. Thus, it is not a single locus marker neither a microsatellite sequence, not even … one single microsatellite gene (cited) . We are afraid that this misconception, although limited to a possible application of otherwise unquestioned experimental data, can lead to erroneous conclusions. Nevertheless, since it looks that ref 1 didn’t like the sentence of the abstract referring to the different applications of aTBP, with specific focus on population genetics , we have changed it to please him/her and make it even more fitting to the experimental data.

REF2 remarks : With respect to ref 2 criticism about poor attention to the overall writing of the manuscript …and his/her recommendation for English editing .., please note that the text have been revised by Prof. Khidir Hilu of Virginia Tech, USA and Prof. Sara Patterson, Emeritus at Wisconsin University USA.

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Decision Letter 2

Tzen-Yuh Chiang

21 Jul 2020

aTBP: a versatile tool for fish genotyping

PONE-D-20-08308R2

Dear Dr. Breviario,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: After reviewing the manuscript titled “aTBP: a versatile tool for fish genotyping”, I feel that the manuscript has been significantly improved and satisfied with previous revisions. The data and analysis generally appear to be sound, the results are clear and interesting. In my opinion, this manuscript does meet criteria, and thus I feel should be published.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

Acceptance letter

Tzen-Yuh Chiang

23 Jul 2020

PONE-D-20-08308R2

aTBP: a versatile tool for fish genotyping

Dear Dr. Breviario:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Seabass and pangasius aTBP analysis, complete dataset.

    (XLSX)

    Click here for additional data file. (11.9KB, xlsx)
    S2 Table. Salmonidae aTBP analysis, complete dataset used for PCA.

    (XLSX)

    Click here for additional data file. (25.3KB, xlsx)
    S1 Data. COI sequences, aligned and unaligned, of different fish species.

    (TXT)

    Click here for additional data file. (48.6KB, txt)
    S1 Fig

    (TIF)

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    Attachment

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    Attachment

    Submitted filename: Rebuttal letter_RESPONSE TO REWIEVERS_DBfinal.docx

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    Attachment

    Submitted filename: Comment-0613.docx

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    Attachment

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    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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