756 Chiang Mai J. Sci. 2016; 43(4)

Chiang Mai J. Sci. 2016; 43(4) : 756-766 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Discovery of Insertion-deletion Polymorphism for Identification on Species (Pangasianodon gigas, Pangasianodon hypophthalmus) Kriangsak Mengumphan [a], Nantaporn Sutthi[b], Doungporn Amornlerdpison[a] and Supamit Mekchay* [b] [a] Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiang Mai 50290, Thailand. [b] Department of and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 15 October 2015 Accepted: 20 January 2016

ABSTRACT The (Pangasianodon gigas) and striped catfish (Pangasianodon hypophthalmus) are important fresh water catfish for aquaculture in Thailand. The two species are difficult to distinguish by morphological characteristics, especially in the larvae state. Thus, the aim of this study was to develop DNA markers for identifying these species. Ten potential of amplified fragment length polymorphism (AFLP) were sequenced and six sequence characterized amplified region (SCAR) markers were developed. However, only three markers (Pg1, Pg2 and Hf1) were clear PCR banding in 50 P. gigas, 50 P. hypophthalmus from six locations (10 samples/species/location) and 130 hybrid catfish (P. gigas×P. hypophthalmus). The nucleotide showed insertion and deletion (Ins/Del) polymorphism. The variation of these markers showed with 89.8 % of concordant and 89.49 % of the accuracy test to clearly separated P. gigas from another catfish species. Similarly, P. hypophthalmus showed 86.0 % of concordant and 84.56 % with the accuracy test. Whereas, in the 119 hybrid catfish were showed low concordant and accuracy test as 55.0% and 48.40%, respectively. These results indicated that the three markers have high potential to separate P. gigas and P. hypophthalmus species. However, additional markers are needed to ensure 100% accuracy for identify parental species and hybrids.

Keywords: Pangasiidae catfish, identification, SCAR marker, insertion-deletion polymorphism

1. INTRODUCTION Fish of the family Pangasiidae, such as critically endangered in the wild [2]. These Pangasianodon hypophthalmus Sauvage 1878, are particular two species are closely related and widely cultured in the lower Mekong basin artificial hybrids are used for aquaculture [1]. Some Pangasiidae catfish, Pangasianodon purposes. In term of fish species identification, gigas Chevey 1913, or the Mekong giant catfish, the adult of Pangasiid catfish is traditionally are important conservation targets as they are based on external morphological characters Chiang Mai J. Sci. 2016; 43(4) 757

[3], including body shape, color pattern, scale a short period of time [18]. Conversion of size and count, number and relative position AFLP markers into single-locus markers, of fins, number and type of fin rays, or SCAR is relatively easy and fast [19]. In the various relative measurements of body parts present investigation, we evaluated the [4]. However, in some cases morphology is potential of species-specific SCAR markers of limited value for species identification [5]. that were developed from AFLP approach In the early life stages (egg and larvae) it is for identify P. gigas and P. hypophthalmus. usually more difficult to identify species using morphological characteristics alone [4-6], 2. MATERIALS AND METHODS this creates problems for fish farmers, as 2.1 Sampling and DNA Extraction they cannot distinguish the larvae of different A total of 230 adult Pangsiid catfish species or between pure lines and hybrids. specimens consisting of fifty P. gigas, fifty

Moreover, the flesh of some species are P. hypophthalmus and 130 hybrid catfish (F1 and × similar in appearance and texture; when F2 hybrids were crossbred from P. gigas there is the possibility of substituted cheaper P. hypophthalmus) were collected from variety species for more expensive one this creates of locations (Table 1). Genomic DNA was other problems for markets. DNA-based extracted from fin clip of adult fish samples methods can be useful in helping solve these using the standard TNES-urea phenol- types of problems [7]. chloroform protocol [20]. The concentration Molecular genetic markers continue to be of DNA samples were measured with developed that help improve species a Nanodrop 2000c spectrophotometer identification and clarify relationships. (Thermo Scientific, USA) and genomic DNA Several molecular method such as restriction was measured on 1% of agarose gel fragment length polymorphism (RFLP) [8]), electrophoresis for checking DNA quality. multiplex polymerase chain reaction (PCR) [9]) and real-time PCR [10] have been 2.2 Conversion of AFLP to SCAR adopted in studies involving species Markers identification of food products. The AFLP Following methods described in detail [11] is a PCR based DNA molecular by Sutthi et al. [21], polymorphic bands, one method, which combines the advantage of each specific to Pangasianodon gigas and RFLP and random amplification of Pangasianodon hypophthalmus, were excised polymorphic DNA (RAPD). AFLP is highly from the polyacrylamide gel. The selected reproducible and reliable for the assessment fragment were ligated into pGEM®-T vector of genetic variation among and within (Promega, USA) cloning vector. The ligation populations [12,13]. AFLP does not require reaction was performed in 5 μl of volume, any previously known genetic information, containing 2.5 μl of 2× ligation buffers, and is thus applicable to less well-studied 0.5 μl of vector, 1.5 μl of PCR product fish species [14, 15] for which there is no and 0.5 μl of T4 DNA ligase. The reaction established polymorphic markers, or for was incubated overnight at 4°C and then which there is limited sequence information recombination plasmids were transformed [16,17]. Other advantages of AFLP are the into E. coli strain (DH5α) by heat shock capability to produce multi-locus fingerprints method. The cells were then spread on in a single analysis [11], and the capability to Luria-Bertani (LB) selection medium screen a large number of specimens within containing ampicillin (50 μ/ml), IPTG and 758 Chiang Mai J. Sci. 2016; 43(4)

X-gal, incubated at 37°C for 16 h. The Five gels (with ethidium bromide) in 1 × TAE white colonies, expected to contain an insert, buffer. The gel was photographed under were picked from each plate along with a blue UV- transilluminator. The positive clone colony and confirmed by PCR analysis. was cultured overnight at 37°C in 5 ml To identify clones containing an insert, LB-broth ampicillin. The plasmid DNA one blue and five white colonies of each was extracted by using GenElute™ DNA fragment were picked and suspended Plasmid Miniprep Kit (Qiagen) for sequence in 30 μl of 1 × PCR buffer. PCR reactions analysis. The clone DNA fragments were were performed as standard protocol and sequenced carried out on a CEQ 8000 M13 primers (forward: 5′-TTGTAAAACG Genetic Analysis System (Beckman Coulter) ACGGCCAGT-3′, reverse: 5′-CAGGAAA using Dye Terminator Cycle Sequencing CAGCTATGACC-3′) were used to amplify (GenomeLab™ DTCS) Quick Start Kit the insert DNA fragment with 59°C annealing (Beckman Coulter). Base on the sequence of temperature and 70°C for elongation step. the cloned DNA fragments, six sequences Aliquots of 5 μl of PCR product were were designed as SCAR markers (Table 2). electrophoresed on a 1 % (w/v) agarose

Table 1. Detail of samples, population names, locations and sample size of P. gigas and P. hypophthalmus. population names Locations sample size Maejo University, Chiang Mai province 10 Chiang Mai Inland Fisheries Research and 10 Development Center, Chiang Mai province Chiang Rai Inland Fisheries Research and 10 Pangasianodon gigas Development Center, Chiang Rai Province Jaran farm, Chiang Rai Province 10 Ingkong farm , Phayao Province 10 Maejo University, Chiang Mai province 10 Chiang Mai Inland Fisheries Research and 10 Development Center, Chiang Mai province Pangasianodon hypophthalmus Chiang Rai Inland Fisheries Research and 10 Development Center, Chiang Rai Province Jaran farm, Chiang Rai Province 10 CP farm, Can Tho, Vietnam 10 Hybrid catfish Maejo University, Chiang Mai province 130

(F1 and F2 hybrids were crossbred from P. gigas×P. hypophthalmus) Chiang Mai J. Sci. 2016; 43(4) 759

Table 2. Loci, AFLP primer combinations, SCAR primer sequence and product size range of six primers were use for PCR analysis.

Locus AFLP primer combination SCAR Primer sequence (5′-3′) Product size designed according to nucleotide (bp) sequences of the AFLP markers Pg1 EcoRI-ACG / TaqI-CAC F: TGCAACGAACAAACAAAGTTTC 75 bp R: ACAAAAAGGTCAGGGAACATC Pg2 EcoRI- ACG / TaqI-CAT F: GCTGTCAATCTTCCCAAGAG 125 bp R: TGACGATAAAAGGGGACAGC Ph1 EcoRI- AAG / TaqI-CGA F: CTCTAAGTTTCCCTCCACCA 150 bp R: CAAGATGCTCCAGTATCTCC Hb1 EcoRI- AAG / TaqI-CGT F: GGATCATACCCAGTACAAGT 111 bp R: AAGACTAAGTTGTATTCTTTTAG Hb2 EcoRI- AAC / TaqI-CAT F: AGCTGCCCTTTGCCCTTAT 222 bp R: AATAACCTTTAGTGCTGTACC Hf1 EcoRI- ACG / TaqI-CAT F: CAGTGGAGAAAAAAGTGGAC 197 bp R: GATGAGATTGTGAATTTAGAAG

F = Forward primer, R = Reverse primer

β β β 2.3 PCR Detection of Species-specific logit(LP)= In (Pi/1-Pi) = 0+ 1X1+ 2X2 β Following confirmation of the SCAR + …+ nX n + e (1*) product amplification profile, the specificity and reproducibility of SCAR markers was Where LP = logistic probability of a verified using DNA from all the 50 P. gigas catfish to be predicted as P. gigas or non and 50 P. hypophthalmus individual genotypes. P. gigas (P. hypophthalmus, F1, and F2 hybrid) The PCR amplification included an initial ° β denaturation step at 94 C for 4 min, followed 0 = value of y-intercept by 35 cycles of 94°C for 30 s, annealing at ° ° β β β 58-60 C for 30 s and extension at 72 C 30 s 1, 2,…, n = regression coefficient of ° and final extension was performed at 72 C molecular markers X1, X2…, Xn for 5 min. The PCR were separated on 6-8% polyacrylamide gel electrophoresis and stained X1, X2…, Xn = molecular marker X1, with silver staining. The different bands of X2…, Xn P. gigas and P. hypophthalmus from SCAR markers were sequenced again. eij = residual error term

The best-fit model was selected using a 2.4 Statistical Analysis backward elimination regression, which tends The individual samples of allele and to be the preferred method of exploratory genotype frequencies were analyzed using analyses. Predicted probability to be assigned logistic regression model as follows: of P. gigas species used as follows: 760 Chiang Mai J. Sci. 2016; 43(4)

LP* LP* P*i = e /1+e NCBI (http://blast.ncbi.nlm.nih.gov/Blast.). We converting the AFLP markers to six

Where P*i = predicted probabilities test single-locus SCAR markers, including Pg1, of P. gigas species: if probability ≥ 0.5, it is Pg2, Ph1, Hb1 and Hb2 and Hf1. Six pairs of P. gigas species; if probability < 0.5 it is non SCAR primers were designed and synthesized P. gigas species. according to the above nucleotide sequences (Table 2). From six initially tested SCAR e = value of exponential constant markers, three markers (Pg1, Pg2 and Hf1) gave clear PCR banding patterns. The * LP = ln(Pi/1-Pi) from equation (1*) individual patterns distinguish P. gigas and P. hypophthalmus. The fragment sizes of Pg1, Finally, molecular markers were tested Pg2 and Hf1 markers were 75, 125, and 197 with sensitivity, specificity and accuracy as bp, respectively (Table 2). The amplification described by Zhu et al. [22] as follows: results for this set of SCAR markers showing the insertion-deletion (Ins/Del) polymorphism Sensitivity = number of true positive are illustrated in Figure 1. Nucleotide sequence assessment/number of all positive assessment alignment suggested the presence of Ins/Del polymorphisms in these regions. The Ins/Del Specificity = number of true negative was responsible for length variation in P. gigas assessment/number of all negative assessment and P. hypophthalmus (Figure 2, 3). There are no previous reports of Ins/Del variation in Accuracy = number of correct P. gigas and P. hypophthalmus catfish species. assessments/number of all assessments The Ins/Del has been found previously in expressed sequences (ESTs) in Atlantic salmon Separate logistic regressions were done (Salmo salar) [23], common carp (Cyprinus for other species (P. hypophthalmus and hybrids) carpio) [24] and from mtDNA in Atlantic using similar equations to above. salmon (Salmo salar) [25]. Verma and Serajuddin [26] found Ins/Del in 28S rRNAs 3. RESULTS AND DISCUSSIONS (D8) genes in four fresh water catfish (Ompok 3.1 SCAR Development pabda, O. pabo, O. bimaculatus and Wallago attu). Ten polymorphic bands were found to be significant associated with specifics 3.2 Detection of SNP Polymorphism species using logistic regression procedure [21]. Clones of three specific markers (Pg1, Screening from twenty primer combinations Pg2 and Hf1) from P. gigas and P. hypophthalmus of sixteen Pangasiid catfish (P. gigas, were sequenced. The nucleotide sequence of

P. hypophthalmus, F1 and F2 hybrid) generated a the Pg1, Pg2 and Hf1 from P. gigas compared total 487 AFLP fragment and 335 markers with P. hypophthalmus showed the insertion- [21]. Ten polymorphic bands recovered from deletion (Ins/Del) polymorphism as 15 bp the AFLP gels were cloned and sequenced. (TTCCTCCTATAGTGT), 8 bp (GTATCA No homologous sequences were found in the GG) and 18 bp (CCATTTTTTTTTCCC GenBank database after using BLASTn from TGG), respectively (Figure 2). Chiang Mai J. Sci. 2016; 43(4) 761

Figure 1. Pg1 Pg2 and Hf1 markers amplified in P. gigas and P. hypophthalmus. (a) Species- specific band of Pg1 marker from P. gigas (1-5) about 75 bp and P. hypophthalmus (6-10) about 60 bp (b) Species-specific band of Pg2 marker was from P. gigas (1-5) about 133 bp and P. hypophthalmus (6-10) about 125 bp (c) Species-specific band of Pg1 marker was from P. gigas (1-5) about 215 bp and P. hypophthalmus (6-10) about 197 bp and M = DNA ladder 100 bp.

Figure 2. The insertion-deletion polymorphism (gray mark) of three specific markers (Pg1, Pg2 and Hf1) from P. gigas (A) and P. hypophthalmus (B) were sequenced analysis. 762 Chiang Mai J. Sci. 2016; 43(4)

Figure 3. Genotyping profile of three markers (Pg1, Pg2 and Hf1) in hybrid catfish (Pangasianodon gigas x Pangasianodon hypophthalmus). Lane M = 100 bp DNA marker, lane 1-10 = variation of homozygous (AA and BB) and heterozygous (AB) genotype.

3.3 Genetic Variation 0.252), respectively. Similarly, P. hypophthalmus The reliability of our SCAR markers was from hatchery stocks in Bangladesh showed tested using samples of 50 P. gigas and 50 an averaged of He values as 0.141 [28]. P. hypophthamus collected from six locations All allele frequencies for the three markers and 130 hybrid (F1 and F2) individuals were none significantly different according to (Table 1). Three primers (Pg1, Pg2 and Hf1) Chi-square test in purebred. Thus, these were tested for twice using the same set of markers were in Hardy-Weinberg equilibrium DNA samples of each species and identical (HWE) (Table 3). Nonetheless, 130 individuals running condition in every analysis. The allele of hybrid catfish (P. gigas x P. hypophthalmus), frequencies of a common allele for P. gigas only 119 individuals had clear band and thus in each marker (allele A) ranged from were analyzable (Figure 3). For this set of 89.534 to 96.875% (Table 3). Whereas, the hybrid samples the allele frequencies of A and common allele for P. hypophthalmus in each B at three loci ranged from 37.401 to 55.859 marker (allele B) ranged from 85.185 to and 44.140 to 62.598%, respectively. The

93.519%. The average observed (Ho) and average Ho and He values of three SCAR expected (He) heterozygosities of three SCAR markers were 0.334 (0.228 to 0.438) and 0.484 markers for P. gigas were 0.138 (0.062 to (0.468 to 0.493), respectively. Only the Pg2 0.209), and 0.126 (0.060 to 0.187), respectively marker was in HWE (Table 3). These result

(Table 3). However, our He values was lower indicated that, hybrids catfish are not in HWE, than P. gigas that caught from the Mekong it may be the reason from mixed samples

River (He = 0.410) [27]. On the other hand, between F1 and F2 generations. And the the average observed (Ho) and expected (He) population size is always finite and the heterozygosities for P. hypophthalmus were frequency of an allele may fluctuate from 0.148 (0.092 to 0.222), and 0.164 (0.121 to generation to generation. Chiang Mai J. Sci. 2016; 43(4) 763

Table 3. Genetic variation based on three SCAR markers of the Pangasianodon gigas and Pangasianodon hypophthalmus.

Fish species No. of Markers Alleles frequency Ho He P- values HWE samples f(A) f(B) P. gigas Pg1 92.857 7.142 0.142 0.132 0.865 * 50 Pg2 96.875 3.125 0.062 0.060 0.975 * Hf1 89.534 10.465 0.209 0.187 0.745 * Average 93.089 6.911 0.138 0.126 P. hypopthalmus Pg1 14.814 85.185 0.222 0.252 0.679 * 50 Pg2 6.481 93.519 0.129 0.121 0.878 * Hf1 6.482 93.518 0.092 0.121 3.221 * Average 9.259 90.740 0.148 0.164 Hybrid Pg1 55.859 44.140 0.335 0.493 0.001 - 119 Pg2 43.461 56.538 0.438 0.491 0.417 * Hf1 37.401 62.598 0.228 0.468 4.36E-08 - Average 45.574 54.425 0.334 0.484 Ho= observed heterozygosity, He = expected heterozygosity, Significant difference (P-values <0.05), df = 2, The asterisk * = data in Hardy-Weinberg Equilibrium (HWE).

3.4 The Predicted and Accuracy Tested may be from the whole samples between of Three Markers F1 and F2 hybrids and their homozygotes Logistic regression equations based on frequency quite high, it seems to be gene of three markers (Pg1, Pg2 and Hf1) was estimated hybrids was segregated. However, in pure to for identify P. gigas, P. hypophthalmus line (P. gigas and P. hypophthalmus) markers and hybrid catfish are shown as Table 4. have almost fixed differences in allele The results found that, the percentage of frequencies (allele A and B). concordant for three markers were higher Previous studies of Pangasiid catfish in P. gigas (89.8%) and P. hypophthalmus (86.0%) have normally focused on genetic diversity than hybrid catfish (55.0%). Similarly, using microsatellites markers and the percentage of accuracy of the three mitochondria DNA [27, 29-32]. However, markers tested for P. gigas, P. hypophthalmus microsatellites may not detect genetic and hybrids were 89.49, 84.56 and 48.40, differences among closely related breeds [33]. respectively. Moreover, percentage of Sriphairoj et al. [34] distinguished P. gigas, sensitivity and specificity are shown in P. hypophthalmus, bocourti, and Table 5. Pangasius larnaudii using an AFLP approach The concordance and accuracy tests were and SSCP technique to screen potential high indicating that P. gigas could be clearly species-specific markers. Our study is the separated from another catfish species, first report of using Ins/Del polymorphism P. hypophthalmus, and vice versa. As expected, to distinguish P. gigas and P. hypophthalmus according to Mendelian inheritance, 119 catfish species. Higher reliability in species hybrid catfish showed that low concordance identification would be possible if we could and accuracy test scores (Table 4, 5). The low expand number of markers beyond the potential of three markers in hybrid catfish three identified here. 764 Chiang Mai J. Sci. 2016; 43(4)

Table 4. Estimates value of logistic regression, P-value and odds ratio for identify Pangasianodon gigas, Pangasianodon hypophthalmus and hybrid.

Species Variant Parameter P-value(Pr>X2) Odds ratio ± P. gigas Intercept 0.8516 0.2977 0.0042 - ± Pg1 -0.00445 0.5895 0.9940 0.996 ± Pg2 -2.6722 0.6601 <.0001 0.069 ± Hf1 -1.2598 0.4724 0.0077 0.284 Percent Concordant = 89.8, Percent Discordant 2.7, Percent Tied = 7.5 ± P. hypophthalmus Intercept -6.8871 1.1416 <.0001 - ± Pg1 0.8143 0.3544 0.0216 2.258 ± Pg2 1.8408 0.4644 <.0001 6.301 ± Hf1 1.1822 0.4388 0.0071 3.262 Percent Concordant = 86.0, Percent Discordant 6.5, Percent Tied = 7.5 ± Hybrids Intercept 0.0799 0.2469 0.7462 - ± Pg1 -0.6141 0.2474 0.0130 0.541 ± Pg2 0.2028 0.2330 0.3841 1.225 ± Hf1 0.4577 0.2176 0.0354 1.580 Percent Concordant = 55.0, Percent Discordant 34.9, Percent Tied = 10.1

Table 5. Percentage of sensitivity, specificity and accuracy test of molecular markers for P. gigas, P. hypophthalmus and hybrid.

species n % sensitivity % specificity % accuracy test P-value P. gigas 50 80.952 91.525 89.497 <.0001 P. hypophthalmus 50 64.150 91.566 84.566 <.0001 Hybrid 119 79.032 8.421 48.401 0.011

4. CONCLUSIONS ACKNOWLEDGEMENTS In present study, we demonstrate the This research is supported by Chiang Mai successful development of three species- University Post-Doctoral Fellowship. specific three Ins/Del markers to distinguish We gratefully thank the Thailand Research P. gigas from P. hypophthalmus. These markers Fund through the Royal Golden Jubilee Ph.D. were effective in differentiating P. gigas and Program (PHD/ 0149/ 2554) for partially P. hypophthalmus. The markers cannot, however, supported financial. And we would like to be used to separate the hybrids catfish from thank The Plabuk Integration Knowledge the pure lines (P. gigas and P. hypophthalmus). Base, Faculty of Fisheries Technology and These markers provide a powerful, easy and Aquatic Resource, Maejo University and rapid method for ensuring traceability pure the Center of Excellence on Agricultural lines for the aquaculture industry. However, Biotechnology, Science and Technology additional markers are needed to ensure 100% Postgraduate Education and Research accuracy for identify parental species and Development Office, Office of Higher hybrids. Education Commission, Ministry of Chiang Mai J. Sci. 2016; 43(4) 765

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