Iranian Journal of Fisheries Sciences 11(2) 415-423 2012 Identification of bester hybrids (female huso Linnaeus, 1758 and male ruthenus Linnaeus, 1758) using AFLP molecular technique

Yarmohammadi M.*1, 2; Shabani A.2; Pourkazemi M.1; Baradaran Noveiri S.1 Received: January 2011 Accepted: June 2011

Abstract In this study Amplified Fragment Length Polymorphism (AFLP) was applied to species identification of bester hybrids. Hybrids identification was performed by comparison of electrophoresis profiles with parental species. The simultaneous occurrence of diagnostic bands fixed in the parental species, genetic distance and identification and cluster analyses (UPGMA) allow a correct identification. We used 8 primer combinations (Eco+3, Mse+4) and a total of 250 bands (size range 40-1000 bp) were generated. Primer combinations of (E- AAT, M-CGAT) and (E-AAG, M-CGAT) produced diagnostic bands in hybrids and parental species. Moreover, the results of genetic identification showed that Bester hybrids are more similar to (Huso huso) (0.68) in comparison with sterlet (Acipenser ruthenus) (0.45). The results suggested that this technique could be suitable for precise identification of species and inter-generic hybrids like bester.

Keywords: AFLP, Hybrid identification, Bester, Beluga

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______1-Genetics Department, International Research Institute, P.O. Box 41635-346, Rasht, Iran. 2- Fisheries Department, Gorgan Agricultural Sciences and Natural Resources University, PO Box: 336, Gorgan, Iran. *Corresponding author’s email: [email protected]

416 Yarmohammadi et al., Identification of bester hybrids using AFLP … Introduction Fishes of the order , widely as taxonomical markers (Alarcon comprising the (Acipenseridae) and Alvarez, 1999). Among molecular and (Polyodontidae) have techniques, the AFLP (Amplified luxury and expensive (Flynn et al., Fragment Length Polymorphism) 2006). Because of the high commercial technique allows obtaining a very high demands for sturgeons and due to their number of dominant nuclear markers. The endangered status, the high occurrence of results are similar to those obtained by sturgeon hybridization, both in the wild Random Amplified Polymorphic DNA and in aquaculture, the necessity of (RAPDs) (Williams et al., 1990), however, valuable techniques for hybrid the AFLP markers exhibit a higher (98- identification in both manufactured 99%) reproducibility (Questiau et al., products for commercial monitoring, and 1999) and a stronger mendelian live samples to guarantee correct segregation. This technique has received restocking programs is felt (Congiu et al., increasing attention for study of 2002). During the recent decade, sturgeon hybridization (Tranah et al. 2003). At the farming has been focused and many hybrid identification level AFLP has countries are doing extensive activities on successfully been used to differentiate farm meat and caviar production (Congiu populations of sturgeon (Congiu et al. et al., 2002). Bester which is produced by 2003) and other (O'Hanlon et al., crossing beluga (Huso huso) female and 1999; Bensch et al., 2002). sterlet (Acipenser ruthenus) male, is The goals of the present study were suitable for aquaculture because it shows to investigate the utility of AFLP to find better growth rates than its males and markers that recognize bester hybrid from earlier sexual maturation than its female its parental species that could easily be parents (Burtsev, 1997). In recent years, applied in any molecular biology Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021 the bester has been cultured not only in laboratory. Russia but also in other countries, such as Germany, Hungary and Japan (Omoto et Materials and methods al., 2005) Samples Earlier, hybrid identification was 12 individuals from three species were largely based on morphometric characters, analyzed to verify the usefulness of the which could be biased or inaccurate AFLP technique for identification 1 A. measures of hybrid status (Tranah et al., ruthenus (male parent), 1 H. huso (female 2003). Recently molecular advances have parent), 5 H. huso as control group of improved the ability of hybrid progeny and 5 besters (female H. huso× identification and so it could greatly male A. ruthenus). All samples were enhance the ability of susceptible provided at Propagation and Genetic population's management. However, departments of International sturgeon protein markers, such as immunological or Research Institute, Rasht. Iran. electrophoresis methods have been used DNA extraction

Iranian Journal of Fisheries Sciences, 11(2), 2012 417

DNA was extracted using Ammonium 94oC, 30 sec annealing at (56oC) and 1 min Acetate procedure modified from Roche extension at (72oC) followed by 22 cycles. DNA extraction Kit. A small part of the For selective amplification, preliminary caudal fin was homogenized in 600 μl STE trials were carried out using 8 different buffer (50 mM Tris, 20 mM EDTA, 100 primer combinations. Selective mM Nacl, pH 8) and 20 μl SDS 20% and amplification PCR was performed in 15 μl incubated with proteinase K overnight, volumes, with 3 μl diluted (1:10) followed by centrifugation at 12000 rpm preamplification product as template, 1X

for 10 min. The supernatant was PCR buffer with 1.5 mM MgCl2, 0.2 mM transferred to new tubes and an equal of each dNTPs (Cina Gene, Tehran, Iran), volume of 100% ethanol (-20 C) was 10 pmol Mse+4 and ECO+3 primers: PCR added. DNA was pelleted by consisted of 1 min denaturation (94oC), 10 centrifugation (12000 rpm, 10 min) and cycles of 30 s (94oC), 30 s (63oC) and 2 washed twice with 70% ethanol before min (72oC) with the annealing temperature being air-dried and re-suspended in 100 μl decreasing from 63oC by 1oC increments autoclaved nanopure water. The quality in cycles 2- 10. This was followed by 26 and quantity of extracted DNA was cycles of 30 s (94oC), 30 s (54oC) and 2 assessed by 1.0% agarose gel min (72oC). electrophoresis and spectrophotometer Electrophoresis of AFLP fragments (Cecil, CE2040, Italy), respectively After polymerase chain reaction, an equal (Chakmedooz, 2005). volume of loading buffer (98% formamide, AFLP reactions 10 mM EDTA, 0.1% bromophenol blue, AFLP procedures including all adapter 0.1% xylene cyanol) was added. The and primer sequences were used from Vos reaction mixture was then heated for 10 et al. (1995) with some modification. An min at (95oC) and immediately cooled on aliquot of total DNA (250 ng) was ice. The gel was pre-electrophoresed at Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021 digested using 10 U MseI and ECORI in (55oC), 95 W for 20 min, then 5μl of the Tango buffer (Fermentas, France) in a 20 amplified DNA was loaded and run on a μl reaction at 37oC for 90 min. Ligation 6% polyacrylamide gel (19:1 was started immediately by a 5 μl mix of acrylamide/bis; 7.5 M Urea; 1X TBE MseI (50 mM) and ECORI (5 mM) buffer) with 1X TBE buffer on a vertical

adapters, T4 DNA Ligase and 1X ligation gel electrophoresis system (BIO-RAD buffer. Ligation was carried out at 37oC for sequi Gen, GT, 38 ×30 cm/ PowerPAC 3 h. Preamplification PCR was performed 5000) at 85 W and 50oC for 90 min. After in 15 μl volumes, with 5 μl diluted electrophoresis, the bands were visualized restricted/ligated DNA, 1X PCR buffer, using silver staining. Clear and 1.5 mM Mgcl2, 0.2 mM of each dNTPs, unambiguous bands in length ranging from 10 pmol MseI + C and ECORI+A primers. 40 to 1000 bp were considered as usable. PCR was carried out with an Eppendorf Fragment size (bp) was determined with thermocycler (Mastercycle ep gradiant, 96 ladder VIII (Roche) and scored manually. plus, Germany) with 30 sec denaturation at Data analysis

418 Yarmohammadi et al., Identification of bester hybrids using AFLP … Only the recognizable AFLP markers Determination of AFLP primers for were scored. The scored AFLP bands were H.huso, A.ruthenus and bester dark, consistent and repeatable in The resolution of AFLP pattern is individuals across polymerase chain determined by the genome complexity, the reaction and gels. The binary matrix was GC content and the number of selective built up from AFLP patterns, attributing nucleotides in the AFLP primer "1" to presence and "0' to the absence combinations (Zhang et al. 2004). Based (Zhang et al., 2004). Genetic distance and on the AFLP analysis, 8 primer genetic identification was constructed combinations of E+3 and M+4 were using the Gene Alex and cluster analysis, chosen and tested for AFLP analyses of then performed to create a dendogram this study and produced 250 score able using the unweight Pair- Group Method bands between 40 to 1000 bp from the with an Arithmetic mean (UPGMA) by combination of E-AAT, M-CGAT NTSYS software (version 2.02e). produced more AFLP markers (14 bands) Potential species – specific bands should than the other combination and 7 bands be indicated by a high frequency in one were present in sterlet but hybrids were parental species and the hybrid and a low absent in beluga (Fig.1) and the frequency in the other parental species and combination E-AAT, M-CACA produced the hybrids should share specific bands less AFLP markers (5 bands, table 1). The from two parents. combination of E-AAG, M-CACA Results produced no bands.

Table 1: Number of amplified and polymorphic bands scored from AFLP analysis in beluga, sterlet and Bester hybrids

No. of Total no. of No. of Percentage of No. of species- Percentage of Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021 individuals bands amplified polymorphic polymorhic specific bands species- studied bands bands specific bands 12 250 69 28 7 12

Table 2: Pairwise Population Matrix of Nei (1978) Genetic Distance

Iranian Journal of Fisheries Sciences, 11(2), 2012 419

Figure 1: AFLP analysis with use of M-CGAT, E-AAG primer combination s: Male sterlet, b: Female beluga , H1-H5: Hybrid bester, M: PUC Mix Marker, 8 (Fermentas, France)

These 7 bands occurred in all hybrids, between sterlet and beluga, the lowest making them strong candidates for species genetic distance (0.37) was between markers in H. huso and A. ruthenus. beluga and bester, and the genetic distance Species relatedness between sterlet and bester was (0.79). The genetic distances are given in table 2. The pairwise population matrix of The pairwise population matrix of Nei Nei (1978) genetic identity ranged from (1978) genetic distance between 3 species 0.66 to 1. The genetic identity between ranged from 0.377 to 0.830. The highest beluga and bester was (0.68) and it was genetic distance (0.83) was obtained 0.45 between bester and sterlet (table 3). Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021

Table 3: Pairwise Population Matrix of Nei (1978) Genetic Identity

In UPGMA dendogram, three groups were relationship to beluga and bester in the formed: (1) A. ruthenus (2) Huso huso and neighbor- joining tree and sterlet as a (3) Bester hybrids. The neighbour-joining distinct cluster from beluga and bester tree between species showed close (Fig. 2).

420 Yarmohammadi et al., Identification of bester hybrids using AFLP …

Sterlet

Bluga

BlugaC2

BlugaC4

BlugaC1

BlugaC3

BlugaC5

Bester2

Bester1

Bester3

Bester4

Bester5

0.19 0.39 0.60 0.80 1.00 Coefficient Figure 2: UPGMA dendogram base on AFLP fingerprinting of 12 sturgeons analyzed: 1 male starlet (S), 1 female beluga (b), 5 control beluga larvae and 5 bester hybrid larvae (H1-H5). M: Molecular Ladder

Discussion Several polymerase chain reaction-based of genetic constitution of the organism molecular methods of genotype analysis under investigation, and AFLP have been developed for identification of fingerprinting techniques show high

Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021 fish (Grewe and et al., 1993). Sequence reproducibility and a large number of analysis of species-specific clearly separate markers (Vos et al. 1995). mitochondrial/ribosomal DNA fragments These advantages make it suitable for and multiplex PCR of species - conserved distinguishing genetic differences at the DNA fragments are efficient for fish species level and below (Vos et al., 1995). species identification (Bartlet and AFLP has successfully been applied to Davidson, 1991; Rocha-Olivares, 1998; identification and typing of bacteria and Gharrett et al., 2001; Noell et al., 2001, plants (Coeny et al., 1999; Wong et al., Wasko et al., 2001). Compared with the 1999; Tyrka, 2002), marine animals DNA sequence method above, DNA (Ogden and Thorpe, 2001) and sturgeons fingerprinting is quicker and cheaper. In (Congiu et al., 2002). addition, the experimental requirements Han and Ely (2002) demonstrated are lower (Cordes et al., 2001; Sebastio that AFLP is a useful technique for the and Neri, 2001). Large numbers of assessment of hybrid variations of fish. molecular markers by AFLP can be Furthermore, the species-specific patterns generated without any previous knowledge produced by AFLP can be used for

Iranian Journal of Fisheries Sciences, 11(2), 2012 421

identification of closely related species. In detectable (Birstein and Doukakis, 2000), the present study, the AFLP patterns for but in many cases in lower growth stages parent and larvae from control group of of bester and H. huso, they are beluga were 98-100% identical, and AFLP morphologically similar to each other patterns of bester larvae with sterlet and (Kozlov, 1993). beluga parents could easily be identified. Congiu et al. (2002) identified There were a total of 69 AFLP loci based species- specific markers for natural on the size of AFLP marker, and the species of A. naccarii, A. baerii and A. number of AFLP markers for each species transmantanus and also commercial varied from 5 to 14, but only 7 markers samples of meat and caviar of 10 sturgeon were fixed to all species. The reliability of species and interspecific hybrid of A. AFLP analysis in species identification of naccarii × A. baerii with AFLP procedure. sturgeon hybrids which was confirmed by The result of the present genetic analysis the fact of the highest genetic similarity showed that bester is genetically similar to (corresponding to the lowest genetic female parent or beluga that is in distance) was recognized between H. huso accordance with the results of and bester hybrids on the basis of 0.68 of morphological characteristics and data different AFLP markers. The AFLP from bester hybrids growth (Kozlov, technique proved to be highly reliable and 1993). reproducible on sturgeon samples. Although, band sharing among species Acknowledgements was found to be lower than within species, We are grateful to everyone who assisted the AFLP profiles detected a relevant us in this research. Our appreciation goes intergeneric variation allowing to Mr. Hossein Mohammadi Parashkooh identification of samples from unknown and Mahmood Mohseni for producing origins. bester and sample preparations.

Downloaded from jifro.ir at 1:08 +0330 on Monday September 27th 2021 The resolution power of AFLP may decrease for species in which the analyzed Refrences phylogenetic distance is lower. However, Alarcon, J. A. and Alvarez, M. C., 1999, H. huso and bester hybrids are two closely- Genetic identification of sparid related species (according to their genetic species by isozyme distance and similarity in this research), markers:application to interspecific and they are clearly distinguishable by the hybrids. Aquaculture, 173, 95-103. present approach. Therefore, since the Bartlet, S. E., Davidson W. S.,1991. AFLP technique allows random analysis of Identification of Thunnus tuna species the entire genomic DNA, it should be by the polymerase chain reaction and possible to find diagnostic markers by direct sequence analysis of their simply performing further amplifications mitochondrial cytochrome b genes. with different selective bases (Congiu et Canadian Journal Fish.Aquatic al., 2002). However, the species of A. Science, 48, 309-317. ruthenus and H. huso are morphologically

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