296 4ο Γιεθνές Σσνέδριο “Υδροβιολογίας – Αλιείας”, Βόλος 9-11 Ιοσνίοσ 2011 ΑΝΙΥΝΕΤ΢Η ΓΟΝΙΔΙΑΚΩΝ ΣΟΠΩΝ ΠΟ΢ΟΣΙΚΩΝ ΙΔΙΟΣΗΣΩΝ (QTL) ΢ΣΗΝ Σ΢ΙΠΟΤΡΑ (Sparus aurata L.) Λοσκοβίηες Δ.1,4*, Σαρροπούλοσ Ε.2, Μπαηαργιάς Κ.3, Αποζηολίδες Α.4, Κωηούλας Γ.2, Τζιγγενόποσλος Κ.2, Χαηδεπλής Δ.1 1 2 3 4 Αιεμάλδξεην Τερλνινγηθό Δθπαηδεπηηθό Ίδξπκα Θεζζαινλίθεο (Α.Τ.Δ.Η.Θ.), Σρνιή Τερλνινγίαο Γεωπνλίαο, Τκήκα Εωηθήο Παξαγωγήο, Τ.Θ. 141, 57400, Σίλδνο Διιεληθό Κέληξν Θαιαζζίωλ Δξεπλώλ, Ηλζηηηνύην Θαιάζζηαο Βηνινγίαο θαη Γελεηηθήο, Τ.Θ. 2214, Γνύξλεο Πεδηάδνο, 715 00 Ζξάθιεην Κξήηεο Τερλνινγηθό Δθπαηδεπηηθό Ίδξπκα Μεζνινγγίνπ, Τκήκα Υδαηνθαιιηεξγεηώλ θαη Αιηεπηηθήο Γηαρείξηζεο, Νέα Κηίξηα, 30200, Μεζνιόγγη Αξηζηνηέιεην Παλεπηζηήκην Θεζζαινλίθεο, Γεωπνληθή Σρνιή, Τνκέαο Εωηθήο Παξαγωγήο, Δξγαζηήξην Ηρζπνθνκίαο-Αιηείαο, Λεωθόξνο Γεωξγηθήο Σρνιήο, Θεζζαινλίθε Περίληψη Ζ ηζηπνύξα απνηειεί έλα νηθνλνκηθήο ζεκαζίαο είδνο ζηελ Μεζνγεηαθή πδαηνθαιιηέξγεηα. Πξαγκαηνπνηήζακε κία αλάιπζε κέξνπο ηνπ γνληδηώκαηνο γηα ηελ ραξηνγξάθεζε γνληδηαθώλ ηόπωλ πνζνηηθώλ ηδηνηήηωλ (QTL) πνπ επεξεάδνπλ ην ζωκαηηθό βάξνο θαη ην θύιν ζε απηό ην εξκαθξόδηην είδνο. Πξνεγνύκελε εξεπλεηηθή κειέηε έδεημε ηελ ύπαξμε δύν ζηαηηζηηθά ζεκαληηθώλ QTL ζηελ νκάδα ζύλδεζεο 21 (LG21). Σηελ παξνύζα εξγαζία πξνζζέζακε πεξηζζόηεξε πιεξνθνξία ζηα γνλνηππηθά δεδνκέλα καο ώζηε λα δηεμάγνπκε κηα εθηελέζηεξε κειέηε ηεο γελεηηθήο βάζεο ηωλ παξαπάλω ηδηνηήηωλ. Γηα ην ζθνπό απηό ρξεζηκνπνηήζεθαλ δείγκαηα από δέθα παηξνγνληθέο εηεξνζαιείο νηθνγέλεηεο ηζηπνύξαο θαη ζπλνιηθά 74 πιεξνθνξηαθνί κηθξνδνξπθνξηθνί δείθηεο γηα ηελ θαηαζθεπή ελλέα νκάδωλ ζύλδεζεο, ζπλνιηθνύ κήθνπο 495,3 cM θαη κέζε απόζηαζε δεηθηώλ 8,2 cM. Δθαξκόζζεθαλ νη ζηαηηζηηθέο κέζνδνη ηεο αλάιπζεο δηαζηήκαηνο θαη γξακκηθήο παιηλδξόκεζεο, αιιά θαη ηεο αλάιπζεο πνιιαπιώλ QTL ώζηε λα απμεζεί ε δπλαηόηεηα ραξηνγξάθεζεο. Σπλνιηθά εληνπίζηεθαλ ηέζζεξα QTL γηα βάξνο θαζώο θαη έμε QTL γηα θύιν (ηξία δεπγάξηα ζε ηξεηο δηαθνξεηηθέο νκάδεο ζύλδεζεο) ζύκθωλα κε ην κνληέιν ηωλ δύν ζπλδεδεκέλωλ QTL. Τν πνζνζηό ηεο θαηλνηππηθήο παξαιιαθηηθόηεηαο πνπ εμεγείηαη από ηα QTL γηα βάξνο θπκάλζεθε από 9,3% κέρξη 17,2%, ππνγξακκίδνληαο έηζη ηελ δπλαηόηεηα γηα ρξεζηκνπνίεζή ηνπο ζηελ ‘ππνβνεζνύκελε από δείθηεο’ επηινγή (MAS). Τξεηο νκάδεο ζύλδεζεο παξνπζίαζαλ ζηαηηζηηθή ζεκαληηθόηεηα γηα ηελ ύπαξμε QTL πνπ επεξεάδνπλ ηόζν ην βάξνο όζν θαη ην θύιν, γεγνλόο πνπ δείρλεη ηελ γελεηηθή ζπζρέηηζε ζε έλα βαζκό ηωλ δύν απηώλ ηδηνηήηωλ ζηελ ηζηπνύξα. Δπίζεο, ε κειέηε ηνπ κνξηαθνύ κεραληζκνύ πνπ νδεγεί ζηελ αιιαγή ηνπ θύινπ κπνξεί λα πξνζθέξεη λέα γλώζε ζην πνιύπινθν ζύζηεκα θαζνξηζκνύ ηνπ θύινπ αιιά θαη ηεο εμέιημεο απηνύ. Λέξεις κλειδιά: QTL, Sparus aurata, σδατοκαλλιέργειες * Σπγγξαθέαο επηθνηλωλίαο: Λνπθνβίηεο Γεκήηξηνο ([email protected]) QUANTITATIVE TRAIT LOCI FOR BODY GROWTH AND SEX DETERMINATION IN THE HERMAPHRODITE TELEOST FISH Sparus aurata L. Loukovitis D.1,4*, Sarropoulou E.2, Batargias C.3, Apostolidis A.P.4, Kotoulas G.2, Tsigenopoulos C.S.2, Chatziplis D.1 1 2 Αlexander Technological Educational Institute of Thessaloniki (A.T.E.I.Th), School of Agricultural Technology, Department of Animal Production, Laboratory of Animal Breeding and Genetics, Sindos, 57400, Greece Hellenic Center for Marine Research, Institute of Marine Biology and Genetics, Gournes Pediados, Heraklion, 71003, Crete, Greece 4th International Symposium “Hydrobiology – Fisheries”, Volos 9-11 June 2011 297 4ο Γιεθνές Σσνέδριο “Υδροβιολογίας – Αλιείας”, Βόλος 9-11 Ιοσνίοσ 2011 3 Technological Educational Institute of Messolonghi, School of Agricultural Technology, Department of Aquaculture and Fisheries, Laboratory of Molecular Population and Quantitative Genetics, Messolonghi, 30200, Greece 4 Aristotle University of Thessaloniki, Faculty of Agriculture, Department of Animal Production, Laboratory of Ichthyology and Fisheries, Thessaloniki, 54124, Greece Abstract Gilthead seabream (Sparus aurata L.) is an important marine foodfish in the Mediterranean aquaculture. A partial-genome scan was conducted to map quantitative trait loci (QTL) affecting body weight and sex in this sequential hermaphrodite species. Fish from ten paternal half-sib families were included and 47 informative microsatellite markers were used to construct five linkage groups (2, 3, 6, 9 and 21) of S. aurata genome with a total sex-average length of 237.5 cM (Kosambi) and mean marker interval of 5.9 cM. Interval mapping by linear regression was applied and extended to a multiple-QTL analysis approach to increase mapping power. In total, two growth-related QTL were found and four sex-linked QTL (two pairs in two different LGs) were detected under the two-QTL model of analysis. The proportion of phenotypic variation explained by the two body weight QTL was 9.3 and 17.2 %, revealing their potential for use in marker-assisted selection (MAS). In conclusion, two out of five examined linkage groups showed significant evidence for QTL affecting both growth and sex, suggesting that the two traits are genetically correlated to some extent in seabream. Studying the molecular mechanism of sex reversal may also elucidate the complex mechanistic of the sex determination process and its evolution. Keywords: QTL, Sparus aurata, aquaculture. *Corresponding author: Loukovitis Dimitrios ([email protected]) 1. Introduction Quantitative genetic variation characterizes most traits of economic importance in livestock, including growth-related traits and disease resistance. Variation in such complex traits is often controlled by a number of different loci, named Quantitative Trait Loci (QTL), as well as environmental factors. The identification of QTL in nonmodel species like the gilthead seabream (Sparus aurata) is of importance in order to unravel specific biological questions which cannot be addressed in model-organisms. In addition, by identifying QTL in commercial important species will enhance the application of marker-assisted breeding for genetic improvement of productive traits. Simulation studies have shown that the utilization of marker information might be extremely helpful toward time- and cost-efficient breeding programs by increasing the accuracy of selection and decreasing the generation interval, compared to selection based only upon records of individuals and their relatives (Smith and Simpson 1986, Lande and Thomson 1990). In the present study, we used the seabream genetic map (Franch et al. 2006) to conduct a partial genome scan for the identification of possible QTL affecting body weight and sex. 2. Materials and Methods Ten paternal half-sib families were used to search for body weight- and sexassociated QTL. Pedigree structure was obtained after parentage analysis with nine microsatellite markers. Overall, 10 male and 48 female brooders contributed to the population structure having a total number of 409 offspring, with 360 (88%) being males and 49 (12%) being females. Genomic DNA was isolated from fin clips, by standard proteinase K digestion, following the salting out procedure as described in Miller et al. (1988). Forty-seven (47) informative microsatellite markers were chosen from the published genetic linkage map (Franch et al. 2006) of S. aurata, to cover five linkage groups totally (2, 3, 6, 9 and 21). These were genotyped on all samples (parents 4th International Symposium “Hydrobiology – Fisheries”, Volos 9-11 June 2011 298 4ο Γιεθνές Σσνέδριο “Υδροβιολογίας – Αλιείας”, Βόλος 9-11 Ιοσνίοσ 2011 and offspring) using an ABI PRISM® 3700 DNA analyzer (Applied Biosystems, CA, USA). The construction of the linkage groups, the order and the distance between the genotyped microsatellite markers was done from the data using the CRIMAP 3.0 software (Green et al. 1990). Given the seabream family structure in our study, the QTL detection method was based on half-sib interval mapping analysis through a linear regression approach (Knott et al. 1996), testing also for the existence of multiple QTL on the same linkage group by fitting a two-QTL model to the analysis. The web-based software GridQTL 1.3.2 was used to perform the analysis. Furthermore, an estimation of the percentage of within-family variance explained (PVE) by the QTL was also calculated. 3. Results The 47 microsatellites were used to construct five linkage groups of S. aurata genome with a total sex-average length of 237.5 cM (Kosambi) and mean marker interval of 5.9 cM. Mapping of markers was consistent with the existing LG map of seabream, although their order and distance were not totally preserved. QTL analysis for body weight revealed one genome-wide significant QTL and one chromosome-wide significant QTL on linkage groups 21 and 9, respectively (Table 1). The percentage of within-family variance explained (PVE across all 10 families) by the QTL on LG21 was 17.2%. On the other hand, fitting one-QTL model in the analysis for sex failed to detect significant QTL on any of the linkage groups tested. When assuming a two-QTL model, however, two linkage groups (LG9 and LG21) showed chromosome-wide significant evidence for the presence of two linked QTL affecting the trait of sex (Table 2). Table 1. Estimates of location, significance and percentage of within-family variation explained (PVE) for body weight-related QTL in Sparus aurata. 5% significance threshold LG Position (cM) F ratio Chromosome-wide Genome-wide PVE (%) 9 35 2.31 2.20 3.03 9.3 21 20 3.42 2.19 3.03 17.2 Table 2. Estimates of location, significance for sex-related QTL in Sparus aurata. Position (cM) F ratio 2 QTL LG QTLA QTLB vs. 0 QTL 9 17 49 3.14 21 6 20 3.45 and percentage of within-family variation explained (PVE) 5% significance threshold 2 QTL vs. 1 QTL 4.43 4.33 Chromosome-wide Genome-wide 2.81 2.86 4.0 4.0 PVE (%) 11.7 19.3 4. Discussion Body weight was significantly associated at a genome-wide scale with one QTL positioned on linkage group 21. The fraction of phenotypic variance explained by this QTL was 17.2 %, showing clearly its potential for use in marker-assisted selection (MAS). In addition, preliminary results from ANOVA revealed that allelic substitution at the microsatellite markers located near the QTL had large effects on body weight. Certain allelic combinations showed significantly improved performances of this commercially important trait. 4th International Symposium “Hydrobiology – Fisheries”, Volos 9-11 June 2011 299 4ο Γιεθνές Σσνέδριο “Υδροβιολογίας – Αλιείας”, Βόλος 9-11 Ιοσνίοσ 2011 Previous data in the sequential hermaphrodite fish Sparus aurata suggest that the mechanism of sex reversal is controlled by social activities and group dynamics (Zohar et al. 1978). In our study, however, we found for the first time linkage between sex and four loci (two pairs) located on LGs 9 and 21. Moreover, these linkage groups showed also significant evidence for QTL affecting growth, suggesting either the linkage between QTL (in each LG) or possibly the existence of a single QTL on each linkage group with pleiotropic effects. Whatever the case is, we give evidence that the two traits are genetically correlated to some extent in seabream. Consequently, based on our results, we believe that there might be a genetic component involved in S. aurata sex change apart from environmental factors. 5. Acknowledgments This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. References Franch R., Louro B., Tsalavouta M., Chatziplis D., Tsigenopoulos C.S. (2006) A genetic linkage map of the hermaphrodite teleost fish Sparus aurata L. Genetics, 174: 851-861. Green P., Falls K., Crooks S. (1990) Documentation for CRIMAP, Version 2.4. Washington University School of Medicine, St. Louis, pp 70. Knott S. A., Elsen J.M., Haley C.S. (1996) Methods for multiple marker mapping of quantitative trait loci in half-sib populations. Theoretical and Applied Genetics, 93: 71-80. Lande R., Thomson R. (1990) Efficiency of MAS in the improvement of quantitative characters. Genetics, 124: 743-756. Miller S. A., Dykes D.D., Polesky H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16: 1215. Smith C., Simpson P.S. (1986) The use of genetic polymorphism in livestock improvement. J. Anim. Breed. Genet., 103: 205-217. Zohar Y., Abraham M., Gordin H. (1978) The gonadal cycle of the captivity-reared hermaphroditic teleost Sparus aurata (L.) during the first two years of life. Annales de Biologie Animale Biochimie Biophysique, 18: 877-882. This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. European Union European Social Fund 4th International Symposium “Hydrobiology – Fisheries”, Volos 9-11 June 2011