Searching the genome of beluga (Huso huso) for sex markers based on targeted Bulked Segregant Analysis (BSA)




In sturgeon aquaculture, where the main purpose is caviar production, a reliable method is needed to separate fish according to gender. Currently, due to the lack of external sexual dimorphism, the fish are sexed by an invasive surgical examination of the gonads. Development of a non-invasive procedure for sexing fish based on genetic markers is of special interest. In the present study we employed Bulked Segregant Analysis (BSA) methodology to search for DNA markers associated with the sex of the beluga sturgeon (Huso huso).DNA bulks (male and female) were created by combining equal amounts of genomic DNA from 10 fish of both sexes. A total of 101 decamer primers associated with the sex-specific sequences in non-sturgeon species was used for targeted screening of the bulks, resulting in 2846 bands that all of them were present in both sexes. Our results showed that sex chromosomes are weakly differentiated in the sturgeon genome and comprised sequences not complementary to the sex-specific primers in non-sturgeon species.


Abe H., Kanehara M, Terada T., Ohbayashi F., Shimada T., Kawai S.,Suzuki M., Sugasaki T.,& Oshiki T.(1998)Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyxmoriand the wild silkworm, B. mandarina, and their retrotransposableelementrelated nucleotide sequences. Genes & genetic systems,73:243-254.AgrawalV, SharmaK, GuptaS, Kumar R, Prasad M(2007)Identification of sex in Simmondsiachinensis(Jojoba) using RAPD markers. Plant Biotechnology Reports, 1(4):207-210.Alstrom-Rapaport C., Lascoux M, Wang YC, Roberts G. &TuskanGA (1998)Identification of a RAPD Marker Linked to Sex Determination in the Basket Willow(Salix viminalisL.). Journal of Heredity, 89:44-49.Alves-Costa F.A.&Wasko A.P.(2010)Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinusmacrocephalusthrough mRNAdifferential display and real time PCR analyses. Genetica138 ,321331.Banerjee N.S., Manoj P.&Das M.R.(1999)Male sex associated marker in PiperlongumL. Current science, 77(5):693-695.Bardakci F.(2000)The Use of Random Amplified Polymorphic DNA (RAPD) Markers in Sex Discrimination in Nile Tilapia, Oreochromisniloticus(Pisces: Cichlidae). Turkish Journal of Biology, 24, 169-175.Bemis W.E., Findies E.K. &Grande L (1997) An overview of Acipenseriforms. Environmental Biology of Fishes, 48(1-4), 25-71. Billard R, Lecointre G (2000)Biology and conservation of sturgeon and paddlefish. Reviews in Fish Biology and Fisheries, 10, 355-392. Birstein V.J.(1993)Sturgeons and Paddlefishes -Threatened Fishes in Need of Conservation. Conservation Biology, 7, 773-787.Casas,L., Sanchez,L.&Orban,L.,(2011)Sex-associated DNA markers from turbot. Marine BiologyResearch,7,378-387.Cesoniene L., Daubaras R. &Gelvonauskis B.(2007)Evaluation of genetic diversity and genetic relationships among female Lithuanian accessions of Kolomikta kiwi. Journal of Fruit and Ornamental Plant Research, 15:95-102.Chapman F.A., Van Eenennaam J.P., Doroshov S.I.(1996)The reproductive condition of white sturgeon, Acipensertransmontanus, in San Francisco Bay, California. Fishery Bulletin, 94:628-634.Chen J., Wang Y., Yue Y., Xia X., Du Q. &Chang Z (2009)A novel male-specific DNA sequence in the common carp, Cyprinuscarpio. Molecular and cellular probes, 23:235-239. da Silva, E. M., Wong, M. S. L., Martins, C., &Wasko, A. P. (2012)Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Bryconamazonicus(Teleostei: Characiformes: Characidae). Fish Physiology and Biochemistry, 38:1487-1496.Deputy,J.C., Ming,R, MaH., Liu Z., Fitch M.M.,Wang M., Manshardt R&Stiles J.I.(2002)Molecular markers for sex determination in papaya (CaricapapayaL.). Theoretical and Applied Genetics, 106, 107-111.Devlin, R. H., &Nagahama, Y. (2002). Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture, 208, 191-364.Di Stilio, V. S., Kesseli, R. V., &Mulcahy, D. L. (1998). A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silenedioica. Genetics, 149, 2057-2062.Doroshov, S. I., Moberg, G. P., & Van Eenennaam, J. P. (1997). Observations on the reproductive cycle of cultures white sturgeon, Acipensertransmontanus. Environmental Biology of Fishes, 48:265-278.Duan, W., &Fuerst, P. A. (2001). Isolation of a sex-linked DNA sequence in cranes. Journal of Heredity, 92, 392-397.Esfandiyari, B., Davarynejad, G. H., Shahriari, F., Kiani, M., &Mathe, A. (2012). Data to the sex determination Khodaparast et al.,191in Pistaciaspecies using molecular markers. Euphytica, 185, 227-231.Feist, G., Van Eenennaam, J. P., Doroshov, S. I., Schreck, C. B., Schneider, R. P., & Fitzpatrick, M. S. (2004). Early identification of sex in cultured white sturgeon, Acipensertransmontanus, using plasma steroid levels. Aquaculture, 232, 581-590.Flynn, S. R., Matsuoka, M., Reith, M., Martin-Robichaud, D. J., &Benfey, T. J. (2006). Gynogenesis and sex determination in shortnose sturgeon, AcipenserbrevirostrumLesuere. Aquaculture, 253(1), 721-727.Fontana, F., & Colombo, G. (1974). The chromosomes of Italian sturgeons. Experientia, 30(7), 739-742.Fopp-Bayat, D. (2010). Meiotic gynogenesis revealed not homogametic female sex determination system in Siberian sturgeon (AcipenserbaeriBrandt). Aquaculture, 305, 174-177.Gill, G. P., Harvey,C. F., Gardner, R. C., & Fraser, L. G. (1998). Development of sex-linked PCR markers for gender identification in Actinidia. Theoretical and Applied Genetics, 97, 439-445.Griffiths, R., &Tiwari, B. (1993). The isolation of molecular genetic markers for the identification of sex. Proceedings of the National Academy of Sciences, 90, 8324-8326.Gunter, L. E., Roberts, G. T., Lee, K., Larimer, F. W., &Tuskan, G. A. (2003). The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalisL. Journal of heredity, 94(2), 185-189.Hale, M., McCormick, C., Jackson, J., &DeWoody, J. A. (2009). Next-generation pyrosequencing of gonad transcriptomes in the polyploid lake sturgeon (Acipenserfulvescens): the relative merits of normalization and rarefaction in gene discovery. BMC genomics, 10, 203.Harshbarger, J.C., Coffey, M.J.&Young, M.Y(2000)Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis,Missouri, USA. Marine Environmental Research 50, 247-250.Hett, A.K., Ludwig, A(2005)SRY-related (Sox) genes in the genome of European Atlantic sturgeon (Acipensersturio). Genome48: 181-186.Hett, A. K., Pitra, C., Jenneckens, I., & Ludwig, A. (2005). Characterization of sox9 in European Atlantic sturgeon (Acipensersturio). Journal of Heredity, 96, 150-154.Horng, Y. M., Chen, Y. T., Wu, C. P., Jea, Y. S., & Huang, M. C. (2004). Cloning of Taiwan water buffalo male-specific DNA sequence for sexing. Theriogenology, 62, 1536-1543.Horng, Y. M., & Huang, M. C. (2003). Male-specific DNA sequences in pigs. Theriogenology, 59(3), 841-848.Horng, Y. M., Wu, C. P., Wang, Y. C., & Huang, M. C. (2006). A novel molecular genetic marker for gender determination of pigeons. Theriogenology, 65, 1759-1768.Hosseini, F.S., ShahsavandHassani, H., Arvin, M.J., Baghizadeh, A. &MohammadiNejad, G.(2011)Sex determination of jojoba (Simmondsiachinensiscv. Arizona) by random amplified polymorphic DNA (RAPD) molecular markers.African Journal ofBiotechnology10:470-474.Hsu, H.A., Wang, P.H., Chao, M.C., Chan, F.T., Wang, L.M., Lin, P.I., Chang, C.H., Yuan, H.W. &Ding, S.T.(2009)Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk. Theriogenology72:755764.Ii, Y., Uragami, A., Uno, Y., Kanechi, M. and Inagaki, N (2012)RAPD-based analysis of differences between male and female genotypes of Asparagus officinalis. HorticultureScience(Prague)39:33-37.Iturra, P., Medrano, J. F., Bagley, M., Lam, N., Vergara, N., & Marin, J. C. (1998). Identification of sex chromosome molecular markers using RAPDs and fluorescent in situ hybridization in rainbow trout. Genetica, 101:209-213.Jiang, C. &Sink, K.C (1997)RAPD and SCAR markers linked to the sex expression locus M in asparagus. Euphytica94,329333.George, J., Karun, A., Manimekalai, R., Rajesh, M. K., &Remya, P. (2007). Identification of RAPD markers linked to sex determination in 192Searching the Genome of...palmyrah (BorassusflabelliferL.). Current science, 93(8), 1075-1077.Keyvanshokooh, S., &Gharaei, A. (2010). A review of sex determination and searches for sexspecific markers in sturgeon. Aquaculture Research, 41, e1-e7.Keyvanshokooh, S., Kalbassi, M.R., Hosseinkhani, S., Vaziri, B (2009)Comparative proteomics analysis of male and female Persian sturgeon (Acipenserpersicus) gonads. Animal ReproductionScience,111:361-368.Keyvanshokooh, S., Pourkazemi, M., &Kalbassi, M. R. (2007). The RAPD technique failed to identify sexspecific sequences in beluga (Husohuso). Journal of Applied Ichthyology, 23(1):1-2.Khadka, D.K., Nejidat, A., Tal, M. &Golan-Goldhirsh, A (2002)DNA markers for sex: Molecular evidence for gender dimorphism in dioeciousMercurialisannuaL.. MolecularBreeding,9,251257.Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P. & Lovell-Badge, R (1991)Male development of chromosomally female micetransgenic for Sry. Nature, 351,117-121.Kovacs, B., Egedi, S., Bartfai, R., Orban, L. (2001) Male-specific DNA markers from African catfish (Clariasgariepinus). Genetica,110,267276.Lemos, E.G.M., Silva, C.L.S.P. and Zaidan, H.A (2002)Identification of sex in Carica papayaL. using RAPD markers. Euphytica,127,179184.Liao, L., Lui, J., Dai, Y., Li, Q., Xia, M., Chen, Q., Yin, H., Qiu, G. and Liu, X (2009)Development and application of SCAR markers for sex identification in the dioecious species Ginkgo bilobaL. Euphytica,169:4955. Linsen, Z., Qiang, Z., Jinxia, M., Jian, X. &Hua, Z (1999)RAPD analysison genomic DNA of male and female plants of Acer negundoL.Journal ofForestResearch, 10,152-154.Logan, S.H.,Johnston, W.E., Doroshov, S.I (1995)Economics of Joint Production of Sturgeon (AcipensertransmontanusRichardson) and Roe for Caviar. Aquaculture, 130:299-316.Longdou, L., Qibai, X., Meili, J., Wujun, G. &Ruili, L (2006)Identification of RAPD marker linked to the sex in dioeciousGinkgo Biloba. GeneticasiBiologieMoleculara, 7:105-110.McCormick, C. R., Bos, D. H., &DeWoody, J. A. (2008). Multiple molecular approaches yield no evidence for sexdetermining genes in lake sturgeon (Acipenserfulvescens). Journal of Applied Ichthyology, 24, 643-645.Michelmore, R., Paran, I., &Kesseli, R. V. (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences, 88(21), 9828-9832.Nasrollahzadeh, A. (2010). Caspian Sea and its Ecological Challenges. Caspian Journal of Environmental Sciences, 8(1), 97-104.Omoto, N., Maebayashi, M., Adachi, S., Arai, K.&Yamauchi, K (2005)Sex ratios of triploids and gynogeneticdiploids induced in the hybrid sturgeon, the bester (Husohusofemale x Acipenserruthenusmale). Aquaculture,245,39-47.Penman, D. J., &Piferrer, F. (2008). Fish gonadogenesis. Part I: genetic and environmental mechanisms of sex determination. Reviews in Fisheries Science, 16:16-34.Persson, H. A., &Nybom, H. (1998). Genetic sex determination and RAPD marker segregation in the dioecious species sea buckthorn (HippophaerhamnoidesL.). Hereditas, 129:45-51.Polley,A., Seigner, E. &Ganal, M.W (1997) Identification ofsex in hop (Humuluslupulus) using molecular markers. Genome,40,357-361.Pourkazemi, M. (2006). Caspian Seasturgeon conservation and fisheries: past present and future. Journal of Applied Ichthyology, 22:12-16.Rana, S., Shirkot, P. and Yadav, M.C (2009)A female sex associated Randomly Amplified Polymorphic DNA marker in dioeciousHippophaesalicifolia. Genes, Genomes and Genomics, 3:96-101.Reddy, S. R., Krishna, R. B., & Reddy, K. J. (2012). Sex determination of Papaya (Caricapapaya) at seedling stage Khodaparast et al.,193through RAPD Markers. Research in Biotechnology, 3(1).Roy, S. K., Gangopadhyay, G., & Mukherjee, K. K. (2012). Determination of sex in Zamia fischeriMiq., an endangered gymnosperm. International Journal of Biodiversity and Conservation, 4, 287-293.Ruas, C.F., Fairbanks, D.J., Evans, R.P., Stutz, H.C., Andersen, W.R. &Ruas, P.M (1998)Male-Specific DNA in the dioecious species Atriplexgarrettii(Chenopodiaceae). American Journal ofBotany, 85,162167.Russell, S.R., Sanchez-Soriano, N., Wright, C.R., Ashburner, M (1996)TheDichaete gene of Drosophila melanogasterencodes a SOX-domain protein required for embryonic segmentation. Development, 122:69-76.Sakamoto, K., Abe, T., Matsuyama, T., Yoshida, S., Ohmido, N., Fukui, K. &Satoh, S (2005)RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativaL.. Genome, 48:931936.Samantaray, S., Geetha, K. A., Hidayath, K. P., &Maiti, S. (2010). Identification of RAPD markers linked to sex determination in guggal [Commiphorawightii(Arnott.)] Bhandari. Plant Biotechnology Reports, 4:95-99.Samantaray, S., Phurailatpam, A., Bishoyi, A. K., Geetha, K. A., &Maiti, S. (2012). Identification of sex-specific DNA markers in betel vine (Piper betle L.). Genetic Resources and Crop Evolution, 59:645-653.Sharma, A., Zinta, G., Rana, S., &Shirko, P. (2010). Molecular identification of sex in Hippophaerhamnoides L. using isozyme and RAPD markers. Forestry Studies in China, 12:62-66.Shirkot, P.,Sharma, D.R. and Mohapatra, T (2002)Molecular identification of sex in Actinidiadeliciosavar. deliciosaby RAPD markers. ScientiaHorticulturae, 94:3339. Shirkot, P.,Sharma, D.R. &Mohapatra, T.(2002)Molecular identification of sex in Actinidiadeliciosavar. deliciosaby RAPD markers. ScientiaHorticulturae, 94,3339. Shokrollahi, B., &Aryapour, H. (2011). A novel male-specific DNA sequence for river buffaloes. African Journal of Microbiology Research, 5:284-288.Sim, M. C., Lim, P. E., Gan, S. Y., &Phang, S. M. (2007). Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophyticthalli of Gracilariachangii(Rhodophyta). Journal of Applied Phycology, 19: 763-769.Sinclair, A., Berta, P., Palmer, M., Hawkins, J., Griffiths, B. & Palmer, M. S. (1990). A gene from the human sex-determining region encodes a protein with. Nature, 346:240-4.Singh, M., Kumar, S., Singh, A.K. &Kalloo, G.(2002) Female sex associated marker in pointed ground (Trichosanthes dioica Roxb.). Current Science, 82, 131132.Torjek, O., Bucherna, N., Kiss, E., Homoki, H., Finta-Korpelova, Z., Bocsa, I., Nagy, I. &Heszky, L.E (2002)Novel male-specific molecular markers (MADC5, MADC6) in hemp. Euphytica, 127:209218.Underwood, A. P., &Bianco, A. E. (1999). Identification of a molecular marker for the Y chromosome of Brugiamalayi. Molecular and biochemical parasitology, 99:1-10.Urasaki, N.,Tokumoto, M.,Tarora, K.,Ban, Y., Kayano, T.,Tanaka, H., Oku, H., Chinen, I. &Terauchi, R.(2002)A male and hermaphrodite specific RAPD marker for papaya (Carica papayaL.). Theoretical and Applied Genetics, 104:281-285.VanEenennaam, J.I.&Doroshov, S.I (1998)Effects of age and body size on gonadal development of Atlantic sturgeon. Journal ofFish Biology, 53:624-637. Van EenennaamAL, Murray JD, Medrano JF (1998)Mitotic analysis of the North American white sturgeon, AcipensertransmontanusRichardson (Pisces Acipenseridae), a fish with a very high chromosome number. Genome, 41,266-271.Van Eenennaam, A.L., Van Eenennaam, J.P., Medrano, J.F.&Doroshov, S.I (1999)Evidence of female heterogametic genetic sex 194Searching the Genome of...determination in white sturgeon. Journal ofHeredity, 90,231-233.Xia, X., Zhao, J., Du, Q., Zhi, J. &Chang, J (2011)Cloning and identification of a female-specific DNA marker in Paramisgurnusdabryanus. Fish Physiology andBiochemistry, 37,53-59.Xiang-feng, L., Zheng-hong, S., &Xue-cheng, Z. (1998). Application of RAPD in genetic diversity study onGracilarialemaneiformisIII. Phase and sex related markers. Chinese Journal of Oceanology and Limnology, 16(1), 147-151.Xu,W.J., Wang, B.W. &Cui, K.M (2004)RAPD and SCAR markers linked to sex determination in EucommiaulmoidesOliv.Euphytica, 136:233238.WebbM A.H., Feist G.W., Foster E.P.,Schreck C B,& Fitzpatrick M.S.(2002)Potential classification of sex and stage of gonadal maturity of wild white sturgeon using blood plasma indicators. Transactions of the American Fisheries Society, 131, 132-142.Wright, E.M., Snopek, B., Koopman, P (1993)Seven new members of the Sox genefamily expressed during mouse evelopment. Nucleic Acids Research, 21:744.Wu, C.P., Horng, Y.M., Wang, R.T., Yang, K.T. &Huang, M.C (2007)A novel sex-specific DNA marker in Columbidae birds. Theriogenology, 67,328333. Wuertz, S., Gaillard, S., Barbisan, F., Carle, S., Congiu, L., Forlani, A., Aubert, J., Kirschbaum, F., Tosi, E., Zane, L., & Grillasca, J.P (2006)Extensive screening of sturgeon genomes by techniques revealed no sex-specific random screening marker. Aquaculture, 258,685-688.Yarmohammadi, M., Pourkazemi, M., Ghasemi, A., Hassanzadeh Saber, M., &Chakmehdouz, F (2011)AFLP reveals no sex-specific markers in Persian sturgeon (Acipenserpersicus) or beluga sturgeon (Husohuso) from the southern Caspian Sea, Iran. Progress inBiologicalScience,1:55-60.Younis, R.A.A.,Ismail, O.M. &Soliman, S.S (2008)Identification of Sex-specific DNA Markers for Date Palm (PhoenixdactyliferaL.) Using RAPD and ISSR Techniques. ResearchJournal ofAgriculture and BiologicalSciences,4:278-284.