Searching the genome of beluga(Husohuso) for sex markers based on targeted Bulked SegregantAnalysis (BSA)

Searching the genome of beluga(Husohuso) for sex markers based on targeted Bulked SegregantAnalysis (BSA)

 M. Khodaparast¹, S. Keyvanshokooh^1*, M. Pourkazemi², S.J.Hosseini³, H. Zolgharnein⁴

 1-Dept. of Fisheries, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran

2- Dept. of Genetics, International Sturgeon Research Institute,Rasht, Iran

3- Persian Gulf Research and Studies Center, Persian Gulf University,Boushehr, Iran

4- Dept. of Marine Biology,Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran

^*Corresponding author'sEmail: Keyvan56@yahoo.com

ABSTRACT

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 SegregantAnalysis (BSA) methodology to search for DNA markers associated with the sex of the beluga sturgeon (Husohuso).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.

INTRODUCTION

The extant sturgeon species (family Acipenseridae) are considered to be one of the most primitive groups of fishes that evolved approximately 250 million years ago (Bemis et al., 1997). Six sturgeon species, belonging to two genera (Huso and Acipenser), are found in the Caspian Sea and its drainage basin which provide today the bulk of the world’s caviar yield (Pourkazemi, 2006; Nasrollahzadeh, 2010).Sturgeons mature very late in life and their populations are declining worldwide caused by overfishing as well as pollution and habitat degradation (Birstein, 1993; Billard&Lecointre 2000). Beluga sturgeon (Husohuso) is regarded as one of the most important commercially species in the Caspian Sea and hasbeen overfished nearly to extinction in pursuit of their caviar (Pourkazemi, 2006). Nowadays, production of sturgeon for both meat and caviar will increasingly have to rely on aquaculture (Logan et al., 1995; Keyvanshokooh&Gharaei, 2010). In sturgeon aquaculture, where the main purpose is caviar production, a reliable method is needed to separate fish according to gender. Males are destined to the meat market while females remain in culture for many more years under conditions of optimal growth and development. The availability of monosex populations of caviar-producing females would significantly enhance the economic viability of domestic caviar production systems (Logan et al., 1995).

None of the sturgeon species exhibit external sexual dimorphism, and it is not possible to distinguish male fish from females by morphological markers at larval, juvenile and even adult stages (Keyvanshokoohet al., 2009). Blood plasma sex steroid levels in sturgeon remain low until the beginning of gonadal development (Doroshovet al., 1997). Although an examination of plasma steroids could be used to sex sturgeon (Webb et al., 2002), these steroid indicators are influenced by age, husbandry conditions and water temperature (Feistet al., 2004).Currently, sturgeon producers wait 3-4 years before fish are sexed by an invasive surgical examination of the gonads (Doroshovet al., 1997). Although survival rate is nearly 100% (Feistet al., 2004), the development of a non-invasive procedure for sexing sturgeon is of special interest. One effective solution is to use DNA markers to diagnose the sex. Such markers will be present in species where one sex possesses a unique chromosome or DNA sequence (Griffith &Tiwari, 1993; Devlin &Nagahama, 2002;Keyvanshokooh&Gharaei, 2010).

Regarding the failure of randomly screening methodologies to find a sex-specific marker in various sturgeon species (Wuertzet al., 2006; Keyvanshokoohet al., 2007; McCormick et al., 2008; Yarmohammadiet al., 2011), we conducted a targeted search based on previously identified sex-specific sequences in non-sturgeon species to compare male and female DNA. The search for sex-specific sequences in beluga sturgeon was performed  using bulked segregant analysis (BSA) methodology in conjunction withtherandom amplified polymorphic DNA (RAPD) assay. BSA is based on grouping together of individuals that share a common trait and studying the genomic regions related with that trait against a randomized background of unlinked loci (Michelmoreet al., 1991). This approach has been used in identifying sex-specific sequences in some species (Griffith &Tiwari, 1993; Iturraet al., 1998; Kovacs et al., 2001).

MATERIALSAND METHODS

Fish Sampling and DNA Extraction

Fin tissue samples were obtained from 10 adult beluga sturgeon of each sex. The fish were caught as broodstock from the Iranian Caspian Sea coastline and transferred to the Shahid Dr.BeheshtiSturgeon Fish Propagation and Rearing Complex, Rasht, Iran. Sex identification was carried out by observation of testes and ovaries of necropsied spawners.

The CTABmethod was used to obtain genomic DNA. The quantity and quality of extracted DNA was assayed using a spectrophotometer and 1% agarose electrophoresis. Two DNA pools (male and female) were created by combining equal amounts of genomic DNA from each fish.

Polymerase Chain Reaction (PCR) and Electrophoresis

BSA methodology was employed in conjunction with the RAPD assay to screen  genetic markers associated with the sex of beluga sturgeon. Searching through databases forsex-specific sequences in non-sturgeon species (including animal and plant species), a total of 101 RAPD primers (Metabion, Germany) was found (Table 1) and was used for targeted amplifications.

Amplifications were performed in 20-µlreaction volumes containing 15 ng of DNA, 0.5 µM primer, 400 µM each of dNTPs, 1 unit Taq polymerase (Cinnagen, Iran), 1X PCR buffer, and 1.5 mM MgCl₂. PCR consisted of 3 min denaturation at 94ºC, followed by 30 cycles of 30 sec at 94ºC, 30 sec at 40ºC, and 30 sec at 72ºC, with a final extension at 72ºCfor 5 min. PCR products were separated and analyzed in gels of 6% polyacrylamide stained with silver nitrate (Keyvanshokoohet al., 2007).

RESULTSAND DISCUSSION

A set of 101 RAPD primers yielded a total of 2486 scoreable bands that were present in both sexes. Only two primers (primers no. 29 and 82; Table 1) produced different band patterns on pools; each primer produced a band that was present only in the female pool. Following reconfirmation of the bulk polymorphism, the individual DNA samples used to create both bulks were screened using the primer no. 29 and no. 82. The polymorphic bands produced by the primers were found in one of the 10 fishes from both sexes.

We were unable to identify a sex-specific marker in beluga sturgeon associated with previously identified sex-specific sequences in non-sturgeon species using BSA methodology.Using RAPD, AFLP (amplified fragment length polymer-phism), and ISSR (inter-simple sequence repeats) techniques,Wuertzet al. (2006) focused on the identification of genomic sex-specific markers in four sturgeon species (A. baerii; A. naccarii; A. gueldenstaedtii and A. ruthenus). Although 1100-9230 bands screened per species, no sex-specific markers were detected. Similar result has been obtained by searching the genome of beluga sturgeon (Keyvansh-okoohet al., 2007)  using bulked segregant analysis (BSA; separate pooling of DNA from males and females). They used a total of 310 randomly amplified polymorphic DNA primers to screen the bulks, resulting in 4146 bands that were present in both sexes. Using the RAPD technique, McCormick et al. (2008) did also failed to find a sex-specific marker in lake sturgeon (Acipenserfulvescens).Searching the genome of the Persian sturgeon (Acipenserpersicus) and beluga by using AFLP, Yarmohammadiet al. (2011) also observed no sex-specific sequence. With regard to these failures, McCormick et al. (2008) mentioned that an environmental sex-determining system may exist in sturgeon. In theory, the lack of sex-specific markers in the search could be due to the lack of genetic sex-determining mechanisms. Although heteromorphic sex chromo-somes have not been identified in sturgeon (Fontana & Colombo, 1974; Van Eenennaamet al., 1998), but we do know that a female heterogametic genetic sex determination is in operation in beluga (Omoto et al., 2005) and some other sturgeon species studied  to date(Van Eenennaamet al., 1999; Flynn et al., 2005; Fopp-Bayat, 2010). Based on this proved assumption, the female sturgeon should carry sex-specific DNA sequences.Moreover, hermaphroditism in sturgeon is very infrequently observed (Chapman et al., 1996; Van Eenennaam&Doroshov 1998; Harshbargeret al., 2000) and the sex ratio in adult populations of sturgeon is 1 ♂: 1 ♀ (Chapman et al., 1996). Enviro-nmental sex determination produces variations in sex ratios when there are systematic fluctuations in the environ-mental factors influencing sex (Penman &Piferrer, 2008). In fact, the failure in search for this class of DNA markers could be due to the size of genome, the number of markers screened, and the proportion of the genome that is sex-specific in species studied (Keyvanshokoohet al., 2007; Penman &Piferrer, 2008).

Of the 2846 bands amplified  using the primers which were sex-specific in other species, none werelinked to a sex-determining gene in beluga sturgeon. One general approach to identify sex-specific DNA markers is based on candidate genes, where genes or sequences that are sex-determining or sex-linked in one species are searched for in the target species. Members of the Sox gene family are known to be involved in numerous developmental processes and sex determination in vertebrates (Koopmanet al., 1991; Wright et al., 1993; Russelet al., 1996). Sox proteins are characterized by a conserved high mobility group (HMG)-box domain, which is responsible for DNA binding and bending (Sinclair et al., 1990). Based on this approach and using highly degenerate primers that amplified a broad range of HMG boxes, 22 different sequences coding for 8 Sox genes (Sox2, Sox3, Sox4, Sox9, Sox11, Sox17, Sox19, and Sox21) were shown to be present in the genome of European Atlantic sturgeon (Acipensersturio) (Hett& Ludwig, 2005; Hettet al., 2005). Similarly, sequences with homology to Sox gene family (Sox2, Sox4, Sox17, and Sox21) were detected in lake sturgeon (A. fulvescens) (McCormick et al., 2008). However, although Sox genes were found in the genomes of A. sturio and A. fulvescens, none were associated with the sex sequences in any of these species. Regarding the afforementioned studies and our results, it seems that sex-specific DNA present in beluga sturgeon may be comprised sequences which are not conserved and complementary to sex-specific genes in other species.

In conclusion, targeted screening of beluga sturgeon genome based on primers which were sex-specific in non-sturgeon species failed to detect sex-specific sequences. With regard to failure in search for this class of DNA markers, it is proposed that sex chromosomes are weakly differentiated in the sturgeon genome. With recent advances in genomic and proteomic approaches, gene expression profiling could be considered as an alternative approach (Wuertzet al., 2006).For example, regarding the great potential of next-generation sequencing to rapidly identify genes of interest in sturgeon (Hale et al., 2009), this approach could be used in search for sturgeon sex markers.  

Table 1 Sex-specific primers of non-sturgeon species used for targeted screening of sex-linked sequences in beluga sturgeon