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)

Document Type : Research Paper

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.

Keywords

Full Text

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

(Received: Sept. 21. 2013, Accepted: Jan. 25. 2014)

ABSTRACT

Keywords:Beluga, Husohuso, sex marker, genetics,bulked segregant analysis (BSA)

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

No.	Sequence (3’→5’)	Species	Reference	No.	Sequence (3’→5’)	Species	Reference
1	AGGTGACCGT	Gracilariachangii	Simet al., 2007	52	CTGCTGGGAC	Ginkgo biloba	Liao et al., 2009
2	CAATCGCCGT	Oncorhynchusmykiss	Iturraet al., 1998	53	TGAGCGGACA	Cannabis sativa	Torjeketal., 2002
3	GTGGTCCGCA	Oncorhynchusmykiss	Iturraet al., 1998	54	TCGTCGAAGG	Spilornischeelahoya	Hsu et al., 2009
4	GGCTATAGGG	Eucommiaulmoides	Xuet al., 2004	55	TTGCTCACGG	pigs	Horng&Huang, 2003
5	GAGACGCACA	Commiphorawightii	Samantarayet al., 2010	56	GTTGCGATCC	Brugiamalayi	Underwood &Bianco, 1999
6	AAGCGACCTG	Commiphorawightii	Samantarayet al., 2010	57	CAGGCCCTTC	Actinidiadeliciosavar. deliciosa	Shirkot etal., 2002
7	GTTGCGATCC	Commiphorawightii	Samantarayet al., 2010	58	AATCGGGCTG	Actinidiadeliciosavar. deliciosa	Shirkot etal., 2002
8	CATAATCAAC	Actinidiachinensis	Gill et al., 1998	59	AGCCAGCGAA	Actinidiadeliciosavar. deliciosa	Shirkot et al., 2002
9	TCGCAATTCG	Actinidiachinensis	Gill et al., 1998	60	CTCACGTTGG	Actinidiadeliciosavar. deliciosa	Shirkot et al., 2002
10	ACTTCGCCAC	Hippophaerhamnoides	Sharma et al., 2010	61	CCCAAGGTCC	Psetta maxima	Casas et al., 2011
11	ACGCGAACCT	Paramisgurnusdabryanus	Xia et al., 2011	62	GGAAGCCAAC	Bryconamazonicus	Da Silva et al., 2012
12	CTCGAACCCC	Streptopeliaorientalis	Wu et al., 2007	63	CTGAGACGGA	Simmondsiachinensis	Agrawal et al., 2007
13	CACACTCCAG	Bubalusbubalis	Horng et al., 2004	64	TAGCGTCGAC	Iranian river buffalo	Shokrollahi&Aryapour, 2011
14	GCACCGAGAG	Carica papaya	Lemos et al., 2002	65	TTGGTACCCC	Hippophaesalicifolia	Ranaet al., 2009
15	CACCATCGTG	Cyprinuscarpio	Chen et al., 2009	66	CTAGAGGCCG	Salixviminalis L	Alstrom-Rapaport et al., 1998
16	GATGACCGCC	Actinidiadeliciosavar. deliciosa	Shirkot et al., 2002	67	CCGCATCTAC	Bombyxmori	Abe et al., 1998
17	TGCGTGCTTG	Oreochromisniloticus	Bardakci, 2000	68	TGTGGACTGG	Bombyxmori	Abe et al., 1998
18	GGTCCCTGAC	Borassusflabellifer	George et al., 2007	69	CCAGAACGGA	Bombyxmori	Abe et al., 1998
19	TGATCCCTGG	Ginkgo biloba	Longdou et al., 2006	70	CGCGTGCCAG	Bombyxmori	Abe et al., 1998
20	GTGAGGCGTC	Actinidiakolomikta	Cesonieneet al., 2007	71	GGTGCGCACT	Atriplexgarrettii.	Ruaset al., 1998
21	CAAACGTCGG	Actinidiakolomikta	Cesonieneet al., 2007	72	TGGGGGACTC	Cannabis sativa	Sakamoto et al., 2005
22	TTGGCACGGG	Carica papaya	Urasaki et al., 2002	73	CCTTGACGCA	Cannabis sativa	Sakamoto et al., 2005
23	AGGAGTCGGA	Simmondsiachinensis	Hosseini et al., 2011	74	CCACAGCAGT	Cannabis sativa	Sakamoto et al., 2005
24	GGGCCACTCA	Simmondsiachinensis	Hosseini et al., 2011	75	AACGGTGACC	Cannabis sativa	Sakamoto et al., 2005
25	GTCCCGACGA	Trichosanthesdioica	Singh et al., 2002	76	CCTGATCACC	Cannabis sativa	Sakamoto et al., 2005
26	GAAACGGGTG	Acer negundo	Linsen etal., 1999	77	TGAGCCTCAC	Humuluslupulus	Polleyet al., 1997
27	GTGACGTAGG	Acer negundo	Linsen etal., 1999	78	GGCGAAGGTT	Humuluslupulus	Polleyet al., 1997
28	CAGCACCCAC	Acer negundo	Linsen etal., 1999	79	CGACCAGAGC	Gracilarialemaneiformis	Xiang-fenget al., 1998
29	CAAACGTCGG	Acer negundo	Linsen etal., 1999	80	CCGGCCTTAG	Gracilarialemaneiformis	Xiang-fenget al., 1998
30	GTTGCGATCC	Acer negundo	Linsen et al., 1999	81	TTCCCCGCGC	Gracilarialemaneiformis	Xiang-fenget al., 1998
31	GGACTGGAGT	Acer negundo	Linsen et al., 1999	82	TTCCCCGACC	Gracilarialemaneiformis	Xiang-fenget al., 1998
32	TGCGCCCTTC	Acer negundo	Linsen et al., 1999	83	GTGATCGCAG	Phoenixdactylifera L.	Younis et al., 2008
33	CATCCGTGCT	Hippophaerhamnoides	Persson&Nybom, 1998	84	TCGGCGATAG	Phoenixdactylifera L.	Younis et al., 2008
34	TGTCATCCCC	Piper longum	Banerjee et al., 1999	85	GGTCTACACC	Phoenixdactylifera L.	Younis et al., 2008
35	GGGTAACGCC	Silenedioica	Di Stilio et al., 1998	86	CATCCCCCTG	ZamiafischeriMiq.	Roy et al., 2012
36	AACGCGTCGG	Leporinusmacrocephalus	Alves-Costa &Wasko, 2010	87	GGACTGGAGT	ZamiafischeriMiq.	Roy et al., 2012
37	TTGGTACCCC	Grusamericana	Duan&Fuerst, 2001	88	TTCACGGTGG	Asparagus officinalis	Ii et al., 2012
38	ACTTCGCCAC	pigeons	Horng et al., 2006	89	CTGGCTCAGA	Salixviminalis L.	Gunter et al., 2003
39	GTTTCGCTCC	Mercurialisannua	Khadka et al., 2002	90	GTCCACACGG	Oreochromisniloticus	Bardakci, 2000
40	GACGGATCAG	asparagus	Jiang &Sink, 1997	91	CCACAGCAGT	Oreochromisniloticus	Bardakci, 2000
41	GTGACGTAGG	Piper betle	Samantarayet al., 2012	92	CAGCACCCAC	Oreochromisniloticus	Bardakci, 2000
42	AATCGGGCTG	Piper betle	Samantarayet al., 2012	93	AAAGCTGCGG	Oreochromisniloticus	Bardakci, 2000
43	ACCAGGGGCA	Piper betle	Samantarayet al., 2012	94	TGAGTGGGTG	Oreochromisniloticus	Bardakci, 2000
44	GAACGGACTC	Piper betle	Samantarayet al., 2012	95	TGCGAGAGTC	Carica papaya	Reddy et al., 2012
45	GCCTGATTGC	Pistacia species	Esfandiyariet al., 2012	96	GGGCGGTACT	Carica papaya	Reddy et al., 2012
46	GAAACGGGTG	Gracilariachangii	Simet al., 2007	97	ACCGCCTGCT	Carica papaya	Reddy et al., 2012
47	TCCGCTCTGG	Gracilariachangii	Simet al., 2007	98	AGCCTGAGCC	Carica papaya	Reddy et al., 2012
48	TTCGAGCCAG	Gracilariachangii	Simet al., 2007	99	GGGCCACTCA	Caricapapaya L.	Deputy et al., 2002
49	CCGCATCTAC	Gracilariachangii	Simet al., 2007	100	GGGTGTGTAG	Caricapapaya L.	Deputy et al., 2002
50	GACGGATCAG	Gracilariachangii	Simet al., 2007	101	CTGATGCGTG	Caricapapaya L.	Deputy et al., 2002
51	CACACTCCAG	Gracilariachangii	Simet al., 2007

ACKNOWLEDGMENTS

This research was funded by Iran National Science Foundation (Sandoogh-e HemayatazPajooheshgaranvaFannavaran-e

Keshvar)and supported by Khorramshahr University of Marine Science and Technology.

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