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


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

 M. Khodaparast1, S. Keyvanshokooh1*, 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

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: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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