Determination of genomic sex in salmonids

• US 5,480,774 A
• Filed: 10/14/1993
• Issued: 01/02/1996
• Est. Priority Date: 10/14/1993
• Status: Expired due to Fees

• Current Assignee: A/F Protein, Inc.
• Original Assignee: A/F Protein, Inc.
• Inventors: Hew, Choy L., Du, Shao J.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/137,252
• Time in Patent Office: 810 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/270, 435/810, 435/183, 536/23.1, 536/23.51, 536/24.31, 536/24.33, 935/76, 935/77, 935/80, 935/9, 548/303.7, 436/56, 436/57, 436/800
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The invention provides a method for determining the genomic sex of various salmonids (family salmonidae). In particular, the invention provides the nucleic acid sequence of a pseudogene, designated GH-Ψ, which is linked to a sex determining locus on the Y chromosome, and may be used as a marker for determination of the sex of the fish.

Claims

1. A method of determining genomic sex of a member of the genus Oncorhynchus of the salmonid family by detecting a presence or absence of a growth hormone pseudogene (GH-Ψ

), said method comprising;

obtaining DNA from a salmonid species in which only males carry the GH-Ψ

pseudogene; and

detecting the GH-Ψ

, where said detecting includes either the amplification of a select subsequence specific to the pseudogene or duplex formation of a nucleic acid hybridizing specifically to the pseudogene and to no other genome of the salmonid species.

2. The method of claim 1 wherein said amplification is by polymerase chain reaction.

3. The method of claim 2 wherein said polymerase chain reaction utilizes a pair of PCR primers competent to amplify a DNA sequence which includes a subsequence of exon 5 and intron 5 of the growth hormone gene and pseudogene, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 4, intron 4, or exon 5 and the other primer of said pair binds selectively to conserved regions of exon 6, said conserved regions present in both the growth hormone gene and the growth hormone pseudogene.

4. The method of claim 2 wherein said polymerase chain reaction utilizes a pair of PCR primers competent to amplify the DNA sequence between about base 4870 and base 5019 of Sequence Id No:

1, designated GH-I, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 5, and the other primer of said pair binds selectively to conserved regions of intron 5 or exon 6.

5. The method of claim 4 where said pair of PCR primers consists of an oligonucleotide of sequence 5'"'"'-CCTGGATGACAATGACTCTCA-3'"'"' (SEQ ID No.:

4) and an oligonucleotide of sequence 5'"'"'-CTACAGAGTGCAGTTGGCCTC- 3'"'"' (SEQ ID No.;

     5).

6. The method of claim 1 wherein said amplification is by ligase chain reaction.

7. The method of claim 1 wherein said presence or absence of a growth hormone pseudogene is the detection of a deletion of about 149 base pairs between exons 5 and 6 in the growth hormone genes of salmonids.

8. The method of claim 1 wherein said nucleic acid is a nucleic acid probe which hybridizes specifically to Sequence Id No.:

2, but not Sequence Id No.;

1 or Sequence Id No.;

3 in 2x SSC, 0.1% SDS at 42°

C.

9. The method of claim 8 wherein said probe hybridizes specifically to the region between about base 4843 and base 4863 of Sequence Id No:

2 in 2x SSC, 0.1% SDS at 42 C.

10. The method of claim 9 wherein said probe is labeled with a marker selected from the group consisting of:

a fluorophore, a lumiphore, a chromogen, a radioactive label, horseradish peroxidase, biotin, or dioxigenin.

11. The method of claim 1 wherein said salmonid species is selected from the group consisting of Oncorhynchus tshawytscha, and Oncorhynchus kisutch.

12. A composition comprising a pair of PCR primers competent to amplify a DNA sequence which includes a subsequence of exon 5 and intron 5 of a growth hormone gene and a pseudogene from a salmonid, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 4, intron 4, or exon 5 and the other primer of said pair binds selectively to conserved regions of exon 6, said conserved regions present in both the growth hormone gene and the growth hormone pseudogene.

13. The composition of claim 12 wherein said pair of PCR primers are competent to amplify a DNA subsequence between about base 4870 and base 5019 of Sequence Id No.:

1, designated GH-I, which includes a subsequence of exon 5 and intron 5 of the GH gene or pseudogene, said primers binding selectively to exon 5 and exon 6.

14. The composition of claim 13 where said pair of PCR primers consists of an oligonucleotide of sequence 5'"'"'-CCTGGATGACAATGACTCTCA-3'"'"' (SEQ ID No.:

4) and an oligonucleotide of sequence 5'"'"'-CTACAGAGTGCAGTTGGCCTC-3'"'"' (SEQ ID No.;

     5).

15. A composition of claim 12 wherein the member of the genus Oncorhynchus is selected from the group consisting of O. tshawytscha and O. kisutch.

16. A nucleic acid probe which specifically detects a GH-Ψ

pseudogene wherein said probe is a nucleic acid which hybridizes specifically to Sequence Id No.;

2, but not Sequence Id No.;

1 or Sequence Id No.;

3 in 2x SSC, 0.1% SDS at 42°

C.

17. The probe of claim 16 wherein said probe hybridizes to the region between about base 4843 and base 4863 of Sequence Id No:

2 in 2x SSC, 0.1% SDS at 42°

C.

18. The probe of claim 17 wherein said probe is labeled with a marker selected from the group consisting of:

a fluorophore, a lumiphore, a radioactive label, horseradish peroxidase, biotin, or dioxigenin.

19. A kit useful for determining the genomic sex of a member of the genus Oncorhynchus of the salmonid family by detecting the presence or absence of a growth hormone pseudogene which comprises a container containing an isolated oligonucleotide for the amplification of a unique subsequence of the growth hormone pseudogene or hybridizes specifically to the growth hormone pseudogene.

20. The kit of claim 19 wherein said nucleic acid is a pair of PCR primers competent to amplify a DNA subsequence which includes a portion of exon 5 and intron 5 of the GH gene or pseudogene from a salmonid, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 4, intron 4, or exon 5 and the other primer of said pair binds selectively to conserved regions of exon 6.

21. The kit of claim 20 wherein said nucleic acid is a pair of PCR primers able to amplify a DNA sequence between about base 4870 and base 5019 of Sequence Id No.:

1, which includes a portion of exon 5 and intron 5 of the GH gene or pseudogene, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 5 and the other primer of said pair binds selectively to conserved regions of intron 5 or exon 6.

22. The kit of claim 21 where said pair of PCR primers consists of an oligonucleotide of sequence 5'"'"'-CCTGGATGACAATGACTCTCA-3'"'"' (SEQ ID No.:

4) and an oligonucleotide of sequence 5'"'"'-CTACAGAGTGCAGTTGGCCTC-3'"'"' (SEQ ID No.;

     5).

23. The kit of claim 19 wherein said nucleic acid is a nucleic acid probe which hybridizes specifically to Sequence Id No.:

2, but not Sequence Id No.;

1 or Sequence Id No.;

3 in 2x SSC, 0.1% SDS at 42°

C.

24. The kit of claim 23 wherein said probe hybridizes specifically to a region between about base 4843 and base 4863 of Sequence Id No:

2 in 2x SSC, 0.1% SDS at 42°

C.

25. The kit of claim 24 wherein said probe is labeled with a marker said marker is selected from the group consisting of:

a fluorophore, a lumiphore, a radioactive label, a chromogen, horseradish peroxidase, biotin, or dioxigenin.

26. The kit of claim 19 wherein said salmonid is selected from the group consisting of Oncorhynchus tshawytscha, and Oncorhynchus kisutch.