Sex-specific markers for prawns

Abstract

Disclosed is a primer pair comprising nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, where the primer pair is a PCR-based molecular marker, and where the marker is a sex-specific marker of prawns. Use of the PCR-based molecular marker of the present invention for determining the sex of prawns is disclosed. Disclosed is a method of establishing a male parsexual culture of prawns, the method comprising a PCR-based sex determination wherein the PCR-based sex determination comprises the use of a PCR-based molecular marker of the invention.

Description

Technical Field

[0001] The present invention relates to sex-specific markers for shrimp. More specifically, the present invention relates to a sex-specific marker for shrimp derived from Macrobrachium rosenbergii ( Macrobrachium rosenbergii / M. rosenbergii A polymerase chain reaction (PCR)-based sex-specific molecular marker (SMM) can be used to determine the sex of shrimp, particularly Macrobrachium rosenbergii, including but not limited to sex determination during early growth stages. This sex-specific marker is used to establish monosex culture. Background Art

[0002] Shrimp farming is a significant aquaculture activity worldwide. Several crustacean species, including the freshwater giant prawn Macrobrachium rosenbergii, exhibit a bimodal growth pattern, with males exhibiting superior growth compared to females. Male populations of Macrobrachium rosenbergii have been shown to produce higher marketable yields and grow faster than female populations. Consequently, harvesting requires a shorter timeframe, resulting in higher revenue per unit area.

[0003] Macrobrachium rosenbergii possesses androgenic glands, which are responsible for sexual differentiation. Previous studies have demonstrated the role of androgenic glands in male sexual differentiation by observing primary and secondary sexual characteristics in Macrobrachium rosenbergii following androgenic gland removal or transplantation. Complete functional sex reversal from male to neo-female and from female to neo-male was achieved by bilateral androgenic gland ablation and transplantation, respectively.

[0004] Administration of double-stranded RNA, in which one of the strands comprises a nucleotide sequence complementary to at least a portion of the open reading frame of the insulin-like factor gene of the androgenic gland of Macrobrachium rosenbergii, to post-larval males results in silencing of the expression of the insulin-like factor gene in these freshwater shrimp.

[0005] Silencing the expression of the insulin-like factor gene in males of Macrobrachium rosenbergii temporarily blocks the regeneration of the male masculine appendage and inhibits spermatogenesis, which is accompanied by a delay in the molting interval and a reduction in body mass accumulation. Thus, silencing the insulin-like factor gene in males of the freshwater shrimp Macrobrachium rosenbergii results in fully functional sex reversal from male to neofemale (i.e., genotypically male but phenotypically female, which can mate with actual males to produce all-male offspring).

[0006] However, means of generating male monosex populations of M. rosenbergii were found to be inefficient and involved lengthy procedures, such as manually isolating male M. rosenbergii before introducing double-stranded RNA into males to avoid unnecessary introduction of double-stranded RNA into females.

[0007] In summary, there is still a need for efficient, technically improved and economically beneficial methods to produce monosex male populations of shrimp, particularly Macrobrachium rosenbergii. Summary of the Invention

[0008] The present invention relates to a primer pair comprising the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer pair is a PCR-based molecular marker, and wherein the marker is a sex-specific marker for shrimp.

[0009] Furthermore, the present invention relates to use of a PCR-based molecular marker for determining the sex of shrimp, wherein the PCR-based molecular marker is a primer pair comprising SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] Still further, the present invention relates to a method for establishing male-only culture of shrimp, the method comprising PCR-based sex determination, wherein the PCR-based sex determination comprises using the PCR-based molecular markers as described above.

[0011] Additional aspects, features, and advantages of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of the preferred embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be fully understood from the detailed description given hereinafter and the accompanying drawings, which are given by way of illustration only and therefore do not limit the present invention, in which: In the attached figure: Figure 1 : is an explanatory diagram showing PCR bands obtained by polymerase chain reaction (PCR) amplification of gDNA from a population of juvenile Macrobrachium rosenbergii. Figure 1 In the table, (i) posM refers to the male positive control, (ii) posF refers to the female positive control, (iii) neg refers to the negative control, (iv) 100 bp refers to the DNA ladder, and (v) LT13 to LT22, LT06, and LT08 to LT10 refer to samples of juvenile Macrobrachium rosenbergii populations.

[0013] Figure 2 : is an explanatory diagram showing PCR bands obtained by PCR amplification of gDNA of an adult population of Macrobrachium rosenbergii. Figure 2 In the table, (i) posM refers to male positive control, (ii) posF refers to female positive control, (iii) neg refers to negative control, (iv) 100 bp refers to DNA ladder, and (v) SW1 to SW15 refer to samples of adult populations of Macrobrachium rosenbergii. DETAILED DESCRIPTION

[0014] This document discloses a detailed description of preferred embodiments of the present invention. However, it should be understood that these embodiments are merely illustrative of the present invention, which may be embodied in various forms. Therefore, the details disclosed herein should not be construed as limiting, but rather as a basis for the claims and for teaching those skilled in the art. Numerical values ​​or ranges used in this specification should not be construed as limiting.

[0015] The present invention relates to sex-specific markers for shrimp. More specifically, the present invention relates to a polymerase chain reaction (PCR)-based sex-specific molecular marker derived from Macrobrachium rosenbergii, which can be used to determine the sex of shrimp, particularly Macrobrachium rosenbergii, including but not limited to sex determination during early growth stages. The sex-specific marker is used to establish monosex culture.

[0016] A first aspect of the present invention discusses the use of PCR-based molecular markers for determining the sex of shrimp, particularly Macrobrachium rosenbergii, wherein the PCR-based molecular marker is a primer pair comprising the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. It should be understood that SEQ ID NO: 1 is a forward primer of 23 nucleotides in length, and SEQ ID NO: 2 is a reverse primer of 24 nucleotides in length. The forward and reverse primers have 3' and 5' terminal sequences that are complementary to the nucleic acid substrate.

[0017] The primer pairs comprising the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as the PCR-based molecular markers of the present invention are synthesized using, but not limited to, an oligo synthesizer. The synthesis scale can be as small as 10 nmol under desalting and purification scale.

[0018] Furthermore, when PCR amplification of genomic deoxyribonucleic acid (gDNA) of Macrobrachium rosenbergii is performed using the PCR-based molecular markers of the present invention, female Macrobrachium rosenbergii will produce a PCR band of 148 base pairs (bp), while male Macrobrachium rosenbergii will not produce a band. The PCR technique used can be any PCR-based technique known to those skilled in the art. While detection of PCR-based markers is preferably accomplished using PCR-based techniques, it will be apparent to those skilled in the art that other techniques (such as, but not limited to, DNA-DNA hybridization, microarray technology, or DNA melting curves) can be used alone or in combination with PCR amplification to detect marker sequences.

[0019] The control primer pair comprises the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, wherein the control primer pair serves as a positive control. It should be understood that SEQ ID NO: 3 is a forward primer of 18 nucleotides in length, and SEQ ID NO: 4 is a reverse primer of 20 nucleotides in length. The forward primer and the reverse primer have 3' and 5' terminal sequences that are not complementary to the nucleic acid substrate.

[0020] PCR amplification of genomic DNA (gDNA) of Macrobrachium rosenbergii using the control primer pair will generate a PCR band of 234 bp on any gDNA of Macrobrachium rosenbergii, regardless of the sex of the species.

[0021] A second aspect of the present invention discusses a method for establishing male-only culture of shrimp, particularly Macrobrachium rosenbergii, comprising PCR-based sex determination using the PCR-based molecular markers of the present invention. PCR-based sex determination using the PCR-based molecular markers of the present invention is used to distinguish between male and female Macrobrachium rosenbergii during, but not limited to, the early growth stages of the species. Subsequently, male Macrobrachium rosenbergii are selected and / or isolated for culture.

[0022] Next, double-stranded RNA (one of the strands comprising a nucleotide sequence complementary to at least a portion of the open reading frame of the insulin-like factor gene of the androgenic gland of Macrobrachium rosenbergii) is administered to male Macrobrachium rosenbergii, resulting in silencing of insulin-like factor gene expression in male Macrobrachium rosenbergii. Administration of the double-stranded RNA to male Macrobrachium rosenbergii is performed by, but is not limited to, injection, immersion, transdermal administration, or feeding.

[0023] Silencing the expression of the insulin-like factor gene in males of the prawn Macrobrachium rosenbergii temporarily blocks the regeneration of male appendages and inhibits spermatogenesis, which is accompanied by a delay in the molting interval and a reduction in body mass accumulation. Thus, silencing the insulin-like factor gene in males of the prawn Macrobrachium rosenbergii results in fully functional sex reversal from male to neofemale (i.e., genetically male but phenotypically female, which can mate with males to produce all-male offspring).

[0024] The following examples are constructed to illustrate the invention in a non-limiting sense.

[0025] Example 1 – Sex determination of a population of Macrobrachium rosenbergii Polymerase chain reaction amplification (PCR amplification) of gDNA of a Macrobrachium rosenbergii population was performed using the primer pair of the present invention (SEQ ID NO: 1 and SEQ ID NO: 2) and the control primer pair (SEQ ID NO: 3 and SEQ ID NO: 4) to determine the sex of the Macrobrachium rosenbergii population.

[0026] The steps and parameters involved in the PCR reaction are as follows: Table 1 shows the parameters (i.e., volume and concentration) of the substances used in the PCR reaction. Table 2 also shows the steps and parameters involved in the PCR reaction (i.e., temperature, duration, and number of cycles). Table 3 shows the PCR bands obtained from Figure 3 , obtained when gDNA from a population of juvenile Macrobrachium rosenbergii was amplified using the primer pair of the present invention (SEQ ID NO: 1 and SEQ ID NO: 2) and the control primer pair (SEQ ID NO: 3 and SEQ ID NO: 4). Table 4 shows the PCR bands obtained from Figure 4 , obtained when gDNA from a population of adult Macrobrachium rosenbergii was amplified using the primer pair of the present invention (SEQ ID NO: 1 and SEQ ID NO: 2) and the control primer pair (SEQ ID NO: 3 and SEQ ID NO: 4).

[0027]

[0028]

[0029]

[0030] Based on the results obtained in Table 3, it is apparent that PCR amplification of gDNA from female juvenile Macrobrachium rosenbergii using the PCR-based molecular markers of the present invention generated a PCR band of 148 bp in size. Meanwhile, PCR amplification of gDNA from male juvenile Macrobrachium rosenbergii using the PCR-based molecular markers of the present invention did not generate a PCR band of 148 bp in size. Therefore, this demonstrates that the PCR-based molecular markers of the present invention are suitable for determining the sex of Macrobrachium rosenbergii at the juvenile stage during PCR amplification.

[0031]

[0032] Based on the results obtained in Table 4, it is apparent that PCR amplification of gDNA from adult female Macrobrachium rosenbergii generated a PCR band of 148 bp. Meanwhile, PCR amplification of gDNA from adult male Macrobrachium rosenbergii did not generate a PCR band of 148 bp. Therefore, this demonstrates that the PCR-based molecular markers of the present invention accurately determine the sex of adult Macrobrachium rosenbergii during PCR amplification.

[0033] In addition, the sex of the adult Macrobrachium rosenbergii was determined by visual inspection, wherein the visual inspection of the sex of the adult Macrobrachium rosenbergii was performed by observing the sex organs located at the fifth leg of male Macrobrachium rosenbergii and the third leg of female Macrobrachium rosenbergii. Subsequently, the sex of the adult Macrobrachium rosenbergii determined by visual inspection was compared with the sex of the adult Macrobrachium rosenbergii determined by PCR amplification of gDNA of a population of adult Macrobrachium rosenbergii using the PCR-based molecular markers of the present invention.

[0034] Based on the results obtained in Table 4, it is apparent that the sex of adult Macrobrachium rosenbergii determined by PCR amplification using the PCR-based molecular markers of the present invention is consistent with the sex of adult Macrobrachium rosenbergii determined by visual inspection. Therefore, this demonstrates that the PCR-based molecular markers of the present invention are suitable for determining the sex of Macrobrachium rosenbergii at the adult stage during PCR amplification. This further demonstrates that the PCR-based molecular markers of the present invention are suitable for determining the sex of Macrobrachium rosenbergii at the juvenile stage during PCR amplification, even when visual inspection to confirm sex consistency is not possible at this stage.

[0035] In summary, the PCR-based molecular markers of the present invention can overcome conventional disadvantages because they are suitable for determining the sex of penaeid shrimp, particularly Macrobrachium rosenbergii, which helps avoid the unnecessary introduction of double-stranded RNA into female Macrobrachium rosenbergii. Furthermore, the PCR-based molecular markers of the present invention facilitate efficient methods for generating male-only populations of penaeid shrimp, particularly Macrobrachium rosenbergii.

[0036] The terminology used herein is for the purpose of describing specific example embodiments only and is not intended to be limiting. As used herein, the singular forms "a / an" and "the" may also be intended to include the plural forms, unless the context clearly indicates otherwise. The terms "comprises / comprising," "including," and "having" are inclusive and, thus, specify the presence of recited features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0037] The method steps, processes, and operations described herein should not be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as a certain order of performance. It should also be understood that additional or alternative steps may be employed. The use of the expression "at least" or "at least one" indicates the use of one or more elements as may be employed in one of the embodiments to achieve one or more desired objectives or results.

Claims

1. A primer pair comprising the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer pair is a PCR-based molecular marker, and wherein the marker is a sex-specific marker for shrimp.

2. The primer pair as claimed in claim 1, wherein the prawn is Macrobrachium rosenbergii.

3. Use of a PCR-based molecular marker for determining the sex of shrimp, wherein the PCR-based molecular marker is a primer pair comprising SEQ ID NO: 1 and SEQ ID NO:

2.

4. The use of PCR-based molecular markers as claimed in claim 3, wherein the prawn is Macrobrachium rosenbergii.

5. A method for establishing male-only culture of shrimp, the method comprising PCR-based sex determination, wherein the PCR-based sex determination comprises using the PCR-based molecular marker according to claim 1.

6. The method for establishing male-only culture of shrimp according to claim 5, wherein the shrimp is Macrobrachium rosenbergii.

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