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    • 专利摘要:
    The invention relates to a sturgeon sexing method, comprising: a) a step of extracting the genomic DNA contained in a blood sample of said sturgeon some months old, b) a step of qPCR comprising the detection and the quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a), c) a step of qPCR comprising the detection and the quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity, (d) the calculation of the ratio of the quantity obtained in (b) in relation to the average quantity obtained in (c), (e) the comparison of the ratio obtained in (d) with a threshold value, in which where the ratio obtained in d) is greater than the threshold value, then said sturgeon is generally a male, and when the ratio obtained in d) is below the threshold value, then said sturgeon is generally a female.
    公开号:FR3055905A1
    申请号:FR1658514
    申请日:2016-09-13
    公开日:2018-03-16
    发明作者:Sandrine Gaillard；Remy Julien Simide
    申请人:Universite de Toulon；
    IPC主号:
    专利说明:

    © Publication number: 3,055,905 (use only for reproduction orders)
    ©) National registration number: 16 58514 ® FRENCH REPUBLIC
    NATIONAL INSTITUTE OF INDUSTRIAL PROPERTY
    COURBEVOIE
    ©) Int Cl 8 : C 12 Q 1/68 (2017.01), A 01 K61 / 00
    A1 PATENT APPLICATION
    ©) Date of filing: 13.09.16. © Applicant (s): UNIVERSITE DE TOULON— FR. (30) Priority: ©) Inventor (s): GAILLARD SANDRINE and SIMIDE REMY JULIEN. (43) Date of public availability of the request: 16.03.18 Bulletin 18/11. ©) List of documents cited in the report preliminary research: Refer to end of present booklet (© References to other national documents ©) Holder (s): UNIVERSITE DE TOULON. related: ©) Extension request (s): @) Agent (s): CABINET PLASSERAUD.
    METHOD OF EARLY STURGEON SEXING.
    FR 3 055 905 - A1 sturgeon, comprising:
    a) a step of extracting genomic DNA contained in a blood sample from said sturgeon a few months old,
    b) a qPCR step comprising the detection and quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a),
    c) a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity,
    d) the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c),
    e) comparing the ratio obtained in d) to a threshold value, in which when the ratio obtained in d) is greater than the threshold value, then said sturgeon is generally a male, and when the ratio obtained in d) is less than the threshold value, then said sturgeon is generally a female.
    i
    The present invention relates to a method of early sexing of sturgeon, especially Siberian.
    In aquaculture, knowing the sex of the fish is an important problem, in particular to improve growth rates, which can depend on sex. For sturgeon farmers, this question is even more crucial. Sturgeon farming is expensive because it is a large fish that requires a lot of space and food. The trade in sturgeon meat is therefore hardly profitable. Only the caviar trade, which of course concerns females, is lucrative. It is therefore important to be able to distinguish females from males very early on: this is called early sexing.
    In sturgeons, early sexing is difficult because there is no sexual dimorphism: males and females are similar in morphological level. It is necessary to wait between 2 and 10 years approximately, before the gonads of the sturgeons are differentiable. Moreover, current sexing techniques require waiting until the gonads are differentiated; they are based on gonad ultrasound, hormone testing and biopsy or gonad surgery.
    Thus, no current sturgeon aquaculture, developed in the world, performs early sexing (i.e. a few months).
    Application US2013 / 0244261 discloses a hormone dosing technique earlier than current techniques. It is based on the quantification in serum, plasma or blood, of hormones from the superfamily of TGF-β, in sturgeons aged from one to several years.
    Surprisingly, the inventors have succeeded in implementing an early sexing method for sturgeon, especially Siberian. This method is based on the measurement of the length of telomeres, by qPCR.
    Telomeres play a major role in maintaining the integrity of the genome. They are at the ends of the chromosomes, and have a characteristic sequence TTAGGG repeated n times. Until now, telomeres have never been used as a sexing tool for a species.
    However, the inventors have identified that the length of the telomeres can be used as a marker for the early sexing of sturgeons. Indeed, the highest values are observed for males, while the lowest values are observed for females.
    The method proposed in the invention is thus reliable, and can be implemented in sturgeons only a few months old.
    The invention therefore relates to a method of sexing sturgeon, in particular Siberian, comprising:
    a) a step of extracting the genomic DNA contained in a blood sample from said sturgeon a few months old,
    b) a qPCR step comprising the detection and quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a),
    c) a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity,
    d) the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c), then
    e) comparing the ratio obtained in d) to a threshold value, in which when the ratio obtained in d) is greater than the threshold value, then said sturgeon is generally a male, and when the ratio obtained in d) is less than the threshold value, then said sturgeon is generally a female.
    This method is called the sexing method according to the invention.
    The sturgeon is a fish in the Acipenseridae family. This family includes the Acipenserinae subfamily, which itself includes the genus Acipenser, and the Scaphirhynchinae subfamily. Preferably, the sturgeon targeted by the method according to the invention is the Siberian sturgeon (Acipenser baerii).
    "Sexing method" of a fish means a method of determining the sex of the fish. The sexing method according to the invention is an early sexing method, i.e. which aims to determine the sex in fish a few months old from a blood sample.
    By "a few months old sturgeon" is meant a sturgeon several months old, preferably 2 to 6 months, preferably approximately 3 months.
    The sturgeon sexing method according to the invention comprises a step of extracting the genomic DNA contained in a blood sample of said sturgeon a few months old: this is step a).
    The step can be done by any method known in the prior art. In particular, it can be carried out using any commercial kit for this purpose, in particular containing proteinase K, such as the NucleoSpin 8 Blood kit from Mâcherey-Nagel.
    Then, once the genomic DNA has been extracted, there follows a step b) of qPCR, which comprises the detection and quantification of the telomeric sequence (TTAGGG) n (n being an integer) present in the genomic DNA obtained in a ).
    Conventionally, the technique used to measure the length of telomeres is the Southern Blot. However, such a technique is expensive and time-consuming to implement. The qPCR technique proposed in the present invention provides rapidity and reliability of the measurements.
    This step of qPCR is notably carried out using the following tell and Tel2 telomeric primers:
    Tell: 5'CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3 '(SEQ ID NO: 1) and Tel2: 5'GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3' (SEQ ID NO: 2).
    Thus, preferably, step b) is carried out using the primers of sequences SEQ ID
    NO: 1 and 2.
    Preferably, quantitative PCR (qPCR) uses the SYBR Green technology (fluorescent marker), in particular with the LightCycler 480 thermocycler from Roche.
    Simultaneously, before or after step b), there is step c), which comprises a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity. Preferably, steps b) and c) are simultaneous.
    By "reference gene" is meant a constitutional gene which mainly codes for a protein essential for basic cellular functions. Preferably, the quantity of the reference gene according to the invention is representative of the quantity of genomic DNA (genome) extracted in step a). For example, the reference gene may be the gene coding for beta actin, alpha actin or the gene coding for 18S ribosomal RNA. Preferably, the two reference genes used in the invention are beta actin and the gene coding for 18S ribosomal RNA. These reference genes are of major interest in the context of the invention. Indeed, they make it possible to obtain an average quantity of gene, and thus to standardize the length of the telomeres.
    Preferably, and as indicated above, quantitative PCR (qPCR) uses the SYBR Green technology (fluorescent marker), in particular with the Roche LightCycler 480 thermocycler.
    Preferably, the two reference genes used are beta actin and the gene coding for the ribosomal RNA 18S (“gene 18S”), and step c) of qPCR is carried out using the following primers Actl and Act2 for beta actin, and the following 18S1 and 18S2 for the 18S gene:
    Actl: 5'TATCCTGACCCTGAAGTACCCAATC-3 '(SEQ ID NO: 3) and Act2: 5'-CACGCAGCTCATTGTAGAAGGTGTG-3' (SEQ ID NO: 4)
    18S1: 5'-TGAGAAACGGCTACCACATCC-3 '(SEQ ID NO: 5) and 18S2: 5'-GCCTCGAAAGAGTCCTGTATTG-3' (SEQ ID NO: 6).
    Thus, preferably, step c) is carried out using the primers of sequences SEQ ID NO: 3 to 6.
    Preferably, the amplification conditions of step b) and / or of step c), preferably of steps b) and c), are the following:
    - a first heating phase, in order to denature the DNA (initial denaturation step); followed by
    - a second phase of several cycles, each cycle comprising:
    o a denaturation phase, then o a priming hybridization phase, and o an elongation phase.
    At the end of step c), the average quantity of each of the two reference genes is thus obtained.
    This average quantity is then used to normalize the measured quantity relative to the length of the telomeres of step b): this is step d).
    More specifically, step d) includes the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c). This ratio corresponds to the relative length of the telomeres.
    This ratio is then compared to a threshold value; this is step e). During this comparison:
    - if the ratio obtained in d) is greater than the threshold value, then said sturgeon is a male, and
    - if the ratio obtained in d) is less than the threshold value, then said sturgeon is a female.
    By "threshold value" is thus meant the limit value of the ratio obtained in d) above which the sturgeon from which the sample comes is sexed as male according to the method of the invention.
    Preferably, as indicated in the example, the threshold value of the ratio is equal to 9.
    A subject of the invention is also a method for increasing the proportion of female sturgeons (in particular Siberians) present in a sturgeon aquaculture of a few months old (in particular Siberian) comprising the sexing method according to the invention, followed by a step f) selection of females.
    One such method of increasing the proportion of female sturgeon may include selecting females from a given population, or extracting males from that population.
    The method for increasing the proportion of female sturgeon present in aquaculture of sturgeon a few months old, thus comprises the following stages:
    a) a step of extracting the genomic DNA contained in a blood sample from each of the sturgeons a few months old,
    b) a qPCR step comprising the detection and quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a),
    c) a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity,
    d) the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c),
    e) comparing the ratio obtained in d) to a threshold value, in which when the ratio obtained in d) is greater than the threshold value, then said sturgeon is a male, and when the ratio obtained in d) is less than the threshold value, then said sturgeon is female, then
    f) selection of females identified in e).
    Indeed, as demonstrated in the example, the sexing method according to the invention makes it possible to sexually sturgeon, in particular Siberian, early, to select the females and to extract at least 40% of the males. This ultimately makes it possible to increase the female: male sex ratio, and thus to increase the proportion of females present in aquaculture.
    The invention is illustrated using the example below.
    Figure 1. Mustache boxes representing the length of telomeres between young males and females.
    The average is indicated by the cross, the lower bar represents the first quartile, the intermediate bar the median and the upper bar the third quartile. The whiskers represent the thresholds 5 and 95%.
    EXAMPLE: Protocol for the analysis of the length of telomeres for sexing juvenile Siberian sturgeon
    The sex ratio of the Siberian sturgeon is 1 male for 1 female. The aquacultures dedicated to the production of caviar seek to determine the sex of the individuals as early as possible in order to focus the production efforts on the females.
    Sexing of Siberian sturgeons is currently carried out on individuals whose gonads are developed by an ultrasound or, more rarely, a hormonal assay. The age of these fish, around 3 years, depends on environmental conditions. The error rate of sexing by ultrasound is around 10%, depending on the level of maturity of the gonads and the acuity of the experimenter.
    The early sexing technique according to the invention makes it possible to enrich batches of Siberian sturgeon 3 months old in females.
    The sample is taken from 3-month-old Siberian sturgeons by taking a blood sample from the tail vein.
    The sample is centrifuged (10 min, 3000 g), the plasma is eliminated and 1 ml of absolute ethanol is added to the erythrocyte pellet before storage at -80 ° C until use. Genomic DNA is extracted using the NucleoSpin 8 Blood kit (Macherey-Nagel) following the supplier's recommendations. The concentration of each sample is then adjusted to 10 ng.pl ' 1 , storage is done at -80 ° C until use. The analysis is carried out in relative quantitative PCR, using SYBR Green technology on a LightCycler 480 real-time thermal cycler (Roche). The reaction mixture (final volume of 10 μΐ) consists of 5 μΐ of master mix (Roche), 1 μΐ of each primer (1 μΜ final), 1 μΐ of ultra pure water and 2 μΐ of DNA (20 pg final ). Each sample is processed in duplicate and the 384-well plates used all contain a negative control and a calibrator to standardize the analyzes between them.
    The amplification protocol and the primers binding to the telomeric sequences (Tel primers) are taken from the publication by Cawthon (2002). Thus, the primers Tel are Tell and Tel2, of sequences SEQ ID NO: 1 and 2.
    Two reference genes are used, B-actin and 18S, the respective primers of which are Act1 and Act2; 18S1 and 18S2, of sequences SEQ ID NO: 3 to 6.
    For all plates, the fusion curves of each amplifier, the negative control and the calibrator are checked. A single peak must be observed on each fusion curve, no amplification should be found for the negative control and a standard value will be obtained from the calibrator. The actual effectiveness of each pair of primers is defined using dilution ranges from a pool of samples.
    These efficiencies make it possible to determine the telomeric signal which is expressed relative to the two references. The ratio obtained using LC480 software version 1.5 allows the relative length of the telomeres to be estimated for each individual.
    In order to test this methodology, individuals were removed at the age of 3 months, microchipped, then, after the development of their gonads, sexed conventionally by ultrasound. A total of 111 males and 101 females were thus obtained.
    The length of the telomeres is generally shorter in females than in males (Student's t test, p = 0.03; Figure l.A). Thus, by setting a threshold below 9 for the ratio, 25% of individuals are preserved and have a sexual ratio of 3 females for 1 male (elimination of 50% of male individuals).
    A part of these individuals were analyzed in a double-blind fashion one year apart (Figure 1.B). Once again the length of the telomeres is generally shorter in females than in males (Student's t test, p = 0.03).
    Eliminating 40% of the fish with the longest telomeres makes it possible to immediately extract 50% of the male individuals for 28% of the female individuals, giving in the end a sexual ratio of 60% of females for 40% of males.
    权利要求:
    Claims (7)
    [1" id="c-fr-0001]
    Claims
    1. Sturgeon sexing method, comprising:
    a) a step of extracting T genomic DNA contained in a blood sample from said sturgeon a few months old,
    b) a qPCR step comprising the detection and quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a),
    c) a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity,
    d) the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c),
    e) comparing the ratio obtained in d) to a threshold value, in which when the ratio obtained in d) is greater than the threshold value, then said sturgeon is generally a male, and when the ratio obtained in d) is less than the threshold value, then said sturgeon is generally a female.
    [2" id="c-fr-0002]
    2. Method according to claim 1, characterized in that the sturgeon is a Siberian sturgeon.
    [3" id="c-fr-0003]
    3. Method according to claim 1 or 2, characterized in that the sturgeon is 2 to 6 months old, preferably about 3 months.
    [4" id="c-fr-0004]
    4. Method according to one of claims 1 to 3, characterized in that step b) is carried out using the primers of sequences SEQ ID NO: 1 and 2.
    [5" id="c-fr-0005]
    5. Method according to any one of claims 1 to 4, characterized in that step c) is carried out using the primers of sequences SEQ ID NO: 3 to 6.
    [6" id="c-fr-0006]
    6. Method according to any one of claims 1 to 5, characterized in that the threshold value of the ratio is equal to 9.
    ίο
    [7" id="c-fr-0007]
    7. Method for increasing the proportion of female sturgeons present in a sturgeon aquaculture of a few months old, thus comprises the following stages:
    a) a step of extracting the genomic DNA contained in a blood sample from each of the sturgeons a few months old,
    b) a qPCR step comprising the detection and quantification of the telomeric sequence (TTAGGG) n present in the genomic DNA obtained in a),
    c) a qPCR step comprising the detection and quantification of two reference genes present in the genomic DNA obtained in a), then the calculation of their average quantity,
    d) the calculation of the ratio of the quantity obtained in b) to the average quantity obtained in c),
    e) comparing the ratio obtained in d) to a threshold value, in which when the ratio obtained in d) is greater than the threshold value, then said sturgeon is a male, and when the ratio obtained in d) is less than the threshold value, then said sturgeon is female, then
    f) selection of females identified in e).
    1/1
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    法律状态:
    2017-09-27| PLFP| Fee payment|Year of fee payment: 2 |
    2018-03-16| PLSC| Search report ready|Effective date: 20180316 |
    2018-08-30| PLFP| Fee payment|Year of fee payment: 3 |
    2020-10-16| ST| Notification of lapse|Effective date: 20200906 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1658514|2016-09-13|
    FR1658514A|FR3055905B1|2016-09-13|2016-09-13|PRECISE SEXING METHOD OF STURGEON|FR1658514A| FR3055905B1|2016-09-13|2016-09-13|PRECISE SEXING METHOD OF STURGEON|
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