Method for determining the sex of solea senegalensis

Abstract

The invention relates to a method for determining the sex of Solea senegalensis. The invention is based on the differential amplification between males and females of a region of exon 14 of the follicle-stimulating hormone receptor (fshr) gene of Solea senegalensis in order to determine sex in individuals of this species. Amplification of this region is preferably carried out using loop-mediated isothermal amplification (LAMP). Primers for said differential amplification via LAMP are also provided.

Description

[0001] METHOD FOR DETERMINING THE SEX OF SOLEA SENEGALENSIS.

[0002] DESCRIPTION

[0003] TECHNICAL SECTOR

[0004] The invention falls within the field of biotechnology and is of particular interest for its application in aquaculture, as it allows for improved efficiency and productivity in marine fish farming. Specifically, it provides a method for determining the sex of the Senegalese sole, Solea senegalensis, a species of high commercial interest. This method utilizes molecular techniques such as loop-mediated isothermal amplification (LAMP).

[0005] BACKGROUND OF THE INVENTION

[0006] Aquaculture is one of the most widely accepted options among managers and scientists for supplying food to the population, given that ocean catches are insufficient to meet growing market demand. Controlling aquaculture production is characterized, among other major challenges, by the need to identify the sex of individuals early on (before sexual maturity), thus facilitating the production of populations biased towards the sex of the individuals that offer the greatest profitability.

[0007] Sexual differentiation systems in fish can be based on external factors, environmental causes such as temperature, size, or age, or even the presence of parasites. Genetic mechanisms based on a single factor are also known, determining sex according to the genes present at a single locus.

[0008] Of all the sex determination mechanisms, the most widely observed in nature and the best known are the XX-XY and WZ-ZZ systems, where homogametic individuals are female and male, respectively [1]. In fish, with the exception of Neotropical fish, only 7% of fish species have shown heteromorphic sex chromosomes. Regarding flatfish species, the system has been studied in 14 of them, with the XX / XY and ZZ / ZW systems found for sex determination. Specifically, the XX / XY system has been described in the flounder, although there are no clearly heteromorphic chromosomes [2].

[0009] The Senegalese sole (Solea senegalensis Kaup, 1858) (Soleoidei: Soleidae) is a species of the order Pleuronectiformes, characterized by its dorsoventrally flattened body, reduced body cavity, and asymmetry in adulthood. It exhibits a pelagic lifestyle in its larval and juvenile stages, and a benthic or demersal lifestyle as an adult along continental margins. It is distributed along the Atlantic coast from Senegal to the United Kingdom and extends into the Mediterranean, reaching the Tunisian coast. The catch rate of this species has increased from approximately 14 million tons in 1950 to 65 million tons in 2016. The proven commercial value of the species has placed it among the most sought-after species in the aquaculture market, although its introduction into aquaculture systems and captive breeding programs is not without its challenges.The development of Senegalese sole aquaculture requires the control of reproduction and pathologies during the growth phase.

[0010] On the one hand, the larval growth rate is low, and problems in the metamorphosis process make it difficult to obtain marketable adults. Male S. senegalensis mature at around two years of age, and females a year later, generally before reaching the minimum market size (24 cm). Once first mature, reproduction occurs in annual cycles, with the main spawning period in southern Iberia from February to May, and a secondary spawning event in autumn. Males can remain mature year-round, with some variations that may be related to fluctuations in the females' maturity stage.

[0011] On the other hand, adult males raised in captivity exhibit sexual dysfunction. [3] In male individuals of the first generation of offspring, proper spermatogenesis is observed, and they present good androgen levels. However, the reasons for this infertility, and therefore the failure of the reproductive process in captivity, are, on the one hand, a reduced sperm production compared to captured wild individuals and, on the other hand, the absence of an adequate sexual courtship process on the part of these males.

[0012] Production optimization has not been possible also due to the high variability in growth rates [3]. This may be due to the variation in the sex ratio between batches and the sexual growth dimorphism in favor of females.

[0013] Despite recent advances in the biology and breeding of Senegalese sole, several aspects remain to be discovered. One of these concerns the lack of information on the sex determination mechanism and the process of sex differentiation. This knowledge is necessary for the development of sex control strategies that favor the most desired sex through endocrine therapy or in combination with chromosome manipulation. Female Senegalese sole grow faster and mature later than males [3], and therefore, female-biased populations would be suitable for aquaculture of this species.

[0014] Therefore, one of the major challenges currently being addressed is the design of molecular tools that facilitate the early identification of the sex of individuals.

[0015] The Senegalese sole has 21 pairs of chromosomes and lacks heteromorphic sex chromosomes [4] that can be identified by their morphology. However, the XX / XY system has been described as responsible for sex determination in the species [2]. Given the absence of a sex chromosome and the difficulty of identifying a sex-determining gene in the species, it was postulated that the larger metacentric chromosome, originating from the fusion of two acrocentric chromosomes, was a proto-sex chromosome [5, 6] because it contains the dmrt1-dmrt2-dmrt3 genes, which have been related to the process of sex determination in other species (e.g., in the Z chromosome of Cynoglossus semilaevis Günther, 1873) [7].

[0016] On the other hand, the sequencing of the complete genome of S. senegalensis and its comparison between males and females has allowed the identification of 41 differential allelic variants between males and females in the follicle-stimulating hormone receptor (FSH) gene [8]. These differential single nucleotide polymorphisms (SNPs) between males and females coincide with the XX / XY system described for the species and also cause differences in gene expression of the FSH gene (higher in males than in females). The identification of the FSH gene as a determinant of sex differentiation in the Senegalese sole opens the door to the design of a molecular assay that allows for easy and robust diagnosis of the sex of the individuals to be analyzed, as has already been proposed in other fish species of different orders (Mugil cephalus, Mugiliformes) [9].

[0017] Molecular assays for sex determination in fish are typically based on PCR or quantitative PCR, which has historically restricted their application to specialized laboratories equipped with the necessary materials and instruments. Furthermore, the average time required for these diagnoses, assuming optimized laboratory processes, is usually four hours (including DNA extraction, amplification of the diagnostic marker, and obtaining results through visualization on an agarose gel or interpretation of quantitative PCR results - qPCR). The total time for the sex determination process can extend to several days or weeks if the time elapsed from sample submission to receipt and processing at the reference laboratory is taken into account.Therefore, it is necessary to identify the DNA quickly and easily, minimizing the work with numerous samples to reduce laboratory working time and minimize the probability of DNA contamination or degradation.

[0018] Recently, loop-mediated isothermal amplification (LAMP) has joined the set of molecular techniques used for the amplification of DNA fragments and the identification of species.

[0019] LAMP is a molecular technique that synthesizes DNA copies of any region of the genome flanked by two pairs of primers (i.e., a single-stranded DNA sequence that serves as the initiator of DNA synthesis). LAMP uses the enzyme Bst polymerase from the bacterium Bacillus stearothermophilus and takes advantage of its 5'-3' polymerase activity and displacement activity, which allows the synthesis of the new DNA strand through a self-cyclic double-strand displacement system. This technique, unlike the widely used PCR, only requires a single incubation temperature for the synthesis of DNA fragments, without the need to modify the reaction temperature.This amplification method is based on the use of four specific primers that recognize six amplification sites in the DNA sequence, thus exhibiting high specificity and sensitivity to the region being amplified. It can even function with very few DNA copies per reaction (10-15 fg, that is, orders of magnitude more sensitive than PCR). Furthermore, the results of the LAMP reaction can be determined using colorimetric techniques (a simple color change in the reaction indicates the presence of amplification products in the test tube), eliminating the logistical requirements of electrophoretic techniques associated with PCR. This represents a significant advantage over other isothermal amplification techniques such as recombinase-assisted amplification (RAA), an isothermal technique that requires loading the sample onto additional devices based on immunohistochemical techniques.Finally, the reagents used in LAMP can be stored at room temperature (by dehydrating the primers and adding preservatives), which greatly facilitates their transport and shipping. For these reasons, LAMP is currently used in various disciplines such as medicine, microbiology, and parasitology, largely for species identification. However, despite the advantages already described, the application of isothermal techniques has barely been developed in fish. BRIEF EXPLANATION OF THE INVENTION.

[0020] The present invention is based on the amplification of a specific 191 bp region within exon 14 of the follicle-stimulating hormone receptor (FSH) gene located on chromosome 12 of the Senegalese sole, Solea senegalensis, for sex determination in individuals of this species. Amplification of this region can be carried out by any method known in the prior art, but preferably by loop-mediated isothermal amplification (LAMP).

[0021] The method described in the present invention describes the application of amplification techniques to this region of the genome in individuals of unknown sex, resulting in the synthesis of nucleotide sequences corresponding to said region in a differential manner in males and females. Specifically, the present invention provides a set of primers for LAMP amplification that allows amplification of this region only in males; no amplification product is obtained when this technique is applied to samples obtained from female individuals.

[0022] To ensure the correct implementation of the method of the invention, a preferred embodiment is described as the amplification of an additional region that is amplified in both males and females. This region serves as a positive control for the quality of the DNA used and reduces the probability of errors in sex assignment of the analyzed samples. Two regions are proposed for use as positive controls: a 208 bp region of the 18S ribosomal subunit and a region within exon 14 of the follicle-stimulating hormone receptor (fs / ?r) gene, which will be designated FSHR_X. For the purposes of the present invention, any other region of the genome that is amplified in both sexes may be used as a reference.

[0023] Therefore, the invention describes the differential amplification of a specific region of the fshr gene, and the amplification of another region of the Solea senegalensis genome as a positive control, as part of a method for determining the sex of individuals of this species from previously isolated biological samples.

[0024] According to this embodiment of the invention, an individual is categorized as male when an amplification product of the 191 bp region of the aforementioned fshr gene is detected, and the individual is categorized as female when no amplification product is detected.

[0025] Preferably, this amplification is performed simultaneously or sequentially with the amplification of a control region, the latter preferably being either the aforementioned 18S ribosomal subunit region or the region within exon 14 of the follicle-stimulating hormone receptor (fs / ?r) gene designated as FSHR_X. In this particular embodiment, an individual is categorized as male when the amplification product of both regions is detected (FSHR+ / 18S+ or FSHR+ / FSHR_X, depending on the control used). In this preferred embodiment of the invention involving a positive control, the individual is categorized as female when the amplification product of only the control region is detected (FSHR- / 18S+ or FSHR- / FSHR_X+, depending on the control used).

[0026] The invention provides specific primer sequences designed to perform the amplification of the regions of interest, the specific 191 bp region within exon 14 of the fshr gene, the 208 bp region of the 18S ribosomal subunit and the FSHR_X region also within exon 14 of the fshr gene, by means of LAMP, such that the differential amplification of the fshr gene region allows the identification of males and females and the amplification of the 18S or FSHR_X region serves as a positive control.

[0027] It is worth noting that amplification of the complete fshr gene sequence does not allow differentiation between males and females of S. senegalensis since it does not show a differential amplification pattern, resulting in amplification product in both sexes.

[0028] Within the fshr gene, not every region is useful for carrying out the purpose of the present invention, the determination of sex in S. senegalensis, and analyses performed with other regions of this gene provide evidence of this (as shown in example 1).

[0029] The specific 191 bp region within the fshr gene detailed in the present invention, when subjected to amplification processes using techniques such as LAMP, results in differential amplification depending on the sex of the individual, allowing for the identification of males and females. The primers described in the present invention are expressly designed to amplify this specific region, although the invention is not limited to them and also includes other primers equally capable of amplifying this region differentially between males and females.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1: Sequence of exon 14 of the fshr gene for males and females, with color coding indicating the location of the primers that delimit the LAMP-amplified fragment and underlined nucleotides indicating the location of the PCR primers. It should be noted that, in the case of the female sequence, the LAMP and PCR primers do not align well in females (hence, not all of them are represented in the female sequence).

[0032] Figure 2. Agarose gel electrophoresis (2%) of the amplification products of the FSHR, FSHR_X, and 18S markers. Samples 1–5 are female, while samples 6–10 are male. M: size marker. Samples 11 and 12 are reactions where water was added instead of DNA (negative controls). The samples where amplification of the molecular marker occurred (FSHR in males and FSHR_X and 18S in both sexes) show the characteristic LAMP ladder pattern.

[0033] Figure 3. Microtubes with the LAMP amplification products of the FSHR, FSHR_X, and 18S markers under natural light (a) and ultraviolet light (b). Samples 1 to 5 are female, while 6 to 10 are male. Samples 11 and 12 are reactions where water was added instead of DNA (negative controls).

[0034] Figure 4. Agarose gel electrophoresis (2%) of PCR amplification products of the FSHR signature fragment. Samples 1 to 5 are female, 6 to 10 are male, and 11 and 12 are reactions where water was added instead of DNA. M: size marker.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The invention relates to a method of identifying the sex of individuals of the species S. senegalensis based on the application of amplification techniques, such as LAMP, to a specific region of the genome, specifically, a 191 bp region within exon 14 of the follicle-stimulating hormone receptor (fshr) gene.

[0037] The fshr gene of S. senegalensis, with accession number in GenBank Gene ID: 122759636, NCBI Reference Sequence: NC_058041.1, consists of 9,769 base pairs and corresponds to the SEQ. ID. No. 1 sequence shown below:

[0038] SEQ. ID. No. 1:

[0039] AGTTAAGATACTCCTTAGATGATGAGGGGGACACGGTTAATGATAATGCTCATGGTGGT AAACGGGGCAATAGCTTCAGTGCCAGGTTCAGAAGTGGACAGTGAAGCTGGATTTGAG GCCAGCTTGGCTAAACGAACTGTGAACAGCTACCGCCTGAGAGCTTGGGCCACAGAGA TTCCTTCTAACATCTCCAGCAGCATCCAACACCTGTGAGTACACAAACGCTTAACAACAC ACTCCAGCAACATTCCGAGTCAAATGTTAGATTTAAAAAAGTTGTCGTAGCCACTGCAGA TGTTTACCTGCTGAGCCACTGCTGCCCAGTTTCTCCTTGTTGAAATGTAAGTTATTTTGT CACAACTTCCTCTTCTGTTTGGCATCTGCACAGCAAAAGAAAAAACAGTAGAAGTTTGTA

[0040] TGGAGTGTTGGTGTAATTCTTCCTGATAGCGACACCACATTCTGTCCAATTCTTCTATAC

[0041] ACTTTCCTTCAAATTTATTATTAATCCTTCAACCAAAAAACACAGGCTACTTTCGCCTTCA

[0042] AACTGTTGTGAATTTTATGTTGAATGACATTGATTTTCTGGGGGTTTATTCTGTATTATTT

[0043] TTGCATTTATTAATTTCTAAGCTCATTTGGGGATTTTCAGGCTCCTACTAGAGCCTGAAG

[0044] CGCACATGTAAACCATGACACTAACTTGTAGTTGCTCGTATGGTCCAAGGTCCAAAAAC

[0045] AACACATTCTTTATTTCATGCAACACAGTTAAATAGCATAATTTGTTACATTGTGAAGCTG

[0046] TGAAGGACTCATGTGCTGCAAATTATCTTTACTCTCATTTGCATGTATGCAAACCATGTC

[0047] TGCTCATCTTTATTGAGACCCAGTGACCTGCATTCTTTTTTTTTAGTGTCACTTAAACACA

[0048] ATGTTGGAATGACAACATTAGGGTCCATGTGATATACAAGAAGGTACCAACAGGTGAAT

[0049] TTATTCCAGCAGCTATAGACTGTAGCTTTTACCTCCAAAGATGTGGTGGTCAGAGATCA

[0050] GAGGTGGTGCAAGTGTTATAGGTGTTTTTGACACAGTGACACAAAGCTAAATGAGGCAA

[0051] GTGAGGGTGACTGCCACTTGGAGTAAACACTGACCTGTTGTTTTTGCCATTAAAGCATTT

[0052] TGAAGGAATCTCAAGGCCAGGAGGGGGAGGAGGGTGGAGCAGTGGGGGCCAGCATTA

[0053] AGATGTGGACAGCCTGGAGTTTTTGCATGAACATGCACCTAGACAGAACAGGGAGCAG

[0054] ATATGCATAATCAGAGACATACAGAGAGAGGGAGAAGGAGAAGGGAAAATGTTTGTCAA

[0055] AAGTAGCAACTAATTCCCAAAAGACTTTCAGTTGGCAATGTGTGTGTTCCGACCCGTTCT

[0056] GTTCTGCTTGAAGGTCAGGGCTAAAGAACAGTATGCAGCAACGTAGTACAGTTTCAGAG

[0057] CCCACCACCAAATGCAAAATGAGACTAAAACCGCCAAATTCAGTGAATTAAAGGCCCTG

[0058] GCCTTCTTGCGCACTTCTGTCATTGATTAATGAAAACGCATAAAGTTGAGAATGTGAGAA

[0059] ACGAACCCGCTCACATCCCTGACACACAACAACACTGACCCCCAGTGACTGAGGATTTC

[0060] TGTGCTGTGATCACACATCTGTGTCGTTGTATCTTCATCAACAGGGTGGTGACGAACAC

[0061] AAAGGTCAGCGTGATTCCCCAGGGGACCCTCAGTGGCCTACAGCTCCTCAGAAGAGTG

[0062] TGAGTGTGTGCATGTCTGTCACTCATGTTTACACTTTGATGTGGAACGTAATGAACTGTG

[0063] CAGGTCAAAACAATTAATATACGGATCGTGCAGACAGAGGAAAAGTGTGTGTGTGTGTG

[0064] GGGGGGCTATGGCCAGGCCTGATGGGCAAATATTCTGTGCACATATTTGATGATGTTTC

[0065] TTTGAATAAAAAAAACCCATTACATTTACATGTATCAGTAAAGCTAGAATAATCAGATTAT

[0066] ATATTAAATTACTGGAATGGACTTGTAAAATACAATAAATTTGTCCTCTATATACTTAATTG

[0067] TGAACGATTATTTATCTACAAATCAGATATTATTCTATGTTATATGCATGCATTCAGTCAG

[0068] AGACTCTATTTACAGCTTTACAGAGGGAGACTCTTTACTCTTCAGATCATATATATAAGTA

[0069] TTAAATGATAAATCCAGACTTCCAAAATCAAATTGTTGTACAATTGCTTTGGTTTCCTAA

[0070] TTTCTTAGTGTTCCACAGTTTATATCACATTGCATGTGTCCGGCTCTCTCTTATAATTTAA

[0071] TCTTTTGACCTGCAATTGTAAAAACTACATTTCCCATATGTGTTTGTATGAAGACACAAAG

[0072] GTGTGCACTGTTAAGTTTGTGTGCATTTTGCTTGTGATTAAAGAACCACAGATACTGAAA

[0073] ACATGATCTGTATCACAATTTACTGTATCTTCTTCATATATTATAACACCCTCCTCTACCC

[0074] ACAACCTTAAAGCATCATATTGGGGAACAACATTCTGAAGAGTATCGACCCGTTTGCTTT CGCCAACCTCCCTCAGCTCAGTGATATGTGAGTATACAGTTAGCAAAGAAACCATTGTG

[0075] AACATGTTTTATACAACGTGCTGGTGTGTTTAGTCTCTAGCGTGGAACATGAAGAGCTG

[0076] CAAACTGTGAGAAAGTAGATCGCCATACAGTGGCAGACAAAATATATCAAAGAATAAGT

[0077] GTGAATGAATACCTGACAGCTGGATGTTTGTAATATTTTGTATTGTTGTTTCCTCTTAGCT

[0078] TAATCTCTGAAAAATCTGGCTTTGGAAAGTATTGGAGCTTTTTCTTTCTCCAATCTCCCAG

[0079] AACTCACTGAGCTGTAAGTATTAAGGTCAGAGGTTAGGATTATTTAATTAATGATGTATAT

[0080] TGATTTGTACATGTATTTTTCCTCAGGACCATAACAAATTCAAAAAACCTACGTTCCATCC

[0081] ATCCAGATGCATTCGGGAACATGATGAAACTACAGTATCTGTGAGTTCTCGCCTGTGCA

[0082] TAAAACTGAAATCTTTATAACTTGTGCTTTTGTGTGTAACTGTTTGTGTTCAAATTGGGTT

[0083] AGGGGTGACAGGCCTATTTCTGACAGTCTTCCAACACAAAAATACATTGTTTGTATCTTT

[0084] TGTAGCATCTCAAAATAGTGTTATGCTATAGTCTCCCTGACATTAGCATAACGATTACTG

[0085] CTGTGTTTTCCTGGTTTTGAAAAACTAGGACACATACTTTAAATAGAGCAGTATACAGTG

[0086] GAGGAAAATGCATTCCTTAAAGTACCTCTGCAAACAAACATCGTATTTTTAATGTATACCT

[0087] CAAATGTAGCTGATATTGCTAAATGTTCTCAACATCATTTCATACATTCAGTTGACTTAAG

[0088] AAAAAAAGGGGCAAAGGCTGTACCATCTTGGGCATATATATAGTACATATATACATATAT

[0089] AAAGTACCTATATATACTGTATATATATTCAGTTTTGGGAGACAATACAACTCATTATTGT

[0090] CTCATTTCAAACCAGTTGGTCTCAATAATGAGGGTTTTTTTAATGAGTGCCACTGAATAA

[0091] GGCTGAATGTGTCATATGTTTTTGCCAGACATTTTATCTGGAAGAGGGAGCCCTTTAAAT

[0092] GTCCGATCTTTCTCTCTGGCAGCCAAGCTTCTCTCTGAAGTCTCTTCTGAGTCTGTCTTT

[0093] TCACGCCGTTTATTTATTTGTTTATCTGTAAAAATGAATGCTTCGTTGTCTGATTTTTTTTA

[0094] TCTGTTGTTCGTGAGGCCATGGTGTGAACATTGTAACATTTGGTGGTTCATTTGCACGTC

[0095] ACAGGACCATCACCAACACTGGACTCAGCATTTTTCCAGACTTAACCAAGATCTCCTCC

[0096] ACAGCCTTCGACTTTCTGTTGTAAGTCCCATGTATGCACAGATGTATGCTCAGACATACA

[0097] CCCACACATGAGTCCCACATTCATCTGACTGTATACACTCCTATGCACAAATAATGAAAT

[0098] ATGCCCAGAAGTCACACCATGATACAGTATATATAACACTGCAGTCAAAGCCTACACAG

[0099] TGTGCACTCTACACAAGCCAGAATATCAGAGATGGAAACCATCTGTTCCTCTCCACCAA

[0100] CCTGCCTTAAAATGTGTCTTTCAACTCAATGCCATGCCTTCCTCTGAAGGGAATTTGATT

[0101] GGAAGGGGATCGTAGCATGTATGCCTGATATTTTGCACCTTCAGTGTATATACTCATAAG

[0102] CTTCACGTGTTTCTGATCTTTGTGCTGCTTAGGTGACCTGCAGGATAACAGCGGAATAAC

[0103] AAGAGTCCCCCCCAATGCCTTCAGAGGCCTCTGCACTCAAACTATCTCAGAGATGTAAG

[0104] AGATCCATTGTTATTTTTTTCTTCCTCTTCTTTTCAGAAACACACAACCCTCACACAGCTCC

[0105] GAGTAAGCAGTACAGTGATGTTCCCTGAAATTGCTGTCTGAGCTCTGTCTTTTCCT

[0106] AATTGTCTCCTTTCCCCATCACATTTCTGTAACATACCCCGGCATATATTACCTTATTTTA

[0107] AATGAAGTTAAAATTGAGCAGTTCCATTGTAAGCTACTTAATATTACTTTATTTTACTTTTG

[0108] CTGTTCTTTACAGTTGAACGGGGATTTTATTGTCATAAAGGATAAATTCAATTAAATTGAT

[0109] ATGACAGCTTCCTGACTGCTGCAGTCAGGGCCTAAATCTGTCTCATCAACTTATAAAGTA GTCACAGTTACATTAAACTACCCAATATATATTGAATAGCTTAACTTAACATCCACAGCA

[0110] TTTTTGCCTCTGTCAAAATAATCCAATATTATTATACAAAACAAAATAGAAACAGAGAAGC

[0111] TCCACTGCATAGTAAGCACTTCTAATTGCTTAAATGTATTACACTGTTAATGTATTACACT

[0112] ACTTTCATATCAATACACTATGATTCATTTTGCTTCTATGGACACAAGACCATTGTTGGTG

[0113] CCCAAATCAGCCAATTTTCTCCAGGTCCAAGTGATTTGTCTGAAGTAACCAAACCTAATA

[0114] GTGAATAAGTGACTCCAGGACTCTCATTTGTAATGGAATTGTTTTAAACTGTTATAGTGC

[0115] TAAAAAAACAATCAAAATCTAACGACTCCATCAAAGAAATATAGTTTGCCCACAGTTTCTC

[0116] GCGTTCAGACTGATAAAAACCCATAAAAGAAAAAAAGAGGAAAAGACGTCTTACATAATG

[0117] AATTTCATACAGCTTTAGTTACTGTGAGCAAAGAAGCATTTATAGTTCAGAGACGTTTAC

[0118] AGTTGGAAATATTCTGTGTTGGAGCCACATGACTAATTTTGCTACTCCCTCTGATGACAG

[0119] ACGGCTCAACAGAAATGGCATCAAGGAGGTGGCAAGAGGCGCCTTCAATGGTACAAAG

[0120] CTGAAAAGAGTGTGAGTCACAAGCAGAGACACAATGACAATCAGTGGATTTACAAAAAG

[0121] GAAAACATATTTAATTTCTCGTTCTCCCTCCCTCAGATACCTAAAAGACAACCAAGAACTTA

[0122] CTCACATCAATACCAACGCCTTTGGGAGATCCAGTGGGTTGGTGGTACTGTAAGCATGA

[0123] CATCTAACTCATCTAACTCATGTCTGCATGATTCACCCTCTCATCATCACTTATTAAAGT

[0124] CAGGATGTAGGAGAATACAAGACACAACATGGTGTGTGGGAAGACTCAAAAGAGAAAA

[0125] GAAAAGGCTTAGAAACGTGCCAGATTTGGAGTAAAAATAAATCGAGAGCAGGTAATTTC

[0126] AAATCTAAAGAGTCCTGTTTATTCTTCAAACAAAATGACACCTTTTTCCATTGAAGTGTGT

[0127] TCAGACAAGTCGTCAAGCTATTTTCAAGTGTTTCTTTTGCCGACATAAAACCTTCAT

[0128] GTGTCATTTCTTAAACCTTCTGGAGTCTGTCAACGAATACACTGAGAATGTTGTATATGC

[0129] TGTAATATAGCATTATTTATATATCACACAGAGTAAAAAAAATAGTTTCATATGTTCATGT

[0130] GAGGCTTTTTTTTACAAAGCACATTTCTTATTCATGCCTGTAGACGATACATTTTTTAAAA

[0131] ATCTATAAAACTGATCATGAAAATGCTTGAAAACAGAATTATAAATATTTTTTGAATGATTT

[0132] ACAGGCATAAGTAAATAAGATGGAACCACAGGTTCCTCCATTATATCAACTTCTGAAACT

[0133] TTTATACTTAAATTGACAGGTGTAAATTTGGAAGGAGGCAATACCACCTAGTAGTAATGATA

[0134] TTGGTCCAATAGTGCTCAAAAACACTGAATGAAATACAAACCACATACAGTCCAATTATA

[0135] AAAAAATTACATTTTACACAAATGCCTGATTAAAAATGTGGGGTGTTTATTTCTTCTTTCT

[0136] AAGAGCAGAACATATACTGTATACAGTATATATATGAGTAATAGAGTTGGAACTTTGTTTT

[0137] TGAAATGGCAGGTTCAGACTCGGTTGTAAATGCATCAGCAAAAATGTGTTGTGTGTGTG

[0138] TTAACATTCTGGTAGTTCATAAGACGTCTGTATTGAACTGATATTTGTATATCCCTAGTGT

[0139] AAATATGCTATTTACAGACAACCATAAAATCATACATAGTTAAGTAAAACAAAATCAACTA

[0140] AATTTAGTGCCCATGAGCTGCTGAAGTGCACCTATACCTCATTTCCTCCTCTGCTAACTC

[0141] CTCATTGTTATTGTCTGCAGGGACATCTCTCAGACAGCCATCAGCTCCCTGCCACACAA

[0142] CATCCTTGGTGGACTGCATATACTGATCGCAGAGTCTGCCTTCCACCTCAAAGAGCTTC

[0143] CTCCTCTGCAGCTCTTCACCAAACTGCAGCAGGCCAACCTAACATACTCGTCACACTGC

[0144] TGCGCCTTCAAGAACGTGCACAGGAACAGGTACAGGCTGTCAAGAAGTGTCTAAGCCA CACTGCAATGGTTTCTGAAGCTTTTTATTTAACTGGTTTATTGCTGAGGAGGAGTGATGT

[0145] CTATGTCGTGTTTGTGGCTTCGCTTAGCATTAAGAAATAATAATTAGAGTACATAACTTTA

[0146] TCAGCTCCGCACAACTTTCTCTGTGTTACTCAGTAGATCAATGATTAGTACTTGTGTCGC

[0147] CCGGTGACATCCCCACACTGCACACAGGCAGGGATTTGTTTTATATCAAGACAAATGGA

[0148] GGCAAAAGCAAGATTGCACAGATTAGAGATGATGCTCCTAACCCTCGCCCACCACTGTA

[0149] TACACACAAATGTTCCCTCACTCACGCCTGATAGTATTCCTTGATAGATGTAACATGTAC

[0150] TTTCTGTTCACAAAAACTCGACAGAGGCAGAATAATAACATCAACGCTCACCAAAGGTCA

[0151] CATACTGCAGCTTTAAGATCAATAATTATCTTGTATGTTTAATCCACACAAAAACTATAGA

[0152] TTAAAAATAGTATTATGAAGCACAATGCATTAGATGCAGCTCTATTGACAAAACATAACA

[0153] GAAGTAAATATAACATTATGGGTATGTATTTTCATATAATCATGACCCCATTACTACTATT

[0154] AACCACAACAGGATGCCTGCTCACCACAGTGTGGAAAATAAAATAAAAAAACAAATAAAA

[0155] AAATACAGTTGAGAAAAGAGAAGAGCGTTTGTTTACCTGCACATTGTGTGTGTGTTGTTT

[0156] GTTAGGACGAGGTGGAATCCCCTGTGCTCGCTCCCCGAGGCTCGGGATAACATTTATTT

[0157] CTACAGAGACCACTGCTCCAATGCCACGGCCATCACCTGCAGCCCGCTGCCAGATGAG

[0158] TTTACTCCCTGTGAGGATGTCATGTCCACCACCTTCCTACGGATCCTCATCTGGATCATC

[0159] TCTATCCTCACACTGCTTGGAAACGGGGTAGTACTCCTTGTGTTGTTAGGTATGTTCATG

[0160] TCAGATCTATTATTTACCTCCCTTGTGCATGTGTCTACACTCATTCATTTCCATTTCATTC

[0161] TTGACGCATGCAACTAATGCCATTCCAAGTACTTTTTTACATACTAACATAATTAGGTTTT

[0162] AATCTCTTTTACACCAACCACAAGATATTTTAATGTTAAAACTGGTTTATATTTAATACCAA

[0163] GACTCGCTGATTTGTACTTTTTTCCACCATTGTGTTGACAGGCAGCCGTGCCAAACTCA

[0164] CTGTTCCCCGTTTCCTCATGTGCCACTTGGCCTTTGCTGACCTCTGCATGGGTGTCTAC

[0165] CTGGTAGTCATAGCAAGTGTGGACACGATCACTCGAGGCCAGTATTACAACCATGCTAT

[0166] TGAATGGCAGAATGGCCCAGGCTGCAATGCTGCGGGCTTCTTTACGGTGTGTACACAC

[0167] ATATAGACTCCACATAATATAACAGGTCATCGTTGTATGGGTTTGACTAAAAAGATGTTT

[0168] GTGTGTTTGACAGGTGTTTGCCAGTGAGTTATCAGTGTTCACGTTAACTGCGATCACCC

[0169] TAGAGCGCTGGCACACCATCAAATATGCTCTGCGGCTGGACTGCAAAATCCGCCTGAG

[0170] ACACGCATGTCTAATCATGTCGGTGGGCTGGATCTTCTCATCTGTGGCTGCTTTGCTGC

[0171] CCACGGTCGGGGTCAGTAGCTACAGCAAGGTGAGCTTTTAGACAATGCATTCTCTGCTT

[0172] CACTTCATCTGTGATGTCCGGTTTGTGATGACCTGCACCAATTGAGACTGTCTTGTGTTT

[0173] TCATGTATGCTACAGGTGAGTATCTGCCTGCCCATGGACGTGGAGTTTCTGGTGGCTCA

[0174] GGTCTACATTGTGTCTCTCCTCCTCCTCAACATCCTGGCCTTCTTCACTGTGTGTGGCT

[0175] GTTACCTCAGCATCTATCTGACCGTTCGCAACCCCTCATCAATGCCAGCCCACGCTGAC

[0176] ACCAGCGTGGCCCAACGCATGGCCATTCTCATCTTCACTGACTTTGTCTGCATGGCTCC

[0177] CATCTCCTTTTTTGCCATCTCAGCTGCCCTCAAGCTCCCTCTAATCACCATTTCAGATTC

[0178] CAAACTCCTGCTTGTCCTCTTCTACCCAATCAATTCATGCTCAAACCCCTTCCTGTATGC

[0179] CTTCTTTACTCGCACCTTCAGGCAGGACTTCTTTCTCTTTACATCTCGCTTCGGCATCTT TAAGACCCGGGCGCAGATCTACCGGACAGAGAGTATGTCCTGTCAGCTGTCAGGACGT

[0180] ACCAGTGTGGTGGTGTACTCAGTGGCGACTGGACTGAGTTTCGATGGGAAAACAGAAA

[0181] GATGAGAATCTTTCAAAACCCCACAGAAGTTATATTTGTTGCTAGAATATCAGCAGTAGG

[0182] GTGCTAATTCTCTCGCTAGAGAAATACAGAGATAGTGACAGAATAGATAACTCCTGAAG

[0183] GTCCTCTTGTACTCTCCCACGTGTTGTGAAGAGGATAATCACACTTTATAATCCTGATAA

[0184] CAATGTATAATTGTTTCATTTCTGTGTATCTCAACTGTGTTGCCAATATATTTTAACTCAT

[0185] GACACATCTGTGAGTGTACAACAGCTAAACTGTTGTTCTTTAGGCACACACACAAAAAAA

[0186] TGCAGTGTCTGTAAAGGTTCTCAGTCATCTAGGTCATAGTCATTTTCACATTAACCCTAA

[0187] CATTTACCTCTCATCCAAGATTAACTGAAGAAGATTCTTGGATGTAAGGGATATAAGTGA

[0188] ATTCTGTAATGTTGTGTCTGAATTATATTATATCATTTCAACTACATTTAATCTGTTGTAGC

[0189] TCTTCGTGCCACATGTGTGCCCCACTGCCCTTCAGTTTCTCCATATGTTTACAAGAAGGA

[0190] CATGGCATAACTGTGATGTTATATTGTTTATTGTTTGTAAAAAACAGTTCTAAGTGTATCT

[0191] ATGTCTTTTGCACAAAATGTATCTCTAGTTTCCACATCGTTTAATAAAGTTGCTTGATA TTG

[0192] The sequence of exon 14 of the male fshren gene (FSHR_E14_SNPs_males), corresponds to the SEQ sequence. ID. No. 2 which is set out below:

[0193] SEQ. ID. No. 2:

[0194] GGTGAGTATCTGCCTGCCCATGGACGTGGAGTTTCTGGTGGCTCAGGTCTACATGTGT

[0195] CTCTCCTCCTCCTCAACATCCTGGCCTTCTTCACTGTGTGTGGCTGTTACCTCAGCATCT

[0196] ATCTGAACATTCGCAACCCCTCATCAATGCCAGCCCCCGCTGACACCAGGGTGGCCCA

[0197] ACGCATGGCCATTCTCATCTTCACTGACTTTGTCTGCATGGCTCCCATCTCCTTTTTTGC

[0198] CATCTCAGCTGCCCTCAAGCACCCTCTAATCACCATTTCAGATTCCAAACTCCTGCTTGT

[0199] CCTCTTCTACCCAATCAATTCATGCTCGAACCCCTTCCTGTAGCCTTCTTTACTCGCAC

[0200] CTTCAGGCAGGACTTCTTTCTCTTTACATCTCGCTTCGGCATCTTCCAAGACCCAGGCAC

[0201] GGAGCTACCGGACAGAGAGTATGTCCCGTCAGCTGTTAGGACGTACCAGTGTGGTGGT GTACTCAGTGAAGACTGGACTGAGTTTTGATGGGAAAACAGAAAGATGA

[0202] The sequence of exon 14 of the fshr gene in females (FSHR_E14_SNPs_females) and corresponds to the SEQ sequence. ID. No. 3 which is set out below:

[0203] SEQ. ID. No. 3:

[0204] GGTGAGTATCTGCCTGCCCATGGACGTGGAGTTTCTGGTGGCTCAGGTCTACATGTGT

[0205] CTCTCCTCCTCCTCAACATCCTGGCCTTCTTCACTGTGTGTGGCTGTTACCTCAGCATCT

[0206] ATCTGAACATTCGCAACCCCTCATCAATGCCAGCCCCCGCTGACACCAGGGTGGCCCA ACGCATGGCCATTCTCATCTTCACTGACTTTGTCTGCATGGCTCCCATCTCCTTTTTTGC CATCTCAGCTGCCCTCAAGCTCCCTCTAATCACCATTTCAGATTCCAAACTCCTGCTTGTGT CCTCTTCTACCCAATCAATTCATGCTCGAACCCCTTCCTGTACGCCTTCTTTACTCGCAC CTTCAGGCAGGACTTCTTTCTCTTTATCTCGCTTCGGCATCTTTAAGACCCGGGCGC AGATCTACCGGACAGAGAGTATGTCCCGTCAGCTGTTAGGACGTACCAGGTGGTGGT GTACTCAGTGGCGACTGGACTGAGTTTTGATGGGAAAACAGAAAGATGA

[0207] This region exhibits differential nucleotide positions between male and female S. senegalensis, allowing the design of specific primers that amplify fragments of this DNA region differentially.

[0208] Specifically, the primers provided in the present invention allow amplification of fragments of this region in males but not in females. Primers are described for differential amplification using the LAMP technique, as well as for verifying the results obtained by LAMP using a second differential amplification technique by conventional PCR. In both cases, the primers provided in the present invention allow differential amplification that will occur only in males due to the differential alignment between sexes of the primers used.

[0209] The present invention proposes the amplification of a specific 191-base-pair region within the fshr gene, and more specifically within exon 14 of said gene, which exhibits sequence differences between males and females, with 16 different nucleotide positions. The amplification product of this region of the fshr gene, and more specifically, the amplification product of this region in males, will also be referred to throughout this invention as the “FSHR marker.”

[0210] LAMP amplification

[0211] Within SEQ. ID. No. 2, the specific 191 bp sequence of exon 14 of the fshr gene in males (FSHR_E14_SNPs_males) that is amplified by LAMP corresponds to the SEQ. ID. No. 4 sequence or “FSHR marker” shown below:

[0212] SEQ. ID. No. 4

[0213] CATGCTCGTCTGTCTCTCTCTCTCTCTCTCTCAGGACTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTCTCTCTCICCICCICCICCICCICUGICCICUGICCICUGICCIanlls GTATGTCCGTCAGTGTGITGTGTGTGTGTGTGTGTGTGTGICCTICTITIES ACTGTGHTGGGGGGGGGGGGGGGGGGGGGGGGH position of the SEQ sequence. ID. No. 4 present in both males and females is SEQ. ID. No. 5, which is set out below:

[0214] SEQ. ID. No. 5

[0215] CATGCTCGAACCCCTTCCTGTACGCCTTCTTTACTCGCACCTTCAGGCAGGACTCTTT CTCTTTACATCTCGCTTCGGCATCTTTAAGACCCGGGCGCAGATCTACCGGACAGAGAG TATGTCCCGTCAGCTGTTAGGACGTACCAGTGGTGGTGTACTCAGTGGCGACTGGA CTGAGGGGTTGTT

[0216] Primers

[0217] The primers used to differentially amplify, only in males, the region of the fshr gene corresponding to the sequence described in SEQ. ID. No. 4 using the LAMP amplification technique are those corresponding to the sequences SEQ. ID. No. 6 to SEQ. ID. No. 9 described in Table 1 below.

[0218] Table 1: Position, length and sequence of the primers, indicating the characteristic polymorphic position and determining factor of amplification position in the reference sequence in males (in bold and underlined).

[0219] F3: external primer; B3: external primer; FIP: internal primer consisting of F1c+F2; BIP: internal primer consisting of B1c+B2.

[0220] Positive control

[0221] On the other hand, to ensure the correct implementation of the method of the invention, the extension of a region that is amplified in both sexes is proposed. Any region of the genome that is amplified in both sexes can be used as a reference, but the present invention proposes two regions as a positive control.

[0222] The first is the 209 bp region of the 18S ribosomal subunit, also referred to in this document as the “18S marker”.

[0223] The primers for amplifying the 18S marker using LAMP (SEQ. ID. No. 15 to SEQ. ID. No. 18) were designed according to the sequence of the 18S subunit of the μsomal DNA SEQ. ID. No. 14, with accession number in GenBank EF126042, which is set out below.

[0224] SEQ. ID. No. 14:

[0225] TCTGGTTGATTCTGCCAGTAGCATATGCTTGTCTCAAAGACTAAGCCATGCAAGTCTAAG TACACACGGCCGGTACAGTTAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTG ATCGCTCCCAAGTTTACTTGGATAACTGTGGCAATTCTAGAGCTAATACATGCCAACGA GCGCTGACCTCCGGGGATGCGTGCATTTATCAGACCCAAAACCCATGCGGGGTGTCCC

[0226] GCTCCTCGGGGCGGGCGCCCCGGCCGCTTTGGTGACTCTAGATAACCTCGAGCCGAT CGCTGGCCCTCCGTGGCGGCGACGTCTCATTCGAATGTCTGCCCTATCAACTTTCGAT GGTACTTTCTGTGCCTACCATGGTGACCACGGGTAACGGGGAATCAGGGTTCGATTCC GGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAAT

[0227] TACCCACTCCCGACTCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAG GCCCTGTAATTGGAATGAGTACACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAA GTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATCTTAAAGTTGCTGCA GTTAAAAAGCTCGTAGTTGGATCTCGGGATCGAGCTGGCGGTCCGCCGCGAGGCGAG CTACCGCCTGTCCCAGCCCCTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGT CCCGCGGGGTCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGGT CGCCTGAATACCGCAGCTAGGAATAATGGAATAGGACTCCGGTTCTATTTTGTGGGTTT TCTCTCTCTGAACTGGGGCCATGATTAAGAGGGACGGCCGGGGGCATTCGTATTGTGC CGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGGCGAAAGCGAAAGCATTTGCCA AGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTC GTAGTTCCGACCATAAACGATGCCAACTAGCGATCCGGCGGCGTTATTCCCATGACCC GCCGGGCAGCGTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGCAA AGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTA ATTTGACTCAACACGGGAAATCTCACCCGGCCCGGACACGGAAAGGATTGACAGATTG ATAGCTCTTTCTCGATTCTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGA TTTGTCTGGTTAATTCCGATAACGAACGAGACTCCGGCATGCTAAATAGTTACGCGGCCCCCGTGCGGTCGGCGTCCAACTTCTTAGAGGGACAAGTGGAATTCAGCCACACGAGAT TGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCCACACT

[0228] GAGTGGATCAGTGTGTGTCTACCCTTCGCCGAGAGGCGCGGGTAACCCGCTGAACCCC ACTCGTGATAGGGATTGGGGATTGCAATTATTTCCCATGAACGAGGAATTCCCAGTAAG CGCGGGTCATAAGCTCGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGC TACTACCGATTGGATGGTTTAGTGAGGTCCTCGGATCGGCCCCGCCGGGGTCGGTTTC GGTCCTGGCGGAGCGCCGAGAAGACGATCAAACTTGACTATCTAGAGGAAGTAAAAGT

[0229] CGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTA

[0230] Of the total sequence, the primers of the invention allow amplification, in both males and females, of a 209 bp region of the 18S ribosomal subunit. The primers used to amplify the 209 bp region of the 18S ribosomal subunit using the LAMP amplification technique correspond to sequences SEQ. ID. No. 15 to SEQ. ID. No. 18, which are described in Table 2 below:

[0231] Table 2: Primer sequence.

[0232] F3: external primer; B3: external primer; FIP: internal primer consisting of F1c+F2; BIP: internal primer consisting of B1c+B2.

[0233] The 209 bp fragment corresponding to the sequence of the 18S ribosomal DNA subunit is amplified in both sexes, so it serves as a marker to identify false negatives in the FSHR marker.

[0234] The LAMP primers of sequence SEQ. ID No. 6 to 9 and 15 to 18 were designed using the Primer Explorer v.4 tool and the recommendations specified in your manual.

[0235] The second option proposed as a positive control is the amplification of a region also located in exon 14 of the fshr gene, which is shared by both males and females, hence its potential use as a positive control. This region is 203 bp long and is referred to in this dissertation as the “FSHR_X marker”.

[0236] The amplified sequence is SEQ. ID. No. 19 or “FSHR_X marker” shown below.

[0237] SEQ. ID. No. 19:

[0238] TGCTTGTCCTCTTCTACCCAATCAATTCATGCTCGAACCCCTTCCTGTACGCCTTCTTTA CTCGCACCTTCAGGCAGGACTTCTTTCTCTTTACATCTCGCTTCGGCATCTTTAAGACCC GGGCGCAGATCTACCGGACAGAGAGTATGTCCCGTCAGCTGTTAGGACGTACCAGTGT GGTGGTGTACTCAGTGGCGACTGGA

[0239] The primers used to amplify this region using the LAMP amplification technique correspond to sequences SEQ. ID No. 20 to SEQ. ID. No. 23, which are described in Table 3 below:

[0240] Table 3: Primer sequence.

[0241] PCR amplification

[0242] As an alternative to LAMP amplification, the present invention provides a conventional PCR amplification method that also allows for the identification of sex in S. senegalensis by differential amplification of a region within exon 14 of the fshr gene. This methodology is used herein to confirm the results obtained from LAMP amplification and to verify the sex of individuals by a second method.

[0243] Primers

[0244] Within SEQ. ID. No. 2, the specific 333 bp sequence of exon 14 of the fshr gene in males, which is amplified by PCR, corresponds to the sequence SEQ. ID. No. 24 shown below: SEQ. ID. No. 24

[0245] ACTGTGTGTGGCTGTTACCTCAGCATCTATCTGAACATTCGCAACCCCTCATCAATGCC AGCCCCCGCTGACACCAGGGTGGCCCAACGCATGGCCATTCTCATCTTCACTGACTTT GTCTGCATGGCTCCCATCTCCTTTTTTGCCATCTCAGCTGCCCTCAAGCACCCTCTTAATTC ACCATTTCAGATTCCAAACTCCTGCTTGTCCTCTTCTACCCAATCAATTCATGCTCGAAC CCCTTCCTGTACGCCTTCTTTACTCGCACCTTCAGGCAGGACTCTTTCTCTTTACATCT CGCTTCGGCATCTTCAAAGACCCAGGCACGGAGCTAC

[0246] The region that matches with the position of the SEQ sequence. ID. No. 24 present in both males and females is SEQ. ID. No. 25 which is set out below:

[0247] SEQ.ID. No. 25

[0248] ACTGTGTGTGTGGCTGTTACCTCAGCATCTATCTGAACATTCGCAACCCCTCATCAATGCC AGCCCCCGCTGACACCAGGGTGGCCCAACGCATGGCCATTCTCATCTTCACTGACTTT GTCTGCATGGCTCCCATCTCCTTTTTTGCCATCTCAGCTGCCCTCAAGCTCCTCCTCTAATC ACCATTTCAGATTCCAAACTCCTGCTTGTCCTCTTCTACCCAATCAATTCATGCTCGAAC CCCTTCCTGTACGCCTTCTTTACTCGCACCTTCAGGCAGGACTCTTTCTCTTTACATCT CGCTTCGGCATCTTTAAGACCCGGGCGCAGATCTAC

[0249] The primers used to differentially amplify, only in males, the fshr gene region of SEQ. ID. No. 24 using the PCR amplification technique are those corresponding to the sequences SEQ. ID. No. 26 to SEQ. ID. No. 27 described below:

[0250] SEQ. ID. No. 26: GTAGCTCCGTGCCTGGGTCTCG

[0251] SEQ. ID. No. 27: ACTGTGTGTGGCTGTTACCTCAGCATCTATC

[0252] Therefore, a first aspect of the invention is the in vitro use of the exon 14 region of the follicle-stimulating hormone receptor (FSHR) gene located on chromosome 12 of S. senegalensis, corresponding to SEQ. ID. No. 4, for sex determination in S. senegalensis individuals. Specifically, the in vitro amplification product of the SEQ. ID. No. 4 sequence is proposed as a biomarker for sex determination in S. senegalensis individuals. The presence of this biomarker indicates that the individual is male.

[0253] In a second aspect of the invention, primers for the differential amplification of SEQ. ID. No. 4 using LAMP amplification techniques are described.

[0254] In the present invention, “differential amplification” refers to the amplification of one of the two sequences without the amplification of the other, taking into account that both sequences occupy the same position within the fshr gene.

[0255] The primers of the present invention allow LAMP amplification of the 191 bp region of the fshr gene for only one of the two possible sequences at that position, SEQ. ID. No. 4, without producing amplification of the other. This results in sex-differential amplification of this region of the genome. Preferably, the primers are designed to specifically amplify SEQ. ID. No. 4, the sequence corresponding to male individuals, and not amplify SEQ. ID. No. 5, which is present in individuals of both sexes.

[0256] Specifically, primers for use in LAMP are described that correspond to the sequences SEQ. ID. No. 6 to SEQ. ID. No. 9 capable of amplifying SEQ. ID. No. 4, which identifies males, while not allowing amplification of SEQ. ID. No. 5, which is present in individuals of both sexes.

[0257] Primers for the differential amplification of SEQ. ID. No. 24 using conventional PCR techniques are also described. Specifically, primers for PCR use are described that correspond to the sequences SEQ. ID. No. 26 to SEQ. ID. No. 27, capable of amplifying the SEQ. ID. No. 24 sequence, which identifies males, while not amplifying SEQ. ID. No. 25, which is present in individuals of both sexes.

[0258] A third aspect of the invention describes a method for determining the sex of S. senegalensis individuals comprising the differential amplification of the SEQ. ID. No. 4 sequence from a previously isolated biological sample, wherein the amplification product of SEQ. ID. No. 4 is indicative of samples from male individuals and there is no amplification product in samples from female individuals.

[0259] DNA extraction from the previously isolated biological sample can be performed using any protocol that guarantees obtaining DNA of sufficient quantity and quality for a molecular assay.

[0260] In a preferred embodiment of the method of the invention, the amplified sequence is SEQ. ID. No. 4. In that case, the method for determining the sex of Solea senegalensis individuals comprises the differential amplification of the SEQ. ID. No. 4 sequence from a previously isolated biological sample, wherein the amplification product of SEQ. ID. No. 4 is indicative that the sample comes from a male individual, while the absence of amplification product is indicative that the sample comes from a female individual.

[0261] In a further preferred embodiment, the amplification of SEQ. ID. No. 4 is carried out by the primers of the invention corresponding to the sequences SEQ. ID. No. 6 to SEQ. ID. No. 9 previously described.

[0262] In another preferred embodiment of the method of the invention, it comprises the amplification of another S. senegalensis DNA sequence as a positive control. Preferably, the sequence amplified as a positive control is SEQ ID. No. 14, corresponding to a region of the 18S ribosomal subunit. Alternatively, the sequence amplified as a positive control is SEQ ID. No. 19, corresponding to a region of the fshr gene.

[0263] In a preferred embodiment of the method of the invention, amplification is performed using LAMP. Unlike other PCR-based techniques, LAMP diagnosis does not require electrophoresis of the amplified fragments, but rather relies on the color change (positive reaction) or lack thereof (negative reaction) of a staining agent added to the isothermal reaction. Therefore, it does not require specialized laboratory equipment and allows for determining the sex of sole in one hour, outside of specialized laboratories and without the need for electrophoresis in agarose gels.

[0264] In a preferred embodiment of this aspect of the invention, the result of LAMP amplification is determined using electrophoretic, fluorescence, or colorimetric techniques. Electrophoretic techniques allow for the visualization, on an agarose gel, of samples where DNA amplification of any of the described markers has occurred, as they are identified by a characteristic ladder-like pattern of LAMP products. In negative reactions, no band is visualized as a result of the nonspecific alignment of the primers on the template DNA.

[0265] Fluorescence or colorimetry techniques allow direct observation of the results of the LAMP amplification reaction. In tubes where DNA synthesis occurs (positive reaction), magnesium pyrophosphate ions are produced, which interact with a fluorescent agent and cause a color change. When there is no DNA synthesis (negative reaction), the color of the microtube remains unchanged.

[0266] The sex of Senegalese flounder individuals is identified based on the amplification results for the FSHR marker.

[0267] When using FSHR as the sole marker, a positive amplification result (FSHR+) is diagnostic, characteristic, and definitive for male Senegalese flounder, while FSHR-, or the absence of amplification, is diagnostic, characteristic, and definitive for females of this species. In other words, samples containing amplification product are assigned as originating from male individuals, and samples without amplification product are assigned as originating from female individuals.

[0268] If, in addition to the FSHR marker, a control marker such as 18S is used, a positive result for both FSHR (FSHR+) and 18S (18S+) amplification is diagnostic, characteristic, and definitive for male Senegalese flounder, while FSHR- and 18S+ will be diagnostic, characteristic, and definitive for females of this species. A negative result for the control marker (18S-) would require repeating the DNA extraction and / or amplification reaction.

[0269] Therefore, the method of the invention allows assigning as corresponding to male individuals, those samples where there is amplification product of SEQ. ID. No. 4 and SEQ. ID. No. 14, and as corresponding to female individuals, those samples where there is amplification product only of SEQ. ID. No. 14. Samples in which amplification of neither of the two sequences has occurred are considered amplification errors, do not allow assigning the sex to the individual and the method must be repeated.

[0270] If, in addition to the FSHR marker, a control marker such as FSHR_X is used, a positive result for both FSHR (FSHR+) and FSHR_X (FSHR_X+) amplifications is diagnostic, characteristic, and definitive for male Senegalese sole, while FSHR- and FSHR_X+ will be diagnostic, characteristic, and definitive for females of this species. A negative result for the control marker (FSHR_X-) would require repeating the DNA extraction and / or amplification reaction.

[0271] Therefore, the method of the invention allows assigning as corresponding to male individuals, those samples where there is amplification product of SEQ. ID. No. 4 and SEQ. ID. No. 19, and as corresponding to female individuals, those samples where there is amplification product only of SEQ. ID. No. 19. Samples in which amplification of neither of the two sequences has occurred are considered amplification errors, do not allow assigning the sex to the individual and the method must be repeated.

[0272] The method of the invention also allows for the differential amplification, by PCR, of the exon 14 region of the fshr gene of S. senegalensis, corresponding to SEQ. ID. No. 24 in males, between males and females, and describes the use of specific primers for this method (SEQ. ID. No. 26 and SEQ. ID. No. 27). In this case, samples containing amplification product are assigned as originating from male individuals, and samples without amplification product are assigned as originating from female individuals.

[0273] A fourth aspect of the invention describes primers for the amplification of SEQ ID. No. 14, the 18S ribosomal DNA subunit, by LAMP. Specifically, primers for use in LAMP are described that correspond to sequences SEQ ID. No. 15 to SEQ ID. No. 18. Their use as a positive control within the method of the invention described above is also described.

[0274] A fifth aspect of the invention describes primers for the amplification of SEQ ID. No. 19 using LAMP. Specifically, primers for use in LAMP are described that correspond to sequences SEQ ID No. 20 to SEQ ID No. 23. Their use as a positive control within the method of the invention described above is also described.

[0275] A sixth aspect of the invention describes a kit for determining the sex of Senegalese flounder using any of the methods described in the invention, which includes the means and reagents necessary for the differential amplification between males and females of DNA fragments obtained from DNA samples isolated from S. senegalensis.

[0276] Specifically, a kit for the differential amplification of SEQ. ID. No. 4 is described. In a preferred embodiment of the kit, it is designed for the amplification of SEQ. ID. No. 4 in males using the LAMP technique and for the subsequent interpretation of the results using electrophoretic, fluorescence, or colorimetric techniques.

[0277] In a particular embodiment of the kit of the invention, it comprises means and reagents necessary for amplification by LAMP, preferably including the specific primers SEQ. ID. No. 6 to 9, and the means and reagents for developing the amplification result by electrophoretic, fluorescence or colorimetric techniques.

[0278] In a preferred embodiment of the kit of the invention, it further comprises primers for the amplification of SEQ. ID. No. 14 by LAMP as a positive control, preferably the primers corresponding to sequences SEQ. ID. No. 15 to SEQ. ID. No. 18.

[0279] In another preferred embodiment of the kit of the invention, it further comprises primers for the amplification of SEQ. ID. No. 19 by LAMP as a positive control, preferably the primers corresponding to sequences SEQ. ID. No. 20 to SEQ. ID. No. 23. In a particular embodiment of the kit of the invention, it comprises media and reagents necessary for the differential amplification of SEQ. ID. No. 24 by PCR only in males, and more preferably including the specific primers for SEQ. ID. No. 24 that correspond to SEQ. ID. No. 26 and 27, and the media and reagents for developing the amplification result by electrophoretic techniques.

[0280] A seventh aspect of the invention describes the use of the kit of the invention in any of its embodiments for the determination of the sex of Solea senegalensis individuals.

[0281] The term "comprises" may also be interpreted, in a particular embodiment, as "consists of." The term "comprises" and its variants are not intended to exclude other technical features, additives, components, or steps.

[0282] For experts in the field, other objects, advantages, and features of the invention will become apparent partly from the description and partly from the practice of the invention.

[0283] PREFERRED EMBODIMENT OF THE INVENTION

[0284] For the experiments included in this report, all individuals were reproductive adults of known sex from the marine aquaculture facility of the University of Cádiz. Their sex is known because they are used annually for spawning (females) or egg fertilization (males). These individuals exhibited sexual dimorphism, which allowed for comparison of the results obtained from sex identification using LAMP. It should be noted that sexual dimorphism in this species appears in reproductive adults. Only when the reproductive gonads develop can it be determined by direct observation whether an individual is male or female.

[0285] EXAMPLE 1: Identification of the differential amplification sequence between sexes in the qen fshr.

[0286] Although the sequencing of the complete genome of S. senegalensis and its comparison between males and females has allowed the identification of 41 differential allelic variants in the fshr gene, it is crucial to have a method that allows the simple and rapid identification of these differential single nucleotide polymorphisms (SNPs) between males and females without the need to sequence the entire gene.

[0287] To this end, the possibility of using molecular methods to discriminate between the two forms in which this gene is present—that corresponding to male individuals and that corresponding to female individuals—is considered. The present invention identifies a specific region within the 9,769 base pairs of this gene, composed of 191 bp, where differential amplification of the sequence occurs between males and females. As will be explained in detail later, the amplification is preferably performed using LAMP. The 191 bp sequence of the fshr gene is unique within the complete 9,769 base pair sequence of said gene, as can be verified from the results of the amplification of other regions of this gene, which, a priori, could be candidates for the sex determination method of Solea senegalensis described in the present invention.Table 4: fshr gene sequences proposed for differential amplification by LAMP. Candidate sequences for LAMP amplification are indicated based on their start and stop positions within the sequence of exon 14 of the fshr gene (corresponding to SEQ. ID. No. 2 in males and SEQ. ID. No. 3 in females).

[0288] + there is amplification product; - there is no amplification product; * amplification in some individuals and not in others, the amplification result obtained among the analyzed individuals does not correspond to the known sex of the individual, so it is not indicative of the sex of the individual.

[0289] Specific primers were designed for all target regions for amplification by LAMP using the Primer Explorer v.4 tool And the recommendations specified in your manual.

[0290] As can be seen, the amplification results of the different regions of the fshr gene, even within exon 14 of said gene, do not allow discrimination between males and females, except in the last case, marked in bold, which corresponds to the differential amplification described in the present invention. The amplification product of this specific region (from position 318 to 508 of exon 14 of the fs / ?r gene) is the only one that allows differentiation between males (+) and females (-). This region corresponds to sequence SEQ. ID. No. 4 in males and SEQ. ID. No. 5 in females.

[0291] EXAMPLE 2: Determination of the sex of S. senegalensis by LAMP: interpretation of the results by electrophoresis, colohmethic and fluorescent techniques.

[0292] To extract DNA from the samples to be analyzed, approximately 100 mg of tissue (muscle, skin, any organ) is disaggregated into a 1.5 ml microtube to which 100 ml of 100 mM NaOH has been previously added. The tissue, mechanically disaggregated using a lancet, is incubated at 90°C for 10 minutes. The resulting extract is allowed to cool, and a 1:50 dilution is prepared for subsequent use in the molecular assay.

[0293] Molecular diagnosis of the sex of Senegalese sole is performed in two 200 µl microtubes. In both microtubes, LAMP reactions are performed to amplify the FSHR and 18S markers. Both reactions are composed of the same reagents and concentrations described below except for the primers used, which will differ depending on the marker to be amplified: 0.4 mM dNTP, 1 M betaine, 8 units of Bst, 1x buffer (20 mM Tris-HCl, 10 mM (NH4)2S₄, 50 mM KCl, 2 mM MgS₂C₅H₁₀O₅, 0.1% Tween® 20, pH 8.8), 1-2 µM of the inner primers (Forward Inner / Backward Inner FIP / BIP; FSHR: SEQ. ID. Nos. 8 and 9; 18S: SEQ. ID. Nos. 13 and 14) and a concentration of the outer primers (F3 / B3) between five and ten times lower (FSHR: SEQ. ID. Nos. 6 and 7; 18S: SEQ. ID. No. 11) and 12). To each reaction, between 2 and 5 µl of the DNA extract obtained in the previous step is added and the reaction is completed to a final volume of 25 µl.Both microtubes are incubated at temperatures between 60°C-65°C for 30-60 minutes.

[0294] The amplification product can be analyzed using two complementary and non-exclusive methodologies.

[0295] On one hand, 5-10 lp of the amplification product are loaded onto a 1-2% agarose gel and electrophoresis is performed at 100V / 30 minutes. After this time, the samples where amplification of the tested molecular marker is present show the characteristic ladder pattern of LAMP reactions. Positive reactions for FSHR are obtained in males and for 18S in both sexes, and negative reactions for FSHR in females (Figure 2).

[0296] On the other hand, 5 ml of Sybr Green I Nucleic Acid Stain (Life Technologies) are added to the LAMP reactions at a 1:50 dilution. This orange fluorescent agent reacts with the pyrophosphate reagents produced during DNA synthesis and changes color to a bright yellow in those reactions where amplification has occurred, indicating that the sample belongs to a male. Similarly, microtubes with a positive reaction fluoresce when irradiated with ultraviolet light (male), unlike those where there is no amplification (female) (Figure 3).

[0297] Following the same protocol as in the previous example, LAMP reactions are performed to amplify the FSHR marker and FSHR_X as a positive control instead of 18S.

[0298] Both reactions are composed of the same reagents and concentrations described below except for the primers used, which will differ depending on the marker to be amplified: 0.4 mM dNTP, 1 M betaine, 8 units of Bst, 1x buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween® 20, pH 8.8), 1-2 µM of the inner primers (Forward Inner / Backward Inner FIP / BIP; FSHR: SEQ. ID. No. 8 and 9; FSHR_X: SEQ. ID. No. 22 and 23) and a concentration of the outer primers (F3 / B3) between five and ten times lower (FSHR: SEQ. ID. No. 6 and 7; FSHR_X: SEQ. ID. No. 20 and 21).

[0299] The amplification product can be determined by agarose gel electrophoresis as described in the previous example. Positive reactions for FSHR are obtained in males and for FSHR_X in both sexes, and negative reactions for FSHR are obtained in females (Figure 2).

[0300] On the other hand, Sybr Green I Nucleic Acid Stain was used as described in the previous example. Microtubes where there is a positive reaction emit fluorescence when irradiated with ultraviolet light (male), unlike those where there is no amplification (female) (Figure 3).

[0301] EXAMPLE 3: Validation of the sex of S. senegalensis by PCR: interpretation of the results by electrophoresis.

[0302] The results obtained by LAMP were verified by conventional PCR using the specific primers SEQ. ID. No. 26 and SEQ. ID. No. 27 previously described in the present invention. For this purpose, the same fragment of the FSHR marker was amplified using the PCR primers specific to male Senegalese flounder.

[0303] The PCR is performed in a 200 ml microtube to which the following reagents and concentrations are added: 0.4 mM dNTP, 5 Taq units, 1x buffer, and 1-2 µM of the primers. Between 2 and 5 µl of the DNA extract obtained in the previous step are added to each reaction, and the reaction is completed to a final volume of 25 µl. The PCR cycle for this marker consists of an initial denaturation (95°C / 5 mins) followed by 32 cycles of 94°C / 45 secs, 60-65°C / 45 secs, and 72°C / 45 secs, ending with an extension at 72°C / 50 mins.

[0304] The PCR results can be visualized on an agarose gel by loading 5-10 pl of the amplification product onto a 1-2% concentration agarose gel and performing electrophoresis at 100V / 30 minutes.

[0305] After this time, samples showing amplification of the tested molecular marker exhibit a band around 333 bp on the agarose gel (Figure 4) and are classified as males. Samples showing no amplification are classified as females.

[0306] References

[0307] 1. D. Chalopin et al. Transposable elements and early evolution of sex chromosomes in fish. Chromosom. Res. 23, 545-560 (2015).

[0308] 2. MJ Molina-Luzon et al., Chromosomal manipulation in Senegalese sole (Solea senegalensis Kaup, 1858): induction of triploidy and gynogenesis. J. Appl. Genet. 56, 77-84 (2014). 3. MT Dinis et al., A review on the cultivation potential of Solea senegalensis in Spain and in Portugal. Aquaculture. 176, 27-38 (1999).

[0309] 4. L. Vega et al., Cytogenetic and isoenzymatic characterizations of the tongue Solea senegalensis Kaup, 1858. Bulletin of the Inst. Spanish Oceanogr. 18, 245–250 (2002). 5. S. Portela-Bens et al. Integrated gene mapping and synteny studies provide insights into the evolution of a sex proto-chromosome in Solea senegalensis . Chromosome. 126, 261–277 (2016).

[0310] 6. ME Rodriguez et al., Evolution of the proto sex-chromosome in Solea senegalensis. Int. J. Mol. Sci. 20 (2019), doi:10.3390 / ijms20205111. 7. S. Chen et al., Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat. Genet. 46, 253–260 (2014).

[0311] 8. R. De la Herran etal., A chromosome-level genome assembly enables the identification of the follicle stimulating hormone receptor as the master sex determining gene in Solea senegalensis. BioRVIX Prepr, 32 (2022). 9. Curzon AY, et al. A novel c.1759T>G variant in follicle-stimulating hormone-receptor gene is concordant with male determination in the flathead grey mullet (Mugil cephalus). G3 (Bethesda). 2021 Feb 9;11(2):jkaa044. doi: 10.1093 / g3journal / jkaa044. PMID: 33589926; PMCID: PMC8022982.

Claims

CLAIMS 1. Use of the in vitro amplification product of the SEQ. ID. No. 4 sequence as a biomarker for the determination of the sex of Solea senegalensis individuals.

2. A method for determining the sex of Solea senegalensis individuals comprising the differential amplification of the SEQ. ID. sequence No. 4 from a previously isolated biological sample, wherein the amplification product of SEQ. ID. No. 4 is indicative that the sample comes from a male individual, while the absence of amplification product is indicative that the sample comes from a female individual.

3. Primers for differential amplification of SEQ. ID. No. 4 using LAMP that correspond to the sequences SEQ. ID. No. 6 to SEQ. ID. No.

9.

4. The method according to claim 2 characterized in that the amplification is carried out by means of LAMP.

5. The method according to the preceding claim, characterized in that the primers according to claim 3 are used for the amplification of SEQ. ID. No.

4.

6. The method according to any of claims 2 and 4 to 5, characterized in that it further comprises the amplification of the SEQ. ID. No. 14 or SEQ. ID. No. 19 sequence of Solea senegalensis DNA as a positive control.

7. The method according to the preceding claim, characterized in that the primers used for the amplification of SEQ ID. No. 14 by LAMP correspond to the sequences SEQ ID. No. 15 to SEQ ID. No.

18.

8. The method according to claim 6 characterized in that the primers used for the amplification of SEQ. ID. No. 19 by LAMP correspond to the sequences SEQ. ID. No. 20 to SEQ. ID. No.

23.

9. The method according to any of claims 2 or 4 to 8, characterized in that the amplification result is determined by electrophoretic, fluorescence, or colorimetric techniques.

10. The method according to any of claims 2 or 4 to 8, characterized in that the sample containing amplification product of SEQ. ID. No. 4 and SEQ. ID. No. 14 or SEQ. ID. No. 19 is assigned as originating from a male individual and the sample containing Amplification product only of SEQ. ID. No. 14 or SEQ. ID. No. 19 is assigned as coming from a female individual.

11. Kit for determining the sex of Solea senegalensis individuals using the method according to claim 5 comprising media and reagents necessary for the differential amplification of SEQ. ID. No. 4 using LAMP, the primers according to claim 3, and media and reagents for developing the amplification result using electrophoretic, fluorescence or colorimetric techniques.

12. The kit according to the preceding claim further comprising the primers of sequence SEQ. ID. No. 15 to SEQ. ID. No. 18 or of sequence SEQ. ID. No. 20 to SEQ. ID. No.

23.

13. Use of the kit according to claim 11 or 12 for the determination of the sex of Solea senegalensis individuals.

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