Specific molecular markers, primer sets, kits, methods and applications for catfish sex identification

Abstract

This invention provides a method for identifying catfish ( Silurus asotus This invention discloses a specific molecular marker for sex identification. The molecular marker is a DNA fragment located on the Y chromosome of catfish, and its nucleotide sequence is shown in SEQ ID No. 3. A DNA fragment partially homologous to this fragment is located on chromosome 8, and its nucleotide sequence is shown in SEQ ID No. 4. This invention is the first to screen male-specific fragments from catfish genome information and design specific primers for genetic sex identification of catfish. The genetic sex PCR identification method based on this molecular marker is applicable to genetic sex identification and screening of catfish at different growth stages and in different populations in different watersheds in the field, in laboratories, or in aquaculture enterprises. Since female catfish grow faster than males, applying this method to assist in the production of all-female fry helps improve the efficiency of aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding, and particularly relates to specific molecular markers for catfish sex identification and a method for genetic sex identification of catfish based on these molecular markers. Background Technology

[0002] Many fish exhibit significant sexual dimorphism in growth, such as females of carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), tongue sole (Cynoglossus semilaevis), flounder (Hippoglossushippoglossus), and wolf perch (Dicentrarchus labrax), whose growth rate is significantly faster than that of males (Bye and Lincoln, 1986; Bjornsson, 1995; Saillant et al., 2001; Chen et al., 2013). Conversely, males of fish such as Nile tilapia (Oreochromis niloticus), channel catfish (Ictalurus punctatus), yellow catfish (Pelteobagrus fulvidraco), and bluefin tuna (Thunnus maccoyii) grow faster than females (Beardmore et al., 2001; Wang et al., 2009; Chen et al., 2014; Farley et al., 2014; Sun et al., 2014). Furthermore, some ornamental fish exhibit sexual dimorphism in terms of individual morphology or coloration, such as guppies (Poecilia reticulata), anglerfish (Rhodeus ocellatus), and swordtails (Xiphophorushelleri) (Turan et al., 2010). Therefore, the production and cultivation of parthenogenetic fry can significantly improve the economic benefits and commercial value of these species. However, sex determination in breeding practice is not easy for several reasons: most fish have low levels of sex chromosome differentiation, making it impossible to identify sex chromosome composition through cytological methods; secondly, the external morphological differences between male and female individuals are small in the early stages of development, making them indistinguishable; and thirdly, sex differentiation is easily affected by environmental factors, leading to inconsistencies between genotype and phenotype. Therefore, the development of sex-linked molecular markers in different fish species has significant application value in aquaculture.

[0003] In recent years, with the development of biotechnology and the completion of genome sequencing for a large number of farmed fish species, significant advancements have been made in the study of African catfish (Clarias gariepinus) (Kovacs et al., 2000), channel catfish (Waldbieser et al., 2001), rainbow trout (Felip et al., 2005; Yano et al., 2012), tongue sole (Chen et al., 2007), common carp (Chen et al., 2009), Nile tilapia (Lee et al., 2011; Sun et al., 2014), Amazonian peccary carp (Bryconamazonicus) (da Silva et al., 2012), yellow catfish (Dan et al., 2013), turbot (Psettamaxima) (Vale et al., 2014), large yellow croaker (Larimichthys crocea) (Lin et al., 2017), and golden croaker (Scatophagus). Sex-linked molecular markers have been successfully developed in farmed fish such as croaker (Mustapha et al., 2018) and yellow croaker (Nibea albiflora) (Sun et al., 2018), laying the foundation for sex control and providing important technical support for increasing aquaculture yield and improving economic value.

[0004] The catfish (Silurus asotus), also known as the native catfish, belongs to the order Siluriformes, family Siluridae, and genus Silurus, and is widely distributed throughout Asia. Catfish possess advantages such as rapid growth, strong adaptability to low temperatures, adaptability to different diets, easy availability of seedlings, and strong disease resistance. However, its production cannot be significantly increased by relying on natural river resources; artificial breeding is the only way to meet societal demand. Large-scale catfish farming in my country currently reaches an annual production of 365,000 tons (FAO 2020). Like many farmed fish, catfish exhibit significant sexual dimorphism, with females growing significantly faster than males (Wei Gang, 1995). All-female monosex farming can improve farming efficiency. Furthermore, obtaining super-male and all-male catfish is beneficial for basic research. Therefore, screening for sex-specific molecular markers in catfish for genetic sex determination has both significant scientific value and broad application prospects. Summary of the Invention

[0005] The primary objective of this invention is to provide a specific molecular marker for sex identification in catfish.

[0006] A second objective of the present invention is to provide amplification primers for the specific molecular markers.

[0007] A third objective of this invention is to provide a kit for identifying the sex of catfish based on the specific molecular markers or the primers.

[0008] A fourth objective of this invention is to provide a method for identifying the sex of catfish based on the specific molecular marker or the primer.

[0009] The fifth objective of this invention is to provide the application of the specific molecular marker or the primer in catfish sex identification.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A specific molecular marker for sex identification in catfish includes partially homologous DNA fragments from the catfish's autosome 8 and Y chromosome, whose nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0012] The primers for amplifying the specific molecular marker described above have the following nucleotide sequences: upstream primer F, as shown in SEQ ID No. 1; and downstream primer R, as shown in SEQ ID No. 2.

[0013] A kit for identifying the sex of catfish based on the aforementioned specific molecular markers or primers, wherein the kit contains the aforementioned primers, the upstream primer being F, the nucleotide sequence of which is shown in SEQ ID No. 1; and the downstream primer being R, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0014] A method for identifying the sex of catfish based on the above-mentioned specific molecular markers or primers includes the following steps:

[0015] A. PCR amplification: Genomic DNA was extracted from the fin rays of the catfish to be tested. Using the obtained genomic DNA as a template, PCR amplification was performed using the upstream primer (5'-ACCTGGGCAAACACCTAGAGA-3') shown in SEQ ID NO.1 and the downstream primer (5'-GTTGCCAGTTCCAAAACAAGC-3') shown in SEQ ID NO.2. The obtained PCR amplification products were compared with the above-mentioned catfish sex chromosome-specific molecular markers.

[0016] B. Result Interpretation: A 321bp autosomal-specific band was amplified from the genomic DNA of the catfish under test, with its nucleotide sequence shown in SEQ ID NO.4. This indicates that the catfish under test is genetically female, with a sex chromosome genotype of XX. Simultaneously, a 321bp autosomal-specific band (nucleotide sequence shown in SEQ ID NO.4) and a 363bp Y-chromosome-specific band (nucleotide sequence shown in SEQ ID NO.3) were amplified from the genomic DNA of the catfish under test. This indicates that the catfish under test is genetically male, with a sex chromosome genotype of XY.

[0017] The application of the above-mentioned specific molecular markers or primers in catfish sex identification includes the following steps:

[0018] A. The offspring of XX female fish and XY male fish were masculinized, and the above molecular marker method was used to screen out the transformed male fish with genotype XX;

[0019] B. Mating male XX fish with normal female XX fish will produce all-female XX fish, which can then be mass-produced as all-female commercial fish.

[0020] The present invention has the following beneficial effects:

[0021] There are many methods for screening molecular markers, such as RAPD, AFLP, and whole-genome sequencing. Many methods have been tried in the development of sex-specific molecular markers, but the results have been unsatisfactory. For example, one method involved sequencing only the whole genome of XX female fish and then comparing short reads obtained through male-female resequencing to a reference genome of the female fish. This resulted in short molecular markers and a number of false positives. Ultimately, this invention screens for sex-specific molecular markers in catfish by combining whole-genome sequencing of both male and female individuals with resequencing of mixed male-female pools. Using the male genome as a reference, the identified male-specific regions are located on the sex chromosomes, effectively eliminating false positives. Furthermore, compared to resequencing multiple individuals, this method significantly reduces costs while ensuring accurate test results. This invention provides the first specific molecular marker for identifying the sex of catfish with high accuracy. Specific primers were designed for genetic sex identification of catfish. The genetic sex PCR identification method based on this molecular marker is applicable to the genetic sex identification and screening of catfish at different growth stages and in different populations in different watersheds in the field, in laboratories, or in aquaculture enterprises. It is characterized by high efficiency, speed, accuracy, and stability. Since female catfish grow faster than males, the established molecular marker-assisted method for producing all-female fry helps improve the efficiency of aquaculture. Attached Figure Description

[0022] Figure 1This is a sequence alignment diagram of the Y chromosome-specific molecular marker and the autosomal homologous sequence described in this invention. The primer positions are marked with green boxes, the black background represents the consistent sequence of the Y chromosome and autosomal DNA fragments, and "-" indicates the deletion sequence.

[0023] Figure 2 The results of genetic sex identification of 8 female catfish (XX) and 8 male catfish (XY) using the Y chromosome-specific molecular markers described in this invention are shown. M represents the DNA molecular weight standard DL2000.

[0024] Figure 3 To apply the technical route of molecular marker-assisted selection to produce genetically all-female fish as described in this invention, FAD (Fadrozole), aromatase inhibitor, and MAS (marker-assisted selection) are used. Detailed Implementation

[0025] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0026] Experimental methods not specified in this invention are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition, by J. Sambrook et al., translated by He Fuchu et al., Science Press, 2017), or as recommended by the manufacturer.

[0027] I. Obtaining Specific Molecular Markers for Catfish Sex Identification

[0028] 1. Phenotypic sex determination

[0029] Gonads of 5-month-old catfish were fixed overnight in Born's solution. The fixed gonadal material was dehydrated with alcohol gradient, cleared with xylene, embedded in paraffin as usual, and then cross-sectioned with a section thickness of 5 μm. After that, it was stained with HE, observed under an optical microscope, and photographed.

[0030] 2. Genomic DNA extraction and library construction

[0031] Whole-genome DNA was extracted from each sample using the standard phenol-chloroform extraction method, and Illumina, Nanopore, and Hi-C sequencing libraries were constructed using standard procedures.

[0032] 3. Whole genome sequencing and pooled resequencing

[0033] The whole genomes of a male and a female catfish were sequenced using Illumina, Nanopore, and Hi-C sequencing technologies. The sequencing results were assembled using software such as NextDenovo, NextPolish, 3dDNA, and Juicer to obtain chromosome-level genomes with sizes of 748.02 Mb and 745.84 Mb, respectively. In addition, resequencing was performed on mixed-sex pools (8 females and 8 males) using Illumina sequencing technology, yielding data of 49.9 Gb and 52.6 Gb, respectively.

[0034] 4. Screening of sex-specific molecular markers

[0035] The third-generation sequencing data and resequencing data of male and female catfish obtained from sequencing were aligned to the XY male reference genome using Minimap2 and Bowtie2, respectively. The alignment results were processed using SAMtools software to obtain a BAM file. Read coverage analysis identified a male-specific region (no female read coverage, and male read coverage was half that of other regions). This region was located on chromosome 5, indicating that chromosome 5 is the sex chromosome of catfish. Primers were designed for this male-specific region, and PCR amplification was performed in the genomes of both male and female catfish individuals to screen for sex-specific molecular markers. The results showed that one primer pair (upstream primer F sequence (5'-ACCTGGGCAAACACCTAGAGA-3') as shown in SEQ ID NO.1, and downstream primer R sequence (5'-GTTGCCAGTTCCAAAACAAGC-3') as shown in SEQ ID NO.2) exhibited significant sex specificity, amplifying a 321 bp band in female individuals and a 363 bp male-specific band in male individuals, in addition to a 321 bp DNA band. This indicates that the catfish follows an XX / XY genetic sex determination system (females are XX, males are XY).

[0036] 5. Cloning and sequence analysis of sex-specific molecular markers

[0037] The agarose gel containing the shared male-female band and the male-specific band was cut off with a blade, then recovered. The recovered product was ligated into the pMD-19T vector, transformed into competent E. coli cells, and positive clones were screened using the blue-white screening method. Single colonies of white spots were picked, and positive clones were identified by PCR and then sequenced. The shared male-female DNA fragment (sequence shown in SEQ ID NO.3, named Sa-A) and the male-specific DNA fragment (sequence shown in SEQ ID NO.4, named Sa-Y) obtained from sequencing were compared. The sequence alignment results are as follows: Figure 1As shown, there are three main differences between the sequences of Sa-A and Sa-Y: Compared with Sa-A, Sa-Y ① inserts a 28bp sequence at 39bp; ② inserts a 5bp sequence at 89bp; ③ inserts a 9bp sequence at 115bp.

[0038] II. Molecular marker methods for genetic sex determination in catfish

[0039] Molecular marker methods for genetic sex determination in catfish include the following steps:

[0040] A. PCR Amplification: A small amount of catfish tail fin was cut and genomic DNA was extracted. The extracted genomic DNA was amplified by PCR using catfish sex identification primers SEQ ID NO.1 and SEQ ID NO.2 (PCR reaction system: 2×PCR mix Buffer 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of genomic DNA template, 21 μL of ddH2O, mixed and centrifuged. PCR amplification program: 95℃ 3 min; 95℃ 30 s, 60℃ 30 s, 72℃ 30 s, 35 cycles; 72℃ 10 min, 4℃ 10 min); the obtained PCR products were subjected to 1.5% agarose gel electrophoresis.

[0041] B. Result Interpretation: If only a 321bp autosomal-specific band (i.e., Sa-A as described in this invention) is amplified from the genomic DNA of the catfish to be tested, then the catfish to be tested is determined to be genetically female, with a sex chromosome genotype of XX; if both a 321bp autosomal-specific band and a 363bp Y chromosome-specific band (i.e., the Y chromosome-specific molecular marker Sa-Y as described in this invention) are amplified from the genomic DNA of the catfish to be tested, then the catfish to be tested is determined to be genetically male, with a sex chromosome genotype of XY.

[0042] The above method was used to perform genetic sex identification on 8 female catfish (XX) and 8 male catfish (XY). The results are shown in the figure. Figure 2 In all XX individuals, only an autosomal-specific band of 321 bp was amplified, while in all XY individuals, both an autosomal-specific band of 321 bp and a Y-chromosome-specific band of 363 bp were amplified, consistent with the theoretical results.

[0043] III. Verification of the universality of sex-specific molecular markers in catfish

[0044] To verify the accuracy of the molecular markers involved in this patent for genetic sex identification in catfish, we applied the above method to identify the genetic sex of 106 catfish (46 phenotypic females and 60 phenotypic males) from three locations: the Beibei section of the Jialing River, the Hechuan section of the Jialing River, and the Zhengzhou section of the Yellow River. In all the female catfish tested, only a 321 bp autosomal-specific band was amplified, while in all the male catfish tested, both a 321 bp autosomal-specific band and a 363 bp Y-chromosome-specific band were amplified simultaneously, indicating that the phenotype and genotype of these 106 catfish tested were completely consistent (Table 1). The results show that the sex-specific molecular markers developed in this experiment are applicable to wild catfish in different river basins and have good universality.

[0045] Table 1. Genetic sex identification results of catfish from three different populations.

[0046]

[0047]

[0048] IV. Method for producing all-female catfish based on sex-specific molecular markers

[0049] The method for genetic sex identification of catfish based on the sex-specific molecular markers of the present invention, and for producing all-female catfish, includes the following steps ( Figure 3 ):

[0050] A. Five days after hatching, the offspring of female catfish of XX and male catfish of XY were subjected to FAD masculinization treatment. The molecular marker method for genetic sex identification of catfish described in this invention was used to screen out the transformed male catfish with genotype XX.

[0051] B. By mating male XX fish with normal female XX fish, all-female XX fish can be obtained, which can then be used to produce large quantities of all-female commercial fish.

[0052] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for identifying the sex of catfish, characterized in that, Includes the following steps: A. PCR amplification: Genomic DNA was extracted from the fin rays of the catfish to be tested. Using the obtained genomic DNA as a template, PCR amplification was performed using the F primer shown in SEQ ID NO.1 and the R primer shown in SEQ ID NO.

2. The obtained PCR amplification product was compared with a catfish sex chromosome-specific molecular marker. The catfish sex chromosome-specific molecular marker is a DNA fragment located on the catfish Y chromosome, the nucleotide sequence of which is shown in SEQ ID No.

3. A DNA fragment partially homologous to this fragment is located on chromosome 8, the nucleotide sequence of which is shown in SEQ ID No.

4. B. Result Interpretation: If only a 321 bp autosomal-specific band is amplified from the genomic DNA of the catfish to be tested, the catfish to be tested is determined to be genetically female, with a sex chromosome genotype of XX; If both a 321 bp autosomal-specific band and a 363 bp Y chromosome-specific band are amplified from the genomic DNA of the catfish to be tested, the catfish to be tested is determined to be genetically male, with a sex chromosome genotype of XY.

2. A molecular marker-assisted breeding method for the production of all-female commercial fish, characterized in that, The molecular marker is a DNA fragment located on the catfish Y chromosome, the nucleotide sequence of which is shown in SEQ ID No.

3. A DNA fragment partially homologous to this fragment is located on chromosome 8, the nucleotide sequence of which is shown in SEQ ID No.

4. The procedure includes the following steps: A. The offspring of female catfish with XX genotype and male catfish with XY genotype are subjected to masculinization treatment, and the sex of catfish is determined by the method described in claim 1 to screen out the transformed male catfish with genotype XX. B. By mating male XX fish with normal female XX fish, all-female XX fish can be obtained, which can then be used to mass-produce all-female commercial fish.

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