Primer, method and application for detecting sex-specific SNP molecular markers of catfish from different sources

Abstract

The present invention discloses a primer for detecting sex-specific SNP molecular markers of catfish from different sources. The primers include a forward primer F and a reverse primer R. The sequence of the forward primer F is shown in SEQ ID NO:3, and the sequence of the reverse primer R is shown in SEQ ID NO:4. The SNP molecular marker is located at the 11,946th position of the sequence shown in SEQ ID NO:1, and its base is A or C, wherein the CC genotype is female fish, and the A / C genotype is male fish. A method for identifying the sex of catfish from different sources using the primers is also disclosed, as well as the application of the primers or methods in sex identification of catfish from different sources. The present invention develops primers for detecting the SNP molecular markers. The primers can be used for genetic sex identification of catfish, especially catfish from different sources, and provide great production application value for the monosexual breeding of catfish.

Description

Technical Field

[0001] The invention belongs to the technical field of sex identification of catfish, and particularly relates to a primer, method and application for detecting sex-specific SNP molecular markers of catfish from different sources. Background Art

[0002] Clarias fuscus, also known as catfish, belongs to the order Siluriformes, family Clarias, and genus Clarias. This species has tender meat, delicious taste, and high economic value. It is a freshwater fish that is farmed on a large scale in southern my country. Clarias fuscus has special auxiliary respiratory organs that can adapt to extremely low dissolved oxygen environments. It is a wide-temperature Clarias fish with strong disease resistance, simple breeding, short cycle, high yield, low cost and easy transportation. At present, a large-scale breeding industry has been formed in Guangxi, Guangdong and Fujian, becoming a freshwater breeding species with good development prospects.

[0003] The catfish belongs to the male heterogametic XX / XY sex determination type, with obvious sexual dimorphism. Under the same breeding conditions, the growth rate of male fish is much higher than that of female fish of the same age. When the cultured catfish reaches the market size, the female catfish is in the stage of gonad development, with an enlarged abdomen and a gonad index much higher than that of male fish, resulting in the price of female fish being about 4 yuan / kg lower than that of male fish of the same size. Therefore, researching and breeding all-male catfish varieties can greatly save breeding costs and improve the quality and economic benefits of commercial fish. However, male and female catfish do not show morphological differences in the embryonic and juvenile stages, and sex identification cannot be performed based on body size and surface characteristics of reproductive organs. The process of achieving single-sex variety breeding can be accelerated by locating and cloning candidate genes for sex determination. However, candidate genes for sex determination of catfish have not yet been reported, which brings great difficulties to the study of gonad differentiation and single-sex seed breeding of catfish, and also greatly restricts the development of the industry of this species.

[0004] The problems existing in the prior art are:

[0005] (1) In scientific research, the physiological sex of catfish can be identified through dissection and other methods, but dissection not only leads to individual death, but is also inefficient and cannot achieve large-scale identification, limiting its application in practical production;

[0006] (ii) Current biotechnology can identify sex in vivo, but existing SNP molecular markers are located in the flanking regions of sex-determining candidate genes. The accuracy of such flanking markers varies in different groups (e.g., different families, different geographical groups), which affects the reliability of sex identification results and limits their widespread application;

[0007] Therefore, the main problem with the existing technology is that the detection site is not completely linked to sex, resulting in a high error rate in the detection and limited applicability. The most ideal solution is to develop the sex-determining candidate gene into a sex-specific marker and further verify its accuracy. Summary of the invention

[0008] The purpose of the present invention is to provide a primer for detecting sex-specific SNP molecular markers of catfish from different sources.

[0009] The present invention also aims to provide a method for identifying the sex of catfish from different sources.

[0010] The last object of the present invention is to provide the application of the above primers or methods in sex identification of catfish from different sources and in single-sex breeding of catfish.

[0011] The above-mentioned first purpose of the present invention can be achieved by the following technical scheme: a primer for detecting sex-specific SNP molecular markers of bearded catfish from different sources, the primer comprising a forward primer F and a reverse primer R, wherein the sequence of the forward primer F is shown in SEQ ID NO:3, and the sequence of the reverse primer R is shown in SEQ ID NO:4, and the primer is used to detect sex-specific SNP molecular markers of bearded catfish from different sources, the SNP molecular marker is located at the 11,946th position of the sequence shown in SEQ ID NO:1, and its base is A or C, wherein the CC genotype is female fish, and the A / C genotype is male fish.

[0012] The invention develops a sex determination gene for Silurus catfish, and the base sequence of the gene is shown as SEQ ID NO:1.

[0013] The invention determines the interval where the sex determination candidate gene is located through the method of sex QTL positioning and whole genome sequencing of catfish, and clones and studies the sex determination candidate gene.

[0014] The present invention discovered a gene associated with gender in the located interval, and its base sequence is shown in SEQ ID NO:1.

[0015] The applicant of the present invention further used the catfish genome resequencing technology and transcriptome data comparison to screen SNP molecular markers on sex-related genes that are strongly correlated with sex.

[0016] The sex-specific SNP molecular marker of the catfish is located at position 11,946 of the sequence shown in SEQ ID NO: 1, and its base is A or C, wherein the CC genotype is female fish, and the A / C genotype is male fish.

[0017] That is, the SNP molecular marker of the present invention is located at position 11,946 of the gene sequence shown in SEQ ID NO: 1, wherein the female fish has a CC genotype and the male fish has an A / C genotype.

[0018] Therefore, the present invention provides a SNP molecular marker associated with the sex of catfish, wherein the SNP molecular marker is located at the 11,946th position of the above-mentioned catfish sex determination candidate gene, and its base is A or C, wherein the female fish has a CC genotype and the male fish has an A / C genotype.

[0019] Finally, the present invention also designs primers for detecting sex-specific SNP molecular markers of bearded catfish from different sources, wherein the primers include a forward primer F and a reverse primer R, wherein the sequence of the forward primer F is shown in SEQ ID NO:3, and the sequence of the reverse primer R is shown in SEQ ID NO:4.

[0020] Specifically:

[0021] Forward primer F: 5'-ACCTGTATGTCTGTGGACTGTG-3' (i.e., the sequence shown in SEQ ID NO: 3);

[0022] Reverse primer R: 5'-GCCATTCTTGAAGTACCGCTTG-3' (ie, the sequence shown in SEQ ID NO: 4).

[0023] The above second object of the present invention can be achieved by the following technical scheme: A method for identifying the sex of catfish from different sources, comprising the following steps:

[0024] (1) extracting DNA from the fin tissue of the catfish to be tested;

[0025] (2) using the above primers and the catfish DNA extracted in step (1) as a template, performing PCR amplification;

[0026] (3) The amplified products were detected by capillary electrophoresis, and the male and female individuals were identified based on the sequencing peak pattern, where the heterozygous peak pattern was for male individuals and the homozygous peak pattern was for female individuals.

[0027] Therefore, the present invention develops intragenic SNP molecular markers and primers based on genome resequencing and transcriptome data comparison, and develops a method for auxiliary identification of the genetic sex of the catfish using the primers.

[0028] Preferably, during PCR amplification in step (2), the PCR reaction system used includes: 30 ng / μL DNA template: 1.0 μL, 2×EasyPCR Super Mix: 12.5 μL, 10 mM forward primer F and reverse primer R, 1 μL each, dd H2O 9.5 μL, a total of 25 μL.

[0029] Preferably, during PCR amplification in step (2), the PCR reaction procedure is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, 35 cycles, and extension at 72°C for 10 min.

[0030] The third object of the present invention can be achieved by the following technical solution: application of the primers in sex identification of catfish from different sources.

[0031] Furthermore, the present invention also discloses the application of the above primers in the monosex breeding of catfish.

[0032] The invention also provides application of the method in sex identification of catfish from different sources.

[0033] Furthermore, the present invention also discloses the application of the above method in the monosex breeding of catfish.

[0034] In summary, the present invention successfully identified sex-determining candidate genes based on whole genome sequencing and QTL mapping technology, and further developed intragenic SNP molecular markers and primers for amplifying the SNP molecular markers. These primers can be used for genetic sex identification of catfish from different sources, providing great production application value for the monosex breeding of catfish.

[0035] The present invention has the following advantages:

[0036] (1) The present invention locates and clones a candidate gene for sex determination in catfish, and further obtains a SNP molecular marker related to the sex of catfish in the gene;

[0037] (2) The present invention designs intragenic SNP molecular markers and primers for amplifying the SNP molecular markers based on the located sex-determining candidate genes, which can be used for auxiliary screening of sex identification of female and male catfish, thereby providing great production application value for the single-sex breeding of catfish;

[0038] (3) The sex-specific SNP molecular marker of the catfish designed by the present invention is located on the exon of the candidate gene for sex determination. Compared with the problem (ii) of the prior art, the SNP molecular marker has a strong degree of linkage with sex, which improves the accuracy and applicability;

[0039] (4) The present invention realizes batch detection of the sex of catfish from any group in a common laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0041] Figure 1This is a density distribution diagram of the variant sites on the genome that are consistent with female homozygosity and male heterozygosity after the whole genome resequencing of male and female catfish in Example 2;

[0042] Figure 2 This is an IGV view of the molecular marker test based on transcriptome data comparison of catfish in Example 2, left: homozygous site of female individuals; right: heterozygous site of male individuals; the base pointed by the arrow is the target detection site, the bottom sequence represents the reference sequence, the gray column indicates consistency with the reference sequence, and the presence of SNP is emphasized by colored columns, A: green; T: red; G: orange; C: blue;

[0043] Figure 3 This is the sequencing peak diagram after PCR amplification of catfish gDNA using SNP molecular marker primers in Example 3. The sequencing uses forward primer F. Top: heterozygous peak type of male individuals; bottom: homozygous peak type of female individuals; the base pointed by the arrow is the target detection site. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical solution of the present invention. The reagents or materials used in the embodiments, unless otherwise specified, are all from commercial channels.

[0045] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0046] Example 1

[0047] This example locates the sex determination interval and sex determination candidate gene of Silurus fulgens, and the base sequence of the gene is shown in SEQ ID NO: 1.

[0048] The base sequence of the gene is shown below (as shown in SEQ ID NO: 1).

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] (as shown in SEQ ID NO: 1).

[0058] Note: Bold indicates the start codon and stop codon, the wavy line indicates the CDS sequence, the bold slash in square brackets indicates the two alleles A and C, and the bold underline indicates the designed upstream and downstream primers.

[0059] The molecular marker is located at position 11,946 of the gene (as shown in SEQ ID NO: 1), the female fish has a CC genotype, and the male fish has an A / C genotype.

[0060] The gene associated with the sex of the catfish was constructed by the following method:

[0061] First, a high-quality female catfish reference genome was constructed. Genomic DNA was extracted from the muscle genome, and CCS sequencing was performed using the PacBio sequencing platform. After obtaining contigs, HiFiasm was preliminarily assembled. Single-base correction was performed using the PacBio sequencing data and Illumina sequencing data of the assembled original genome, and the Hi-C data was aligned to the preliminarily assembled genome. The interaction map was constructed using the Juicer software, and manual error correction was performed using JuiceBox. Finally, a female fish reference genome at the 28 chromosome level was obtained.

[0062] Next, the structure of coding genes was predicted, including homolog prediction and denovo prediction, and the gonad transcriptome data of catfish published by the research group (sra database: SRR1382323-SRR13823244, SRR13823246-SRR13823249; GSA database: CRA008348) were used for comparison and Cufflinks assembly to obtain transcripts. Then, MAKER software was used to integrate non-redundant coding gene sets, and functional annotations were performed through the exogenous protein database.

[0063] Based on the sex-related QTL interval located by the high-density genetic linkage map of Clarias fuscus published by the research team earlier (Linet al., A high-density genetic linkage map and QTL mapping for sex in Clarias fuscus. Aquaculture 561, 2022), the sex-linked SNP markers in the QTL positioning interval were aligned to the reference genome of Clarias fuscus using BLASTN, and the genes in the interval were screened according to the genome annotation results. Near the LOD peak on chr13, a gene with the function of determining male sex was retrieved (its base is shown in SEQ ID NO: 1), and the CDS sequence of the gene was cloned and verified (wavy line part).

[0064] Example 2

[0065] This example develops SNP molecular markers and primers within sex determination candidate genes based on genome resequencing and transcriptome data comparison.

[0066] A total of 48 sample genomes of male and female catfish (17 females and 17 males from Guangxi mixed families, 3 females and 3 males from Guangdong, and 3 females and 3 males from Fujian) and two mixed pools (mixed families from Guangxi) were resequenced and compared with the reference genome of female catfish in Example 1. After quality control filtering, a total of 8,539,125 SNP sites were obtained (see Figure 1 ), and preliminary screening was performed according to the conditions of female homozygosity and male heterozygosity, and SNPs that met the screening conditions within the interval (>0.7) were used as candidate SNPs.

[0067] The candidate SNP sites were further compared with the gonadal transcriptome data published by the research group (SRA database: SRR1382323-SRR13823244, SRR13823246-SRR13823249; GSA database: CRA008348) (see Figure 2 ), the SNP molecular markers that met a high ratio (>0.9) on the gene (such as the 11,946th position shown in SEQ ID NO: 1, the two alleles are A and C) were developed as sex-specific markers.

[0068] Primer6 software was used to design primers from a partial sequence of the sex determination candidate gene in the catfish genome sequence (catfish sex-specific molecular marker). The catfish sex-specific molecular marker sequence is as follows:

[0069]

[0070] The horizontal lines represent the forward primer and the reverse primer (the forward sequence, which is the base sequence of the reverse primer R after reverse complementation), and the square brackets and bold slashes represent the target mutation to be detected. The female fish has the CC genotype, and the male fish has the A / C genotype.

[0071] The designed specific amplification primers are as follows:

[0072] Forward primer F: 5'-ACCTGTATGTCTGTGGACTGTG-3', as shown in SEQ ID NO: 3;

[0073] Reverse primer R: 5′-GCCATTCTTGAAGTACCGCTTG-3′, as shown in SEQ ID NO:4.

[0074] In order to verify the accuracy of the SNP molecular marker and primer, this example is further described by the verification experiment in the following Example 3, as follows.

[0075] Example 3

[0076] The method for sex identification of Silurus catfish provided in this embodiment comprises the following steps: amplifying specific fragments in the genomic DNA of male and female individuals by PCR amplification, specifically comprising: designing the primers in Example 2, extracting the genomic DNA of the Silurus catfish to be tested, using the primers in Example 2 and the extracted genomic DNA as a template to perform PCR amplification, detecting the amplified product by capillary electrophoresis (specifically, ABI 3730XL gene sequencer, sequencing by Sangon Biotech (Shanghai) Co., Ltd.) and identifying the male and female individuals according to the sequencing peak results.

[0077] The implementation object of this embodiment is Silurus fuliginosa, with a total of 97 samples, including 50 females and 47 males (42 females and 37 males from Beiliu group in Guangxi, 8 females and 10 males from Nanning group in Guangxi). After the physiological sex was identified by dissection, the sex identification experiment of the above primers was carried out.

[0078] (1) DNA extraction

[0079] Fin rays of about 1 square centimeter were cut from the living catfish (this process did not cause any substantial harm to the living catfish, and the catfish individuals were observed to grow healthily after being released after sampling, the same below), preserved in 95% alcohol, and the catfish individuals were released after sampling.

[0080] Tissue DNA was extracted using a commercially available universal DNA extraction kit or conventional phenol imitation method.

[0081] (2) Amplification of sex-specific markers in catfish

[0082] The forward primer F and reverse primer R of the above primers were used as amplification primers, the extracted catfish DNA was used as a template, and PCR amplification was performed using EasyTaq DNA polymerase.

[0083] Forward primer F: 5'-ACCTGTATGTCTGTGGACTGTG-3', as shown in SEQ ID NO: 3;

[0084] Reverse primer R: 5'-GCCATTCTTGAAGTACCGCTTG-3', shown in SEQ ID NO:4.

[0085] PCR reaction system: 30 ng / μL DNA template: 1.0 μL, 2×EasyPCR Super Mix: 12.5 μL, 10 mM forward primer F and reverse primer R each 1 μL, dd H2O 9.5 μL, a total of 25 μL.

[0086] PCR reaction program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 sec, 58°C annealing for 30 sec, 72°C extension for 30 sec, 35 cycles, 72°C extension for 10 min.

[0087] (3) Gender-specific fragment detection

[0088] The amplified product in step (2) was subjected to base detection on an ABI 3730XL gene sequencer using forward primer F.

[0089] Figure 3 This is an example of the peak type of capillary electrophoresis detection of catfish individuals using F primer. The sequencing used forward primer F. Top: heterozygous peak type of male individuals; bottom: homozygous peak type of female individuals, that is, the heterozygous peak type is male individuals, and the homozygous peak type is female individuals.

[0090] Results: 47 male fish peaks and Figure 3 The heterozygous type in 50 female fish was consistent with Figure 3 The homozygous type in the test results is consistent, and the sequencing results are highly consistent with the results of the anatomical experiment. Therefore, using the primers and methods of the present invention, the accuracy rate of male and female identification is 100%.

[0091] In order to further verify the accuracy and applicability of the SNP molecular markers and primers, this example is further described by the verification test of catfish from more sources in the following Example 4, as follows.

[0092] Example 4

[0093] The method for identifying sex of catfish from different sources provided in this embodiment has the same implementation steps as those in Example 3.

[0094] The implementation object of this embodiment is Silurus catfish, with a total of 233 samples, including 118 females and 115 males (60 females and 60 males from Beiliu group in Guangxi, 48 females and 41 males from Nanning group in Guangxi, 5 females and 6 males from Guangdong group, and 5 females and 8 males from Fujian group). After the physiological sex was identified by dissection, the sex identification experiment using the above primers was carried out.

[0095] The extraction of DNA, amplification of sex-specific markers of Silurus catfish, PCR reaction system, PCR reaction procedure and sex-specific fragment detection are the same as those in Example 4.

[0096] The amplified product was subjected to base detection on the ABI 3730XL gene sequencer using forward primer F.

[0097] Table 1 is a statistical table of the results of capillary electrophoresis detection of catfish individuals using F primers. The forward primer F was used for sequencing. 1 represents ovary, 2 represents testis, heterozygous peak type is male individual, and homozygous peak type is female individual.

[0098] Table 1 Statistical table of the results of capillary electrophoresis detection of catfish individuals using primer F

[0099]

[0100]

[0101]

[0102]

[0103] The results showed that the sequencing results of 115 male fish showed an A / C heterozygous peak type; the peak type of 118 female fish showed a CC homozygous peak type (Table 1). The sequencing results were highly consistent with the results of the anatomical experiment. Therefore, using the primers and methods of the present invention, the accuracy rate of male and female identification is 100%.

[0104] Therefore, further, the above primers can be made into a kit or biological preparation for the identification of the sex of the catfish and parthenogenesis breeding.

[0105] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A method for identifying the sex of catfish from different sources, characterized in that: The following steps are involved: (1) Extracting DNA from the fin tissue of the catfish to be tested; (2) using primers for detecting sex-specific SNP molecular markers of catfish from different sources and using the catfish DNA extracted in step (1) as a template to perform PCR amplification; (3) The amplified products were detected by capillary electrophoresis, and the male and female individuals were identified based on the sequencing peak pattern. The heterozygous peak pattern was for male individuals, and the homozygous peak pattern was for female individuals. The primers in step (2) include a forward primer F and a reverse primer R, wherein the sequence of the forward primer F is shown in SEQ ID NO:3, and the sequence of the reverse primer R is shown in SEQ ID NO:

4. The primers are used to detect sex-specific SNP molecular markers of catfish from different sources, wherein the SNP molecular marker is located at position 11,946 of the sequence shown in SEQ ID NO:1, and its base is A or C, wherein the CC genotype is female fish, and the AC genotype is male fish.

2. The method for identifying the sex of catfish from different sources according to claim 1, characterized in that: During PCR amplification in step (2), the PCR reaction system used includes: 30 ng / μL DNA template: 1.0 μL, 2×EasyPCR Super Mix: 12.5 μL, 10 mM forward primer F and reverse primer R, 1 μL each, dd H2O 9.5 μL, a total of 25 μL.

3. The method for identifying the sex of catfish from different sources according to claim 1, characterized in that: During PCR amplification in step (2), the PCR reaction procedure is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and extension at 72°C for 10 min.

4. Use of primers for detecting sex-specific SNP molecular markers of bearded catfish from different sources in sex identification of bearded catfish from different sources, the primers comprising a forward primer F and a reverse primer R, wherein the sequence of the forward primer F is shown in SEQ ID NO:3, and the sequence of the reverse primer R is shown in SEQ ID NO:4, and the primers are used to detect sex-specific SNP molecular markers of bearded catfish from different sources, wherein the SNP molecular marker is located at position 11,946 of the sequence shown in SEQ ID NO:1, and its base is A or C, wherein the CC genotype is female fish, and the AC genotype is male fish.

5. Application of primers for detecting sex-specific SNP molecular markers of bearded catfish from different sources in parthenogenetic breeding of bearded catfish, the primers comprising a forward primer F and a reverse primer R, wherein the sequence of the forward primer F is as shown in SEQ ID NO:3, and the sequence of the reverse primer R is as shown in SEQ ID NO:4, and the primers are used to detect sex-specific SNP molecular markers of bearded catfish from different sources, wherein the SNP molecular marker is located at position 11,946 of the sequence shown in SEQ ID NO:1, and its base is A or C, wherein the CC genotype is female fish, and the AC genotype is male fish.

6. Application of the method according to any one of claims 1 to 3 in sex identification of catfish from different sources.

7. Use of the method according to any one of claims 1 to 3 in the parthenogenesis of catfish.

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