Molecular Marker for the Sex of Mastacembelus armatus and Its Application

Abstract

This application relates to a molecular marker for the sex of Mastacembelus armatus and its application. The molecular marker includes nucleotide sequences that differ between male and female Mastacembelus armatus. Using these nucleotide sequences can effectively detect, identify, and authenticate the sex of Mastacembelus armatus, and provide technical support for the creation of unisexual germplasm and sex-controlled breeding of Mastacembelus armatus.

Description

Technical Field

[0001] This application relates to the technical field of Mastacembelus armatus sex detection, and specifically to Mastacembelus armatus sex molecular markers and their applications. Background Art

[0002] Mastacembelus armatus (abbreviated as M. armatus) belongs to the order SymbRanchiFoRmes, the family Mastacembelidae, and the genus Mastacembelus. Mastacembelus armatus has a long and narrow body, similar to that of an eel, with a flat and thin tail, and belongs to small freshwater benthic fish. Its body color is blackish gray or blackish brown, the abdomen is grayish white or grayish yellow, there are blocky reticulations and water ripples on both sides of the body surface, there is a black longitudinal band in the middle of the back, and the front part of the back is composed of many thorn-like short spines. Adult females have a short and thick body with a yellowish color, while adult males have a thick and slender body with a dark black color.

[0003] Mastacembelus armatus has potential development prospects and market value, but currently, some basic scientific information about the breeding technology of Mastacembelus armatus, especially its fecundity, is seriously lacking. In 2013, the exploration of artificial breeding and reproduction technology of Mastacembelus armatus began, successfully breeding Mastacembelus armatus seedlings, and dividing the embryonic development process of Mastacembelus armatus into 8 stages and 22 periods, such as fertilized eggs, embryonic swelling, cleavage stage, blastula stage, gastrula stage, neurula stage, organogenesis stage, and hatching stage, and also studied the artificial induced spawning and artificial insemination technology of Mastacembelus armatus, and explored the optimal hatching temperature of Mastacembelus armatus.

[0004] However, it is found in the artificial breeding production of Mastacembelus armatus that there is a phenomenon of gender ratio imbalance in the breeding process of this fish, resulting in a much higher proportion of physiological female fish of commercial specifications than physiological male fish. This not only restricts the normal breeding work of Mastacembelus armatus, but also seriously reduces the economic benefits of aquaculture production, becoming a bottleneck restricting the large-scale development of the Mastacembelus armatus industry. The traditional artificial breeding and variety breeding methods of Mastacembelus armatus are less efficient and cannot meet the requirements of aquaculture and production. Summary of the Invention

[0005] In this application, nucleotide sequences with male-female differences are screened from the whole genome sequence of wild population Mastacembelus armatus, which can be developed as its sex molecular markers. Using these nucleotide sequences can effectively detect, identify, and authenticate the sex of Mastacembelus armatus, and provide technical support for the creation of Mastacembelus armatus single-sex germplasm and sex-controlled breeding.

[0006] Therefore, the embodiments of this application at least disclose the following technical solutions:

[0007] In the first aspect, the embodiments disclose sex-linked molecular markers of giant spiny loach, including: sex-difference nucleotide sequences of 18549253 to 18549792 nt of the reference genome Y chromosome CM033531.1 of giant spiny loach (M.armatus); sex-difference nucleotide sequences of 18549276 to 18549794 nt of the reference genome Y chromosome CM033531.1 of giant spiny loach (M.armatus); sex-difference nucleotide sequences of 24467692 to 24468634 nt of the reference genome Y chromosome CM033531.1 of giant spiny loach (M.armatus); at least one of sex-difference nucleotide sequences of 20716821 to 207177735 nt of the reference genome Y chromosome CM033531.1 of giant spiny loach (M.armatus).

[0008] In the second aspect, the embodiment discloses a nucleic acid molecule, which comprises: a sex difference nucleotide sequence of 18549253 to 18549792 nt of the reference genome Y chromosome CM033531.1 of the giant spiny loach (M.armatus); a sex difference nucleotide sequence of 18549276 to 18549794 nt of the reference genome Y chromosome CM033531.1 of the giant spiny loach (M.armatus); a sex difference nucleotide sequence of 24467692 to 24468634 nt of the reference genome Y chromosome CM033531.1 of the giant spiny loach (M.armatus); at least one of the sex difference nucleotide sequences of 20716821 to 207177735 nt of the reference genome Y chromosome CM033531.1 of the giant spiny loach (M.armatus).

[0009] In the third aspect, the embodiment discloses a primer, which includes an upstream primer and a downstream primer for PCR amplification of the nucleic acid molecule in the second aspect.

[0010] In a fourth aspect, the embodiment discloses a kit, which includes the primers described in the third aspect and other reagents required for PCR amplification.

[0011] In a fifth aspect, the embodiment discloses a method for detecting the sex of a giant spiny loach, comprising: using the primers described in the third aspect to perform PCR amplification on the genomic DNA of the giant spiny loach to be detected; and determining the sex of the giant spiny loach based on the PCR amplification product.

[0012] In a sixth aspect, the embodiment discloses a method for screening giant spiny loach, which includes determining the sex of the giant spiny loach according to the detection method described in the fifth aspect, and screening according to the sex.

[0013] In a seventh aspect, the embodiments disclose the use of the molecular markers described in the first aspect, the nucleic acid molecules described in the second aspect, the primers described in the third aspect, or the kits described in the fourth aspect, and the use is selected from any of the following:

[0014] 1) At least one of the detection, identification, and determination of the sex of Mastacembelus armatus;

[0015] 2) Screening and breeding of Mastacembelus armatus. Description of the Drawings

[0016] Figure 1 Pictures of the gonads of wild Mastacembelus armatus from Jiujiang, Jiangxi, and the results of HE staining of tissue sections provided for the examples. In the figure: A1 is a picture of the gonad of a female fish, and A2 is a picture of the gonad of a male fish. Picture a1 is the ovary, and picture a2 is the testis. Pictures a1 and a2 are the display images under a 4x microscope. O is the oocyte, and SZ is the sperm.

[0017] Figure 2 Gel picture of the quality detection of genomic DNA extracted from wild Mastacembelus armatus from Jiujiang, Jiangxi, provided for the examples. Lanes 1-10 are the results of JX1F, JX2F, JX3F, JX4F, JX5F, JX1M, JX2M, JX3M, JX4M, and JX5M in sequence.

[0018] Figure 3 Electrophoresis picture of the quality detection of genomic DNA of wild Mastacembelus armatus from Nanchang, Jiangxi, and Nanning, Guangxi, provided for the examples.

[0019] Figure 4 Electrophoresis picture of the PCR amplification products for the genetic sex identification of wild Mastacembelus armatus from Jiujiang, Jiangxi, using four sex-specific molecular markers (Marker1-4) provided for the examples.

[0020] Figure 5 Sequence diagram of the specific band amplified by PCR of the molecular marker (Marker1) in male individuals provided for the examples. The red boxes are the positions of the upstream and downstream primers respectively.

[0021] Figure 6 Sequence diagram of the specific band amplified by PCR of the molecular marker (Marker2) in male individuals provided for the examples. The red boxes are the positions of the upstream and downstream primers respectively.

[0022] Figure 7 Sequence diagram of the specific band amplified by PCR of the molecular marker (Marker3) in male individuals provided for the examples. The red boxes are the positions of the upstream and downstream primers respectively.

[0023] Figure 8Sequence diagram of the specific band amplified by PCR of the molecular marker (Marker4) provided for the example in male individuals. The red boxes indicate the positions of the upstream and downstream primers respectively.

[0024] Figure 9 Electrophoretogram of the PCR amplification products of the genetic sex identification of the wild population in Nanchang, Jiangxi by the four sex-specific molecular markers (Marker1-4) provided for the example.

[0025] Figure 10 Electrophoretogram of the PCR amplification products of the genetic sex identification of the wild population in Nanning, Guangxi by the molecular marker (Marker1) provided for the example.

[0026] Figure 11 Electrophoretogram of the PCR amplification products of the genetic sex identification of the wild population in Nanning, Guangxi by the molecular marker (Marker2) provided for the example.

[0027] Figure 12 Electrophoretogram of the PCR amplification products of the genetic sex identification of the wild population in Nanning, Guangxi by the molecular marker (Marker3) provided for the example.

[0028] Figure 13 Electrophoretogram of the PCR amplification products of the genetic sex identification of the wild population in Nanning, Guangxi by the molecular marker (Marker4) provided for the example. Detailed implementation manners

[0029] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and particularly are all conventional experimental methods and can be known from the prior art. The test samples and test processes used in the following embodiments include the following content (if the specific experimental conditions are not indicated in the embodiments, usually according to the conventional conditions or according to the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels).

[0030] Development of Molecular Markers Linked to the Sex of Mastacembelus armatus

[0031] In the present application, the large spiny eels in the wild population were re-sequenced, the quality of the test raw data was filtered, the differences between its sequenced genome and the reference genome were compared, and multiple molecular markers linked to the sex of the large spiny eel were mined and obtained. Specifically, it includes the following steps.

[0032] 1. Phenotypic sex identification

[0033] The wild giant loach needed for this step comes from Jiujiang, Jiangxi, with a total of 10 tails, including 5 females and 5 males. The average weight of females is 80±3.57g, and the average weight of males is 148.67±22.18g. It was raised in water at 22-28℃, and after confirming that there was no infection on its body surface and normal activity, it was used for subsequent experimental arrangements. The phenotypic sex of its individuals was determined by whether it could squeeze out sperm and eggs, tissue sections and HE staining for sex identification. Sample collection was carried out in the following steps: each individual was placed in an ice box for anesthesia, and after anesthesia, the fish body was disinfected with 75% ethanol to remove dirt, and then cut along the genital pore, and the gonads were placed in 2.0mL centrifuge tubes containing Bonn's solution; skin and other tissues not required for research were removed, muscle tissue was cut, and placed in 1.5mL centrifuge tubes containing anhydrous ethanol, and all samples were stored in a -20℃ refrigerator.

[0034] The gonadal tissue of the giant loach placed in Bonn's solution was fixed overnight, preserved in 70% ethanol, and sliced ​​and HE stained. The specific operation steps are as follows: Embedding: After taking out the sample preserved in 70% ethanol, place it in 75%, 85%, 95% ethanol for 30 minutes each, and 100% ethanol for 1 hour. Then place it in a solution of xylene: anhydrous ethanol (1:1) for 3 minutes; xylene for 3 minutes; xylene: paraffin (1:1, in a 65℃ constant temperature oven) for 10 minutes; paraffin for 2 hours, change the paraffin solution once in the middle, and finally embed. Slicing: After embedding, place the paraffin block on the cold stage of the tissue embedding machine, take out the wax block after solidification, and then install it on the slicer for conventional paraffin continuous sectioning, with a thickness of 4 to 8μm. Spread the paraffin strip in a 41℃ water bath for 2 minutes, and then use an adhesive slide to fish out the strip. Dry the slice on a 42℃ drying machine overnight and finally store it at room temperature. Staining: First, dewax the sections according to the following procedure: xylene, 30 minutes, 2 times; 100% ethanol, 15 minutes, 2 times; 100% ethanol: xylene (1:1), 15 minutes; 100% ethanol 3 minutes, 2 times; 90% ethanol, 3 minutes; 70% ethanol, 3 minutes; 50% ethanol, 3 minutes; distilled water, 3 minutes; hematoxylin, 2 minutes; ultrapure water, 2 to 3 times; eosin, 1 minute, ultrapure water, 2 to 3 times; then reverse the above steps, 1 minute each time. After the xylene evaporates, the sections are sealed with neutral resin, and finally observed and photographed under a microscope.

[0035] like Figure 1 As shown, during the breeding season, the female gonads are larger than the male gonads, contain eggs, and are golden or light yellow; while the male gonads are slender and soft, and are pink or milky white.

[0036] 2. Extraction of genomic DNA

[0037] Extract the genomic DNA of experimental samples using Omega's MicRoElute Genomic DNA Kit and ONREW's Tissue DNA Extraction CZ Kit. The specific experimental steps are carried out according to the instructions. After extraction, use 1% agarose gel electrophoresis to detect the quality of the extracted DNA, and use a ultra-micro nucleic acid and protein detector to detect its concentration and purity.

[0038] The genomic DNA of Mastacembelus armatus individuals was gently placed in an ultraviolet imager for observation after being detected by 1% agarose gel electrophoresis. The results showed that the bands were clear and complete, and the DNA quality was qualified. The concentration measurement by the ultra-micro nucleic acid and protein detector showed that the OD260 / OD280 of the genome was between 1.8 and 2.0, which could be used for subsequent sequencing operations on the machine. The detection results and the DNA quality detection gel map are shown in Table 1 and Figure 2 .

[0039] Table 1 Detection results of genomic DNA

[0040] Sample Name Concentration (ng / μL) Volume (μL) Total Amount (μg) Library Construction Type JX1F 12.2 60 0.732 DNBSEQWGS JX2F 36.2 60 2.172 DNBSEQWGS JX3F 34.5 60 2.07 DNBSEQWGS JX4F 39.1 60 2.346 DNBSEQWGS JX5F 38.7 60 2.322 DNBSEQWGS JX1M 43.2 60 2.592 DNBSEQWGS JX2M 46.9 60 2.814 DNBSEQWGS JX3M 11.8 60 0.708 DNBSEQWGS JX4M 18.8 60 1.128 DNBSEQWGS JX5M 39.6 60 2.376 DNBSEQWGS

[0041] 3. Library construction and whole-genome resequencing

[0042] A total of 10 wild Mastacembelus armatus from Jiujiang, Jiangxi were used for library construction and whole-genome resequencing, including 5 females and 5 males. After extracting the genomic DNA of these 10 Mastacembelus armatus, library construction and sequencing were carried out.

[0043] Libraries were constructed separately according to the samples and sequenced using the DNBSEQ platform. A total of 1.168 GB of Raw Reads were obtained from the sequencing of all samples, and the average sequencing volume of Raw Reads was 0.117 GB; 1.146 GB of Clean Reads were obtained after filtering, and the average sequencing volume of Clean Reads was 0.115 GB; 175.176 GB of Raw Bases were obtained, and the average sequencing volume of Raw Bases was 17.518 GB; 171.845 GB of Clean Bases were obtained after filtering, and the average sequencing volume of Clean Bases was 17.184 GB. The detailed data statistics are shown in Table 2. The average value of Q20 was 99.46%, the average value of Q30 was 96.53%, and the average GC content was 40.865%, indicating that the overall sequencing quality was high. Through the statistics and analysis of the sequencing data results, it can be seen that the GC content was normal and the error rate of the generated data was low. In Table 2, Raw Reads is the number of Reads sequenced; Raw Base is the number of bases sequenced; Clean Reads is the number of Reads after filtering; Clean Bases is the number of bases after filtering; Q20% is the proportion of bases with a quality value greater than 20 (error rate less than 1%) in the total number of bases; Q30% is the proportion of bases with a quality value greater than 30 (error rate less than 0.1%) in the total number of bases; GC is the proportion of G bases and C bases in the total number of bases.

[0044] Table 2 Statistical Table of Sequencing Data Volume and Data Filtering Results

[0045] Sample RawReads (bp) RawBases (bp) CleanReads (bp) CleanBases (bp) Q20(％) Q30(％) GC (%) JX1F 157,118,420 23,567,763,000 153,815,390 23,072,308,500 99.55 97.13 41.16 JX2F 111,273,234 16,690,985,100 108,998,800 16,349,820,000 99.51 96.88 40.87 JX3F 100,940,594 15,141,089,100 99,370,566 14,905,584,900 99.39 96.02 40.92 JX4F 102,860,726 15,429,108,900 101,298,360 15,194,754,000 99.35 95.8 40.82 JX5F 113,708,074 17,056,211,100 111,400,694 16,710,104,100 99.46 96.5 40.73 JX1M 119,641,292 17,946,193,800 117,387,080 17,608,062,000 99.5 96.79 41.08 JX2M 108,707,384 16,306,107,600 106,823,728 16,023,559,200 99.48 96.68 40.77 JX3M 148,156,648 22,223,497,200 144,832,386 21,724,857,900 99.52 96.98 40.75 JX4M 110,147,876 16,522,181,400 108,253,984 16,238,097,600 99.42 96.21 40.8 JX5M 95,285,890 14,292,883,500 93,450,834 14,017,625,100 99.42 96.3 40.75

[0046] 4. Filtering of Raw Data Quality

[0047] Base calling was performed on the raw image data obtained from re-sequencing through the CASAVA software, and these raw sequence data were further converted into the FASTQ format. However, these raw data often contain sequencing adapter sequences and low-quality Reads. Therefore, before further analysis, the data needs to be strictly filtered. We used the SOAPnuke (v2.1.0) software developed by BGI Shenzhen Co., Ltd. to remove Reads contaminated by adapters, Reads with too low base quality (i.e., the proportion of bases with a base quality value less than or equal to 12 reaches 50%), and Reads containing too many N bases (proportion exceeding 10%). The obtained high-quality CleanData was used for subsequent analysis (Q20 of CleanData after filtering was greater than 90%, and Q30 was greater than 80%).

[0048] 5. Alignment with the Reference Genome

[0049] Using the BWA alignment software, the mem alignment strategy was used to align the paired-end sequencing data to the Mastacembelus armatus reference genome, which contains 23 pairs of autosomes, the Y chromosome, and the Y chromosome (NCBI accession numbers: GCA_019455535.1, GCA_019455525.1), and the default alignment parameters were used for the alignment. To improve the accuracy and reliability of molecular marker detection and reduce the interference caused by alignment errors to molecular marker detection, only paired-end alignments and the sequencing Reads that were uniquely aligned to the reference genome were selected for molecular marker detection analysis. After alignment, samtools (version 1.14) was used to sort the aligned sequences.

[0050] The filtered data mentioned above was aligned to the reference genome, and the total number of aligned Reads (including single-end alignment and paired-end alignment), the number of correctly paired-end aligned Reads, and their proportions for each sample were counted. The results showed that the average PrimaRymapped was 99.66%, and the average PRopeRlypaiRed mapped was 97.31%. The detailed statistics of the alignment rate results are shown in Table 3. In Table 3, TotalReads is the total number of sequencing Reads; PrimaRymapped is the total number of aligned Reads, including single-end alignment and paired-end alignment; PrimaRymapped (%) is the proportion of aligned Reads in the total Reads, including single-end alignment and paired-end alignment; PRopeRlypaiRedmapped is the number of correctly paired-end aligned Reads; PRopeRlypaiRedmapped (%) is the proportion of the number of correctly paired-end aligned Reads.

[0051] Table 3 Summary of the statistics of the alignment rate results between the resequencing results and the reference genome

[0052]

[0053]

[0054] 6. Mining sex-linked molecular markers

[0055] After the alignment was completed, in this step, the deepvaRiant software was used to detect molecular markers, and samtools mpileup and a python program were used to recheck the genotypes of molecular markers to comprehensively determine the Indel sites. Mastacembelus armatus has an XY sex determination mechanism. Indels that are located on the sex chromosomes and meet the conditions that male samples are heterozygous and female samples are homozygous were selected as potential sex-linked molecular markers.

[0056] It was found that the sex-linked molecular markers disclosed in the examples include: the sex-differentiating nucleotide sequences of 18549253-18549792 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; the sex-differentiating nucleotide sequences of 18549276-18549794 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; the sex-differentiating nucleotide sequences of 24467692-24468634 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; the sex-differentiating nucleotide sequences of 20716821-207177735 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0057] In some embodiments, the sex-linked molecular markers of Mastacembelus armatus include the sex-differentiating nucleotide sequences of 18549253-18549792 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0058] In some embodiments, the sex-linked molecular markers of Mastacembelus armatus include the sex-differentiating nucleotide sequences of 18549276-18549794 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0059] In some embodiments, the sex-linked molecular markers of Mastacembelus armatus include the sex-differentiating nucleotide sequences of 24467692-24468634 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0060] In some embodiments, the sex-linked molecular markers of Mastacembelus armatus include the sex-differentiating nucleotide sequences of 20716821-207177735 nt on the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0061] On the one hand, the embodiment discloses a nucleic acid molecule. The nucleic acid molecule includes at least one of the sex-differential nucleotide sequences of 18549253-18549792 nt of the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; the sex-differential nucleotide sequences of 18549276-18549794 nt of the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; the sex-differential nucleotide sequences of 24467692-24468634 nt of the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus; and the sex-differential nucleotide sequences of 20716821-207177735 nt of the Y chromosome CM033531.1 of the reference genome of Mastacembelus armatus.

[0062] Verification and Identification of Molecular Markers Linked to the Sex of Mastacembelus armatus

[0063] Using the same method as above, the genomic DNA of wild Mastacembelus armatus samples (5 females and 5 males) from Jiujiang, Jiangxi was extracted as a template for PCR amplification and Sanger sequencing to preliminarily verify the accuracy of the sequencing results. Then, population verification was carried out on 15 wild Mastacembelus armatus populations from Nanchang, Jiangxi and 30 wild Mastacembelus armatus populations from Nanning, Guangxi. According to the PCR amplification products, the linkage relationship between the molecular markers mined above and the sex of Mastacembelus armatus was judged. The specific process includes the following steps:

[0064] 1. Extract genomic DNA

[0065] The concentrations of the genomic DNA extracted from 15 wild Mastacembelus armatus from Nanchang, Jiangxi and 30 wild Mastacembelus armatus from Nanning, Guangxi were measured using a ultra-micro nucleic acid and protein detector, and the ratios of OD260 / OD280 were all in the range of 1.8-2.0. At the same time, 11 individuals were randomly selected from each group and observed under ultraviolet light after electrophoresis detection. The results are as Figure 3 shown. The bands are clear and the fragments are complete, which can be used as templates for PCR reaction amplification.

[0066] 2. PCR

[0067] It has been determined that the sex determination mechanism of Mastacembelus armatus is XX / XY. Therefore, taking 23 pairs of autosomes, the Y chromosome and the Y chromosome as the reference genomes (NCBI accession numbers: GCA_019455535.1, GCA_019455525.1), the female sequencing data and the male sequencing data were respectively aligned to the reference genomes. The genomic region that is only covered by the male sequencing data and not covered by the female sequencing data was defined as the male-specific fragment. 77 pairs of primer sequences were designed using PRemieR5.0, and the primer sequences were synthesized jointly by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. and Guangzhou Aiji Biotechnology Co., Ltd.

[0068] The PCR reaction was carried out in a 20 μL system: 10 μL of GReenTaqmix, 1 μL of each Marker, 1 μL of DNA template, and 7 μL of ddH2O. Then, it was centrifuged and mixed evenly. The PCR tube containing the mixture was placed in a PCR amplifier and subjected to PCR amplification according to the following procedure: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s; annealing at 60 °C for 30 s; extension at 72 °C for 30 s; 35 cycles; repair extension at 72 °C for 5 min. After the PCR amplification was completed, it was detected by 1% agarose gel electrophoresis to check whether there was a target band. Immediately, the PCR product was sent to Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing.

[0069] Such as Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 As shown, the Sanger sequencing results of 8 loci (Molecular Marker 1, Molecular Marker 2, Molecular Marker 6, Molecular Marker 7, Molecular Marker 9, Molecular Marker 10, Molecular Marker 12, Molecular Marker 13) were consistent with the phenotypic sex and were completely linked to sex. These 8 loci were homozygous in females and heterozygous in males.

[0070] A total of 45 samples (30 females and 15 males) from 15 wild Macrognathus aculeatus populations (10 females and 5 males) in Nanchang, Jiangxi and 30 wild Macrognathus aculeatus populations (20 females and 10 males) in Nanning, Guangxi were further verified. As the results showed, 8 loci (Molecular Marker 1, Molecular Marker 2, Molecular Marker 6, Molecular Marker 7, Molecular Marker 9, Molecular Marker 10, Molecular Marker 12, Molecular Marker 13) were homozygous genotypes in females and heterozygous genotypes in males, and were completely linked to sex. See Table 4 for details. In the table, F is the upstream primer, R is the downstream primer, EX is the enhanced primer, Ho represents homozygous, He represents heterozygous, P HWE represents the Hardy-Weinberg equilibrium index, and PIC represents the polymorphic information index.

[0071] Table 4 Verification results of four sex-differentiated sequences for 45 wild Macrognathus aculeatus

[0072]

[0073] As shown in Table 4, the genomic DNA of wild Mastacembelus armatus was amplified by PCR using primer pairs F1 and R1. If the electrophoresis band of the amplification product is 539 bp, the corresponding individual is a male individual; if there is no electrophoresis band in the amplification product, the corresponding individual is a female individual. The genomic DNA of wild Mastacembelus armatus was amplified by PCR using primer pairs F2 and R2. If the electrophoresis band of the amplification product is 518 bp, the corresponding individual is a male individual; if there is no electrophoresis band in the amplification product, the corresponding individual is a female individual. The genomic DNA of wild Mastacembelus armatus was amplified by PCR using primer pairs F3 and R3. If the electrophoresis bands of the amplification product are 483 bp and 942 bp, the corresponding individual is a male individual; if the electrophoresis band of the amplification product is 483 bp, the corresponding individual is a female individual. The genomic DNA of four wild Mastacembelus armatus was amplified by PCR using primer pairs F4 and R. If the electrophoresis band of the amplification product is 1084 bp, the corresponding individual is a male individual; if the electrophoresis band of the amplification product is 914 bp, the corresponding individual is a female individual.

[0074] As shown in the electrophoresis results and sequencing results in the figure, these four primers can all identify the genetic sex of wild Mastacembelus armatus in Jiujiang, Jiangxi.

[0075] Based on this, on the one hand, the embodiment discloses a primer. The primer includes an upstream primer and a downstream primer for PCR amplification of the nucleic acid molecule described in the second aspect.

[0076] In some embodiments, the primer includes at least one of the following:

[0077] The DNA molecule shown in SEQ ID NO.1 and the DNA molecule shown in SEQ ID NO.2;

[0078] The DNA molecule shown in SEQ ID NO.3 and the DNA molecule shown in SEQ ID NO.4;

[0079] The DNA molecule shown in SEQ ID NO.5 and the DNA molecule shown in SEQ ID NO.6;

[0080] The DNA molecule shown in SEQ ID NO.7 and the DNA molecule shown in SEQ ID NO.8.

[0081] On the one hand, the embodiment discloses a kit. The kit includes the primer and other reagents required for PCR amplification.

[0082] On the one hand, the embodiment discloses a method for detecting the sex of Mastacembelus armatus. The method includes: performing PCR amplification on the genomic DNA of the Mastacembelus armatus to be tested using the primer described in the third aspect; determining the sex of the Mastacembelus armatus according to the nucleotide sequence of the PCR amplification product.

[0083] In some embodiments, the steps of determining the sex of the large spiny eel according to the nucleotide sequence of the PCR amplification product include: if the electrophoresis band of the amplification product is 539 bp, the corresponding individual is a male individual; if there is no electrophoresis band in the amplification product, the corresponding individual is a female individual.

[0084] In some embodiments, the steps of determining the sex of the large spiny eel according to the nucleotide sequence of the PCR amplification product include: if the electrophoresis band of the amplification product is 518 bp, the corresponding individual is a male individual; if there is no electrophoresis band in the amplification product, the corresponding individual is a female individual.

[0085] In some embodiments, the steps of determining the sex of the large spiny eel according to the nucleotide sequence of the PCR amplification product include: if the electrophoresis bands of the amplification product are 483 bp and 942 bp, the corresponding individual is a male individual; if the electrophoresis band of the amplification product is 483 bp, the corresponding individual is a female individual.

[0086] In some embodiments, the steps of determining the sex of the large spiny eel according to the nucleotide sequence of the PCR amplification product include: if the electrophoresis band of the amplification product is 1084 bp, the corresponding individual is a male individual; if the electrophoresis band of the amplification product is 914 bp, the corresponding individual is a female individual.

[0087] On the other hand, the embodiments also disclose a screening method for the large spiny eel. The screening method includes determining the sex of the large spiny eel according to the detection method provided in the above embodiments and screening according to its sex.

[0088] On the other hand, the embodiments disclose the applications of the molecular marker described in the first aspect, the nucleic acid molecule described in the second aspect, the primer described in the third aspect or the kit described in the fourth aspect, and the applications are selected from any of the following:

[0089] 1) At least one of the detection, identification and authentication of the sex of the large spiny eel;

[0090] 2) Screening and breeding of the large spiny eel.

[0091] Genetic Sex Identification of Wild Populations in Nanchang, Jiangxi Using Sex-Specific Molecular Markers

[0092] In order to verify the universality of the four sex-specific molecular markers screened in this experiment, the four sex-specific molecular markers preliminarily screened were used to verify 15 wild large spiny eels in the wild population of Nanchang, Jiangxi (10 females and 5 males). The results of genetic sex identification were 10 females and 5 males.

[0093] As Figure 9 shown in Table 5, the identification results of the four sex-specific molecular markers were consistent, and the coincidence rate between the phenotypic sex and the genetic sex identification results was 100%.

[0094] Table 5 Genetic sex identification of wild population in Nanchang, Jiangxi using four sex-specific molecular markers

[0095]

[0096] Genetic Sex Identification of Wild Populations in Nanning, Guangxi Using Sex-Specific Molecular Markers

[0097] The screened molecular markers were used to conduct genetic sex identification on the wild population in Nanning, Guangxi (20 females and 10 males), a total of 30 wild Macrognathus aculeatus. The results of genetic sex identification were 20 females and 10 males, and the results identified by these four sex-specific molecular markers were consistent. However, since the sizes of the amplified bands of Marker4 were relatively close, it was difficult to distinguish them by the naked eye.

[0098] As Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 and Table 6 show, the coincidence rate between the phenotypic sex and the results of genetic sex identification was 100%.

[0099] Table 6 Genetic sex identification of wild population in Nanning, Guangxi using four sex-specific molecular markers

[0100]

[0101] As mentioned above, the above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application.

Claims

1. A method for detecting the sex of a giant loach, the method comprising: PCR amplification of the genomic DNA of the giant spiny loach to be tested is performed using the primer combination shown in SEQ ID NO.5 and SEQ ID NO.6; If the electrophoresis bands of the amplified product are 483bp and 942bp, the corresponding individual is a male individual; if the electrophoresis band of the amplified product is 483bp, the corresponding individual is a female individual.

2. A method for screening giant spiny loach, comprising determining the sex of the giant spiny loach according to the detection method according to claim 1, and screening according to the sex of the giant spiny loach.

Images