Sex molecular markers and their application in giant spiny loach

Abstract

The present application relates to a sex molecular marker of a giant spiny loach and its application. The molecular marker includes a nucleotide sequence formed by single nucleotide mutations at multiple positions of the giant spiny loach's X chromosome. These molecular markers can be used to effectively detect, identify and authenticate the sex of the giant spiny loach, and provide technical support for the creation of monosex germplasm and sex-controlled breeding of the giant spiny loach.

Description

Technical Field

[0001] The present application relates to the technical field of sex detection of giant spiny loach, and in particular to molecular markers of sex of giant spiny loach and their application. Background Art

[0002] The armatus (Mastacembelus armatus, M. armatus for short) belongs to the order Symbranchiformes, family Mastacembelidae, genus Mastacembelus. The armatus is narrow and long, like an eel, with a thin and flat tail. It is a small freshwater bottom-dwelling fish. Its body color is black-gray or black-brown, its abdomen is grayish white or grayish yellow, and it has blocky mesh patterns and water ripples on both sides of its body. There is a black longitudinal band in the middle of its back, and there are many short thorn-like spines on the front of its back. Adult females are short and thick with a yellowish color, while adult males are stout and slender with a dark black color.

[0003] The giant spiny loach has potential development prospects and market value, but there is currently a serious lack of basic scientific information on its breeding technology, especially its fertility. In 2013, we began to explore the artificial breeding and reproduction technology of the giant spiny loach, successfully bred giant spiny loach seedlings, and divided the embryonic development process of the giant spiny loach into 8 stages and 22 periods, including fertilized eggs, embryonic bulges, cleavage stages, blastocyst stages, gastrula stages, neurula stages, organ formation stages, and membrane emergence stages. We also studied the artificial induction of labor and artificial insemination techniques for the giant spiny loach, and explored the optimal incubation temperature for the giant spiny loach.

[0004] However, in the artificial breeding of giant spiny loach, it was found that the sex ratio of the fish was unbalanced during reproduction, resulting in a much higher proportion of physiological female fish than physiological male fish of commercial size. This not only restricted the normal breeding of giant spiny loach, but also seriously reduced the economic benefits of breeding and production, becoming a bottleneck to curb the large-scale development of the giant spiny loach industry. Traditional artificial breeding and variety breeding methods of giant spiny loach are inefficient and cannot meet the requirements of breeding and production. Summary of the invention

[0005] This application screened multiple SNP sites significantly linked to sex from the sequenced whole genome of wild populations of giant loach, which can be developed as its sex molecular markers. These molecular markers can be used to effectively detect, identify and identify the sex of giant loach, and provide technical support for the creation of monosex germplasm and sex-controlled breeding of giant loach.

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

[0007] In a first aspect, the embodiments disclose sex-linked molecular markers of giant spiny loach, including: a nucleotide sequence formed by a single nucleotide mutation T>C at 18030271 nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation C>T at 21146890 nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation G>T at 23800863 nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation C>T at 23543793 nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; A nucleotide sequence formed by a single nucleotide mutation G>A at 22836394nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); a nucleotide sequence formed by a single nucleotide mutation C>G at 22786222nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); a nucleotide sequence formed by a single nucleotide mutation T>C at 20845609nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); and a nucleotide sequence formed by a single nucleotide mutation A>G at 20814783nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus).

[0008] In a second aspect, the embodiment discloses a nucleic acid molecule, comprising: a nucleotide sequence in which a single nucleotide mutation T>C occurs at 18030271 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence in which a single nucleotide mutation C>T occurs at 21146890 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence in which a single nucleotide mutation G>T occurs at 23800863 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence in which a single nucleotide mutation C>T occurs at 23543793 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); >A; a nucleotide sequence formed by a single nucleotide mutation G>A at 22836394nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); a nucleotide sequence formed by a single nucleotide mutation C>G at 22786222nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); a nucleotide sequence formed by a single nucleotide mutation T>C at 20845609nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); and a nucleotide sequence formed by a single nucleotide mutation A>G at 20814783nt of the reference genome X chromosome CM033555.1 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, which comprises: 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 according to the nucleotide sequence of 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 an application 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, wherein the application is selected from any of the following:

[0014] 1) at least one of detection, identification and identification of the sex of the giant loach;

[0015] 2) Screening and breeding of giant spiny loach. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The gonad images and tissue section HE staining results of wild giant loach in Jiujiang, Jiangxi provided in the examples. In the figure: A1 is the gonad image of female fish, A2 is the gonad image of male fish. Figure a1 is the ovary, and figure a2 is the testis. Figure a1 and figure a2 are the displayed images under 4 times microscope, O is oocyte, SZ is sperm.

[0017] Figure 2 This is a gel image of the quality detection of genomic DNA extracted from wild giant loach in Jiujiang, Jiangxi Province provided in the example. Lanes 1 to 10 are the results of JX1F, JX2F, JX3F, JX4F, JX5F, JX1M, JX2M, JX3M, JX4M and JX5M.

[0018] Figure 3 The electrophoresis diagrams for the quality detection of genomic DNA of wild giant spiny loach in Nanchang, Jiangxi and wild giant spiny loach in Nanning, Guangxi provided in the examples.

[0019] Figure 4 The sequencing verification results of SNP1 and SNP2 provided in the embodiment for wild giant loach in Jiujiang, Jiangxi. The top of the figure shows 5 female samples, and the bottom shows 5 male samples. The red box is the SNP site, double peaks represent heterozygous, and single peaks represent homozygous.

[0020] Figure 5 The sequencing verification results of SNP6 and SNP7 provided in the embodiment for wild giant loach in Jiujiang, Jiangxi. The top of the figure shows 5 female samples, and the bottom shows 5 male samples. The red box is the SNP site, and the double peak indicates heterozygous, and the single peak indicates homozygous.

[0021] Figure 6 The verification results of SNP9 and SNP10 provided in the embodiment for wild giant loach in Jiujiang, Jiangxi. The top of the figure shows 5 female samples, and the bottom shows 5 male samples. The red box is the SNP site, and the double peak indicates heterozygous, and the single peak indicates homozygous.

[0022] Figure 7 The verification results of SNP12 and SNP13 provided in the embodiment for wild giant loach in Jiujiang, Jiangxi. The top of the figure shows 5 female samples, and the bottom shows 5 male samples. The red box is the SNP site, and the double peak indicates heterozygous, and the single peak indicates homozygous. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in this application are all conventional reagents and can be obtained from commercial sources; the methods not specifically described in detail are all conventional experimental methods, which can be learned from the prior art. The test samples and test processes used in the following examples include the following (if the specific experimental conditions not specified in the examples are usually in accordance with conventional conditions, or in accordance with the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can be obtained from commercial sources).

[0024] Development of sex-linked molecular markers for giant spiny loach

[0025] The present application resequences wild populations of giant spiny loach, performs quality filtering on the original test data, compares the differences between the sequenced genome and the reference genome, and mines and obtains multiple molecular markers linked to the sex of giant spiny loach. The specific steps include the following steps.

[0026] 1. Phenotypic sex identification

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 2. Extraction of genomic DNA

[0031] The experimental sample DNA was extracted according to the Omega MicRoElute Genomic DNA Kit and the ONREW Tissue DNA EXt Raction CZ Kit. The specific experimental steps were carried out according to the instructions. After the extraction was completed, the quality of the extracted DNA was detected by 1% agarose gel electrophoresis, and its concentration and purity were detected by ultra-micro nucleic acid protein detector.

[0032] The genomic DNA of the individual spiny loach was tested by 1% agarose gel electrophoresis and then gently placed in a UV imager for observation. The results showed that the bands were clear and complete, and the DNA quality was qualified; the concentration was measured by an ultra-trace nucleic acid protein detector, showing that the OD260 / OD280 of the genome was between 1.8 and 2.0, which can be used for subsequent sequencing operations. The test results and DNA quality test gel images are shown in Table 1 and Figure 2 .

[0033] Table 1 DNA test results

[0034] Sample name Concentration (ng / μL) Volume (μL) Total amount (μg) Database type JX1F 12.2 60 0.732 DNBSEQWGS JXj 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 JX2 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

[0035] 3. Library construction and whole genome resequencing

[0036] A total of 10 wild giant loaches from Jiujiang, Jiangxi, were used for library construction and whole genome resequencing, including 5 females and 5 males. Genomic DNA was extracted from these 10 giant loaches, and then library construction and sequencing were performed.

[0037] Libraries were constructed according to the samples and sequenced using the DNBSEQ platform. A total of 1.168GB of RawReads were obtained for all samples, and the average sequencing amount of RawReads was 0.117GB; a total of 1.146GB of CleanReads were obtained after filtering, and the average sequencing amount of CleanReads was 0.115GB; a total of 175.176GB of RawBases were obtained, and the average sequencing amount of RawBases was 17.518GB; a total of 171.845GB of CleanBases were obtained after filtering, and the average sequencing amount of CleanBases was 17.184GB. Detailed data statistics are shown in Table 2. The average Q20 value is 99.46%, the average Q30 value is 96.53%, and the average GC content is 40.865%. The overall sequencing quality is high. Through the statistics and analysis of the sequencing data results, it can be seen that the GC content is normal and the error rate of the generated data is low. In Table 2, Raw Reads is the number of sequenced Reads; RawBase is the number of sequenced bases; CleanReads is the number of filtered Reads; CleanBases is the number of filtered bases; Q20% is the ratio of bases with a quality value greater than 20 (error rate less than 1%) to the total number of bases; Q30% is the ratio of bases with a quality value greater than 30 (error rate less than 0.1%) to the total number of bases; GC is the ratio of G bases and C bases to the total number of bases.

[0038] Table 2 Statistics of sequencing data volume and data filtering results

[0039] 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 JXj 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 JX2 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

[0040] 4. Raw data quality filtering

[0041] The raw image data obtained by resequencing were base-identified by CASAVA software, and the raw sequence data were further converted into FASTQ format. However, these raw data often contain sequencing adapter sequences and low-quality reads, so the data need to be strictly filtered before further analysis. We used SOAPnuke (v2.1.0) software developed by Shenzhen BGI Co., Ltd. to remove reads contaminated by adapters, reads with low base quality (i.e., bases with base quality values ​​less than or equal to 12 account for 50%), and reads containing too many N bases (more than 10%). The obtained high-quality CleanData data was used for subsequent analysis (Q20 of CleanData data after filtering was greater than 90%, and Q30 was greater than 80%).

[0042] 5. Reference genome alignment

[0043] The BWA alignment software and mem alignment strategy were used to align the paired-end (PaiR-end) sequencing data to the reference genome of the giant loach, which contains 23 pairs of autosomes, chromosome X and chromosome Y (NCBI accession numbers: GCA_019455535.1, GCA_019455525.1). The default alignment parameters were used. In order to improve the accuracy and reliability of SNP detection and reduce the interference of alignment errors on SNP detection, only paired-end alignment and unique sequencing reads aligned to the reference genome were selected for SNP detection analysis. After alignment, samtools (version 1.14) was used to sort the aligned sequences.

[0044] The filtered data were aligned to the reference genome, and the total number of reads on the alignment (including single-end alignment and double-end alignment) and the number of reads with correct double-end alignment and their proportions were counted for each sample. The results showed that the average PRimaRymapped was 99.66%, and the average PRomaiRed 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 reads on the alignment, including single-end alignment and double-end alignment; PRimaRymapped (%) is the proportion of reads on the alignment to the total number of reads, including single-end alignment and double-end alignment; PRomaiRedmapped is the number of reads with correct double-end alignment; PRomaiRedmapped (%) is the proportion of reads with correct double-end alignment.

[0045] Table 3 Summary of the statistical results of the resequencing results and the reference genome alignment rate

[0046]

[0047]

[0048] 6. Mining sex-linked SNPs

[0049] After the alignment is completed, this step uses deepvaRiant software to detect SNPs, and uses samtoolsmpileup and python programs to perform a secondary review of the SNP genotype to comprehensively determine the SNP site. The giant loach has an XY type sex determination mechanism, and SNPs on the sex chromosomes that meet the requirements of heterozygous male samples and homozygous female samples are selected as potential sex-linked molecular markers. Table 4 shows the verified sex-linked SNP site information. In Table 4, CHROM is the Genebank number of the chromosome, Location is the location information, and REF / ALT is the reference genome / sequencing genome.

[0050] Table 4 Verification of information related to sex-linked SNPs

[0051] CHROM Location REF>ALT SNP Name CM033555.1 18030271 T>C SNP1 CM033555.1 21146890 C>T SNP2 CM033555.1 23800863 G>T SNP6 CM033555.1 23543793 C>A SNP7 CM033555.1 22836394 G>A SNP9 CM033555.1 22786222 C>G SNP10 CM033555.1 0845609 T>C SNP12 CM033555.1 20814783 A>G SNP13

[0052] Based on this, on the one hand, the embodiment discloses a sex-linked molecular marker for giant spiny loach, including: a nucleotide sequence formed by a single nucleotide mutation T>C at 18030271nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation C>T at 21146890nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation G>T at 23800863nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1; a nucleotide sequence formed by a single nucleotide mutation G>T at 23543793nt of the giant spiny loach (M. armatus) reference genome chromosome X CM033555.1 C>A formed nucleotide sequence; 22836394nt of the reference genome X chromosome CM033555.1 of the giant loach (M.armatus) has a single nucleotide mutation G>A formed nucleotide sequence; 22786222nt of the reference genome X chromosome CM033555.1 of the giant loach (M.armatus) has a single nucleotide mutation C>G formed nucleotide sequence; 20845609nt of the reference genome X chromosome CM033555.1 of the giant loach (M.armatus) has a single nucleotide mutation T>C formed nucleotide sequence; and 20814783nt of the reference genome X chromosome CM033555.1 of the giant loach (M.armatus) has a single nucleotide mutation A>G formed nucleotide sequence at least one of the following.

[0053] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation T>C at 18030271 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0054] In some embodiments, at 18030271 nt of chromosome X CM033555.1 of the reference genome of giant spiny loach (M. armatus), the individual with the TT genotype is female, and the individual with the TC genotype is male.

[0055] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation C>T at 21146890 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0056] In some embodiments, the sex-linked molecular marker for giant spiny loach includes 21146890 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with CC genotype are females, and individuals with CT genotype are males.

[0057] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence in which a single nucleotide mutation G>T occurs at 23800863 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0058] In some embodiments, the sex-linked molecular marker for giant spiny loach includes 23800863 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with the GG genotype are females, and individuals with the GT genotype are males.

[0059] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation C>A at 23543793 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0060] In some embodiments, the sex-linked molecular marker for giant spiny loach includes 23543793 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with CC genotype are females, and individuals with CA genotype are males.

[0061] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation G>A at 22836394 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0062] In some embodiments, the sex-linked molecular markers of giant spiny loach include 22836394 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with the GG genotype are females, and individuals with the GA genotype are males.

[0063] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence in which a single nucleotide mutation C>G occurs at 22786222 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0064] In some embodiments, the sex-linked molecular marker for giant spiny loach includes 22786222 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with CC genotype are females, and individuals with CG genotype are males.

[0065] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation T>C at 20845609 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0066] In some embodiments, the sex-linked molecular marker for giant spiny loach includes 20845609 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with the TT genotype are females, and individuals with the TC genotype are males.

[0067] In some embodiments, the sex-linked molecular marker of giant spiny loach includes a nucleotide sequence formed by a single nucleotide mutation A>G at 20814783 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome.

[0068] In some embodiments, the sex-linked molecular markers of giant spiny loach include 20814783 nt of chromosome X CM033555.1 of the giant spiny loach (M. armatus) reference genome, and individuals with the GG genotype are females, and individuals with the GA genotype are males.

[0069] In one aspect, the embodiment discloses a nucleic acid molecule. The nucleic acid molecule comprises: a nucleotide sequence formed by a single nucleotide mutation T>C at 18030271 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence formed by a single nucleotide mutation C>T at 21146890 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence formed by a single nucleotide mutation G>T at 23800863 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus); a nucleotide sequence formed by a single nucleotide mutation C>A at 23543793 nt of the reference genome X chromosome CM033555.1 of the giant loach (M. armatus). Column; nucleotide sequence formed by single nucleotide mutation G>A at 22836394nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); nucleotide sequence formed by single nucleotide mutation C>G at 22786222nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); nucleotide sequence formed by single nucleotide mutation T>C at 20845609nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus); and nucleotide sequence formed by single nucleotide mutation A>G at 20814783nt of the reference genome X chromosome CM033555.1 of the giant spiny loach (M.armatus).

[0070] Validation and identification of sex-linked molecular markers in Triplophys maximus

[0071] The same method as above was used to extract the genomic DNA of wild giant loach (5 females and 5 males) from Jiujiang, Jiangxi Province as a template for PCR amplification and Sanger sequencing. The accuracy of the sequencing results was preliminarily verified. Then, a population of 15 wild giant loaches from Nanchang, Jiangxi Province and a population of 30 wild giant loaches from Nanning, Guangxi Province were used for population verification. The linkage relationship between the molecular markers mined above and the sex of giant loaches was determined based on the PCR amplification products. The specific process includes the following steps:

[0072] 1. Extraction of genomic DNA

[0073] The genomic DNA of wild giant loach (15) from Nanchang, Jiangxi and wild giant loach (30) from Nanning, Guangxi was extracted and measured using an ultra-micro nucleic acid protein detector. The OD260 / OD280 ratios were both within the range of 1.8 to 2.0. At the same time, 11 individuals were randomly selected for electrophoresis and observed under ultraviolet light. The results are as follows: Figure 3 As shown, the bands are clear and the fragments are complete, and can be used as templates for PCR amplification.

[0074] 2. PCR

[0075] The sex determination mechanism of the giant loach has been determined to be XX / XY, so the 23 pairs of autosomes, X chromosomes and Y chromosomes were used as reference genomes (NCBI accession numbers: GCA_019455535.1, GCA_019455525.1), and the female sequencing data and male sequencing data were aligned to the reference genome. The genomic regions that were only covered by male sequencing data and not covered by female sequencing data were defined as male-specific fragments. 77 pairs of primer sequences were designed using PRemieR5.0, and the primer sequences were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. and Guangzhou Aiji Biotechnology Co., Ltd.

[0076] The PCR reaction was carried out according to the 20μL system: GReenTaqmix 10μL, PRimeR 1μL each, DNA template 1μL, ddH2O 7μL. Then centrifuge and mix, place the PCR tube containing the mixed solution in the PCR amplification instrument, and perform PCR amplification according to 94℃ pre-denaturation, 3min; 94℃ denaturation, 30s; 60℃ annealing, 30s; 72℃ extension, 30s; 35 cycles; 72℃ repair extension, 5min. After the PCR amplification is completed, use 1% agarose gel electrophoresis to check whether there is a target band, and then send the PCR product to Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing.

[0077] like Figure 4 , Figure 5 , Figure 6 , Figure 7 As shown, the SangeR sequencing results of 8 loci (SNP1, SNP2, SNP6, SNP7, SNP9, SNP10, SNP12, SNP13) are consistent with the phenotypic sex and are completely linked to sex. These 8 loci are homozygous in females and heterozygous in males.

[0078] A total of 45 samples (30 females and 15 males) from 15 wild giant loaches (10 females and 5 males) in Nanchang, Jiangxi and 30 wild giant loaches (20 females and 10 males) in Nanning, Guangxi were further verified. As shown in the results, 8 loci (SNP1, SNP2, SNP6, SNP7, SNP9, SNP10, SNP12, SNP13) were homozygous genotypes in females and heterozygous genotypes in males, and were completely linked to sex, as shown in Table 5. 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 represents HWE It is expressed as the Hartmann balance index and PIC is expressed as the polymorphic information index.

[0079] Table 5 Validation results of eight SNPs on 45 wild giant loaches

[0080]

[0081] Based on this, in one aspect, the embodiment discloses a primer, which includes an upstream primer and a downstream primer for PCR amplification of the nucleic acid molecule described in the second aspect.

[0082] In some embodiments, the primer comprises at least one of the following:

[0083] Primer pair 1: a DNA molecule as shown in SEQ ID NO.1, a DNA molecule as shown in SEQ ID NO.2, and a DNA molecule as shown in SEQ ID NO.3;

[0084] Primer pair 2: a DNA molecule as shown in SEQ ID NO.4, a DNA molecule as shown in SEQ ID NO.5, and a DNA molecule as shown in SEQ ID NO.6;

[0085] Primer pair 6: a DNA molecule as shown in SEQ ID NO.7, a DNA molecule as shown in SEQ ID NO.8, and a DNA molecule as shown in SEQ ID NO.9;

[0086] Primer pair 7: a DNA molecule as shown in SEQ ID NO.10, a DNA molecule as shown in SEQ ID NO.11, and a DNA molecule as shown in SEQ ID NO.12;

[0087] Primer pair 9: a DNA molecule as shown in SEQ ID NO.13, a DNA molecule as shown in SEQ ID NO.14, and a DNA molecule as shown in SEQ ID NO.15;

[0088] Primer pair 10: a DNA molecule as shown in SEQ ID NO.16, a DNA molecule as shown in SEQ ID NO.17, and a DNA molecule as shown in SEQ ID NO.18;

[0089] Primer pair 12: a DNA molecule as shown in SEQ ID NO.19, a DNA molecule as shown in SEQ ID NO.20, and a DNA molecule as shown in SEQ ID NO.21;

[0090] Primer pair 13: the DNA molecule shown as SEQ ID NO.22, the DNA molecule shown as SEQ ID NO.23 and the DNA molecule shown as SEQ ID NO.24.

[0091] In one aspect, the embodiment discloses a kit, which includes the primers and other reagents required for PCR amplification.

[0092] In one 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 according to the nucleotide sequence of the PCR amplification product.

[0093] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation T>C at 18030271 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then at 18030271 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the TT genotype is female, and the individual with the TC genotype is male.

[0094] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation C>T at 21146890 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 21146890 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the CC genotype is female, and the individual with the CT genotype is male.

[0095] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation G>T at 23800863 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 23800863 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the GG genotype is female, and the individual with the GT genotype is male.

[0096] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation C>A at 23543793 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 23543793 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the CC genotype is female, and the individual with the CA genotype is male.

[0097] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation G>A at 22836394 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 22836394 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the GG genotype is female, and the individual with the GA genotype is male.

[0098] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation C>G at 22786222 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 22786222 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with CC genotype is female, and the individual with CG genotype is male.

[0099] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation T>C at 20845609 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 20845609 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the TT genotype is female, and the individual with the TC genotype is male.

[0100] In some embodiments, the step of determining the sex of the giant spiny loach according to the nucleotide sequence of the PCR amplification product comprises: if the PCR amplification product is a nucleotide sequence comprising a single nucleotide mutation A>G at 20814783 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, then the sex-linked molecular marker of the giant spiny loach includes 20814783 nt of the giant spiny loach (M. armatus) reference genome X chromosome CM033555.1, the individual with the GG genotype is female, and the individual with the GA genotype is male.

[0101] In another aspect, the embodiment also discloses a method for screening giant spiny loach, which includes determining the sex of the giant spiny loach according to the detection method provided in the above embodiment, and screening according to the sex.

[0102] In another aspect, the embodiment discloses an application 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, wherein the application is selected from any of the following:

[0103] 1) at least one of detection, identification and identification of the sex of the giant loach;

[0104] 2) Screening and breeding of giant spiny loach.

[0105] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in 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: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 1, wherein the primer pair 1 comprises: a DNA molecule as shown in SEQ ID NO.1, a DNA molecule as shown in SEQ ID NO.2, and a DNA molecule as shown in SEQ ID NO.3; The PCR amplification product contains the corresponding giant spiny loach ( M.armatus ) Reference genome X chromosome CM033555.1 nucleotide sequence 18030271 nt, in the giant spiny loach ( M.armatus ) Referring to 18030271 nt of chromosome X CM033555.1 of the reference genome, individuals with the TT genotype are females, and individuals with the TC genotype are males.

2. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 2, wherein the primer pair 2 is: a DNA molecule as shown in SEQ ID NO.4, a DNA molecule as shown in SEQ ID NO.5, and a DNA molecule as shown in SEQ ID NO.6; The PCR amplification product includes a nucleotide sequence corresponding to the 21146890 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 21146890 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the CC genotype is female, and the individual with the CT genotype is male.

3. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 6, wherein the primer pair 6 is: a DNA molecule as shown in SEQ ID NO.7, a DNA molecule as shown in SEQ ID NO.8, and a DNA molecule as shown in SEQ ID NO.9; The PCR amplification product comprises a nucleotide sequence corresponding to the 23800863 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 23800863 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the GG genotype is female, and the individual with the GT genotype is male.

4. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 7, wherein the primer pair 7 is: a DNA molecule as shown in SEQ ID NO.10, a DNA molecule as shown in SEQ ID NO.11, and a DNA molecule as shown in SEQ ID NO.12; The PCR amplification product includes a nucleotide sequence corresponding to the 23543793 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 23543793 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the CC genotype is female, and the individual with the CA genotype is male.

5. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 9, wherein the primer pair 9 is: a DNA molecule as shown in SEQ ID NO.13, a DNA molecule as shown in SEQ ID NO.14, and a DNA molecule as shown in SEQ ID NO.15; The PCR amplification product includes a nucleotide sequence corresponding to the 22836394th nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 22836394th nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the GG genotype is female, and the individual with the GA genotype is male.

6. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 10, wherein the primer pair 10 is: a DNA molecule as shown in SEQ ID NO.16, a DNA molecule as shown in SEQ ID NO.17, and a DNA molecule as shown in SEQ ID NO.18; The PCR amplification product includes a nucleotide sequence corresponding to the 22786222 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 22786222 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the CC genotype is female, and the individual with the CG genotype is male.

7. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 12, wherein the primer pair 12 is: a DNA molecule as shown in SEQ ID NO.19, a DNA molecule as shown in SEQ ID NO.20, and a DNA molecule as shown in SEQ ID NO.21; The PCR amplification product is a nucleotide sequence comprising the 20845609 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 20845609 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the TT genotype is female, and the individual with the TC genotype is male.

8. A method for detecting the sex of a giant loach, the method comprising: The genomic DNA of the giant spiny loach to be tested is amplified by PCR using primer pair 13, wherein the primer pair 13 is: a DNA molecule as shown in SEQ ID NO.22, a DNA molecule as shown in SEQ ID NO.23, and a DNA molecule as shown in SEQ ID NO.24; The PCR amplification product is a nucleotide sequence comprising the 20814783 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach. At the 20814783 nt of the X chromosome CM033555.1 of the reference genome of the giant spiny loach, the individual with the AA genotype is female, and the individual with the GA genotype is male.

9. A method for screening giant spiny loach, comprising determining the sex of the giant spiny loach according to the detection method according to any one of claims 1 to 8, and screening based on the sex of the giant spiny loach.

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