A SNP marker related to fast growth trait of mastacembelus giganteus and application thereof

Abstract

The application belongs to the field of biological genetic genes and aquatic biological technology, discloses a SNP marker related to the rapid growth character of Mastacembelus cone and application thereof, the SNP marker is shown as SEQ ID NO:1, and the base position of 1305 in the palmitoyl protein thioesterase 1 gene sequence is C or G. The application further provides a primer pair and a kit. The application analyzes the growth character of a mixed family population of Mastacembelus cone through whole genome association analysis, determines one SNP allele related to the rapid growth of Mastacembelus cone, and establishes a method for rapidly, accurately and effectively detecting fast-growing individuals of Mastacembelus cone by using the SNP marker, which can identify individuals of Mastacembelus cone with excellent growth character in an early stage, so as to shorten the breeding cycle of Mastacembelus cone, and has important significance for industrial screening and breeding of fast-growing Mastacembelus cone varieties.

Description

Technical Field

[0001] This invention belongs to the field of biological genetics and aquatic organism technology, specifically relating to a SNP marker related to the rapid growth trait of the giant spiny loach and its application. Background Technology

[0002] Giant spiny loach ( Mastacembelus armatus It belongs to the order Symbranchiformes, family Mastacembelidae, genus Mastacembelidae. Mastacembelus The spiny loach (Scopterocarpus spp.) is a unique freshwater fish species in my country, commonly known as the "pig's saw" or "knife loach." It is mainly distributed in Guangdong, Guangxi, Fujian, and Hainan provinces in my country, as well as Southeast Asia. Rich in nutrients and with delicious meat, it is very popular among consumers. In recent years, the farming of spiny loach has increased annually, and farming techniques have become increasingly mature. However, its growth stability is poor. Significant differences in growth rate exist within the same batch of spiny loach, seriously affecting its yield and market sales. Therefore, there is an urgent need to improve the farmed spiny loach species, increase its growth rate, and cultivate new, fast-growing varieties.

[0003] Despite the rapid development of spiny loach aquaculture in recent years, problems such as slow growth rate and significant growth variability still exist, severely limiting the increase in spiny loach production. Traditional breeding methods for spiny loach suffer from low selection rates and long cycles. Traditional methods for improving fish growth rate mainly rely on phenotypic selection, that is, selecting larger adult fish as candidate broodstock for the following year's fry production. This method is simple and quick, and can effectively improve fish growth rate, but it also has significant drawbacks, namely, it is highly susceptible to human interference. Furthermore, fish growth is greatly influenced by various factors, and relying solely on phenotypic selection carries a degree of uncertainty.

[0004] In recent years, with the rapid development of high-throughput sequencing technology and the continuous decline in sequencing costs, genome selection breeding has been widely applied. Currently, genome selection has been used in the breeding of various fish species, including large yellow croaker and tongue sole. Genome selection is mainly based on single nucleotide polymorphism (SNP) marker information to assist in fish breeding. SNP markers are the most common type of variation, with a huge number, high genome-wide coverage, high diversity, and strong genetic stability. In recent years, SNP markers have been widely used in the analysis of important animal traits and genetic breeding. The genome of the large spiny loach was analyzed in 2020; it has 24 chromosomes and a total genome size of approximately 0.7 Mb. Although using molecular markers for assisted breeding of the large spiny loach is an effective means to solve this problem, the specific application of SNP markers in assisted breeding of the large spiny loach faces various technical difficulties, and no relevant research or reports have been found yet, making it difficult to meet the needs of the aquaculture industry. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a SNP marker associated with the rapid growth trait of the spiny loach and its application. Through genome-wide association analysis, the growth traits of mixed families of spiny loach were analyzed. Further multiplex genome resequencing and comprehensive selection identified a single SNP allele in palmitoyl protein thioesterase 1 (PPT1) associated with rapid growth in spiny loach. A rapid, accurate, and effective method for detecting fast-growing parents of spiny loach using SNP markers was established. This method enables the early identification of spiny loach individuals with superior growth traits, shortening the breeding cycle and holding significant importance for industrial screening and breeding of fast-growing spiny loach varieties.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A SNP marker associated with the rapid growth trait of the giant spiny loach, the SNP marker being located at position 1305 in the palmitoyl protein thioesterase 1 (PPT1) gene sequence, as shown in SEQ ID NO:1, wherein the base position at position 1305 shown in SEQ ID NO:1 is C or G.

[0007] A primer pair comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:2 and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3. Forward primer SEQ ID NO:2: 5'-GAGAAAGGAAATGACATAAGACTGC-3'; Reverse primer SEQ ID NO:3: 5'-CCCGAGAATGGTTCAACAAA-3'.

[0008] A kit comprising the primer pair for detecting the SNP markers associated with the rapid growth trait of the giant spiny loach.

[0009] The application of the aforementioned SNP markers, primer pairs, or kits in the screening or detection of parental growth superiority of the spiny loach, or in the application of molecular marker-assisted breeding of the spiny loach.

[0010] A method for screening parent individuals of the growth-advantageous giant spiny loach, comprising using the aforementioned kit or primer pair to detect SNP markers associated with the rapid growth trait of the giant spiny loach, and screening for parent individuals with CC homozygous phenotype, including the following steps: S1. Cut off the tail fin rays of the spiny loach to be tested and preserve them in 95% alcohol; S2. Extract genomic DNA from the fin rays of the spiny loach to be tested; S3. The genomic DNA of the above-mentioned spiny loach fin rays was amplified by PCR using primer pairs SEQ ID NO:2 and SEQ ID NO:3 to obtain PCR amplification products; S4. Perform Sanger sequencing on the PCR amplification products; S5. Analyze the sequencing results to determine whether the SNP marker of the spiny loach to be tested is a CC homozygous parent individual with the SNP marker.

[0011] The PCR reaction system consisted of 25 μL, including 12.5 μL of 2×Taq MasterMix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of upstream and downstream primers, 100 ng of template DNA, and ddH2O added to a final volume of 25 μL.

[0012] The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for a total of 35 cycles; and finally extension at 72℃ for 5 min.

[0013] The present invention has at least the following beneficial effects: (1) This invention analyzes the growth traits of mixed families of large spiny loach through genome-wide association analysis, and then screens multiple candidate SNP loci associated with individual weight growth traits from the genetic information of related traits by comprehensively selecting the results of genome resequencing analysis. Then, it further verifies in another large spiny loach population, and finally determines that one SNP locus located in the PPT1 gene sequence is associated with the rapid growth trait, and obtains the SNP marker associated with the rapid growth of large spiny loach. The accuracy of the SNP marker has been verified in subsequent industrial actual testing and breeding processes.

[0014] (2) The SNP markers related to the rapid growth trait of the giant spiny loach provided by the present invention can be used to determine the superior growth individuals of the giant spiny loach by PCR amplification and sequencing. The operation is simple and convenient and the results are accurate and reliable. (3) The SNP sites, primers and kits provided by this invention have broad application prospects in marker-assisted breeding of giant spiny loach. The SNP molecular markers are not affected by factors such as individual age and sex, and can be used for early screening of giant spiny loach, which can significantly shorten the breeding time and maximize the breeding benefits.

[0015] (4) The SNP sites, primer pairs and kits related to the rapid growth trait of giant spiny loach provided by the present invention can identify male and female giant spiny loach at the molecular level. By comprehensively utilizing analysis methods such as genome assembly and comparison, they can be applied to the large-scale breeding and production of giant spiny loach in multiple populations, thus meeting the needs of industrial production. Attached Figure Description

[0016] Figure 1 Manhattan plot of body weight trait in genome-wide association analysis of *Gnaphalium affine*, an embodiment of the present invention; Figure 2 This is a QQ plot of the body weight trait in genome-wide association analysis of *Scoideus spp.*, as described in an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the significant correlation between SNP markers and growth traits in an embodiment of the present invention. Detailed Implementation

[0017] To illustrate the present invention in more detail, the following embodiments are provided. It should be emphasized that the following embodiments are for illustrative purposes only and are not intended to limit the scope or content of the present invention.

[0018] The following is in conjunction with the appendix Figure 1-3 Specific implementation examples further illustrate the technical solution of the present invention in detail, so that those skilled in the art can fully understand the technical solution of the present invention. Unless otherwise specified, all reagents or materials used in the implementation examples of the present invention are derived from commercial sources.

[0019] Example 1 The SNP markers related to the rapid growth trait of the giant spiny loach provided in this embodiment and their application are based on 12-month-old mixed families of giant spiny loach raised at Guangdong Lianyi Aquatic Technology Development Co., Ltd.

[0020] The screening process for SNP markers associated with growth traits of the giant spiny loach is as follows: 175 experimental spiny loaches were randomly selected, and growth traits of each fish, including total length, body length, weight, body height, body width, and head length, were measured and recorded. Simultaneously, caudal fins of each fish were collected, preserved in 95% ethanol, and stored at -20°C. Fin DNA was extracted using a marine animal tissue DNA extraction kit (Tiangen, China). All DNA samples were analyzed by 1.2% agarose gel electrophoresis, and the results were verified using a gel imaging system (Bio-Rad, USA) to ensure genomic DNA integrity. Qualified DNA samples were then concentrated using a Qubit 3.0 spectrometer and uniformly adjusted to 50 ng / μL. Samples were then used for library construction according to standard procedures and sequenced using the Illumina platform.

[0021] The raw sequencing data were first subjected to data quality control. Then, Bowtie v2.0 software was used to align all sequencing reads to the *Gastropoda spp.* reference genome (GCA_019455535.1), followed by SNP detection using samtools software. A total of 13,844,114 high-quality SNP loci were obtained. Figure 2The SNPs are distributed on all 24 chromosomes. Genome-wide association studies (GWAS) were performed using a mixed linear model (MLM) in Tassel software. Based on Bonferroni correction, the threshold for genome-wide significant SNP markers was set to -log10>=6, yielding SNP markers that were highly significantly associated with the body weight trait. Figure 1 Further comprehensive selection and screening yielded one SNP marker located on chromosome 11. Figure 2 ).

[0022] After the above screening, an SNP marker related to the rapid growth trait of the giant spiny loach was obtained. The SNP site is located in the coding sequence of the PPT1 gene, specifically at position 1305 of the palmitoyl protein thioesterase 1 (PPT1) gene sequence, as shown in SEQ ID NO:1. The base position at position 1305 shown in SEQ ID NO:1 is C or G.

[0023] The primer pair provided in this embodiment includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3. Forward primer SEQ ID NO:2: 5'-GAGAAAGGAAATGACATAAGACTGC-3'; Reverse primer SEQ ID NO:3: 5'-CCCGAGAATGGTTCAACAAA-3'.

[0024] The kit provided in this embodiment contains the primer pairs (including forward and reverse primers) and can be used to detect SNP markers associated with the rapid growth trait of the giant spiny loach.

[0025] The SNP markers, primer pairs, and kits provided in this embodiment can all be used in the screening or detection of dominant spiny loach parents, or in the molecular marker-assisted breeding of spiny loach.

[0026] The method for screening parent individuals of the growth-advantageous giant spiny loach provided in this embodiment involves using the aforementioned kit or primer pair to detect SNP markers associated with the rapid growth trait of the giant spiny loach, and screening out parent individuals with CC homozygous phenotypes. The method includes the following steps: S1. Cut off the tail fin rays of the spiny loach to be tested and preserve them in 95% alcohol; S2. Extract genomic DNA from the fin rays of the spiny loach to be tested; S3. The above-mentioned genomic DNA of the giant spiny loach was amplified by PCR using primer pairs SEQ ID NO:2 and SEQ ID NO:3 to obtain the PCR amplification product; S4. Perform Sanger sequencing on the PCR amplification products; S5. Analyze the sequencing results to determine whether the SNP marker of the spiny loach to be tested is a CC homozygous parent individual with the SNP marker.

[0027] The PCR reaction system is 25 μL, including 12.5 μL of 2×Taq MasterMix, 1 μL each of upstream and downstream primers, 100 ng of template DNA, and ddH2O added to a final volume of 25 μL.

[0028] The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for a total of 35 cycles; and finally extension at 72℃ for 5 min.

[0029] In this embodiment, palmitoyl protein thioesterase 1 is derived from the fin rays of the giant spiny loach. This palmitoyl protein thioesterase 1 is present in the giant spiny loach's body for a long time and is one of the components of the giant spiny loach organism. Therefore, the gene sequence of palmitoyl protein thioesterase 1 is also present in the resolved giant spiny loach genome; and the SNP marker provided by this invention is located within the gene sequence of palmitoyl protein thioesterase 1.

[0030] This invention identifies a single SNP allele in the PPT1 gene sequence that is associated with rapid growth in the spiny loach, and establishes a rapid, accurate, and effective method for detecting fast-growing individuals of the spiny loach using SNP markers. This method can identify individuals of the spiny loach with excellent growth traits at an early stage, thereby shortening the breeding cycle of the spiny loach.

[0031] Example 2 The SNP markers and their applications related to the rapid growth trait of the giant spiny loach provided in this embodiment are based on Example 1, and further demonstrate and apply the SNP molecular markers related to the growth trait of the giant spiny loach.

[0032] The validation experiment used two populations of spiny loach raised in different regions. 500 experimental fish were randomly selected from each population, and the weight trait of each fish was measured and recorded. 80 individuals with extreme phenotypes (the 40 largest and 40 smallest individuals in the population) were selected as experimental samples. At the same time, tail fin samples of each fish were collected and preserved for DNA extraction, and then the effectiveness of the SNP markers was verified.

[0033] First, Samtools was used to obtain 500bp sequences upstream and downstream of the SNP marker. Then, Primer 5 was used to design amplification primer pairs, as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0034] The total PCR amplification reaction volume was 25 μL, including 12.5 μL of 2×Taq MasterMix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of upstream and downstream primers, 100 ng of template DNA, and ddH2O added to a total volume of 25 μL.

[0035] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; then 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 35 cycles; and finally 72℃ final extension for 5 min.

[0036] After the PCR reaction, the products were subjected to 1.2% agarose gel electrophoresis. They were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing using an ABI 3730XL sequencer to detect the SNP genotypes of 80 individuals. Figure 3 The results showed that there were significant differences in body weight among individuals with different genotypes at this SNP locus in the giant spiny loach. Among them, the CC genotype was more common in the large population and less common in the small population, while the GG genotype was less common in the large population and more common in the small population. After further comprehensive selection, the base position at position 1305 in the palmitoyl protein thioesterase 1 gene sequence was determined to be either C or G, which is a significantly associated SNP locus with the rapid growth trait in the giant spiny loach.

[0037] Then, different populations of large spiny loach aged 4-12 months were selected from other aquaculture units. 200-500 loaches were selected from each population, and the largest populations were selected for SNP locus detection. It was found that the SNP locus in the large spiny loach samples from each population was significantly associated with the rapid growth trait of large spiny loach.

[0038] Based on the SNP sites and detection and screening methods provided in the above embodiments, their genotypes can be determined by a pair of primers. The operation is simple and reliable, and molecular marker-assisted breeding of giant spiny loach can be carried out on an industrial scale.

[0039] Small-scale practical applications under confidential conditions have shown that the SNP markers related to the rapid growth trait of giant spiny loach provided in the above embodiments of the present invention can identify giant spiny loach with rapid growth traits at the molecular level. They can be widely used to identify the genetic sex of giant spiny loach covering various wild and farmed populations in the Pearl River Basin. The primer pair can simultaneously and rapidly and accurately identify the rapid growth trait of giant spiny loach, which can meet the needs of large-scale breeding and industrial production of multiple populations of giant spiny loach distributed in different regions. Moreover, it is low in cost and easy to implement.

[0040] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art are all within the scope of protection of the present invention. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection is determined by the claims.

Claims

1. The application of primer pairs or kits in the screening or detection of parental species of *Scourgette maxima* with superior body weight growth, or in marker-assisted breeding of *Scourgette maxima* with body weight, wherein the primer pairs are used to detect SNP markers associated with body weight traits in *Scourgette maxima*; wherein the SNP marker is located at position 1305 in the palmitoyl protein thioesterase 1 (PPT1) gene sequence, as shown in SEQ ID NO:1, and the base position at position 1305 shown in SEQ ID NO:1 is C or G; The primer pair includes a forward primer and a reverse primer, the nucleotide sequence of which is shown in SEQ ID NO:2 and the nucleotide sequence of which is shown in SEQ ID NO:

3. Forward primer SEQ ID NO:2: 5'-GAGAAAGGAAATGACATAAGACTGC-3'; Reverse primer SEQ ID NO:3: 5'-CCCGAGAATGGTTCAACAAA-3'; The kit includes the primer pair.

2. A method for screening parent species of the giant spiny loach with superior body weight growth, characterized in that, Using the kit or primer pair described in claim 1, the SNP markers associated with the weight growth trait of *Scoidea spp.* as described in claim 1 were detected, and parental individuals with CC homozygous phenotypes were screened out.

3. The screening method according to claim 2, characterized in that, It includes the following steps: S1. Cut off the tail fin rays of the spiny loach to be tested and preserve them in 95% alcohol; S2. Extract genomic DNA from the fin rays of the spiny loach to be tested; S3. The genomic DNA of the above-mentioned spiny loach fin rays was amplified by PCR using primer pairs SEQ ID NO:2 and SEQ ID NO:3 to obtain PCR amplification products; S4. Perform Sanger sequencing on the PCR amplification products; S5. Analyze the sequencing results to determine whether the SNP marker of the target giant spiny loach is a CC homozygous parent individual with the SNP marker described in claim 2.

4. The screening method according to claim 3, characterized in that, The PCR reaction system is 25 μL, including 12.5 μL of 2×TaqMasterMix, 1 μL each of upstream and downstream primers, 100 ng of template DNA, and ddH2O added to a final volume of 25 μL.

5. The screening method according to claim 4, characterized in that, The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 55℃ for 15 s, extension at 72℃ for 30 s, for a total of 35 cycles; and finally extension at 72℃ for 5 min.

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