SNP (Single Nucleotide Polymorphism) marker related to rapid growth traits of mastacembelus armatus and application thereof

Abstract

The invention belongs to the technical field of biological genetic genes and aquatic organisms, and discloses an SNP (Single Nucleotide Polymorphism) marker related to rapid growth traits of Mastacembelus armatus and application of the SNP marker, the SNP marker is shown as SEQ ID NO: 1, and the base site at the 1305th site in a palmitoyl protein thioesterase 1 gene sequence is C or G. The invention also provides a primer pair and a kit. According to the invention, growth traits of a mastacembelus armatus mixed family group are analyzed through whole genome association analysis, an SNP allele related to rapid growth of mastacembelus armatus is determined, and a method for rapidly, accurately and effectively detecting rapid-growing individuals of mastacembelus armatus by using the SNP marker is established. The method can be used for identifying the mastacembelus armatus individuals with excellent growth traits in the early stage so as to shorten the breeding period of the mastacembelus armatus, and is of great significance to industrial screening and cultivation of mastacembelus armatus varieties which grow quickly.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic genes and aquatic biology, and particularly relates to a SNP marker related to the rapid growth trait of giant spiny loach and an application thereof. Background Art

[0002] Giant loach Mastacembelus armatus ) belongs to the genus Mastacembelidae of the order Symbranchiformes Mastacembelus ), is a characteristic freshwater fish in my country, commonly known as pig saw, knife loach, etc. The big-spined loach is mainly distributed in Guangdong, Guangxi, Fujian, Hainan and other places in my country as well as Southeast Asia. It is rich in nutrition and has delicious meat, and is very popular among consumers. In recent years, the breeding of big-spined loach has increased year by year, and the breeding technology has become more mature, but its growth stability is relatively poor. There are obvious differences in the growth rate of the same batch of big-spined loach, which seriously affects the production and market sales of big-spined loach. Therefore, there is an urgent need to improve the breeding varieties of big-spined loach, increase the growth rate of big-spined loach, and cultivate new fast-growing varieties of big-spined loach.

[0003] Although the aquaculture of giant loach has developed rapidly in recent years, there are still problems such as slow growth rate and large growth differences, which seriously limit the increase in the production of giant loach. The traditional breeding methods for giant loach selection have problems such as low selection rate and long cycle. The traditional method to improve the growth rate of fish is mainly phenotypic selection, that is, selecting larger adult fish as candidate parents for seed production in the next year. The operation is simple and fast, and can improve the growth rate of fish, but there is also an obvious problem, that is, it is subject to greater human interference. Moreover, the growth of fish itself is greatly affected by various factors, and there is a certain blindness in selecting based solely on phenotype.

[0004] In recent years, with the rapid development of high-throughput sequencing technology, the cost of sequencing has continued to decline, and genomic selection breeding has been widely used. At present, genomic selection has been applied in the breeding of many fish species such as large yellow croaker and semi-smooth tongue sole. Genomic selection is mainly based on single nucleotide polymorphism (SNP) marker information to assist fish breeding. SNP markers are the most common type of variation, with a huge number, high genome 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 giant loach was analyzed in 2020. Its genome has 24 chromosomes and a total genome size of about 0.7 Mb. Although the use of molecular markers for assisted breeding of giant loaches is an effective means to solve this problem, how to specifically use SNP markers for assisted breeding of giant loaches has many specific technical difficulties. At present, no relevant research or reports have been seen, and it is difficult to meet the needs of the aquaculture industry. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a SNP marker associated with the rapid growth trait of giant spiny loach and an application thereof, analyze the growth traits of a mixed family population of giant spiny loach through whole genome association analysis, further perform multiple genome resequencing analysis and comprehensive selection, determine a SNP allele associated with the rapid growth of giant spiny loach in palmitoyl protein thioesterase 1 (PPT1), and establish a method for quickly, accurately and effectively using SNP markers to detect the parents of fast-growing individuals of giant spiny loach, which can identify giant spiny loach individuals with excellent growth traits at an early stage, shorten the breeding cycle of giant spiny loach, and is of great significance for the industrial screening and breeding of fast-growing spiny loach varieties.

[0006] To achieve the above object, the present invention provides the following technical solutions: A SNP marker associated with the rapid growth trait of giant spiny loach, wherein the SNP marker is located at the 1305th position in the palmitoyl protein thioesterase 1 (PPT1) gene sequence, as shown in SEQ ID NO:1, and the base position at the 1305th position 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 comprises the primer pair and is used for detecting the SNP marker associated with the fast growth trait of giant spiny loach.

[0009] The SNP marker or primer pair or kit is used in screening or detecting growth-advantage giant spiny loach parents, or in giant spiny loach weight molecular marker-assisted breeding.

[0010] A method for screening a parent of a growth-advantage giant loach, which uses the kit or the primer pair to detect SNP markers related to the fast growth trait of the giant loach, and screens out parent individuals with CC homozygous type, comprising the following steps: S1. Cut the caudal fin ray of the giant loach to be tested and store it in 95% alcohol; S2. extracting genomic DNA from the fin rays of the giant spiny loach to be tested; S3. Using primers SEQ ID NO: 2 and SEQ ID NO: 3, PCR amplification was performed on the genomic DNA of the fin ray of the above-mentioned giant spiny loach to obtain a PCR amplification product; S4. performing Sanger sequencing on the PCR amplification product; S5. Analyze the sequencing results to determine whether the genotype detection of the SNP marker of the giant spiny loach to be tested is a CC homozygous parent individual having the SNP marker.

[0011] The PCR reaction system was 25 μL, including 12.5 μL of 2×Taq MasterMix (Nanjing Novogene Biotechnology Co., Ltd.), 1 μL of upstream and downstream primers, 100 ng of template DNA, and ddH 2 0 to 25 μL system.

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

[0013] The present invention has at least the following beneficial effects: (1) The present invention analyzes the growth traits of a mixed family population of giant spiny loach through whole genome association analysis, and then screens multiple candidate SNP sites associated with individual weight growth traits from the genetic information of related traits through comprehensive selection of genome resequencing analysis results. Then, it is further verified in another giant spiny loach population, and finally a SNP site located in the PPT1 gene sequence is determined to be associated with the rapid growth trait, thereby obtaining a SNP marker associated with the rapid growth of giant spiny loach; the accuracy of the SNP marker is 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 through PCR amplification and sequencing. The operation is simple and convenient, and the results are accurate and reliable. (3) The SNP loci, primers and kit provided by the present invention have broad application prospects in molecular marker-assisted breeding of giant spiny loach. The SNP molecular marker is not affected by factors such as individual age and gender, 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 loci, primer pairs and kits associated with the rapid growth trait of giant spiny loach provided by the present invention can identify male and female giant spiny loaches at the molecular level. By comprehensively utilizing analysis methods such as genome assembly and alignment, they can be applied to large-scale breeding and production of multiple populations of giant spiny loaches to meet the needs of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The Manhattan plot of the body weight trait of the whole genome association analysis of giant spiny loach in the embodiment of the present invention; Figure 2 This is a QQ graph of the body weight trait of the whole genome association analysis of the giant loach in the embodiment of the present invention; Figure 3 This is a schematic diagram showing that SNP markers are significantly correlated with growth traits in an embodiment of the present invention. DETAILED DESCRIPTION

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

[0018] The following is combined with Figure 1-3 The specific implementation case further describes 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. All reagents or materials used in the implementation case of the present invention, unless otherwise specified, are from commercial channels.

[0019] Example 1 The SNP markers and applications thereof associated with the rapid growth trait of giant spiny loach provided in this embodiment are based on giant spiny loach of a 12-month-old mixed family raised in Guangdong Lianyi Aquatic Technology Development Co., Ltd. as research subjects.

[0020] The screening process for SNP markers related to the growth traits of giant spiny loach is as follows: 175 experimental giant loaches were randomly selected, and the growth traits of each fish were measured and recorded, including total length, body length, weight, body height, body width and head length. At the same time, the tail fin of each fish was collected and stored in 95% alcohol and stored at -20°C. Fin ray DNA was extracted using the Marine Animal Tissue DNA Extraction Kit (Tiangen, China). All DNA was detected by 1.2% agarose gel electrophoresis, and the electrophoresis results were judged by a gel imaging system (Bio-Rad, USA) to ensure the integrity of genomic DNA. The concentration of qualified DNA samples was then measured using Qubit 3.0 and uniformly adjusted to 50 ng / μL. The samples were then library constructed according to the standard process and then sequenced using the Illumina platform.

[0021] The obtained raw sequencing data were first subjected to data quality control, and then all sequencing reads were aligned to the reference genome of the giant loach (GCA_019455535.1) using Bowtie v2.0 software, and then SNP detection was performed using samtools software. A total of 13,844,114 high-quality SNP sites were obtained ( Figure 2), which are distributed on all 24 chromosomes. The mixed linear model (MLM) of Tassel software was used for genome-wide association studies (GWAS) analysis. Based on the Bonferroni correction, the threshold of genome-wide significant SNP markers was set to -log10>=6, and SNP markers with extremely significant association with weight traits were obtained ( Figure 1 ), further comprehensive selection and screening resulted in a SNP marker located on chromosome 11 ( Figure 2 ).

[0022] After the above screening, a SNP marker related to the rapid growth trait of giant spiny loach was obtained. The SNP site is located in the coding sequence of the PPT1 gene, specifically at the 1305th position in the palmitoyl protein thioesterase 1 (PPT1) gene sequence, as shown in SEQ ID NO:1. The base position at the 1305th position 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, which comprises the primer pair (including a forward primer and a reverse primer), can be used to detect SNP markers associated with the rapid growth trait of giant spiny loach.

[0025] The SNP markers, primer pairs and kits provided in this embodiment can be used in screening or detecting growth-advantage giant spiny loach parents, or in molecular marker-assisted breeding of giant spiny loach body weight.

[0026] The method for screening the growth-advantage giant loach parents provided in this embodiment is to use the kit or primer pair to detect the SNP markers related to the fast growth trait of the giant loach, and screen out the parent individuals with CC homozygous type, which comprises the following steps: S1. Cut the caudal fin ray of the giant loach to be tested and store it in 95% alcohol; S2. extracting genomic DNA from the fin rays of the giant spiny loach to be tested; S3. Using primers SEQ ID NO: 2 and SEQ ID NO: 3, PCR amplification was performed on the genomic DNA of the above-mentioned giant loach to obtain a PCR amplification product; S4. performing Sanger sequencing on the PCR amplification product; S5. Analyze the sequencing results to determine whether the genotype detection of the SNP marker of the giant spiny loach to be tested is a CC homozygous parent individual having the SNP marker.

[0027] The PCR reaction system was 25 μL, including 12.5 μL of 2×Taq MasterMix, 1 μL of upstream and downstream primers, 100 ng of template DNA, and ddH 2 0 to 25 μL system.

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

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

[0030] The embodiment of the present invention determines a SNP allele in the PPT1 gene sequence that is associated with the rapid growth of giant spiny loach, and establishes a method for quickly, accurately and effectively detecting fast-growing individuals of giant spiny loach using SNP markers, which can identify giant spiny loach individuals with excellent growth traits at an early stage to shorten the breeding cycle of giant spiny loach.

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

[0032] The verification experiment used two populations of giant loach raised in different areas. 500 experimental fish were randomly selected from each population, and the weight trait indicators of each fish were measured and recorded. 80 individuals with extreme phenotypes (the largest 40 and the smallest 40 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 validity of the SNP markers was verified.

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

[0034] The total PCR amplification reaction system was 25 μL, including 12.5 μL of 2× Taq MasterMix (Nanjing Novogene Biotechnology Co., Ltd.), 1 μL of upstream and downstream primers, 100 ng of template DNA, and ddH 2 0 to 25 μL system.

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

[0036] After the PCR reaction was completed, the product was subjected to 1.2% agarose gel electrophoresis. Then it was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing using ABI 3730XL sequencer to detect the SNP genotypes of 80 individuals. Figure 3 The results showed that: in the giant loach, there was a significant difference in body weight between individuals with different genotypes at this SNP site; among them, individuals with CC genotype accounted for a larger proportion in the largest population and a smaller proportion in the smallest population; while individuals with GG genotype accounted for a smaller proportion in the largest population and a larger proportion in the smallest population; after further comprehensive selection, it was determined that the base position 1305 in the palmitoyl protein thioesterase 1 gene sequence was C or G, which was a SNP site significantly associated with the rapid growth trait of the giant loach.

[0037] Then, different groups of giant loaches aged 4-12 months raised by other breeding units were selected, 200-500 loaches were selected from each group, and the groups with the largest weight were selected for SNP site detection. It was found that in the giant loach samples from each group, the SNP site was significantly correlated with the rapid growth trait of giant loach.

[0038] According to the SNP sites and detection and screening methods provided in the above embodiments, the genotype can be determined by a pair of primers respectively, the operation is simple and reliable, and the molecular marker-assisted breeding of giant spiny loach can be carried out industrially.

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

[0040] The above implementation cases are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above implementation cases. The technicians under the implementation cases all belong to the protection scope of the present invention. Ordinary technicians in the relevant technical field can achieve the purpose of the present invention based on the above disclosure of the present invention. Any improvements and modifications made based on the concept of the present invention fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A SNP marker associated with the rapid growth trait of giant spiny loach, characterized in that: 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.

2. A primer pair, characterized in that: It 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'.

3. A kit, characterized in that: It comprises the primer pair according to claim 2.

4. Use of the SNP marker according to claim 1, the primer pair according to claim 2, or the kit according to claim 3 in screening or detecting growth-advantage giant spiny loach parents, or in molecular marker-assisted breeding of giant spiny loach body weight.

5. A method for screening a parent of a growth-advantage giant loach, characterized in that: The kit according to claim 3 or the primer pair according to claim 2 is used to detect the SNP marker associated with the rapid growth trait of the giant spiny loach according to claim 1, and parent individuals with CC homozygous type are screened.

6. The screening method according to claim 5, characterized in that It includes the following steps: S1. Cut the caudal fin ray of the giant loach to be tested and store it in 95% alcohol; S2. extracting genomic DNA from the fin rays of the giant spiny loach to be tested; S3. Using primers SEQ ID NO: 2 and SEQ ID NO: 3, PCR amplification was performed on the genomic DNA of the fin ray of the above-mentioned giant spiny loach to obtain a PCR amplification product; S4. performing Sanger sequencing on the PCR amplification product; S5. Analyze the sequencing results to determine whether the genotype detection of the SNP marker of the giant spiny loach to be tested is a CC homozygous parent individual having the SNP marker of claim 1.

7. The screening method according to claim 6, characterized in that: The PCR reaction system is 25 μL, including 12.5 μL of 2×TaqMasterMix, 1 μL of upstream and downstream primers, 100 ng of template DNA, and ddH2O is added to the 25 μL system.

8. The screening method according to claim 7, characterized in that The PCR reaction procedure is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, for a total of 35 cycles; and final extension at 72°C for 5 min.

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