Portunus trituberculatus growth-related SNP (Single Nucleotide Polymorphism) molecular marker and application thereof in breeding

Abstract

The invention belongs to the field of biological genetic breeding of portunus trituberculatus, and particularly relates to a group of SNP (Single Nucleotide Polymorphism) molecular markers related to growth of portunus trituberculatus and application of the SNP molecular markers in breeding. The SNP molecular marker comprises SNP22419687A > G, SNP16656164A > G, SNP5522418T > G, SNP6078644A > G, SNP6196760C > T and SNP25148584A > G. The SNP molecular marker is stable in amplification and good in genetic typing effect, is obviously associated with the growth traits of the portunus trituberculatus, and can promote the molecular marker of the portunus trituberculatus to assist in selective breeding.

Description

Technical Field

[0001] The invention belongs to the field of biological genetic breeding of swimming crabs, and in particular relates to a group of growth-related SNP molecular markers of swimming crabs and applications thereof in breeding. Background Art

[0002] Since the introduction of selective breeding technology in aquaculture, a large number of aquatic animals have been purposefully bred to obtain desirable economic traits such as rapid growth, disease resistance, and stress resistance. With the advancement of high-throughput sequencing technology, more and more genetic variations, especially single nucleotide polymorphisms (SNPs), have been used for genetic improvement of economic traits, effectively shortening breeding time and costs, and greatly promoting the development of aquaculture. For most aquaculture species, genetic improvement of growth traits has always been the focus of breeding programs. Growth traits are quantitative traits controlled by multiple genes, have moderate heritability, and directly affect aquaculture production. Therefore, identifying SNPs associated with growth traits can provide valuable molecular markers for assisted breeding.

[0003] The three-tuberculate swimming crab (Portunus trituberculatus) is an important aquaculture species, mainly distributed in coastal waters of China, Japan, South Korea and Southeast Asia. Artificial breeding of three-tuberculate swimming crab began in the early 1990s and has become one of the main marine aquaculture species in China. In recent years, intensive farming, environmental degradation, overfishing and frequent diseases have led to slow growth of three-tuberculate swimming crabs, weakened disease resistance, and a gradual decrease in wild resources. Growth traits are important indicators of aquatic animals and directly affect the production and profits of the aquaculture industry. However, growth traits are a complex quantitative trait, which is usually controlled by multiple micro-effect genes. Traditional breeding methods are based on the improvement of families or individuals with excellent traits, resulting in long breeding cycles and low genetic benefits.

[0004] At present, research on the identification of molecular markers related to the growth traits of swimming crabs has been reported, but the number of markers is relatively small and cannot meet the needs of molecular marker-assisted breeding of swimming crabs. Therefore, it is necessary to further use high-throughput sequencing technology to develop high-quality SNP markers and perform association analysis with the growth traits of swimming crabs, in order to screen more growth-related markers at the whole genome level. Summary of the invention

[0005] The purpose of the present invention is to provide a group of growth-related SNP molecular markers of swimming crabs trituberculate and their application in breeding, so as to provide valuable genetic markers for molecular marker-assisted breeding of swimming crabs trituberculate.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A group of growth-related SNP molecular markers of Portunus trituberculatus, including at least one of SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T or SNP25148584A>G;

[0008] in,

[0009] The SNP22419687A>G is located at position 123 of the nucleotide sequence shown in SEQ ID NO.1, and the site is A / G;

[0010] The SNP16656164A>G is located at position 129 of the nucleotide sequence shown in SEQ ID NO.2, and the site is A / G;

[0011] The SNP5522418T>G is located at position 88 of the nucleotide sequence shown in SEQ ID NO.3, and the site is T / G;

[0012] The SNP 6078644A>G is located at position 142 of the nucleotide sequence shown in SEQ ID NO. 4, and the site is A / G;

[0013] The SNP 6196760C>T is located at position 76 of the nucleotide sequence shown in SEQ ID NO.5, and the site is C / T;

[0014] The SNP25148584A>G is located at the 122nd position of the nucleotide sequence shown in SEQ ID NO.6, and the site is A / G.

[0015] Further, the SNP22419687A>G is associated with the carapace length and body weight traits of the swimming crab;

[0016] The SNP16656164A>G is associated with the carapace length, total carapace width, body weight and carapace width traits of the swimming crab;

[0017] The SNP5522418T>G is associated with the carapace length, total carapace width, body height, body weight and carapace width traits of the swimming crab;

[0018] The SNP 6078644A>G is associated with the carapace length, body height and carapace width traits of the swimming crab;

[0019] The SNP6196760C>T and SNP25148584A>G are both associated with the carapace length, body height, body weight and carapace width traits of the swimming crab.

[0020] The present invention also provides primers for the above-mentioned growth-related SNP molecular markers of the swimming crab trituberculate, wherein:

[0021] The primers for SNP22419687A>G are as follows:

[0022] SNP22419687A>GF: sequence as shown in SEQ ID NO.7, SNP22419687A>GR: sequence as shown in SEQ ID NO.8;

[0023] The primers for SNP16656164A>G are as follows:

[0024] SNP16656164A>GF: sequence as shown in SEQ ID NO.9, SNP16656164A>GR: sequence as shown in SEQ ID NO.10;

[0025] The primers for SNP16656164A>G are as follows:

[0026] SNP5522418T>GF: sequence as shown in SEQ ID NO.11, SNP5522418T>GR: sequence as shown in SEQ ID NO.12;

[0027] The primers for SNP6078644A>G are as follows:

[0028] SNP6078644A>GF: sequence as shown in SEQ ID NO.13, SNP6078644A>GR: sequence as shown in SEQ ID NO.14;

[0029] The primers for SNP6196760C>T are as follows:

[0030] SNP6196760C>TF: sequence as shown in SEQ ID NO.15, SNP6196760C>TR: sequence as shown in SEQ ID NO.16;

[0031] The primers for SNP25148584A>G are as follows:

[0032] SNP25148584A>GF: the sequence is shown in SEQ ID NO.17, SNP25148584A>GR: the sequence is shown in SEQ ID NO.18.

[0033] The present invention also provides the use of the above-mentioned Swimming crab growth-related SNP molecular markers or primers of the Swimming crab growth-related SNP molecular markers in the genetic breeding of growth traits of Swimming crabs.

[0034] Furthermore, the application comprises the following steps:

[0035] (1) extracting genomic DNA of the tested swimming crab population;

[0036] (2) using the DNA of step (1) as a template, and using the primers of the growth-related SNP molecular markers of the above-mentioned swimming crab trituberculate to perform multiplex PCR amplification and high-throughput sequencing;

[0037] (3) The genotype data of the multiplex PCR amplification products are analyzed as follows to selectively retain the target individuals:

[0038] When amplifying the sample to be tested, if the primers with SNP22419687A>G can amplify the AA and AG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length and weight traits associated with the AA and AG genotypes;

[0039] When amplifying the sample to be tested, if the primers with SNP16656164A>G can amplify the AG and GG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, total carapace width, body weight and carapace width traits associated with the genotypes AG and GG;

[0040] When amplifying the sample to be tested, if the primers with SNP5522418T>G can amplify the TG and GG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, total carapace width, body height, weight and carapace width traits associated with the genotypes TG and GG;

[0041] When amplifying the sample to be tested, if the primers with SNP6078644A>G can amplify the genotypes AA, AG and GG, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, body height and carapace width traits associated with the genotypes AA, AG and GG;

[0042] When amplifying the sample to be tested, if the primer SNP6196760C>T can amplify the TC and TT genotypes, it indicates that the sample to be tested is a strain of swimming crab with superior carapace length, body height, weight and carapace width traits associated with genotypes TC and TT.

[0043] When amplifying the sample to be tested, if the primer SNP25148584A>G can amplify the AA and AG genotypes, it indicates that the sample to be tested is a strain of swimming crab with superior carapace length, body height, weight and carapace width traits associated with genotypes AA and AG.

[0044] The inventors used the high-quality SNP molecular markers obtained from the simplified genome sequencing of the swimming crab in the laboratory to conduct growth trait association analysis on a cultured population, and further discovered 6 SNP molecular markers related to growth in the genome of the swimming crab. The SNP molecular markers have stable PCR amplification, good genotyping effect, and are significantly associated with the growth traits of the swimming crab, which will promote molecular marker-assisted selection breeding of the swimming crab. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the sequencing scatter plot of the molecular marker SNP6078644A>G of the swimming crab (Pontus trituberculatus). DETAILED DESCRIPTION

[0046] The present invention is described in detail below in conjunction with the examples, so that the technical personnel of the relevant technical field can understand the present invention. It is necessary to point out here that the examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The non-essential improvements and adjustments made to the present invention by the skilled person in the relevant field according to the above invention content should still belong to the scope of protection of the present invention. At the same time, the raw materials mentioned below that are not described in detail are all commercially available products; the process steps or preparation methods that are not mentioned in detail are all process steps or preparation methods known to those skilled in the art.

[0047] Example 1 Obtaining SNP molecular markers related to growth of swimming crabs

[0048] 1. Sample source and measurement of growth trait phenotypic values

[0049] The experimental materials include a total of 244 samples, which are the third-generation marker population (F3C) tracked and investigated by the inventors using SSRs and SNPs. They were collected from the same breeding pond in the Huanghua three-spotted swimming crab farm in September 2022.

[0050] Each sample of swimming crab was weighed and phenotypic traits were measured, including five important growth traits: full carapace width (FCW), carapace width (CW), carapace length (CL), body height (BH) and body weight (BW). The average values ​​were FCW: 144.43±11.58 mm; CW: 65.95±5.67 mm; CL: 67.68±5.61 mm; BH: 34.75±2.84 mm, BW: 152.43±33.36 g.

[0051] Full carapace width (FCW): the straight-line distance between the ninth pair of outermost teeth;

[0052] Carapace width (CW): the distance between the vertical lines of the eighth pair of lateral teeth;

[0053] Carapace length (CL): the straight-line distance between the base and the middle frontal spine;

[0054] Body height (BH): the straight-line distance from the middle of the abdomen to the middle of the carapace;

[0055] Body weight (BW): The wet weight of the whole body. Before weighing, use filter paper to absorb the moisture on the body surface.

[0056] 2. DNA extraction from muscle tissue of swimming crab samples

[0057] The marine animal genome extraction kit (Tiangen, Beijing) was used to extract the muscle tissue DNA of the above 244 individual swimming crabs. The specific operation steps were referred to the kit instructions. The DNA concentration of each individual was measured using a nucleic acid concentration meter Nano Drop 2000, and the quality and integrity of the DNA was detected by 1% agarose gel electrophoresis. The DNA of all individuals was diluted to 50 ng / μl for later use.

[0058] 3. Target region resequencing for SNP genotyping

[0059] DNA samples of 244 swimming crabs were sent to Shanghai Yihe Applied Biotechnology Co., Ltd., and target region resequencing combined with multiplex PCR technology was used to design specific primers for the 6 SNP sites of the DNA samples (as shown in Table 1), and high-throughput sequencing and genotyping were performed. The specific steps include: using the DNA of the sample to be tested as a template, adding the corresponding PCR amplification primer pairs and single-base extension primers to carry out PCR amplification reactions, and after three rounds of PCR amplification, high-throughput sequencing of the amplicons. Bioinformatics methods are used to distinguish different samples from the sequencing results, and finally the mutation information of each site in each sample is obtained. Figure 1 Shown is the sequencing scatter plot of the molecular marker SNP6078644A>G of the swimming crab (P. trituberculatus), which shows that its PCR amplification is stable and the genotyping effect is good.

[0060] Table 1 Primer sequences of SNP molecular markers

[0061]

[0062]

[0063] 4. Association analysis between SNP molecular markers and growth traits

[0064] In the association analysis between SNP molecular markers and growth traits, in order to reduce the existence of false positives, firstly, the Q value corresponding to 244 samples of swimming crabs was calculated using Structure software and used as a covariate; then, the general linear model (GLM) program in TASSEL2.1 software was used to perform association analysis on the phenotypic data of growth traits of swimming crabs and the genotypic data of SNP molecular markers, and the explanation rate of SNP molecular markers for phenotypic variation was calculated; finally, the false discovery rate (FDR) was calculated using the Benjamini-Hochberg method, and FDR correction was performed on the significant association sites with P<0.05. When the FDR value was also less than 0.05, it indicated that the corresponding SNP site was significantly associated with the growth trait.

[0065] Through the above association analysis, 6 SNP molecular markers significantly associated with the growth traits of swimming crabs were finally screened out. The results of the association analysis are shown in Table 2, among which SNP22419687A>G is associated with the carapace length and weight traits of swimming crabs; SNP16656164A>G is associated with the carapace length, total carapace width, weight and carapace width traits of swimming crabs; SNP5522418T>G is associated with the carapace length, total carapace width, body height, weight and carapace width traits of swimming crabs; SNP6078644A>G is associated with the carapace length, body height and carapace width traits of swimming crabs; SNP6196760C>T is associated with the carapace length, body height, weight and carapace width traits of swimming crabs; SNP25148584A>G is associated with the carapace length, body height, weight and carapace width traits of swimming crabs.

[0066] Table 2 Association between SNP markers and growth traits of Swimming crab

[0067]

[0068] 5. Multiple comparisons between genotypes

[0069] SPSS software was used to perform multiple comparisons (LSD) of the phenotypic values ​​of five growth traits between different genotypes for the six SNP markers significantly associated with growth. The results of the multiple comparisons are shown in Table 3.

[0070] Table 3 Multiple comparisons of different genotypes of SNP molecular markers in phenotypic traits of swimming crab (P. trituberculatus)

[0071]

[0072] Note: Different letters indicate significant differences

[0073] From the above data, we can see that the average growth traits corresponding to the TG genotype in SNP5522418T>G are significantly greater than those of the GG genotype (P<0.05). In SNP6078644A>G, the AG genotype corresponds to significantly smaller carapace width (FCW), body height (BH) and body weight (BW) than the AA genotype and the GG genotype (P<0.05), while there is no significant difference between the AA genotype and the GG genotype (P>0.05); the carapace width (CW) and carapace length (CL) corresponding to the AA genotype are significantly greater than those of the AG genotype and the GG genotype (P<0.05), while there is no significant difference between the AG genotype and the GG genotype (P>0.05); Overall, the AA genotype is a dominant genotype. In SNP6196760C>T marker, there was no significant difference between TC genotype and TT genotype in the full carapace width (FCW) trait (P>0.05), but there were significant differences in the other four growth traits (P<0.05), and the average growth trait corresponding to TC genotype was greater than that of TT genotype. In SNP16656164A>G, the average growth trait corresponding to GG genotype was significantly greater than that of AG genotype (P<0.05). In SNP25148584A>G, the average growth trait corresponding to AA genotype was significantly greater than that of AG genotype (P<0.05), while in SNP22419687A>G, the opposite was true (the average growth trait corresponding to AG genotype was significantly greater than that of AA genotype (P<0.05)).

[0074] In summary, the six SNP molecular markers are associated with the five growth traits of swimming crab, namely, carapace width, carapace length, body height and weight, to varying degrees. The six SNP molecular markers can be used as ideal markers for the breeding of swimming crab with carapace length as the breeding target; SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6196760C>T and SNP25148584A>G can be used as ideal markers for the breeding of swimming crab with weight as the breeding target; SNP5522418T>G, SNP6078644A>G >G, SNP6196760C>T and SNP25148584A>G can be used as ideal markers for body height in the breeding of swimming crabs; SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T and SNP25148584A>G can be used as ideal markers for carapace width in the breeding of swimming crabs; SNP16656164A>G and SNP5522418T>G can be used as ideal markers for full carapace width in the breeding of swimming crabs.

[0075] Example 2 Application of SNP molecular markers in genetic breeding of growth traits of swimming crabs

[0076] The six SNP molecular markers of the present invention can be applied to the genetic breeding of growth traits of swimming crabs, and the specific steps are as follows:

[0077] (1) Extracting genomic DNA from the tested population of swimming crabs (same as "2. Extraction of DNA from muscle tissue of swimming crab samples" in Example 1);

[0078] (2) using the DNA described in step (1) as a template, and using primers of growth-related SNP molecular markers of Swimming crab trituberculatus described in Table 1 to perform multiplex PCR amplification and high-throughput sequencing;

[0079] (3) The genotype data of the multiplex PCR amplification products are analyzed as follows to selectively retain the target individuals:

[0080] When amplifying the sample to be tested, if the primers with SNP22419687A>G can amplify the AA and AG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length and weight traits associated with the AA and AG genotypes;

[0081] When amplifying the sample to be tested, if the primers with SNP16656164A>G can amplify the AG and GG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, total carapace width, body weight and carapace width traits associated with the genotypes AG and GG;

[0082] When amplifying the sample to be tested, if the primers with SNP5522418T>G can amplify the TG and GG genotypes, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, total carapace width, body height, weight and carapace width traits associated with the genotypes TG and GG;

[0083] When amplifying the sample to be tested, if the primers with SNP6078644A>G can amplify the genotypes AA, AG and GG, it indicates that the sample to be tested is a strain of the swimming crab with superior carapace length, body height and carapace width traits associated with the genotypes AA, AG and GG;

[0084] When amplifying the sample to be tested, if the primer SNP6196760C>T can amplify the TC and TT genotypes, it indicates that the sample to be tested is a strain of swimming crab with superior carapace length, body height, weight and carapace width traits associated with genotypes TC and TT.

[0085] When amplifying the sample to be tested, if the primer SNP25148584A>G can amplify the AA and AG genotypes, it indicates that the sample to be tested is a strain of swimming crab with superior carapace length, body height, weight and carapace width traits associated with genotypes AA and AG.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A group of growth-related SNP molecular markers of Portunus trituberculatus, characterized in that: The growth-related SNP molecular markers of the swimming crab include at least one of SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T or SNP25148584A>G; in, The SNP22419687A>G is located at position 123 of the nucleotide sequence shown in SEQ ID NO.1, and the site is A / G; The SNP16656164A>G is located at position 129 of the nucleotide sequence shown in SEQ ID NO.2, and the site is A / G; The SNP5522418T>G is located at position 88 of the nucleotide sequence shown in SEQ ID NO.3, and the site is T / G; The SNP 6078644A>G is located at position 142 of the nucleotide sequence shown in SEQ ID NO. 4, and the site is A / G; The SNP 6196760C>T is located at position 76 of the nucleotide sequence shown in SEQ ID NO.5, and the site is C / T; The SNP25148584A>G is located at the 122nd position of the nucleotide sequence shown in SEQ ID NO.6, and the site is A / G.

2. The growth-related SNP molecular marker of Swimming crab (Portunus trituberculatus) according to claim 1, characterized in that: The SNP22419687A>G is associated with the carapace length and body weight traits of the swimming crab; The SNP16656164A>G is associated with the carapace length, total carapace width, body weight and carapace width traits of the swimming crab; The SNP5522418T>G is associated with the carapace length, total carapace width, body height, body weight and carapace width traits of the swimming crab; The SNP 6078644A>G is associated with the carapace length, body height and carapace width traits of the swimming crab; The SNP6196760C>T and SNP25148584A>G are both associated with the carapace length, body height, body weight and carapace width traits of the swimming crab.

3. The primers for the growth-related SNP molecular markers of the swimming crab (P. trituberculatus) according to claim 1, characterized in that: The primers for SNP22419687A>G are as follows: SNP22419687A>GF: sequence as shown in SEQ ID NO.7, SNP22419687A>GR: sequence as shown in SEQ ID NO.8; The primers for SNP16656164A>G are as follows: SNP16656164A>GF: sequence as shown in SEQ ID NO.9, SNP16656164A>GR: sequence as shown in SEQ ID NO.10; The primers for SNP16656164A>G are as follows: SNP5522418T>GF: sequence as shown in SEQ ID NO.11, SNP5522418T>GR: sequence as shown in SEQ ID NO.12; The primers for SNP6078644A>G are as follows: SNP6078644A>GF: sequence as shown in SEQ ID NO.13, SNP6078644A>GR: sequence as shown in SEQ ID NO.14; The primers for SNP6196760C>T are as follows: SNP6196760C>TF: sequence as shown in SEQ ID NO.15, SNP6196760C>TR: sequence as shown in SEQ ID NO.16; The primers for SNP25148584A>G are as follows: SNP25148584A>GF: the sequence is shown in SEQ ID NO.17, SNP25148584A>GR: the sequence is shown in SEQ ID NO.

18.

4. Use of the growth-related SNP molecular marker of the swimming crab of claim 1 in genetic breeding of growth traits of the swimming crab of claim 1.

5. Use of the primers for the growth-related SNP molecular markers of the swimming crab of claim 3 in genetic breeding of growth traits of the swimming crab of claim 3.

Images