Molecular markers of snp associated with growth in portunus trituberculatus and their application in breeding

Abstract

The application belongs to the biological genetic breeding field of Portunus trituberculatus, and particularly relates to a set of growth-related SNP molecular markers of Portunus trituberculatus and application thereof in breeding. The SNP molecular markers include SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T and SNP25148584A>G, which are stable in amplification, have good genotyping effect, and are significantly associated with the growth traits of Portunus trituberculatus, and will promote the molecular marker-assisted selection breeding of Portunus trituberculatus.

Description

Technical Field

[0001] This invention belongs to the field of biological genetics and breeding of the swimming crab *Portunus trituberculatus*, specifically involving a set of growth-related SNP molecular markers of *Portunus trituberculatus* and their application in breeding. Background Technology

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

[0003] The three-spined swimming crab (Portunus trituberculatus) is an important aquaculture species, mainly distributed in the coastal waters of China, Japan, South Korea, and Southeast Asia. Artificial breeding of the three-spined swimming crab began in the early 1990s and it has become one of the major marine aquaculture species in China. In recent years, intensive farming, environmental degradation, overfishing, and frequent diseases have led to slow growth, weakened disease resistance, and a gradual decline in wild resources. Growth traits are important indicators for aquatic animals, directly affecting the production and profits of aquaculture. However, growth traits are complex quantitative traits, usually controlled by multiple minor genes. Traditional breeding methods, based on improving families or individuals with superior traits, result in long breeding cycles and low genetic gains.

[0004] Currently, studies on the identification of molecular markers related to growth traits in *Portunus trituberculatus* have been reported, but the number of markers is relatively small and cannot meet the needs of marker-assisted breeding of *Portunus trituberculatus*. Therefore, it is necessary to further utilize high-quality SNP markers developed using high-throughput sequencing technology to conduct association analysis with the growth traits of *Portunus trituberculatus*, in order to screen more growth-related markers at the whole-genome level. Summary of the Invention

[0005] The purpose of this invention is to provide a set of growth-related SNP molecular markers for the swimming crab *Portunus trituberculatus* and their applications in breeding, so as to provide valuable genetic markers for molecular marker-assisted breeding of *Portunus trituberculatus*.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A group of growth-related SNP molecular markers for swimming crabs, 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, at a position of A / G;

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

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

[0012] The SNP6078644A>G is located at position 142 of the nucleotide sequence shown in SEQ ID NO.4, at position A / G;

[0013] The SNP6196760C>T is located at position 76 of the nucleotide sequence shown in SEQ ID NO.5, at a position of C / T;

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

[0015] Furthermore, the SNP22419687A>G is associated with the shell length and weight traits of the swimming crab (Portunus trituberculatus).

[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 SNP6078644A>G is associated with the shell length, body height and shell width traits of the swimming crab.

[0019] Both SNP6196760C>T and SNP25148584A>G are associated with the shell length, body height, weight, and shell width traits of the swimming crab *Portunus trituberculatus*.

[0020] This invention also provides primers for the above-mentioned growth-related SNP molecular markers of the swimming crab *Portunus trituberculatus*, 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: sequence as shown in SEQ ID NO.17; SNP25148584A>GR: sequence as shown in SEQ ID NO.18.

[0033] This invention also provides the application of the above-mentioned growth-related SNP molecular markers or primers of growth-related SNP molecular markers of swimming crab in the genetic breeding of growth traits of swimming crab.

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

[0035] (1) Extract genomic DNA from the population of the three-spined swimming crab to be tested;

[0036] (2) Using the DNA described in step (1) as a template, multiplex PCR amplification and high-throughput sequencing were performed using the primers of the above-mentioned growth-related SNP molecular markers of swimming crab.

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

[0038] If the primers SNP22419687A>G can amplify the AA and AG genotypes when amplifying the sample to be tested, it indicates that the sample to be tested is a swimming crab strain with superior shell length and body weight traits associated with genotypes AA and AG.

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

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

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

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

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

[0044] The inventors used high-quality SNP molecular markers obtained from previous simplified genome sequencing of *Portunus trituberculatus* in the laboratory to conduct a growth trait association analysis on a cultured population, further identifying six growth-related SNP molecular markers in the *Portunus trituberculatus* genome. These SNP molecular markers exhibited stable PCR amplification, good genotyping results, and significant association with growth traits in *Portunus trituberculatus*, which will promote marker-assisted selection breeding of *Portunus trituberculatus*. Attached Figure Description

[0045] Figure 1 Sequencing scatter plot of the molecular marker SNP6078644A>G for the swimming crab. Detailed Implementation

[0046] The present invention will now be described in detail with reference to embodiments to facilitate understanding of the invention by those skilled in the art. It is particularly important to note that the embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Non-essential improvements and adjustments made to the invention by those skilled in the art based on the above description should still fall within the scope of protection of the invention. Furthermore, all raw materials mentioned below, unless otherwise specified, are commercially available products; and all process steps or preparation methods not mentioned in detail are process steps or preparation methods known to those skilled in the art.

[0047] Example 1: Obtaining growth-related SNP molecular markers in the swimming crab *Portunus trituberculatus*

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

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

[0050] Each *Portunus trituberculatus* sample was weighed and its 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; and BW: 152.43 ± 33.36 g.

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

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

[0053] Carapace length (CL): The straight-line distance between the base of the carapace and the medial spinous process of the forehead;

[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 total wet weight. Before weighing, use filter paper to absorb the water on the surface of the body.

[0056] 2. Extraction of DNA from muscle tissue of *Portunus trituberculatus* samples

[0057] DNA was extracted from the muscle tissue of 244 individuals of the swimming crab *Portunus trituberculatus* using a marine animal genome extraction kit (Tiangen, Beijing). Specific procedures were followed according to the kit instructions. The DNA concentration of each individual was determined using a Nano Drop 2000 nucleic acid analyzer, and the quality and integrity of the DNA were assessed by 1% agarose gel electrophoresis. The DNA from all individuals was diluted to 50 ng / μl for later use.

[0058] 3. Target region resequencing for SNP genotyping

[0059] DNA samples from 244 swimming crabs (Portunus trituberculatus) were sent to Shanghai Yihe Applied Biotechnology Co., Ltd. Targeted region resequencing combined with multiplex PCR was employed. Specific primers were designed for six SNP sites in the DNA samples (as shown in Table 1), followed by high-throughput sequencing and genotyping. The specific steps included: using the DNA sample as a template, adding the corresponding PCR amplification primer pairs and single-base extension primers for PCR amplification. After three rounds of PCR amplification, the amplicon was sequenced using high-throughput sequencing. Bioinformatics methods were used to differentiate between different samples based on the sequencing results, ultimately obtaining mutation information for each site in each sample. Figure 1 The scatter plot shown is a sequencing data of the molecular marker SNP6078644A>G in the swimming crab *Portunus trituberculatus*. It can be seen that its PCR amplification is stable and the genotyping effect is good.

[0060] Table 1 Primer sequences for 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, to reduce the presence of false positives, firstly, the Q-values ​​for 244 *Portunus trituberculatus* samples were calculated using Structure software and used as covariates. Then, the General Linear Model (GLM) program in TASSEL 2.1 software was used to perform association analysis between the phenotypic data of growth traits and the genotypic data of SNP molecular markers, and the explanatory power of SNP molecular markers for phenotypic variation was calculated. Finally, the Benjamini-Hochberg method was used to calculate the false discovery rate (FDR), and significant association sites with P < 0.05 were corrected for FDR. 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, six SNP molecular markers that were significantly associated with the growth traits of *Portunus trituberculatus* were finally screened. The association analysis results are shown in Table 2. Among them, SNP22419687A>G was associated with the carapace length and weight of *Portunus trituberculatus*; SNP16656164A>G was associated with the carapace length, total carapace width, weight, and carapace width of *Portunus trituberculatus*; SNP5522418T>G was associated with the carapace length, total carapace width, body height, weight, and carapace width of *Portunus trituberculatus*; SNP6078644A>G was associated with the carapace length, body height, and carapace width of *Portunus trituberculatus*; SNP6196760C>T was associated with the carapace length, body height, weight, and carapace width of *Portunus trituberculatus*; and SNP25148584A>G was associated with the carapace length, body height, weight, and carapace width of *Portunus trituberculatus*.

[0066] Table 2. Association between SNP molecular markers and growth traits of the swimming crab (Portunus trituberculatus).

[0067]

[0068] 5. Multiple comparisons between genotypes

[0069] Multiple comparisons (LSD) of phenotypic values ​​of five growth traits among different genotypes were performed using SPSS software for six SNP markers that were significantly associated with growth. The results of the multiple comparisons are shown in Table 3.

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

[0071]

[0072] Note: Different letters indicate significance.

[0073] The data above show that in SNP5522418T>G, the average growth trait value of the TG genotype was significantly greater than that of the GG genotype (P<0.05). In SNP6078644A>G, the total nail width (FCW), body height (BH), and body weight (BW) of the AG genotype were significantly smaller than those of the AA and GG genotypes (P<0.05), while there was no significant difference between the AA and GG genotypes (P>0.05); the nail width (CW) and nail length (CL) of the AA genotype were significantly greater than those of the AG and GG genotypes (P<0.05), while there was no significant difference between the AG and GG genotypes (P>0.05); overall, the AA genotype is a dominant genotype. In SNP 6196760C>T, the TC and TT genotypes showed no significant difference in total forearm width (FCW) (P>0.05), but significant differences were observed in the other four growth traits (P<0.05). Furthermore, the mean growth trait value for the TC genotype was greater than that for the TT genotype. In SNP 16656164A>G, the mean growth trait value for the GG genotype was significantly greater than that for the AG genotype (P<0.05). In SNP 25148584A>G, the mean growth trait value for the AA genotype was significantly greater than that for the AG genotype (P<0.05), while the opposite was true for SNP 22419687A>G (the mean growth trait value for the AG genotype was significantly greater than that for the AA genotype (P<0.05)).

[0074] In summary, the six SNP molecular markers were associated with five growth traits of *Portunus trituberculatus*: carapace width, carapace length, body height, and body weight. All six SNP molecular markers can serve as ideal markers for selecting superior *Portunus trituberculatus* breeds with carapace length as the selection target. SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6196760C>T, and SNP25148584A>G can serve as ideal markers for selecting superior *Portunus trituberculatus* breeds with body weight as the selection target. SNP5522418T>G and SNP6078644A>G are also suitable for selecting superior *Portunus trituberculatus* breeds with body weight as the selection target. >G, SNP6196760C>T, and SNP25148584A>G can be used as ideal markers for breeding high-quality Swimming Crab with body height as the breeding target; SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T, and SNP25148584A>G can be used as ideal markers for breeding high-quality Swimming Crab with carapace width as the breeding target; SNP16656164A>G and SNP5522418T>G can be used as ideal markers for breeding high-quality Swimming Crab with total carapace width as the breeding target.

[0075] Example 2: Application of SNP molecular markers in the genetic breeding of growth traits in the swimming crab (Portunus trituberculatus).

[0076] The six SNP molecular markers described in this invention can be applied to the genetic breeding of growth traits in the swimming crab *Portunus trituberculatus*. The specific steps are as follows:

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

[0078] (2) Using the DNA described in step (1) as a template, multiplex PCR amplification and high-throughput sequencing were performed using the primers for the growth-related SNP molecular markers of the swimming crab described in Table 1.

[0079] (3) The genotype data of the multiplex PCR amplification products were analyzed as follows, and the target individuals were selectively retained:

[0080] If the primers SNP22419687A>G can amplify the AA and AG genotypes when amplifying the sample to be tested, it indicates that the sample to be tested is a swimming crab strain with superior shell length and body weight traits associated with genotypes AA and AG.

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

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

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

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

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

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of growth-related SNP molecular markers in the genetic breeding of growth traits in *Portunus trituberculatus*, wherein the growth-related SNP molecular markers include at least one of SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T, or SNP25148584A>G; in, The SNP22419687A>G is associated with the shell length and weight traits of the swimming crab, and it is located at position 123 of the nucleotide sequence shown in SEQ ID NO.1, at site A / G; The SNP16656164A>G is associated with the shell length, total shell width, body weight, and shell width traits of the swimming crab, and it is located at position 129 of the nucleotide sequence shown in SEQ ID NO.2, at site A / G; The SNP5522418T>G is associated with the shell length, total shell width, body height, body weight, and shell width traits of the swimming crab, and it is located at position 88 of the nucleotide sequence shown in SEQ ID NO.3, at the site T / G; The SNP6078644A>G is associated with the shell length, body height and shell width traits of the swimming crab, and it is located at position 142 of the nucleotide sequence shown in SEQ ID NO.4, at site A / G; Both SNP6196760C>T and SNP25148584A>G are associated with the shell length, body height, weight, and shell width traits of the swimming crab *Portunus trituberculatus*. SNP6196760C>T is located at position 76 of the nucleotide sequence shown in SEQ ID NO. 5, at a C / T site; SNP25148584A>G is located at position 122 of the nucleotide sequence shown in SEQ ID NO. 6, at an A / G site.

2. Application of primers for growth-related SNP molecular markers in the genetic breeding of growth traits in *Portunus trituberculatus*, wherein the growth-related SNP molecular markers include at least one of SNP22419687A>G, SNP16656164A>G, SNP5522418T>G, SNP6078644A>G, SNP6196760C>T, or SNP25148584A>G; in, The SNP22419687A>G is associated with the shell length and weight traits of the swimming crab, and it is located at position 123 of the nucleotide sequence shown in SEQ ID NO.1, at site A / G; The SNP16656164A>G is associated with the shell length, total shell width, body weight, and shell width traits of the swimming crab, and it is located at position 129 of the nucleotide sequence shown in SEQ ID NO.2, at site A / G; The SNP5522418T>G is associated with the shell length, total shell width, body height, body weight, and shell width traits of the swimming crab, and it is located at position 88 of the nucleotide sequence shown in SEQ ID NO.3, at the site T / G; The SNP6078644A>G is associated with the shell length, body height and shell width traits of the swimming crab, and it is located at position 142 of the nucleotide sequence shown in SEQ ID NO.4, at site A / G; Both SNP6196760C>T and SNP25148584A>G are associated with the shell length, body height, weight, and shell width traits of the swimming crab *Portunus trituberculatus*. SNP6196760C>T is located at position 76 of the nucleotide sequence shown in SEQ ID NO. 5, at a C / T site; SNP25148584A>G is located at position 122 of the nucleotide sequence shown in SEQ ID NO. 6, at an A / G site. 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 SNP5522418T>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: sequence as shown in SEQ ID NO.17; SNP25148584A>GR: sequence as shown in SEQ ID NO.18.

Images