A SNP molecular marker combination related to the body weight of pufferfish hippocampus, a liquid chip and application thereof

Abstract

This invention relates to the field of aquatic breeding technology, and in particular to a combination of SNP molecular markers related to the body weight of the bloated seahorse, a liquid-phase chip, and its applications. The SNP molecular marker combination provided by this invention is obtained by sequencing, identifying, and screening bloated seahorse samples, and possesses advantages such as high minimum allele frequency (MAF), high detection rate, and high genotyping accuracy. These characteristics ensure extremely high reliability and universality in testing bloated seahorse populations from different batches and sources. Using the SNP molecular marker combination provided by this invention, the body weight of bloated seahorses can be estimated, enabling early, non-destructive determination of breeding values ​​for body weight genomic estimation, shortening the generation interval in breeding, and significantly accelerating the genetic improvement process of seahorse growth traits.

Description

Technical Field

[0001] This invention relates to the field of aquatic breeding technology, and in particular to a combination of SNP molecular markers, a liquid-phase chip, and applications related to the body weight of the bloated seahorse. Background Technology

[0002] The abdominal seahorse (Hippocampus abdominalis) is one of the largest, fastest-growing, and most disease-resistant species in the seahorse genus, possessing extremely high medicinal and ornamental value. In the intensive farming of abdominal seahorses, growth traits (especially weight and body length) are the most critical economic indicators determining the farming cycle, feed conversion rate, and final market price. Developing superior breeds with fast growth rates and large individual sizes is of significant industrial importance for improving farming efficiency and shortening the farming cycle.

[0003] Currently, the breeding of superior bloated seahorses mainly relies on traditional phenotypic selection, which involves visually observing or measuring the weight and body length of individuals to select parent stock. However, this traditional breeding model has significant limitations: 1) Highly influenced by the environment: Quantitative traits (such as weight and body length) are significantly affected by environmental factors (such as water temperature, feed, and stocking density). Relying solely on phenotype often fails to accurately reflect an individual's genetic potential, resulting in low breeding accuracy. 2) Long breeding cycle: Phenotypic testing and breeding can only be carried out after the seahorses reach sexual maturity or commercial size, making it impossible to predict in the early stages and lengthening the generation interval. 3) Slow genetic progress: Due to the difficulty in accurately capturing the genetic effects of small-scale polygenic genes, the accumulation of genetic gain for superior traits is slow, making it difficult to achieve breakthrough improvements in germplasm resources in the short term.

[0004] With the development of molecular biology techniques, genomic selection (GS) technology based on whole-genome information has become a revolutionary tool in the field of plant and animal breeding. Single nucleotide polymorphism (SNP) markers, due to their wide distribution in the genome, high stability, and ease of high-throughput detection, have become the preferred choice for marker-assisted breeding. By mining SNP molecular markers that are closely linked or significantly associated with growth traits, and combining them with genomic breeding value estimation methods, it is possible to: 1) Early selection: The growth potential of an individual can be predicted during the seedling stage, without waiting until adulthood; 2) Improved accuracy: Selection is carried out directly using genotype information, eliminating environmental interference and greatly improving the accuracy of breeding; 3) Accelerated breeding process: The generation interval is significantly shortened, and the fixation of superior traits is accelerated.

[0005] Although genomic selection technology has been applied in pigs, cattle, chickens, and some major economic fish species, there is currently a lack of a validated SNP molecular marker combination that covers the entire genome and is highly specific to growth traits in the breeding of the bloated seahorse. Therefore, in order to overcome the bottlenecks of traditional breeding, it is urgent to develop a comprehensive, effective, and highly targeted SNP molecular marker combination for growth traits in the bloated seahorse. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a combination of SNP molecular markers related to the body weight of the bloated hippocampus, a liquid-phase chip, and its applications. The SNP molecular marker combination provided by this invention can significantly accelerate the genetic progression of growth traits in the bloated hippocampus, offering the industry a precise and efficient molecular breeding tool.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a combination of SNP molecular markers associated with the body weight of the bloated hippocampus, the combination of SNP molecular markers comprising 11,000 SNP molecular markers; the location information of the SNP molecular markers on the bloated hippocampus reference genome is shown in Table 1 of the specification; the version number of the bloated hippocampus reference genome in the NCBI database is GCA_018466805.1.

[0008] Table 1 SNP molecular marker location information Note: In the position column, the part before the colon is the chromosome, and the part after the colon is the location of the SNP molecular marker on that chromosome.

[0009] Preferably, the SNP molecular marker combination comprises 11,000 nucleic acid molecules containing the SNP molecular marker and its upstream and downstream sequences; the nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO.1-SEQ ID NO.11,000.

[0010] This invention provides a liquid-phase chip for breeding bloated seahorses, wherein the genotyping sites of the liquid-phase chip include SNP molecular markers in the SNP molecular marker combination described in the above technical solution.

[0011] Preferably, the liquid-phase chip includes a probe array for detecting SNP molecular marker combinations as described in the above technical solutions.

[0012] This invention provides the application of the SNP molecular marker combination described in the above technical solution or the liquid phase chip described in the above technical solution in targeted capture sequencing of the distended hippocampus.

[0013] This invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above-mentioned technical solutions in the analysis of body weight traits in the abdominal distended hippocampus.

[0014] This invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above technical solution in the breeding of bloated hippocampus, and the breeding traits include body weight.

[0015] This invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above technical solution in calculating the genomic estimation breeding value of the body weight of the bloated hippocampus.

[0016] Preferably, the calculation method includes calculation using the GBLUP model.

[0017] Preferably, the software used for the calculation includes r-Asreml.

[0018] Beneficial effects: The SNP molecular marker combination provided by this invention is a combination of SNP molecular markers related to the body weight of bloated hippocampi, obtained through sequencing, identification, and screening of bloated hippocampus samples. It boasts advantages such as high minimum allele frequency (MAF), high detection rate, and high genotyping accuracy. These characteristics ensure extremely high reliability and universality in testing bloated hippocampus populations from different batches and sources. Using the SNP molecular marker combination provided by this invention, the body weight of bloated hippocampi can be estimated, enabling early, non-destructive determination of breeding values ​​for body weight through genomic estimation, shortening the breeding generation interval, and significantly accelerating the genetic improvement process of hippocampal growth traits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 The distribution of high-quality SNP sites in the bloated hippocampus genome (120K window size); the right-hand caption shows the density of SNP sites. Figure 2 This is a distribution map of the secondary allele frequencies of the SNP sites in the chip described in this invention; Figure 3 This is a graph showing the overall situation of variant detection; each point represents a sample. Figure 4 The results show the comparison of average body weight between different genders and the GEBV group. Detailed Implementation

[0021] This invention provides a combination of SNP molecular markers associated with the body weight of the bloated hippocampus, the combination of SNP molecular markers comprising 11,000 SNP molecular markers; the location information of the SNP molecular markers on the bloated hippocampus reference genome is shown in Table 1 of the specification; the version number of the bloated hippocampus reference genome in the NCBI database is GCA_018466805.1.

[0022] In one embodiment, the SNP molecular marker combination includes 11,000 nucleic acid molecules containing the SNP molecular marker and its upstream and downstream sequences; the nucleotide sequences of the nucleic acid molecules are shown as SEQ ID NO.1-SEQ ID NO.11,000.

[0023] The SNP molecular marker combination provided by this invention is a combination of SNP molecular markers related to the weight of bloated hippocampi obtained through sequencing, identification, and screening of bloated hippocampus samples. It has advantages such as high minimum allele frequency (MAF), high detection rate, and high genotyping accuracy. The liquid-phase chip prepared using the aforementioned SNP molecular marker combination achieves a 91.9% capture efficiency for genome-wide SNPs, while reducing detection costs by two times compared to whole-genome resequencing. Based on the genotypes of different SNP molecular markers, the genome-estimated breeding value (GEBV) can be calculated to assess the weight of the tested bloated hippocampi. Experimental results show that, in both female and male populations, individuals in the high GEBV group have significantly higher weights than those in the low GEBV group.

[0024] Based on the above advantages, this invention provides a liquid-phase chip for breeding bloated seahorses, wherein the genotyping sites of the liquid-phase chip include the SNP molecular markers in the SNP molecular marker combination described in the above technical solution. This invention does not impose special requirements on the preparation method of the liquid-phase chip; methods well known to those skilled in the art can be used.

[0025] In one embodiment, the liquid-phase chip includes a probe array for detecting SNP molecular marker combinations as described in the above technical solutions.

[0026] Based on the above advantages, the present invention provides the application of the SNP molecular marker combination described in the above technical solution or the liquid phase chip described in the above technical solution in targeted capture sequencing of the distended hippocampus.

[0027] Based on the above advantages, the present invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above technical solution in the analysis of body weight traits in the bloated hippocampus.

[0028] Based on the above advantages, the present invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above technical solution in the breeding of bloated hippocampus, and the breeding traits include body weight.

[0029] This invention provides the application of the SNP molecular marker combination or the liquid phase chip described in the above technical solution in calculating the genomic estimation breeding value of the body weight of the bloated hippocampus.

[0030] As one implementation method, the calculation includes calculation using the GBLUP model. This invention does not have specific requirements for the GBLUP model method; methods well-known to those skilled in the art can be used. As one implementation method, a batch of bloated seahorses can be used as a reference population. The genotypes of the SNP molecular markers described in the above technical solution for each sample in the reference population and the sample to be tested can be detected. Then, a genotype association matrix is ​​constructed. Finally, based on the genotype association matrix and the genotypes of the SNP molecular markers in the sample to be tested, the estimated breeding value of body weight is calculated using the GBLUP model.

[0031] As one implementation, the software used for the calculation includes r-Asreml.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a combination of SNP molecular markers related to the weight of a distended hippocampus, a liquid-phase chip, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 1. Population Construction and Data Collection: An experimental population of 874 age-matched, bloated seahorses from Rizhao Lanmu Fishery Aquaculture Co., Ltd. was constructed. Individual samples were collected from each seahorse in the population, including: measuring body weight and sex to establish a phenotypic database; and preserving fin tissue in anhydrous ethanol for subsequent DNA extraction.

[0034] 2. Sequencing and Sequence Alignment: Genomic DNA was extracted from the samples to be tested and a next-generation sequencing library was constructed. High-throughput sequencing was performed using an MGIDNBSEQ-T7 sequencer (target depth: ≈6 Gb / sample). After obtaining the sequencing data, it was aligned to the specified bloated hippocampal reference genome (NCBI database reference genome version number GCA_018466805.1) using the BWA algorithm. SNP calling was then performed using GATK software to obtain the initial SNP locus dataset.

[0035] 3. SNP Screening and Imputation: The initial SNP dataset was subjected to multi-dimensional quality control using VCFtools software (v0.1.13). Low-quality sites with sequencing depth <10×, missing rate ≥5%, minimum allele frequency (MAF) ≤0.05, and those severely deviating from Hardy-Weinberg equilibrium (HWE, P value <0.0001) were filtered out, resulting in 3,869,385 high-quality SNP sites. For the filtered data, the Beagle algorithm was further used for genotype imputation, ultimately obtaining a high-quality genotype file containing complete genetic information of the reference population. Finally, 11,000 SNP sites uniformly distributed across the genome were extracted using the `-bp-space 40345` command in Plink (v2.0) software (Table 1). The distribution of the SNP sites is as follows: Figure 1 As shown, the frequency distribution of the second allele is as follows: Figure 2 As shown.

[0036] Example 2 Chengdu Tiancheng Future Technology Co., Ltd. was commissioned to prepare a liquid-phase chip covering 11,000 SNP sites screened in Example 1, which contains a probe combination capable of specifically capturing 11,000 SNP sites in the reference genome of the bloated hippocampus (GCA_018466805.1).

[0037] The prepared liquid-phase chip was used to perform SNP genotyping on 1040 candidate seahorses from a non-reference population obtained from Rizhao Lanmu Fishery Aquaculture Co., Ltd. The results showed that the genotyping detection rate of all 1040 samples exceeded 90%, and the sample qualification rate reached 100.00%. Statistical analysis indicated that the overall average SNP detection rate of the project was as high as 99.12%. Specific variation detection statistics are as follows... Figure 3 As shown.

[0038] Example 3 Performance of SNP liquid-phase microarrays in assessing genetic parameters of reference populations and predicting candidate populations Using the 874 hippocampi from Example 1 as a reference population (including phenotype and genotype), a genotype association matrix (G matrix) was constructed using the A.mat function in the "sommer" package of the R language. Using the GBLUP model, combined with the probit linking function, the 3,869,385 high-quality SNP loci screened in Example 1 and the 11,000 SNP loci obtained from the final screening were analyzed using r-Asreml v4.2 software to estimate their additive genetic variance and heritability.

[0039] Using the 874 seahorses in Example 1 as a reference population, and combining the genotypes of the 1040 bloated seahorses detected in Example 2, the estimated genomic breeding value (GEBV) of individuals in Example 2 was calculated using the r-Asreml v4.2 software based on the GBLUP model, and the sex was recorded at the same time.

[0040] The GBLUP model is shown in Equation II: Formula II: ; in, This represents the phenotypic value, which in this embodiment is body weight; For fixed effects (sex and batch), sex and batch are substituted as factor variables into Equation II; For fixed effects, the values ​​are calculated by the software after model fitting. The genotype association matrix is ​​obtained using the genotype matrix (e.g., AA / Aa / aa represent 2 / 1 / 0 respectively) based on the calculation method proposed by Vanraden (2008), and the random effects term is substituted in. These are random effects values ​​(genomically estimated breeding values), automatically calculated and obtained by the software. The residual is automatically calculated and obtained by the software. The heritability assessment equation is shown in Equation II: Formula II: ; in, Represents heredity. Represents the variance of additive effects. This represents the residual variance.

[0041] Table 2 shows the comparison results of the liquid-phase chip described in this invention and whole-genome SNP loci in terms of genetic parameter estimation. The data shows that the liquid-phase chip achieves a 91.9% capture efficiency for growth trait genetic parameters compared to whole-genome SNPs, while reducing detection costs by a factor of 2 compared to whole-genome resequencing.

[0042] Table 2 Genetic parameters estimated using liquid-phase microarrays

[0043] Note: All SNPs are the 3,869,385 high-quality SNP sites obtained in the initial screening of Example 1, and the SNPs of this invention are the 11,000 SNP sites obtained in the final screening of Example 1.

[0044] To evaluate the predictive accuracy of GEBV, individuals in the top 10% and bottom 10% of GEBV rankings from Example 2 were selected, and their average body weight differences were calculated and compared. The results showed that, regardless of whether they were female or male, individuals in the high GEBV group had significantly higher body weights than those in the low GEBV group. This result confirms the reliability and effectiveness of the liquid-phase chip described in this invention. Specific results are as follows... Figure 4 As shown.

[0045] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of SNP molecular markers associated with the body weight of a distended hippocampus, characterized in that, The SNP molecular marker combination includes 11,000 SNP molecular markers; the location information of the SNP molecular markers on the bloated hippocampus reference genome is shown in Table 1 of the specification; the version number of the bloated hippocampus reference genome in the NCBI database is GCA_018466805.

1.

2. The SNP molecular marker combination according to claim 1, characterized in that, The SNP molecular marker combination comprises 11,000 nucleic acid molecules containing the SNP molecular marker and its upstream and downstream sequences; the nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO.1-SEQ ID NO.11,000.

3. A liquid-phase chip for breeding bloated seahorses, characterized in that, The genotyping sites of the liquid-phase chip include the SNP molecular markers in the SNP molecular marker combination described in claim 1 or 2.

4. The liquid phase chip according to claim 3, characterized in that, The liquid-phase chip includes a probe array for detecting the SNP molecular marker combination as described in claim 1 or 2.

5. The application of the SNP molecular marker combination as described in claim 1 or 2 or the liquid phase chip as described in claim 3 or 4 in targeted capture sequencing of the distended hippocampus.

6. The application of the SNP molecular marker combination as described in claim 1 or 2 or the liquid phase chip as described in claim 3 or 4 in the analysis of body weight traits in the bloated hippocampus.

7. The application of the SNP molecular marker combination of claim 1 or 2 or the liquid phase chip of claim 3 or 4 in the breeding of bloated hippocampus, wherein the breeding trait includes body weight.

8. The application of the SNP molecular marker combination as described in claim 1 or 2 or the liquid phase chip as described in claim 3 or 4 in calculating the genomic estimation breeding value of the body weight of the bloated hippocampus.

9. The application according to claim 8, characterized in that, The calculation method includes calculation using the GBLUP model.

10. The application according to claim 8 or 9, characterized in that, The software used for the calculations includes r-Asreml.

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