A high-temperature condition under the feed coefficient of lateolabrax japonicus snp molecular marker combination and its application and lateolabrax japonicus breeding method

Abstract

This application discloses a combination of SNP molecular markers related to the feed conversion ratio of spotted bass under high temperature conditions, its application, and a method for breeding spotted bass. The SNP molecular marker combination includes: a first SNP molecular marker SEQ ID NO.1, a second SNP molecular marker SEQ ID NO.2, and a third SNP molecular marker SEQ ID NO.3, with the polymorphism of the 201st base from the 5' end being either A or G. When the genotype of the SNP molecular marker combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA, it is a dominant genotype combination. Under high temperature conditions, the feed conversion ratio of spotted bass is lower than that of other genotypes, providing a basis for further exploration of the genetic mechanism of feed conversion ratio in spotted bass and early assisted breeding.

Description

Technical Field

[0001] This application relates to the fields of aquatic biotechnology and molecular breeding, specifically to a combination of SNP molecular markers related to the feed conversion ratio of spotted bass under high temperature conditions, their application, and a method for breeding spotted bass. Background Technology

[0002] Spotted sea bass ( Lateolabrax maculatus The spotted sea bass (Siniperca chuatsi) is an important aquaculture species in both marine and freshwater environments in my country. Against the backdrop of global warming and intensive, high-density aquaculture, persistently high summer temperatures have become a key bottleneck restricting the sustainable development of the spotted sea bass farming industry. High temperatures inhibit the feeding and growth metabolism of spotted sea bass, redistributing energy originally intended for individual growth to maintain basal metabolism and antioxidant defense, resulting in an abnormally high feed conversion ratio (FCR). FCR is the total amount of feed consumed per unit of body weight gain in farmed animals; a lower FCR indicates higher feed conversion efficiency, making it a core economic trait for measuring feed utilization efficiency in farmed individuals. Currently, feed costs account for over 70% of the spotted sea bass farming industry, and even small fluctuations in FCR can have a significant impact on economic benefits. Therefore, to reduce FCR, existing technologies mainly focus on nutritional interventions (such as adding functional amino acids or probiotics) and environmental regulation. In terms of genetic breeding, with the development of high-throughput sequencing technology, molecular marker-assisted selection (MAS) has accelerated the fish breeding process; however, current MAS studies on spotted sea bass mainly focus on growth and disease resistance traits, with relatively few studies related to FCR.

[0003] Although genome-wide association analysis (GWAS) based on genome resequencing has been successfully applied in FCR studies of various fish species, systematic mining and validation of molecular markers related to FCR in spotted bass remains a gap. Existing technologies have the following limitations: (1) Lack of specific molecular markers for FCR traits under high-temperature conditions: There is a lack of high-confidence targets that can be used for molecular-assisted breeding of superior spotted bass strains with low FCR under high-temperature conditions; (2) Traditional FCR breeding is extremely difficult: FCR is a complex quantitative trait, which is affected by multiple physiological processes. In actual aquaculture, it is extremely difficult to accurately measure the feed intake and FCR of individual fish, which seriously restricts the large-scale development of traditional breeding and conventional association analysis; (3) Neglect of high-order multi-gene interaction effects: Complex traits are often driven by multiple genes with minor effects and gene-gene interactions. Traditional single SNP marker analysis is difficult to capture the epistatic effects that determine complex phenotypes, resulting in limited predictive accuracy and breeding value of molecular markers.

[0004] Therefore, the development of SNP molecular marker combinations related to low FCR in spotted bass under high temperature conditions is of great significance for the breeding of new spotted bass varieties. It can provide early auxiliary identification of the low FCR trait in spotted bass under high temperature conditions and its application in breeding, making the screening of SNP molecular marker combinations more accurate, further improving the feed conversion efficiency of spotted bass, reducing the cost of artificial breeding of spotted bass, and filling the research gap related to FCR in spotted bass. Summary of the Invention

[0005] The purpose of this application is to develop a combination of SNP molecular markers related to the feed conversion ratio of spotted bass under high temperature conditions, its application, and a method for spotted bass breeding. This method can provide early auxiliary identification of the low FCR trait in spotted bass under high temperature conditions and its application in breeding, making the screening of SNP molecular marker combinations more accurate, further improving the feed conversion efficiency of spotted bass, reducing the cost of artificial breeding of spotted bass, and filling the research gap related to FCR of spotted bass.

[0006] To solve this technical problem: This patent provides a combination of SNP molecular markers related to the feed conversion ratio of perch under high temperature conditions. The combination of SNP molecular markers consists of three SNP molecular markers, wherein: The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 201st base from the 5' end is A / G; The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, and the polymorphism of the 201st base from the 5' end is G / A; The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, and the polymorphism of the 201st base from the 5' end is G / A; When the genotype of the SNP molecular marker combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA, it is the dominant genotype combination, and the feed conversion ratio of spotted bass is lower than that of other genotypes under high temperature conditions.

[0007] Furthermore, the reference genome for spotted sea bass is designated PRJCA015925.

[0008] Furthermore, the first SNP molecular marker is located at 16891626 bp on chromosome 23 of the spotted perch.

[0009] Furthermore, the second SNP molecular marker is located at 16902618 bp on chromosome 23 of the spotted perch.

[0010] Furthermore, the third SNP molecular marker is located at 1517639 bp on chromosome 4 of the spotted perch.

[0011] This application also provides the application of a SNP molecular marker combination related to the feed conversion ratio of spotted bass under high temperature conditions in spotted bass breeding. The SNP molecular marker combination consists of three SNP molecular markers, wherein: The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 201st base from the 5' end is A / G; The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, and the polymorphism of the 201st base from the 5' end is G / A; The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, and the polymorphism of the 201st base from the 5' end is G / A; When the genotype of the SNP molecular marker combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA, it is the dominant genotype combination, and the feed conversion ratio of spotted bass is lower than that of other genotypes under high temperature conditions.

[0012] This patent also provides a method for breeding spotted bass, comprising the following steps: Step S1: SNP molecular marker detection was performed on spotted sea bass to obtain the genotype of the SNP molecular marker combination. The SNP molecular marker combination consists of three SNP molecular markers. The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, with a polymorphism of A / G at the 201st base from the 5' end; the nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, with a polymorphism of G / A at the 201st base from the 5' end; the nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, with a polymorphism of G / A at the 201st base from the 5' end. Step S2: Screening is performed based on the genotypes of SNP molecular marker combinations to obtain dominant genotype combinations; the screening is based on the feed conversion ratio of spotted bass, and the dominant genotype combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA. Step S3: Breed by pairing male and female parents of spotted bass with the same homozygous dominant genotype combination. After genotype screening, offspring with a lower feed conversion ratio of spotted bass under high temperature conditions are obtained, and the genotype combination of the offspring is consistent with that of the male and female parents.

[0013] Compared with existing technologies, it has the following beneficial technical effects: This application provides a molecular marker combination of SNPs associated with the feed conversion ratio (FCR) of spotted bass under high-temperature conditions, its application, and a method for spotted bass breeding. Based on individuals from two different spotted bass populations, SNP loci associated with the FCR phenotype were screened through mixed grouping analysis. A core SNP set associated with the FCR phenotype was screened using a combination of Euclidean distance and sliding window analysis. Then, the core SNP sets screened from the two different spotted bass populations were cross-referenced, identifying 169 SNPs that were significantly associated with the FCR phenotype in both populations. A chi-square test was used to identify 7 SNPs that showed significant differences in both genotype and allele frequencies. The GMDR combination was then used to predict the interactions of the 7 loci, and a three-way ANOVA was performed for validation. Finally, a genotype combination containing three core SNP loci with optimal discriminative power was predicted. In the validation results, when the genotype combinations of these three SNP molecular markers were AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, and GG-AA-AA, the model output a negative score, indicating that the above genotype combinations were dominant. Individuals with these genotypes, even after controlling for environmental and size factors, still exhibited relatively stable high feed conversion efficiency (low FCR) at high temperatures. This model provides a certain statistical reference for judging low FCR in spotted bass under high-temperature conditions. Based on the above findings, this application provides the group of SNP loci and dominant genotype combinations, as well as the validation method for this model. These molecular markers and related analytical methods provide preliminary data references and a feasible technical approach for further exploration of the FCR genetic mechanism in spotted bass and early assisted breeding, serving as a supplementary analytical tool to traditional breeding methods. Attached Figure Description

[0014] The above description of this application and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0015] Figure 1 This is the optimal three-SNP interaction combination model in the GMDR model of the embodiment; Figure 2 The following is a distribution of individuals with positive / negative effects in the FCR extreme population of spotted bass in the example after nonparametric testing (where a is the distribution of FCR of spotted bass with different genotype combinations, the horizontal axis is the genotype combination, and the vertical axis is the FCR of spotted bass; b is the distribution of different genotype combinations in terms of sample size, the horizontal axis is different genotype combinations, and the vertical axis is the sample size, in units of (tails)). Figure 3 The above is a distribution of positive / negative effect individuals in the sea bass validation population after nonparametric testing in the example, based on genotype combinations (where a is the distribution of FCR of sea bass with different genotype combinations, the horizontal axis is the genotype combination, and the vertical axis is the sea bass FCR; b is the distribution of different genotype combinations in terms of sample number, the horizontal axis is different genotype combinations, and the vertical axis is the sample number, in units of (tails)). Detailed Implementation

[0016] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0017] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: As used in this application, the "phenol-chloroform extraction method" is a classic DNA extraction method that separates and purifies DNA by utilizing the differences in the solubility of DNA and proteins in different solvents. Phenol denatures and dissolves proteins while inhibiting nuclease activity, thus protecting DNA from degradation. Chloroform enhances the separation of the aqueous and organic phases, ensuring that DNA remains in the upper aqueous phase while denatured proteins precipitate in the lower organic phase. Isoamyl alcohol reduces foam formation and aids in phase separation, resulting in more stable layering.

[0018] The specific steps of the phenol-chloroform extraction method include: 1) Lysis: Take muscle tissue and add 550 μL TNES (125 mM NaCl; 10 mM Tris-HCl, pH 7.4; 10 mM EDTA; SDS 0.5%; Urea 4 M), 6 μL proteinase K (20 mg / mL), incubate in a metal bath at 56℃ for 20-30 min, then use small scissors to cut the tissue into small pieces, and incubate in a water bath at 56℃ overnight until the lysate is clear.

[0019] 2) Protein extraction: Add 300 μL each of 1:1 balanced phenol:chloroform / isoamyl alcohol (24:1), invert and mix for 20 min, then centrifuge at 13000 rpm for 20 min; the solution separates into layers, the upper layer is the aqueous phase containing DNA; the lower layer is the organic phase; the middle layer is the protein layer; use a wide-mouth pipette tip to aspirate the supernatant into a new 1.5 mL EP tube.

[0020] 3) Repeat the previous step until there is no protein layer between the aqueous phase and the organic phase.

[0021] 4) Removal of residual phenol: Take the supernatant, add 600 μL of chloroform / isoamyl alcohol (24:1), mix by inversion for 20 min, centrifuge at 13000 rpm for 10 min, and take the supernatant for later use. This step can be repeated.

[0022] 5) Precipitate DNA: Add 700 μL of isopropanol and 50 μL of saturated NaAc solution to the solution in step 4), mix well, place in a -20℃ freezer for 3 h, then centrifuge at 13000 rpm for 10 min and discard the supernatant. 6) Washing the precipitate: Add 1 mL of pre-cooled 70% ethanol to the precipitate from step 5), gently tap the tube wall to suspend the precipitate, then centrifuge at 13000 rpm for 10 min and discard the supernatant. Add 1 mL of pre-cooled anhydrous ethanol and repeat the washing step.

[0023] 7) Dissolve DNA: Place the washed DNA precipitate in a fume hood to dry. After the ethanol has completely evaporated, add TE buffer to dissolve the precipitate.

[0024] 8) Digest RNA: Add RNase and digest in a 37°C metal bath for 1 hour.

[0025] 9) Quality control: DNA concentration was determined using NanoDrop 2000, followed by DNA quality detection using 1.5% agarose gel electrophoresis.

[0026] 10) Store the extracted DNA sample at -20°C.

[0027] As used in this application, "constructing a genome library" is a systematic process of fragmenting the whole genome DNA of a target organism, adding sequencing adapters, and then amplifying it by PCR for subsequent research and analysis. Genome library construction typically follows a standard workflow of "sample collection - genomic DNA extraction and purification - DNA fragmentation - DNA fragment end repair - adding sequencing adapters - fragment selection - PCR amplification - library quality control - sequencing." Currently, genome file construction can be performed entirely using commercially available DNA library construction kits.

[0028] As used in this application, "BSA analysis" stands for Bulked Segregant Analysis, which is a molecular marker-assisted gene mapping technique. It involves selecting parents with contrasting traits to construct a segregating population, selecting a certain number of individuals with extreme phenotypes to construct a pool, and using the differences in DNA molecular markers between the two pools to achieve QTL mapping. It is suitable for the preliminary mapping of major genes for both qualitative and quantitative traits.

[0029] As used in this application, the "Genome Warehouse (GWH)" is a public genome data repository used to store, manage, and share genome assembly data from different species, including genome sequences, genome annotation information, and other related metadata. It supports genomes at different assembly levels (such as complete or draft genomes) as well as partial genomes (such as mitochondria, chloroplasts, etc.). The specific URL is https: / / ngdc.cncb.ac.cn / gwh / .

[0030] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, in particular, meaning that one of ordinary skill in the art can directly and without doubt determine how the technical solution of this application can be implemented after reading the claims, description and drawings of this application.

[0031] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, description, and drawings of this application, those skilled in the art can still arrive at the only correct understanding by reading the claims, description, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this application.

[0032] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0033] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0034] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0035] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0036] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0037] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0038] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0039] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0040] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0041] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0042] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0043] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0045] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of this application are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0046] Sample material source In this embodiment, the juvenile spotted sea bass were artificially bred by Yantai Hailan Blue Seed Industry Research Institute Co., Ltd. (Yantai City, Shandong Province).

[0047] Example This patent provides a combination of SNP molecular markers related to the feed conversion ratio of spotted bass under high temperature conditions, its application, and a method for breeding spotted bass, including the following steps: S1. Artificial rearing and feed conversion ratio determination of juvenile spotted sea bass, the specific steps are as follows: (1) A group of artificially bred spotted sea bass was collected from Yantai by Hailan Seed Industry Research Institute Co., Ltd., including three sizes: small size (Group 1) 45 fish with an average weight of 1.92±0.65 g, medium size (Group 2) 64 fish with an average weight of 15.7±3.9 g, and large size (Group 3) 64 fish with an average weight of 31.2±5.9 g.

[0048] (2) The above three groups of juvenile sea bass were first placed in a temperature of 21℃ and a volume of 12 m³. 3 They were temporarily acclimatized in a cylindrical tank with circulating water for about 21 days. During the acclimatization period, they were fed commercially available pellet feed of appropriate size at 10:00 am and 3:00 pm every day, and the natural light cycle was simulated.

[0049] Subsequently, the water temperature was increased by 1°C daily until it reached 28°C, at which point the temperature increase was stopped, and each individual sea bass was transferred to a separate tank (0.02 m). 3In a recirculating aquaculture system, after a 5-day single-tank acclimatization period at 28℃, a formal 50-day single-tank culture experiment was initiated. During the experiment, the daily feed intake and uneaten feed amount for each individual were recorded, and initial and final growth traits were statistically analyzed. After the experiment, the feed conversion ratio (FCR) for each individual was calculated using the following formula (1): FCR = Actual total food intake / (Final weight - Initial weight) Formula (1); The units for actual total food intake are g, final body weight is g, and initial body weight is g.

[0050] S2. Genotyping of the spotted bass population, the specific steps are as follows: (1) The sea bass population that has been artificially cultured in step S1 above is anesthetized with tricaine mesylate (MS-222, commercially available). After disinfecting the scissors, a portion of the muscle tissue of each sea bass is cut off and cut into 3mm × 3mm pieces. The pieces are immediately frozen with liquid nitrogen and stored at -80℃ for later use.

[0051] (2) From the second and third groups with relatively stable physiological states in step S1 above, 20 individuals with extreme FCR phenotypes were selected respectively based on the FCR calculated in step S1 above, for a total of 40 individuals. That is, 10 spotted bass with high FCR and 10 spotted bass with low FCR in the second group, and 10 spotted bass with high FCR and 10 spotted bass with low FCR in the third group. These individuals were used to construct mixed pools with extreme FCR phenotypes, and finally 4 mixed pools with extreme FCR phenotypes were obtained.

[0052] (3) The muscle tissues of the four FCR extreme phenotype individuals in the pool of (2) above were used to extract whole genome DNA from the muscle tissues in (1) of S2. The genotype DNA of 10 individuals in each group was mixed in equal amounts to obtain DNA pools of four FCR extreme phenotype individuals (equal amount 1000 ng). Then, the genome library was constructed according to the existing technology to obtain the genome libraries of the four FCR extreme phenotype pools. Then, whole genome resequencing was performed to obtain the DNA resequencing data of the four FCR extreme phenotype pools, which were used for subsequent BSA analysis.

[0053] (4) Use Fastp software to perform quality control on the resequencing data of the four FCR extreme phenotype pools obtained in step (3) above, and filter out low-quality data for subsequent analysis. The filtering criteria for low-quality data are: a) remove reads containing partial adapter dimers; b) delete reads with a quality parameter Q below 30; c) remove reads with a length less than 50 bp. Finally, clean reads (resequencing data after quality filtering) are obtained.

[0054] (5) Use BWA software to align the clean reads obtained in step (4) above to the reference genome of the spotted sea bass (from the Genome Warehouse (GWH) genome database, sequencing project number (BioProject number) PRJCA015925, released in April 2023, genome size 597611097 bp), generate a sam file, and then use SAMtools software to convert the aligned sam file into a bam file format and sort it.

[0055] (6) Use GATK software to perform mutation detection and genotyping on the bam file obtained in step (5) above, then extract SNP mutations and generate vcf file.

[0056] (7) Using VCFtools software, the VCF files of the two extreme pools with high and low FCR in the second group were merged, and the VCF files of the two extreme pools with high and low FCR in the third group were merged, and SNPs were filtered. The filtering criteria were: a) maximum deletion rate less than 0.9; b) minor allele frequency greater than 0.05; c) minimum quality value Q greater than 30; d) only biallelic loci. Then, the filtered data were filled using Beagle software. After filling, a total of 4,796,662 SNPs were obtained in the second group and a total of 4,300,049 SNPs were obtained in the third group.

[0057] (8) The allele frequency differences between high and low FCR pools in the second group and the third group were further quantified using the Euclidean distance (ED) method. A sliding window analysis (window size 20 kb, step size 10 kb) was then used to screen the top 1% of regions with the highest average ED values. Ultimately, 584 candidate regions related to spotted bass FCR were obtained in the second group and 581 candidate regions related to spotted bass FCR were obtained in the third group.

[0058] (9) Extract and annotate the SNP information in the candidate regions related to the FCR of the spotted bass obtained in step (8) above. Among them, 41,284 SNPs related to the FCR of the spotted bass are obtained in the second group and 40,200 SNPs related to the FCR of the spotted bass are obtained in the third group.

[0059] Further cross-matching of the SNPs related to the FCR of the spotted bass obtained in the second group and the SNPs related to the FCR of the spotted bass obtained in the third group yielded 169 common SNPs distributed across 14 genes.

[0060] (10) From the first group in step S1 above, based on the FCR calculated in step S1 above, select 12 individuals with extreme FCR phenotypes, namely 6 spotted bass with high FCR and 6 spotted bass with low FCR in the first group. Select 12 individuals with extreme FCR phenotypes in the first group (6 with high FCR and 6 with low FCR) and 20 individuals with extreme FCR phenotypes in the mixed pool in the second and third groups above S2 (10 with high FCR and 10 with low FCR), for a total of 52 spotted bass individuals as the SNP genotyping verification population. And perform whole genome resequencing according to the methods shown in steps (3) to (7) above to obtain the whole genome resequencing data of a total of 52 spotted bass individuals with extreme FCR phenotypes.

[0061] (11) The whole genome resequencing data of the above 52 FCR extreme phenotype individuals were used to perform genotyping verification of the 169 SNPs in steps (8) to (9) above. Based on the genotyping and chi-square test results, 10 sites were found from the 169 candidate SNP sites that met the criteria of significant differences in allele frequency chi-square test (P<0.05) or significant differences in genotype frequency chi-square test (P<0.05). Among them, 7 sites showed significant differences in both genotype frequency and allele frequency between extreme phenotype groups (P<0.05).

[0062] S3. Establish a linear model and verify the SNP. The specific steps are as follows: (1) Use the 7 significant SNP genotyping data obtained in step S2 (11) above to perform generalized multifactor dimensionality reduction (GMDR) analysis.

[0063] a) Since FCR is affected by environmental factors such as initial weight and batch size, a generalized linear model (GLM) is first established, as shown in the following formula (2): Formula (2); in, Let be the predicted FCR value for the i-th individual; Wi This refers to the individual's initial weight. B i For group batches; β 0 , β 1 , β 2 These are the regression coefficients; i This is the error term.

[0064] b) Then, calculate the residual value (the difference between the actual value and the model prediction value) for each individual, as shown in the following formula (3): Formula (3); in, Yi Let be the actual FCR value of the i-th individual.

[0065] c) For each genotype combination consisting of 3 core SNPs, calculate the average residual of all individuals within that combination. Formula (4) is as follows: Formula (4); in, n G This indicates the number of individuals containing this genotype combination. If A value >0 indicates that the average FCR of individuals with this genotype combination is higher than the population expectation, classifying it as a positive effect (Class 1, high-risk / disadvantageous combination). <0 indicates that the average FCR of individuals in this genotype combination is lower than the population expectation, and is classified as a negative effect (Class 0, low risk / advantage combination).

[0066] For the seven candidate SNPs, all genotype combinations of order 1, order 2, and order 3 were exhaustively enumerated, and 10-fold cross-validation was used. Cross-validation consistency, training balance accuracy, and sign test p-value were calculated for each combination. The highest cross-validation consistency was used as the primary criterion, combined with balance accuracy and significance. Significant higher-order interaction models located at the three core SNP loci were successfully predicted. The results are shown in Table 1 and [Table data would be inserted here]. Figure 1 As shown.

[0067] The first SNP molecular marker (SNP1) is located at 16891626 bp on chromosome 23 of the spotted perch, and its nucleotide sequence is shown in SEQ ID NO.1. The polymorphism of the 201st base is A / G.

[0068] The second SNP molecular marker (SNP2) is located at 16902618 bp on chromosome 23 of the spotted perch. The nucleotide sequence is shown in SEQ ID NO.2, and the polymorphism of the 201st base is G / A.

[0069] The third SNP molecular marker (SNP3) is located at 1517639 bp on chromosome 4 of the spotted perch. Its nucleotide sequence is shown in SEQ ID NO.3, and the polymorphism of the 201st base is G / A.

[0070] Table 1 shows that when the genotype combinations of these three SNP molecular markers are AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, and GG-AA-AA, the model outputs a negative score. This means that the above eight genotype combinations are the dominant genotype combinations, and individuals with these genotypes can still exhibit relatively stable high feed conversion efficiency (low FCR) under high temperature conditions, even after controlling for environmental and size factors.

[0071] Table 1. GMDR score table for three SNP molecular marker site combinations ;

[0072] (2) In order to further verify the accuracy of the GMDR model in step S3 (1) above, 109 spotted bass individuals (including the FCR phenotype of 109 spotted bass individuals) were selected from the single-tank culture experiment individuals that had completely passed step S1 above. Among them, 12 spotted bass individuals in the first group, 60 spotted bass individuals in the second group, and 37 spotted bass individuals in the third group underwent whole genome resequencing. At the same time, the whole genome DNA of some muscle tissue in step S2 (1) was extracted using the phenol-chloroform extraction method in the existing technology. Then, whole genome resequencing was performed according to the methods shown in step S2 (3) to step S2 (7) above, and the whole genome resequencing data of 109 spotted bass individuals (i.e., including SNP information) was obtained.

[0073] The GMDR model obtained in step S3 (1) above is mapped to the genotype and phenotype of the spotted bass population (as shown in Table 1 and...). Figure 1 The effectiveness of the model was verified by introducing three-way ANOVA and nonparametric tests.

[0074] The results are shown in Table 2. Figure 2 and Figure 3 As shown, the GMDR model was successfully verified to have an accuracy of 80.77% at the extreme population level of 52 FCR (i.e., 12 in the first group, 20 in the second group, and 20 in the third group, for a total of 52); and an accuracy of 72.55% at the full population level of 109 (i.e., the spotted bass population in step S3 (2), 12 in the first group, 60 in the second group, and 37 in the third group, for a total of 109). All three factors of variance results were significant.

[0075] Table 2 GMDR Model Accuracy Statistics ;

[0076] Note: The expected value is the individual that meets the 8 dominant genotype combinations or the 6 inferior genotype combinations in Table 1; the actual value is the value of 0 or 1 after calculating the individual residual according to formula (3).

[0077] In summary, this application provides a combination of SNP molecular markers related to the feed conversion ratio (FCR) of spotted bass under high-temperature conditions, its application, and a method for spotted bass breeding. Based on individuals from two different spotted bass populations, SNP loci associated with the FCR phenotype were screened through mixed grouping analysis. A core SNP set associated with the FCR phenotype was screened using a combination of Euclidean distance and sliding window analysis. Then, the core SNP sets screened from the two different spotted bass populations were cross-referenced, resulting in 169 SNPs that were significantly associated with the FCR phenotype in both populations. A chi-square test was used to identify 7 SNPs that showed significant differences in both genotype and allele frequencies. Finally, GMDR (Generalized Multivariate Diagnostic and Regression) combinations were used to predict the interactions of the 7 loci, and a three-way ANOVA was performed for validation. Ultimately, a genotype combination containing three core SNP loci with optimal discriminative power was predicted. In the validation results, when the genotype combinations of these three SNP molecular markers were AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, and GG-AA-AA, the model output a negative score, indicating that the above genotype combinations were dominant. Individuals with these genotypes, even after controlling for environmental and size factors, still exhibited relatively stable high feed conversion efficiency (low FCR) at high temperatures. This model provides a certain statistical reference for judging low FCR in spotted bass under high-temperature conditions. Based on the above findings, this application provides the group of SNP loci and dominant genotype combinations, as well as the validation method for this model. These molecular markers and related analytical methods provide preliminary data references and a feasible technical approach for further exploration of the FCR genetic mechanism in spotted bass and early assisted breeding, serving as a supplementary analytical tool to traditional breeding methods.

[0078] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various application aspects. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features are more numerous than expressly stated in each claim. Rather, the claimant aspect reflected in the claims lies in non-all features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0079] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0080] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0081] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A combination of SNP molecular markers related to the feed conversion ratio of spotted bass under high temperature conditions, characterized in that, The SNP molecular marker combination consists of three SNP molecular markers, wherein: The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 201st base from the 5' end is A / G; The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, and the polymorphism of the 201st base from the 5' end is G / A; The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, and the polymorphism of the 201st base from the 5' end is G / A; When the genotype of the SNP molecular marker combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA, it is a dominant genotype combination, and the feed conversion ratio of the spotted bass is lower than that of other genotypes under high temperature conditions.

2. The SNP molecular marker combination related to the feed conversion ratio of spotted bass under high temperature conditions according to claim 1, characterized in that, The reference genome of the spotted bass is project numbered PRJCA015925; The first SNP molecular marker is located at 16891626 bp on chromosome 23 of the spotted perch; The second SNP molecular marker is located at 16902618 bp on chromosome 23 of the spotted perch; The third SNP molecular marker is located at 1517639 bp on chromosome 4 of the spotted perch.

3. The application of a SNP molecular marker combination related to the feed conversion ratio of spotted bass under high temperature conditions in spotted bass breeding, characterized in that, The SNP molecular marker combination consists of three SNP molecular markers, wherein: The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, and the polymorphism of the 201st base from the 5' end is A / G; The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, and the polymorphism of the 201st base from the 5' end is G / A; The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, and the polymorphism of the 201st base from the 5' end is G / A; When the genotype of the SNP molecular marker combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA, it is a dominant genotype combination, and the feed conversion ratio of the spotted bass is lower than that of other genotypes under high temperature conditions.

4. A method for breeding spotted bass, characterized in that, Includes the following steps: Step S1: Detect the SNP molecular markers of the spotted sea bass to obtain the genotype of the SNP molecular marker combination. The SNP molecular marker combination consists of three SNP molecular markers. The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO.1, with a polymorphism of A / G at the 201st base from the 5' end; the nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO.2, with a polymorphism of G / A at the 201st base from the 5' end; the nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO.3, with a polymorphism of G / A at the 201st base from the 5' end. Step S2: Screening is performed based on the genotypes of the SNP molecular marker combinations to obtain dominant genotype combinations; the screening is based on the feed conversion ratio of the spotted bass, and the dominant genotype combination is any one of AA-GG-GG, AG-GG-GG, GG-GG-GG, AG-GA-GG, AG-GG-AA, GG-AA-AG, GG-AA-GG, or GG-AA-AA; Step S3: Breed by pairing male and female parents of spotted bass with the same homozygous dominant genotype combination. After genotype screening, offspring with a lower feed conversion ratio of the spotted bass under high temperature conditions are obtained. The genotype combination of the offspring is consistent with that of the male and female parents.

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