A large yellow croaker fast-growing trait associated snp marker combination and breeding application

Abstract

The application provides a large yellow croaker fast-growing trait associated SNP marker combination and breeding application, which is prepared from probes of 10 SNP molecular markers for detecting the fast-growing trait of the large yellow croaker, and the location of each SNP molecular marker on a chromosome, an altered allele, and reference gene information are shown in Table 1. The SNP marker combination can be used to select the best large yellow croaker individuals with fast-growing potential in a certain scale in the offspring in advance, and the selected individuals are bred and cultivated to the parents, so that the large yellow croaker individuals do not need to be bred and cultivated in the breeding process, and the breeding cost can be greatly reduced. The SNP marker combination can solve the problem that the growth speed of the large yellow croaker cannot be detected in a non-lethal manner in the traditional breeding mode, and can ensure the smooth development of the fast-growing trait breeding work of the large yellow croaker.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fish genetic breeding, and relates to the field of fish molecular marker assisted breeding, in particular to a large yellow croaker fast growth trait associated SNP marker combination and breeding application. BACKGROUND

[0002] The large yellow croaker is one of the important economic fish in China, and is widely distributed in the southeast coastal areas of China. It is delicious and nutritious, and is deeply loved by consumers, with high market demand and breeding value. With the development of aquaculture technology and the innovation of breeding mode, how to breed fast-growing large yellow croakers has become the focus of the breeding industry. Traditional breeding methods mainly rely on phenotype selection, which has achieved certain results to some extent, but has many limitations, such as long-term observation and record, high cost and low efficiency.

[0003] In recent years, with the progress of molecular biology and genomics technology, the application of assisted selection based on molecular markers in breeding is becoming more and more widely. Molecular marker assisted breeding can select individuals in the early development stage by analyzing the association between DNA level markers and target traits, greatly improving the breeding efficiency. Single nucleotide polymorphism (SNP) refers to the variation of a single nucleotide in the genome. SNP markers have become an ideal molecular marker tool due to their high density distribution and stable genetic characteristics in the genome. By identifying SNP markers significantly associated with fast growth traits of large yellow croakers and applying them to breeding practice, the efficiency and accuracy of selection can be significantly improved.

[0004] Although there have been some studies on the genome of large yellow croaker, the research on SNP marker screening and application of fast growth traits of large yellow croaker is still limited. Therefore, it is urgent to develop new molecular marker tools to promote the development and popularization of fast growth breeding technology of large yellow croaker. SUMMARY

[0005] The present application provides a large yellow croaker fast growth trait associated SNP marker combination and corresponding breeding application, which can realize low-cost and high-efficiency genetic improvement of fast growth traits of large yellow croaker, and can be used for genotype identification, growth trait association analysis and genome selection breeding of large yellow croaker breeding population, so as to save cost and improve breeding efficiency and accuracy.

[0006] In order to achieve the above purpose, the technical scheme provided by the present application is as follows:

[0007] In the first aspect of the present application, a large yellow croaker fast growth trait associated SNP marker combination is provided. By whole genome resequencing of fast-growing and slow-growing large yellow croaker populations, whole genome association analysis (GWAS) is used to identify SNP markers significantly associated with fast growth traits of large yellow croakers. Figure 1The 10 SNP markers are located in the nucleotide sequences shown in Table 1.

[0008] Table 1 Nucleotide sequences where the SNP markers are located

[0009]

[0010] The specific meanings of the English table headers in Table 1 above are as follows: SNP represents the number of single nucleotide polymorphism markers; chromosome represents the chromosome number; Positions represents the physical position of the SNP marker on the chromosome; REF represents the reference allele of the SNP site; ALT represents the changed allele of the SNP site; P represents the significant value of the SNP marker in the whole genome association analysis. MAF represents the frequency of the minor allele, MarkerR 2 , i.e. the phenotypic explanation rate PVE, represents the proportion of phenotypic difference explained by each SNP marker.

[0011] The 10 sites are as follows in the sequence of 50bp before and after the SNP site in the genome, with the SNP site in brackets.

[0012] LG1_15601822

[0013] TTTAATGAATAGATCTATTTCAATCAGAAGAGACAAGTGAAGGAAGTGAA(G / A)ATGATTGTTTATTATACAACTCTCTGACCCAATTTAAATGTCATTTTG (SEQ ID NO. 1)

[0014] LG2_16815030

[0015] TGTATCTGCCTTGTAATTCAGATCCCTTCCAAAATGTAATGGATCCTGCC(T / A)TGACCCTTACTTACCCTTAAATTTGGGAAATCCTGCTCACAGACAAACAA (SEQ ID NO. 2)

[0016] LG9_2408311

[0017] TGAAACTGTGGCTAGACACAAAGAAAGAAGAAATCACATAGGTACAGTAA(C / G)AGCGCACACATTGTTTGTGACGTTCAGGTGACTGCAGCATGAGGAACATG (SEQ ID NO. 3)

[0018] LG10_18792519

[0019] CAGTTATAAAAACAGATGAGTGCGCAACAAGATGATGTTATTAAAAGAGC(T / G)C ATTTGATGGACACACAGCTACTGTCCAGCCAGACCAGTTAACCCACATC(SEQ ID NO.4)

[0020] LG16_6992719

[0021] CTCCCATCTGTCTGCCAATGAAGTGCAACATGCTTTTAAAGACTAGGTCC(A / T)CT CTTCGTGCCTGACCAAGTCCCACAATTAGAGTTGAATATGATAATTCC(SEQ ID NO.5)

[0022] LG16_17593706

[0023] GGTTCGTCTCGAATTGCTGCTCCAACAAGAAACTAGCAGTAATGATAGTA(G / A)C TGGTGGTATCTTCCCAGGACCCCACGCTACCAGATTCAATGTTTTTCTG(SEQ ID NO.6)

[0024] LG18_10424432

[0025] ATAGCCCAAAGGCCAACATTGCTCAGCTAATGCAGGATTGCATAACAATG(G / A)A AGACCCACTGGGCAAAATTTCAAAATTAGCATTAAGGTGGCTGCCAGTA(SEQ ID NO.7)

[0026] LG18_25441716

[0027] TCATCATTTTCAATAACGTCAGAAAAAATGAAAAAGTCACAGAGCGAGAG(G / T)GTGAAAACTGCAGGGAAAGGAGTGGTTTACATCAGTTAAAGGCCAGACAG(SEQ ID NO.8)

[0028] LG19_18697561

[0029] AAATGTGCCAAAGCCAGCACACCAGGCATCACATCATTAAGTTTGCCAAC(A / G)G CTTGGTCATAGTGTCACTGATAGAAAATAAATCAAATTAAAAAATAAAA(SEQ ID NO.9)

[0030] LG19_19564455

[0031] TTTCAACATATCACATTTTAATTTTATATGTACATATGGATTAACTACG(A / T)CTCA CCTTAAAAGTGGATAACGTCTCATCTTTATACACACTTTTGTTTTGG (SEQ ID NO. 10).

[0032] In a second aspect, the present invention provides a chip carrying the aforementioned combination of SNP markers associated with the fast-growing trait of large yellow croaker.

[0033] Preferably, the chip is a solid-state chip or a liquid-phase chip.

[0034] The third aspect of the present invention provides the use of the above-mentioned large yellow croaker fast-growing trait associated SNP marker combination or chip in large yellow croaker fast-growing trait genotype identification or fast-growing trait breeding.

[0035] A fourth aspect of the present invention provides a method for breeding large yellow croaker for fast-growing traits, comprising the following steps:

[0036] (1) Collect and keep alive the basic population of large yellow croaker for breeding, and cultivate it to the parent, as the candidate parent, with a male-female ratio of 3:1;

[0037] (2) Using a non-lethal sampling method, collect fin ray samples from the distal end of the dorsal fin of large yellow croaker parents, store them in anhydrous ethanol, and replace the anhydrous ethanol every 12 hours within 24 hours;

[0038] (3) Extracting DNA from parental fin samples, genotyping the above-mentioned SNP markers, and ensuring that the genotyping of all SNP markers is successful;

[0039] (4) Based on the genotyping results, the RandomForest model was used to calculate the breeding value (GEBV) of the parent individuals using the R language package randomForest. The larger the GEBV value of the candidate parent individual, the faster the growth rate;

[0040] (5) The individuals ranked in the top 10% of GEBV values ​​were selected as breeding parents, and the male-female ratio was controlled at 3:1 for artificial breeding to obtain a new germplasm of fast-growing large yellow croaker.

[0041] Research results show that the average body weight of the fast-growing large yellow croaker bred using the present invention is increased by more than 20% compared with the non-selected breeding group.

[0042] Therefore, the fifth aspect of the present invention also provides the application of the above-mentioned large yellow croaker fast-growing trait associated SNP marker combination in the genetic improvement of growth rate.

[0043] Functions and effects of the invention

[0044] (1) The 10 SNP marker combinations of the present invention can be used to quickly and accurately select and identify fast-growing large yellow croaker breeding parents in the early stages, which not only improves the breeding efficiency but also greatly reduces the number of large yellow croaker seed stocks and effectively reduces the breeding costs.

[0045] (2) By screening out SNP markers that are significantly associated with the rapid growth traits of large yellow croaker, the best large yellow croaker individuals with rapid growth potential of a certain size can be selected in advance from the breeding offspring, and then cultured and preserved and cultivated to the parent. There is no need to culture and preserve a large number of offspring seedlings during the breeding process, which can greatly reduce the breeding cost.

[0046] (3) The use of the SNP marker combination of the present invention can solve the problem that the traditional breeding method cannot achieve non-lethal detection of the growth rate of large yellow croaker, and can ensure the smooth development of the breeding work of large yellow croaker with fast-growing traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Manhattan plots of genome-wide association analyses are shown. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below with reference to the examples and accompanying drawings. However, the following examples should not be considered as limiting the scope of the present invention.

[0049] Example 1 Genotyping Detection of Fast-Growing and Slow-Growing Large Yellow Croaker Populations

[0050] In 2022, a genotyping experiment was conducted on the fast-growing and slow-growing populations of large yellow croaker. The specific steps are as follows:

[0051] (1) According to body weight, large yellow croaker were divided into fast-growing (441.41±61.92g) and slow-growing (213.14±25.02g) groups, with 200 fish in each group;

[0052] (2) Extracting genomic DNA from the two populations of large yellow croaker and performing whole-genome resequencing;

[0053] (3) The detection of SNP marker combinations in the above two groups is shown in Table 2. As shown in Table 2, there are significant differences in the allele frequencies of the 10 SNP loci between the fast-growing and slow-growing groups (P < 0.05), further proving that the SNP markers of the present invention can be used for the fast-growing trait selection of large yellow croaker.

[0054] Table 2 Detection of SNP marker combinations in fast-growing and slow-growing large yellow croaker populations

[0055]

[0056]

[0057] Example 2 Cultivation of new germplasm of fast-growing large yellow croaker

[0058] In 2023, the SNP marker combination of the present invention was used to carry out fast-growing breeding of large yellow croaker, and a fast-growing F1 generation of large yellow croaker germplasm was successfully cultivated. The specific steps are as follows:

[0059] (1) More than 800 large yellow croakers with good body shape and no body damage were selected as candidate parents, with a male-female ratio of 3:1.

[0060] (2) Fin ray samples were collected, genomic DNA was extracted, and genotyping was performed using a SNP liquid-phase breeding chip.

[0061] (3) The RandomForest model was used to calculate the GEBV of candidate parents using the R language package randomForest. The individuals with the top 10% GEBV values ​​were selected as breeding parents, with a male-female ratio of 3:1 (Table 3).

[0062] (4) By May 2023, more than 1 million fast-growing F1 generation large yellow croaker germplasm seedlings will be artificially bred.

[0063] (5) In May 2024, 60 individuals from the fast-growing F1 generation and the non-selected line were randomly selected and weighed. The average body weight increased by more than 20% (Table 4).

[0064] Table 3 Breeding values ​​of breeding parents

[0065]

[0066]

[0067] Table 4 Body mass values ​​of large yellow croaker in breeding and non-breeding populations

[0068]

[0069]

[0070] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and practice described. Accordingly, all such variations and modifications are intended to be included within the scope of the application as defined in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. Use of a chip loaded with a combination of SNP markers associated with the fast-growing trait of large yellow croaker in the identification of fast-growing trait genotypes or breeding for fast-growing traits of large yellow croaker, characterized in that: The SNP marker combination consists of the following 10 SNP molecular markers: The 50 bp sequence before and after the SNP sites of the 10 SNP molecular markers on the genome are shown below, where the SNP sites are in brackets: LG1_15601822 TTTAATGAATAGATCTATTTCAATCAGAAGAGACAAGTGAAGGAAGTGAA(G / A)ATGATTGTTTATTATACAACTCTCTGACCCAATTTAAATGTCATTTTG, LG2_16815030 TGTATCTGCCTTGTAATTCAGATCCCTTCCAAAATGTAATGGATCCTGCC(T / A)TGACCCTTACTTACCCTTAAATTTGGGAAATCCTGCTCACAGACAAACAA, LG9_2408311 TGAAACTGTGGCTAGACACAAAGAAAGAAGAAATCACATAGGTACAGTAA(C / G)AGGCCACACATTGTTTGTGACGTTCAGTGACTGCAGCATGAGGAACATG, LG10_18792519 CAGTTATAAAAACAGATGAGTGCGCAACAAGATGATGTTATTAAAAGAGC(T / G)CATTTGATGGACACACAGCTACTGTCCAGCCAGACCAGTTAACCCACATC, LG16_6992719 CTCCCATCTGTCTGCCAATGAAGTGCAACATGCTTTTAAAGACTAGGTCC(A / T)CTCTTCGTGCCTGACCAAGTCCCACAATTAGAGTTGAATATGATAATTCC, LG16_17593706 GGTTCGTCTCGAATTGCTGCTCCAACAAGAAACTAGCAGTAATGATAGTA(G / A)CTGGTGGTATCTTCCCAGGACCCCACGCTACCAGATTCAATGTTTTTCTG, LG18_10424432 ATAGCCCAAAGGCCAACATTGCTCAGCTAATGCAGGATTGCATAACAATG(G / A)AAGACCCACTGGGCAAAATTTCAAAATTAGCATTAAGGTGGCTGCCAGTA, LG18_25441716 TCATCATTTTCAATAACGTCAGAAAAAATGAAAAAGTCACAGAGCGAGAG(G / T)GTGAAAACTGCAGGGAAAGGAGTGGTTTACATCAGTTAAAGGCCAGACAG, LG19_18697561 AAATGTGCCAAAGCCAGCACACCAGGCATCACATCATTAAGTTTGCCAAC(A / G)GCTTGGTCATAGTGTCACTGATAGAAAATAAATCAAATTAAAAAATAAAA, LG19_19564455 TTTCAACATATCACATTTTAATTTTATATGTACATATGGATTAACTACG(A / T)CTCACCTTAAAAGTGGATAACGTCTCATCTTTATACACACTTTTGTTTTGG, The fast-growing trait is body mass.

2. The use according to claim 1, characterized in that The chip is a liquid phase chip.

3. A method for breeding large yellow croaker for fast-growing traits, characterized in that: The method is carried out using the SNP marker combination according to claim 1, comprising the following steps: (1) Collect and keep alive the basic population of large yellow croaker for breeding, and cultivate it to parents as candidate parents; (2) Samples were collected using a non-lethal sampling method and stored in anhydrous ethanol, with the anhydrous ethanol replaced every 12 hours within 24 hours; (3) Extract DNA from parental samples and perform genotyping on the above-mentioned SNP markers, ensuring that all SNP markers are successfully genotyped; (4) Calculate the breeding value (GEBV) of the parent individuals based on the genotyping results; (5) The parent individuals of large yellow croaker were ranked according to their GEBV, and the top-ranked individuals were selected as breeding parents for artificial breeding. The male-female ratio was controlled at 3:1, and the offspring obtained were the new fast-growing large yellow croaker germplasm. The fast-growing trait is body mass.

4. The fast-growing trait breeding method for large yellow croaker according to claim 3, wherein In step (1), the male-female ratio of the candidate parents is 3:1; In step (2), a non-lethal sampling method is used to collect fin ray samples from the distal end of the dorsal fin of the large yellow croaker parent; In step (3), DNA is extracted from the parental fin ray sample; In step (4), the RandomForest model is used to calculate the breeding value GEBV of parent individuals using the R language package randomForest.

5. The fast-growing trait breeding method for large yellow croaker according to claim 3, wherein in, Males and females were ranked according to GEBV, and the top 10% of the ranked individuals were selected as the breeding parent group, with a male-to-female ratio of 3:1.

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