Prawn anti-flow trait associated snp marker combination and application

Abstract

The application provides a large yellow croaker anti-flow trait associated SNP marker combination and breeding application, the combination is prepared from 13 SNP molecular marker combination probes for detecting the muscle elasticity trait of the large yellow croaker, the position of each SNP molecular marker on the chromosome, the changed allele of the site, and the reference gene information are shown in Table 1. Through the SNP marker combination of the application, the best large yellow croaker individual with anti-flow potential can be selected in advance in the breeding offspring, breeding and cultivation to the parent, without breeding and cultivation of a large number of offspring seedlings in the breeding process, so that the breeding cost can be greatly reduced. The SNP marker combination of the application can solve the problem that the muscle elasticity of the large yellow croaker cannot be detected by non-lethal detection in the traditional breeding mode, and can ensure the smooth development of the large yellow croaker anti-flow trait breeding work.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic breeding, and relates to the field of fish molecular marker-assisted breeding technology, specifically to a combination of SNP markers associated with the flow resistance trait of large yellow croaker and its breeding application. Background Technology

[0002] Large yellow croaker is currently the largest-scale marine cage-cultured fish species in China, with an annual production exceeding 280,000 tons. With breakthroughs in artificial breeding technology, genetic breeding research on large yellow croaker has also gradually unfolded. To date, four new varieties of large yellow croaker have been developed: "Minyou No. 1," "Donghai No. 1," "Yongdai No. 1," and "Fufa No. 1." However, existing new varieties of large yellow croaker still cannot meet the current needs of the deep-sea aquaculture industry, severely restricting its high-quality development. Promoting the development of the deep-sea aquaculture industry for large yellow croaker is an effective way to improve the quality and efficiency of large yellow croaker farming, upgrade the industry, and implement a comprehensive food concept, drawing food from rivers, lakes, and seas, and promoting the development of my country's deep-sea fisheries. "New varieties of deep-sea aquaculture fish" has been included in the "2022 Major Technological Needs for Agricultural and Rural Industry Development." Therefore, to meet the needs of the deep-sea aquaculture large yellow croaker seed industry, it is necessary to cultivate new varieties of large yellow croaker that are highly suitable for deep-sea aquaculture, thereby promoting the high-quality development of my country's deep-sea aquaculture industry for large yellow croaker.

[0003] Deep-sea aquaculture is an emerging farming method, typically conducted in deep-sea areas at depths exceeding 10 meters and 3 km offshore, where water exchange is good, the farming area is large, and fish have ample space to move. The strength of large yellow croaker's resistance to currents is a crucial indicator of its suitability for deep-sea aquaculture. Traditional testing methods for current resistance can damage the fish's body surface, potentially leading to death; therefore, traditional methods cannot be used for selecting breeding populations. Instead, selection of large yellow croaker with strong current resistance must be conducted at the genotypic level. However, whole-genome selection is too costly. Therefore, marker-assisted breeding (MABB) is a more feasible approach. As is well known, phenotypic traits are influenced by both environmental and genetic factors. Therefore, it is necessary to select large yellow croaker with strong current resistance under the same farming conditions (identical environmental factors). By analyzing their genotypes, SNP marker combinations associated with current resistance can be identified. Then, marker-assisted breeding techniques can be used to select large yellow croaker with strong current resistance. Therefore, a marker-assisted breeding technique specifically designed to improve the current resistance of large yellow croaker is needed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a combination of SNP markers associated with the current resistance trait in large yellow croaker and its corresponding breeding applications. This enables low-cost and efficient genetic improvement of the current resistance trait in large yellow croaker and can be used for genotyping of large yellow croaker breeding populations, association analysis of current resistance traits, and genomic selection breeding.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] In a first aspect, this invention provides a combination of SNP markers associated with the current resistance trait in large yellow croaker, which is based on whole-genome resequencing of both current-resistant and non-current-resistant large yellow croaker populations and utilizes genome-wide association analysis (GWAS). Figure 1 The samples were obtained through screening using the Manhattan diagram. They include 16 SNP markers, and the nucleotide sequences containing these SNP markers are shown in Table 1.

[0007] Table 1 shows the nucleotide sequences containing SNP markers.

[0008]

[0009] The specific meanings of the English and Chinese headers in Table 1 above are as follows: Mark represents the single nucleotide polymorphism (SNP) marker number; Chr (chromosome) represents the chromosome number; Position represents the physical location of the SNP marker on the chromosome; REF represents the reference allele for the SNP site; ALT represents the allele of the altered SNP site; P represents the significance value of the SNP marker in the genome-wide association study; MAF represents the frequency of the minor allele; MarkerR2, or phenotypic explained rate (PVE), represents the proportion of phenotypic difference explained by each SNP marker.

[0010] The 16 sites are shown below in the 50bp sequence before and after the genome, with SNP sites in parentheses.

[0011] (1)LG2_15637606

[0012] GCTGCTGCTGCTGCTGCCACTGGTCACAATGCATGTTTTGCAACTCATTT(A / C)AT ACAGATGTTTTTAAAAAGAAAAAGAAAAACACACCAAAACTCGGGACA(SEQ ID NO.1);

[0013] (2)LG4_11158806

[0014] AAAAAAGCCATAAGGATTGTAATAATGTAGGTTTTTCACAAAACACGC(C / T)A ACACAATAAAGTTGGTGAATCTTGTAGAATAGAGCACAGCACAAATAAT(SEQ ID NO.2);

[0015] (3)LG4_16110143

[0016] GAATCCCAACAGAAAGCAATGTTACCTTAAGTATAATGCGTTTGGTAGCC(C / T)AC TAGGTTGCCAACCCCGCAATTTAGTTCTTTGCAGCTTTTTATTCATGG(SEQ ID NO.3);

[0017] (4)LG4_17909643

[0018] TGTATGAGGTGGAACAGAGGGAGACCACAGCTCTCCAAAAACTCTACTGT(A / G)GGCCAGGACACACTGTGCAATTCAGATAAGTCACTGATAACTACTCAGAG(SEQ ID NO.4);

[0019] (5)LG5_8367699

[0020] TCAAATGTGAGCACAAATTACCAGTTAGTACACCACTCTTTGCCAACATT(C / T)TT TTATTTTGGTTTTTTTCTCTGTCTTTTCCCCAGTGGTCTTCCAGCT(SEQ ID NO.5);

[0021] (6)LG5_16555811

[0022] TTCTTTGCTTAAAGAGCCTGAGCGGGGCAGGAGGCTTTGGTTGATGTTTC(A / G)T ATGAATAGAATTCCAGAACAGAAATAATCTATTTTTTTTTGGGT(SEQ ID NO.6);

[0023] (7)LG7_14490445

[0024] TTTGCCGGTTATGGCCTCTTAAAGGTCCAGTGTGTACGATTTCAGGGGCT(C / A)TG TTTACAGAACATGACAGGAATGGAACATAACCTGTATAGATATTTATC(SEQ ID NO.7);

[0025] (8)LG8_27200376

[0026] TAGTATTTCTACCACAATTCATATTCAAGAGATTTTCAGAGACCCGTCAA(G / A)GT TTAAATAAATAGCTTCTTACTTTAGGCTTGAAGAGAGACAGGAAGGAT(SEQ ID NO.8);

[0027] (9)LG8_27667693

[0028] CACAACTAAAGAAAGAGTTGATCCTATGAAACGCCAAGCTTTTTGAGCTG(T / C)G GCATGGAAAATGGCACTGATATGCAACGAGTGGAGTTTGTGTCACTGCT(SEQ ID NO.9);

[0029] (10)LG8_27759293

[0030] TGTCGAACGCCGCGATGAGAAAAAAAAAGCCGGCTGTGTTCTCGCTGTCG(T / C)CGTACAATAACAGGTGTGTGCACAGGTGCGTGTACGTGTGTGCTGATTAG(SEQ ID NO.10);

[0031] (11)LG12_3539141

[0032] ATATATTTTTTTTACGAGCATGTGTGTAATCTCAGCGGACAGGTGTGAAA(G / A)GG ATGCTTGTAGATGATGATGTTGTTTGACAAGATGCAACAGCGAATTTA(SEQ ID NO.11);

[0033] (12)LG12_11690127

[0034] TCTTGTGTCTGTGCTTGTTAAAATCATAAGACTCAGCCCCCCTCTTATGAT(G / A)AAATTCAATAAAAATATCTGATTAATGCAAAAGCACAATGTTAAATATGC (SEQ ID NO. 12);

[0035] (13)LG19_28477706

[0036] GTCACAGAGTCCCATCAGGTATGATTTTTGTGCCGTTCGTGAGCTTAAAT(G / C)TA CCAACTTTACTTTGCTTAAGAAGAACTCTGACCTGTTTGCACCGCAGG(SEQ ID NO.13);

[0037] (14)LG22_20801417

[0038] CTCATCAACATGCATGACTGCTCAGCCCTGTGTGTGTCTGTGTGTGTGTG(C / T)GC ACTGTATTGTGTTTAGTATAACATGGTTTTGTCTTGTGCTGGTGGACT(SEQ ID NO. 14);

[0039] (15)LG24_14857586

[0040] AGTTGAACTGTGGTTGTTTCATGTATCTGGGTTGCCTGCGTCGCTTGGGA(A / G)A AATGACAAAGCAAAGATAAAATGATCCCAGCACTTATCAAATACTGCGC (SEQ ID NO. 15);

[0041] (16)LG24_15510376

[0042] TTGCTGTATGCCTACATATATATCTGCGATCTCGCACGGCACAAAGTCAC(A / G)CA CCACTGAGTATTTCACAGATTTTGGGGATACTGGATCATATTTTATGA (SEQ ID NO. 16).

[0043] In a second aspect, the present invention provides a chip loaded with the above-described large yellow croaker anti-flow trait associated SNP marker group.

[0044] Preferably, the chip is a solid-state chip or a liquid-phase chip.

[0045] In a third aspect, the present invention provides the application of the above-described SNP marker combination or chip for the large yellow croaker's resistance to current trait in the identification of large yellow croaker's resistance to current trait genotype or in the breeding of large yellow croaker's resistance to current trait.

[0046] In a fourth aspect, the present invention provides a method for breeding large yellow croaker with resistance to flooding, comprising the following steps:

[0047] (1) Collect and keep alive the basic population of large yellow croaker for breeding, and cultivate it to parent stock as candidate parent stock, with a female-to-male ratio of 3:1;

[0048] (2) A non-lethal sampling method was used to collect fin rays from the end of the dorsal fin of the parent large yellow croaker, which were stored in anhydrous ethanol and replaced every 12 hours within 24 hours.

[0049] (3) Extract DNA from the fin samples of the parents, perform genotyping on the above SNP markers, and ensure that the genotyping of all SNP markers is successful;

[0050] (4) Based on the genotyping results, the Bayes Lasso model was used, and the breeding value GEBV of the parent individuals was calculated using the R language package BGLR.

[0051] (5) Based on the GEBV of the parent large yellow croaker individuals, sort them and select the top 10% of individuals as breeding parents for artificial breeding. The male-female ratio is controlled at 3:1. The offspring obtained are the new germplasm of the current-resistant large yellow croaker.

[0052] The results showed that the proportion of large yellow croaker resistant to currents bred using this invention increased from 64% to 88%.

[0053] Therefore, in a fifth aspect, the present invention also provides the application of the above-mentioned large yellow croaker flow resistance trait-associated SNP marker combination in flow resistance genetic improvement.

[0054] The role and effect of invention

[0055] (1) By using the 16 SNP marker combinations of the present invention, breeding parents of large yellow croaker with strong resistance to current can be selected quickly and accurately, which greatly improves the breeding efficiency.

[0056] (2) The SNP marker combination of the present invention can select the best large yellow croaker individuals of a certain size with resistance to current in advance from the offspring, carry out breeding and preservation and cultivate to the parent, without the need to carry out breeding and preservation of a large number of offspring seedlings during the breeding process, which can greatly reduce the breeding cost.

[0057] (3) The SNP marker combination of the present invention can solve the problem that the non-disabling (lethal) detection of the flow resistance ability of large yellow croaker cannot be achieved under the traditional breeding method, and can ensure the smooth progress of the breeding work of large yellow croaker flow resistance trait. Attached Figure Description

[0058] Figure 1 The Manhattan plot shows the genome-wide association analysis;

[0059] Figure 2 The data shows the proportion of the current-resistant large yellow croaker population in the selected and non-selected groups (* represents P < 0.05). Detailed Implementation

[0060] The present invention will now be described in detail with reference to embodiments and accompanying drawings. However, the following embodiments should not be construed as limiting the scope of the present invention.

[0061] Example 1: Detection of SNP marker combinations in large yellow croaker populations with different current resistance capabilities

[0062] In 2021, genotyping experiments were conducted on different populations of large yellow croaker with varying resistance to currents, following the steps below:

[0063] (1) Using a flow-resistant device with a flow rate of 1 m / s and a flow resistance of 30 min as the screening conditions, the large yellow croaker population was divided into a flow-resistant population and a non-flow-resistant population, with no less than 150 fish in each population.

[0064] (2) Extract genomic DNA from individuals of large yellow croaker in the anti-current and non-anti-current groups and perform whole-genome resequencing.

[0065] (3) The success rate of SNP marker gene typing included in this invention is 100%.

[0066] (4) The detection of the SNP marker combinations included in this invention in different large yellow croaker populations with different current resistance is shown in Table 2:

[0067] Table 2. Detection of SNP marker combinations in large yellow croaker populations with different current resistance capabilities.

[0068]

[0069]

[0070] Example 2: Cultivation of New Germplasm for Flood-Resistant Large Yellow Croaker

[0071] In 2022, this invention was used to conduct breeding work on the resistance to currents of large yellow croaker, and a new F1 generation of large yellow croaker with strong resistance to currents was obtained. The specific steps are as follows:

[0072] (1) More than 600 large yellow croakers were selected from the breeding base population as candidate parents, with a female-to-male ratio of 3:1.

[0073] (2) Collect fin rays from the posterior dorsal fin of the candidate parent large yellow croaker, preserve them in anhydrous ethanol, and replace the anhydrous ethanol every 12 hours within 24 hours.

[0074] (3) Extract genomic DNA from the sample and perform genotyping using SNP liquid phase breeding chip.

[0075] (4) The genotyping success rate of the SNP marker combination of the present invention is 100%.

[0076] (5) Using the Bayes-Lasso model, the breeding value (GEBV) of the parent individuals was calculated using the R language package BGLR. The GEBV of the candidate parent individuals was calculated (the smaller the value, the stronger the resistance to flooding). The individuals were sorted by sex and then by their GEBV values. The top 10% of the individuals were selected as the breeding parent population, with a sex ratio of 3:1 (see Table 3).

[0077] Table 3 Breeding Values ​​of Parent Lines

[0078]

[0079]

[0080] (6) In April 2022, approximately 480,000 F1 generation large yellow croaker seedlings with strong resistance to current were obtained through artificial spawning and cultivation.

[0081] (7) From June 2022 to May 2024, phenotypic evaluations were conducted on the F1 generation of large yellow croaker fry, juveniles, and broodstock (more than 200 fish). Among them, the proportion of resistant-current population in the large yellow croaker fry (average weight 2.03g) increased from 38% to 50% at a current velocity of 0.15m / s (P<0.05); the proportion of resistant-current population in juveniles (average weight 38g) increased from 30% to 38% at a current velocity of 0.5m / s (P<0.05); and the proportion of resistant-current population in broodstock (average weight 575g) increased from 64% to 88% at a current velocity of 1m / s (P<0.05), demonstrating significant breeding results. Figure 2 ).

[0082] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of SNP marker combinations associated with the current resistance trait in large yellow croaker for genotypic identification or breeding of current resistance traits, characterized in that, The SNP marker combination consists of the following 16 SNP molecular markers. The SNP sites of these 16 SNP molecular markers are shown in the following 50bp sequences on the genome, with the SNP sites in parentheses: (1) LG2_15637606 GCTGCTGCTGCTGCTGCCACTGGTCACAATGCATGTTTTGCAACTCATTT(A / C)ATACAGATGTTTTTAAAAAGAAAAAGAAAAACACACCAAAACTCGGGACA; (2) LG4_11158806 AAAAAAGAGCCATAAGGATTGTAAATAATGTAGGTTTTTCACAAACACGC(C / T)AACACAATAAAGTTGGTGAATCTTGTAGAATATAGCACAGCACAAATAAT; (3) LG4_16110143 GAATCCCAACAGAAAGCAATGTTACCTTAAGTATAATGCGTTTGGTAGCC(C / T)ACTAGGTTGCCAACCCCGCAATTTAGTTCTTTGCAGCTTTTTATTCATGG; (4) LG4_17909643 TGTATGAGGTGGAACAGAGGGAGACCACAGCTCTCCAAAAACTCTACTGT(A / G)GGCCAGGACACACTGTGCAATTCAGATAAGTCACTGATAACTACTCAGAG; (5) LG5_8367699 TCAAATGTGAGCACAAATTACCAGTTAGTACACCACTCTTTGCCAACATT(C / T)TTTTATTTTGGTTTTTTTTTCTCTGTCTTTTCCCCAGTGGTCTTCCAGCT; (6) LG5_16555811 TTCTTTGCTTAAAGAGCCTGAGCGGGGCAGGAGGCTTTGGTTGATGTTTC(A / G)TATGAATAGAATTCCAGAACAGAAAATAATCTATTTTATTTTGTTTGGGT; (7) LG7_14490445 TTTGCCGGTTATGGCCTCTTAAAGGTCCAGTGTGTACGATTTCAGGGGCT(C / A)TGTTTTACAGAACATGACAGGAATGGAACATAACCTGTATAGATATTTATC; (8) LG8_27200376 TAGTATTTCTACCACAATTCATATTCAAGAGATTTTCAGAGACCCGTCAA(G / A)GTTTAAATAAATAGCTTCTTACTTTAGGCTTGAAGAGAGACAGGAAGGAT; (9)LG8_27667693 CACAACTAAAGAAAGAGTTGATCCTATGAAACGCCAAGCTTTTTGAGCTG(T / C)GGCATGGAAAATGGCACTGATATGCAACGAGTGGAGTTTGTGTCACTGCT; (10)LG8_27759293 TGTCGAACGCCGCGATGAGAAAAAAAAAGCCGGCTGTGTTCTCGCTGTCG(T / C)CGTACAATAACAGGTGTGTGCACAGGTGCGTGTACGTGTGTGCTGATTAG; (11)LG12_3539141 ATATATTTTTTTTACGAGCATGTGTGTAATCTCAGCGGACAGGTGTGAAA(G / A)GGATGCTTGTAGATGATGATGTTGTTTGACAAGATGCAACAGCGAATTTA; (12)LG12_11690127 TCTTGGTCTGTGCTTGTTAAAATCATAAGACTCAGCCCCCCTCTTATGAT(G / A)AAATTCAATAAAAATATCTGATTAATGCAAAAGCACAATGTTAAATATGC; (13)LG19_28477706 GTCACAGAGTCCCATCAGGTATGATTTTTGTGCCGTTCGTGAGCTTAAAT(G / C)TACCAACTTTACTTTGCTTAAGAAGAACTCTGACCTGTTTGCACCGCAGG; (14)LG22_20801417 CTCATCAACATGCATGACTGCTCAGCCCTGTGTGTGTCTGTGTGTGTGTG(C / T)GCACTGTATTGTGTTTAGTATAACATGGTTTTGTCTTGTGCTGGTGGACT; (15)LG24_14857586 AGTTGAACTGTGGTTGTTTCATGTATCTGGGTTGCCTGCGTCGCTTGGGA(A / G)AAATGACAAAGCAAAGATAAAATGATCCCAGCACTTATCAAATACTGCGC; (16)LG24_15510376 TTGCTGTATGCCTACATATATATCTGCGATCTCGCACGGCACAAAGTCAC(A / G)CACCACTGAGTATTTCACAGATTTTGGGGATACTGGATCATATTTTATGA.

2. A breeding method for the current resistance trait of large yellow croaker, characterized in that, The process using the SNP marker combination described in claim 1 includes the following steps: (1) Collect and preserve the basic population of large yellow croaker for breeding, and cultivate it into parent stock as candidate parent stock; (2) Samples were taken using a non-lethal sampling method, stored in anhydrous ethanol, and the anhydrous ethanol was replaced every 12 hours within 24 hours. (3) Extract DNA from parental samples, perform genotyping on the above SNP markers, and ensure that the genotyping of all SNP markers is successful; (4) Calculate the breeding value (GEBV) of the parent individuals based on the genotyping results; (5) Sort the large yellow croaker parent individuals according to their GEBV, select the top-ranked individuals as breeding parents for artificial breeding, and control the male-to-female ratio at 3:

1. The offspring obtained are the new germplasm of the current-resistant large yellow croaker.

3. The breeding method for the flow resistance trait of large yellow croaker according to claim 2, characterized in that, In step (1), the female-to-male ratio of the candidate parents is 3:1; In step (2), a non-lethal sampling method was used to collect fin rays samples from the end of the dorsal fin of the parent large yellow croaker; In step (3), DNA is extracted from the parent fin sample; In step (4), the Bayes Lasso model is used, and the breeding value GEBV of the parent individuals is calculated using the R language package BGLR.

4. The breeding method for the flow resistance trait of large yellow croaker according to claim 2, characterized in that, in, Males and females were sorted according to their GEBV levels, and the top 10% of individuals were selected as the breeding parent group, with a male-to-female ratio of 3:1.

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