Application of functional gene ghrb recombination variant molecular markers in hybrid bream breeding

Abstract

This invention discloses a functional gene grb Application of recombinant mutant molecular markers in hybrid bream breeding, this functional gene grb Recombinant mutant molecular markers are used to identify hybrid bream germplasm or screen backcross progeny of hybrid bream with fast growth traits, among which functional genes... grb Recombinant variant molecular markers are located in functional genes grb The base mutations at positions 2079, 2083, 2101, and 2161 are as follows: position 2079 is C or A, position 2083 is A or G, position 2101 is C or A, and position 2161 is G or C. This is the functional gene of the present invention. grb Recombinant mutant molecular markers can not only quickly identify the germplasm of hybrid bream, but also accurately screen backcross progeny of hybrid bream with fast growth traits, providing technical support for precision hybridization breeding and providing screening sites for the development of breeding chips.

Description

Technical Field

[0001] This invention belongs to the field of aquatic molecular breeding, specifically relating to a functional gene. ghrb Application of recombinant mutant molecular markers in hybrid bream breeding. Background Technology

[0002] Growth traits are complex economic traits influenced by multiple factors and are also one of the key traits of interest in aquaculture breeding. Traditionally, selection was primarily based on the phenotypic factor of individual size. With the development of high-throughput sequencing technology, researchers have discovered through genome resequencing that SNP loci associated with growth traits can serve as auxiliary screening markers. However, these molecular markers do not directly link growth traits to regulatory mechanisms, making the breeding process for obtaining superior germplasm with fast growth traits often lengthy and risky. Therefore, screening for functional gene molecular markers associated with growth traits can better address these bottlenecks.

[0003] Distant hybridization, as an important fish genetic breeding technique, can rapidly obtain offspring with hybrid vigor by integrating two different genomes. However, there is limited analysis of the theoretical genetic background of hybrid vigor in these offspring, and no stable recombination variation points of functional genes related to advantageous economic traits have been found. Therefore, it is not possible to theoretically better utilize hybrid vigor economic traits to establish precision hybridization breeding techniques.

[0004] Currently, one-step and multi-step breeding methods based on the genetic and reproductive patterns of fish, using distant hybridization, are important techniques in fish genetic breeding. These techniques can create high-quality diploid hybrid fish strains and also obtain polyploid fish strains, which can then be used to produce high-quality triploid fish. For high-quality diploid fish formed by one-step hybridization, such as *Brucea bream* and *Culter alburnus*, intergeneric distant hybridization (reciprocal crosses) yielded diploid *Culter alburnus* F1 and *Culter alburnus* F1, integrating the genomes of both parents. When judging hybrid germplasm, the primary method is chromosome karyotype, combined with morphological changes; alternatively, genetic variation detection using conserved sequences such as 5S rDNA can be used to identify hybrid germplasm from the perspectives of amplified band size and locus variation; alternatively, species-specific sequence probes can be used to determine hybrid germplasm through chromosome in situ hybridization, such as the red crucian carp-specific 263 probe used to detect hybrid germplasm with red crucian carp as the original parent. For high-quality diploid fish formed by multi-step hybridization, multiple crosses are performed with the original parents based on the first generation of hybridization. The heterozygosity of the genome decreases, making it unsuitable for identification by chromosome karyotype. Stable gene loci of variation have not yet been found. Most identification methods rely on morphological changes or breeding origin, which are relatively crude. There are currently no stable molecular markers that can be used for germplasm identification.

[0005] Hybrid offspring acquire desirable traits due to hybrid vigor, such as rapid growth, disease resistance, and tolerance to low oxygen levels. However, the genetic basis for these advantageous traits is rarely elucidated, making it difficult to reliably screen and identify hybrid individuals with these traits, or to obtain precise molecular markers to aid subsequent hybridization breeding. Therefore, there is an urgent need to find a functional gene recombination variation molecular marker to assist in the breeding of hybrid bream, enabling rapid identification of hybrid bream germplasm and rapid screening of individuals with rapid growth traits in backcross offspring. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a functional gene. ghrb Application of recombinant mutant molecular markers in hybrid bream breeding.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0008] A functional gene ghrb Application of recombinant mutant molecular markers in hybrid bream breeding: The functional gene ghrb recombinant mutant molecular marker is used to identify hybrid bream germplasm or screen backcross progeny of hybrid bream with fast growth traits; the functional gene... ghrb The recombinant variant is marked by a base mutation at positions 2079, 2083, 2101, and 2161 of the functional gene ghrb, wherein the base at position 2079 is C or A, the base at position 2083 is A or G, the base at position 2101 is C or A, and the base at position 2161 is G or C.

[0009] The above-described application, and further improvements, refers to the functional gene. ghrb When using recombinant mutant molecular markers to identify hybrid bream germplasm, the following steps are included:

[0010] (1.1) Extract genomic DNA from the fish species to be tested; the fish species to be tested is either the hybrid topmouth bream or the blunt snout bream;

[0011] (1.2) PCR amplification of the genomic DNA of the fish to be tested was performed using specific primers to obtain the amplification products;

[0012] (1.3) Sequencing the amplified products to obtain a sequencing peak diagram;

[0013] (1.4) Genotyping is performed on the sequencing peak diagram. If functional genes ghrb If the recombinant variant molecular markers all show bimodal peaks in the sequencing peak diagram, then the fish being tested is a hybrid topmouth bream. If the functional gene... ghrb If the recombinant mutant molecular markers are all single peaks in the sequencing peak diagram, then the fish species to be tested is blunt snout bream;

[0014] The above-described application, and further improvements, refers to the functional gene. ghrb When using recombinant mutant molecular markers to screen for backcross progeny of hybrid bream with fast growth traits, the following steps are included:

[0015] (2.1) Extract genomic DNA from the fish to be tested; the fish to be tested are the offspring of a backcross of hybrid bream;

[0016] (2.2) PCR amplification of the genomic DNA of the fish to be tested was performed using specific primers to obtain the amplification products;

[0017] (2.3) Sequencing the amplified products to obtain a sequencing peak diagram;

[0018] (2.4) Genotyping is performed on the sequencing peak diagram. If functional genes ghrb If the recombinant variant molecular markers all show bimodal peaks in the sequencing peak diagram, then the fish being tested is a backcross progeny of a hybrid bream with a fast-growing trait.

[0019] In a further improvement to the above application, the maternal parent of the backcross offspring of the hybrid bream is the hybrid bream, and the paternal parent of the backcross offspring of the hybrid bream is the blunt-snout bream.

[0020] The above-described application, and further improvements, refers to the functional gene. ghrb The nucleotide sequence is shown in SEQ ID NO.1.

[0021] In a further improvement to the above application, the specific primer includes an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] This invention provides a functional gene ghrb Application of recombinant mutant molecular markers in hybrid bream breeding, this functional gene ghrb When recombinant mutant molecular markers are used for germplasm identification or breeding screening, they can not only quickly identify the germplasm of hybrid bream, but also accurately screen backcross progeny of hybrid bream with fast growth traits. This can provide technical support for precision hybridization breeding and provide screening sites for the development of breeding chips. Attached Figure Description

[0024] Figure 1 The image shows the morphological characteristics of the hybrid bream and blunt snout bream in Example 1 of this invention.

[0025] Figure 2 Functional gene of Example 1 of the present invention ghrbA map showing the amino acid distribution of recombinant mutant molecular markers on proteins.

[0026] Figure 3 The functional gene of the hybrid bream in Example 1 of this invention ghrb Sequencing peak diagram of the recombinant region.

[0027] Figure 4 Functional genes of blunt snout bream in Example 1 of this invention ghrb Sequencing peak diagram of the recombinant region.

[0028] Figure 5 This is a growth curve diagram of the hybrid bream and blunt snout bream in Example 1 of the present invention.

[0029] Figure 6 This is a route diagram for the backcrossing of hybrid bream in Example 2 of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0031] Example 1

[0032] A functional gene ghrb The application of recombinant molecular markers in hybrid bream breeding, when the functional gene ghrb recombinant molecular marker is used to identify hybrid bream germplasm, includes the following steps:

[0033] (1) Functional genes ghrb Acquisition of recombinant mutant molecular markers

[0034] (1.1) Using *Culter alburnus* as the female parent and *Culter chinensis* as the male parent, intergeneric distant hybridization was performed to obtain *Culter alburnus* F1; using female diploid fish from *Culter alburnus* F1 as the female parent and *Culter alburnus* as the male parent, backcrossing was performed to obtain hybrid *Culter chinensis*, such as... Figure 1 As shown. Among them, the hybrid bream has been approved by the National Aquatic Original and Improved Variety Approval Committee and obtained the new aquatic variety certificate, with the variety registration number GS-02-003-2014.

[0035] This hybrid topmouth bream has silvery-gray scales on its sides, a slightly darker back, and a pale silvery-white belly. Its body is elongated and laterally compressed, with a raised back. The head is relatively small, rising rapidly in an arc, with a superior mouth and a strong, upturned lower jaw positioned diagonally in front of the mouth. This hybrid topmouth bream combines the physical characteristics of both parents, featuring a high back and an upturned mouth. Furthermore, it exhibits herbivorous feeding habits, rapid growth, and high-quality meat, demonstrating hybrid vigor and making it a high-quality new freshwater aquaculture species.

[0036] (1.2) Six hybrid bream of the same age and six bream of the same age were taken, and transcriptome sequencing was performed on growth-related tissues (hypothalamus, liver and muscle). Then, in-depth analysis was performed on recombination variant sites on the exons of functional genes expressed in these tissues. A total of 1073 recombination variant sites were present in all samples of bream but not in bream, mainly concentrated on chromosome NC_063061.1 (981 sites).

[0037] Functional genes ghrb As a growth hormone receptor, it is one of the key genes in the classic growth hormone action pathway. Functional genes in hybrid bream. ghrb Sixteen recombination mutation sites were found on the cDNA, three in the 3'UTR region and thirteen in the open reading frame (ORF) coding region. Among the recombination mutation sites in the coding region, there were six synonymous mutation sites and seven missense mutation sites. The seven missense mutation sites are located in functional genes... ghrb The positions 723 (A→T), 974 (A→C), 2079 (C→A), 2083 (A→G), 2101 (C→A), 2161 (G→C), and 2328 (C→G) are shown in Table 1. For functional genes... ghrb Predictions were made about the conserved domains of the protein, and missense mutations at positions 2079 (C→A), 2083 (A→G), 2101 (C→A), and 2161 (G→C) were found within the conserved domain (GHBP domain), which may affect the function of the protein, such as... Figure 2 As shown. Analysis of differential gene expression levels in the transcriptome revealed... ghrb The gene is highly expressed primarily in the liver and muscle, and less so in the hypothalamus. Furthermore, comparing the two fish species, there was no significant difference in gene expression in the liver between the hybrid topmouth bream and the bluntnose bream, but the expression level of this gene in the muscle of the hybrid topmouth bream was much higher than that of the bluntnose bream, corresponding to the rapid growth phenotype of the hybrid topmouth bream, as shown in Table 2. Therefore, functional genes... ghrb The recombinant variants were marked by base mutations at positions 2079, 2083, 2101, and 2161 of the functional gene ghrb, corresponding to bases C / A, A / G, C / A, and G / C.

[0038] Table 1 Functional genes in hybrid bream ghrb Distribution of recombinant variant sites

[0039]

[0040] Table 2 Functional Genes ghrb Expression changes in three tissues of blunt snout bream and hybrid topmouth bream

[0041]

[0042] Functional genes ghrb The nucleotide sequence is shown in SEQ ID NO.1, specifically as follows:

[0043]

[0044] (2) Germplasm identification of hybrid bream

[0045] (2.1) Extraction of genomic DNA from hybrid bream and blunt snout bream

[0046] Three batches of hybrid bream (30 bream from each of the three different batches) were randomly selected. Caudal fin tissue was cut and placed in 1.5 mL centrifuge tubes. Whole-genome DNA was extracted using a genomic DNA extraction kit. The obtained DNA samples were stored at -20℃, and the OD500 of the extracted DNA was measured using a microplate reader. 260 / OD 280 The ratio and concentration were determined, and DNA templates with a ratio of approximately 1.8 were selected for subsequent experiments.

[0047] (2.2) PCR amplification

[0048] Based on the distribution of recombination variant sites in stable missense mutations, according to functional genes ghrb Specific primers were designed based on the corresponding genomic sequence, including upstream primer F and downstream primer R; using the genomic DNA of the hybrid bream to be tested as a template, PCR amplification was performed using upstream primer F and downstream primer R to obtain PCR products.

[0049] The upstream primer F is shown in SEQ ID NO.2, specifically as follows:

[0050] 5'-GGAAGAGCAGGAAAGAATGAAAC-3'.

[0051] The downstream primer R is shown in SEQ ID NO.3, specifically as follows:

[0052] 5'-CCTTCTGTTGGGTGGTTGGTG-3'.

[0053] PCR amplification system: The total reaction volume is 20 μL, including 10 μL of 2×Rapid Taq Master Mix (Dye), 1 μL of Primer F, 1 μL of Primer R, 7 μL of Water, and 1 μL of genomic DNA.

[0054] PCR amplification program: 95℃ for 5 min; 35 cycles, including 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s; end at 72℃ for 10 min.

[0055] (2.3) Sanger sequencing analysis

[0056] The PCR products were purified by agarose gel electrophoresis and then recovered from the gel. The purified PCR products were then directly subjected to Sanger sequencing using upstream primer F as the sequencing primer to obtain a sequencing peak diagram. Germplasm was identified based on the sequencing peak diagram results. If functional genes... ghrb If the sequencing peaks at positions 2079 (C / A), 2083 (A / G), 2101 (C / A), and 2161 (G / C) are all bimodal, then the detected germplasm is a hybrid of the common bream. Figure 3 As shown; if the above functional genes ghrb If all four sequencing peaks are single peaks (C / A / C / G), then the detected germplasm is *Brucea buergeriana*. Figure 4 As shown.

[0057] A comparative analysis of the growth rates of hybrid topmouth bream and bluntnose bream was conducted, and the results are as follows: Figure 5 As shown in Table 3.

[0058] Table 3. Comparison of growth rates between hybrid topmouth bream and blunt snout bream.

[0059]

[0060] Combination Figure 5 As shown in Table 3, the average weight of first-year-old blunt-snout bream reached 287.5±24.6g, and the average weight of first-year-old (12-month-old) hybrid topmouth bream reached 312.9±33.5g, with a higher weight growth rate of 8.8%. The average weight of second-year-old (24-month-old) blunt-snout bream reached 895.5±90.3g, and the average weight of second-year-old hybrid topmouth bream reached 1086.6±115.2g, with a higher weight growth rate of 21.3%. Clearly, the hybrid topmouth bream exhibited the hybrid vigor trait of rapid growth, proving the existence of functional genes. ghrb Recombinant variant molecular markers are also associated with growth traits.

[0061] Example 2

[0062] A functional gene ghrb Application of recombinant mutant molecular markers in hybrid bream breeding, when functional genes ghrb When using recombinant mutant molecular markers to identify backcross progeny of hybrid bream with the fast growth trait, the following steps are included:

[0063] (1) Backcrossing of hybrid bream

[0064] Using hybrid topmouth bream as the female parent and blunt-snout bream as the male parent, backcrossing was performed to obtain a backcross offspring population of hybrid topmouth bream, such as... Figure 6 As shown, this hybrid bream (i.e., the offspring of a backcross of a hybrid mandarin fish) is still a hybrid source of the bream.

[0065] (2) Extraction of genomic DNA from the backcross progeny of hybrid bream

[0066] Thirty backcross progeny of hybrid bream were randomly selected, and the tail fin tissue was cut and placed in a 1.5 mL centrifuge tube. Whole genomic DNA was extracted using a genomic DNA extraction kit. The obtained DNA samples were stored at -20℃, and the OD260 / OD280 ratio and concentration of the extracted DNA were measured using an enzyme-linked immunosorbent assay (ELISA) reader. DNA templates with a ratio of around 1.8 were selected for subsequent experiments.

[0067] (3) PCR amplification

[0068] Using the genomic DNA of the backcross progeny of the hybrid bream as a template, PCR amplification was performed using the specific primers in Example 1 to obtain the PCR product.

[0069] PCR amplification system: The total reaction volume is 20 μL, including 10 μL of 2×Rapid Taq Master Mix (Dye), 1 μL of Primer F, 1 μL of Primer R, 7 μL of Water, and 1 μL of genomic DNA.

[0070] PCR amplification program: 95℃ for 5 min; 35 cycles, including 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s; end at 72℃ for 10 min.

[0071] (4) Sanger sequencing typing

[0072] The PCR products were purified by agarose gel electrophoresis and then recovered from the gel. The purified PCR products were then directly sequenced using the upstream primer F from Example 1 as the sequencing primer. The backcross progeny of the hybrid bream exhibiting rapid growth traits was identified based on the sequencing peak results. (The text then abruptly shifts to a seemingly unrelated topic: functional genes...) ghrb The sequencing peaks at positions 2079 (C / A), 2083 (A / G), 2101 (C / A), and 2161 (G / C) were all bimodal, and this was named the heterozygous type. This heterozygous basal type was a backcross progeny of a hybrid bream with fast growth traits; if the above functional genes ghrb The sequencing peaks at all four sites were single peaks (C / A / C / G), and were named homozygous.

[0073] The weight of individuals with different subtypes was measured, and the results are shown in Table 4.

[0074] Table 4 Functional genes in the backcross progeny of hybrid bream ghrb Correlation between recombinant variant molecular marker typing and body weight

[0075]

[0076] In Table 4, ** indicates that the weight of heterozygous individuals is significantly higher than that of homozygous individuals. P <0.01).

[0077] As shown in Table 4, the body weight of individuals corresponding to the heterozygous variety was significantly higher than that of individuals corresponding to the homozygous variety, proving that the functional gene ghrb recombination variant molecular marker can be used to screen for high-quality hybrid bream with the fast growth advantage from the hybrid bream-Culter alburnus source.

[0078] As can be seen from the above results, in this invention, functional genes ghrb When recombinant mutant molecular markers are used for germplasm identification or breeding screening, they can not only quickly identify the germplasm of hybrid bream, but also accurately screen backcross progeny of hybrid bream with fast growth traits. This can provide technical support for precision hybridization breeding and provide screening sites for the development of breeding chips.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A functional gene ghrb The application of recombinant mutant molecular markers in the breeding of hybrid bream is characterized by, The functional genes ghrb Recombinant mutant molecular markers are used to identify hybrid bream germplasm or screen backcross progeny of hybrid bream with fast growth traits; the functional genes ghrb Recombinant variant molecular markers are located in functional genes ghrb Mutations at positions 2079, 2083, 2101, and 2161, wherein position 2079 is C or A, position 2083 is A or G, position 2101 is C or A, and position 2161 is G or C; the functional gene ghrb The nucleotide sequence is shown in SEQ ID NO.1; The functional genes ghrb When recombinant variant molecular markers are used to identify hybrid bream germplasm, the fish species to be tested must be either hybrid bream or blunt snout bream. The functional genes ghrb When recombinant mutant molecular markers are used to screen backcross progeny of hybrid bream with fast growth traits, the fish to be tested are backcross progeny of hybrid bream; the maternal parent of the backcross progeny of hybrid bream is hybrid bream, and the paternal parent of the backcross progeny of hybrid bream is blunt-snout bream.

2. The functional gene according to claim 1 ghrb The application of recombinant mutant molecular markers in the breeding of hybrid bream is characterized by, The functional genes ghrb When using recombinant mutant molecular markers to identify hybrid bream germplasm, the following steps are included: (1.1) Extract genomic DNA from the fish to be tested; (1.2) PCR amplification of the genomic DNA of the fish to be tested was performed using specific primers to obtain the amplification products; (1.3) Sequencing the amplified products to obtain a sequencing peak diagram; (1.4) Genotyping is performed on the sequencing peak diagram. If functional genes ghrb If the recombinant variant molecular markers all show bimodal peaks in the sequencing peak diagram, then the fish being tested is a hybrid topmouth bream. If the functional gene... ghrb If the recombinant mutant molecular markers are all single peaks in the sequencing peak diagram, then the fish species to be tested is blunt snout bream.

3. The functional gene according to claim 1 ghrb The application of recombinant mutant molecular markers in the breeding of hybrid bream is characterized by, The functional genes ghrb When using recombinant mutant molecular markers to screen backcross progeny of hybrid bream with fast growth traits, the following steps are included: (2.1) Extract genomic DNA from the fish to be tested; (2.2) PCR amplification of the genomic DNA of the fish to be tested was performed using specific primers to obtain the amplification products; (2.3) Sequencing the amplified products to obtain a sequencing peak diagram; (2.4) Genotyping is performed on the sequencing peak diagram. If functional genes ghrb If the recombinant variant molecular markers all show bimodal peaks in the sequencing peak diagram, then the fish being tested is a backcross progeny of a hybrid bream with a fast-growing trait.

4. The functional gene according to any one of claims 2 to 3 ghrb The application of recombinant mutant molecular markers in the breeding of hybrid bream is characterized by, The specific primers include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

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