DNA molecule module, primer pair for screening alkali-tolerant fast-growing grass carp and application thereof

Abstract

The application discloses a DNA molecular module for screening alkali-tolerant fast-growing grass carps, a primer pair and application thereof, and belongs to the field of biomolecular markers. In view of the problems of long breeding cycle of grass carps and unsuitability of large parent fish for alkali tolerance test, the application provides the DNA molecular module for screening alkali-tolerant fast-growing grass carps, which is composed of eight SNP molecular markers, namely, SNP01-SNP08 molecular markers (SEQ ID NO. 1-8), and a primer pair (SEQ ID NO. 9-18) for amplifying the above molecular module. The DNA molecular module and the primer pair are used for identifying the alkali-tolerant fast-growing grass carp type from the genetic level, avoiding the problems of contingency and inaccuracy of judging whether the grass carp has the alkali-tolerant fast-growing characteristics by the survival state, and can be used for early selection, shortening of the breeding time, simple operation, high breeding accuracy and efficiency, and promoting the breeding process of the alkali-tolerant fast-growing grass carp germplasm.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular markers, specifically relating to a highly efficient and low-damage DNA molecular module, primer pair, and their applications for screening alkali-resistant, fast-growing grass carp. Background Technology

[0002] Currently, the shortage of freshwater aquaculture resources has become a significant limiting factor for the development of aquaculture. Developing and utilizing saline-alkali water and soil resources is one of the effective ways to expand the space for aquaculture. High alkalinity easily leads to alkali poisoning and ammonia poisoning in fish, seriously affecting their survival, growth, and reproduction. Selecting and breeding new, fast-growing, and high-quality fish species that can adapt to saline-alkali water bodies, developing saline-alkali fisheries, and implementing "using fish to control alkali" are important measures to realize the grand food concept of "obtaining food from rivers, lakes, and seas" and "obtaining protein from saline-alkali lands."

[0003] Grass carp is an important freshwater aquaculture species with a wide distribution and high yield. Evaluation of grass carp's alkali tolerance from physiological indicators and proteomics perspectives revealed that it can generally adapt to artificially prepared water with an alkalinity of 20 mM sodium bicarbonate, indicating potential for breeding new alkali-tolerant and fast-growing varieties. However, grass carp have a long breeding cycle, typically reaching sexual maturity in 4-6 years. Furthermore, the competitive feeding behavior caused by intensive feed distribution can lead to the selection of fast-growing, nutritionally oriented individuals through traditional breeding processes targeting growth traits, making it difficult to fix the genetic characteristics of economically important traits. Therefore, existing conventional breeding methods are relatively inefficient in improving the alkali tolerance and growth performance of grass carp.

[0004] With the rapid development of high-throughput sequencing technology, modern breeding in aquaculture is entering a stage of precision molecular design breeding centered on genomic selection (GS). Most economic traits in fish are regulated by multiple genes and possess a "modular" characteristic. Genomic selection breeding constructs multi-gene aggregated molecular modules by detecting molecular marker sets and genotypes of single nucleotide polymorphisms (SNPs) closely linked to the target trait, enabling rapid screening of target economic traits. Summary of the Invention

[0005] This invention addresses the problems of long breeding cycles for grass carp and the unsuitability of large broodstock for alkali tolerance testing by providing a DNA molecular module, primer pairs, and their applications for screening alkali-tolerant, fast-growing grass carp.

[0006] One of the objectives of this invention is to provide a DNA molecular module for screening alkali-tolerant, fast-growing grass carp, wherein the DNA molecular module consists of 8 SNP molecular markers, namely SNP01-SNP08 molecular markers. The SNP01 molecular marker is located at base 2320551 on chromosome 4 of grass carp. This base position exhibits T / C polymorphism, and the genotype is TT, TC, or CC. The SNP02 molecular marker is located at base 29280354 on chromosome 4 of grass carp. This base position exhibits T / C polymorphism, and the genotype is TT, TC, or CC. The SNP03 molecular marker is located at base 1646710 on chromosome 10 of grass carp. This base position exhibits A / G polymorphism, and the genotype is AA, AG, or GG. The SNP04 molecular marker is located at base 15475728 on chromosome 14 of grass carp. This base position exhibits A / C polymorphism, and the genotype is AA, AC, or CC. The SNP05 molecular marker is located at base 15475886 on chromosome 14 of grass carp. This base position exhibits C / T polymorphism, and the genotype is CC, CT, or TT. The SNP06 molecular marker is located at base 15,475,888 on chromosome 14 of grass carp. This base position exhibits A / T polymorphism, and the genotype is AA, AT, or TT. The SNP07 molecular marker is located at base 15475972 on chromosome 14 of grass carp. This base position exhibits T / A polymorphism, and the genotype is TT, TA, or AA. The SNP08 molecular marker is located at base 9539862 on chromosome 24 of grass carp. This base position exhibits G / A polymorphism, with genotypes of GG, GA, or AA.

[0007] In a preferred embodiment of the present invention, the nucleotide sequences of 400 bp upstream and downstream of the SNP01 molecular marker site are SEQ ID NO.1; The nucleotide sequences 400 bp upstream and downstream of the SNP02 molecular marker site are SEQ ID NO.2; The nucleotide sequences 400 bp upstream and downstream of the SNP03 molecular marker site are SEQ ID NO.3; The nucleotide sequences 400 bp upstream and downstream of the SNP04 molecular marker site are SEQ ID NO.4; The nucleotide sequences 400 bp upstream and downstream of the SNP05 molecular marker site are SEQ ID NO.5; The nucleotide sequences 400 bp upstream and downstream of the SNP06 molecular marker site are SEQ ID NO.6; The nucleotide sequences 400 bp upstream and downstream of the SNP07 molecular marker site are SEQ ID NO.7; The nucleotide sequences 400 bp upstream and downstream of the SNP08 molecular marker site are SEQ ID NO.8.

[0008] The second objective of this invention is to provide a primer pair for amplifying the above-mentioned DNA molecular module, wherein the primer pair includes primer pairs for amplifying the above-mentioned SNP01-SNP08 molecular markers respectively. The primer pair used to amplify the SNP01 molecular marker includes the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10; The primer pair used to amplify the SNP02 molecular marker includes the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.12; The primer pair used to amplify the SNP03 molecular marker includes the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14; The primer pair used to amplify the molecular markers SNP04, SNP05, SNP06, or SNP07 includes the forward primer shown in SEQ ID NO. 15 and the reverse primer shown in SEQ ID NO. 16; The primer pair used to amplify the SNP08 molecular marker includes the forward primer shown in SEQ ID NO.17 and the reverse primer shown in SEQ ID NO.18.

[0009] A third objective of this invention is to provide a kit for screening alkali-tolerant, fast-growing grass carp, the kit comprising the aforementioned primer pairs.

[0010] The fourth objective of this invention is to provide a method for screening alkali-tolerant, fast-growing grass carp broodstock, the method comprising the following steps: S1: Extract genomic DNA from the grass carp to be tested; S2: Using the grass carp genomic DNA obtained in S1 as a template, PCR amplification was performed using the primer pairs described above. S3: The amplification products obtained in S2 were subjected to Sanger sequencing analysis, and the alkali-tolerant fast-growing grass carp were screened based on the sequencing results.

[0011] In a preferred embodiment of the present invention, the PCR amplification system in S2 is as follows: 2 μL DNA template, 0.1 μL forward primer at a concentration of 10 μM, 0.1 μL reverse primer at a concentration of 10 μM, 12.5 μL Premix Ex Taq, and 8.5 μL ddH2O.

[0012] In a preferred embodiment of the present invention, the PCR amplification conditions in S2 are as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 30 s, 72°C extension for 30 s, for a total of 30 cycles; 72°C extension for 7 min.

[0013] In a preferred embodiment of the present invention, the criteria for screening alkali-tolerant, fast-growing grass carp based on sequencing results in step S3 are as follows: For the SNP01 molecular marker, when the genotype is TT, the grass carp being tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP02 molecular marker, when the genotype is TT, the grass carp being tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP03 molecular marker, when the genotype is AA, the grass carp to be tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP04 molecular marker, when the genotype is AA, the grass carp being tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP05 molecular marker, when the genotype is CC, the grass carp to be tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP06 molecular marker, when the genotype is AA, the grass carp to be tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP07 molecular marker, when the genotype is TT, the grass carp being tested is determined to be an alkali-tolerant, fast-growing grass carp. For the SNP08 molecular marker, when the genotype is GG, the grass carp being tested is identified as an alkali-tolerant, fast-growing grass carp.

[0014] The fifth objective of this invention is to provide the application of the above-mentioned DNA molecular modules, primer pairs or kits in screening alkali-tolerant and fast-growing grass carp germplasm.

[0015] In a preferred embodiment of the present invention, the criterion for determining the fast-growing grass carp is as follows: based on the grass carp weight index, grass carp individuals with a weight ≥ average weight + standard deviation are classified as fast-growing grass carp, and individuals with a weight < average weight - standard deviation are classified as slow-growing grass carp; the average weight refers to the average weight of a grass carp group of ≥400 fish.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: Addressing the problems of long breeding cycles for grass carp and the unsuitability of large broodstock for alkali tolerance testing, this invention provides a DNA molecular module for screening alkali-tolerant, fast-growing grass carp. This module consists of eight SNP molecular markers, namely SNP01-SNP08 (SEQ ID NO. 1-8). It also provides primer pairs (SEQ ID NO. 9-18) for amplifying the aforementioned DNA molecular module, and a method for screening alkali-tolerant, fast-growing grass carp germplasm. This method causes minimal damage to the fish, eliminates the need for alkali tolerance testing, and only requires the removal of a small amount of fin rays, exhibiting precision, efficiency, and low damage. Furthermore, this method can significantly shorten the grass carp breeding cycle (by at least one generation, or 4-6 years), accelerating the breeding of new alkali-tolerant, fast-growing grass carp strains.

[0017] The DNA molecular module provided by this invention utilizes the difference in mutated bases to perform Sanger assays on PCR amplification products, each 400 bp in length, and performs genotyping based on the sequencing results. Specifically, for the SNP01 molecular marker, when the genotype is TT; for the SNP02 molecular marker, when the genotype is TT; for the SNP03 molecular marker, when the genotype is AA; for the SNP04 molecular marker, when the genotype is AA; for the SNP05 molecular marker, when the genotype is CC; for the SNP06 molecular marker, when the genotype is AA; for the SNP07 molecular marker, when the genotype is TT; and for the SNP08 molecular marker, when the genotype is GG, the grass carp being tested is identified as an alkaline-tolerant, fast-growing grass carp.

[0018] The DNA molecular module and primer pairs provided by this invention for screening alkali-tolerant and fast-growing grass carp are identified at the gene level, avoiding the randomness and inaccuracy of judging whether grass carp have alkali-tolerant and fast-growing characteristics based on their survival status. Furthermore, this method is not limited by age or sex, can be used for early selection, shortens the breeding time, is easy to operate, and has high breeding accuracy and efficiency, thus promoting the breeding process of new alkali-tolerant and fast-growing grass carp strains. Attached Figure Description

[0019] Figure 1 Weight distribution diagrams for fast-growing and slow-growing groups of different grass carp populations; Figure 2 The distribution of dominant genotypes in the fast-growing and slow-growing groups of different grass carp populations is shown in the figure. Detailed Implementation

[0020] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0022] Example 1: A DNA molecular module and primer pair for screening alkali-tolerant, fast-growing grass carp 1. A DNA molecular module for screening alkali-tolerant, fast-growing grass carp Genome resequencing was performed on alkali-tolerant grass carp from Heilongjiang Province (provided by Nashui Agricultural Development Co., Ltd., Zhaodong City, Heilongjiang Province) and fast-growing grass carp (Husu No. 1) purchased from Suzhou Shenhang Ecological Technology Development Co., Ltd. Based on the resequencing results, DNA molecular modules were screened. These modules consisted of eight SNP molecular markers: SNP01-SNP08. The nucleotide sequences of the SNP01 marker site (400 bp upstream and downstream) are SEQ ID NO.1; the SNP02 marker site (400 bp upstream and downstream) are SEQ ID NO.2; the SNP03 marker site (400 bp upstream and downstream) are SEQ ID NO.3; the SNP04 marker site (400 bp upstream and downstream) are SEQ ID NO.4; the SNP05 marker site (400 bp upstream and downstream) are SEQ ID NO.5; and the SNP06 marker site (400 bp upstream and downstream) are SEQ ID NO. NO.6; the nucleotide sequences of 400 bp upstream and downstream of the SNP07 molecular marker site are SEQ ID NO.7; the nucleotide sequences of 400 bp upstream and downstream of the SNP08 molecular marker site are SEQ ID NO.8.

[0023] The specific steps for screening to obtain DNA molecular modules are as follows: Forty grass carp were randomly selected from both the alkali-tolerant grass carp population in Heilongjiang and the fast-growing grass carp population of Husu No. 1, and their weight was measured. The growth type of the grass carp was determined according to the following criteria: grass carp with a weight ≥ average weight + standard deviation were classified as fast-growing grass carp, and grass carp with a weight < average weight - standard deviation were classified as slow-growing grass carp. The average weight refers to the average weight of the grass carp population ≥ 400.

[0024] Fin tissues were excised from all grass carp individuals, and grass carp DNA was extracted from the fin tissues using the phenol-chloroform method. The extracted grass carp DNA, after passing quality tests, was sent to Beijing Novogene Technology Co., Ltd. for whole-genome resequencing. The raw data was filtered using FASTP software and aligned to the reference genome using BWA software. The alignment results were then subjected to SAMTOOLS to remove duplicates, and population SNPs were detected using SAMTOOLS. A Bayesian model was used to detect polymorphic sites in the population. After filtering and screening, a total of 4,369,521 high-quality SNPs were obtained. The filtering parameters were: sequencing depth <3, MAF (Minor allele frequency) >0.05, Miss >0.1. Finally, ANNOVAR software was used to annotate the SNPs to obtain the functional gene information closest to the SNP sites. Combining the grass carp phenotype with the resequencing and correlation analysis results, eight SNP molecular markers related to the alkali-tolerant growth of grass carp were screened out, namely SNP01-SNP08.

[0025] The method for detecting grass carp DNA is as follows: The grass carp DNA extracted above was subjected to PCR amplification. The PCR amplification products were detected by 1% agarose gel electrophoresis. The qualified products were then subjected to Sanger sequencing.

[0026] The PCR amplification system consisted of: 2 μL DNA template, 0.1 μL forward primer (10 μM), 0.1 μL reverse primer (10 μM), 12.5 μL Premix Ex Taq, and 8.5 μL ddH2O. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 30 cycles; and 72℃ extension for 7 min.

[0027] 2. Primer pairs used to amplify DNA molecular modules Using 400bp sequences upstream and downstream of the SNPs extracted from the reference genome as templates, specific primers were designed using the Primer-BLAST online tool on the ncbi website to obtain primer pairs for amplifying DNA molecular modules. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] The primer pair for amplifying the SNP01 molecular marker includes the forward primer shown in SEQ ID NO. 9 and the reverse primer shown in SEQ ID NO. 10; the primer pair for amplifying the SNP02 molecular marker includes the forward primer shown in SEQ ID NO. 11 and the reverse primer shown in SEQ ID NO. 12; the primer pair for amplifying the SNP03 molecular marker includes the forward primer shown in SEQ ID NO. 13 and the reverse primer shown in SEQ ID NO. 14; the primer pair for amplifying the SNP04, SNP05, SNP06, or SNP07 molecular markers includes the forward primer shown in SEQ ID NO. 15 and the reverse primer shown in SEQ ID NO. 16; and the primer pair for amplifying the SNP08 molecular marker includes the forward primer shown in SEQ ID NO. 17 and the reverse primer shown in SEQ ID NO. 18.

[0029] Example 2: A method for screening alkali-tolerant, fast-growing grass carp germplasm. 1. Selection of test materials The test material was selected from 400 grass carp from a random population in Lianhuan Lake, Heilongjiang Province (20 mM carbonate alkalinity) in 2024, with a culture period of 4 months.

[0030] 2. Detection of physiological indicators and acquisition of DNA The weight index of the grass carp population obtained above was measured, and the growth type of grass carp was determined according to the following criteria: grass carp individuals with a weight ≥ average weight + standard deviation were classified as fast-growing grass carp, and individuals with a weight < average weight - standard deviation were classified as slow-growing grass carp; the average weight refers to the average weight of a grass carp population of ≥400 individuals.

[0031] Fin tissue was excised from all the above-mentioned grass carp individuals, and grass carp DNA was extracted from the fin tissue using the phenol-chloroform method. After the extracted grass carp DNA passed the test, it was sent to Beijing Novogene Technology Co., Ltd. for Sanger sequencing.

[0032] The method for detecting grass carp DNA is as follows: The grass carp DNA extracted above was amplified by PCR using the primers shown in Table 1. The PCR amplification products were detected by 1% agarose gel electrophoresis. The qualified products were then subjected to Sanger sequencing.

[0033] The PCR amplification system consisted of: 2 μL DNA template, 0.1 μL forward primer (10 μM), 0.1 μL reverse primer (10 μM), 12.5 μL Premix Ex Taq, and 8.5 μL ddH2O. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 30 cycles; and 72℃ extension for 7 min.

[0034] Table 1

[0035] 3. Genotyping Based on the above Sanger sequencing results, the DNA molecular module was genotyped, and the results are shown in Table 2. For the SNP01 molecular marker site, the fast-growing group was mainly composed of individuals with the TT genotype, while the slow-growing group was mainly composed of individuals with the TC genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TC or CC genotypes. For the SNP02 molecular marker site, the fast-growing group was dominated by individuals with the TT genotype, while the slow-growing group was dominated by individuals with the TC genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TC or CC genotypes. For the SNP03 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AG genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AG genotype. For the SNP04 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AC genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AC or CC genotypes. For the SNP05 molecular marker site, the fast-growing group was dominated by individuals with the CC genotype, while the slow-growing group was dominated by individuals with the CT genotype. This indicates that individuals carrying the CC genotype at this site have better alkali-tolerant growth performance than individuals carrying the CT or TT genotypes. For the SNP06 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AT genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AT or TT genotypes. For the SNP07 molecular marker site, the fast-growing group was dominated by individuals with the TT genotype, while the slow-growing group was dominated by individuals with the TA genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TA or AA genotypes. For the SNP08 molecular marker site, the fast-growing group was dominated by individuals with the GG genotype, while the slow-growing group was dominated by individuals with the GA genotype. This indicates that individuals carrying the GG genotype at this site have better alkali-tolerant growth performance than individuals carrying the GA or AA genotypes.

[0036] Table 2

[0037] Example 3: Application of a DNA molecular module and primer pair for screening alkali-tolerant, fast-growing grass carp germplasm. 1. Selection of test materials The test material was selected from a random grass carp population (number of fish: 400) raised in Lianhuan Lake, Heilongjiang Province (20 mM carbonate alkalinity) in 2024, with a culture period of 4 months.

[0038] 2. Detection of physiological indicators and acquisition of DNA The weight index of the grass carp population obtained above was measured, and the growth type of grass carp was determined according to the following criteria: grass carp individuals with a weight ≥ average weight + standard deviation were classified as fast-growing grass carp, and individuals with a weight < average weight - standard deviation were classified as slow-growing grass carp; the average weight refers to the average weight of a grass carp population of ≥400 individuals.

[0039] Fin tissue was excised from all the above-mentioned grass carp individuals, and grass carp DNA was extracted from the fin tissue using the phenol-chloroform method. After the extracted grass carp DNA passed the test, it was sent to Beijing Novogene Technology Co., Ltd. for whole genome resequencing.

[0040] The method for detecting grass carp DNA is as follows: The grass carp DNA extracted above was amplified by PCR using the primers shown in Table 1. The PCR amplification products were detected by 1% agarose gel electrophoresis. The qualified products were then subjected to Sanger sequencing.

[0041] The PCR amplification system consisted of: 2 μL DNA template, 0.1 μL forward primer (10 μM), 0.1 μL reverse primer (10 μM), 12.5 μL Premix Ex Taq, and 8.5 μL ddH2O. The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 30 cycles; and 72℃ extension for 7 min.

[0042] 3. Genotyping Based on the above Sanger sequencing results, the DNA molecular module was genotyped, and the results are shown in Table 3. For the SNP01 molecular marker site, the fast-growing group was mainly composed of individuals with the TT genotype, while the slow-growing group was mainly composed of individuals with the TC genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TC or CC genotypes. For the SNP02 molecular marker site, the fast-growing group was dominated by individuals with the TT genotype, while the slow-growing group was dominated by individuals with the TC genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TC or CC genotypes. For the SNP03 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AG genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AG genotype. For the SNP04 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AC genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AC or CC genotypes. For the SNP05 molecular marker site, the fast-growing group was dominated by individuals with the CC genotype, while the slow-growing group was dominated by individuals with the CT genotype. This indicates that individuals carrying the CC genotype at this site have better alkali-tolerant growth performance than individuals carrying the CT or TT genotypes. For the SNP06 molecular marker site, the fast-growing group was dominated by individuals with the AA genotype, while the slow-growing group was dominated by individuals with the AT genotype. This indicates that individuals carrying the AA genotype at this site have better alkali-tolerant growth performance than individuals carrying the AT or TT genotypes. For the SNP07 molecular marker site, the fast-growing group was dominated by individuals with the TT genotype, while the slow-growing group was dominated by individuals with the TA genotype. This indicates that individuals carrying the TT genotype at this site have better alkali-tolerant growth performance than individuals carrying the TA or AA genotypes. For the SNP08 molecular marker site, the fast-growing group was dominated by individuals with the GG genotype, while the slow-growing group was dominated by individuals with the GA genotype. This indicates that individuals carrying the GG genotype at this site have better alkali-tolerant growth performance than individuals carrying the GA or AA genotypes.

[0043] Table 3

[0044] This invention analyzes the weight detection indicators of different grass carp groups in Examples 2 and 3, and the results are as follows: Figure 1 The figure shows the weight distribution of fast-growing and slow-growing groups in random grass carp populations in Wuhan and Zhaodong. In the Wuhan population, the average weight of the fast-growing group was 184.17 g, and the average weight of the slow-growing group was 31.25 g, with the fast-growing group's average weight being 589.34% of the slow-growing group's. In the Zhaodong population, the average weight of the fast-growing group was 81.87 g, and the average weight of the slow-growing group was 4.34 g, with the fast-growing group's average weight being 1886.41% of the slow-growing group's. Therefore, the DNA molecular module and primer pairs provided by this invention can be used to identify the growth type (fast-growing and slow-growing) of grass carp.

[0045] The present invention performs statistical analysis on the genotyping results obtained in Examples 2 and 3, and the results are as follows: Figure 2 As shown, for the aforementioned grass carp alkali-tolerant and fast-growing related DNA molecular module, in both random grass carp populations in Wuhan and Zhaodong, the number of dominant genotypes enriched in the fast-growing group was significantly higher than that in the slow-growing group. Therefore, the DNA molecular module and primer pairs provided by this invention can be used to screen alkali-tolerant and fast-growing grass carp.

[0046] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A SNP molecular marker composition for screening alkali-tolerant, fast-growing grass carp, characterized in that, The SNP molecular marker composition is composed of SNP01-SNP08 molecular markers; The nucleotide sequence of the SNP01 molecular marker is shown in SEQ ID NO.1, and the 401st base is either T or C; The nucleotide sequence of the SNP02 molecular marker is shown in SEQ ID NO.2, and the 401st base is either T or C; The nucleotide sequence of the SNP03 molecular marker is shown in SEQ ID NO.3, wherein the 401st base is A or G; The nucleotide sequence of the SNP04 molecular marker is shown in SEQ ID NO.4, and the 401st base is A or C; The nucleotide sequence of the SNP05 molecular marker is shown in SEQ ID NO.5, and the 401st base is either T or C. The nucleotide sequence of the SNP06 molecular marker is shown in SEQ ID NO.6, and the 401st base is either T or A. The nucleotide sequence of the SNP07 molecular marker is shown in SEQ ID NO.7, and the 401st base is either T or A. The nucleotide sequence of the SNP08 molecular marker is shown in SEQ ID NO.8, and the 401st base is G or A; The SNP01 molecular marker is located at base 2320551 on chromosome 4 of grass carp. This base position exhibits T / C polymorphism, with genotypes of TT, TC, or CC. The SNP02 molecular marker is located at base 29280354 on chromosome 4 of grass carp. This base position exhibits T / C polymorphism, with genotypes of TT, TC, or CC. The SNP03 molecular marker is located at base 1646710 on chromosome 10 of grass carp. This base position exhibits A / G polymorphism, and the genotype is AA, AG, or GG. The SNP04 molecular marker is located at base 15475728 on chromosome 14 of grass carp. This base position exhibits A / C polymorphism, and the genotype is AA, AC, or CC. The SNP05 molecular marker is located at base 15475886 on chromosome 14 of grass carp. This base position exhibits C / T polymorphism, and the genotype is CC, CT, or TT. The SNP06 molecular marker is located at base 15475888 on chromosome 14 of grass carp. This base position exhibits A / T polymorphism, and the genotype is AA, AT, or TT. The SNP07 molecular marker is located at base 15475972 on chromosome 14 of grass carp. This base position exhibits T / A polymorphism, and the genotype is TT, TA, or AA. The SNP08 molecular marker is located at base 9539862 on chromosome 24 of grass carp. This base position exhibits G / A polymorphism, with genotypes of GG, GA, or AA.

2. A primer pair for amplifying the SNP molecular marker composition of claim 1, characterized in that, The primer pairs include primer pairs used to amplify the SNP01-SNP08 molecular markers of claim 1, respectively; The primer pair used to amplify the SNP01 molecular marker includes the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10; The primer pair used to amplify the SNP02 molecular marker includes the forward primer shown in SEQ ID NO.11 and the reverse primer shown in SEQ ID NO.12; The primer pair used to amplify the SNP03 molecular marker includes the forward primer shown in SEQ ID NO.13 and the reverse primer shown in SEQ ID NO.14; Primer pairs used to amplify SNP04, SNP05, SNP06, or SNP07 molecular markers include the forward primer shown in SEQ ID NO.15 and the reverse primer shown in SEQ ID NO.16; The primer pair used to amplify the SNP08 molecular marker includes the forward primer shown in SEQ ID NO.17 and the reverse primer shown in SEQ ID NO.

18.

3. A kit for screening alkali-tolerant, fast-growing grass carp, characterized in that, The kit includes the primer pair as described in claim 2.

4. A method for screening alkali-tolerant, fast-growing grass carp broodstock, characterized in that, The method includes the following steps: S1: Extract genomic DNA from the grass carp to be tested; S2: Using the grass carp genomic DNA obtained in S1 as a template, perform PCR amplification using the primer pairs described in claim 3; S3: The amplification products obtained in S2 were subjected to Sanger sequencing analysis, and the alkali-tolerant fast-growing grass carp were screened based on the sequencing results; The criteria for screening alkali-tolerant, fast-growing grass carp based on sequencing results are as follows: For the SNP01 molecular marker, the genotype is TT; For the SNP02 molecular marker, the genotype is TT; For the SNP03 molecular marker, the genotype is AA; For the SNP04 molecular marker, the genotype is AA; For the SNP05 molecular marker, the genotype is CC; For the SNP06 molecular marker, the genotype is AA; For the SNP07 molecular marker, the genotype is TT; For the SNP08 molecular marker, the genotype is GG. If the above genotypes are met simultaneously, the grass carp to be tested is determined to be an alkali-tolerant, fast-growing grass carp; the alkali-tolerant condition is 20 mM carbonate alkalinity.

5. The method according to claim 4, characterized in that, The PCR amplification system described in S2 is as follows: 2 μL DNA template, 0.1 μL forward primer (10 μM), 0.1 μL reverse primer (10 μM), 12.5 μL Premix ExTaq, and 8.5 μL ddH2O.

6. The method according to claim 4, characterized in that, The PCR amplification conditions described in S2 are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; 72℃ extension for 7 min.

7. The application of the SNP molecular marker composition of claim 1, the primer pair of claim 2, or the kit of claim 3 in screening alkali-tolerant, fast-growing grass carp germplasm; the alkali tolerance condition is 20 mM carbonate alkalinity.

8. The application according to claim 7, characterized in that, The criteria for judging fast-growing grass carp are as follows: based on the grass carp weight index, grass carp individuals with a weight ≥ average weight + standard deviation are classified as fast-growing grass carp, and individuals with a weight < average weight - standard deviation are classified as slow-growing grass carp; the average weight refers to the average weight of a grass carp population of ≥400 fish.

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