SNP markers associated with rapid growth traits in South China carp and their application

Abstract

The present invention belongs to the field of animal molecular biology DNA marker technology and application technology, and specifically discloses SNP molecular markers related to the growth traits of South China carp. The present invention screens out SNP markers and typing information related to the fast growth traits of South China carp by performing whole-genome resequencing and genome-wide association analysis on fast-growing individuals and slow-growing individuals in the same South China carp population. Specific primers are designed based on the sequence of the molecular marker, which can quickly and accurately detect sites related to growth. By detecting the SNP marker, fast-growing South China carp can be effectively selected under the same breeding conditions. It can be effectively used in molecular marker-assisted breeding of South China carp, providing technical support for breeding.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular biology DNA marker technology and application technology, and particularly relates to SNP markers related to the rapid growth trait of South China carp and applications thereof. Background Art

[0002] South China carp (Cyprinus carpio rubrofuscus) belongs to the Cypriniformes order, Cyprinidae family, and Cyprinus genus. South China carp is distributed in water systems south of the Nanling Mountains, such as the Pearl River, Yuanjiang River, and Hainan Island, and is a key aquaculture species in southern China. Rice-field culture has become a prominent feature of the Guangdong and Guangxi regions, maximizing resource utilization while significantly increasing farmers' profits. As one of the world's most important economic aquatic species, improving the yield and quality of carp has always been a key goal of breeding. However, long-term inbreeding in breeding has significantly reduced genetic diversity in populations, severely degenerating germplasm, and causing some individuals to grow slowly. Therefore, developing reliable breeding techniques to obtain fast-growing South China carp and eliminate slow-growing individuals would not only save feed costs but also shorten the breeding cycle, significantly reducing South China carp aquaculture costs. Given the low efficiency and time-consuming and labor-intensive nature of traditional parent or population selection, molecular markers have become a powerful tool for assisted breeding. With the rapid development of sequencing technology and the decline in sequencing costs, whole genome sequencing technology has been widely used in the development of molecular markers for important traits of aquatic animals. Compared with traditional microsatellite and simplified genome technologies, whole genome resequencing can cover the entire genome site, greatly improving the precision and accuracy of data analysis. In the prior art, researchers have used whole genome resequencing technology to analyze candidate genes related to growth or sex traits in pearl oysters and constructed a high-density genetic map. And based on genome resequencing data, a SNP site (g26854_18684937) related to Pacific oyster growth and shell shape was identified. In summary, molecular marker development technology based on whole genome sequencing has become an important research method for breeding work.

[0003] Currently, studies have reported molecular markers associated with carp growth. For example, a simplified genome screen identified the key growth-related gene Adrb2a in the Yellow River carp. Using 250K microarray technology, candidate genes Kiss2, Igf1, Smtlb, and Npffr1 were identified for growth in the Yellow River carp. However, molecular markers associated with growth traits remain underdeveloped in South China carp breeding. Therefore, the development of SNP markers associated with rapid growth in South China carp is crucial for accelerating molecular breeding of South China carp.

[0004] The G-protein-coupled receptor 75 gene (GPR75) encodes a membrane protein receptor containing 540 amino acids, which has the typical structural characteristics of G protein-coupled receptors, namely 7 transmembrane domains, the N-terminus is located outside the cell, and the C-terminus is located inside the cell. At present, the metabolic functions of GPR75 in the brain and nervous system, cardiovascular and weight-related have been preliminarily elucidated in humans and mice. Based on the exome sequencing of 645,000 individuals, current scholars have found that individuals carrying GPR75 protein truncating mutations have an average BMI (Body Mass Index) that is 1.8 kg / m lower than that of normal individuals. 2 To further verify the effect of the GPR75 gene on body weight, the researchers fed mice with heterozygous or homozygous mutations of GPR75 and normal mice with a high-fat diet and found that the normal mice doubled their weight within 14 weeks, while the heterozygous mutation mice (Gpr75 + / - ) weighed 25% less than normal mice, while mice with the homozygous mutation (Gpr75 - / - ) weigh 44% less than normal mice. However, no relevant research has yet shown whether the GPR75 gene can also regulate the growth of South China carp.

[0005] Based on the above content, this application is filed. Summary of the Invention

[0006] The present invention obtains SNP molecular markers related to the growth of South China carp by utilizing molecular genetics and molecular biology methods. The SNP molecular markers can be used for breeding new varieties (lines) of fast-growing South China carp.

[0007] The purpose of the first aspect of the present invention is to provide SNP molecular markers related to growth traits of South China common carp.

[0008] The second aspect of the present invention aims to provide a primer set for amplifying the SNP molecular marker of the first aspect of the present invention.

[0009] The third aspect of the present invention aims to provide a detection reagent, gene chip or kit.

[0010] The fourth aspect of the present invention aims to provide applications of the SNP molecular marker of the first aspect of the present invention, the primer set of the second aspect of the present invention, and / or the detection reagent, gene chip or kit of the third aspect of the present invention.

[0011] The fifth aspect of the present invention aims to provide a method for screening the growth rate of South China common carp.

[0012] The purpose of the sixth aspect of the present invention is to provide an application of the method of the fifth aspect of the present invention in breeding excellent-growing South China carp varieties.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, SNP molecular markers associated with growth traits of South China carp are provided, wherein the SNP molecular markers include at least one of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762;

[0015] in,

[0016] The SNP site of SNP17_4687 is located at position 51 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / T;

[0017] The SNP site of SNP17_4688 is located at position 58 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / T;

[0018] The SNP site of SNP17_4689 is located at position 68 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C;

[0019] The SNP site of SNP17_4691 is located at position 94 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / A;

[0020] The SNP site of SNP17_4692 is located at position 96 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is A / T;

[0021] The SNP site of SSNP17_4694 is located at position 124 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / A;

[0022] The SNP site of SNP17_4695 is located at position 137 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C;

[0023] The SNP site of SNP17_4758 is located at position 1247 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C;

[0024] The SNP site of SNP17_4761 is located at position 1378 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / G;

[0025] The SNP site of SNP17_4762 is located at position 1379 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is A / T.

[0026] In some embodiments of the present invention, when the genotype of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 is CC, CC, TT, TT, AA, CC, TT, TT, CC, AA, it is a rapid growth factor. For the growing South China carp, when the genotypes of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 are CT, CT, CT, AT, AT, AC, CT, CT, CG, AT respectively, it is a slow-growing South China carp.

[0027] The present invention obtains 10 SNP molecular markers with breeding value based on whole genome resequencing, which can be applied to molecular marker-assisted breeding of South China carp, accelerate the selection of South China carp varieties with excellent growth traits, increase production efficiency, and provide a reference basis for the breeding work of South China carp.

[0028] The second aspect of the present invention provides a primer set for amplifying the SNP molecular marker of the first aspect of the present invention.

[0029] In some embodiments of the present invention, the primer set includes primer pair 1 and primer pair 2, and the nucleotide sequence of primer pair 1 is as follows:

[0030] F: 5'-TGCTGTCCATTCACGGCT-3';

[0031] R: 5'-TGGTAGACCCTCTGGATTGC-3';

[0032] The nucleotide sequence of the primer pair 2 is as follows:

[0033] F: 5'-TCATTTGGTGATGCTATACTTC-3';

[0034] R: 5'-TGCTAACGGCAAGTCTCA-3'.

[0035] In some embodiments of the present invention, the primer pair 1 is used to amplify SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694 and SNP17_4695, and the primer pair 2 is used to amplify SNP17_4758, SNP17_4761 and SNP17_4762.

[0036] The third aspect of the present invention provides a detection reagent, gene chip or kit comprising the primer set according to the second aspect of the present invention.

[0037] In some embodiments of the present invention, the detection reagent, gene chip or kit further comprises a buffer used in PCR.

[0038] In some embodiments of the present invention, the buffer used in the PCR is any reagent required for PCR amplification, such as dNTP, Taq enzyme, MgCl2, etc.

[0039] The fourth aspect of the present invention provides the use of the SNP molecular marker of the first aspect of the present invention, the primer set of the second aspect of the present invention, and / or the detection reagent, gene chip or kit of the third aspect of the present invention in any one of (1) to (6):

[0040] (1) Assisted selection or breeding of South China carp;

[0041] (2) preparing products to assist in the selection or breeding of South China carp;

[0042] (3) Determine or identify the growth rate of South China carp;

[0043] (4) preparing products for judging or identifying the growth rate of South China carp;

[0044] (5) Management, development and utilization of South China carp germplasm resources;

[0045] (6) Protection and evaluation of South China carp varieties.

[0046] In some embodiments of the present invention, the breeding is to screen and obtain fast-growing South China carp varieties (lines).

[0047] In some embodiments of the present invention, the breeding or assisted breeding includes at least one of assisted major effect gene selection, molecular assisted breeding, whole genome selection breeding, South China carp variety identification, genetic map construction, gene mapping, species evolution analysis and germplasm resource identification.

[0048] The fifth aspect of the present invention provides a method for screening the growth rate of South China carp, by detecting the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the South China carp to be tested, and determining the growth rate of the South China carp to be tested according to the genotype.

[0049] In some embodiments of the present invention, the DNA of the South China carp to be tested is used as a template, and PCR amplification is performed using the primer set of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention to obtain a PCR amplification product; the PCR amplification product is sequenced and analyzed to determine the gene of the SNP molecular marker of the first aspect of the present invention in the genome of the South China carp to be tested.

[0050] In some embodiments of the present invention, when the genotype of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 is CC, CC, TT, TT, AA, CC, TT, TT, CC, AA, it is a rapid growth factor. For the growing South China carp, when the genotypes of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 are CT, CT, CT, AT, AT, AC, CT, CT, CG, AT respectively, it is a slow-growing South China carp.

[0051] In some embodiments of the present invention, the DNA of the South China common carp can be extracted using conventional means in the art, including the phenol-chloroform method and various DNA extraction kits.

[0052] In some embodiments of the present invention, the reaction procedure for PCR amplification is pre-denaturation at 90-94°C for 3-6 min; denaturation at 90-94°C for 40-60 s, annealing at 56-60°C for 50-110 s, extension at 70-72°C for 60-70 s, 28-35 cycles; extension at 70-72°C for 5-10 min.

[0053] In some embodiments of the present invention, the sequencing includes sequencing using the Sanger method, second-generation sequencing, etc.

[0054] The sixth aspect of the present invention provides the application of the method of the fifth aspect of the present invention in breeding excellent growth South China carp varieties.

[0055] The beneficial effects of the present invention are:

[0056] This study screened for SNP markers and their genotyping information related to the rapid growth trait of South China carp by performing whole-genome resequencing and genome-wide association analysis on fast-growing and slow-growing individuals from the same population of South China carp. Specific primers designed based on the sequence of the molecular markers can quickly and accurately detect growth-related sites. By detecting these SNP markers, fast-growing South China carp can be effectively selected under the same breeding conditions. This can be effectively used in molecular marker-assisted breeding of South China carp, providing technical support for breeding.

[0057] By detecting this SNP marker, the genotype of the South China carp parents can be identified according to actual breeding needs, and the South China carp parents with suitable genotypes can be selected for breeding, so as to obtain South China carp varieties with excellent growth traits (i.e., fast-growing South China carp offspring (fry)), save breeding time, reduce costs, increase accuracy, and accelerate the breeding process of South China carp.

[0058] The genotype obtained by the present invention is based on the base mutation generated within the gene, so there is no genetic exchange and no need for further verification of the phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the SNP distribution density map.

[0060] Figure 2 Schematic diagram of the gene structure and the location of the SNP mutation sites screened according to the embodiments of the present invention.

[0061] Figure 3 The Manhattan diagram provided in the embodiment; the markers therein are the molecular markers screened by the present invention. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below through specific examples.

[0063] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0064] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0065] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0066] Example 1

[0067] This example provides screening of SNP molecular markers that can be used for breeding new varieties (lines) of fast-growing South China carp, as follows:

[0068] (1) Sample collection: Approximately 1,000 6-month-old South China carp were randomly selected from the breeding base of the Tropical and Subtropical Fish Genetics and Breeding Center of the Ministry of Agriculture and Rural Affairs (Guangzhou, Guangdong Province). After weighing, the 35 largest individuals (573.76 ± 110.47 g) and the 35 smallest individuals (12.39 ± 4.07 g) were selected. A small amount of tail fin was clipped from each individual and stored in anhydrous ethanol solution at -20°C for subsequent whole-genome resequencing.

[0069] (2) DNA extraction, detection, and library preparation: Whole-genome DNA was extracted from all samples using a genome extraction kit (Axgeny, UE-MN-MS-GDNA-50). After testing the DNA concentration and quality, a whole-genome resequencing library was constructed using a DNA library construction kit. The library fragment size and concentration were detected using an Agilent 2100 Bioanalyzer. Libraries that passed the quality test were sequenced on the machine, with a sequencing depth of 20× for each sample. The sequencing quality is summarized in Table 1.

[0070] (3) Data analysis: The raw data were removed from the machine using fastp software to remove the connectors, filter reads containing too much N or a large number of low base quality and PCR duplicates. The filtered data were aligned to the South China carp genome using BWA software. The Haplotypecaller in GATK4.0 was used to detect variant sites, and then vcftools was used to filter according to allele frequency and depth, and a total of 11,038,745 SNP sites were obtained. The results of PCA cluster analysis showed that there was no obvious grouping of the population. The mixed linear model of GEMMA software was used for genome-wide association analysis, and 34 SNP sites significantly associated with growth were preliminarily screened (P<10E-7.04). Annovar software was used to annotate SNPs. The F value of each window was counted using the vcftools sliding box. ST , window 100 000 bp, step size 5 000 bp. Then select the windows with more than 5 SNP sites and ranked in the top 100. Calculate the F of each site in the window that meets the screening conditions. ST The top 1% (FST = 0.405) was used as the screening threshold. Combining P value and FST, the 10 candidate SNP sites with the most significant association with growth were screened out. The SNP site information is shown in Table 2 and Figures 1 to 3 The locations of the SNP sites are shown in bold in SEQ ID NO: 1, and the SNP molecular marker genotyping and corresponding growth traits are shown in Table 3.

[0071] Table 1 Resequencing data statistics

[0072]

[0073]

[0074]

[0075] Table 2 Information of SNP sites

[0076]

[0077] Table 3 SNP molecular marker genotyping and corresponding growth traits

[0078]

[0079]

[0080]

[0081] Note: The bold font indicates SNP sites.

[0082] Example 2

[0083] This example provides a primer pair that can be used to amplify SNP molecular markers associated with the growth rate of South China common carp.

[0084] Because the 10 loci are concentrated on chromosome 17, and the GPR75 gene has been reported to be associated with metabolism and body weight, two pairs of specific primers were designed based on the sequence information of the carp GPR75 gene from Ensembl (nucleotide sequence as SEQ ID NO: 2, amino acid sequence as SEQ ID NO: 3) to verify the accuracy of the loci. The primers amplify fragments encompassing the promoter region, UTR region, and intron region of GPR75. The primer sequences are shown in Table 4.

[0085] Table 4 Primer information

[0086]

[0087] Note: In the table, primers a are used to verify the SNP sites: SNP17_4687 to SNP17_4695; primers b are used to verify the SNP sites: SNP17_4758 to SNP17_4762.

[0088] Example 3

[0089] This embodiment provides a method for breeding South China carp, comprising the following steps:

[0090] (1) Extraction of sample genomic DNA: Same as step (2) in Example 1.

[0091] (2) Amplify the sequence containing the SNP site using the primer pair of Example 2

[0092] The primers provided in Example 2 were used to perform PCR amplification on the DNA of the tested South China carp. The PCR reaction system (25 μL) was as follows: 12.5 μL of Premix Taq (Takara), 9.5 μL of ddH2O, 1 μL each of the forward and reverse primers, and 1 μL of DNA template.

[0093] The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 53°C or 57°C for 1 min, extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 5 min.

[0094] The amplified products were detected by 1% agarose gel electrophoresis and then sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for bidirectional sequencing.

[0095] (3) Typing determination

[0096] According to the sequencing peak graph, the genotype of the mutation site was determined, and it was determined whether the tested South China carp was a fast-growing South China carp or a slow-growing South China carp (Table 3).

[0097] Example 4

[0098] To verify whether the markers were growth-related molecular markers, this example randomly selected one 6-month-old population of South China common carp from each of the Ruyuan breeding base in Shaoguan City, Guangdong Province, and one from the Jiangmen breeding base in Guangdong Province. Growth data, including length and weight, were measured, and fin rays were collected and preserved. Whole-genome DNA was extracted from the 15 heaviest and 15 smallest individuals from each population for subsequent SNP verification. The assay procedure was the same as in Example 3.

[0099] The statistics of genotype results are shown in Table 5.

[0100] Table 5 SNP site validity verification

[0101]

[0102]

[0103] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A SNP molecular marker associated with growth traits of South China carp, characterized in that: The SNP molecular markers include SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 and SNP17_4762; in, The SNP site of SNP17_4687 is located at position 51 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / T; The SNP site of SNP17_4688 is located at position 58 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / T; The SNP site of SNP17_4689 is located at position 68 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C; The SNP site of SNP17_4691 is located at position 94 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / A; The SNP site of SNP17_4692 is located at position 96 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is A / T; The SNP site of SNP17_4694 is located at position 124 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / A; The SNP site of SNP17_4695 is located at position 137 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C; The SNP site of SNP17_4758 is located at position 1247 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is T / C; The SNP site of SNP17_4761 is located at position 1378 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is C / G; The SNP site of SNP17_4762 is located at position 1379 from the 5' end of the sequence shown in SEQ ID NO: 1 of the GPR75 gene, and its polymorphism is A / T.

2. The SNP molecular marker according to claim 1, characterized in that When the genotypes of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 are CC, CC, TT, TT, AA, CC, TT, TT, CC, AA, it is a fast-growing South China carp. When the genotypes of SNP17_4687, SNP17_4688, SNP17_4689, SNP17_4691, SNP17_4692, SNP17_4694, SNP17_4695, SNP17_4758, SNP17_4761 or SNP17_4762 are CT, CT, CT, AT, AT, AC, CT, CT, CG, AT, respectively, it is a slow-growing South China common carp.

3. A primer set for amplifying the SNP molecular marker according to claim 1 or 2.

4. The primer set according to claim 3, characterized in that The primer set includes primer pair 1 and primer pair 2, and the nucleotide sequence of primer pair 1 is as follows: F: 5'-TGCTGTCCATTCACGGCT-3'; R: 5'-TGGTAGACCCTCTGGATTGC-3'; The nucleotide sequence of the primer pair 2 is as follows: F: 5'-TCATTTGGTGATGCTATACTTC-3'; R: 5'-TGCTAACGGCAAGTCTCA-3'.

5. A detection reagent, gene chip or kit comprising the primer set according to claim 3 or 4.

6. The detection reagent, gene chip or kit according to claim 5, characterized in that: The detection reagent, gene chip or kit also includes a buffer used in PCR.

7. Use of the SNP molecular marker according to claim 1 or 2, the primer set according to claim 3 or 4, and / or the detection reagent, gene chip or kit according to claim 5 or 6 in any one of (1) to (4): (1) Assisted selection or breeding of South China carp; (2) preparing products to assist in the selection or breeding of South China carp; (3) Determine or identify the growth rate of South China carp; (4) Prepare products for judging or identifying the growth rate of South China carp.

8. A method for screening the growth rate of South China carp, by detecting the genotype of the SNP molecular marker according to claim 1 or 2 in the genome of the South China carp to be tested, and determining the growth rate of the South China carp according to the genotype.

9. The method according to claim 8, characterized in that The method comprises the following steps: using the DNA of the South China carp to be tested as a template, performing PCR amplification using the primer set described in claim 3 or 4 or the detection reagent, gene chip or kit described in claim 5 or 6 to obtain a PCR amplification product; sequencing and analyzing the PCR amplification product to determine the gene of the SNP molecular marker described in claim 1 or 2 in the genome of the South China carp to be tested.

10. Use of the method according to claim 8 or 9 in breeding excellent growth South China carp varieties.

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