SNP (Single Nucleotide Polymorphism) marker related to growth performance of fast-growing cyprinus carpio, primer and application of SNP marker

Abstract

The invention belongs to the technical field of animal molecular biology DNA (deoxyribonucleic acid) marking technology and application, discloses an SNP (single nucleotide polymorphism) marker related to growth performance of fast-growing cyprinus carpio, a primer and application of the SNP marker, and particularly discloses an SNP molecular marker related to growth traits of cyprinus carpio, the sequence of the SNP molecular marker is as shown in SEQ ID NO: 1, the SNP site is located at the 1001st site from the 5'terminal of the sequence as shown in SEQ ID NO: 1, and the polymorphism of the SNP molecular marker is T / A. The invention provides the SNP marker of the cyprinus carpio, by detecting the SNP marker, the cyprinus carpio with better growth traits can be effectively selected under the same culture condition, and the SNP marker can be effectively used for molecular marker-assisted breeding of the cyprinus carpio. Further, genotype identification can be carried out on the cyprinus carpio parents according to actual breeding requirements, the cyprinus carpio parents with proper genotypes are selected for breeding, cyprinus carpio offspring (fry) which grows fast can be obtained, and the cyprinus carpio breeding process is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular biology DNA marker technology and application technology, and specifically relates to a SNP marker related to the growth performance of fast-growing South China carp, a primer and an application thereof. Background Art

[0002] Integrated rice-fish farming is a typical ecological cycle agricultural development model. It combines rice cultivation with aquaculture, and uses ecological principles such as biological mutualism and complementary resource utilization to build a rice-fish rotation system. Under the premise of stable rice production, it produces high-quality pollution-free aquatic products, greatly improves the comprehensive economic benefits of rice fields, and protects and improves the ecological environment of rice fields. The main fish species farmed in rice fields in Guangdong, my country are South China carp (Cyprinus carpio rubrofuscus), which is a subspecies of Cypriniformes, Cyprinidae, Cyprinus, and C. carpio Linnaeus. The South China carp farmed in traditional rice fields has not been systematically selected and bred, and there are many fish that stagnate in growth. In order to increase the economic benefits of South China carp farmed in rice fields and increase the yield of South China carp, it is necessary to select and breed new varieties (lines) of fast-growing South China carp.

[0003] With the rapid development of high-throughput sequencing technology, the application of genome-wide association study (GWAS) in aquatic animal genetic breeding is becoming more and more common, and has become an important method for studying quantitative traits of fish. Genome-wide association analysis is to obtain the nucleic acid polymorphism (single nucleotide polymorphism, SNP) sites of individuals at the whole genome level through resequencing, gene chips, etc., and then explore the associated genes that affect phenotypic traits (such as disease, growth, etc.). It has the advantages of high resolution, wide source of research materials, rich variation that can be captured, high accuracy and time saving. In the early stage, this research team screened a group of candidate SNP markers related to growth traits through resequencing, and further verified it by expanding the population, aiming to find SNP markers closely related to the growth of South China carp, laying the foundation for the molecular marker-assisted breeding of fast-growing South China carp new varieties. Summary of the invention

[0004] The invention obtains SNP molecular markers related to growth of South China carp by using molecular genetics and molecular biology methods. The SNP molecular markers can be used for breeding new varieties (lines) of fast-growing South China carp.

[0005] The purpose of the first aspect of the present invention is to provide SNP molecular markers related to growth traits of South China carp.

[0006] 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.

[0007] The third aspect of the present invention is to provide a kit.

[0008] The purpose of the fourth aspect of the present invention is to provide 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 kit of the third aspect of the present invention.

[0009] The fifth aspect of the present invention aims to provide a method for screening the growth rate of South China carp.

[0010] 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 South China carp varieties and evaluating the variation of South China carp populations.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a SNP molecular marker associated with growth traits of South China carp, the sequence of the SNP molecular marker is shown in SEQ ID NO: 1, the SNP site is located at the 1001st position from the 5' end of the sequence shown in SEQ ID NO: 1, and its polymorphism is T / A.

[0013] In some embodiments of the present invention, when the genotype of the SNP site is AA, the growth trait of South China carp is a growth disadvantage, and when the genotype of the SNP site is TT, the growth trait of South China carp is a growth advantage (fast growth rate and low population variation coefficient).

[0014] 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.

[0015] In some embodiments of the present invention, the nucleotide sequence of the primer set is as follows:

[0016] F: 5'-AAACAAACTGGCTCCCAGAG-3' (SEQ ID NO: 2);

[0017] R: 5'-GGAACTAGTGGAAGTGGTTG-3' (SEQ ID NO: 3).

[0018] The third aspect of the present invention provides a kit comprising the primer set of the second aspect of the present invention.

[0019] In some embodiments of the present invention, the kit further comprises a buffer used in PCR.

[0020] 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.

[0021] 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 kit of the third aspect of the present invention in any one of (1) to (6):

[0022] (1) Assisted breeding or breeding of South China carp;

[0023] (2) preparing products to assist in the selection or breeding of South China carp;

[0024] (3) Determine or identify the growth rate of South China carp;

[0025] (4) Preparing products for judging or identifying the growth rate of South China carp;

[0026] (5) Evaluate the population variation of South China carp;

[0027] (6) Prepare products for evaluating the population variation of South China common carp.

[0028] In some embodiments of the present invention, the breeding is to screen for South China carp varieties (lines) with excellent weight, total length, body length, body height and head length and low population variation coefficient.

[0029] 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.

[0030] 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 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.

[0031] In some embodiments of the present invention, when the genotype of the SNP site is TT, the South China common carp has excellent weight, total length, body length, body height and head length and a low population variation coefficient.

[0032] In some embodiments of the present invention, the DNA of the South China carp can be extracted by conventional means in the art, including the phenol-chloroform method and various DNA extraction kits.

[0033] 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 30-35 s, annealing at 56-60°C for 30-35 s, extension at 70-72°C for 80-90 s, 30-35 cycles; extension at 70-72°C for 5-10 min.

[0034] In some embodiments of the present invention, the reaction system for PCR amplification includes 1-3 mmol / LMgCl2, 0.1-0.2 mmol / L dNTP, 0.05-0.1 U / μL Taq enzyme, and 0.2-0.3 μmol / L of each upstream and downstream primer.

[0035] In some embodiments of the present invention, the sequencing includes sequencing using the Sanger method.

[0036] The sixth aspect of the present invention provides the application of the method of the fifth aspect of the present invention in breeding excellent South China carp varieties and evaluating the variation of South China carp populations.

[0037] The beneficial effects of the present invention are:

[0038] The present invention provides a SNP marker (SNP27368615) of South China carp. By detecting the SNP marker, South China carp with better growth traits (faster growth and low population variation coefficient) can be effectively selected under the same breeding conditions, and can be effectively used for molecular marker-assisted breeding of South China carp. Then, the genotype of South China carp parents can be identified according to actual breeding needs, and South China carp parents with suitable genotypes can be selected for breeding, so that South China carp offspring (fry) with faster growth can be obtained, breeding time can be saved, cost is low, accuracy is high, the breeding process of South China carp can be accelerated, and the economic benefits of paddy field breeding of South China carp can be increased.

[0039] 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 further verification of the phenotype is required. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Agarose gel electrophoresis of PCR amplification products of some samples.

[0041] Figure 2 This is the Sanger sequencing peak graph, from left to right are the TT genotype peak graph, AT genotype peak graph and AA genotype peak graph, in the figure, the arrow points to the SNP site. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below through specific examples.

[0043] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] This embodiment provides SNP molecular markers that can be used for breeding new varieties (lines) of fast-growing South China carp.

[0048] We selected a group of 4-month-old South China carp from the same batch of breeding and pond culture for sampling. We selected 14 extremely large individuals (weight ≥ 25g) and 14 extremely small individuals (weight ≤ 10g). We took the tail fins of the samples and sent them to BGI for resequencing. The resequencing results screened out that SNP27368615 (nucleotide sequence as SEQ ID NO: 1) had a significant difference in distribution frequency between the two groups.

[0049]

[0050]

[0051] Note: The bold bases in the brackets represent the SNP mutation point. The mutation type of this site is T to A. The underlined parts correspond to the detection primer sequences of the mutation point.

[0052] As can be seen from Table 1, the distribution frequencies of AA genotype in the extremely large and extremely small groups are 0% and 57.14%, respectively, while the distribution frequencies of TT genotype in the extremely large and extremely small groups are 78.57% and 0%, respectively, and the distribution frequencies of AT genotype in the extremely large and extremely small groups are 21.43% and 42.86%, respectively. It is speculated that AA genotype is a growth disadvantage genotype and TT genotype is a growth advantage genotype.

[0053] Table 1 Distribution of SNP27368615 marker genotypes in extreme samples

[0054] Group AA AT TT Extremely large individuals 0 3 11 Extremely small individuals 8 6 0

[0055] Example 2

[0056] This example provides a primer pair that can be used to amplify SNP molecular markers associated with the growth rate of South China carp.

[0057] According to the gene fragment where the SNP27368615 marker is located, a pair of primers P were designed, the upstream primer sequence was F: 5'-AAACAAACTGGCTCCCAGAG-3' (SEQ ID NO: 2); the downstream primer sequence was R: 5'-GGAACTAGTGGAAGTGGTTG-3' (SEQ ID NO: 3).

[0058] Example 3

[0059] This embodiment provides a method for breeding South China carp, comprising the following steps:

[0060] (1) Extraction of sample genomic DNA

[0061] 1) Take 3 mg of the fin tissue of the fish to be tested, cut it into pieces, add 0.5 mL of lysis buffer (10 mmol / L Tris-HCl, 0.1 mol / L EDTA, 0.5% SDS, 30 mg / L RNase and 100 mg / L proteinase K, pH 8.0), and digest at 55°C for 1 hour;

[0062] 2) Add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) mixture, mix well, let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, take the supernatant, extract once with chloroform, let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant;

[0063] 3) Add 2 volumes of anhydrous ethanol, let stand at room temperature for 10 minutes to precipitate DNA, and centrifuge at 12,000 rpm for 10 minutes;

[0064] 4) The precipitate was washed once with 70% ethanol, centrifuged at 12,000 rpm for 2 minutes, the supernatant was aspirated, and the mixture was allowed to stand and dry at room temperature for 10 minutes. 50 μL TE (10 mmol / L Tris-HCl and 1 mmol / L EDTA, pH 8.0) was added to dissolve the DNA, and the mixture was stored at -20°C for later use.

[0065] (2) Amplify the sequence containing the SNP site using the primer pair of Example 2

[0066] The primers provided in Example 2 were used to perform PCR amplification on the DNA of the South China carp to be tested. The reaction system of PCR amplification included 1-3 mmol / LMgCl2, 0.1-0.2 mmol / L dNTP, 0.05-0.1 U / μL Taq enzyme, and 0.2-0.3 μmol / L of upstream and downstream primers respectively.

[0067] The reaction procedure of PCR amplification is pre-denaturation at 90-94°C for 3-6 min; denaturation at 90-94°C for 30-35 s, annealing at 60°C for 30-35 s, extension at 70-72°C for 80-90 s, 30-35 cycles; extension at 70-72°C for 5-10 min.

[0068] (3) Typing

[0069] Using the PCR system to amplify the genomic DNA of South China carp, a 280bp specific amplification band can be obtained. The amplification diagram of some samples is shown in Figure 1 Then the PCR product was sequenced directly using the Sanger method. Check the sequencing peak diagram ( Figure 2 ) to determine the genotype of the mutation site. If the site is the TT genotype, it is a dominant genotype for body weight, total length, body length, body height and head length (that is, the South China carp with this genotype grows fast and has a low coefficient of population variation), while if the site is the AA genotype, it is a disadvantageous genotype.

[0070] Example 4

[0071] In order to verify whether the marker is a growth-related molecular marker, the present invention reconstructs the simultaneous reproduction and the one-month-old South China carp cultured in the same pond, randomly samples and puts them into the rice field for 4 months of culture, and measures the weight (accuracy 0.01g) and total length, body length, body height, head length, tail peduncle length and tail peduncle height of the test fish before and after rice field culture, with an accuracy of 0.01cm. The number of random samples before culture is 271, and the number of random samples after culture is 280. The fin rays of the test fish are clipped for sample genotyping.

[0072] The growth-related statistical results are shown in Table 2. It can be seen that before breeding, there were no significant differences in weight, total length, body height, head length, caudal peduncle length and caudal peduncle height among the three genotypes of test fish (P>0.05). After 4 months of breeding, the TT genotype was significantly higher than the AA and AT genotypes in weight, total length, body length, body height and head length (P<0.05), but there was no significant difference in caudal peduncle length and caudal peduncle height (P>0.05), indicating that the TT genotype was an advantageous genotype in weight, total length, body length, body height and head length, while AA and AT were disadvantageous genotypes, among which the mean values ​​of the AA genotype samples in weight, total length, body length, body height and head length were lower than those of the AT genotype. It shows that the growth identification results of South China carp using the method of Example 3 are consistent with the identification results obtained by directly measuring the weight, body length, body height, etc. of South China carp, indicating that the SNP site of SEQ ID NO: 1 can provide a reliable basis for the identification of the growth of South China carp, and can be used to quickly and accurately screen out South China carp with excellent growth traits.

[0073] Table 2 Correlation analysis between SNP27368615 genotype and growth traits

[0074]

[0075] Note: The same shoulder letters indicate no significant difference (P>0.05), and different shoulder letters indicate significant difference (P<0.05).

[0076] In order to verify whether a fast-growing South China carp population can be quickly obtained by selecting TT genotype parents in production, the present invention selects 20 pairs of South China carp parents whose loci are all TT type, and another 20 pairs of South China carp parents whose loci are all AT type, and obtains two offspring populations through natural reproduction, which are respectively referred to as breeding population and non-breeding population. When the fish are bred to one month old, they are marked with PIT electronic chips and randomly placed in the same rice field for mixed breeding. After 4 months of breeding, 100 samples are randomly selected to measure the weight and body length of the breeding population and the non-breeding population.

[0077] The growth data results are shown in Table 3. The results show that the weight and length of the breeding group are significantly higher than those of the non-breeding group (P>0.05), with growth rates of 8.71% and 5.15% respectively, and the coefficient of variation of the weight and length of the breeding group are lower than those of the non-breeding group, indicating that the offspring of the selected TT genotype parents have a fast growth rate and a low population variation coefficient, and can be used as a dominant genotype for the breeding of fast-growing South China carp populations. This suggests that in production, by testing South China carp parents and selecting TT samples as parents, faster-growing South China carp offspring can be obtained, and the production and breeding process of South China carp can be accelerated.

[0078] Table 3 Growth data of two groups cultured in the same pond for 4 months

[0079]

[0080]

[0081] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A SNP molecular marker associated with growth traits of South China carp, characterized in that: The sequence of the SNP molecular marker is shown in SEQ ID NO:

1. The SNP site is located at the 1001st position from the 5' end of the sequence shown in SEQ ID NO: 1, and its polymorphism is T / A.

2. The SNP molecular marker according to claim 1, characterized in that: When the genotype of the SNP site is AA, the growth trait of South China carp is a growth disadvantage. When the genotype of the SNP site is TT, the growth trait of South China carp is a growth advantage.

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 nucleotide sequences of the primer set are as follows: F: 5′-AAACAAACTGGCTCCCAGAG-3′; R: 5'-GGAACTAGTGGAAGTGGTTG-3'.

5. A kit comprising the primer set according to claim 3 or 4.

6. The kit according to claim 5, characterized in that The 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 kit according to claim 5 or 6 in any one of (1) to (6): (1) Assisted selection or breeding of South China carp; (2) preparing products to assist in the selection or breeding of South China carp; (3) Identify the growth rate of South China carp; (4) preparing a product for identifying the growth rate of South China carp; (5) Evaluate the population variation of South China carp; (6) Prepare products for evaluating the population variation of South China common 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 to be tested 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 of claim 3 or 4 or the kit of 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 of claim 1 or 2 in the genome of the South China carp to be tested; Preferably, when the genotype of the SNP site is TT, the South China common carp has excellent body weight, total length, body length, body height and head length and a low population variation coefficient.

10. Use of the method according to claim 8 or 9 in breeding excellent South China carp varieties and evaluating the variation of South China carp populations.

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