A SNP marker related to growth performance of fast-growing south china carp, primer and application thereof

Abstract

The application belongs to the field of animal molecular biology DNA marker technology and application technology, discloses a SNP marker related to the growth performance of fast-growing southern catfish, primers and application thereof, and specifically discloses a SNP molecular marker related to the growth trait of southern catfish, the sequence of the SNP molecular marker is shown as SEQ ID NO:1, the SNP site is located at the 1001th position from the 5' end of the sequence shown as SEQ ID NO:1, and the polymorphism is T / A. The application provides a SNP marker of southern catfish, through detection of the SNP marker, better southern catfish with growth traits can be effectively selected under the same breeding conditions, and the SNP marker assisted breeding of southern catfish can be effectively used. Furthermore, according to actual breeding requirements, the genotypes of southern catfish parents can be identified, suitable genotype southern catfish parents can be selected for breeding, and southern catfish offspring (fish fry) with faster growth can be obtained, and the southern catfish breeding process can be accelerated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal molecular biology DNA marker technology and application technology, and particularly relates to a SNP marker related to the growth performance of fast-growing South China carp, primers and application thereof. BACKGROUND

[0002] Rice-fish integrated farming is a typical ecological circular agricultural development model, which organically combines rice planting and aquaculture, utilizes the principles of biological reciprocity and resource complementary utilization, and constructs a rice-fish rotation system. Under the premise of stable rice yield, high-quality and non-polluting aquatic products are produced, which greatly improves the comprehensive economic benefit of rice fields and protects and improves the ecological environment of rice fields. The fish species for rice farming in Guangdong, China is mainly South China carp (Cyprinus carpio rubrofuscus), which is a subspecies of Cyprinus carpio Linnaeus in the order Cypriniformes, family Cyprinidae, genus Cyprinus. Traditional rice-farmed South China carp has many growth-stagnant fish due to lack of systematic breeding. In order to increase the economic benefit of rice-farmed South China carp and improve the yield of South China carp, it is necessary to breed new varieties (lines) of fast-growing South China carp.

[0003] With the rapid development of high-throughput sequencing technology, Genome-wide association study (GWAS) is increasingly applied to genetic breeding of aquatic animals, and has become an important method for studying fish quantitative traits. Genome-wide association study is to obtain nucleic acid polymorphism (single nucleotide polymorphism, SNP) sites at the whole genome level of individuals through resequencing, gene chip and other methods, and then to mine associated genes affecting phenotypic traits (such as disease, growth, etc.). It has the advantages of high resolution, wide source of research materials, rich variations captured, high accuracy and time saving. In the previous study, our research team screened a batch of candidate SNP markers related to growth traits by resequencing, and further verified them by expanding the population, aiming to find SNP markers closely related to the growth of South China carp, and lay a foundation for molecular marker-assisted breeding of fast-growing South China carp new varieties. SUMMARY

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

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

[0006] The second aspect of the present application aims to provide a primer set for amplifying the SNP molecular marker of the first aspect of the present application.

[0007] The third aspect of the present application aims to provide a kit.

[0008] The fourth aspect of the present application aims to provide an application of the SNP molecular marker of the first aspect of the present application, the primer set of the second aspect of the present application and / or the kit of the third aspect of the present application.

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

[0010] The sixth aspect of the present application aims to provide an application of the method of the fifth aspect of the present application in breeding South China carp varieties with excellent growth and evaluating the variation of a South China carp population.

[0011] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0012] The first aspect of the present application provides a SNP molecular marker related to the growth trait 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 1001th position from the 5' end of the sequence shown in SEQ ID NO: 1, and the polymorphism is T / A.

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

[0014] The second aspect of the present application provides a primer set for amplifying the SNP molecular marker of the first aspect of the present application.

[0015] In some embodiments of the present application, 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 application provides a kit comprising the primer set of the second aspect of the present application.

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

[0020] In some embodiments of the present application, the buffer used in the PCR is any reagent required for PCR amplification, such as dNTP, taq enzyme, MgCl2, etc.

[0021] In a fourth aspect of the present application, the SNP molecular marker of the first aspect of the present application, the primer set of the second aspect of the present application and / or the kit of the third aspect of the present application are used in any one of (1) to (6):

[0022] (1) assisted selection or breeding of C. sinensis;

[0023] (2) preparation of a product for assisted selection or breeding of C. sinensis;

[0024] (3) determining or identifying the growth rate of C. sinensis;

[0025] (4) preparation of a product for determining or identifying the growth rate of C. sinensis;

[0026] (5) evaluating the population variation of C. sinensis;

[0027] (6) preparation of a product for evaluating the population variation of C. sinensis.

[0028] In some embodiments of the present application, the selection is to screen C. sinensis varieties (lines) with excellent body weight, total length, body length, body height and head length and low population variation coefficient.

[0029] In a fifth aspect of the present application, a method for screening the growth rate of C. sinensis is provided, which comprises detecting the genotype of the SNP molecular marker of the first aspect of the present application in the genome of the C. sinensis to be tested, and determining the growth rate of the C. sinensis to be tested according to the genotype.

[0030] In some embodiments of the present application, the DNA of the C. sinensis to be tested is used as a template to perform PCR amplification using the primer set of the second aspect of the present application or the kit of the third aspect of the present application, so as to obtain a PCR amplification product; and the PCR amplification product is subjected to sequencing analysis to determine the gene of the SNP molecular marker of the first aspect of the present application in the genome of the C. sinensis to be tested.

[0031] In some embodiments of the present application, when the genotype of the SNP site is TT, it is C. sinensis with excellent body weight, total length, body length, body height and head length and low population variation coefficient.

[0032] In some embodiments of the present application, the DNA of the C. sinensis can be extracted by conventional means in the technical field, including phenol chloroform method and various DNA extraction kits.

[0033] In some embodiments of the present application, the reaction procedure of PCR amplification is 90-94℃ pre-denaturation for 3-6min; 90-94℃ denaturation for 30-35s, 56-60℃ annealing for 30-35s, 70-72℃ extension for 80-90s, 30-35 cycles; 70-72℃ extension for 5-10min.

[0034] In some embodiments of the present application, the reaction system of PCR amplification comprises 1-3mmol / L MgCl2, 0.1-0.2mmol / L dNTP, 0.05-0.1U / μL Taq enzyme, and 0.2-0.3μmol / L of each of the upstream and downstream primers.

[0035] In some embodiments of the present application, the sequencing comprises sequencing using sanger method.

[0036] In the sixth aspect of the present application, the method of the fifth aspect of the present application is applied in breeding excellent growth Southern catfish varieties and evaluating Southern catfish population variation.

[0037] The present application has the following advantages:

[0038] The present application provides a SNP marker (SNP27368615) of Southern catfish. By detecting the SNP marker, Southern catfish with better growth traits (faster growth, lower population variation coefficient) can be effectively selected under the same breeding conditions, and the SNP marker assisted breeding of Southern catfish can be effectively used. Furthermore, according to the actual breeding needs, the genotypes of Southern catfish parents can be identified, and the Southern catfish parents with suitable genotypes can be selected for breeding, so that Southern catfish offspring (fish fry) with faster growth can be obtained, the breeding time is saved, the cost is low, the accuracy is high, the breeding process of Southern catfish is accelerated, and the economic benefits of rice field breeding of Southern catfish are increased.

[0039] The genotype obtained by the present application is based on the base mutation generated in the gene, so there is no genetic exchange, and no further phenotype verification is required. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 2 is an agarose gel electrophoresis diagram of PCR amplification products of some samples.

[0041] Figure 2 Figure 3 is a sanger sequencing peak diagram, from left to right, respectively, TT genotype peak diagram, AT genotype peak diagram and AA genotype peak diagram, and the arrow points to the SNP site. DETAILED DESCRIPTION

[0042] The content of the present application is further described in detail through specific examples.

[0043] It should be understood that the embodiments are only used for illustrating the present application but not for limiting the scope of the present application.

[0044] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. The specific conditions not noted in the embodiments are implemented according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all the conventional products which can be purchased in the market.

[0045] The features and performances of the present application will be further described in details below in combination with the embodiments.

[0046] Embodiment 1

[0047] The present embodiment provides a SNP molecular marker which can be used for the selection of a new variety (strain) of fast-growing South China Tilapia.

[0048] The 4-month-old South China Tilapia breeding groups in the same batch of breeding and in the same pond were sampled. 14 tail fin samples of extreme large individuals (body weight ≧ 25 g) and 14 tail fin samples of extreme small individuals (body weight ≦ 10 g) were selected, and the samples were sent to Huada Gene for resequencing. The SNP27368615 (nucleotide sequence as shown in SEQ ID NO: 1) was screened in the resequencing results, and the distribution frequencies of the SNP27368615 in the two groups were significantly different.

[0049]

[0050]

[0051] Note: The bold bases in the square brackets represent the SNP mutation point. The mutation type of this site is T mutated into A. The underlined part corresponds to the detection primer sequence of the mutation point.

[0052] As shown in Table 1, the distribution frequencies of AA genotype in the extreme large individual group and the extreme small individual group are 0% and 57.14% respectively, while the distribution frequencies of TT genotype in the extreme large individual group and the extreme small individual group are 78.57% and 0% respectively, and the distribution frequencies of AT genotype in the extreme large individual group and the extreme small individual group 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 body group 0 3 11 Extremely small body group 8 6 0

[0055] Embodiment 2

[0056] The present embodiment provides a primer pair which can be used for amplifying a SNP molecular marker related to the growth rate of South China Tilapia.

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

[0058] Example 3

[0059] The present example provides a selection method for Ctenopharyngodon idellus, which comprises the following steps:

[0060] (1) Extraction of sample genomic DNA

[0061] 1) 3 mg of fin tissue of the fish to be detected is cut and added with 0.5 mL of lysis solution (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 then digested at 55°C for 1 hour;

[0062] 2) An equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) mixture is added, mixed, and then placed at room temperature for 5 minutes, followed by centrifugation at 12000 rpm for 10 minutes, and then the supernatant is taken and extracted with chloroform again, and then placed at room temperature for 5 minutes, followed by centrifugation at 12000 rpm for 10 minutes, and then the supernatant is taken;

[0063] 3) 2 volumes of anhydrous ethanol are added, and then the DNA is precipitated by placing at room temperature for 10 minutes, followed by centrifugation at 12000 rpm for 10 minutes;

[0064] 4) The precipitate is washed with 70% ethanol once, centrifuged at 12000 rpm for 2 minutes, and then the supernatant is absorbed and dried at room temperature for 10 minutes, and then 50 μL of TE (10 mmol / L Tris-HCl and 1 mmol / L EDTA, pH 8.0) is added to dissolve the DNA, and then the DNA is stored at -20°C for standby use.

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

[0066] The DNA of the Ctenopharyngodon idellus to be detected is subjected to PCR amplification by using the primer pair provided in Example 2, and the reaction system of the PCR amplification comprises 1-3 mmol / L MgCl2, 0.1-0.2 mmol / L dNTP, 0.05-0.1 U / μL Taq enzyme, and 0.2-0.3 μmol / L of the upstream and downstream primers.

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

[0068] (3) Genotyping determination

[0069] The genomic DNA of the South China Tilapia is amplified by using the PCR system, and a specific amplification band of 280 bp can be obtained, and part of the amplification diagram of the sample is shown in Figure 1 Then, the PCR product is directly sequenced by using the sanger method. The sequencing peak diagram is viewed Figure 2 ), the mutation site genotype is determined, if the site is TT genotype, it belongs to the dominant genotype of body weight, total length, body length, body height and head length (i.e. the South China Tilapia with this genotype belongs to the South China Tilapia with fast growth speed and low population variation coefficient), and if the site is AA genotype, it belongs to the inferior genotype.

[0070] Example 4

[0071] In order to verify whether the marker is a growth-related molecular marker, the present application re-constructs the 1-month-old South China Tilapia which is bred in the same period and in the same pond, and randomly samples are put into the rice field for 4-month breeding. The body weight (precision 0.01 g) and total length, body length, body height, head length, tail handle length and tail handle height of the test fish before and after the rice field breeding are measured, and the precision is 0.01 cm. The number of random samples before breeding is 271, and the number of random samples after breeding is 280. The test fish is cut for fin bar for sample genotyping.

[0072] The growth-related statistical results are shown in Table 2. It can be seen that before breeding, the test fish of the three genotypes has no significant difference in body weight, total length, body height, head length, tail handle length and tail handle height (P>0.05). After 4-month breeding, the TT genotype is significantly higher than the AA and AT genotypes in body weight, total length, body length, body height and head length (P<0.05), but has no significant difference in tail handle length and tail handle height (P>0.05), indicating that the TT genotype belongs to the dominant genotype of body weight, total length, body length, body height and head length, and the AA and AT genotypes belong to the inferior genotype, wherein the average of the AA genotype sample in body weight, total length, body length, body height and head length is lower than that of the AT genotype. It is shown that the growth identification result of the South China Tilapia by the method of Example 3 is consistent with the identification result by directly measuring the body weight, body length, body height, etc. of the South China Tilapia, indicating that the SNP site of SEQ ID NO: 1 can provide a reliable basis for the growth identification of the South China Tilapia, and can be used for quickly and accurately screening the South China Tilapia with excellent growth traits.

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

[0074]

[0075] Note: The same superscript letter means no significant difference (P>0.05), and different superscript letter means significant difference (P<0.05).

[0076] In order to verify whether the fast-growing South China Sea carp population can be quickly obtained by selecting and retaining TT genotype parents in production, 20 pairs of South China Sea carp parents with TT type at this site and 20 pairs of South China Sea carp parents with AT type at this site are selected, and two offspring populations are obtained by natural breeding, which are called breeding population and non-breeding population, respectively. PIT electronic chip is labeled at 1 month of age and randomly placed in the same rice field for mixed culture. After 4 months of culture, 100 tail samples are randomly selected to measure the weight and length of the breeding population and the non-breeding population.

[0077] The growth data results are shown in Table 3, and the results show that the weight and length of the breeding population are significantly higher than those of the non-breeding population (P>0.05), the growth rates are 8.71% and 5.15%, respectively, and the coefficients of variation of the weight and length of the breeding population are lower than those of the non-breeding population, indicating that the offspring of the selected TT genotype parents have fast growth rate and low population variation coefficient, which is an advantageous genotype for fast-growing South China Sea carp breeding. It is suggested that by detecting South China Sea carp parents and selecting and retaining TT samples as parents in production, South China Sea carp offspring with faster growth can be obtained, and the production and breeding process of South China Sea carp can be accelerated.

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

[0079]

[0080]

[0081] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary skilled persons in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a primer set for amplifying a SNP molecular marker related to growth traits of South China Sea Cyprinus carpio and / or a kit comprising the primer set in any one of (1) to (4): (1) South China Sea Cyprinus carpio assisted selection; (2) preparation of a product of South China Sea Cyprinus carpio assisted selection; (3) identifying the growth rate of South China Sea Cyprinus carpio; (4) preparation of a product for identifying the growth rate of South China Sea Cyprinus carpio; the sequence of the SNP molecular marker is shown as SEQ ID NO: 1, the SNP site is located at the 1001th position from the 5' end of the sequence shown as SEQ ID NO: 1, and the polymorphism is T / A; the selection in (1) to (2) is screening of a South China Sea Cyprinus carpio breed with excellent body weight, total length, body length, body height and head length and low population variation coefficient.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the primer set is as follows: F: 5'-AAACAAACTGGCTCCCAGAG-3'; R: 5'-GGAACTAGTGGAAGTGGTTG-3'.

3. Use according to claim 2, characterized in that, The kit further comprises a buffer used in PCR.

4. A method for screening the growth rate of South China Sea Cyprinus carpio by detecting the genotype of the SNP molecular marker in claim 1 in the genome of the South China Sea Cyprinus carpio to be tested, and determining the growth rate of the South China Sea Cyprinus carpio to be tested according to the genotype.

5. The method of claim 4, wherein, The method comprises the following steps: using the DNA of the South China Sea Cyprinus carpio to be tested as a template, using the primer set in any one of claims 1 to 3 or the kit in any one of claims 1 to 3 to perform PCR amplification to obtain a PCR amplification product; and performing sequencing analysis on the PCR amplification product to determine the genotype of the SNP molecular marker in the genome of the South China Sea Cyprinus carpio to be tested in claim 1.

6. The method of claim 5, wherein, When the genotype of the SNP site is TT, it is a South China Sea Cyprinus carpio with excellent body weight, total length, body length, body height and head length and low population variation coefficient.

7. Use of the method in any one of claims 4 to 6 in breeding a South China Sea Cyprinus carpio breed with excellent growth; the breeding is screening of a South China Sea Cyprinus carpio breed with excellent body weight, total length, body length, body height and head length and low population variation coefficient.

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