Molecular markers related to growth traits of Hehua carp and their application

Abstract

The present invention belongs to the field of animal molecular biology DNA marker technology and application technology, discloses molecular markers and applications related to the growth traits of Hehua carp, and specifically discloses SNP molecular markers related to the growth traits of Hehua 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 A / G. The present invention provides a SNP marker for Hehua carp, by detecting the SNP marker, Hehua carp with better growth traits can be effectively selected under the same breeding conditions, and can be effectively used for molecular marker-assisted breeding of Hehua carp. Then, the genotype of Hehua carp parents can be identified according to actual breeding needs, and the appropriate genotype Hehua carp parents can be selected for breeding, so that Hehua carp offspring (fry) with faster growth can be obtained, and the breeding process of Hehua carp can be accelerated.

Description

Technical Field

[0001] The invention belongs to the field of animal molecular biology DNA marker technology and application technology, and in particular relates to molecular markers related to growth traits of grass carp and applications thereof. Background Art

[0002] Common carp (Cyprinus carpio Linnaeus) is an important economic fish species widely cultured in Eurasia (Biermann and Geist, 2019). Due to factors such as geographical isolation and artificial selection, there are many local populations of common carp. Hehua carp refers to warm-water carp cultured in rice fields in southern China. It is named "Hehua carp" because the fish meat has the fragrance of Hehua flowers after eating Hehua flowers. Hehua carp culture in rice fields in the mountainous areas of northern Guangdong in my country has a long history. The integrated rice-fish farming model has the advantages of "promoting rice with fish, stabilizing grain production and increasing efficiency, quality and safety, and ecological protection", and has become one of the important ways to develop the rural economy. The survey found that the Hehua carp cultured in traditional rice fields has mixed germplasm, slow growth rate, and different body shapes because it has not been systematically selected. In order to increase the economic benefits of Hehua carp cultured in rice fields and improve the yield of Hehua carp, the existing Hehua carp materials in northern Guangdong were selected and bred based on growth rate and short round body shape.

[0003] While carrying out conventional breeding research, the development and application of molecular marker-assisted breeding technology can accelerate the breeding process of Cyprinus carpio rubrofuscus in this study. SNP refers to the polymorphism caused by single nucleotide variation in the genomic DNA sequence. Non-synonymous SNPs located in the gene coding region can lead to changes in amino acids, thereby affecting the function of proteins. In particular, SNPs occurring in the structural and functional regions are particularly important, and ultimately cause changes in biological phenotypes; SNPs located in the promoter region and intron region may also have an impact on gene expression regulation, indirectly regulating biological phenotypic traits. SNP markers are the first choice for finding markers related to the growth of Cyprinus carp because of their large number, reliable typing results, easy automated batch detection, and easy computer analysis results. In the early stage, this research team screened a number of candidate markers related to growth traits through resequencing, and further verified them by expanding the population, aiming to find genetic markers related to the growth of Cyprinus carp, and provide basic data for the molecular marker-assisted breeding of fast-growing new varieties of Cyprinus carp. Summary of the invention

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

[0005] The purpose of the first aspect of the present invention is to provide SNP molecular markers related to the growth traits of Grain 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 Grain 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-growing Grain Carp varieties.

[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 the growth traits of grass 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 A / G.

[0013] In some embodiments of the present invention, when the genotype of the SNP site is GG, the growth trait of the Hehua carp is a growth disadvantage, and when the genotype of the SNP site is AA, the growth trait of the Hehua carp is a growth advantage.

[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'-TCACATCATGCTCCCCCAAC-3' (SEQ ID NO: 2);

[0017] R: 5'-TAAGCTTCCCAATCCTCTGC-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 (4):

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

[0023] (2) preparing products for assisting breeding or breeding of grass carp;

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

[0025] (4) Prepare products for judging or identifying the growth rate of grass carp.

[0026] In some embodiments of the present invention, the breeding is to screen and obtain a variety (line) of Hehua carp with excellent weight, total length and body length.

[0027] The fifth aspect of the present invention provides a method for screening the growth rate of Grain Carp, by detecting the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the Grain Carp to be tested, and determining the growth rate of the Grain Carp to be tested according to the genotype.

[0028] In some embodiments of the present invention, the DNA of the grass 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 grass carp to be tested.

[0029] In some embodiments of the present invention, when the genotype of the SNP site is AA, the grass carp has excellent weight, total length and body length.

[0030] In some embodiments of the present invention, the DNA of the grass carp can be extracted by conventional means in the art, including the phenol-chloroform method and various DNA extraction kits.

[0031] 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.

[0032] 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.

[0033] In some embodiments of the present invention, the sequencing includes sequencing using the Sanger method.

[0034] The sixth aspect of the present invention provides application of the method of the fifth aspect of the present invention in breeding excellent-growing Grain Carp varieties.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides a SNP marker (SNP30621285) of a grass carp. By detecting the SNP marker, grass carp with better growth traits (heavier weight and faster growth) can be effectively selected under the same breeding conditions, and can be effectively used for molecular marker-assisted breeding of grass carp. Furthermore, the genotype of grass carp parents can be identified according to actual breeding needs, and grass carp parents with suitable genotypes can be selected for breeding, so that grass carp offspring (fry) with faster growth can be obtained, breeding time can be saved, cost is low, accuracy is high, and the breeding process of grass carp can be accelerated.

[0037] 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

[0038] Figure 1 Agarose gel electrophoresis of PCR amplification products of some samples.

[0039] Figure 2 This is the Sanger sequencing peak graph, from left to right are the AA genotype peak graph, the AG genotype peak graph and the GG genotype peak graph, in the figure, the arrow points to the SNP site. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below through specific examples.

[0041] 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.

[0042] 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.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1

[0045] This embodiment provides SNP molecular markers that can be used for breeding new varieties (lines) of fast-growing Hehua carp.

[0046] We selected 4-month-old Hehua carp from the same batch of breeding and pond 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 showed that SNP30621285 (SEQ ID NO: 1) had a significant difference in distribution frequency between the two populations.

[0047]

[0048]

[0049] Note: The bold bases in the brackets represent the SNP mutation point. The mutation type of this site is A to G. The underlined parts correspond to the detection primer sequences of the mutation point.

[0050] As can be seen from Table 1, the AA genotype only exists in the extremely large individual group, accounting for 78.57% of the sample distribution frequency, and is not distributed in the extremely small individual group; the GG genotype is not distributed in the extremely large individual group, but has a distribution frequency of 57.14% in the extremely small individual group; the AG genotype is distributed in both the extremely large individual group and the extremely small individual group. It is speculated that the AA genotype is a growth advantage genotype and the GG genotype is a growth disadvantage genotype.

[0051] Table 1 Distribution frequency of SNP30621285 marker genotypes in extreme samples

[0052] Group AA AG GG Extremely large individuals 11 3 0 Extremely small individuals 0 6 8

[0053] Example 2

[0054] This embodiment provides a primer pair that can be used to amplify SNP molecular markers associated with the growth rate of grass carp.

[0055] According to the gene fragment where the SNP30621285 marker is located, a pair of primers P were designed, the upstream primer sequence was F: 5'-TCACATCATGCTCCCCCAAC-3' (SEQ ID NO: 2); the downstream primer sequence was R: 5'-TAAGCTTCCCAATCCTCTGC-3' (SEQ ID NO: 3).

[0056] Example 3

[0057] This embodiment provides a method for breeding grass carp, comprising the following steps:

[0058] (1) Extraction of sample genomic DNA

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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.

[0063] (2) Amplify the sequence containing the SNP site using the primer pair of Example 2

[0064] The primers provided in Example 2 were used to perform PCR amplification on the DNA of the tested grass carp. 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.

[0065] 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.

[0066] (3) Typing

[0067] Using the PCR system to amplify the genome DNA of Hehua carp, a 300bp specific amplification band can be obtained. The amplification diagram of some samples is shown in Figure 1Then 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 AA genotype, it is a dominant genotype in terms of weight, total length and body length (i.e., the Hehua carp with this genotype is heavier and grows faster), while if the site is the GG genotype, it is a disadvantageous genotype.

[0068] Example 4

[0069] In order to verify whether the marker is a growth-related molecular marker, this embodiment reconstructs a batch of Hehua carp that were bred in the same pond and cultured in the same period. At the age of 1 month, random sampling was carried out and placed in a rice field for culture. After 5 months of culture, random sampling was carried out, and the weight was measured using an electronic balance (accuracy 0.01g), and the total length, body length, body height, head length, tail peduncle length and tail peduncle height were measured using a vernier caliper with an accuracy of 0.01cm. Finally, the fin rays were cut for sample genotyping (the determination method is the same as in Example 3), and the number of random sampling at 1 month old was 270, and the number of random sampling at 5 months old was 280.

[0070] The growth-related statistical results are shown in Table 2. It can be seen that at 1 month of age, the three genotypes have no significant differences in growth data such as weight and total length, and after 5 months of breeding, the AA genotype is significantly higher than the GG genotype in weight, total length and body length. However, there are no significant differences in body height, head length, tail peduncle length and tail peduncle height. It shows that the AA genotype belongs to the dominant genotype of weight, total length and body length (that is, the Hehua carp of this genotype belongs to the Hehua carp with heavier weight and faster growth), while GG belongs to the inferior genotype. It shows that the growth identification results of Hehua carp by the method of Example 3 are consistent with the identification results of the weight, body length, body height, etc. of Hehua carp directly measured, indicating that the SNP site of SEQ ID NO:1 can provide a reliable basis for the identification of the growth of Hehua carp, and can be used to quickly and accurately screen out Hehua carp with excellent growth traits. In production, we can obtain Hehua carp offspring with faster growth by detecting Hehua carp parents and selecting AA samples as parents, and can accelerate the production and breeding process of fast-growing Hehua carp.

[0071] Table 2 Genotype growth data corresponding to SNP30621285 marker

[0072]

[0073]

[0074] Note: The same shoulder letters indicate no significant difference (P>0.05), and different shoulder letters indicate significant difference (P<0.05).

[0075] 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. Use of a primer set for amplifying SNP molecular markers associated with growth traits of grass carp in any one of (1) to (4): (1) Assisted breeding or breeding of Hehua carp; (2) Preparing products for assisting the selection or breeding of Hehua carp; (3) Determine or identify the growth rate of Hehua carp; (4) Preparing products for judging or identifying the growth rate of Hehua 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 A / G; The breeding is to select and obtain Hehua carp varieties with excellent weight, total length and body length; When the genotype of the SNP site is AA, the carp has excellent weight, total length and body length; The AA genotype of Hehua carp is heavier and grows faster.

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

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