A SNP molecular marker associated with growth traits of yellowfin seabream and its application

Abstract

The present invention discloses a single-nucleotide polymorphism (SNP) molecular marker associated with growth traits in yellowfin sea bream (Gambusa spp.). The SNP is located at base 19030066 of chromosome 13 of the yellowfin sea bream (Gambusa spp.), with a C / G mutation type (SNP 13:19030066). The CC genotype is the preferred genotype. The present invention also discloses primers and a kit for detecting the SNP molecular marker, as well as the use of the SNP molecular marker, primers, and kit in breeding yellowfin sea bream with superior growth traits.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal molecular markers, and particularly relates to a SNP molecular marker related to growth traits of yellowfin sea bream and an application thereof. Background Art

[0002] Yellowfin sea bream ( Acanthopagrus latus ) belongs to the order Perciformes ( Perciformes )、Sparidae( Sparidae ), Rhynchops ( Sparus The yellowfin sea bream (Salmo salamander) is a popular fish species in southern my country's coastal areas, particularly Zhejiang, Fujian, Guangdong, Guangxi Zhuang Autonomous Region, and Taiwan Province. Its aquaculture is well-established in Guangdong and Fujian. Yellowfin sea bream (Salmo salamander) is a shallow, warm-water, omnivorous, bottom-dwelling fish. Growth rate is a key factor in determining production costs and economic returns during aquaculture. Genetic selection for growth traits has become an important tool for improving animal growth. Growth is influenced by a variety of factors, including genetics and the external environment.

[0003] Currently, traditional breeding methods are still used to select for growth traits in yellowfin seabream. However, since yellowfin seabream typically takes two years to mature from juveniles, traditional selective breeding techniques require more than five years to achieve significant genetic progress, which is a time-consuming process. In recent years, molecular marker-assisted selection, which targets phenotypic characteristics of fish, has gradually become the mainstream and is widely used in genetic breeding for superior traits.

[0004] Molecular markers are specific DNA fragments that can reflect differences in the genomes of individuals or populations. Single nucleotide polymorphisms (SNPs) refer to the presence of two different bases at a specific nucleotide position within the genome. SNP marker-assisted breeding involves selecting genetic material from individuals with different traits to identify heritable markers associated with those traits. Compared to the long lead times of traditional breeding, SNP marker-assisted breeding is fast, effective, accurate, and reliable, and has therefore been extensively studied by numerous scholars. Association analysis between SNP loci and important traits, identifying molecular markers closely associated with those traits for use in breeding, is a key tool in modern aquatic animal molecular breeding and plays a vital role in the genetics and breeding of commercial aquatic animals. Research and development of molecular markers for yellowfin sea bream, and the development of new varieties with superior growth traits such as fast growth and high yield, are essential for the growth and healthy development of the yellowfin sea bream aquaculture industry. Summary of the Invention

[0005] The first object of the present invention is to provide a SNP molecular marker related to the growth traits of yellowfin sea bream.

[0006] The present invention also aims to provide a primer or kit for detecting the above-mentioned SNP molecular markers.

[0007] The last object of the present invention is to provide the use of the above-mentioned SNP molecular marker primers or kit in breeding yellowfin sea bream with excellent growth traits.

[0008] The above-mentioned first purpose of the present invention can be achieved by the following technical scheme: a SNP molecular marker related to the growth traits of yellowfin sea bream is located at base 19030066 of chromosome 13 of yellowfin sea bream, and its mutation type is C / G, named SNP 13:19030066.

[0009] Furthermore, the growth traits include body weight and body length. Among them, the CC genotype is the preferred genotype, and the growth traits of individuals with this genotype are better than those of CG and GG genotypes.

[0010] The above-mentioned second purpose of the present invention can be achieved by the following technical scheme: a primer for detecting SNP molecular markers related to the growth traits of yellowfin sea bream, comprising an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2.

[0011] Specifically, the sequences of the upstream primer and the downstream primer are as follows:

[0012] Upstream primer: 5′-TCTGAACTAAACACTGATTGCACA-3′;

[0013] Downstream primer: 5'-CTCTGTGGCCTTAACCCCTG-3'.

[0014] The present invention also provides a kit for detecting SNP molecular markers related to the growth traits of yellowfin sea bream, and the kit comprises the above-mentioned primers.

[0015] The last object of the present invention can be achieved by the following technical solution: application of the above-mentioned SNP molecular markers or the above-mentioned primers or the above-mentioned kit in breeding yellowfin seabream with excellent growth traits.

[0016] Furthermore, the present invention also provides a method for breeding yellowfin seabream with excellent growth traits, comprising the following steps:

[0017] (1) Extract fin ray DNA from individual yellowfin seabream to be tested;

[0018] (2) performing PCR amplification on the extracted DNA using the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain an amplified product;

[0019] (3) The amplified products were sequenced and analyzed to determine the genotype of the above-mentioned SNP molecular marker (SNP 13:19030066 SNP site), and then analyzed to determine whether the individual has the potential for excellent growth traits.

[0020] Optionally, in step (3), it is determined whether the genotype of the SNP molecular marker (SNP 13:19030066 site) is the CC genotype, which has better individual growth traits than the CG and GG genotypes.

[0021] The present invention has the following advantages:

[0022] (1) The present invention analyzed the growth traits of a mixed family population of yellowfin sea bream through genome-wide association analysis and screened a candidate SNP site associated with individual weight, named SNP 13:19030066; and further verified that this SNP site was associated with growth traits in another yellowfin sea bream population;

[0023] (2) The SNP markers of the present invention can be determined by a pair of primers, which is simple and convenient to operate, and the results are accurate and reliable;

[0024] (3) The SNP sites, primers and kit provided by the present invention have application prospects in molecular marker-assisted breeding of yellowfin sea bream. The SNP molecular marker is not affected by factors such as individual age and gender, and can be used for early screening of yellowfin sea bream, which can significantly shorten the breeding time. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0026] Figure 1 This is the Manhattan plot of the growth shape of yellowfin sea bream, where A is the Manhattan plot of body weight and B is the Manhattan plot of body length (the red line in the figure represents the genome-wide significance threshold, and the blue line represents the suggestive threshold).

[0027] Figure 2 This is the QQ plot of the whole-genome association analysis of the growth shape of yellowfin sea bream, where A is the QQ plot of body weight and B is the QQ plot of body length.

[0028] Figure 3 The statistical results of weight traits of different genotypes of SNP site SNP 13:19030066 in Example 2 are shown. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand and implement the technical solution of the present invention. The following examples and accompanying drawings are only for illustrative purposes and are not to be construed as limiting the present invention. The reagents or materials used in the examples, unless otherwise specified, are all derived from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0030] To illustrate the present invention in more detail, the following examples are provided. It should be emphasized that the following examples are only for the purpose of explaining the present invention and are not intended to limit the scope or content of the present invention.

[0031] Example 1: Screening of SNP molecular markers associated with growth traits of yellowfin sea bream

[0032] The experimental yellowfin sea bream were raised in Guangdong Haiyuan Agriculture Co., Ltd. and were of mixed family stock at the age of 18 months.

[0033] 400 randomly selected experimental fish were measured and their growth traits were recorded. Tail fin samples were also collected and stored in 95% ethanol at -20°C for DNA extraction and sequencing. Fin ray DNA was extracted using the HiPure Tissue DNA Mini Kit (Magen, China). All DNA was analyzed by 1.5% agarose gel electrophoresis, and the results were assessed using a gel imaging system (Bio-Rad, USA) to ensure genomic integrity. The concentration of qualified DNA samples was then measured using a Qubit 3.0 assay and adjusted to 30 ng / μL. Libraries were then prepared according to standard protocols, and qualified libraries were sequenced using the Illumina platform. After quality control and processing of the obtained raw sequencing data, all sequencing reads were aligned to the reference genome (GenBank: GCA_904848185.1) using Bowtie version 2.0 (Langmead and Salzberg, 2012) software. SNPs were detected and genotyped using the standard process of samtools software, and a total of 15 high-quality SNP sites were obtained ( Figure 1 The general linear model (GLM) of Tassel software was used to conduct genome-wide association studies (GWAS) analysis on the body weight of yellowfin sea bream individuals. Figure 2 Subsequently, based on the Bonferroni correction, the genome-wide significant SNP marker threshold was set to -log10(Pvalue = 0.05 / 49940) = 1.00 × 10e -4 ,See Figure 1 .

[0034] Association analysis revealed one SNP marker that was extremely significantly associated with growth traits, and 14 SNP markers that were significantly associated with growth traits. The specific data are shown in Table 1.

[0035] Table 1 Information on SNP sites significantly associated with growth traits

[0036]

[0037] Note: Chr: chromosome; F: degree of freedom; PVE%: explained phenotypic variation; SNP ID is displayed as chromosome / position; "*" indicates that the SNP is extremely significant, and the reference genome for phenotypic explanation rate is GenBank: GCA_904848185.1.

[0038] From the results in Table 1, it can be seen that SNP 13:19030066 is associated with both the weight and body length of yellowfin sea bream. It is located at the 19030066th base on chromosome 13, that is, the 286th base of the nucleotide sequence shown in SEQ ID NO:3. The mutation type is C / G, named SNP 13:19030066, among which the CC genotype is the preferred genotype with better growth performance.

[0039] Example 2: Verification of SNP molecular markers related to growth traits of yellowfin sea bream

[0040] The verification test selected different yellowfin sea bream populations, and randomly selected 100 experimental fish from them, measured and recorded the weight trait indicators of each fish, and simultaneously collected and preserved the tail fin samples of each fish for DNA extraction. The specific process of DNA extraction was the same as that in Example 1.

[0041] Subsequently, PCR amplification was performed using the extracted DNA as a template and SEQ ID NO: 1 and SEQ ID NO: 2 as primers to obtain a gene fragment containing SNP 13: 19030066. The total volume of PCR amplification was 20 μL, and the specific reaction system is shown in Table 2.

[0042] Table 2 PCR amplification reaction system

[0043]

[0044] The specific amplification procedure was as follows: pre-denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 5 min. 2 μL of the reaction product was subjected to 1% agarose gel electrophoresis, and qualified samples were used for subsequent sequencing to determine the genotype of each individual sample at SNP 13:19030066.

[0045] Figure 3The results of validating the SNP site SNP 13:19030066 are shown.

[0046] Figure 3 The results showed that: in yellowfin sea bream, there were significant differences in body weight among individuals with different genotypes of SNP 13:19030066; among them, CC genotype individuals accounted for a larger proportion in the largest group and a smaller proportion in the smallest group, that is, the number of CC genotype individuals in the largest group was much greater than that in the smallest group; while GG genotype individuals accounted for a smaller proportion in the largest group and a larger proportion in the smallest group.

[0047] In summary, SNP 13:19030066 is significantly associated with the growth traits of yellowfin sea bream, and its genotype can be determined by a pair of primers, which is simple and reliable. This SNP site has application prospects in molecular marker-assisted breeding of yellowfin sea bream.

[0048] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention. Any improvements and modifications based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection shall be subject to the claims.

Claims

1. A primer for detecting SNP molecular markers related to growth traits of yellowfin sea bream, characterized in that: It includes an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID NO: 1, and the sequence of the downstream primer is shown in SEQ ID NO: 2; the SNP molecular marker related to the growth traits of yellowfin sea bream is located at the 19030066 base of chromosome 13 of yellowfin sea bream, and its mutation type is C / G.

2. A kit for detecting SNP molecular markers related to growth traits of yellowfin sea bream, characterized in that: The invention comprises the primers according to claim 1; the SNP molecular marker related to the growth traits of yellowfin sea bream is located at base 19030066 of chromosome 13 of yellowfin sea bream, and its mutation type is C / G.

3. Use of the primers according to claim 1 or the kit according to claim 2 in breeding yellowfin seabream with excellent growth traits; the growth traits are body weight and body length, wherein the growth traits of individuals with CC genotype are better than those of CG and GG genotypes.

4. The method for breeding yellowfin seabream with excellent growth traits according to claim 3, characterized in that: The following steps are involved: (1) Extract fin ray DNA from individual yellowfin seabream to be tested; (2) performing PCR amplification on the extracted DNA using the primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain an amplified product; (3) Sequencing and analyzing the amplified products to determine the genotype of the SNP molecular marker, and then analyzing to determine whether the individual has the potential for excellent growth traits.

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