CN117051130B - SNP molecular marker associated with streptococcus agalactiae resistance of trachinotus ovatus and application thereof - Google Patents

Abstract

The invention discloses an SNP molecular marker associated with the streptococcus agalactiae resistance of trachinotus ovatus, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, wherein the 121 st position is A/C, and when the genotype is CC, the streptococcus agalactiae resistance of the trachinotus ovatus is obviously better than that of the trachinotus ovatus with the genotypes of AC and AA. Also discloses a primer and a kit for amplifying the SNP molecular marker and a detection method for resisting streptococcus agalactiae trachinotus ovatus; the SNP molecular marker, the primer, the kit or the method are applied to selective breeding of the anti-streptococcus agalactiae character of the trachinotus ovatus. By applying the technical scheme of the invention, the breeding material of the trachinotus ovatus can be screened early, the breeding period is shortened, the breeding accuracy is improved, the genetic quality of the trachinotus ovatus is improved, the molecular marker resources of the trachinotus ovatus are enriched, and finally, the efficient breeding of a new variety of high-quality trachinotus ovatus is realized.

Description

A SNP molecular marker associated with the resistance to Streptococcus agalactiae in the oval pompano fish and its its application

Technical field

The invention belongs to the technical field of screening and assisted breeding of aquatic animal molecular markers, and specifically relates to a SNP molecular marker related to the resistance of Streptococcus agalactiae in oval pompano and its application.

Background technique

The pomfret ovate is a cultured fish with high economic value. However, the pomfret ovate aquaculture industry faces many disease threats, among which Streptococcus agalactiae infection is one of the main diseases. Streptococcus agalactiae disease has seriously affected the growth and survival of Pomfret ovata, causing huge economic losses to the breeding industry. Therefore, improving the resistance to Streptococcus agalactiae in Pomfret ovata has extremely important economic and social significance.

In the existing technology, Streptococcus agalactiae infection is mainly prevented and treated through drug treatment and vaccination. However, the results of these methods are not ideal, and there are problems such as drug residues, drug resistance, and environmental pollution. In addition, improving disease resistance in Pomfret ovata through traditional selective breeding methods takes a long time, and the effects are often limited by the complex interaction of genetic and environmental factors.

Gene-wide association analysis (GWAS) is a technology that has been widely used in molecular biology and genetics research in recent years. By analyzing genotype and phenotypic data of a large number of individuals, the relationship between genetic variations and traits can be found. association. However, although GWAS has successfully found association loci for many important traits in many species, there are still relatively few studies on the resistance to Streptococcus agalactiae in Pomfret ovata.

Therefore, carrying out whole-gene association analysis to find SNP molecular markers related to the resistance of Pomfret ovata to Streptococcus agalactiae, and using these markers for selective breeding, is a promising solution to solve the above problems.

Contents of the invention

The first object of the present invention is to provide a SNP molecular marker related to the resistance to Streptococcus agalactiae in Pomfret ovate.

Another object of the present invention is to provide a primer and a kit for amplifying the above-mentioned SNP molecular markers associated with the anti-Streptococcus agalactiae trait of Pomfret ovate and a detection method for Pomfret ovate against Streptococcus agalactiae.

The last object of the present invention is to provide the application of the above-mentioned SNP molecular markers, primers, kits or methods in the selective breeding of ovate pomfret trevally for resistance to Streptococcus agalactiae.

The above-mentioned first object of the present invention can be achieved by the following technical solution: a SNP molecular marker related to the anti-Streptococcus agalactiae trait of the oval pomfret, the nucleotide sequence of the SNP molecular marker is such as SEQ ID NO. As shown in 1, the 121st position is A/C.

The sequence shown in SEQ ID NO.1 is as follows:

TTTAACCTGCTTCTCTTGATACTGAATGGAAATTATAGATGCAATCGTTTTACAGTAGATAGAATATAGAATCTCTTCTTTGTGTTTTCAAGGACACACTCACTTT

TAGTCACAAAAATCTN(A/C)AAATTTTCCAAATTTATCAAAGTTTTGTCAGATTTAGGTTTGTATTAGGATTCAGTCTTTCACATGATTGTTCAACCATAAAAAAA

AAATTCCCTTTTCAGTTAAACATGCAAGCC.

Through the genome-wide association analysis (GWAS) strategy, the present invention discovered a SNP molecular marker located on chromosome 9 of Pomfret ovate that is significantly associated with resistance to Streptococcus agalactiae.

Specifically, the mark is the 121st base of the nucleotide sequence shown in SEQ ID NO.1.

Regarding the nucleotide sequence of SEQ ID NO. 1, the N at base 121 represents A or C, which indicates the presence of a polymorphism at this position.

Statistical analysis showed that compared with the CC genotype, there were significant differences in resistance to Streptococcus agalactiae in Pomfret ovata corresponding to the AC and AA genotypes.

Alternatively, when the genotype is CC, the ability of Pomfret ovata to resist Streptococcus agalactiae is significantly better than that of Pomfret ovata with genotypes AC and AA.

Specifically, when the genotype is CC, the resistance ability of Pomfret ovata to the semi-lethal concentration of Streptococcus agalactiae at 120 hours after infection is significantly better than that of Pomfret ovata with genotypes AC and AA.

The anti-Streptococcus agalactiae properties of the oval pomfret described in the present invention refer to the survival status of the oval pomfret after being infected by intraperitoneal injection with a semi-lethal concentration of Streptococcus agalactiae for 120 hours.

Resistance assessment: Those with obvious infection characteristics of Streptococcus agalactiae and death within 120 hours of injection are considered susceptible to Streptococcus agalactiae, while those with no obvious infection characteristics 120 hours after injection and swimming in good condition are considered resistant to Streptococcus agalactiae.

The core part of the present invention involves the use of specific SNP molecular markers to conduct accurate genotype detection on selected candidate groups in the breeding of oval pompano. Combined with the analysis of other gene loci related to growth traits, disease resistance and stress resistance traits, the present invention particularly recommends selecting individuals whose SNP genotype is CC as a method for breeding oval pomfret with resistance to Streptococcus agalactiae. Preferred parent.

The present invention screens SNP molecular markers that are significantly related to the anti-Streptococcus agalactiae traits of the oval pomfret, and includes the following important steps:

(1) Select the fin ray samples of the oval pomfret trevally infected by intraperitoneal injection of a 120-hour semi-lethal concentration of Streptococcus agalactiae PBS suspension, and efficiently extract DNA from them for deep whole-genome sequencing;

(2) Strictly compare the sequencing data obtained in step (1) with the standard reference genome of Pomfret ovata to obtain genome-wide SNP molecular markers;

(3) Use genome-wide association analysis technology to conduct an in-depth study of the correlation between the Streptococcus agalactiae resistance traits and genotypes of Pomfret ovate, and identify SNP markers that are significantly associated with the Streptococcus agalactiae resistance traits of Pomfret ovate.

Further, the present invention details a precise method for screening SNP molecular markers related to the resistance of Streptococcus agalactiae in Pomfret ovata. The method includes the following key steps:

(1) First, about 2,000 egg-shaped pomfret were selected. These egg-shaped pomfret were all treated with intraperitoneal injection of a PBS suspension of Streptococcus agalactiae at a semi-lethal concentration for 120 hours. Subsequently, the 100 eggs that died first and the ones that died at 120 hours were selected. Fin ray samples were clipped from 100 oval pomfret trevally whose activity was still good, and then DNA was extracted and whole genome resequenced; (2) The sequencing data obtained in step (1) was compared with the standard of oval pomfret trevally. Conduct detailed comparisons with reference genomes to accurately obtain genome-wide SNP information;

(3) Use genome-wide association analysis (GWAS) technology to conduct in-depth research on the correlation between the anti-Streptococcus agalactiae traits of the ovate pomfret and each genotype, and screen out the anti-Streptococcus agalactiae traits of the ovate pomfret. SNP markers with significant associations.

Through the above method, the present invention contributes valuable molecular marker resources to the field of selective breeding of ovate pomfret, achieves precise typing of SNPs, and selects genes significantly related to the Streptococcus agalactiae resistance traits of ovate pomfret through GWAS. SNP. These SNP markers are expected to be widely used in selective breeding for resistance to Streptococcus agalactiae in Pomfret ovate.

The above-mentioned second object of the present invention can be achieved by the following technical solution: primers for amplifying SNP molecular markers associated with the resistance of Streptococcus agalactiae traits of ovate pomfret, the primers include primer SNPLG918597184FA, primer SNPLG918597184FC and primer SNPLG918597184R , wherein the sequence of the primer SNPLG918597184FA is shown in seq id no.2, the sequence of the primer SNPLG918597184FC is shown in seq id no.3, and the sequence of the primer SNPLG918597184R is shown in seq id no.4.

Based on the nucleotide sequence of SEQ ID NO. 1, the present invention designed specific primers for SNP molecular markers, named SNPLG918597184FA, SNPLG918597184FC and SNPLG918597184R. After using these primers for specific PCR amplification, the fluorescence signal is read by a microplate reader, and then the signal is analyzed to obtain the genotype of the SNP molecular marker site of the oval pompano to be tested.

The present invention describes in detail a series of primers related to the anti-Streptococcus agalactiae traits of Pomfret ovate. The design is based on the 120BP genome sequence of the upstream and downstream markers, and the professional SNP primer software tool is used for accurate primer design.

To elaborate, the nucleotide sequences of these specific primers are listed below:

Snplg918597184FA(seq id no.2)：

GAAGGTGACCAAGTTCATGCTACACTCACTTTTAGTCACAAAATCTCA;

Snplg918597184FC(seq id no.3)：

GAAGGTCGGAGTCAACGGATTACACTCACTTTTAGTCACAAAATCTCC;

Snplg918597184r(seq id no.4)：

TGAAAGACTGAATCCTAAATACAAACC.

In order to cooperate with the application of the above-mentioned primers, the present invention also carefully prepares a special kit, which mainly contains the above-mentioned primers.

The invention also proposes a detection method for resistance to Streptococcus agalactiae in Pomfret ovata, which includes the following steps:

(1) Extract genomic DNA from the oval pomfret to be tested;

(2) Use the specific primers or kits described above to perform PCR amplification of the genomic DNA of the ovate pomfret to be tested, thereby obtaining the PCR amplification product;

(3) By analyzing the fluorescence signal of the PCR amplification product, determine the genotype of the ovoid pomfret to be tested, and determine its anti-Streptococcus agalactiae performance based on this.

In order to ensure the accuracy of the detection method, the present invention preferentially recommends that in step (1), cut some fin rays from the oval pomfret to extract total DNA, while ensuring the quality and purity of the obtained DNA sample, that is, A260/A280 The ratio should be in the range of 1.8 to 2.0, and the DNA concentration should not be less than 100 μg/μL.

In step (2), the optimized PCR system for PCR amplification is shown in Table 1 below:

Table 1 PCR system

The PCR amplification procedure is shown in Table 2 below:

Table 2 PCR amplification procedure

In the genotyping process of step (3), after completing the PCR, use a microplate reader to read the obtained fluorescence signal. Subsequently, the professional online software snpdecoder was used to analyze and convert the fluorescence signal. Based on the color difference of the fluorescent signal, the software can output the corresponding genotype.

In this step, analyzing the fluorescence signal of the PCR product to obtain the genotype is a cost-effective strategy that has cost advantages compared with traditional sequencing methods.

Based on the data results, when the genotype of Pomfret ovate is CC, the survival rate after infection with Streptococcus agalactiae is significantly higher than that of individuals with genotype AC or AA. Therefore, in breeding selection, it is wise to give priority to using the ovate pomfret with the CC genotype as the SNP locus as a parent, and avoid using the ovate pomfret with the AC or AA genotype as the SNP locus as the parent.

The above last object of the present invention can be achieved through the following technical solutions: the application of the above SNP molecular markers, the above primers, the above kits or the above methods in the selective breeding of ovate pomfret for resistance to Streptococcus agalactiae.

The present invention not only provides the preparation and application of the above-mentioned SNP molecular markers, primer pairs and kits, but also clarifies their importance and application potential in the selective breeding of ovate pomfret for resistance to Streptococcus agalactiae.

In summary, the present invention provides SNP analysis markers associated with resistance of Pomfret ovata to Streptococcus agalactiae. It involves the technical fields of molecular marker screening and assisted breeding of aquatic animals. In addition to marker screening methods, subsequent genotype identification is also described in detail. This technical solution allows primary screening of ovate pomfret breeding materials, thereby improving the efficiency and accuracy of breeding for resistance to Streptococcus agalactiae traits and at the same time providing a new SNP molecular marker for ovate pomfret.

The invention has the following advantages:

(1) According to the statistical analysis of the embodiment of the present invention, when the genotype of the SNP marker is CC, the resistance of the ovate pomfret to Streptococcus agalactiae is significantly better than that of the ovate pomfret whose genotype is AA or AC;

(2) Therefore, the SNP molecular marker of the present invention can be used to accurately predict the resistance of ovate pomfret to Streptococcus agalactiae, providing a powerful tool for the selective breeding of ovate pomfret;

(3) By applying the technical solution of the present invention, the breeding materials of the oval pomfret can be screened early, the breeding cycle can be shortened, and the accuracy of the selection can be improved, which can help to improve the genetic quality of the oval pomfret and enrich it. Molecular marker resources, and ultimately achieve efficient breeding of new high-quality oval pompano species.

Description of the drawings

The present invention will be further described below with reference to the accompanying drawings and embodiments.

Figure 1 is a Manhattan diagram of Example 1, in which the portion marked with an arrow represents the position of the molecular marker selected in the present invention. This marker is located on chromosome No. 9 of the oval Pomfret trevally.

Implementation

The technical solution of the present invention will be described in detail below with reference to 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 drawings are only for illustrative purposes and should not be construed as limitations of the present invention. Unless otherwise specified, the reagents or materials used in the examples were obtained from commercial sources. Unless otherwise stated, the experimental instruments used are routine laboratory instruments.

In order to illustrate the present invention in more detail, the following examples are provided. It should be emphasized that the following examples are only for explaining the present invention and are not intended to limit the essential scope or content of the present invention.

Example 1: Screening of SNP markers

(1) Streptococcus agalactiae infection experiment:

2000 oval pompano were randomly selected from the population.

The selected pomfret ovate was intraperitoneally injected with a PBS suspension of Streptococcus agalactiae at a semi-lethal concentration for 120 hours. The half-lethal concentration required for the experiment for 120 hours is 2.0×10 ⁶ CFU/mL.

After treatment, the survival status of each ovate pomfret was recorded in detail, and this data was used as a benchmark for evaluating its resistance to Streptococcus agalactiae.

Resistance assessment: Those with obvious infection characteristics of Streptococcus agalactiae and death within 120 hours of injection are considered susceptible to Streptococcus agalactiae, while those with no obvious infection characteristics 120 hours after injection and swimming in good condition are considered resistant to Streptococcus agalactiae.

(2) DNA extraction:

The 100 fish that died first and the 100 fish that were still alive and showing good activity after 120 h were selected.

Fin rays were cut from each oval pompano, with a total weight of 20 mg.

Use the magnetic bead DNA extraction kit and follow the instructions in the kit to add reagents in order to extract DNA from the fin rays.

The extracted DNA was stored in 150 μL of Buffer AE.

DNA samples were examined using 1% agarose gel electrophoresis, and Nanodrop2000 was used to determine DNA concentration and purity. Here, the ratio of A260/A280 is required to be in the range of 1.8 to 2.0, and the DNA concentration is required to be no less than 100 μg/μL.

(3) Library preparation and sequencing:

Qualified DNA samples are first randomly fragmented using a Covaris ultrasonic fragmentation instrument.

After end repair, A-tail addition, sequencing adapter addition, purification, and PCR amplification, the library preparation is completed.

The prepared library was subjected to high-throughput sequencing using an Illumina sequencer.

(4) Quality inspection before library sequencing:

After completing the library construction, use Qubit2.0 for preliminary quantification, and then dilute the library to 1ng/μL.

Use Agilent 2100 to verify the insert size of the library. After confirming the insert size, use the Q-PCR method to accurately determine the effective concentration of the library (effective concentration is required to be >2nM) to ensure the quality of the library.

Qualified libraries will be pooled according to the effective concentration and target sequencing data volume requirements, and sequenced by Illumina PE150.

The whole-genome resequencing technology used in the present invention can efficiently mine rare variants, has high SNP density, and is suitable for whole-genome association analysis.

High-quality sequencing data were aligned to the target reference genome using BWA software (using parameters: mem -t 10 -k 32 -M). Subsequently, the alignment output was sorted by SAMTOOLS software, using the parameter "sort".

SNP screening and association analysis:

Efficient detection of population SNPs was performed using SAMTOOLS and BCFTOOLS software. The specific detection parameters are as follows: samtools-1.3.1 mpileup -q 1 -C 50 -t SP -t DP -m 2 -F 0.002 and bcftools-1.4call -mv -f GQ.

Among the 200 samples, the total number of original SNP sites was 1,744,190, and the number of high-quality SNP sites after screening was 803,713.

GEMMA software, mixed linear model (MLM), was used to screen SNP sites associated with resistance to Streptococcus agalactiae traits.

The specific mathematical model is described as: y = Xα + Zβ + Wμ + e

Among them, y represents the phenotypic trait, X, Z and W are the indicator matrices of their respective effects, α, β and μ are the corresponding effect parameters, and e is the random residual, whose distribution is e~(0, δe^2 ).

According to genome-wide association analysis (GWAS), a SNP marker located on chromosome 9 of Pomfret ovata and associated with resistance to Streptococcus agalactiae was found (shown in Figure 1, the site indicated by the arrow). This SNP is located at the 121st base of the nucleotide sequence shown in SEQ ID NO.1.

The sequence shown in SEQ ID NO.1 is as follows:

TTTAACCTGCTTCTCTTGATACTGAATGGAAATTATAGATGCAATCGTTTTACAGTAGATAGAATATAGAATCTCTTCTTTGTGTTTTCAAGGACACACTCACTTT

TAGTCACAAAAATCTN(A/C)AAATTTTCCAAATTTATCAAAGTTTTGTCAGATTTAGGTTTGTATTAGGATTCAGTCTTTCACATGATTGTTCAACCATAAAAAAA

AAATTCCCTTTTCAGTTAAACATGCAAGCC.

Example 2: SNP genotype verification

Experimental population: Another germplasm population containing 380 ovate pomfret was used to verify the above SNP molecular markers.

Resistance evaluation: same as Example 1;

DNA extraction: Total DNA was extracted from the pectoral fins of Pomfret ovata. The extraction, storage and quality requirements of DNA are consistent with Example 1.

Specific primer design: Use SNP primer software to design specific amplification primers based on the base sequence of approximately 120 bp upstream and downstream of the above SNP marker.

PCR amplification: Use the following primers for PCR amplification:

Snplg918597184FA(seq id no.2):

GAAGGTGACCAAGTTCATGCTACACTCACTTTTAGTCACAAAATCTCA

Snplg918597184FC(seq id no.3):

GAAGGTCGGAGTCAACGGATTACACTCACTTTTAGTCACAAAATCTCC

Snplg918597184r(seq id no.4):

TGAAAGACTGAATCCTAAATACAAACC

The PCR system is shown in Table 1 below:

Table 1 PCR system

The PCR amplification procedure is shown in Table 2 below:

Table 2 PCR amplification procedure

Genotyping:

Fluorescence signal reading: After the PCR amplification is completed, use a microplate reader to read the fluorescence signal of the PCR product.

Fluorescence signal analysis: Use the online software snpdecoder to analyze and convert the fluorescence signal.

Genotype determination: Based on the different colors of the fluorescence signal, the corresponding genotype is analyzed and output. Thus, the genotypes of the SNP sites described in the present invention in each sample can be clarified.

The classification results are shown in Table 3 below.

Table 3 Effects of SNPLG91597184 polymorphism on the ability of Pomfret ovate to resist Streptococcus agalactiae

According to the data in Table 3, it can be observed that in the resequencing samples and the revalidation expanded population, the ovate pomfret with CC genotype showed a higher survival rate when faced with Streptococcus agalactiae infection. Compared with samples of AC and AA genotypes, its survival rate has a significant advantage. This finding provides strong evidence for the significant link between the polymorphism of the SNPLG918597184 molecular marker revealed in SEQ ID NO. 1 and the resistance of Pomfret ovata to Streptococcus agalactiae. Furthermore, through the analysis of genotype frequencies, it was found that when the genotype of Pomfret ovata is CC, its ability to survive infection with Streptococcus agalactiae is significantly better than samples with AC and AA genotypes.

The method proposed by the present invention may also be extended to the development of a kit specifically used to detect the above-mentioned SNP molecular markers. This kit will contain the specific primers mentioned above. Furthermore, when this SNP molecular marker is utilized in the breeding process of Pomfret ovata, it will mainly play a role in genotype detection of breeding candidate groups. By combining other genotypes related to growth, disease resistance, and stress resistance, the ovate pomfret with CC genotype was prioritized as a high-quality parent for breeding against S. agalactiae.

Therefore, the SNP molecular markers, specific primers and possible kits described in the present invention can all play an important role in selectively breeding Pomfret ovate to enhance its resistance to Streptococcus agalactiae.

Therefore, the application of the SNP molecular markers, primers and kits involved in the present invention in assessing the resistance of Pomfret ovata to Streptococcus agalactiae should be considered to be within the protection scope of the present invention.

It is worth emphasizing that although the above-mentioned embodiments describe certain specific implementations of the present invention in detail, they are not meant to limit the patent scope of the present invention. In fact, for technical experts in the field, various modifications and optimizations can still be made without departing from the basic ideas and principles of the present invention. All these changes and optimizations should be regarded as the protection scope of the present invention. within.

Claims (3)

1. The primers for amplifying the SNP molecular markers associated with the resistance of Streptococcus agalactiae in the oval pomfret, characterized by: the primers include primer SNPLG918597184FA, primer SNPLG918597184FC and primer SNPLG918597184R, wherein the sequence of the primer SNPLG918597184FA is such as SEQ ID NO.2 is shown, the sequence of the primer SNPLG918597184FC is shown in SEQ ID NO.3, the sequence of the primer SNPLG918597184R is shown in SEQ ID NO.4, and the nucleotide sequence of the SNP molecular marker is shown in SEQ ID As shown in NO.1, the 121st position is A/C; the SNP molecular marker genotype is CC and the anti-Streptococcus agalactiae ability of the oval pomfret is significantly better than that of the SNP molecular marker genotypes AC and AA. Egg-shaped Pomfret Trevally. 2. A kit, characterized by: comprising the primer according to claim 1. 3. Application of the primer according to claim 1 or the kit according to claim 2 in the selective breeding of the ovate pomfret's resistance to Streptococcus agalactiae traits; the SNP molecular marker genotype is CC's resistance to Streptococcus agalactiae. The ability of Streptococcus agalactiae is significantly better than that of the SNP molecular marker genotypes of Pomfret ovata with AC and AA.