A SNP locus associated with the heat tolerance trait of Chlamys farreri and its application in breeding

Abstract

The present invention provides a SNP site related to the heat tolerance trait of Chlamys farreri and its application in breeding, that is, a SNP site located on chromosome 12 of Chlamys farreri and associated with the heat tolerance trait, and the heat tolerance of Chlamys farreri is evaluated based on the typing of the SNP site, providing a molecular marker for the breeding of new heat-resistant varieties of Chlamys farreri. The SNP site of the present invention is located at the 554th position of the molecular marker with the sequence of SEQ ID NO: 1, which is T / G. The present invention screened and obtained the SNP site related to the heat tolerance trait of Chlamys farreri, and established a method for screening heat-resistant Chlamys farreri. The method of the present invention is more suitable for whole genome selection breeding work, and the parents are quickly screened by the typing of the SNP. In production, individuals with the genotype of TT at this site can be preferentially selected as parents for breeding of heat-resistant scallops, avoiding the introduction of GG-type individuals into the parents.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine biological genetic breeding, and particularly relates to a SNP site related to the temperature tolerance trait of Chlamys farreri and its application in breeding. Background Art

[0002] Chlamys farreri, a scallop belonging to the phylum Mollusca, class Bivalvia, order Pectinida, family Pectinidae, is naturally distributed along my country's northern coastal areas and is a key marine aquaculture shellfish species. In recent years, frequent global extreme weather events and marine heatwaves have led to mass scallop mortality, resulting in significant economic losses. Therefore, the development of new temperature-tolerant varieties has become a key research focus in the aquaculture industry.

[0003] The selection and breeding of new heat-resistant varieties requires the support of theoretical basis, and accurate and efficient screening of heat-resistant individuals is the prerequisite for the selection and breeding of new heat-resistant varieties. The measurement process of traditional heat-resistant indicators such as survival status, physiological and biochemical indicators, and molecular indicators is cumbersome and will sacrifice biological samples. At present, studies have established a live non-destructive detection method for evaluating the heat tolerance of scallops using the heart rate index ABT (Chinese invention patent with publication number CN111280098B), but the measurement efficiency of this technology still needs to be improved. The rapid development of high-throughput sequencing technology and information technology has promoted the research and development of molecular marker technology based on trait-related functional genes.

[0004] SNPs, as a widely used genetic marker, have significant differences in distribution across species and varieties. They have been widely used in genetics theory and breeding research, and molecular marker-assisted breeding has become an important technical support for modern breeding. A number of molecular markers associated with growth traits in aquatic organisms have been identified, providing theoretical support for molecular marker-assisted breeding of aquatic organisms in my country. However, research on SNP loci associated with stress resistance traits remains rare. Therefore, screening and identifying SNP loci associated with heat tolerance in scallops is of great significance for conducting molecular marker-assisted breeding for heat tolerance traits. Summary of the Invention

[0005] The purpose of the present invention is to provide a SNP site related to the temperature tolerance trait of Chlamys farreri and its application in breeding, that is, a SNP site located on chromosome 12 of Chlamys farreri and associated with the temperature tolerance trait, and to evaluate the temperature tolerance of Chlamys farreri based on the typing of the SNP site, so as to provide molecular markers for the selection and breeding of new temperature-resistant varieties of Chlamys farreri.

[0006] The present invention first provides a SNP site, which is located at the 554th position of the molecular marker with the sequence of SEQ ID NO: 1 and is T / G.

[0007] The present invention also provides typing primers for the above-mentioned SNP markers, wherein the sequence of the upstream typing primer GluRsf1 is SEQ ID NO: 2, and the sequence of the downstream typing primer GluRsr1 is SEQ ID NO: 3;

[0008] The SNP site provided by the present invention can be used for parent breeding of temperature-resistant Chlamys farreri.

[0009] The present invention also provides a method for screening heat-resistant scallops. The method uses genomic DNA as a template, uses typing primers with sequences of SEQ ID NO: 2 and SEQ ID NO: 3 for amplification, and performs Sanger sequencing on the PCR products. The typing of SNP sites is compared and screened, and the correlation between the genotype frequency of the site and the heat tolerance trait is analyzed. Site C.14558770 is significantly correlated with the heat tolerance trait of scallops. The genotype of scallops with the TT genotype at this SNP site is significantly higher than that of scallops with the GG genotype (p < 0.05).

[0010] The present invention screened and obtained SNP sites associated with the heat tolerance trait of Chlamys farreri, and established a method for screening heat-tolerant Chlamys farreri. The method of the present invention is more suitable for whole-genome selective breeding. Parents can be quickly screened based on the typing of this SNP. In production, individuals with the TT genotype at this site can be preferentially selected as parents for breeding heat-tolerant scallops, avoiding the introduction of GG genotype individuals into the parents. DETAILED DESCRIPTION

[0011] In the examples of the present invention, genomic DNA from Chlamys farreri was extracted using a conventional phenol-chloroform extraction method and analyzed by agarose gel electrophoresis and a nucleic acid quantification instrument. The Chlamys farreri DNA was amplified using typing primers with the sequences SEQ ID NO: 2 and SEQ ID NO: 3. The typing of the SNP loci was determined by Sanger sequencing and analyzed for association with the temperature tolerance trait, ABT.

[0012] The method of the present invention is described in detail below using Chlamys farreri as an example, but the present invention is not limited to the following description:

[0013] Example 1:

[0014] In July 2021, 90 14-month-old Chlamys farreri were collected from the Laoshan Sea Area in Qingdao for the detection and application of SNP loci related to the heat tolerance traits of Chlamys farreri. The specific steps are as follows:

[0015] 1. Determination and calculation of scallop ABT index

[0016] 1) Scallop Heart Rate Measurement: Chlamys farreri scallops were placed in an 80cm x 120cm x 60cm glass aquarium. A CNY-70 optical sensor was affixed to the shell surface near the heart cavity using seaweed glue and blue tack. Infrared signal changes were amplified, filtered, and recorded using an AMP03 and PowerLab. After all individual sensors were affixed, the scallops were placed in a temperature-controlled circulating water system to collect heart rate signals. Starting from the scallop acclimation temperature (21°C), the temperature was increased at a rate of 1°C / 15 minutes until the heart rate dropped sharply.

[0017] 2) Calculation of the ABT index of scallops: Heart rate data were viewed and analyzed using LabChart software. ABT was calculated by plotting a scatter plot with temperature as the horizontal axis and heart rate as the vertical axis. A straight line was fitted using linear regression. The temperature at the intersection of the two fitted lines was the ABT.

[0018] 2. SNP locus typing statistics

[0019] 1) DNA Extraction: Based on the ABT values ​​of 90 individual scallops, the 15 scallops with the highest ABT were selected as the heat-tolerant group (HT), and the 15 scallops with the lowest ABT were selected as the heat-sensitive group (HS). Genomic DNA from these 30 scallops was extracted using the traditional phenol-chloroform extraction method. The ABT characteristics of the individuals in the heat-tolerant and heat-sensitive groups are shown in Table 1.

[0020] Table 1: Statistics of ABT traits of individuals in the tolerant and sensitive groups

[0021]

[0022] 2) PCR amplification of the target fragment: DNA of the tolerant group and the sensitive group of Chlamys farreri were amplified using typing primers with sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

[0023] Reaction system: 50 ng DNA template; 5 μL 2× Taq PCR Master Mix; 1 μL typing primer (GluRsf1, 2 μM); 1 μL typing primer (GluRsr1, 2 μM); sterile water to 10 μL. PCR program: 95°C for 5 min, 28 cycles of (95°C for 30 s, 56°C for 30 s, 72°C for 40 s), 72°C for 5 min, and storage at 4°C.

[0024] 3) PCR product sequencing: The PCR product was detected by agarose gel electrophoresis, and the target band was clear without any other bands. The remaining product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0025] 4) Genotype Determination and Data Analysis: Based on the sequencing results, sequencing peak patterns were analyzed using BioEdit software to determine the genotype of the SNP locus. The statistical results of the genotype frequency at the C.14558770 locus and the ABT phenotype information for 30 individuals in the tolerant and sensitive groups are shown in Table 2.

[0026] Table 2: ABT traits of different genotypes of Chlamys farreri at C.14558770 locus

[0027]

[0028] The frequencies of the TT, GT, and GG genotypes in the tolerant group (with a higher ABT) were 83.3, 16.7, and 0%, respectively, with the G gene frequency at 16.7% and the T gene frequency at 83.3%. In the sensitive group (with a lower ABT), the frequencies were 0, 23.3, and 76.7%, respectively, with the G gene frequency at 76.7% and the T gene frequency at 23.3%. The above data were analyzed using Fisher's exact test to compare the genotype frequencies between the tolerant and sensitive groups. The results revealed significant differences in the genotype frequencies of the SNP loci (p = 0.000104, significance level 0.01). Within populations, populations with higher ABT (greater temperature tolerance) had a higher frequency of the T genotype. Individuals with the TT genotype had higher ABT than those with the GG genotype, indicating that scallops with the TT genotype were more temperature tolerant.

[0029] During the selective breeding of heat-tolerant scallops, the candidate population of scallops was typed at locus C.14558770. Combined with typing information from other loci associated with heat tolerance, individuals with the TT genotype at locus C.14558770 were preferentially selected as parents for heat-tolerant scallop breeding, while individuals with the GG genotype at this locus were avoided. The experimental results demonstrate that the SNP loci and primers of the present invention are effective for the breeding of heat-tolerant scallops.

Claims

1. Application of a reagent for detecting SNP molecular markers in screening heat-resistant Chlamys farreri individuals; the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO: 1, wherein the 554th base is T / G.

2. The use according to claim 1, characterized in that The reagent is a primer pair, wherein the sequence of the upstream primer is SEQ ID NO: 2, and the sequence of the downstream primer is SEQ ID NO:

3.

3. Use of the primer pair described in claim 2 in preparing a detection kit for screening heat-resistant Chlamys farreri individuals.

4. A method for screening heat-resistant scallop individuals, the method comprising using genomic DNA as a template, using typing primers with sequences of SEQ ID NO: 2 and SEQ ID NO: 3 for amplification, performing Sanger sequencing on the PCR products, comparing and screening the typing of the SNP molecular markers according to claim 1, analyzing the correlation between the locus genotype frequency and the heat resistance trait, and the TT genotype of scallop individuals is significantly higher than that of the GG genotype of scallop individuals.

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