SNP (Single Nucleotide Polymorphism) site related to temperature resistance character of chlamys farreri and application of SNP site in breeding

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site related to the temperature-resistant character of chlamys farreri and an application of the SNP site in breeding, namely the SNP site which is located on a No.12 chromosome of the chlamys farreri and is related to the temperature-resistant character, the temperature resistance of the chlamys farreri is evaluated based on the typing condition of the SNP site, and a molecular marker is provided for breeding of a new temperature-resistant variety of the chlamys farreri. The SNP site is located at the 554th site of the molecular marker with the sequence of SEQ ID NO: 1, and is T / G. According to the invention, the SNP sites related to the temperature resistance character of the chlamys farreri are obtained through screening, and a method for screening the chlamys farreri is established. The method is more suitable for whole genome selective breeding work, parents are rapidly screened according to the typing condition of the SNP, individuals with the genotype being TT at the site can be preferentially selected in production to serve as parents for breeding temperature-resistant scallops for breeding, and GG type individuals are prevented from being brought into the parents.

Description

Technical Field

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

[0002] Chlamys farreri belongs to the phylum Mollusca, class Bivalvia, order Pectinida, family Pectinidae. It is naturally distributed in the northern coastal areas of my country and is one of the important marine aquaculture shellfish in my country. In recent years, the frequent occurrence of extreme weather and marine heat waves around the world has led to the death of a large number of scallops, causing great economic losses. Therefore, the selection and breeding of new temperature-resistant varieties has become the focus and hotspot of aquaculture research.

[0003] The 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 breeding of new heat-resistant varieties. The measurement process of traditional heat-resistant indicators such as survival status, physiological and biochemical indicators, molecular indicators, etc. is cumbersome and will sacrifice biological samples. At present, studies have established a live non-destructive detection method for evaluating the heat resistance 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] SNP, as a widely used genetic marker, has significant differences in distribution among different species and varieties. It has been widely used in genetic theory research and genetic breeding research. Molecular marker-assisted breeding technology has become an important technical support for modern breeding work. A number of molecular markers related to the growth traits of aquatic organisms have been identified, providing theoretical support for the molecular marker-assisted breeding of aquatic organisms in my country. However, there are still few reports on the research of SNP loci related to stress resistance traits. Therefore, screening and identifying SNP loci related to the heat tolerance traits of scallops is of great significance for the molecular marker-assisted breeding of 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 breeding of new temperature-resistant varieties of Chlamys farreri.

[0006] The present invention first provides a SNP site, wherein the SNP site 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 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 shown 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 product, and compares and screens the typing of SNP sites to analyze the correlation between the site genotype frequency and the heat-resistant trait. Site C.14558770 is significantly correlated with the heat-resistant trait of scallops, and the scallop individuals with the TT genotype of the SNP site are significantly higher than those with the GG genotype (p<0.05).

[0010] The present invention screened and obtained SNP sites related to the temperature tolerance trait of Chlamys farreri, and established a method for screening temperature-tolerant Chlamys farreri. The method of the present invention is more suitable for whole genome selection breeding work, and parents are quickly screened by the typing of the SNP. In production, individuals with a genotype of TT at the site can be preferentially selected as parents for breeding of temperature-tolerant scallops, avoiding the introduction of GG-type individuals into the parents. DETAILED DESCRIPTION

[0011] In the embodiment of the present invention, the genomic DNA of Chlamys farreri is extracted by the traditional phenol-chloroform extraction method, and the DNA is detected by agarose gel electrophoresis and a nucleic acid quantifier. The typing primers of SEQ ID NO: 2 and SEQ ID NO: 3 are used to amplify the DNA of Chlamys farreri, and the typing of the SNP site is obtained by Sanger sequencing and the association analysis is performed with the temperature tolerance trait ABT.

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

[0013] Embodiment 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 temperature tolerance traits of Chlamys farreri. The specific steps are as follows:

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

[0016] 1) Determination of scallop heart rate index: Chlamys farreri was placed in a glass tank of 80cm*120cm*60cm, and the CNY-70 optical sensor was fixed on the shell surface close to the heart cavity of the scallop using waterweed glue and blue tack glue. The changes of infrared signals were amplified, filtered and recorded using AMP03 and Powerlab. After all the individual sensors of the scallops were fixed, they were placed in a temperature-controlled circulating water system to collect heartbeat signals. Starting from the scallop domestication temperature (21°C), the temperature was raised at a rate of 1°C / 15min until the heart rate dropped sharply.

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

[0018] 2. SNP site typing statistics

[0019] 1) DNA extraction: According to the ABT values ​​of 90 Chlamys farreri, the 15 scallops with the highest ABT were selected as the temperature tolerance group (HT) and the 15 scallops with the lowest ABT were selected as the heat sensitive group (HS). The genomic DNA of the above 30 Chlamys farreri was extracted by the traditional phenol-chloroform extraction method. The ABT traits of the individuals in the tolerance group and the sensitive group are shown in Table 1.

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

[0021]

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

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

[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) Determination of genotype and data analysis: According to the sequencing results, the sequencing peak graph was observed using BioEdit software to determine the genotype of the SNP site. The statistical results of the genotype frequency of the C.14558770 site and the ABT phenotype information of 30 scallops in the tolerant group and the sensitive group are shown in Table 2.

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

[0027]

[0028] The genotype frequencies of TT, GT, and GG in the tolerant group with higher ABT were 83.3, 16.7, and 0%, respectively, the gene frequency of G was 16.7%, and the gene frequency of T was 83.3%. In the sensitive group with lower ABT, they were 0, 23.3, and 76.7%, respectively, the gene frequency of G was 76.7%, and the gene frequency of T was 23.3%. The above data were analyzed by Fisher's exact test for the genotype frequencies of the samples in the tolerant group and the sensitive group. The results showed that the genotype frequencies of the SNP loci were significantly different (p=0.000104, with a significant level of 0.01). For the population, the population with higher ABT (stronger temperature tolerance) had a higher T genotype frequency. The temperature tolerance trait ABT of the Chlamys farreri individuals with genotype TT was higher than that of the individuals with genotype GG, and the Chlamys farreri individuals with genotype TT had stronger temperature tolerance.

[0029] In the selection and breeding process of heat-resistant scallops, the scallop breeding candidate population is typed at site C.14558770, and combined with the typing information of other sites related to the heat-resistant trait, individuals with TT type at site C.14558770 are preferentially selected as parents for the selection of heat-resistant scallops, and individuals with GG type at this site are avoided as parents. The experimental results show that the SNP site and primer of the present invention can be effectively used for the selection and breeding of heat-resistant scallops.

Claims

1. A SNP site, characterized in that: The SNP site is located at the 554th position of the molecular marker with the sequence of SEQ ID NO: 1, which is T / G.

2. Use of the SNP site described in claim 1 as a molecular marker in screening temperature-resistant Chlamys farreri individuals.

3. A primer pair for genotyping the SNP site according to claim 1, characterized in that: The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO:

3.

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

5. A method for screening heat-resistant Chlamys farreri individuals, wherein the method comprises amplifying the genomic DNA of the individual to be detected by using a genotyping primer pair to determine whether a specific genotype exists in the individual to be detected.

6. The method according to claim 5, characterized in that The genotype is the TT genotype.

7. The method according to claim 5, characterized in that The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO:

3.

8. The method according to claim 5, characterized in that The determination of whether a specific genotype exists in the individual to be detected is to determine the genotype after sequencing the PCR amplification product.

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