Molecular marker C774 related to growth traits of portunus trituberculatus and application of molecular marker C774

Abstract

The invention discloses a molecular marker C774 related to growth traits of portunus trituberculatus and application of the molecular marker C774. The nucleotide sequence of the molecular marker C774 is as shown in SEQ ID NO.1, and the nucleotide sequences of a primer for detecting the molecular marker C774 are as shown in SEQ ID NO.2 and SEQ ID NO.3. The invention further discloses a kit for detecting the molecular marker C774. The molecular marker C774 is an SNP marker, and the growth speed of a portunus trituberculatus individual of which the genotype is TT at the 97th site of the molecular marker C774 is obviously higher than that of a portunus trituberculatus individual of which the genotypes are AT and AA. The molecular marker C774 provided by the invention can be used for accelerating the breeding speed of the growth traits of the portunus trituberculatus and promoting the process of breeding improved varieties of the portunus trituberculatus, is beneficial to the healthy and sustainable development of the industry of the portunus trituberculatus, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker C774 related to the growth traits of Portunus trituberculatus and its application. Background Art

[0002] Portunus trituberculatus belongs to the class Malacostraca, order Decapoda, family Portunidae, and genus Portunus, commonly known as swimming crab or flying crab, and is an important marine cultured crab species in China. The swimming crab has delicious meat and rich nutrition, enjoys a high reputation at home and abroad, and is deeply loved by consumers. Growth traits directly determine the growth rate and body size of an individual, which is directly related to the economic return of farmers. Screening out genes or molecular markers related to rapid growth can select individuals with fast growth rate and high feed conversion rate through molecular marker-assisted selection (MAS) technology, thereby greatly shortening the breeding cycle, increasing the breeding yield, and enhancing the overall economic benefits. Compared with traditional phenotypic selection breeding, molecular breeding technology is more accurate and efficient.

[0003] At present, the molecular marker-assisted selection breeding technology has been widely applied in aquaculture. By exploring molecular markers closely related to growth traits, the breeding efficiency can be significantly improved. However, the effective molecular markers for the growth traits of Portunus trituberculatus are still relatively limited. Therefore, there is an urgent need to develop a molecular marker that can be used for the selection of the growth traits of Portunus trituberculatus. Summary of the Invention

[0004] The purpose of the present invention is to provide a molecular marker C774 related to the growth traits of Portunus trituberculatus and its application. The present invention uses the methods of filtering and alignment analysis of polymorphic sites in sequencing data and PCR sequencing to obtain SNP markers, and then through step-by-step screening and verification of the markers, a new molecular marker C774 related to the growth of Portunus trituberculatus is finally obtained, which is beneficial to the screening and breeding of the rapid growth traits of Portunus trituberculatus.

[0005] To achieve the above object of the invention, the present invention is implemented by the following technical solutions: The present invention provides a molecular marker C774 related to the growth of Portunus trituberculatus, and the nucleotide sequence of the molecular marker C774 is shown in SEQ ID NO.1.

[0006] Further, the molecular marker C774 is an SNP marker.

[0007] Further, the 97th base of the molecular marker C774 is T or A.

[0008] Further, the genotype TT of the 97th base of the molecular marker C774 is the genotype of the rapid growth trait.

[0009] The present invention also provides amplification primers for molecular marker C774, and the nucleotide sequences of the amplification primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0010] The present invention also provides the application of the said molecular marker C774 or the said amplification primers in breeding fast-growing varieties of Portunus trituberculatus.

[0011] Furthermore, the steps for breeding fast-growing varieties of Portunus trituberculatus are as follows: extracting the DNA of the Portunus trituberculatus test sample to be tested and using it as a template, performing PCR amplification with the amplification primers of molecular marker C774, sequencing the PCR product, and if the genotype of the 97th base of C774 in the sequencing result is TT, then selecting the sequencing sample as the parent for breeding fast-growing varieties of Portunus trituberculatus.

[0012] Furthermore, the system for the PCR amplification is as follows: 1 μL of template, 0.2 μL of forward primer, 0.2 μL of reverse primer, 1 μL of Buffer buffer, 0.8 μL of dNTPs, 0.2 μL of HiFi, and 6.6 μL of ddH2O.

[0013] Furthermore, the procedure for the PCR amplification is: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 3 min, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for 10 min.

[0014] The present invention provides a kit for breeding fast-growing varieties of Portunus trituberculatus, and the kit contains the said molecular marker C774 and / or the said amplification primers.

[0015] The present invention provides the application of the said molecular marker C774 in genetic diversity analysis, germplasm identification, and genetic map construction of Portunus trituberculatus.

[0016] Compared with the existing technology, the effects and advantages of the present invention are as follows: 1. The molecular marker C774 related to the fast-growing trait of Portunus trituberculatus provided by the present invention is not restricted by the growth stage of Portunus trituberculatus, and can be used for the breeding of early-stage crab seedlings of Portunus trituberculatus, thereby significantly promoting the breeding process of Portunus trituberculatus and accelerating the speed of breeding improved varieties.

[0017] 2. Using the molecular marker C774 provided by the present invention to detect the fast-growing trait of Portunus trituberculatus, the method is accurate, reliable, and easy to operate, and can effectively and quickly screen out the traits that meet the requirements, assist early breeding, and achieve the breeding of fast-growing varieties of Portunus trituberculatus in a short time and at low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the gel electrophoresis band result of the mixed template PCR product in the present invention.

[0019] Figure 2 This is the partial sequencing result of the C774 molecular marker in the present invention, where 1 is the TT genotype and 3 is the AA genotype. Specific embodiments

[0020] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection required by the present invention is not limited to the scope described in the examples.

[0021] All the swimming crabs (Portunus trituberculatus) used in the present invention were obtained from Changyi Haifeng Aquatic Products Co., Ltd. During the harvest season, 298 swimming crabs were selected to measure their body weights. Subsequently, muscle tissues were taken and stored in liquid nitrogen.

[0022] Example 1 I. Screening of candidate molecular markers related to the fast growth trait of Portunus trituberculatus 1. Sequencing data filtering and alignment The genomic DNA was extracted using the kit from TransGen Biotech. The purity and integrity of the DNA were analyzed by agarose gel electrophoresis, the purity of the DNA was detected using Nanodrop, and finally, the DNA concentration was quantitatively analyzed using Qubit. The insert fragment size of the library was evaluated using Agilent 5300. After meeting the expectations, the effective concentration of the library was accurately determined using Q-PCR technology (ensuring that the library concentration was greater than 2 nM) to ensure the high quality of the library. After the library passed the quality inspection, Illumina NovaseqTM PE150 sequencing was performed at Novogene Technology Co. (Tianjin, China) to achieve an average sequencing depth of 10×. After obtaining the raw sequencing data from Illumina NovaseqTM sequencing, the FastQC (v0.11.9) software package was used to evaluate the quality of the sequencing reads, and the interference information such as adapter information, low-quality bases, and unsequenced bases (represented by N) was removed to obtain the filtered clean data. The clean data of each individual was aligned to the Portunus trituberculatus reference genome using the BWA software. The alignment results were sorted for the sequence reads aligned to the genome using Samtools v1.19, and the PCR duplicate sequences in the sequencing were removed using the Picard's MarkDuplicates (v2.27.5) command.

[0023] 2. Genotyping Mutation detection and base quality filtering were performed using GATK v4.0. VCFTOOLS v0.1.16 was used to perform quality control on SNPs in the Variant Call Format (VCF) to ensure that the detected SNPs met the following principles: (1) the sequencing coverage depth was not less than 6; (2) the deletion ratio at a single locus was less than 0.1; (3) the MAF (Minor Allele Frequency) was greater than 0.05; (4) the chi-square test was used to mark whether the genotype conforms to the Hardy-Weinberg equilibrium (HWE)( P ≥1×10 -6 ).

[0024] 3. Genome-wide association study Using body weight as the phenotype and SNPs as molecular markers, GEMMA v0.98.3 was used to perform trait association analysis using the mixed linear model. The first three principal components of PCA were used as fixed effects, and the individual kinship was used as a random effect to correct for the effects of population structure and individual kinship. The results were introduced with Bonferroni correction, and the significance threshold and suggestive threshold were defined as 0.05 / N and 1 / N (where N represents the number of SNPs used for association analysis), respectively.

[0025] According to the results of the genome-wide association study, markers with differences in body weight were finally screened out, and a total of 47 SNP markers were selected.

[0026] II. Verification of markers related to fast growth analysis Using the 10 swimming crabs with the largest and smallest body weights as materials respectively, the candidate analysis markers for fast growth traits were verified.

[0027] (1) First, primers were designed for the flanking sequences of the marker locus, and at least one primer was more than 70 bp away from the marker locus; (2) Using the primers, PCR amplification was performed using the DNA of swimming crabs with the largest and smallest individuals as templates respectively, and the amplified target bands were sequenced; (3) Using ContigExpress software to analyze the sequencing peak map, the genotype of each individual was counted, and the correlation between the marker and body weight was analyzed using SPSS software.

[0028] The specific operation steps are as follows: 1. PCR amplification The PCR system in the present invention is: 1 μL of template, 0.2 μL of forward primer, 0.2 μL of reverse primer, 1 μL of Buffer buffer, 0.8 μL of dNTPs, 0.2 μL of HiFi, and 6.6 μL of ddH2O.

[0029] After adding the sample according to the above reaction system, PCR amplification was carried out using the following program: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 3 min, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for 10 min.

[0030] 2. Statistical analysis The PCR products were detected by gel electrophoresis, and the bands with appropriate size, no heterozygous bands and clear were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ContigExpress software was used to analyze the sequencing peak map, and the genotypes of each individual in the sequencing bands were counted. The genotype information was imported into the SPSS software, and the chi-square test was used to calculate P the P markers with < 0.01.

[0031] According to Table 1, at locus C774 (abbreviated as C774), the proportion of homozygous genotype TT in fast-growing individuals (large individuals) was 100%; while in small individuals, the proportion of homozygous genotype TT was 40%, and the proportion of homozygous genotype AA was 50%. This set of data P was < 0.05, showing a significant difference. Therefore, it can be considered that the genotype TT at this locus is the genotype of the fast-growing trait.

[0032] Table 1 Genotype results of C774 molecular marker

[0033] Table 2 Amplification primers of C774 molecular marker

[0034] Example 2 The molecular marker C774 obtained in the present invention can be used to assist in breeding fast-growing varieties of Portunus trituberculatus, including the following application steps: extracting the DNA of the test sample of Portunus trituberculatus and using it as a template, performing PCR amplification using the primers C774-F and C774-R of the molecular marker C774, sequencing the PCR products, and if the genotype of the 97th base of C774 in the sequencing results is TT, the sequencing sample is used as the parent for breeding fast-growing varieties of Portunus trituberculatus.

[0035] The system of the PCR amplification was: 1 μL of template, 0.2 μL of forward primer, 0.2 μL of reverse primer, 1 μL of Buffer buffer, 0.8 μL of dNTPs, 0.2 μL of HiFi, and 6.6 μL of ddH2O.

[0036] The procedure for PCR amplification: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 3 min, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for 10 min.

[0037] The above factual cases only represent the technical solutions of the present invention and do not limit the experiments. Although we have improved the experimental scheme, researchers in the same field can still further improve the previously described experimental scheme or make scientific equivalent replacements for the experimental steps. These changes do not cause the essence of the corresponding technical solution to deviate from the spirit and scope of the technical solution required to be protected by the present invention.

Claims

1. A molecular marker C774 related to the growth traits of Portunus trituberculatus, characterized in that, The nucleotide sequence of the molecular marker C774 is shown in SEQ ID NO.

1.

2. The molecular marker C774 related to the growth traits of Portunus trituberculatus according to claim 1, characterized in that, The 97th base of the molecular marker C774 is T or A.

3. The molecular marker C774 related to the growth traits of Portunus trituberculatus according to claim 1, characterized in that, The genotype TT of the 97th base of the molecular marker C774 is the genotype for fast growth trait.

4. The amplification primer for the molecular marker C774 according to claim 1, characterized in that, The nucleotide sequences of the amplification primers are shown in SEQ ID NO.2 and SEQ ID NO.

3.

5. Use of the molecular marker C774 according to claim 1 or the amplification primer according to claim 4 in breeding fast-growing varieties of Portunus trituberculatus.

6. The application according to claim 5, wherein The steps for breeding fast-growing varieties of Portunus trituberculatus are as follows: Extract the DNA of the Portunus trituberculatus test sample to be tested and use it as a template, perform PCR amplification using the amplification primer of the molecular marker C774, sequence the PCR product. If the genotype of the 97th base of C774 in the sequencing result is TT, then select the sequenced sample as the parent for breeding fast-growing varieties of Portunus trituberculatus.

7. The application according to claim 6, wherein The system for the PCR amplification is as follows: 1 μL of template, 0.2 μL of forward primer, 0.2 μL of reverse primer, 1 μL of Buffer buffer, 0.8 μL of dNTPs, 0.2 μL of HiFi, 6.6 μL of ddH2O.

8. The application according to claim 6, wherein The procedure for the PCR amplification: Pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 3 min, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and finally extension at 72 °C for 10 min.

9. A kit for breeding fast-growing varieties of Portunus trituberculatus, characterized in that, The kit contains the molecular marker C774 according to claim 1 and / or the amplification primer according to claim 4.

10. Use of the molecular marker C774 according to claim 1 in genetic diversity analysis, germplasm identification, and genetic map construction of Portunus trituberculatus.

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