A molecular marker C153 related to growth traits of swimming crab (Portunus trituberculatus) and its application

Abstract

The present invention discloses a molecular marker C153 related to the growth traits of swimming crabs (Panthera tricolor) and its application. The nucleotide sequence of the molecular marker C153 is shown in SEQ ID NO.1, and the sequences of the primers used to detect the molecular marker C153 are shown in SEQ ID NO.2 and SEQ ID NO.3. The molecular marker C153 is a SNP marker, and the growth rate of individuals with the genotype GG at the 115th position of the molecular marker C153 is significantly higher than that of swimming crabs (Panthera tricolor) with the genotypes AG and AA. The molecular marker C153 provided by the present invention can be used to accelerate the speed of growth trait selection and breeding of swimming crabs (Panthera tricolor), save a lot of manpower and material resources, promote the process of breeding of improved varieties of swimming crabs (Panthera tricolor), and is conducive to the healthy and sustainable development of the swimming crab industry, and has broad market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding of aquatic animals, and particularly relates to a molecular marker C153 related to growth traits of swimming crabs trituberculate and an application thereof. Background Art

[0002] The swimming crab (Portunidae), commonly known as swimming crab or flying crab, belongs to the class Crustacea, order Decapoda, family Portunidae, and genus Portunus. It is an important marine aquaculture crab in my country. The swimming crab has delicious meat and rich nutrition. It 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 individuals, which are directly related to the economic returns of farmers. By screening out genes or molecular markers related to rapid growth, individuals with fast growth and high feed conversion rate can be selected through molecular marker-assisted breeding (MAS) technology, thereby greatly shortening the breeding cycle, increasing breeding output, and improving overall economic benefits. Compared with traditional phenotypic selection breeding, molecular breeding technology is more accurate and more efficient.

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

[0004] The purpose of the present invention is to provide a molecular marker C153 related to the growth traits of swimming crabs three tubercles and an application thereof. The present invention utilizes the polymorphic site filtering and comparison analysis of sequencing data and PCR sequencing methods to obtain SNP markers, and then gradually screens and verifies the markers to finally obtain a new molecular marker C153 related to the growth of swimming crabs three tubercles. The use of the molecular marker is conducive to the screening and breeding of rapid growth traits of swimming crabs three tubercles.

[0005] In order to achieve the purpose of the above invention, the present invention adopts the following technical solutions:

[0006] The present invention provides a molecular marker C153 related to the growth of Portunus trituberculatus. The nucleotide sequence of the molecular marker C153 is shown in SEQ ID NO.1.

[0007] Furthermore, the molecular marker C153 is a SNP marker, and the 115th base of the molecular marker C153 is G or A.

[0008] Furthermore, the genotype GG of the 115th base of the molecular marker C153 is a rapid growth trait genotype.

[0009] The present invention also provides amplification primers for the molecular marker C153, 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 use of the molecular marker C153 or the amplification primer in breeding fast-growing varieties of swimming crabs.

[0011] Furthermore, the steps for breeding a fast-growing variety of swimming crabs are: extracting DNA from a test sample of swimming crabs of three tubercles and using it as a template, performing PCR amplification using amplification primers of the molecular marker C153, and sequencing the PCR products; if the genotype of the 115th base of C153 in the sequencing result is GG, then selecting the sequenced sample as the parent for breeding a fast-growing variety of swimming crabs of three tubercles.

[0012] Furthermore, the PCR amplification system is: template 1 μL, primer C153-F 0.2 μL, primer C153-R 0.2 μL, buffer 1 μL, dNTPs 0.8 μL, HiFi 0.2 μL, ddH2O 6.6 μL.

[0013] Furthermore, the PCR amplification program was as follows: 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 also provides a kit for breeding fast-growing varieties of swimming crabs, wherein the kit contains the molecular marker C153 and / or the amplification primer.

[0015] The present invention also provides the application of the molecular marker C153 in genetic diversity analysis, germplasm identification and genetic map construction of swimming crab trituberculate.

[0016] Compared with the existing technology, the present invention has the following effects and advantages:

[0017] 1. The molecular marker C153 related to the rapid growth trait of the swimming crab provided by the present invention is not limited by the growth stage of the swimming crab, and can be used for the breeding of early crab seedlings of the swimming crab, thereby significantly promoting the breeding process of the swimming crab and accelerating the speed of breeding improved varieties.

[0018] 2. The molecular marker C153 provided by the present invention is used to detect the rapid growth trait of the swimming crab. The method is accurate and reliable, and the operation detection can effectively and quickly screen out the traits that meet the requirements, assist in early breeding, and realize the short-term and low-cost selection of fast-growing swimming crab varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The gel electrophoresis bands of the mixed template PCR products in the present invention are shown.

[0020] Figure 2 This is the partial sequencing result of the C153 molecular marker in the present invention, where 1 is the GG genotype and 3 is the GA genotype. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0022] The three-tuberculate swimming crabs used in the present invention were all obtained from Changyi Haifeng Aquatic Products Co., Ltd. 298 three-tuberculate swimming crabs were selected in the harvest season to measure their weights, and then muscle tissues were taken and stored in liquid nitrogen.

[0023] Example 1

[0024] 1. Screening of candidate molecular markers associated with rapid growth traits of swimming crab (Panthera trituberculata)

[0025] 1. Sequencing data filtering and alignment

[0026] The genomic DNA was extracted using the kit of Quanshijin Company, the purity and integrity of the DNA were analyzed by agarose gel electrophoresis, the purity of the DNA was detected by Nanodrop, and finally the DNA concentration was quantitatively analyzed by Qubit. The insert size of the library was evaluated using Agilent 5300. After it met the expectations, the effective concentration of the library was accurately determined by Q-PCR technology (ensuring that the library concentration was greater than 2nM) to ensure the high quality of the library. After the library quality inspection was qualified, Illumina NovaseqTM PE150 sequencing was performed at Novogene Technology Co. (Tianjin, China) to achieve an average sequencing depth of 10×. After the raw sequencing data were obtained by Illumina NovaseqTM sequencing, the FastQC (v0.11.9) software package was used to evaluate the quality of the sequencing reads, remove interference information such as adapter information, low-quality bases, and unmeasured bases (represented by N), and obtain filtered clean data. The clean data of each individual were aligned to the reference genome of the swimming crab (P. trituberculatus) using BWA software. The sequence reads aligned to the genome were sorted using Samtools v1.19, and PCR duplicates were removed using Picard's Mark Duplicates (v2.27.5) command.

[0027] 2. Genotyping

[0028] GATK v4.0 was used for variant detection and base quality filtering. 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 of a single site was less than 0.1; (3) the MAF (minimum allele frequency) was greater than 0.05; (4) the chi-square test of whether the marker genotype conformed to the Hardy-Weinberg equilibrium (HWE) ( P ≥1×10 -6 ).

[0029] 3. Genome-wide association analysis

[0030] With body weight as phenotype and SNP as molecular marker, GEMMA v0.98.3 was used to perform trait association analysis using a mixed linear model. The first three principal components of PCA were used as fixed effects, and individual kinship was used as a random effect to correct for the effects of population structure and individual kinship. The results were introduced with a Bonferroni correction, and the significance threshold and suggestive threshold were defined as 0.05 / N and 1 / N, respectively (where N represents the number of SNPs used for association analysis).

[0031] Based on the results of the whole genome association analysis, markers of weight size differences were finally screened out, and a total of 47 SNP markers were selected.

[0032] 2. Rapid Growth-related Analysis Marker Validation

[0033] The candidate analytical markers for rapid growth traits were verified using the ten largest and ten smallest swimming crabs, respectively.

[0034] (1) First, primers are designed on the flanking sequences of the marker site, with at least one primer being more than 70 bp away from the marker site;

[0035] (2) PCR amplification was performed using primers with the DNA of the largest and smallest individual swimming crabs as templates, and the amplified target bands were sequenced;

[0036] (3) ContigExpress software was used to analyze the sequencing peaks, the genotype of each individual was counted, and the correlation between markers and body weight was analyzed using SPSS software.

[0037] The specific steps are as follows:

[0038] 1. PCR amplification

[0039] The PCR system in the present invention is: template 1 μL, primer C153-F 0.2 μL, primer C153-R 0.2 μL, buffer 1 μL, dNTPs 0.8 μL, HiFi 0.2 μL, ddH2O 6.6 μL.

[0040] After adding the sample according to the above reaction system, PCR amplification was performed 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.

[0041] 2. Statistical analysis

[0042] The PCR products were tested by gel electrophoresis, and clear bands with the same size and no impurities were selected and sent to Shanghai Bio-Tech for sequencing. The sequencing peaks were analyzed using ContigExpress software, and the genotypes of each individual in the sequencing bands were counted. The genotype information was imported into SPSS software, and the chi-square test was used to calculate the P Value, select P <0.01 mark.

[0043] According to Table 1, the homozygous genotype GG of locus C15342241 (referred to as C153) in fast-growing individuals (large individuals) accounts for 100%; while in small individuals, the homozygous genotype GG accounts for 50%, and the homozygous genotype AA accounts for 50%. P <0.05, which is significantly different. Therefore, it can be considered that the genotype GG at this site is a fast growth trait genotype.

[0044] Table 1 Genotyping results of C153 molecular markers

[0045]

[0046] Table 2 Amplification primers for C153 molecular marker

[0047]

[0048] Example 2

[0049] The molecular marker C153 obtained by the present invention can be used to assist in the breeding of fast-growing varieties of swimming crabs. The application steps are simply as follows: extracting DNA from a test sample of swimming crabs of the three tubercles and using it as a template, using primers C153-F and C153-R of the molecular marker C153 to perform PCR amplification, sequencing the PCR product, and if the genotype of the 115th base of C153 in the sequencing result is GG, the sequenced sample can be used as a parent for breeding fast-growing varieties of swimming crabs of the three tubercles.

[0050] The above factual cases only represent the technical solutions of the present invention, rather than limiting the experiments. Although we have improved the experimental solutions, researchers in the same field can still make further improvements to the experimental solutions described above or make scientific equivalent replacements for the experimental links. These changes do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A molecular marker C153 related to the growth traits of Portunus trituberculatus, characterized in that: The nucleotide sequence of the molecular marker C153 is shown in SEQ ID NO.1; the 115th base of the molecular marker C153 is G or A; the genotype GG of the 115th base of the molecular marker C153 is a rapid growth trait genotype.

2. Use of the molecular marker C153 described in claim 1 in breeding fast-growing varieties of swimming crabs.

3. The use according to claim 2, characterized in that: The steps of breeding a fast-growing variety of swimming crabs are as follows: extracting DNA from a sample of swimming crabs to be tested and using it as a template, performing PCR amplification using amplification primers of the molecular marker C153, and sequencing the PCR products; if the genotype of the 115th base of C153 in the sequencing result is GG, then selecting the sequenced sample as a parent for breeding a fast-growing variety of swimming crabs.

4. The use according to claim 3, characterized in that: The PCR amplification system is: template 1 μL, primer C153-F 0.2 μL, primer C153-R 0.2 μL, buffer 1 μL, dNTPs 0.8 μL, HiFi 0.2 μL, ddH2O 6.6 μL.

5. The use according to claim 3, characterized in that: The PCR amplification program was as follows: 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 a final extension at 72°C for 10 min.

Images