CN110724749B

CN110724749B - Molecular marker C104 of portunus trituberculatus resistant vibrio parahaemolyticus and application thereof

Info

Publication number: CN110724749B
Application number: CN201911198804.6A
Authority: CN
Inventors: 吕建建; 题兴斌; 阎德平; 陆璇; 刘萍; 李健
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2021-06-04
Anticipated expiration: 2039-12-02
Also published as: CN110724749A

Abstract

The invention provides a molecular marker C104 of portunus trituberculatus parahaemolyticus and application thereof. The nucleotide sequence of the molecular marker C104 is shown as SEQ ID No.1, and the nucleotide sequence of a primer pair for detecting the molecular marker C104 is shown as SEQ ID No.2 and SEQ ID No. 2. The molecular marker C104 is an SNP marker, and the Vibrio parahaemolyticus tolerance genotype is an AA genotype. The molecular marker C104 provided by the invention can be used for accelerating the breeding speed of the portunus trituberculatus for resisting the excellent character of vibrio parahaemolyticus, promoting the breeding process of the improved variety of the portunus trituberculatus, is not limited by the growth stage, is beneficial to the healthy culture and sustainable development of the portunus trituberculatus, and has wide application prospect.

Description

Molecular marker C104 of portunus trituberculatus resistant vibrio parahaemolyticus and application thereof

Technical Field

The invention belongs to the technical field of DNA molecular markers, and particularly relates to a molecular marker C104 of portunus trituberculatus vibrio resistant to parahaemolyticus and application thereof.

Background

Portunus trituberculatus (Portugulus trituberculatus) belongs to Crustacea, decapod, Paralithodes, and Paralithodes, commonly called Portunus trituberculatus, and is an important large-scale marine economic crab in China. The swimming crabs are delicious in meat quality and rich in nutrition, enjoy full names at home and abroad, and are deeply loved by consumers. The vibrio parahaemolyticus is a marine bacterium, mainly comes from marine products such as fish, shrimp, crab, shellfish and seaweed, has strong survival ability, and can survive in seawater for 47 days. If people eat marine products containing vibrio parahaemolyticus, symptoms such as acute diseases, abdominal pain, emesis, diarrhea, etc. can be caused. The vibrio parahaemolyticus resistant character is one of important breeding characters of the portunus trituberculatus, and has important significance for improving the survival rate of the portunus trituberculatus and preventing food poisoning. However, Vibrio parahaemolyticus has the characteristics of wide tolerance and fast propagation cycle, and if breeding is carried out by the traditional breeding method, the progress is too slow, so that the breeding process needs to be accelerated by the aid of a molecular marker-assisted breeding technology.

The molecular marker is a genetic marker based on nucleotide sequence variation of genetic materials among individuals, and is a direct reflection of DNA level genetic polymorphism. The molecular marker has remarkable advantages: most molecular markers are co-dominant, and selection of recessive characters is very convenient; the genome variation is extremely abundant, and the number of molecular markers is almost unlimited; the DNA of different tissues at different stages of biological development can be used for marking analysis; the molecular marker detection method is simple and rapid. At present, molecular markers related to vibrio parahaemolyticus resistance of the portunus trituberculatus are not reported, so that the development of the molecular markers for the vibrio parahaemolyticus resistance has important significance for the healthy culture and breeding of the portunus trituberculatus.

Disclosure of Invention

The invention provides a molecular marker C104 of portunus trituberculatus parahaemolyticus and application thereof, the invention obtains SNP and InDel markers by utilizing methods of polymorphic site filtration, comparison analysis and PCR sequencing of sequencing data, and finally obtains a new molecular marker C104 of portunus trituberculatus parahaemolyticus by gradually screening and verifying the markers, which is beneficial to screening and breeding of the portunus trituberculatus parahaemolyticus resistant character.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a molecular marker C104 of portunus trituberculatus parahaemolyticus, and the nucleotide sequence of the molecular marker C3 is shown in SEQ ID No. 1.

Further, the molecular marker C104 is an SNP marker.

Further, the Vibrio parahaemolyticus tolerant genotype of the molecular marker C104 is AA genotype.

The invention also provides a primer pair for detecting the molecular marker C104 as claimed in claim 1, which is characterized in that the nucleotide sequence of a forward primer in the primers is shown as SEQ ID No.2, and the nucleotide sequence of a reverse primer is shown as SEQ ID No. 3.

The invention also provides application of the molecular marker C104 in screening of the portunus trituberculatus parahaemolyticus tolerant character variety.

The invention also provides application of the molecular marker C104 in genetic diversity analysis, germplasm identification and genetic map construction of the blue crab.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the molecular marker C104 of the portunus trituberculatus parahaemolyticus provided by the invention can not be limited by the growth stage of the portunus trituberculatus, and can be used for breeding early crab seedlings of the portunus trituberculatus, so that the breeding process of the portunus trituberculatus is obviously promoted, and the breeding speed of excellent characters is accelerated.

2. The molecular marker C104 provided by the invention is used for detecting the character of the portunus trituberculatus parahaemolyticus resistance, the method is accurate and reliable, the operation is simple, the character which meets the requirements can be effectively and quickly screened, early breeding is assisted, the variety of the portunus trituberculatus parahaemolyticus resistance is bred in a short time and at low cost, the number of the portunus trituberculatus crabs with excellent quality is increased, the infection probability of the portunus trituberculatus is reduced, the yield is improved, the healthy propagation of the portunus trituberculatus is promoted, the toxic reaction of edible marine products can be further reduced, and the method has important significance for the healthy breeding and development of the portunus trituberculatus.

Drawings

FIG. 1 shows the result of gel electrophoresis of mixed template PCR products according to the present invention.

Fig. 2 is a result of the difference between the two sets of corresponding positions of the sensitive population mixed template and the resistant population mixed template in the present invention, wherein 1 is the sensitive population mixed template, and 2 is the resistant population mixed template.

FIG. 3 is the result of gel electrophoresis band of the product of PCR amplification with two sets of primers with obvious position difference corresponding to the screened sensitive population mixed template and the screened resistant population mixed template.

FIG. 4 shows the sequencing results of the C104 molecular marker part of the present invention, wherein 1 is GG genotype, 2 is AG genotype, and 3 is AA genotype.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples.

The blue crabs used in the invention are all from the experimental base of Changyi Haifeng aquatic products Limited, China aquatic science research institute yellow sea aquatic product institute, healthy and active blue crabs are randomly obtained from a pond by a trawl fishing method, are temporarily arranged in a net basket and are paved with a layer of waterweeds to prevent crab fighting, and finally 300 blue crabs with the weight of 35 +/-3 g are obtained and are placed in 4 culture ponds (500cm multiplied by 300cm multiplied by 150cm) for temporary culture for 7d, the water temperature is kept at 22 +/-1 ℃ during the temporary culture, water is added to 20cm, the salinity is 33, the pH is 8.2+0.5, oxygen is continuously supplied, fresh seawater is replaced at 8 am every day, fresh trash fishes are fed at 5 pm, and the feeding amount is about 1/3 of the total weight of the crabs. And 7d, selecting the portunids with better vitality and shape for subsequent experiments.

During formal experiments, crabs with vigorous activity and perfect body surfaces are placed in 4 cement pools, 50 crabs are placed in each pool, the ph of the water body is 7.9 +/-0.5, the water temperature is kept at 22 +/-1 ℃, and the water depth is 20 cm. Then 100 mul of Vibrio parahaemolyticus (with a concentration of 2.26X 10) is injected into the first basal ganglia membrane of swimming crab swimming feet⁷cfu/ml). Feeding fresh trash fish at 5 pm every day, wherein the feeding amount is about 1/4 of the weight of crabs, and ensuring that no large amount of bait residues at the bottom of the pool influence the water quality. The death time and number of crabs were recorded every 3h in the first 48h, and after 48h, 100. mu.l of Vibrio parahaemolyticus (concentration 5.3X 10) was injected again into the surviving swimming crabs⁷cfu/ml); at the moment, the ph of the water body is 8.0 +/-0.5, and the water temperature is kept at 22 +/-1 ℃; the death time and number were continuously recorded every 3h, and the experiment was stopped until the number of surviving crabs in 4 pools was 20. The first dead 20 crabs are the vibrio parahaemolyticus infection sensitive group, the last alive 20 crabs are the vibrio parahaemolyticus infection tolerant group, and the crabs are dissected to take muscle tissues to be placed in a freezing storage tube and stored in liquid nitrogen.

Example 1

Screening of candidate molecular markers related to vibrio parahaemolyticus-resistant traits

1. Sequencing data filtering and alignment

DNA extraction is carried out by adopting a kit of the whole gold company and by utilizing the principle that silica gel membrane centrifugal columns specifically adsorb DNA. First, approximately 30mg of a tissue sample was put into a 1.5ml sterile enzyme centrifuge tube, 200. mu.l of lysine Buffer 8(LB8) and 20. mu.l of RNaseA (10mg/ml) were added, the mixture was incubated at room temperature for about 10 seconds with shaking for 2min, 20. mu.l of protease K (20mg/ml) was added, the mixture was thoroughly mixed with shaking, incubated at 55 ℃ until complete Lysis, 1.5 times the volume of Binding Buffer 8(BB8) was added, the mixture was added to a centrifugal column, centrifuged at 12000rpm in a high-speed low-temperature refrigerated centrifuge (model Eppendorf 5804R) for 30 seconds, and the waste liquid was discarded. Then 500. mu.l of Clean Buffer 8(CB8) was added, centrifuged at 12000rpm for 30s, the waste liquid was discarded (repeated), 500. mu.l of Wash Buffer 8(WB8) was added, centrifuged at 12000rpm for 30s, the waste liquid was discarded (repeated), and the mixture was left to stand at 12000rpm for 2min to completely remove the remaining WB 8. The column was placed in a clean centrifuge tube, 50. mu.l of Elution Buffer (EB) was added to the center of the column, and the column was allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 1min, and the DNA was eluted. DNA purity and integrity was analyzed by agarose gel electrophoresis; the purity of the DNA (OD260/280 ratio) was measured by Nanodrop, and the DNA concentration was precisely quantified by Qubit.

Equivalently mixing the DNA samples qualified by the test into two mixing tanks which are respectively named as a vibrio parahaemolyticus sensitive DNA mixing tank (CG) and a vibrio parahaemolyticus resistant DNA mixing tank (CT). Randomly breaking a mixed DNA sample into fragments with the length of 350bp by a Covaris crusher, constructing a Library by adopting a TruSeq Library Construction Kit, and completing the preparation of the whole Library by the steps of end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and the like of the DNA fragments. The constructed library was sequenced by illumina hiseq PE 150. And filtering Raw reads obtained by sequencing to obtain Clean reads for subsequent analysis, wherein the sequencing data result is shown in table 1.

TABLE 1 summary of sequencing data quality

The filtered effective data are compared by Burrows-Wheeler alignment tool (BWA) software, and the comparison result is subjected to SAMTOOLS to remove duplication. The comparison result shows that the comparison rate of all samples is more than 85%, the average sequencing depth is more than 25X, and the method can be used for subsequent analysis.

2. Marker detection and annotation

SNP and InDel are detected by a UnifiedGentyper module in Genome analysis toolkit 3.8(GATK) software, with SNP filtration parameters set as: MQ is less than 40, QD is less than 4, FS is more than 60; the InDel filtration parameters are set to QD < 4 and FS > 200, and the total number of the finally obtained SNP and InDel markers is 515 and 019.

3. SNP and InDel frequency difference analysis

And respectively analyzing and calculating SNP-index and InDel-index of two groups of individuals at each site, and filtering polymorphic sites, wherein the filtering standard is as follows:

(1) the SNP/InDel-index frequencies in the two groups of individuals are less than 0.3;

(2) the sites of SNP/InDel deletion in one individual were filtered out.

And simultaneously calculating the frequency difference distribution of SNP and InDel, wherein the directions are as follows: and (2) filtering sites with the delta index less than 0.3 to obtain 270 SNPs and 183 InDel, which are related candidate molecular markers of the vibrio parahaemolyticus, wherein the results of the candidate SNPs and the InDel are shown in Table 2.

TABLE 2 SNPs and InDel detection and annotation statistics

4. Marker screening

And screening candidate SNP and InDel markers, and selecting a site with All-index close to 0 in the individual or selecting a site with All-index close to 1 in the individual as a preferential selection site for next verification. The screening criteria were as follows:

(1) according to the annotation information of SNP loci, on the basis of sorting from high to low according to delta index, loci of synonymous, non-synonymous mutation or upstream and downstream regions are preferentially selected;

(2) according to the annotation information of InDel sites, sites with more than 5 inserted or deleted bases are preferentially selected on the basis of the order from high to low according to Delta index.

Finally, the markers with large frequency difference before and after the stress of the vibrio parahaemolyticus are screened out, and 55 SNP markers and 60 InDel markers are screened out in total.

Second, verifying the molecular marker related to the vibrio parahaemolyticus

Verifying the candidate molecular markers related to the vibrio parahaemolyticus-resistant traits in CG and CT groups by adopting a PCR product sequencing method:

(1) firstly, designing primers on flanking sequences of marker sites, wherein at least one primer is more than 70bp away from the marker sites;

(2) performing PCR amplification by using the designed primers and CG and CT mixed DNA materials as templates respectively, sequencing the PCR products which are successfully amplified, and selecting the primer which is far away from the marking site by using the sequencing primer;

(3) analyzing the sequencing peak map by using ContigExpress software, selecting two groups of CG and CT marks with larger difference in the sequencing peak map at corresponding positions, and continuing to perform PCR amplification and sequencing analysis on the individual DNA template;

(4) and (4) counting the genotype of each individual according to the sequencing result, and analyzing whether the marker is related to the vibrio parahaemolyticus resistant character or not by SPSS software.

The specific operation steps are as follows:

1. PCR amplification

The PCR system of the invention is as follows: mu.l template, 0.2. mu.l forward primer (10. mu.M), 0.2. mu.l reverse primer (10. mu.M), 1. mu.l Buffer, 0.8. mu.l dNTPs, 0.2. mu.l HiFi, 6.6. mu.l ddH2O 6.6.

After the sample is added according to the system, PCR amplification is carried out according to the following reaction conditions: pre-denaturation at 94 ℃ 5 mia; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, repeating for 35 cycles; finally, extending for 10min at 72 ℃; storing at 4 ℃.

2. Electrophoretic detection

Preparing 1% electrophoresis gel by agarose, mixing agarose and TAE in a certain proportion, heating the mixture in a microwave oven until the mixture is dissolved into colorless transparent liquid, pouring the colorless transparent liquid into a gel-making mould, inserting a comb, standing for 20min for solidification, then pulling out the comb, putting the agarose gel into a horizontal electrophoresis tank, arranging a sample application hole at a negative electrode, taking 0.5% TAE as a buffer solution, selecting Genegeen as a nucleic acid coloring agent, sucking 3ul of PCR products of a mixed template out of a sample hole by a liquid-transferring gun, adjusting voltage and current to 120V and 60mA respectively, setting the time to be 30min for carrying out gel electrophoresis, stopping when a dyeing zone strip reaches 2/3, observing and photographing and recording by a gel imaging system after the electrophoresis is finished, selecting a bright sample with a single strip, sending the sample to a producer for DNA sequencing, carrying out data image analysis on a returned sequencing result, selecting two groups of the markers with obvious sequencing difference at corresponding positions for carrying out individual PCR amplification, the amplification products were also subjected to gel electrophoresis and the PCR products were sequenced.

3. Statistical analysis

Selecting a band (shown in figure 1) meeting the requirement according to the position and the brightness of the gel electrophoresis band of the mixed template PCR product, and selecting 23 pairs of SNP primers and 10 pairs of InDel primers for sequencing analysis.

Analyzing a sequencing peak map by using contigeexpress software, selecting two groups of primers (shown in figure 2) with obvious corresponding position difference from a sensitive group mixed template and a resistant group mixed template, continuously using the primers, carrying out PCR amplification by using individuals as templates, keeping amplification conditions unchanged, selecting bright strips with consistent sizes, sending the strips to be sequenced, and carrying out amplification again on the strips with poor amplification effects to complement sequencing results, wherein 15 SNP markers and 3 InDel markers are selected in total, and the sequencing results are shown in figure 3.

Observing the returned individual sequencing result by using contigeexpress software, introducing the genotype information into SPSS software, calculating a P value by using chi-square test, selecting a marker with the P less than 0.01, and finally selecting 3 SNP markers and 1 InDel marker in total.

From table 4, it can be seen that genotypes GG and AA in C104411267 (abbreviated as C104) account for 10% and 50% of the survival group (tolerogenic group), respectively; the proportions of the individuals with the genotypes GG and AA in early dead individuals (sensitive population) are opposite and respectively account for 50% and 5%; the data P of this group, 0.003, was very different, and therefore genotype AA at this site was considered to be a vibrio parahaemolyticus tolerant genotype. The nucleotide sequence of the C104 molecular marker is shown as SEQ ID No.1, and partial sequencing results are shown as FIG. 4, wherein the amplification primers for developing the molecular marker are shown as SEQ ID No.2 and SEQ ID No.3 (Table 4).

TABLE 3 genotype results for C104 molecular markers

TABLE 4 amplification primers for molecular markers

The molecular marker C104 obtained by the invention can be used for assisting in breeding the portunus trituberculatus parahaemolyticus resistant variety, and the application steps are simply as follows: extracting DNA of a portunus trituberculatus test sample and using the DNA as a template, carrying out PCR amplification by using amplification primers C104-F and C104-R of a molecular marker C104, sequencing a PCR product, and selecting the test sample as a parent for cultivating a portunus trituberculatus parahaemolyticus resistant variety if the genotype of C104 in a sequencing result is AA. In addition, the molecular marker C104 can be used for analyzing the genetic diversity of the blue crab, identifying the germplasm and constructing the genetic map of the blue crab.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<120> molecular marker C104 of portunus trituberculatus resistant vibrio parahaemolyticus and application thereof

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Claims

1. A molecular marker C104 of the portunus trituberculatus resistant vibrio parahaemolyticus is characterized in that the nucleotide sequence of the molecular marker C104 is shown in SEQ ID No.1, wherein the 301 st base is G or A.

2. The application of the amplification primer of the molecular marker C104 in the preparation of the reagent for screening the portunus trituberculatus parahaemolyticus resistant varieties is characterized in that the nucleotide sequence of the amplification primer is shown as SEQ ID No.2 and SEQ ID No. 3.