CN114875158A - Molecular marker 27W1 for breeding high-fertility prawn population and application thereof - Google Patents

Molecular marker 27W1 for breeding high-fertility prawn population and application thereof Download PDF

Info

Publication number
CN114875158A
CN114875158A CN202210443045.0A CN202210443045A CN114875158A CN 114875158 A CN114875158 A CN 114875158A CN 202210443045 A CN202210443045 A CN 202210443045A CN 114875158 A CN114875158 A CN 114875158A
Authority
CN
China
Prior art keywords
molecular marker
fertility
litopenaeus vannamei
population
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210443045.0A
Other languages
Chinese (zh)
Inventor
隋娟
栾生
孔杰
代平
孟宪红
罗坤
曹家旺
谭建
陈宝龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Original Assignee
Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences filed Critical Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Publication of CN114875158A publication Critical patent/CN114875158A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a molecular marker 27W1 for breeding a high-fertility prawn group and application thereof. The nucleotide sequence of the molecular marker 27W1 is shown in SEQ ID No.1, wherein the allele frequency of the 301 th base G in the high-fertility group of litopenaeus vannamei is more than or equal to 98.33%. The invention also provides a primer of the molecular marker 27W1, which comprises an amplification primer with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 and an extension primer with nucleotide sequence shown as SEQ ID No. 4. The molecular marker 27W1 for the high fertility of the litopenaeus vannamei can not be limited by the growth stage, can be used for the early breeding of the litopenaeus vannamei, quickly breeds a parent population with excellent reproductive traits and promotes the breeding process of a new high fertility variety of the litopenaeus vannamei.

Description

Molecular marker 27W1 for breeding high-fertility prawn group and application thereof
Technical Field
The invention belongs to the field of molecular marker assisted breeding of aquatic animals, and particularly relates to a molecular marker 27W1 for breeding a high-fertility prawn group and application thereof.
Background
The litopenaeus vannamei (Penaeus vannamei), also called Penaeus vannamei and Pacific white shrimps, belong to Arthropoda, Crustaea, Deeapoda, Penaeidae and Penaeus (Penaeus), are the types of the Penaeus which have the highest culture yield in China and the world and are also one of the types of aquaculture with the highest single yield value. In 2020, the culture yield of China exceeds 180 million tons, the output value exceeds 700 hundred million yuan, and the yield accounts for about 40 percent of the total world yield. The annual shrimp breeding demand of China exceeds 100 million pairs, and the offspring seed demand exceeds 1.5 trillion tails. The litopenaeus vannamei has the characteristics of high egg laying amount and capability of laying eggs for multiple times. However, in the production of offspring seeds, there are great individual differences in the reproductive capacity of female shrimps. During a production cycle, some of the female shrimps never lay eggs, while some of the female shrimps can lay eggs many times. The method has important significance for improving the seedling raising efficiency and saving the production cost by cultivating new species with high fertility (egg laying and egg laying amount). The breeding character belongs to the middle-low heritability character, the traditional breeding method is slow in progress, and the breeding process can be effectively accelerated by means of a molecular marker-assisted breeding technology.
As a third generation molecular marker, a single nucleotide polymorphism marker (SNP) has the characteristics of high abundance, high density, strong stability, co-dominance and the like, and becomes the most widely applied molecular marker technology in economic crustaceans such as shrimps, crabs and the like at present. At present, the development of related SNP markers for propagation of litopenaeus vannamei is less, and the industry lacks markers applicable to molecular marker assisted breeding. Therefore, the development of molecular markers with high fertility has important significance for breeding new varieties of litopenaeus vannamei.
Disclosure of Invention
The invention provides a molecular marker 27W1 for breeding a high-fertility prawn group and application thereof. The molecular marker 27W1 can be used for screening the Litopenaeus vannamei population with high fertility, and has high identification efficiency and accuracy.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a molecular marker 27W1 for breeding a high-fertility prawn population, wherein the nucleotide sequence of the molecular marker 27W1 is shown as SEQ ID No. 1.
Furthermore, in the Litopenaeus vannamei high-fertility population, the 301 st base G of the molecular marker 27W1 is a candidate 27W1_ SNP site.
Further, the allele frequency of the base G in the high-fertility group of litopenaeus vannamei is more than 98%.
Preferably, the allele frequency of the base G in the high-fertility group of the litopenaeus vannamei is more than or equal to 98.33 percent.
The invention also provides a primer of the molecular marker 27W1, wherein the primer comprises an amplification primer with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 and an extension primer with nucleotide sequence shown as SEQ ID No. 4.
The invention also provides application of the molecular marker 27W1 or the primer of the molecular marker 27W1 in screening the Litopenaeus vannamei populations with high reproductive capacity.
Further, the specific steps of screening the Litopenaeus vannamei population with high fertility are as follows: extracting DNA of a test sample in a Litopenaeus vannamei population (a family, a strain or a geographical population), using the DNA as a template, performing flight mass spectrometry typing by using the primer, and selecting the Litopenaeus vannamei population as a parent for breeding the Litopenaeus vannamei with high fertility if the allele frequency of a 301 st base G of a molecular marker in a typing result is more than or equal to 98.33%.
Furthermore, the number of the test samples of the litopenaeus vannamei family or group is more than 30.
Further, in the flight mass spectrometry, the condition of PCR amplification is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 25s, and extension at 72 ℃ for 30s, repeating for 40 cycles; finally, extending for 4min at 72 ℃; storing at 4 ℃.
Further, in the flight mass spectrometry, the condition of the extension reaction is pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 52 ℃ for 5s, and extension at 80 ℃ for 5s, repeating for 40 cycles; finally extending the temperature of 72 ℃ for 3 min; storing at 4 ℃.
The invention also provides application of the molecular marker 27W1 in genetic diversity analysis, germplasm identification and genetic map construction of the litopenaeus vannamei.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the molecular marker 27W1 for the high fertility of the litopenaeus vannamei provided by the invention can be free from the limitation of the growth stage, can be used for the early breeding of the litopenaeus vannamei, quickly breeds a parent population with excellent reproductive traits and promotes the breeding process of a new variety of the litopenaeus vannamei with high fertility.
2. The molecular marker 27W1 provided by the invention is used for detecting the breeding traits of the litopenaeus vannamei, the method is accurate and reliable, the operation is simple, the high-fertility population can be effectively and quickly screened out, the early breeding is assisted, the use efficiency of the litopenaeus vannamei parent shrimps with excellent quality is increased, the yield of the offspring seeds is improved, the healthy breeding of the litopenaeus vannamei is promoted, the method has important significance for the healthy breeding and development of the litopenaeus vannamei, and the method has wide application prospect.
Drawings
FIG. 1 shows the results of flight mass spectrometry typing verification performed in the verification population according to the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
The litopenaeus vannamei used in the invention is from Popple breed science and technology Limited company of yellow sea aquatic product research institute of China aquatic product science and research institute. Healthy female shrimp larvae of 10 months old from 57 families were subjected to unilateral eyestalk excision to promote synchronous ovarian maturation, with 8-10 tails per family, a total of 604 tails, and a body weight of 50 + -5 g. Placing 6 female shrimps with 16m of cut stems 3 Large pool, density of 6-7 tails/m 2 . The male parent shrimps are from the same family, 8-9 shrimps per family, 500 shrimps in total, and the weight of 45 +/-5 g. Male shrimp is put into 3m by family 3 A cement pool with a density of 2-3 tails/m 2 . Feeding the parent shrimps with 3 clamworms and 2 squids every day for nutrition enhancement, wherein the total feeding amount of the parent shrimps per day is 20 percent of the total weight of the parent shrimps, and feeding the parent shrimps for 5 times every day to ensure that the clamworms are visible for 24 hours. And during the strengthening period, keeping the water temperature at 28-29 ℃, the salinity at 30-31 per mill and the pH at 7.8-8.2, continuously supplying oxygen, and changing the water amount by 80% per day. After 20 days of strengthening, female shrimps with full ovaries and orange color are selected and put into a male shrimp pool for natural copulation, and inbreeding is avoided during copulation. The number of eggs laid by all female shrimps and the egg laying amount of each time within 30 days are recorded. Establishing a female shrimp fertility selection index, wherein the calculation formula is as follows:
y i ＝0.5*(a elfi -μ elf )*σ elf -1 +0.5*(a aeni -μ aen )*σ aen -1 (1)
in the formula, y i Is the fertility index of the ith female shrimp; a is elfi And a aeni Respectively the egg laying frequency and the egg laying amount of the ith female tail shrimp; mu.s elf And mu aen Is the average value of the egg laying frequency and the egg laying amount of all female shrimps; sigma elf And σ aen Is the standard error of the egg laying frequency and the egg laying amount of all female shrimps.
The 30-tailed female shrimps with the highest index were selected as the high-fertility group, and the 30-tailed female shrimps with the lowest index (gonadal maturation was never found) were selected as the low-fertility group. Selecting female shrimps with high fertility and low fertility to respectively construct a population with poor fertility. Muscle tissues are dissected and taken out of the cryopreservation tube, and preserved by liquid nitrogen.
Example 1
Screening of candidate molecular markers related to fertility of female shrimps
1. Sequencing data filtering and alignment
Extracting the muscle DNA of the litopenaeus vannamei by adopting a CTAB method. The CTAB method is collectively referred to as the Cetyltrimethylammonium Bromide method (Cetyltrimethylammoniumnium Bromide). 1ml of 1 × CTAB is added into a 1.5ml sterile enzyme centrifuge tube; adding about 20mg of sample into a centrifuge tube, and grinding for 4min by a grinder at 60 Hz; incubating in water bath at 65 deg.C for 60 min; centrifuging at 8000Xg for 5min in a normal temperature high-speed centrifuge; taking 900 mu L of centrifuged sample supernatant to a new 2mL sterile enzyme centrifuge tube; adding 450 μ L chloroform to the supernatant; covering the tube cover tightly, reversing the tube cover and mixing the sample evenly for 30s until the solution is completely emulsified into white; centrifuging at 13000Xg for 10min by a normal-temperature high-speed centrifuge; transferring 800. mu.L of the supernatant to a new 1.5mL sterile enzyme-free centrifuge tube; beckman Agencourt AMPure XP beads, and balancing for 30min in a dark place at room temperature; adding uniformly mixed Beads with the volume 0.6 times of the volume of the supernatant, gently blowing and uniformly mixing for more than 10 times by using a pipette, and standing for 5min at room temperature; placing on a magnetic frame for 5min until the solution is clear, carefully sucking and discarding the supernatant; keeping the 1.5mL centrifuge tube fixed on a magnetic frame, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, removing the supernatant, and paying attention to not disturb the magnetic beads; washing with 80% ethanol for one time; keeping a 1.5mL centrifuge tube fixed on a magnetic frame, and drying the magnetic beads at room temperature for 2-5 min; taking down a 1.5mL centrifuge tube from the magnetic frame, adding 50 μ L10 mM Tris HCl eluent, gently blowing and stirring the eluent uniformly by a pipette, and standing the mixture for 5min at room temperature; and (3) placing a 1.5mL centrifuge tube on a magnetic frame, standing for 5min at room temperature until the solution is clear, carefully sucking about 50 mu L of supernatant, and transferring to a corresponding new sample storage tube to obtain the purified DNA. 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.
And equivalently mixing the DNA samples qualified by the test into two mixing pools which are named as a high-fertility DNA mixing pool (HE) and a low-fertility DNA mixing pool (LE) respectively. The mixed DNA sample is randomly broken into fragments with the length of 500bp by a Covaris 2/E210 crusher, and the whole library preparation is completed 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 2500 in the sequencing mode 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 overview of sequencing data quality control
Figure BDA0003615372790000041
The filtered effective data are compared by Burrows-Wheeler alignment tool (BWA) software, and the comparison result is subjected to SAMTOOLS software to remove PCR duplication. The average sequencing depth is more than 30X, and the method can be used for subsequent analysis.
2. Genomic differential region screening
Different genomic regions were selected with a sliding window strategy: the window is in units of 50kb, and 25kb is the sliding step. Calculating nucleotide diversity difference multiples (pi-Ratio, Ratio mode is pi high fecundity group/pi low fecundity group) and fixed index (Fst) between two isolated groups aiming at the selected specific genome region, and screening significant regions according to the difference P <0.01 between the groups by combining the results of Fst and pi-Ratio respectively, wherein the intersection part is a reliable candidate interval.
3. Marker detection and annotation
The SNP and InDel of the candidate interval are detected by a Unified Genotyper module in Genome Analysis Toolkit (GATK) software, and the filter parameters are set as follows: -Window 4, -filter "QD <2.0| | FS >60.0| | | MQ <40.0", G _ filter "GQ < 20". And finally, the total number of SNP markers in the genome difference interval of the breeding force difference population is 64,046, and the total number of InDel markers is 20,295.
4. SNP frequency difference analysis
Calculating SNP-index and InDel-index of each site of the two colony mixing pools respectively, and calculating the frequency difference distribution of SNP and InDel at the same time, wherein the directions are as follows: Δ (index) ═ index (high fertility trait) -index (low fertility trait).
5. Marker screening
And screening candidate SNP and InDel markers, and selecting a site with a position point delta index close to 1 or close to-1 in the two groups as a preferential selection position for next verification. The screening criteria were as follows:
(1) according to the annotation information of SNP loci, and on the basis of ranking from high to low according to | Delta index |, preferentially selecting the loci of synonymous, non-synonymous mutation or upstream and downstream regions;
(2) according to the annotation information of InDel sites, sites with more than 5 inserted or deleted bases are preferably selected on the basis of ranking from high to low according to the | Delta index |.
Finally, the markers with large frequency difference of the fertility segregating population are screened out, and 374 SNP markers and 26 InDel markers are screened out in total.
TABLE 2 SNPs and InDel detection and annotation statistics
Figure BDA0003615372790000051
Figure BDA0003615372790000061
Second, reproduction related molecular marker verification
The method adopts a flight time mass spectrometry method to verify the candidate molecular markers related to the spawning traits in the high-fertility and low-fertility groups, and comprises the following specific operation steps:
(1) and (3) PCR amplification: designing primers on the flanking sequences of the marker loci, and amplifying a DNA sequence containing a detection target;
(2) single base extension: SNP sequence specific extension primers are added into the PCR amplification products, and single base extension is carried out through the iPLEX technology. For the detection target, different genotypes have only one base difference of the extended terminal target base;
(3) mass spectrum detection: the product after single base extension was purified and transferred to a SpectroCHIP chip for mass spectrometric detection. The DNA is positively charged by laser irradiation and flies in a vacuum tube to be detected, the flying speed is inversely proportional to the mass of each extension product, and finally the base type is judged through the position of a peak, so that the genotyping is realized.
(4) And (4) counting the mutation markers of each individual according to the mass spectrum detection result, and analyzing whether the markers are related to the fertility traits or not through SPSS software.
The specific operation steps are as follows:
1. primer design
Designing primer software, designing PCR amplification primers and single base extension primers of the to-be-detected sites by using Genotyping Tools and Massarray Assay Design software of Sequenom.
2. PCR amplification
The PCR system of the invention is as follows: 1. mu.l of template (10-30 ng/. mu.l), 0.25. mu.l of forward primer (10. mu.M), 0.25. mu.l of reverse primer (10. mu.M), 0.5. mu.l of Buffer, 0.1. mu.l of dNTPs (25mM), MgCl 2 (25mM)0.4μl，HotStar Taq(5U/μl)0.1μl，ddH 2 O 2.4μl。
After the sample is added according to the system, PCR amplification is carried out according to the following reaction conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 25s, and extension at 72 ℃ for 30s, repeating for 40 cycles; finally, extending for 4min at 72 ℃; storing at 4 ℃.
3. Alkaline phosphatase treatment of PCR products
(1) After the PCR reaction is finished, the PCR product is treated by SAP (SAP alkaline sulfate phosphatase) to remove dNTPs in the reaction. The SAP reaction formulations were as follows (single sample taken as an example): SAP Buffer 0.17. mu.l, SAP Enzyme (1.7U/. mu.l) 0.3. mu.l, ddH 2 O 1.53μl。
(2) Add SAP reaction to PCR reaction plate with pipette, add 2. mu.l per well, seal membrane, and centrifuge.
(3) The PCR reaction plate added with the SAP reaction solution is placed into a PCR instrument, and the following reaction program is operated: at 37 ℃ for 40 min; 5min at 85 ℃; storing at 4 ℃.
(4) After the reaction is finished, the PCR reaction plate is taken out and centrifuged for a short time for standby.
4. Extension reaction
(1) An iPlex reaction reagent (taking a single sample as an example) is prepared: 0.12. mu.l of iPlex Buffer (10X), 0.2. mu.l of iPlex Termination mix (10X), 0.041. mu.l of iPlex Enzyme (2U/. mu.l), 0.3. mu.l of SAP Enzyme (1.7U/. mu.l), 10. mu.m 0.804. mu.l of primer solution, ddH 2 O 0.755μl；
(2) Adding the iPLex reaction solution into a PCR reaction plate by using a pipette, adding 2 mu l of the iPLex reaction solution into each hole, sealing a membrane, and centrifuging;
(3) placing the PCR reaction plate into a PCR instrument, and carrying out PCR amplification according to the following reaction conditions: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 52 ℃ for 5s, and extension at 80 ℃ for 5s, repeating for 40 cycles; finally extending the temperature of 72 ℃ for 3 min; storing at 4 ℃.
5. Purification of the product
(1) The resin scraper is covered with resin uniformly and left for 20 min.
(2) And (3) centrifuging the PCR reaction plate after the reaction is finished for 1min at 1000rpm, adding 25 mu l of deionized water into each hole, inverting the plate on the resin plate (the plate is fixed and cannot be shifted), then buckling the plate on the PCR reaction plate in an inverted manner, knocking the plate to enable the resin to fall into the PCR reaction plate, and sealing the membrane.
(3) The PCR reaction plate was inverted 20min with the long axis of the PCR reaction plate as the axis, and centrifuged at 3500rpm for 5min for use.
6. Mass spectrometric detection
(1) Spotting the Nanodipen SpectroCHIP chip, and transferring a detection sample from a PCR reaction plate to a MassARRAY SpectroCHIP chip with a surface covered by a matrix;
(2) detecting MassARRAY Analyzer Compac by mass spectrum;
(3) and analyzing the experimental result by TYPER software to obtain typing data.
7. Statistical analysis
The invention obtains a molecular marker 27W1 by screening, and the nucleotide sequence is shown in SEQ ID No. 1. As shown in Table 3 and FIG. 1, it was found that the allele frequency of the 301 st base G in 27W1 was 98.33% in the high fertility group, while the allele frequency of the base G in the low fertility group was 83.33%, and the proportion of the base G was significantly different (P <0.05) in the fertility segregating population, and therefore, it was considered that the allele frequency of the base G at this site was 98.33% or more, which was the high fertility population. Wherein the amplification primers for developing the molecular marker 27W1 are shown as SEQ ID No.2 and SEQ ID No.3 (Table 4), and the extension primers are shown as SEQ ID No.4 (Table 4).
TABLE 327 typing results for the W1 molecular marker
Figure BDA0003615372790000081
TABLE 4 primers for molecular markers
Figure BDA0003615372790000082
The molecular marker 27W1 obtained by the invention can be used for assisting in selecting high-fertility populations (families, strains or geographical populations), and the application steps are as follows: extracting DNA of more than 30 test samples in the Litopenaeus vannamei population, taking the DNA as a template, and carrying out flight mass spectrometry typing by using amplification primers 27W1-F and 27W1-R and extension primers 27W1-E of a molecular marker 27W1, wherein if the allele frequency of the 301 th base G of the 27W1 locus in the population is more than or equal to 98.33% in a typing result, the population can be selected as a parent for breeding the Litopenaeus vannamei with high reproductive capacity. In addition, the molecular marker 27W1 can be used for analyzing genetic diversity and germplasm identification of the litopenaeus vannamei and constructing a genetic map of the litopenaeus vannamei.
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
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> molecular marker 27W1 for breeding high-fertility prawn group and application thereof
<150> 2022102023356
<151> 2022-03-02
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atatatatat atatgtatat gtgtggtgtg tgtgtgtgtg tgtgtgtgtg gtgtgtgtgt 60
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtatag agagagagcg agcgagagcg 120
agagagagag agagagacag aataaagaag cgagagagag aagatgagaa gaagatcagg 180
ggaggaaggg ggtagggagg gagagattaa gagagatagc gagaggagag gggaagagag 240
ggagggagat ggcagaagag gcagagggag aaaggaaaag ggcggtgagt cgggggaggg 300
gggtaaggca gggaaggggc ggggttactt ctgccccatt cccatgtttc atatttgtca 360
taaaagtttt cttcctgtaa gatttttgaa ggcatgttcc tctgccctct ttctcacatt 420
tcaaaggttt ctgtctggct ctgtcctttc gtgttgtccg ttggtgccct gcgtttgaaa 480
tgcaaaagga aaacataatg gatatccctt aataaatatg ttagtcatta tatatatata 540
tatatatata tatatatata tatatatata tatgtttgta tatatatgta tatatatgtg 600
g 601
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acgttggatg atgaaacatg ggaatggggc 30
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgttggatg agagggagaa aggaaaaggg 30
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggggccccgc cccttccctg ccttacc 27

Claims (10)

1. The molecular marker 27W1 for breeding the high-fertility prawn population is characterized in that the nucleotide sequence of the molecular marker 27W1 is shown as SEQ ID No. 1.
2. The molecular marker 27W1 according to claim 1, wherein the 301 st base G of the molecular marker 27W1 is a candidate 27W1_ SNP site in a high-fertility population of litopenaeus vannamei.
3. The molecular marker 27W1 of claim 2, wherein the allele frequency in the base G Litopenaeus vannamei high-fertility population is > 98%.
4. The primer of molecular marker 27W1 of claim 1, wherein the primer comprises an amplification primer having a nucleotide sequence shown in SEQ ID No.2 and SEQ ID No.3 and an extension primer having a nucleotide sequence shown in SEQ ID No. 4.
5. Use of the primer of the molecular marker 27W1 according to claim 1 or the molecular marker 27W1 according to claim 4 for screening a Litopenaeus vannamei population with high fertility.
6. The use according to claim 5, wherein the specific steps of screening the Litopenaeus vannamei population with high fertility are as follows: extracting DNA of a test sample in a Litopenaeus vannamei colony, using the DNA as a template, and performing flight mass spectrometry typing by using the primer, wherein if the allele frequency of a 301 th base G of a molecular marker in a typing result is greater than 98%, the Litopenaeus vannamei colony is selected as a parent for breeding the Litopenaeus vannamei with high fertility.
7. The use of claim 6, wherein the number of the test samples of the Litopenaeus vannamei population is more than 30.
8. The use according to claim 6, wherein in flight mass spectrometry, the conditions for PCR amplification are pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 25s, and elongation at 72 ℃ for 30s, repeating for 40 cycles; finally, extending for 4min at 72 ℃; storing at 4 ℃.
9. Use according to claim 6, wherein in flight mass spectrometry, the conditions of the extension reaction are pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 52 ℃ for 5s, extension at 80 ℃ for 5s, repeating for 40 cycles; finally extending for 3min at 72 ℃; storing at 4 ℃.
10. The use of the molecular marker 27W1 of claim 1 in genetic diversity analysis, germplasm identification and genetic map construction of litopenaeus vannamei.
CN202210443045.0A 2022-03-02 2022-04-26 Molecular marker 27W1 for breeding high-fertility prawn population and application thereof Pending CN114875158A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210202335 2022-03-02
CN2022102023356 2022-03-02

Publications (1)

Publication Number Publication Date
CN114875158A true CN114875158A (en) 2022-08-09

Family

ID=82672378

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210443045.0A Pending CN114875158A (en) 2022-03-02 2022-04-26 Molecular marker 27W1 for breeding high-fertility prawn population and application thereof

Country Status (1)

Country Link
CN (1) CN114875158A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118272546A (en) * 2024-06-03 2024-07-02 中国水产科学研究院黄海水产研究所 SNP marker g.reelin-936 related to propagation traits of palaemon carinicauda and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118272546A (en) * 2024-06-03 2024-07-02 中国水产科学研究院黄海水产研究所 SNP marker g.reelin-936 related to propagation traits of palaemon carinicauda and application thereof

Similar Documents

Publication Publication Date Title
CN110438242B (en) Portunus trituberculatus microsatellite marked primer and application thereof
CN113789394B (en) Molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof
CN114875158A (en) Molecular marker 27W1 for breeding high-fertility prawn population and application thereof
CN114717333A (en) Molecular marker 9W1 related to prawn reproduction traits and application thereof
CN113249492B (en) SNP marker for evaluating pig eye muscle area and application method thereof
CN110564867B (en) SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof
CN101899526A (en) Method for selecting molecular marker for goat yeaning traits
CN113604587B (en) Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof
CN106701930B (en) Method for detecting sheep FTH-1 gene insertion deletion polymorphism by using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and application thereof
CN111593129B (en) Primer for identifying family of Bai Suowen perch and identification method of family of micropterus salmoides
CN114752684A (en) Molecular marker 7W1 for identifying fertility of prawns and application thereof
CN114686603A (en) Molecular marker 21W2 and application thereof in screening Litopenaeus vannamei populations with high reproductive capacity
CN114592068A (en) Molecular marker X1W1 for high fertility of litopenaeus vannamei and application thereof
CN113584187A (en) Molecular marker A2629 for screening penaeus japonicus with low temperature resistance, amplification primer and application thereof
CN114752685A (en) Molecular marker of Litopenaeus vannamei, and primer and application thereof
CN113801945A (en) Molecular marker C768 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof
CN113736891A (en) Molecular marker G2997 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof
CN113584188A (en) Low-temperature-resistant molecular marker C6101 of penaeus japonicus and application
CN108913784B (en) Detection method of EC12 SNP marker of exopalaemon carinicauda
CN107475400B (en) Method for auxiliary detection of cattle growth traits through MYLK4 gene and special kit thereof
CN113621714B (en) Low-temperature-resistant molecular marker A257 of penaeus japonicus and application thereof
CN110172516B (en) Method for detecting single nucleotide polymorphism of 5&#39; flanking region of cattle lncFAM200B gene and application thereof
CN113930518B (en) Molecular marker C49 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof
CN113981113B (en) InDel marker C142 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof
CN113981112B (en) InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination