CN114717333B - Molecular marker 9W1 related to prawn reproduction traits and application thereof - Google Patents
Molecular marker 9W1 related to prawn reproduction traits and application thereof Download PDFInfo
- Publication number
- CN114717333B CN114717333B CN202210455955.0A CN202210455955A CN114717333B CN 114717333 B CN114717333 B CN 114717333B CN 202210455955 A CN202210455955 A CN 202210455955A CN 114717333 B CN114717333 B CN 114717333B
- Authority
- CN
- China
- Prior art keywords
- molecular marker
- litopenaeus vannamei
- seq
- fertility
- 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.)
- Active
Links
- 239000003147 molecular marker Substances 0.000 title claims abstract description 43
- 241000238557 Decapoda Species 0.000 title claims abstract description 35
- 241000238553 Litopenaeus vannamei Species 0.000 claims abstract description 41
- 230000035558 fertility Effects 0.000 claims abstract description 26
- 108700028369 Alleles Proteins 0.000 claims abstract description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 21
- 238000004949 mass spectrometry Methods 0.000 claims description 12
- 238000012216 screening Methods 0.000 claims description 11
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 6
- 238000012257 pre-denaturation Methods 0.000 claims description 5
- 230000035772 mutation Effects 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 14
- 230000001488 breeding effect Effects 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 4
- 230000001850 reproductive effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108010000178 IGF-I-IGFBP-3 complex Proteins 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 235000013601 eggs Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 241000238421 Arthropoda Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000013011 mating Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241000238366 Cephalopoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000238550 Penaeidae Species 0.000 description 1
- 241000927735 Penaeus Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (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 9W1 related to prawn fertility traits and application thereof. The nucleotide sequence of the molecular marker 9W1 is shown as SEQ ID No.1, wherein the allele frequency of the 301 th base A in the high-fertility population of the litopenaeus vannamei is more than or equal to 80.00%. The invention also provides a primer of the molecular marker 9W1, which comprises amplification primers with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 and extension primers with nucleotide sequences shown as SEQ ID No. 4. The high-fertility molecular marker 9W1 of the litopenaeus vannamei can be used for early breeding of the litopenaeus vannamei without being limited by a growth stage, can be used for rapidly breeding parent populations with excellent reproductive characteristics, and promotes the breeding process of new high-fertility varieties of the litopenaeus vannamei.
Description
Technical Field
The invention belongs to the field of molecular marker assisted breeding of aquatic animals, and particularly relates to a molecular marker 9W1 related to prawn reproduction traits and application thereof.
Background
Litopenaeus vannamei (Penaeus vannamei) is also called Penaeus vannamei and Pacific white shrimp, belongs to Arthropoda (Arthropoda), crustaceans (Crustaeea), deeapoda (Deeapoda), pacific (Penaeidae) and Pacific shrimp (Penaeus), is the type of shrimp with highest cultivation yield in China and the world, and is one of the types of aquaculture with highest single yield. In 2020, the culture yield of China exceeds 180 ten thousand tons, the yield value exceeds 700 hundred million yuan, and the yield is about 40% of the total world yield. The demand of the shrimp seeds per year in China exceeds 100 ten thousand pairs, and the demand of the young shrimp seeds exceeds 1.5 trillion tails. The litopenaeus vannamei has the characteristics of high spawning quantity and capability of spawning for multiple times. However, in offspring seed production, there is a large individual difference in reproductive capacity of female shrimps. In one production cycle, part of the females never lay eggs, while some females can lay eggs multiple times. The cultivation of new varieties with high fertility (spawning and spawning quantity) has important significance for improving the seedling raising efficiency and saving the production cost. The propagation property belongs to low genetic property, the traditional breeding method is slow in progress, and the breeding process can be effectively accelerated by means of molecular marker assisted breeding technology.
As a third generation molecular marker, single Nucleotide Polymorphism (SNP) markers have the characteristics of high abundance, high density, strong stability, co-dominance and the like, and become the most widely applied molecular marker technology in economic crustaceans such as shrimps, crabs and the like at present. At present, the development of SNP markers related to the reproduction of Guan Fanna penaeus vannamei is less, and the industry lacks markers applicable to molecular marker assisted breeding. Therefore, the development of the molecular marker with high fertility has important significance for the cultivation of new varieties of litopenaeus vannamei.
Disclosure of Invention
The invention provides a molecular marker 9W1 related to prawn reproduction traits and application thereof. The molecular marker 9W1 can be used for screening high-fertility product groups of litopenaeus vannamei, and has high identification efficiency and accuracy.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a molecular marker 9W1 related to prawn reproduction traits, and the nucleotide sequence of the molecular marker 9W1 is shown as SEQ ID No. 1.
Further, the 301 st base A of the molecular marker 9W1 is a candidate 9W1_SNP site.
Furthermore, the allele frequency of the base A in the high-fertility population of the litopenaeus vannamei is more than or equal to 80.00%.
The invention also provides a primer of the molecular marker 9W1, which comprises amplification primers with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 and extension primers with nucleotide sequences shown as SEQ ID No. 4.
The invention also provides application of the molecular marker 9W1 or the primer of the molecular marker 9W1 in screening litopenaeus vannamei populations with high fertility.
Further, the specific steps of screening the litopenaeus vannamei colony with high fertility are as follows: extracting DNA of a test sample in the litopenaeus vannamei (family, strain or geographical group) and taking the DNA as a template, performing flight mass spectrometry typing by using the primer, and selecting the litopenaeus vannamei group as a parent for cultivating the high fertility of the litopenaeus vannamei if the allele frequency of the 301 th base A of the molecular marker in the typing result is more than or equal to 80.00%.
Furthermore, the number of the litopenaeus vannamei family or group test samples is more than 30.
Further, in the flight mass spectrometry, the condition of PCR amplification is pre-denaturation at 94 ℃ for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, extension at 72℃for 30s, repeating 40 cycles in total; finally, extending for 4min at 72 ℃; preserving at 4 ℃.
Further, in the flight mass spectrometry, the conditions of the extension reaction are pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s, repeating 40 cycles in total; finally, extending at 72 ℃ for 3min; preserving at 4 ℃.
The invention also provides application of the molecular marker 9W1 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 high-fertility molecular marker 9W1 of the litopenaeus vannamei provided by the invention can be used for early breeding of the litopenaeus vannamei without being limited by a growth stage, can be used for rapidly breeding a parent population with excellent reproductive characteristics, and promotes the breeding process of a new variety of the litopenaeus vannamei with high fertility.
2. The molecular marker 9W1 provided by the invention is used for detecting the reproduction character of the litopenaeus vannamei, the method is accurate and reliable, the operation is simple, the high-fertility population can be effectively and rapidly screened out, the early breeding is assisted, the use efficiency of the good-quality litopenaeus vannamei parent shrimp is increased, the seed yield is improved, the healthy breeding of the litopenaeus vannamei is promoted, and the method has important significance on the healthy breeding and development of the litopenaeus vannamei and has wide application prospect.
Drawings
FIG. 1 shows the results of a mass spectrometry flight typing test performed in a test population according to the present invention.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to specific embodiments.
The litopenaeus vannamei used in the invention is from the Poplar's industrial science and technology Co-operation of the national institute of aquatic products, yellow sea. Single-sided eyestalk excision was performed on 10 month-old healthy female shrimp from 57 families to promote synchronous ovarian maturation, 8-10 tails per family, total 604 tails, body weight 50±5g. 6 female shrimps with cut stems are put into the mould of 6 pieces of 16m 3 Large pond with density of 6-7 tail/m 2 . The male parent shrimps are from the same family, 8-9 tails/family, 500 tails and 45+ -5 g body weight. The male shrimp is put into 3m by taking family as unit 3 Cement pond with density of 2-3 tail/m 2 . The parent shrimps are fed with 3 clamworm meals and 2 squid meals every day for nutrition enhancement, the total daily feeding amount reaches 20% of the total weight of the parent shrimps, and the parent shrimps are fed for 5 times every day, so that the clamworm is ensured to be visible for 24 hours. The water temperature is kept at 28-29 ℃ during the strengthening period, the salinity is 30-31 per mill, the pH is 7.8-8.2, the oxygen is continuously supplied, and the daily water exchange amount is 80 percent. After 20 days of strengthening, selecting female shrimps with full ovaries and red orange, putting the female shrimps into a male shrimp pool for natural mating, and avoiding inbreeding during mating. The number of eggs laid by all females within 30 days was recorded and the amount laid each time. 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)
wherein y is i Is the fertility index of the ith female shrimp; a, a elfi And a aeni The spawning frequency and spawning amount of the i-th female shrimp are respectively; mu (mu) elf Sum mu aen Is the average value of the spawning frequency and spawning amount of all female shrimps; sigma (sigma) elf Sum sigma aen Is the standard error of spawning frequency and spawning amount of all female shrimps.
The 30 females with the highest index were selected as the high-fertility group, and the 30 females with the lowest index (gonadal maturation was never found) were selected as the low-fertility group. Selecting 30 female shrimps with high fertility and low fertility to form a fertility difference population. Dissecting muscle tissue in freezing tube, and storing in liquid nitrogen.
Example 1
1. Female shrimp fertility-related candidate molecular marker screening
1. Sequencing data filtration and alignment
The muscle DNA of the litopenaeus vannamei is extracted by adopting a CTAB method. The CTAB process is known as cetyltrimethylammonium bromide (Cetyltrimethylammonium Bromide). 1ml of 1 XCTAB was added to a 1.5ml sterile enzyme centrifuge tube; about 20mg of sample is taken and added into a centrifuge tube, and the sample is polished for 4min at 60Hz by a grinder; incubating in a water bath at 65 ℃ for 60min; a normal temperature high speed centrifuge, 8000xg, centrifuging for 5min; taking 900 mu L of the centrifuged sample supernatant into a new 2mL sterile enzyme centrifuge tube; adding 450 mu L of chloroform into the supernatant; the tube cap is closed, and the evenly mixed sample is turned upside down for 30s until the solution is completely emulsified into white; a normal temperature high speed centrifuge, 13000xg, centrifuging for 10min; transfer 800 μl of supernatant to a new 1.5mL sterile, aseptic centrifuge tube; beckman Agencourt AMPure XP beads, balancing at room temperature for 30min in dark place; adding the Beads which are 0.6 times of the supernatant and uniformly mixing, lightly blowing and uniformly mixing for more than 10 times by using a pipette, and standing for 5min at room temperature; placing on a magnetic rack for 5min until the solution is clear, carefully sucking and discarding the supernatant; keeping the 1.5mL centrifuge tube fixed on a magnetic rack, adding 200 mu L of freshly prepared 80% ethanol into the tube, standing at room temperature for 30s, and discarding the supernatant, wherein the magnetic beads are not disturbed; repeatedly cleaning with 80% ethanol for one time; keeping a 1.5mL centrifuge tube fixed on a magnetic rack, and drying magnetic beads at room temperature for 2-5 min; taking down a 1.5mL centrifuge tube from a magnetic rack, adding 50 mu L of 10mM Tris HCl eluent, lightly blowing and mixing by using a pipettor, and standing for 5min at room temperature; and placing the 1.5mL centrifuge tube on a magnetic rack, standing at room temperature for 5min until the solution is clear, carefully sucking about 50 mu L of supernatant, and transferring the supernatant into a corresponding new sample storage tube to obtain purified DNA. Analyzing the purity and integrity of the DNA by agarose gel electrophoresis; the purity of the DNA was measured by Nanodrop (OD 260/280 ratio), and the DNA concentration was precisely quantified by Qubit.
The DNA samples that were inspected for pass were equally mixed into two mixing pools, designated as high-fertility DNA mixing pool (HE) and low-fertility DNA mixing pool (LE), respectively. The mixed DNA sample is randomly broken into fragments with the length of 500bp by a covarias S2/E210 breaker, and the DNA fragments are subjected to the steps of end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and the like to complete the preparation of the whole library. The constructed library was sequenced by IlluminaHiSeq 2500 in the sequencing mode PE150. The Raw reads obtained by sequencing were filtered to obtain Clean reads for subsequent analysis, and the sequencing data results are shown in table 1.
Table 1 overview of sequencing data quality control
The filtered effective data were aligned by means of Burows-Wheeler alignment tool (BWA) software and the alignment was subjected to the removal of PCR duplicates by means of SAMTOOLS software. The average sequencing depth is above 30X, and can be used for subsequent analysis.
2. Genomic differential region screening
Different genomic regions were selected in a sliding window strategy: window in units of 50kb, 25kb as sliding step. And calculating nucleotide diversity fold difference (pi-Ratio, taking the Ratio mode of pi high-fertility group/pi low-fertility group) and fixed index (Fst) between two isolated groups according to the selected specific genome region, combining the results of Fst and pi-Ratio, screening the obvious region according to the difference P <0.01 between the groups, and enabling the intersection part to be a reliable candidate region.
3. Tag detection and annotation
SNP and inde of candidate intervals were detected by Unified Genotyper module in Genome Analysis Toolkit (GATK) software, the filter parameters were set to: -Window 4, -filter "QD <2.0||fs >60.0||mq <40.0", -g_filter "GQ <20". Finally screening out 64,046 SNP markers and 20,295 InDel markers in the genome difference interval of the fertility difference population.
4. SNP frequency variance analysis
And respectively calculating SNP-index and InDel-index of the two population pools at each site, and simultaneously calculating the frequency difference distribution of the SNP and the InDel, wherein the directions are as follows: delta (index) =index (high fertility trait) -index (low fertility trait).
5. Marker screening
Candidate SNP and InDel markers are screened, and the sites with delta index close to 1 or close to-1 in two populations are selected as the preferential selection sites for the next verification. The screening criteria were as follows:
(1) According to the annotation information of SNP loci and then according to the delta index, the loci of synonymous, nonsensical mutation or upstream and downstream regions are preferentially selected on the basis of high-to-low ordering;
(2) Based on the annotation information of InDel sites and the high-to-low ordering of the InDel sites, sites with more than 5 insertion or deletion bases are preferentially selected.
Finally, the markers with larger frequency difference of the fecundity segregation population are screened out, and 374 SNP markers and 26 InDel markers are screened out.
TABLE 2 SNPs and InDel detection and annotation statistics
2. Fertility-related molecular marker validation
And verifying candidate molecular markers related to spawning characteristics in high-fertility and low-fertility populations by adopting a time-of-flight mass spectrometry, wherein the specific operation steps are as follows:
(1) And (3) PCR amplification: designing primers at flanking sequences of the marker loci, and amplifying DNA sequences containing detection targets;
(2) Single base extension: SNP sequence specific extension primers are added into PCR amplification products, and single base extension is carried out by the iPLEX technology. Aiming at detection targets, different genotypes only have the difference of one base of an extended terminal target base;
(3) Mass spectrometry detection: the single base extended product was purified and transferred to a SpectroCHIP for mass spectrometry. The DNA is positively charged by laser irradiation, flies in the detected vacuum tube, the flying speed is inversely proportional to the mass of each extension product, and finally, the base type is judged through the position of the peak, so that the genotyping is realized.
(4) And counting mutation marks of each individual according to a mass spectrum detection result, and analyzing whether the marks are related to fertility traits or not through SPSS software.
The specific operation steps are as follows:
1. primer design
Primer design software PCR amplification primers and single base extension primers of the sites to be tested were designed using the software of Sequencom company Genotyping Tools and Massarray Assay Design.
2. PCR amplification
The PCR system in 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 (25 mM), mgCl 2 (25mM)0.4μl,HotStar Taq(5U/μl)0.1μl,ddH 2 O 2.4μl。
After adding samples according to the above system, PCR amplification was performed according to the following reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, extension at 72℃for 30s, repeating 40 cycles in total; finally, extending for 4min at 72 ℃; preserving at 4 ℃.
3. Alkaline phosphatase treatment of PCR products
(1) After the PCR reaction, the PCR product was treated with SAP (shrimp alikaiine phosphtase shrimp alkaline sulfate) to remove dNTPs in the reaction. The SAP reaction solution formulation is as follows (for example, single sample): SAP Buffer 0.17. Mu.l, SAP Enzyme (1.7U/. Mu.l) 0.3. Mu.l, ddH 2 O 1.53μl。
(2) The SAP reaction solution was added to the PCR reaction plate by a pipette, 2. Mu.l was added to each well, membrane sealed, and centrifuged.
(3) The PCR reaction plate added with the SAP reaction liquid is placed into a PCR instrument, and the following reaction procedure is operated: 37 ℃ for 40min;85 ℃ for 5min; preserving at 4 ℃.
(4) After the reaction, the PCR reaction plate is taken out and centrifuged briefly for standby.
4. Extension reaction
(1) Preparation of iPlex reagent (example single sample): iCalex Buffer (10X) 0.12. Mu.l, iPlex Termination mix (10X) 0.2. Mu.l, iCalex Enzyme (2U/. Mu.l) 0.041. Mu.l, SAP Enzyme (1.7)U/. Mu.l) 0.3. Mu.l, 10 μm 0.804. Mu.l of primer used, ddH 2 O 0.755μl;
(2) Adding the iPLex reaction solution into the PCR reaction plate by using a pipetting gun, adding 2 mu l of the solution into each hole, sealing a film, and centrifuging;
(3) The PCR reaction plate is placed in a PCR instrument, and PCR amplification is carried out according to the following reaction conditions: pre-denaturation at 94℃for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s, repeating 40 cycles in total; finally, extending at 72 ℃ for 3min; preserving at 4 ℃.
5. Purification of the product
(1) The resin scraper is uniformly covered with resin and placed for 20min.
(2) The PCR reaction plate after the reaction was centrifuged at 1000rpm for 1min, 25. Mu.l of deionized water was added to each well, the mixture was inverted over the resin plate (note to fix, unable to shift), and then the resin plate was fastened to the PCR reaction plate in reverse, and the resin was dropped into the PCR reaction plate by knocking, and the membrane was sealed.
(3) The long axis of the PCR reaction plate is taken as the axis, the PCR reaction plate is turned over for 20min, and centrifugation is carried out for 5min at 3500rpm for standby.
6. Mass spectrometry detection
(1) Nanodispenser SpectroCHIP chip spotting, transferring the detection sample from the PCR reaction plate to MassARRAY SpectroCHIP chip with surface covered with matrix;
(2) MassARRAY Analyzer Compac mass spectrometry detection;
(3) And (5) analyzing an experimental result by TYPER software to obtain the typing data.
7. Statistical analysis
The invention obtains a molecular marker 9W1, and the nucleotide sequence of the molecular marker is shown as SEQ ID No. 1. As shown in Table 3 and FIG. 1, it can be seen that the allele frequency of base A at position 301 in 9W1 is 80.00% in the high-fertility group, and that the allele frequency of base A in the low-fertility group is 63.33%, and that the ratio of base A has a significant difference in the fertility segregating population (P < 0.05), so that it can be considered that the allele frequency of base A at this site is 80.00% or more is a high-fertility population, and that this site is a candidate 9W1_SNP site. Wherein the development of the molecular marker 9W1 is shown in SEQ ID No.2 and SEQ ID No.3 (Table 4), and the extension primer is shown in SEQ ID No.4 (Table 4).
TABLE 3 typing results of 9W1 molecular markers
TABLE 4 molecular tagged primers
The molecular marker 9W1 obtained by the invention can be used for assisting in selecting a high fertility population (family, strain or geographical population), and the application steps are as follows: extracting DNA of more than 30 test samples in the litopenaeus vannamei colony and taking the DNA as a template, performing flight mass spectrometry typing by using the amplification primers 9W1-F and 9W1-R of the molecular marker 9W1 and the extension primer 9W1-E, and if the allele frequency of the 301 th base A of the 9W1 site in the colony in the typing result is more than or equal to 80.00%, selecting the colony as a parent for cultivating the litopenaeus vannamei with high fertility. In addition, the molecular marker 9W1 can be used for analyzing the diversity of the litopenaeus vannamei, identifying germplasm and constructing a genetic map of the litopenaeus vannamei.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> yellow sea aquatic institute of China aquatic science institute
<120> molecular marker 9W1 related to prawn reproduction traits and application thereof
<150> 2022102023568
<151> 2022-03-02
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 601
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccgcccgcct caccttccgc cctgaactca atgctgccga cagtgaggtg gacgcggtcg 60
ccgtggacgc tgcggtcgga gctgatggag tagaggcgcg ggcgcagggg cggcagacgg 120
gccagcagga gggcggcgtc cacggagatg ctcgggaact ctgccaggac gtctggaagg 180
ctcgggcagt gggcagcgcg ccacttctgg tattgctgcg tgtcctgaag gcgggaggga 240
gagaacagag agtgaggggg gcgtggagga agctgctctt ccgcgggctg tgcgaagggc 300
agtcagtacc cagggagttt ataacactaa agattcgttc agatgcaaag aaaggaaggc 360
aaacttggcc gccggcaaag ggacatggcc accggcggcc cattggaagc gacggcttga 420
gcctgcctcg cccgcctacc gtggccagcg ccgagagccg ggctgcctgc tggttgttgg 480
cggcgcagga ggcgagcagg aacagcaggc cctgcgacgg cggcgcggag aggtccacgt 540
acctcgccag aagctcctgc agcgtcgtcg ccggcagctg cgggtgagga acccacgtac 600
c 601
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
acgttggatg gtgttataaa ctccctgggt 30
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
acgttggatg gagagaacag agagtgaggg 30
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cccctccctg ggtactgac 19
Claims (10)
1. The molecular marker 9W1 related to the propagation property of the prawns is characterized in that the molecular marker 9W1 is a nucleotide sequence shown as SEQ ID No.1, and the nucleotide sequence shown as SEQ ID No.1 has G/A base mutation from the 301 th position of the 5' end.
2. The molecular marker 9W1 of claim 1, wherein the base a at position 301 of the molecular marker 9W1 is a candidate 9w1_snp site.
3. The molecular marker 9W1 of claim 2, wherein the allele frequency of base a in the high fertility population of litopenaeus vannamei is greater than or equal to 80.00%.
4. The primer of the molecular marker 9W1 as set forth in claim 1, wherein the primer comprises an amplification primer with nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.3 and an extension primer with nucleotide sequences shown in SEQ ID No. 4.
5. The use of the primer of the molecular marker 9W1 as defined in claim 4 for screening out a population of Litopenaeus vannamei having high fertility.
6. The use according to claim 5, wherein the specific steps of screening the population of litopenaeus vannamei with high fertility are: extracting DNA of a test sample in the litopenaeus vannamei colony, taking the DNA as a template, carrying out flight mass spectrometry typing by using the primer, and selecting the litopenaeus vannamei colony as a parent for cultivating the high fertility of the litopenaeus vannamei if the allele frequency of the 301 th base A of the molecular marker in the typing result is more than or equal to 80.00%.
7. The use according to claim 6, wherein the number of test samples of the Litopenaeus vannamei population is above 30.
8. The use according to claim 6, wherein in the mass spectrometry in flight, the conditions of PCR amplification are pre-denaturation 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, extension at 72℃for 30s, repeating 40 cycles in total; finally, extending for 4min at 72 ℃; preserving at 4 ℃.
9. The use according to claim 6, wherein in the mass spectrometry in flight, the conditions of the extension reaction are pre-denatured 94 ℃ 30s; denaturation at 94 ℃ for 5s, annealing at 52 ℃ for 5s, extension at 80 ℃ for 5s, repeating 40 cycles in total; finally, extending for 3min at 72 ℃; stored at 4 ℃.
10. Use of the molecular marker 9W1 of claim 1 in genetic diversity analysis, germplasm identification and genetic map construction of litopenaeus vannamei.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210202356 | 2022-03-02 | ||
CN2022102023568 | 2022-03-02 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114717333A CN114717333A (en) | 2022-07-08 |
CN114717333B true CN114717333B (en) | 2023-05-02 |
Family
ID=82244951
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210455955.0A Active CN114717333B (en) | 2022-03-02 | 2022-04-28 | Molecular marker 9W1 related to prawn reproduction traits and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114717333B (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118272546B (en) * | 2024-06-03 | 2024-08-23 | 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 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
CN103416333A (en) * | 2013-07-31 | 2013-12-04 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei base population establishing method based on genetic information and excellent properties |
CN105936937A (en) * | 2016-06-24 | 2016-09-14 | 广东海洋大学 | SNP marker related with low dissolved oxygen survivability of litopenaeus vannamei, screening and applications thereof |
WO2018214187A1 (en) * | 2017-05-23 | 2018-11-29 | 中国科学院南海海洋研究所 | Snp marker related to low-salt tolerance characteristic of litopenaeus vannamei, amplification primer therefor, and application thereof |
-
2022
- 2022-04-28 CN CN202210455955.0A patent/CN114717333B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
CN103416333A (en) * | 2013-07-31 | 2013-12-04 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei base population establishing method based on genetic information and excellent properties |
CN105936937A (en) * | 2016-06-24 | 2016-09-14 | 广东海洋大学 | SNP marker related with low dissolved oxygen survivability of litopenaeus vannamei, screening and applications thereof |
WO2018214187A1 (en) * | 2017-05-23 | 2018-11-29 | 中国科学院南海海洋研究所 | Snp marker related to low-salt tolerance characteristic of litopenaeus vannamei, amplification primer therefor, and application thereof |
Non-Patent Citations (5)
Title |
---|
Araceli Lorena Montes-Dominguez 等.Comparison between cultured and wild Pacific white shrimp (Penaeus vannamei) vitellogenesis: next-generation sequencing and relative expression of genes directly and indirectly related to reproduction.《PeerJ》.2021,第1-17 页. * |
D. C. Ciobanu 等.A major SNP resource for dissection of phenotypic and genetic variation in Pacific white shrimp (Litopenaeus vannamei ).《Animal Genetics》.2009,第41卷第39-47 页. * |
Shengjie Ren 等.Quantitative Genetic Assessment of Female Reproductive Traits in a Domesticated Pacific White Shrimp (Penaeus vannamei) Line in China.《Scientific Reports》.2020,第10卷第1-10 页. * |
刘峻宇 等.凡纳滨对虾 SNP 标记开发与家系亲 缘关系验证分析.《渔业科学进展》.2021,第42卷(第1期),第 108-116 页. * |
王冉 等.凡纳滨对虾繁殖性状的SNP 分子标记筛选的初步研究.《上海海洋大学学报》.2018,第27卷(第6期),第 825-832 页. * |
Also Published As
Publication number | Publication date |
---|---|
CN114717333A (en) | 2022-07-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107164463A (en) | It is a kind of to be used for the SNP marker of measure and/or genetic improvement pig growth traits | |
CN109554486A (en) | SNP marker relevant to grass carp character and its application | |
CN114717333B (en) | Molecular marker 9W1 related to prawn reproduction traits and application thereof | |
CN106906303A (en) | One SNP marker for influenceing quality character of pork and its application | |
CN112289384A (en) | Construction method and application of whole citrus genome KASP marker library | |
CN110079609B (en) | Molecular marker for identifying pullorum disease resistant chicken and application thereof | |
CN109486961B (en) | Construction method of scylla paramamosain high-density genetic map | |
CN111057771B (en) | SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof | |
CN114875158A (en) | Molecular marker 27W1 for breeding high-fertility prawn population and application thereof | |
CN114752685B (en) | Molecular marker for vannamei and primer and application thereof | |
CN114592068B (en) | High-fertility molecular marker X1W1 of litopenaeus vannamei and application thereof | |
CN114686603B (en) | Molecular marker 21W2 and application thereof in screening litopenaeus vannamei populations with high fertility | |
CN101899500B (en) | Method for detecting single nucleotide polymorphism of cattle krupple-like factor (KLF) 7 gene | |
CN109536624B (en) | Fluorescent molecular marker and detection method for discriminating true and false male fish of cynoglossus semilaevis | |
CN114752684B (en) | Molecular marker 7W1 for identifying fertility of prawns and application thereof | |
CN113604587B (en) | Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof | |
CN111593129B (en) | Primer for identifying family of Bai Suowen perch and identification method of family of micropterus salmoides | |
CN114410806A (en) | Litopenaeus vannamei microsatellite marked primer combination and application | |
CN109609681B (en) | Identification method of loblolly pine individual based on chloroplast genome sequence | |
CN113584187A (en) | Molecular marker A2629 for screening penaeus japonicus with low temperature resistance, amplification primer and application thereof | |
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' flanking region of cattle lncFAM200B gene and application thereof | |
CN113981112B (en) | InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof | |
CN113930518B (en) | Molecular marker C49 for identifying ammonia nitrogen tolerance character of portunus trituberculatus 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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |