Micropterus salmoides sex specific SNP molecular marker primer and application thereof

A sex and primer pair technology, applied in the field of California perch sex-specific SNP molecular marker primers, can solve the problem of no California perch sex-specific molecular markers

Pending Publication Date: 2022-06-03
FOSHAN NANHAI BAIRONG AQUATIC SEED +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of sex-specific molecular markers in California sea bass at home and abroad

Method used

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  • Micropterus salmoides sex specific SNP molecular marker primer and application thereof
  • Micropterus salmoides sex specific SNP molecular marker primer and application thereof
  • Micropterus salmoides sex specific SNP molecular marker primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, the acquisition of California sea bass sex-specific SNP molecular markers

[0034] 1) During the breeding season, 24 females and 24 males of California perch were identified by dissecting and observing the gonads, and the tail fins were clipped and the whole genome DNA was extracted.

[0035] 2) 16 female California perch DNAs were mixed in equal amounts into a female mixed pool, and 16 male California perch DNAs were mixed in equal amounts into a male mixed pool.

[0036] A total of 18 samples from the female mixed pool, the male mixed pool, and the remaining 8 female and 8 male samples were subjected to whole-genome sequencing on the Illumina Hiseq X Ten platform. All the clean reads after filtering the original data were subjected to 60bp fragmentation electronic digestion starting from the 5' end of AC, AG, AT, GA, GC and GT six bases one by one, and the starting bases did not meet the requirements. rear displacement. The clean reads of the 8-tailed fem...

Embodiment 2

[0043] Embodiment 2, the application process of California perch sex-specific SNP molecular markers in identification

[0044] 1) Design the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2 according to the sequence shown in SEQ ID NO.3.

[0045] 2) Genomic DNA extraction (chelex100 boiling method)

[0046] Prepare 5% chelex100 solution; take 5mg of caudal fin tissue into a 0.2mL PCR tube, shake the chelex100 solution to evenly suspend the resin particles in the solution, and use a flaring tip to suck 150uL into the PCR tube containing the caudal fin tissue; put it into the PCR tube Add 5uL of 20mg / mL proteinase K solution, shake and mix well, place it on the PCR machine, set the conditions to heat the lid at 105°C, the module at 55°C, 40min; 98°C, 5min; take out immediately after the reaction, and vortex 3 times , 5s each time; put the PCR tube into the centrifuge and centrifuge at 4000rpm for 5min; the obtained supernatant is the DNA solution, which can be ...

Embodiment 3

[0052] Embodiment 3, California sea bass sex-specific SNP molecular marker stability detection

[0053] 1) The California perch body was randomly collected from the market, numbered 1-96, and the sex of the fish was determined by dissection, with 54 female fish and 42 male fish, and fin ray tissue samples were collected and stored in a 96-well plate for cryopreservation.

[0054] 2) Carry out HRM typing according to the application process provided in Example 2.

[0055] HRM typing results are as follows figure 1 and figure 2 shown. figure 1 The medium-dark curve represents the sample whose detected SNP locus is heterozygous, so it is judged as male (XY); the light-colored curve represents the sample whose detected SNP locus is homozygous, so it is judged as female (XX). figure 2 For the detection results of the 96-well plate, the two "S" marks in the figure are the standard curve reference automatically selected by the software, in which the dark blocks represent the sam...

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Abstract

The invention discloses micropterus salmoides sex-specific SNP (Single Nucleotide Polymorphism) molecular marker primers and application thereof. The SNP molecular marker is used for distinguishing whether a sample belongs to homozygote (XX) or heterozygote (XY), so that the sex of the sample micropterus salmoides is identified, and the judgment accuracy is 100%. The invention further discloses a primer pair and a kit for amplifying the SNP molecular marker. In addition, the invention also provides a method for identifying the sex of the micropterus salmoides. The method comprises the following steps: (a) extracting DNA of the to-be-detected micropterus salmoides; (b) by taking the extracted DNA as a sample, amplifying by using the primer pair or the kit to obtain an amplification product; and (c) identifying the genotype of the amplification product by using a high-resolution dissolution curve analysis (Lightscanner) system, if the amplification product is homozygote, determining that the to-be-detected micropterus salmoides is female, and if the amplification product is heterozygote, determining that the to-be-detected micropterus salmoides is male.

Description

technical field [0001] The present invention relates to the technical field of gene detection, in particular to sex-specific SNP molecular marker primers of California perch and application thereof. Background technique [0002] Aquaculture and field investigations have found significant differences between males and females in many fish species, such as carp (Cyprinus carpio), rainbow trout and semilaevis (Cynoglossus semilaevis), where the ovaries mature later than the testes and most nutrients during sexual maturity Substance is converted into body growth, so females are larger than males; in contrast, in fish such as Nile tilapia (Oreochromis niloticus), yellow catfish (Pelteobagrus fulvidraco), and channel catfish (Ictalurus punctatus), the testes mature later than the ovaries, Males grow faster than females, so males are larger. In fish production, parthenogenesis has the advantages of neat specifications and reduced energy consumption for reproduction, and is current...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2600/124C12Q2531/113C12Q2545/114C12Q2563/107Y02A40/81
Inventor 陈柏湘陈科洁尹建雄张美东
Owner FOSHAN NANHAI BAIRONG AQUATIC SEED
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