CN114317769A - Auxiliary breeding method based on high-throughput SNP site screening - Google Patents
Auxiliary breeding method based on high-throughput SNP site screening Download PDFInfo
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Abstract
The invention discloses an auxiliary breeding method based on high-throughput SNP site screening. 1) Acquiring the three-dimensional morphology and hyperspectral information of a target organism by using a hyperspectral four-dimensional imaging instrument; constructing a test model of phenotypic character characteristics; associating the four-dimensional information with the phenotypic character characteristics of the target organism, and constructing an automatic test platform of the target organism; 2) performing high-throughput genome sequencing and whole genome association analysis aiming at a reference group individual of a target organism with a specific phenotypic character to obtain an SNP locus; designing a high-throughput SNP specificity screening chip; 3) carrying out high-throughput detection on a sample to be detected to obtain a genotype sample with a corresponding genotype; 4) screening a parent subset for phenotypic character identification from candidate breeding parents according to the result of SNP locus analysis and genetic relationship information based on SNP clustering; finally determining the selected breeding parents needed by the breeding plan. The invention can obviously save the selection cost and the screening time of the excellent characters of the target organism.
Description
Technical Field
The invention belongs to the field of analysis and detection, and mainly relates to a high-throughput screening method for SNP loci for target organism assisted breeding.
Background
In traditional breeding, target individuals are selected through phenotype observation, the traditional breeding method not only depends on the experience of breeders, but also is very easily influenced by the interaction between the genotype and the environment, and the ideal genotype is difficult to select. The molecular marker technology is an auxiliary means for selection in the process of crop variety improvement, and mainly comprises the foreground selection of target traits and the selection of genetic backgrounds. The molecular marker is used for assisting background selection in the breeding process, so that the breeding time can be obviously shortened.
With the rapid development of genome sequencing technology, the rapid reduction of sequencing cost and the convenience of acquiring a large amount of genome data, how to effectively combine genome data with breeding practice becomes an important proposition before molecular breeding workers. If genome information can be combined with phenotype identification, the screening range of phenotype identification is narrowed by means of genome, and the efficiency of phenotype identification of breeding work can be improved greatly.
Molecular markers suitable for background selection need to satisfy 3 basic conditions: simple, fast and low cost. SNP (Single Nucleotide polymorphism) is widely applied to researches on aspects of animal genetic breeding, gene positioning, cloning, genetic diversity and the like at present by virtue of the characteristics of high polymorphism, strong genetic stability and the like. By screening the SNP markers linked with the excellent characters, the strains with the excellent characters are selected in advance, and the complicated phenotype screening in the later period is avoided, so that the breeding time and the cost are effectively saved.
The liquid drop (Microdroplet) is used as a micro reactor, can greatly accelerate the biochemical reaction rate, and is widely applied to the fields of analysis and detection, chemical sensing, medical detection, chemical synthesis and the like. The sample is dispensed into tens of millions of units using droplets to perform high throughput detection of the sample.
The seed test is an important link in the genetic breeding process, and the seed test has various projects and varieties and relates to different varieties and phenotypic characters. However, it is very important to improve the breeding efficiency how to select the required variety from the data of many species. In recent years, image recognition has matured increasingly with rapid development of automation, optics, and computer technologies. The information acquisition and processing by the image mode has the characteristics of small distortion, easy storage, easy transmission, strong anti-interference capability and the like. For the characters which cannot be automatically identified and collected originally, rapid collection can be realized by the image technology, and meanwhile, the establishment of a new automatic measuring system for the parameters of the related characters of the blood clam is possible by combining automation, optical technology and computer technology. The hyperspectral imaging refers to an imaging mode capable of collecting continuous hundreds of wave bands, and each pixel point on an acquired image corresponds to a spectral curve. The hyperspectral imaging technology integrates the advantages of the imaging technology and the spectrum technology, and the information richness is greatly improved. Therefore, by utilizing the hyperspectral imaging technology and combining artificial intelligence, the high-precision three-dimensional shape modeling of the target organism can be realized, and the body composition of the target organism can be analyzed and detected.
Disclosure of Invention
In order to overcome the defects in the traditional breeding, the invention provides an auxiliary breeding method based on high-throughput SNP locus screening.
An auxiliary breeding method based on high-throughput SNP site screening,
1) acquiring the three-dimensional morphology and hyperspectral information of a target organism by using a hyperspectral four-dimensional imaging instrument; constructing a seed test model of phenotypic character characteristics, wherein the phenotypic character characteristics comprise growth speed and disease resistance; associating the four-dimensional information with the phenotypic character characteristics of the target organism, and constructing an automatic test platform of the target organism;
2) performing high-throughput genome sequencing and whole genome association analysis aiming at a reference group individual of a target organism with a specific phenotypic character to obtain an SNP locus; designing a high-throughput SNP specificity screening chip;
3) carrying out high-throughput detection on a sample to be detected by using the high-throughput SNP specificity screening chip to obtain a genotype sample with a corresponding property;
4) screening a parent subset for phenotypic character identification from candidate breeding parents according to the result of SNP locus analysis and genetic relationship information based on SNP clustering; and combining the phenotype repeated identification results of the parent subsets to finally determine the selected breeding parents required by the breeding plan.
The high-throughput genome sequencing adopts a digital PCR method for amplification, a PCR reaction system is subjected to micro-titration treatment and is divided into water-in-oil micro-droplets with the diameter of 10-100 mu m generally, amplification and signal amplification on a single-copy nucleic acid level are realized, and a high-throughput sequencing library is established for sequencing analysis after amplification products are mixed.
The target organism is blood clam, the line laser scans 3D appearance, the line laser is projected on the surface of a sample to be detected to form a bright line, the bright line is modulated by the depth fluctuation of the surface to deform, the section height data of the sample to be detected is obtained by calculating the pixel coordinate of the bright line on the image surface of a camera, and the laser line scans the sample by moving an electric displacement platform, so that the local three-dimensional appearance data of the sample is obtained; the hyperspectral spectrometer further detects the optical characteristics of the shell substances of the blood clam and the adductor muscles; the integration of the three-dimensional shape data and the spectrum data enhances the analysis of the quality, the physiological information and the variety of the blood clam.
The selected breeding parent is used for cross breeding.
The invention has the beneficial effects that:
the obtained SNP information of the breeding parents can be used for genotype selection of a target organism, candidate breeding parents with excellent properties and far genetic relationship are effectively identified, timely hybridization and transformation are facilitated, and the breeding process is accelerated.
Drawings
FIG. 1 is a flow chart of the practice of the present invention.
FIG. 2 is a schematic view of a droplet generator according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings.
EXAMPLE 1 determination of phenotypic traits of an object organism
The hyperspectral imaging device is shown in CN211205210U (four-dimensional hyperspectral depth imaging system), line laser scans 3D appearance, and line laser is projected on the surface of a sample to be measured to form a bright line. The bright line is modulated by the depth fluctuation of the surface to deform, and the section height data of the sample to be measured is obtained by calculating the pixel coordinates of the bright line on the image surface of the camera. The laser line scans the sample by the movement of the system electric displacement platform, thereby obtaining the local three-dimensional shape data of the sample. The line light morphology is detected efficiently, and the precision is high, is used for short-term test for the appearance of the tegillarca granosa, and the hyperspectral spectrometer can further detect the optical characteristics of the tegillarca granosa shell material and the adductor muscle. The integration of the three-dimensional shape data and the spectrum data can further enhance the analysis capability of the quality, the physiological information and the species of the blood clam.
After acquiring phenotypic character data of the blood clams, training, optimizing and modeling a plurality of migrated depth networks; then, the tegillarca granosa classifier with higher precision is obtained through comparative analysis; finally, the phenotypic parameters of a certain family of scapharca granosa are calculated by a machine vision technology and used for screening the excellent phenotypic characters of the scapharca granosa.
Example 2 PCR amplification and sequencing
The principle of the centrifugal type liquid drop generating device is shown in figure 2, the liquid drop generating device comprises an upper water phase centrifugal unit and a lower oil phase centrifugal unit, an upper water phase system is formed by assembling three micropipette heads and capillaries, an outermost liquid-transferring gun head is formed by cutting and modifying a 1000-microliter pipette head at a gun head bulge, a middle liquid-transferring gun head is formed by cutting and modifying a 200-microliter pipette head at a gun head bulge and is fixed on the inner side of the outermost liquid-transferring gun head by using an O-shaped collar, an innermost liquid-transferring gun head is formed by cutting and modifying a 10-microliter pipette head at a position 0.5cm away from the lowest end, the micropipette heads of three different specifications are mutually nested, and the capillary with the inner diameter of 690 mu m is just fixed in the innermost micropipette head. The lower oil phase system consisted of 1.5ml centrifuge tubes. The outermost pipette tip in the upper aqueous phase system can be fixed in a 1.5ml centrifuge tube of the lower oil phase system and jointly placed in a common centrifuge of a laboratory to generate water-in-oil droplets. In this example, the pipette tips were of the following type, 1000. mu.l pipette tips: axygen T-1000-B; 200 μ l pipette tip: axygen T-200-Y; 10 μ l pipette tip: axygen T-300. The centrifuge tube can use a common centrifuge tube and is matched with a liquid-transfer gun head of the upper water phase.
When the device disclosed by the invention is used for loading the upper-layer water phase, instruments such as a trace loading needle and the like are adopted to directly add a sample into the capillary, so that the problems of liquid leakage or incomplete centrifugation are avoided, and the quantity of generated liquid drops is increased. For example, 10. mu.l of the upper aqueous phase can produce more than 7 ten thousand uniform droplets.
Therefore, the device can distribute the blood samples of the blood clams of different strains into tens of thousands of liquid drops for PCR amplification, the amplification products are mixed to establish a high-throughput sequencing library, high-throughput sequencing is carried out through Illumina Hiseq4000, and the sequencing data can be filtered for subsequent SNP typing.
Example 3 Process of SNP assisted Breeding
The genome information assisted breeding method for carrying out breeding parent selection by utilizing SNP cluster information and PAV variation information comprises the following steps:
(1) obtaining a large amount of genome reads information of candidate breeding parents through genome re-sequencing;
(2) comparing the reference genome with a conventional sequence comparison method to obtain physical position information, and extracting an SNP information data set; filtering the SNP information data set by setting parameters to obtain a high-quality SNP data subset;
(3) on the basis of the high-quality SNP data subset, calculating by a conventional method to obtain a genetic distance matrix of candidate breeding parents, constructing an SNP cluster tree by the conventional method, and judging the distance of the genetic relationship between the candidate breeding parents according to the SNP cluster information of the candidate breeding parents;
(4) forming contigs of candidate breeding parents by Denovo assembly, and then positioning the contigs to a reference genome by a conventional sequence alignment analysis method to obtain the physical positions of the contigs of the corresponding candidate breeding parents;
(5) selecting important related gene loci according to target characters set by a breeding plan, and analyzing SNP locus sequences of corresponding region sequences of target character control genes in candidate breeding parent contigs according to physical position regions of the genes;
(6) screening a parent subset for phenotype identification from a large number of candidate breeding parents according to the result of SNP locus analysis and genetic relationship information based on SNP clustering;
(7) and combining the phenotype repeated identification results of the parent subsets to finally determine the selected breeding parents required by the breeding plan.
Example 4 application to Breeding of Leqing first Scapharca granosa
The invention is explained in more detail below, according to the following steps:
(1) arca granosa group material selection
The method ensures that the selected Arca granosa groups can have enough representativeness (including wild species, breeding species or samples divided according to different regional sources), the groups are divided into a plurality of (3-4) groups, at least K (such as K =20) bodies in each group are more than, and the size of the finally constructed group is more than N (such as N =200) samples. The scheme takes 'le qing I' as a main breeding object.
(2) Determination of phenotypic traits
The line laser is projected on the surface of the sample to be measured to form a bright line, the bright line is modulated by the depth fluctuation of the surface to deform, and the section height data of the sample to be measured is obtained by calculating the pixel coordinates of the bright line on the image surface of the camera. And (3) combining the line laser with the movement of the electric displacement platform, so that the laser line scans the sample to obtain the local three-dimensional shape data of the sample, and the surface property of the blood clam is obtained. The line light morphology is detected efficiently, and the precision is high, is used for short-term test arca granosa appearance, and the hyperspectral spectrometer can further survey the optical characteristics of positions such as arca granosa shell material and adductor muscle. The integration of the three-dimensional shape data and the spectrum data can further enhance the analysis capability of the quality, the physiological information and the variety of the blood clam.
Specifically, the shells of the blood clam are composed of a horny layer and a calcium carbonate crystal layer, and the calcium carbonate crystal layer is a main part of the blood clam. The formation of shell calcium carbonate crystal is mainly controlled by organic matter in shell, especially shell matrix protein, mainly including chitin and protein components. By analyzing the infrared spectrum, the protein components and structures in the shells can be analyzed. Calcium carbonate crystals in the shells of different shellfish species exhibit different microstructural morphologies, which are often found in aragonite and calcite configurations. Therefore, when the spectrum of the shell is detected, the characteristic peak of the calcium carbonate crystal is obvious, and CO 32-has four absorption peaks, namely a symmetrical stretching vibration peak with v1=1082cm-1, an out-of-plane bending vibration peak with v2=854cm-1, an antisymmetric stretching vibration peak with v3=1442cm-1, and an in-plane bending vibration peak with v4=714cm-1 and 699 cm-1. And the spectrum detection of the pearl layer, the muscle prism layer and the oblique prism layer in the shell can judge which microstructure the pearl layer, the muscle prism layer and the oblique prism layer belong to. (according to the characteristic peaks of typical aragonite calcium carbonate crystals, namely v1=1082cm-1, and the characteristic peaks of split calcite, namely v4=712cm-1 and 699 cm-1. the characteristic peaks of typical calcite, namely v1 disappears, v2 is blue-shifted, and the 699cm-1 in v4 disappears.) the size of the shell of the tegillarca granosa can also be used for analyzing the information of the production area and the like. Firstly, extracting phenotypic parameters such as shape, color and texture of the blood clam through a machine vision technology; then, a convolutional neural network capable of efficiently and accurately identifying the quality of the blood clam is obtained through a series of operations such as network design, network training, parameter optimization and the like; and finally, obtaining a high-precision convolutional neural network for refining and classifying the arca granosa with different qualities through transfer learning.
(3) Detection for screening fine variety of scapharca granosa based on SNP marker
In the detection of screening the fine variety of the blood clam based on the SNP marker, firstly, the genome and high-throughput sequencing can be carried out in a high-throughput sequencing mode to obtain the reference genome of the blood clam, all genome resources of a species can be identified at one time, all variation information (SNP and the like) of the species can be fully excavated, and a foundation is laid for the development of subsequent site screening work; secondly, customizing a high-flux or medium-flux SNP detection chip by using the SNP locus obtained by sequencing, finding the SNP locus related to the target character through strategies such as QTL positioning, GWAS, selective elimination and the like, and realizing the screening and character positioning of the molecular marker; and finally, further genotyping large-scale samples by adopting a low-density candidate SNP site customization mode and utilizing a Boo crystal classics IMAP platform, an Agena SNP platform and the like, thereby meeting the requirements of molecular marker assisted breeding in conventional application.
Materials and methods for SNP analysis:
the experimental samples are collected from Arca granosa of different geographical populations or families, 40 Arca granosa are randomly sampled, and the adductor muscle is taken out after the biopsy and stored in a refrigerator at-20 ℃ for later use.
Experiment main medicine reagent
Tris equilibrated phenol, chloroform isoamyl alcohol (24: 1), proteinase K, acrylamide, STE lysate (pH = 8.00), 10% SDS (pH = 8.00), sodium acetate (3M), agarose, meltvector TM HRM Master MIX (ABI).
Extraction of DNA:
the extraction of DNA uses phenol chloroform method, the extracted DNA is detected by ultraviolet spectrophotometer, the ratio of A260/A280 is 1.8-2.0, A260/A230 is more than 2.0, the DNA concentration is adjusted to 30 ng/microliter, and the DNA is put into a freezer with 20 ℃ below zero for standby.
Candidate site screening and primer design
On the basis of constructing a tegillarca granosa transcriptome library in the laboratory, the qualitySNP software is used for comparison analysis, and the software screening rules are as follows: (1) not less than 4 aligned sequences; (2) the sequence containing the mutation site accounts for not less than 25% of the aligned sequences.
Sites used for validation need to satisfy the following conditions: (1) no other mutation sites within 150bp upstream and downstream of the candidate site (2) are selected as transversion or conversion type candidate sites. The primers used in the experiments were designed using Primer Premier5 software, with the design rule that (1) the length of the primers is 18-24bp (2) the annealing temperature for the amplification products is less than 300bp (3) is recommended at 55-61 ℃. (4) The GC content is 40-60%. (5) The product score was greater than 94 points. And (4) carrying out primer design by using the candidate sites.
The obtained primers need to be subjected to temperature gradient PCR to detect the optimal annealing temperature. And (3) carrying out temperature gradient PCR amplification by using a gradient PCR instrument, and taking 3.5 microliters of the product after the temperature gradient PCR for polyacrylamide gel electrophoresis detection.
The SNP chip (56K) is purchased from Affymetrix chip company, synthesized by adopting a unique photoetching in-situ synthesis technology, can detect 56000 SNPs sites, has a mark detection rate of more than 97 percent, and is a 384-standard chip. And carrying out whole genome scanning on the Axiom Maize56K SNP Array platform by adopting clam SNP chips (56K). Sample preparation was done on the Backman automated workstation, hybridization, washing, scanning, etc. were done on GeneTitan.
SNP chip detection process and SNP chip data quality control
SNPs used for SNP arrays are derived from the whole genome sequence of Scapharca subcrenata (GenBank: JABXWC 000000000.1). Extracting genome DNA from blood clam muscle, sequencing and controlling the quality of sequencing sequence in Illumina HiSeq 2000 sequencing platform, and deleting low-quality sequence, joint sequence and uncertain nucleotide (Ns). Calling an analysis tool (GATK) for SNPs through the genome, identifying SNPs using default parameters and by minimum mapping quality values.
And (3) detection flow: performing alkali denaturation on genome DNA by using NaOH solution, performing genome full amplification at constant temperature overnight, performing DNA fragmentation by using enzyme digestion, performing DNA precipitation and heavy suspension, performing SNP chip hybridization, cleaning a chip, performing single base extension dyeing, scanning the chip, and finally reading data by using an iScan scanning system.
And (3) data quality control:
unqualified samples and SNP sites are screened according to the following quality control standards: and (3) only using the sites on the autosome, wherein the minimum allele frequency is more than 0.05, the P value of the Harvard Winberg equilibrium test is more than 10-6, and the detection rate is more than 90% of the SNP sites.
(4) Breeding of good variety
Screening a parent subset for phenotype identification from a large number of candidate breeding parents according to the result of SNP chip analysis and genetic relationship information based on SNP clustering; and combining the phenotype repeated identification results of the parent subsets to finally determine the selected breeding parents required by the breeding plan.
The embodiments in the above description can be further combined or replaced, and the embodiments are only described as preferred examples of the present invention, and do not limit the concept and scope of the present invention, and various changes and modifications made to the technical solution of the present invention by those skilled in the art without departing from the design concept of the present invention belong to the protection scope of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.
Claims (4)
1. An auxiliary breeding method based on high-throughput SNP site screening is characterized in that:
1) acquiring the three-dimensional morphology and hyperspectral information of a target organism by using a hyperspectral four-dimensional imaging instrument; constructing a seed test model of phenotypic character characteristics, wherein the phenotypic character characteristics comprise growth speed and disease resistance; associating the four-dimensional information with the phenotypic character characteristics of the target organism, and constructing an automatic test platform of the target organism;
2) performing high-throughput genome sequencing and whole genome association analysis aiming at a reference group individual of a target organism with a specific phenotypic character to obtain an SNP locus; designing a high-throughput SNP specificity screening chip;
3) carrying out high-throughput detection on a sample to be detected by using the high-throughput SNP specificity screening chip to obtain a genotype sample with a corresponding property;
4) screening a parent subset for phenotypic character identification from candidate breeding parents according to the result of SNP locus analysis and genetic relationship information based on SNP clustering; and combining the phenotype repeated identification results of the parent subsets to finally determine the selected breeding parents required by the breeding plan.
2. The method of claim 1, wherein: the high-throughput genome sequencing adopts a digital PCR method for amplification, a PCR reaction system is subjected to micro-titration treatment and is divided into water-in-oil micro-droplets with the diameter of 10-100 mu m, amplification and signal amplification on a single-copy nucleic acid level are realized, and a high-throughput sequencing library is established for sequencing analysis after amplification products are mixed.
3. The method of claim 1, wherein: the target organism is blood clam, the line laser scans 3D appearance, the line laser is projected on the surface of a sample to be detected to form a bright line, the bright line is modulated by the depth fluctuation of the surface to deform, the section height data of the sample to be detected is obtained by calculating the pixel coordinate of the bright line on the image surface of a camera, and the laser line scans the sample by moving an electric displacement platform, so that the local three-dimensional appearance data of the sample is obtained; the hyperspectral spectrometer further detects the optical characteristics of the shell substances of the blood clam and the adductor muscles; the integration of the three-dimensional shape data and the spectrum data enhances the analysis of the quality, the physiological information and the variety of the blood clam.
4. The SNP assisted breeding method according to claim 1, characterized in that: the selected breeding parent is used for cross breeding.
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| CN117265147A (en) * | 2023-10-08 | 2023-12-22 | 三门县水产技术推广站 | Molecular marker related to vibrio resistance of blood clam |
| CN117802249A (en) * | 2024-03-01 | 2024-04-02 | 中国海洋大学三亚海洋研究院 | Preparation method and application of Dongxing spot whole genome SNP chip |
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| CN110438245A (en) * | 2019-09-18 | 2019-11-12 | 浙江海洋大学 | The SNP marker and its application of clam |
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| CN110438245A (en) * | 2019-09-18 | 2019-11-12 | 浙江海洋大学 | The SNP marker and its application of clam |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117265147A (en) * | 2023-10-08 | 2023-12-22 | 三门县水产技术推广站 | Molecular marker related to vibrio resistance of blood clam |
| CN117265147B (en) * | 2023-10-08 | 2024-05-03 | 三门县水产技术推广站 | Molecular marker related to vibrio resistance of blood clam |
| CN117802249A (en) * | 2024-03-01 | 2024-04-02 | 中国海洋大学三亚海洋研究院 | Preparation method and application of Dongxing spot whole genome SNP chip |
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