CN114107555A - SNP molecular marker combination for detecting purity of wheat variety and application thereof - Google Patents
SNP molecular marker combination for detecting purity of wheat variety and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker combination for detecting the purity of wheat varieties and application thereof. The method can be used for accurately detecting the purity of wheat varieties from different sources by screening a set of 21 SNP markers with high quality and high polymorphism, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the marker is not influenced by environmental conditions, seeds or any type of plant tissues can be used, and the detection result is accurate and has good repeatability and stability.
Description
Technical Field
The invention belongs to the field of agricultural molecular biology, and particularly relates to an SNP molecular marker combination for detecting the purity of wheat varieties and application thereof.
Background
Wheat is a major food crop; wheat is mainly produced from conventional varieties and hybrids. With the continuous development of wheat breeding technology and the large-area popularization and application of wheat, the purity detection of the wheat seeds becomes the essential quality assurance for wheat breeding research and development, seed production and seed transaction.
The purity identification of wheat varieties is mainly dependent on the traditional field plot planting identification method (Grow-out Test) at present. Identification of wheat field planting the variety is planted in a field test cell, and the purity of the wheat variety is identified by observing the difference of the plant morphological characteristics (such as height and size of the plant, tillering, leaf color, leaf shape, seed size, seed coat color and the like) and biological characteristics (such as growth period, photoperiod, disease resistance, drought resistance, seed shattering property and the like) of the plant in different growth periods (seedling stage, growth, flowering stage, mature stage and seeds). The method depends on visual identification of morphological characteristics and biological characteristics of plants in the field, the judgment standard is difficult to accurately quantify, the subjectivity is strong, and the detection sensitivity and the resolution are low; the method is easily influenced by environment and cultivation conditions, and has poor accuracy and stability; long time consumption and poor timeliness; large amount of manpower and material resources are needed, and the cost is high.
The DNA molecular marking method is the most common method for detecting the purity of the crop varieties at present. The DNA molecular marker is a genetic marker for directly reflecting DNA difference (Polymorphism), and mainly comprises SSR (Simple Sequence Repeat, Simple Repeat Sequence) and SNP (Single Nucleotide Polymorphism). SSR has become one of the main methods for detecting purity and variety authenticity at present, has national standards of SSR in various crops such as rice, wheat, soybean and the like, and has the advantages of simple laboratory operation, low cost, good repeatability, real and reliable results and the like when being used in a large area at present. Compared with an SSR labeling method, the SNP labeling method has the advantages of simpler technology, easy automation, high detection flux and high speed; the detection cost of unit data points is low; data results of different detection laboratories can be compared and verified with each other, and the data has universal comparability; is the most common method for rapidly, simply, sensitively, accurately, stably and cheaply identifying the purity of the variety. However, the wheat purity detection method based on SNP markers is rarely reported at present.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an SNP molecular marker combination for detecting the purity of wheat.
The invention also provides a primer group for amplifying the SNP molecular marker combination.
The invention also provides a kit.
The invention also provides a gene chip.
The invention also provides the application of the SNP molecular marker combination, the primer group, the kit and/or the gene chip.
The invention also provides a method for detecting the purity of the wheat variety by using the SNP molecular marker combination.
In the first aspect of the invention, a SNP molecular marker combination for detecting wheat purity is provided, which consists of the following SNP molecular markers:
a first SNP molecular marker, wherein the first SNP molecular marker is located at 534265581 th nucleotide of 1A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is G or A;
a second SNP molecular marker, wherein the second SNP molecular marker is positioned at 643102010 th nucleotide of 1B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is A or C;
a third SNP molecular marker, wherein the third SNP molecular marker is positioned at 3958512 th nucleotide of 1D chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a fourth SNP molecular marker, wherein the fourth SNP molecular marker is positioned at 16865489 th nucleotide of 2A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is C or T;
a fifth SNP molecular marker, wherein the fifth SNP molecular marker is positioned at 12076633 th nucleotide of 2B chromosome of wheat reference genome or at a corresponding site on homologous genome fragment among varieties thereof, and the nucleotide base of the site is T or G;
a sixth SNP molecular marker, which is located at 69251621 th nucleotide of 2D chromosome of wheat reference genome or corresponding site on homologous genome fragment between varieties thereof, and the nucleotide base of the site is A or G;
a seventh SNP molecular marker, wherein the seventh SNP molecular marker is located at 61219689 th nucleotide of 3A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is A or C;
an eighth SNP molecular marker, wherein the eighth SNP molecular marker is located at 749024987 th nucleotide of 3B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a ninth SNP molecular marker, wherein the ninth SNP molecular marker is located at 578414376 th nucleotide of the 3D chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
a tenth SNP molecular marker, wherein the tenth SNP molecular marker is located at 715512695 th nucleotide of the 4A chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
an eleventh SNP molecular marker, wherein the eleventh SNP molecular marker is located at 159349087 th nucleotide of the 4B chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a twelfth SNP molecular marker, wherein the twelfth SNP molecular marker is located at 456087457 th nucleotide of the 4D chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is C or T;
a thirteenth SNP molecular marker, wherein the thirteenth SNP molecular marker is located at 533072137 th nucleotide of 5A chromosome of wheat reference genome or at a corresponding site on homologous genome fragment between varieties thereof, and the nucleotide base of the site is G or A;
a fourteenth SNP molecular marker, wherein the fourteenth SNP molecular marker is located at 601419406 th nucleotide of 5B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
a fifteenth SNP molecular marker, wherein the fifteenth SNP molecular marker is located at 562098034 th nucleotide of 5D chromosome of wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is A or G;
a sixteenth SNP molecular marker, wherein the sixteenth SNP molecular marker is located at 595564056 th nucleotide of the 6A chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is T or C;
a seventeenth SNP molecular marker, wherein the seventeenth SNP molecular marker is positioned at 704974282 th nucleotide of the 6B chromosome of a wheat reference genome or at a corresponding site on an homologous genome fragment between varieties thereof, and the nucleotide base of the site is C or T;
an eighteenth SNP molecular marker, which is located at 454587758 th nucleotide of 6D chromosome of wheat reference genome or corresponding site on homologous genome fragment between varieties thereof, and nucleotide base of the site is C or T;
a nineteenth SNP molecular marker, wherein the nineteenth SNP molecular marker is located at 54723172 th nucleotide of 7A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a twentieth SNP molecular marker, which is located at 706809043 th nucleotide of 7B chromosome of wheat reference genome or at a corresponding site on homologous genome fragment among varieties thereof, wherein the nucleotide base of the site is C or T;
a twenty-first SNP molecular marker, wherein the twenty-first SNP molecular marker is located at 629330949 th nucleotide of 7D chromosome of wheat reference genome or corresponding site on homologous genome fragment among varieties thereof, and the nucleotide base of the site is G or A;
wherein the wheat reference genome is a Triticum aestivum IWGSC RefSeq v1.0 version reference genome.
In a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker combination is proposed.
In some embodiments of the invention, the primer sets each independently comprise two specific primers linked to different fluorophore-tagged sequences and 1 universal primer.
In some embodiments of the invention, the fluorophore tag sequence is selected from FAM, HEX.
In some embodiments of the invention, the primer set comprises: the first SNP primer group is used for amplifying the first SNP molecular marker, and comprises specific primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a universal primer with nucleotide sequence shown as SEQ ID NO. 3;
the second SNP primer group is used for amplifying the second SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 6;
a third SNP primer set used for amplifying the third SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 9;
the fourth SNP primer group is used for amplifying the fourth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.10 and SEQ ID NO.11, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 12;
a fifth SNP primer set used for amplifying the fifth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.13 and SEQ ID NO.14 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 15;
a sixth SNP primer set, which is used for amplifying the sixth SNP molecular marker and comprises specific primer nucleotide sequences shown as SEQ ID NO.16 and SEQ ID NO.17 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 18;
a seventh SNP primer set for amplifying the seventh SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.19 and SEQ ID NO.20, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 21;
the eighth SNP primer group is used for amplifying the eighth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.22 and SEQ ID NO.23 and a universal primer nucleotide sequence shown as SEQ ID NO. 24;
a ninth SNP primer set for amplifying the ninth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.25 and SEQ ID NO.26, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 27;
a tenth SNP primer set for amplifying the tenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.28 and SEQ ID NO.29, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 30;
an eleventh SNP primer set for amplifying the eleventh SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.31 and SEQ ID NO.32, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 33;
a twelfth SNP primer set used for amplifying the twelfth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.34 and SEQ ID NO.35, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 36;
a thirteenth SNP primer set for amplifying the thirteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.37 and SEQ ID NO.38, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 39;
a fourteenth SNP primer set used for amplifying the fourteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.40 and SEQ ID NO.41, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 42;
a fifteenth SNP primer set used for amplifying the fifteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.43 and SEQ ID NO.44, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 45;
a sixteenth SNP primer set used for amplifying the sixteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.46 and SEQ ID NO.47 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 48;
a seventeenth SNP primer set used for amplifying the seventeenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.49 and SEQ ID NO.50 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 51;
the eighteenth SNP primer set is used for amplifying the eighteenth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.52 and SEQ ID NO.53 and a universal primer nucleotide sequence shown as SEQ ID NO. 54;
a nineteenth SNP primer set used for amplifying the nineteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.55 and SEQ ID NO.56, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 57;
a twentieth SNP primer set for amplifying the twentieth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID No.58 and SEQ ID No.59, and further comprises a general primer nucleotide sequence shown as SEQ ID No. 60;
the twenty-first SNP primer group is used for amplifying the twenty-first SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.61 and SEQ ID NO.62, and also comprises a universal primer nucleotide sequence shown as SEQ ID NO. 63.
In a third aspect of the present invention, a kit is provided, which comprises the above primer set.
In a fourth aspect of the present invention, a gene chip is provided, which comprises the above primer set.
In the fifth aspect of the present invention, any one of the following applications of the above SNP molecular marker combination, primer set, kit and/or gene chip is provided:
(1) the application in identification or auxiliary identification of the purity of wheat varieties;
(2) the application in wheat molecular marker assisted breeding;
(3) the application in wheat breeding;
(4) application in the preparation of wheat breeding products.
In a sixth aspect of the present invention, a method for detecting the purity of wheat varieties by using the above-mentioned SNP molecular marker combination is provided, which comprises the following steps:
s1, extracting genome DNA from the wheat sample to be detected;
s2, carrying out polymorphism detection on the SNP molecular marker combination in the genomic DNA extracted in the step S1 to obtain the genotype of the wheat sample to be detected;
s3, statistically analyzing the genotype of the wheat sample individuals to be detected obtained in the step S2, identifying the hybrid plants, and calculating the purity of the varieties according to the number of the hybrid plant individuals and the total detection number.
In some embodiments of the invention, the formula for variety purity is: percent of degree of purity = (total number of tests-number of hybrid plants)/total number of tests × 100%.
In some embodiments of the invention, the number of wheat samples to be tested is greater than 95.
In some embodiments of the present invention, in step S1, the simplified CTAB method (cetyl trimethyl ammonium bromide method) is used to extract genomic DNA from wheat.
In some embodiments of the invention, in step S2, the SNP molecular marker combination is detected using KASP (competitive allele specific PCR) technology.
In some embodiments of the present invention, in step S3, if the genotype of a certain wheat sample to be tested is different from that of other most individuals to be tested at 2 or more SNP sites, the wheat sample to be tested is determined to be a hybrid.
A wheat breeding method comprises the following steps: the method for detecting the purity of the wheat variety is used for selecting the wheat for subsequent breeding.
The molecular marker combination for detecting wheat SNP has at least the following beneficial effects: the method can be used for accurately detecting the purity of wheat varieties (lines) from different sources by screening a set of (1 SNP/chromosome) SNP markers with high quality and high polymorphism, and has wide application universality; the marker is a co-dominant marker, and has high specificity, sensitivity and resolution; the marker is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; different detection laboratories and different data results can be compared and verified with each other, and the data has universal comparability; the invention carries out genotyping on the developed SNP marker by utilizing the KASP technology based on the Douglas Array Tape platform, has simple technology, easy automation, high speed and high automation degree, and greatly reduces the manpower and human errors in a laboratory; the unit data point detection cost is low, the detection flux is high, and the breeding process of the wheat variety is accelerated.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a flow chart of site development according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the genotyping of the SNP molecular marker WH90016 according to the embodiment of the invention;
FIG. 3 is a schematic diagram of the genotyping of the SNP molecular marker WH90021 according to the embodiment of the invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
The embodiment of the invention is as follows: SNP molecular marker combination for wheat variety purity detection
The site design process of the embodiment of the present invention, as shown in fig. 1, collects wheat SNP site information by using a 660K chip of wheat and wheat-related documents, performs site comparison and screening in a bristol wheat genome marker library to obtain 120 SNP sites for wheat variety purity detection, extracts SNP sites and flanking sequences, and obtains 21 SNP molecular markers according to the present application scheme by designing and synthesizing primer sequences of the markers, and then verifying and detecting the markers, specifically as follows:
screening of 121 SNP molecular markers for wheat variety purity detection
By utilizing a 660K chip of wheat and wheat-related documents, SNP loci related to a region where functional genes of the wheat are located are screened, 4 ten thousand functionally-related loci are collected and subjected to locus comparison in a Bristol wheat genome marker library to obtain 2000 wheat verified KASP markers with completely matched loci, and more than 4 ten thousand SNP loci are obtained. Further carrying out sequence comparison on 2000 obtained KASP markers to a reference genome (Triticum aestivum IWGSC RefSeq v 1.0) of wheat to obtain 120 marker sites with the best specificity, designing KASP primers according to the 120 SNP sites, selecting 144 parts of various wheat resources for carrying out genotyping verification, selecting a set of SNP markers with the least markers and capable of distinguishing marker combinations of all materials, and finally selecting 21 SNP markers for detecting the purity of wheat varieties, wherein the 21 SNP markers are high-quality, single-copy and high-polymorphism (PIC values in 144 parts of wheat materials are all higher than > =0.35) and the data detection rate is more than 98%. The basic information of the 21 SNP molecular markers is shown in Table 1.
TABLE 1 physical location of SNP molecular markers for wheat variety purity detection
2 primer design
Design of KASP marker: KASP primer design was performed on the 21 selected SNP molecular markers using BatchPrimer3 (http:// probes. pw. usda. gov/BatchPrimer3 /). Each KASP marker consists of three primers, including two allele-specific primers X (Primer _ X; specific Primer X is named as Primer name-X) and Y (Primer Y; specific Primer Y is named as Primer name-Y) and one universal Primer C (Primer _ C; universal Primer C is named as Primer name-C). The two allele-specific primers are respectively connected with FAM and HEX fluorophore tag sequences. If only FAM fluorescence is detected in the sample, the genotype of the sample is homozygous Allele X (AlleX); if only HEX fluorescence is detected, the genotype of the sample is homozygous Allele Y (Alley _ Y); if FAM and HEX fluorescence is detected simultaneously, the genotype of the sample is heterozygous (with both alleles X and Y). The 21 KASP marker alleles and primer sequences for wheat variety purity testing are shown in table 2.
TABLE 2 primer sequence Listing
3 KASP detection
DNA extraction: extracting genome DNA from wheat and adopting simplified CTAB method.
KASP tag detection procedure: the validation and detection of KASP markers was performed using the Array Tape system of Douglas Scientific. The Array Tape genotyping platform comprises NEXAR for PCR amplification system assembly, sollex for PCR amplification, ARAYA for fluorescent signal scanning, and INTELLICS for data analysis.
And (3) PCR reaction system: automatic assembly of the PCR reaction system was performed using NEXAR, and the PCR reaction system is shown in Table 3 below.
And (3) PCR amplification: PCR amplification was performed using SOELLEX under the following conditions: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extending for 60 seconds for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 30 cycles.
Signal scanning and genotyping: after the PCR reaction is finished, carrying out fluorescent signal scanning on the reaction system by using ARAYA; genotyping and data analysis were then performed with INTELLICS. In the KASP marker genotyping assay, the genotype of the sample is divided into 3 clusters, X, Y and heterozygous genotype clusters (see fig. 2). Wherein X cluster indicates that the sample contains a homozygous X allele at the KASP marker locus (red in the typing map, at the top left of the graph), Y cluster indicates that the sample contains a homozygous Y allele at the KASP marker locus (blue in the typing map, at the bottom right of the graph), and heterozygous genotype cluster indicates that the sample contains X and Y heterozygous alleles at the KASP marker locus (purple in the typing map). Typical KASP marker genotyping profiles are shown in FIGS. 2-3.
TABLE 3 PCR reaction System for KASP detection
421 SNP molecular marker verifications for wheat variety purity detection
Genotyping quality verification of 21 KASP markers for wheat variety purity detection: the 21 KASP markers were verified with 144 triticale diversity material according to the detection method described above.
The results are shown in FIGS. 2-3, FIG. 2 is a WH90016 KASP marker genotyping map, FIG. 3 is a WH90021 KASP marker genotyping map, and it can be seen from the map that the two homozygous and heterozygous clusters of KASP markers are well-typed and compact, the sites are single-copy and the detection rate is higher than 98%, consistent with the sequencing results, and the results of other sites are consistent with WH90016 and WH 90021. The result shows that the genotyping quality of the 21 KASP markers in the scheme of the application can completely meet the accurate detection of the purity of wheat varieties.
5 calculation of wheat variety purity
The calculation of the purity of the wheat variety comprises the following steps:
(1) and (3) carrying out statistical analysis on the genotypes of the 21 SNP molecular markers of 184 wheat (Chinese spring) samples to be detected. And if 2 or more sites in a certain individual to be detected are different from those of other individuals to be detected, judging the individual wheat to be detected to be a hybrid plant. As a result, as shown in Table 4 (Table 4 includes tables 4-1 and 4-2), 177 individuals had the same genotype at 21 SNP sites (SNP molecular markers) and were pure strains; the individual with the sample number of 12 is different from other 183 individuals in WH90005 and WH 9000212 SNP sites and is judged to be a hybrid strain; the individual with the sample number of 24 is different from other 183 individuals in WH90005 and WH 9000212 SNP sites and is judged to be a hybrid strain; the individual with the sample number of 33 was judged to be a hybrid strain, unlike the other 183 individuals at 3 SNP sites such as WH90009 and WH 900013; the individual with the sample number of 44 is judged to be a hybrid strain, unlike the other 183 individuals at 3 SNP sites such as WH90009 and WH 900013; the individual with the sample number of 75 is different from other 183 individuals in WH90005 and WH 9000212 SNP sites and is judged to be a hybrid strain; the individual with the sample number of 138 is different from other 183 individuals in WH90005 and WH 9000212 SNP sites and is judged to be a hybrid strain; the individual with sample number 173 was judged as a heterostrain, since it was different from the other 183 individuals in 4 SNP sites such as WH90009 and WH 900013.
(2) And (4) calculating the purity of the variety according to the number of the individual hybrid strains and the total detection number in the step (1).
The degree of purity was calculated as: percent of degree of purity = (total number of tests-number of hybrid plants)/total number of tests × 100%.
The variety purity of the wheat variety to be detected in China spring is as follows: (184-7)/184 × 100% = 96.20%.
TABLE 4-1
TABLE 4-2
In conclusion, the invention provides 21 SNP molecular marker combinations, which can be used for the accurate detection of the purity of wheat varieties and genetic materials with different sources, have wide application universality, and can trace and conjecture the pollution source of impure samples; the kit has a co-dominant marker and high specificity, sensitivity and resolution; the marker is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; the technology is simple, the automation is easy, the detection flux is high, and the speed is high. The detection cost of unit data points is low; the data results of different detection laboratories can be compared and verified with each other, and the data has universal comparability.
The Douglas Array Tap genotyping platform used in the present invention comprises NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning and INTELLICS for data analysis, and the consumables for its kit are all purchased from LGC company, England.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
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Claims (10)
1. An SNP molecular marker combination for detecting the purity of wheat varieties is characterized by consisting of the following SNP molecular markers:
a first SNP molecular marker, wherein the first SNP molecular marker is located at 534265581 th nucleotide of 1A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is G or A;
a second SNP molecular marker, wherein the second SNP molecular marker is positioned at 643102010 th nucleotide of 1B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is A or C;
a third SNP molecular marker, wherein the third SNP molecular marker is positioned at 3958512 th nucleotide of 1D chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a fourth SNP molecular marker, wherein the fourth SNP molecular marker is positioned at 16865489 th nucleotide of 2A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is C or T;
a fifth SNP molecular marker, wherein the fifth SNP molecular marker is positioned at 12076633 th nucleotide of 2B chromosome of wheat reference genome or at a corresponding site on homologous genome fragment among varieties thereof, and the nucleotide base of the site is T or G;
a sixth SNP molecular marker, which is located at 69251621 th nucleotide of 2D chromosome of wheat reference genome or corresponding site on homologous genome fragment between varieties thereof, and the nucleotide base of the site is A or G;
a seventh SNP molecular marker, wherein the seventh SNP molecular marker is located at 61219689 th nucleotide of 3A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is A or C;
an eighth SNP molecular marker, wherein the eighth SNP molecular marker is located at 749024987 th nucleotide of 3B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a ninth SNP molecular marker, wherein the ninth SNP molecular marker is located at 578414376 th nucleotide of the 3D chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
a tenth SNP molecular marker, wherein the tenth SNP molecular marker is located at 715512695 th nucleotide of the 4A chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
an eleventh SNP molecular marker, wherein the eleventh SNP molecular marker is located at 159349087 th nucleotide of the 4B chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a twelfth SNP molecular marker, wherein the twelfth SNP molecular marker is located at 456087457 th nucleotide of the 4D chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is C or T;
a thirteenth SNP molecular marker, wherein the thirteenth SNP molecular marker is located at 533072137 th nucleotide of 5A chromosome of wheat reference genome or at a corresponding site on homologous genome fragment between varieties thereof, and the nucleotide base of the site is G or A;
a fourteenth SNP molecular marker, wherein the fourteenth SNP molecular marker is located at 601419406 th nucleotide of 5B chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is T or C;
a fifteenth SNP molecular marker, wherein the fifteenth SNP molecular marker is located at 562098034 th nucleotide of 5D chromosome of wheat reference genome or at a corresponding site on homologous genome fragments among varieties thereof, and the nucleotide base of the site is A or G;
a sixteenth SNP molecular marker, wherein the sixteenth SNP molecular marker is located at 595564056 th nucleotide of the 6A chromosome of the wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is T or C;
a seventeenth SNP molecular marker, wherein the seventeenth SNP molecular marker is positioned at 704974282 th nucleotide of the 6B chromosome of a wheat reference genome or at a corresponding site on an homologous genome fragment between varieties thereof, and the nucleotide base of the site is C or T;
an eighteenth SNP molecular marker, which is located at 454587758 th nucleotide of 6D chromosome of wheat reference genome or corresponding site on homologous genome fragment between varieties thereof, and nucleotide base of the site is C or T;
a nineteenth SNP molecular marker, wherein the nineteenth SNP molecular marker is located at 54723172 th nucleotide of 7A chromosome of a wheat reference genome or at a corresponding site on homologous genome fragments among varieties of the wheat reference genome, and the nucleotide base of the site is G or A;
a twentieth SNP molecular marker, which is located at 706809043 th nucleotide of 7B chromosome of wheat reference genome or at a corresponding site on homologous genome fragment among varieties thereof, wherein the nucleotide base of the site is C or T;
a twenty-first SNP molecular marker, wherein the twenty-first SNP molecular marker is located at 629330949 th nucleotide of 7D chromosome of wheat reference genome or corresponding site on homologous genome fragment among varieties thereof, and the nucleotide base of the site is G or A;
wherein the wheat reference genome is a Triticum aestivum IWGSC RefSeq v1.0 version reference genome.
2. A primer group for amplifying the SNP molecular marker combination for detecting the purity of the wheat variety as claimed in claim 1.
3. The primer set of claim 2, wherein the primer set comprises two specific primers and 1 universal primer, wherein the two specific primers are connected with different fluorophore tag sequences.
4. The primer set according to claim 2, wherein the primer set comprises: the first SNP primer group is used for amplifying the first SNP molecular marker, and comprises specific primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a universal primer with nucleotide sequence shown as SEQ ID NO. 3;
the second SNP primer group is used for amplifying the second SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 6;
a third SNP primer set used for amplifying the third SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.7 and SEQ ID NO.8 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 9;
the fourth SNP primer group is used for amplifying the fourth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.10 and SEQ ID NO.11, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 12;
a fifth SNP primer set used for amplifying the fifth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.13 and SEQ ID NO.14 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 15;
a sixth SNP primer set, which is used for amplifying the sixth SNP molecular marker and comprises specific primer nucleotide sequences shown as SEQ ID NO.16 and SEQ ID NO.17 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 18;
a seventh SNP primer set for amplifying the seventh SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.19 and SEQ ID NO.20, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 21;
the eighth SNP primer group is used for amplifying the eighth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.22 and SEQ ID NO.23 and a universal primer nucleotide sequence shown as SEQ ID NO. 24;
a ninth SNP primer set for amplifying the ninth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.25 and SEQ ID NO.26, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 27;
a tenth SNP primer set for amplifying the tenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.28 and SEQ ID NO.29, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 30;
an eleventh SNP primer set for amplifying the eleventh SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.31 and SEQ ID NO.32, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 33;
a twelfth SNP primer set used for amplifying the twelfth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.34 and SEQ ID NO.35, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 36;
a thirteenth SNP primer set for amplifying the thirteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.37 and SEQ ID NO.38, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 39;
a fourteenth SNP primer set used for amplifying the fourteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.40 and SEQ ID NO.41, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 42;
a fifteenth SNP primer set used for amplifying the fifteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.43 and SEQ ID NO.44, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 45;
a sixteenth SNP primer set used for amplifying the sixteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.46 and SEQ ID NO.47 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 48;
a seventeenth SNP primer set used for amplifying the seventeenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.49 and SEQ ID NO.50 and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 51;
the eighteenth SNP primer set is used for amplifying the eighteenth SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.52 and SEQ ID NO.53 and a universal primer nucleotide sequence shown as SEQ ID NO. 54;
a nineteenth SNP primer set used for amplifying the nineteenth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID NO.55 and SEQ ID NO.56, and also comprises a general primer nucleotide sequence shown as SEQ ID NO. 57;
a twentieth SNP primer set for amplifying the twentieth SNP molecular marker, which comprises specific primer nucleotide sequences shown as SEQ ID No.58 and SEQ ID No.59, and further comprises a general primer nucleotide sequence shown as SEQ ID No. 60;
the twenty-first SNP primer group is used for amplifying the twenty-first SNP molecular marker, and comprises specific primer nucleotide sequences shown as SEQ ID NO.61 and SEQ ID NO.62, and also comprises a universal primer nucleotide sequence shown as SEQ ID NO. 63.
5. A kit comprising the primer set according to any one of claims 2 to 4.
6. A gene chip comprising the primer set according to any one of claims 2 to 4.
7. The SNP molecular marker set according to claim 1, the primer set according to any one of claims 2 to 4, the kit according to claim 5 or the gene chip according to claim 6, wherein any one of the following applications is selected from the group consisting of:
(1) the application in identification or auxiliary identification of the purity of wheat varieties;
(2) the application in wheat molecular marker assisted breeding;
(3) the application in wheat breeding;
(4) application in the preparation of wheat breeding products.
8. The method for detecting the purity of wheat varieties by using the SNP molecular marker combination according to claim 1, which comprises the following steps:
s1, extracting genome DNA from the wheat sample to be detected;
s2, carrying out polymorphism detection on the SNP molecular marker combination in the genomic DNA extracted in the step S1 to obtain the genotype of the wheat sample to be detected;
s3, statistically analyzing the genotype of the wheat sample individuals to be detected obtained in the step S2, identifying the hybrid plants, and calculating the purity of the varieties according to the number of the hybrid plant individuals and the total detection number.
9. The method according to claim 8, wherein if the genotype of a wheat sample to be tested is different from that of the other most individuals to be tested at 2 or more SNP sites, the wheat sample to be tested is determined to be a hybrid.
10. A wheat breeding method comprises the following steps: selecting wheat for subsequent breeding by using the method for detecting the purity of the wheat variety as claimed in claim 8 or 9.
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