CN108486265B - Method for identifying type of male sterile cytoplasm of corn based on KASP technology - Google Patents
Method for identifying type of male sterile cytoplasm of corn based on KASP technology Download PDFInfo
- Publication number
- CN108486265B CN108486265B CN201810118136.0A CN201810118136A CN108486265B CN 108486265 B CN108486265 B CN 108486265B CN 201810118136 A CN201810118136 A CN 201810118136A CN 108486265 B CN108486265 B CN 108486265B
- Authority
- CN
- China
- Prior art keywords
- artificial sequence
- dna
- primer
- corn
- kasp
- 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
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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- 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
- C12Q2600/13—Plant traits
-
- 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
Abstract
The invention provides a method for identifying the type of male sterile cytoplasm of corn based on KASP technology. Chloroplast genome corn male sterility S-type cytoplasm identification specific primer 19 pairs, including 17 SNP and 2 InDel site primers; c-type cytoplasm identification specific primer 10 pairs, including 9 SNPs and 1 primer of InDel locus; t-type cytoplasm identification specific primer 7 pairs are SNP primers. The method can be used for identifying germplasm materials of the male sterile cytoplasm and the normal cytoplasm of the corn, identifying hybrid seeds of sterile seed production and conventional seed production, identifying the purity of the sterile material and the like. The application of the method provides technical support for the maize cytoplasmic sterile seed production and sterile material breeding, and provides powerful guarantee for the production of three-line matched seeds of maize; the range of available marker loci of the corn is expanded on the genome level, and a new thought and means are provided for the research of cytoplasmic hereditary property and the like of the corn.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to a method for identifying a corn male sterility cytoplasm type based on a KASP technology.
Background
Corn is the crop which can realize the utilization of heterosis at the earliest time, and the breeding and utilization of hybrid seeds are praised as a revolution in crop production. The corn hybrid seed production method has two methods of conventional method and male sterility, and the male sterility is used for preparing the corn hybrid seed, so that not only is a large amount of labor saved and the cost reduced, but also the purity of the hybrid seed can be improved and the yield can be improved.
Maize Male Sterility (MS) refers to the phenomenon that maize stamens develop abnormally to produce non-functional pollen, while pistils develop normally to pollinate and fruit. Maize male sterility can be broadly divided into Cytoplasmic Male Sterility (CMS) and nuclear male sterility (GMS). Cytoplasmic Male Sterility (CMS) is a nuclear-cytoplasmic interactive male sterile line commonly controlled by nuclear and cytoplasmic genes, easily realizes the matching of a sterile line, a maintainer line and a restorer line, and is a main male sterile type used in corn breeding and fine variety production. To date, more than 200 CMS materials from different cytoplasm sources have been discovered, and the CMS materials are divided into three types, namely S type, C type and T type according to the special effect reaction of male flower fertility restoration, and the male flower fertility reaction and cytoplasm effect of different types of materials are different.
The method for producing the seeds by adopting the CMS has three problems to be solved, namely identifying the type of the cytoplasm of the male sterile line, identifying the purity of the male sterile material, and identifying the hybrid prepared by the sterile line, so that the establishment of the high-throughput method for identifying the type of the cytoplasm of the CMS of the maize has very important significance. Chloroplast genome information is widely applied to research and application of plant variety, germplasm resource identification, genetic relationship evaluation, system evolution, cytoplasm genetic characteristics and the like due to the following advantages. (1) The chloroplast genome is small and relatively conserved, and the complete sequence is easily obtained; (2) chloroplast genes are maternal inheritance, gene exchange and fusion among different individuals rarely occur, and the genes of chloroplast have good colinearity; (3) the chloroplast genome is single copy genes except for the inverted repeat region, and the paralogous gene interference hardly exists; (4) the evolution speed difference of chloroplast coding regions and non-coding regions is obvious, and some high mutation regions exist, so that the problem of the following classification units can be solved. As CMS is closely related to cytoplasm, the identification of the sterile cytoplasm type by adopting the marker locus on the chloroplast genome is a stable, reliable and innovative method. Development of chloroplast SNP or InDel marker loci suitable for CMS material identification, simultaneous use of multiple locus combinations, can enhance identification reliability and reduce uncertainty. The development of marker loci and methods for CMS type identification in maize based on the chloroplast genome has not been reported.
KASP (Kompetitive Allele Specific PCR) is a competitive Allele Specific PCR technical system, has the characteristics of simple test flow, high efficiency, flexibility, easy operation and the like, and is suitable for genotyping detection of SNP/InDel and other two-state marker types. The matched instrument platform is compatible with 96, 384 and 1536 three-format enzyme label plates, and is suitable for the detection of various sample fluxes, wherein the 1536-hole format not only embodies the advantage of high flux, but also reduces the cost.
The method for identifying the male sterility of the corn is reported to have the methods of field fertility observation, cytological difference comparison, fertility restoration specificity identification, marker locus PCR amplification and the like. The first three methods have long identification period and limited range of identification materials; the PCR-based method does not report an identification method for developing an InDel locus aiming at a chloroplast genome at present, is a detection method based on electrophoresis platforms such as agarose and polyacrylamide, and cannot realize high-throughput and automatic identification.
Disclosure of Invention
The invention aims to provide a method for identifying the type of maize male sterile cytoplasm based on KASP technology.
The invention concept of the invention is as follows: (1) and selecting 170 parts of representative test materials of the corns, wherein the types of the representative test materials comprise all heterosis groups in China, sweet and glutinous types, local varieties, CMS sterile types and the like. (2) High concentration, high quality total DNA preparation. (3) Based on the high-throughput sequencing of a second-generation sequencing platform, the size of a constructed library is 500bp, PE140 is obtained, and the sequencing depth is 5 times. (4) Whole genome sequence data processing, chloroplast genome splicing, independent splicing by using two software, screening contigs belonging to chloroplast genome by using BLAST program based on a maize B73 chloroplast genome sequence, assembling and verifying sequence accuracy. (5) And (3) chloroplast genome annotation, polymorphic site determination and annotation of the chloroplast genome by using DOGMA software. (6)170 parts of materials are compared with each other, and 100 SNP/InDel chloroplast genome variation sites are screened out. (7) And (3) determining the identification specific sites of the maize S, C, T type sterile cytoplasm, and analyzing Fst values (genetic differentiation coefficients) among different groups, wherein the Fst values are group specific sites if being more than 0.9. 20S-type specific sites (17 SNP sites and 3 InDel sites), 11C-type specific sites (9 SNP sites and 2 InDel sites) and 7T-type specific sites are obtained by analyzing the S, C, T-type sterile material of the corn respectively. (8) Primer design, using 170 sequencing data for site polymorphism assessment, wherein the 72 sites with MAF (minor axle frequency) value greater than 0.01 are primer design based on KASP platform. (9) Designing, evaluating and verifying primers based on a KASP platform. 65 samples were selected and the designed primers were verified based on the evaluation of the KASP technology system. 65 samples comprise S, C, T three sterile germplasm materials, different heterosis groups and 3 sets of triplet materials (sterile female parent, fertile male parent and F1 hybrid). The material mainly verifies the amplification effect of the primer, verifies whether the marked site strictly follows the maternal inheritance, verifies the consistency with a sequencing result, and verifies whether the marked site is a specific site of a sterile material type. The validation results showed that only the S-sterile 1 pair of InDel site primers and the C-sterile 1 pair of InDel site primers did not meet the above requirements. Finally obtaining 19 pairs of specific primer combinations for identifying the maize male sterility S-type cytoplasm, wherein the specific primer combinations comprise 17 SNP primers and 2 InDel site primers; c-type cytoplasm identification specific primer combination 10 pairs, including primers of 9 SNPs and 1 InDel site; t-type cytoplasm identification specific primer combination 7 pairs of SNP primers. The locus information for identifying the type of maize S, C, T cytoplasmic sterility is shown in tables 1-3, and the primer information is shown in tables 4-6. The technical scheme of the invention is shown in figure 1 for identifying the specific primers of the maize male sterile cytoplasm type based on the KASP technical system.
In order to achieve the aim, the chloroplast genome maize male sterility S-type cytoplasm type specific primer combination 19 pairs suitable for KASP technology provided by the invention comprise 17 SNPs and 2 primers of InDel sites (Table 4); type C cytosolic type specific primer set 10 pairs, including 9 SNPs and 1 primer at InDel site (table 5); the T-type cytoplasmic type specific primer set 7 pairs were SNP site primers (Table 6). The physical location of the molecular marker is determined based on the chloroplast genome sequence of maize variety B73, the version number of the chloroplast genome of maize variety B73 being AGPv 3.
The invention discloses a molecular marker for identifying maize S-type cytoplasmic sterile material, which is developed based on chloroplast genome, wherein the molecular marker is selected from at least one of the following 17 SNP markers and 2 InDel markers, the site information of the molecular marker is shown in Table 1, and the primer information is shown in Table 4.
TABLE 1
Wherein the nucleotide sequences of the InDel markers CPMIDP01 and CPMIDP02 are shown as SEQ ID NO:39-40 respectively.
The KASP primer sequences for detecting the markers CPMSNP 08-CPMIDP 02 are respectively shown as SEQ ID NO:41-98, wherein the KASP primer for detecting the marker CPMSNP08 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer, the sequences are SEQ ID NO:41-43, the KASP primer for detecting the marker CPMSNP09 is SEQ ID NO:44-46, and the like, the KASP primer for detecting the marker CPMIDP01 comprises an upstream primer 1, an upstream primer 2, a downstream primer 1 and a downstream primer 2, the sequences are SEQ ID NO:92-95, the KASP primer for detecting the marker CPMIDP02 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer, and the sequences are SEQ ID NO: 96-98.
The invention discloses a molecular marker for identifying corn C-type cytoplasmic sterile material, which is developed based on chloroplast genome, wherein the molecular marker is selected from at least one of the following 9 SNP markers and 1 InDel marker, the site information of the molecular marker is shown in Table 2, and the primer information is shown in Table 5.
TABLE 2
The KASP primer sequences for detecting the markers CPMSNP 18-CPMIDP 10 are respectively shown as SEQ ID NO. 119-148, wherein the KASP primer for detecting the marker CPMSNP18 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer with the sequences of SEQ ID NO. 119-121, the KASP primer for detecting the marker CPMSNP38 is SEQ ID NO. 122-124, and the like.
The invention discloses a molecular marker for identifying T-type cytoplasmic sterile materials of corn, which is developed based on a chloroplast genome, wherein the molecular marker is selected from at least one of the following 7 SNP markers, the site information of the molecular marker is shown in Table 3, and the primer information is shown in Table 6.
TABLE 3
The KASP primer sequences for detecting the markers CPMSNP 03-CPMSNP 86 are respectively shown as SEQ ID NO. 163-183, wherein the KASP primer for detecting the marker CPMSNP03 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer with the sequence of SEQ ID NO. 163-165, the KASP primer for detecting the marker CPMSNP04 is SEQ ID NO. 166-168, and the like.
The 36 pairs of SNP/InDel primers provided by the invention can realize the acquisition of genotyping data based on a KASP platform. The specific scheme is that according to KASP technical requirements, a primer is designed aiming at the locus provided by the invention, and the primer is a common primer and does not contain a fluorescent group; purchasing a PCR amplification system MasterMix matched with the KASP technology; preparing a reaction system and adding DNA, a primer and MasterMix; operating a reaction program; scanning the fluorescence signal in situ; and (4) analyzing the data to obtain genotype data.
The invention also provides a method for identifying the inbred line and the hybrid of the maize male sterile cytoplasm type. The method specifically comprises the following steps: (1) extracting the total DNA of the detected inbred line or hybrid material; (2) the SNP/InDel primer provided by the invention is used for carrying out PCR amplification and fluorescence scanning based on a KASP genotyping platform to obtain genotype data, and whether the primer is a sterile material or a sterile type is judged.
The invention also provides a method for identifying the type of the male sterile cytoplasm of the corn based on the KASP technology, which comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) KASP reaction: adding KASP Primer mix and KASP ROX standard reaction mix into the DNA template extracted in the step 1) for PCR amplification;
3) the PCR product was analyzed using a fluorescence detector.
The primers are one or more pairs of primers selected from tables 4 to 6, and the KASP Primer mix is a mixture formed by the 5' end of the upstream Primer of each KASP Primer and the downstream universal Primer (downstream Primer) after different fluorescent label sequences are respectively modified.
Preferably, the fluorescent tag sequence is: 5 '-FAM-GAAGGTGACCAAGTTCATGCT-3';
5’-HEX-GAAGGTCGGAGTCAACGGATT-3’。
preferably, the PCR reaction system in step 2) is: DNA template 1.5. mu.L, KASP ROX standard reaction mix (Kbiosciences, Herts UK) 0.5. mu.L, KASP Primer mix 0.014. mu.L, ddH2O0.5 mu L; the concentration of each Primer in the KASP Primer mix was 100. mu.M.
Preferably, the PCR reaction procedure is: 15min at 94 ℃; adopting a falling PCR mode, wherein the temperature is 94 ℃ for 20s, the temperature is 61-55 ℃ for 1min (the temperature is reduced by 0.6 ℃ per cycle), and the temperature is 10 cycles; 30 cycles of 94 ℃ for 20s and 58 ℃ for 1 min.
Preferably, the amplification product is scanned for fluorescence signals using a BMG Pherastar (LGC, Middlesex, UK) instrument to obtain raw data. The raw data was imported into Kraken software (LGC, Middlesex, UK) for analysis to obtain fingerprint data for each data point of each sample. According to the fingerprint data of each locus, each locus respectively listed in tables 4-6 shows an allele in S, C, T sterile material, if the allele is the same as the allele in the tables, the corresponding cytoplasmic sterility type is determined, and if the allele is the other allele, the cytoplasmic sterility type is not determined.
The invention also provides a detection reagent or a kit containing the KASP primer.
The invention also provides application of the KASP primer, or a detection reagent or a kit containing the primer in identifying the type of the male sterile cytoplasm of the corn or breeding sterile materials.
The invention further provides application of the molecular marker or the KASP primer, or a detection reagent or a kit containing the primer in corn variety detection and corn molecular marker assisted breeding.
The invention is characterized in that: (1) chloroplast genome data were isolated from whole genome data: since the chloroplast genome sequence is relatively conserved and the sequencing quality and length are sufficient, in the present invention chloroplast genome data is obtained by a splicing protocol, in combination with alignment with the maize chloroplast reference genome. The method mainly comprises the steps of screening contigs of chloroplast genomes by using a Blast program, assembling the contigs of the chloroplast genomes by using Sequencher software, and comparing and verifying the assembled sequences with maize chloroplast reference genomes (maize variety B73, AGPv3) to confirm so as to provide guarantee for obtaining accurate and reliable chloroplast genome sequences. (2) Digging of chloroplast mutation sites: the accuracy and the high efficiency of obtaining the chloroplast polymorphic sites are ensured through representative sample selection, high-quality sequencing data and accurate data analysis. 170 parts of materials with wide sources and abundant phenotypes and genotypes are selected, based on 170 spliced chloroplast genome sequences, DnaSP 5.0 is utilized to count variation sites and sequence polymorphisms, and finally chloroplast polymorphic sites are determined, wherein different genetic background materials and high-quality gene sequences are important factors for obtaining accurate and reliable chloroplast polymorphic sites. (3) Determining a corn sterile cytoplasm type identification primer based on KASP technology: specific loci of three sterile types are screened by analyzing the variation loci of chloroplast genomes of all types of maize inbred lines (comprising T, C, S three types of CMS materials). Designing a primer combination based on the KASP technology, wherein the primer screening standard is as follows: whether amplification is successful or not, whether maternal inheritance is followed or not, the accuracy of genotype data and the like are respectively evaluated and verified by selecting a targeted material, so that the designed primer is guaranteed to have the characteristics of reliability, accuracy and effectiveness.
The invention collects 170 parts of maize inbred line materials (including three CMS type materials) with wide sources, rich phenotypes and genotypes and strong representativeness to obtain a chloroplast genome sequence, compares nucleotide polymorphisms, develops and identifies specific SNP/InDel loci of maize male sterility S, C and T-type cytoplasm types, designs and evaluates primers based on a KASP technical platform, and establishes a high-throughput and automatic maize male sterility type identification method. The method can be used for identifying the purity of the sterile cytoplasm inbred line, hybrid and sterile materials, and the like, provides technical support for maize cytoplasm sterile seed production and material breeding, and provides powerful guarantee for production of maize three-line matched seeds. The range of available marker loci of the corn is expanded on the genome level, and a new thought and technical means are provided for the research of cytoplasmic hereditary property and the like of the corn.
Drawings
FIG. 1 is a technical scheme diagram obtained by identifying specific primers for maize male sterile cytoplasm types based on KASP technical system.
FIG. 2 is a diagram showing the test results of the cytoplasmic type specific identification primer for maize male sterility S, C, T of the present invention. A is a result diagram of 19 pairs of specific identification primers for the S-type cytoplasm type, B is a result diagram of 10 pairs of specific identification primers for the C-type cytoplasm type, and C is a result diagram of 7 pairs of specific identification primers for the T-type cytoplasm type.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
EXAMPLE 1 acquisition of specific primers suitable for identifying the type of cytoplasmic Male sterile of maize
1. Selecting a sample: 170 parts of widely representative maize inbred lines were selected for whole genome sequencing. The 170 samples comprise corn types such as common corn, waxy corn, sweet corn, pop corn and the like; comprises all heterosis groups in China, four heads of Tang, red bone, Reid, Lanka, improved Reid, improved Lanka, P group and local variety, and T, C, S cytoplasm sterility type materials.
2. Sample preparation: seeds of 170 corn samples were allowed to germinate in the incubator for 5 days, with no light conditions for the first 3 days and light conditions for the next 2 days (i.e., sufficient light was given after emergence). 30 green shoot leaves were selected from each sample and mixed, and ground thoroughly under liquid nitrogen. Total DNA was extracted by CTAB method and RNA was removed. Respectively detecting the quality of the extracted DNA by using an ultraviolet spectrophotometer and agarose electrophoresis, wherein the agarose electrophoresis shows that the DNA has single band and is not degraded; detecting A260/280 by an ultraviolet spectrophotometer to be between 1.8 and 2.0 (DNA has no protein pollution and low RNA content); a260/230 is between 1.8 and 2.0 (the content of DNA salt ions is low); the DNA concentration is greater than 1000 ng/. mu.L.
3. High throughput sequencing of total DNA: breaking 170 parts of corn sample DNA and PCR products by using ultrasound, cutting gel and recovering 400-plus 600bp DNA fragments
The library construction kit constructs a library with the size of 500bp, a sequencing platform of Hiseq 4000PE150 is used for sequencing, the sequencing depth is 5 times, and about 10GB data are obtained on average for each sample.
4. High-throughput sequencing data processing, chloroplast genome splicing: high throughput sequencing data were stitched independently using two software, SPAdes (Bankevich et al, 2012) and soaldenovo 2(Luo et al, 2012), respectively. For each software spliced contig, screening out contigs of the chloroplast genome using Blast program (Altschul et al, 1997); the selected chloroplast genome contigs were assembled using Sequencher. All reads maps were then applied to the spliced chloroplast genome sequence using geneous 8.1(Kearse et al, 2012) to verify that the spliced contig sequence was correct.
5. Chloroplast genome annotation, polymorphic site determination: chloroplast genome annotation was performed using dodma (dual organic genomic Geno Me antator) (Wyman et al, 2004), and BLASTX and BLASTN searches were used to identify the location of the encoding gene. 170 maize chloroplast genomes were aligned using MAFFT software (Katoh and Standard, 2013) and then manually adjusted using Se-al software. The principle of the inverted alignment occurring within the sequence is to pull it apart so as not to cause erroneous data polymorphisms. The variation sites and sequence polymorphisms in two chloroplast genomes are counted by utilizing DnaSP 5.0 (Librado and Rozas,2009), and 100 SNP/InDel polymorphic sites are developed and obtained.
6. Determination of the site suitable for identifying the cytoplasmic type of maize sterility: based on 100 SNP/InDel polymorphic genotype data of 170 parts of corn representative materials, we-Fst-pop function in VCFtools software is utilized to analyze Fst values among different groups, namely genetic differentiation coefficients among different groups, and the site is considered as a specific site if the Fst value is more than 0.9. 20S-type specific sites (17 SNP sites and 3 InDel sites), 11C-type specific sites (9 SNP sites and 2 InDel sites) and 7T-type specific sites are obtained by analyzing S, C, T-type sterile materials of corn respectively.
7. Designing, evaluating and verifying primers based on a KASP platform: the primers for the specific sites are designed based on the KASP technology. SNP and InDel (insertion deletion of less than 26 bp) sites need to design 3 primers, two primers are allele specific primers, and one primer is a universal reverse primer. For InDel sites with inserts larger than 26bp, two pairs of primers were designed for insertions and deletions, respectively. 65 samples were selected and the designed primers were verified based on the evaluation of the KASP technology system. 65 samples comprise S, C, T three sterile germplasm materials, different heterosis groups and 3 sets of triplet materials (sterile female parent, fertile male parent and F1 hybrid). The material mainly verifies the amplification effect of the primer, verifies whether the marked site strictly follows the maternal inheritance, verifies the consistency with a sequencing result, and verifies whether the marked site is a specific site of a sterile material type.
8. The primer verification test process comprises the following steps: the DNA extraction adopts a mode of extracting DNA by mixing strains, 30 green leaves of single strains are mixed, and the specific steps of DNA extraction are carried out according to the identification standard of the DNA molecules of the corn (Wanfengge, etc., 2014, the SSR marking method of the corn variety identification technical specification, and the agricultural industry standard of the people's republic of China). The DNA mass and concentration were measured with a NanoDrop 2000(Thermo Scientific) UV spectrophotometer, and the working solution concentration was adjusted to 20 ng/. mu.L based on the measurements. The PCR reaction system was 1. mu.L, and included 1.5. mu.L of total DNA (oven dried), 0.5. mu.L of KASP ROX standard reaction mix (Kbiosciences, Herts UK), 0.014. mu.L of primer mixture, and 0.5. mu.L of deionized water. The reaction program is executed in a water bath PCR instrument (Hydrocycler HC-64), and the reaction program is 94 ℃ for 15 min; circulating for 10 times, adopting falling PCR mode, at 94 deg.C for 20s, at 61 deg.C for 1min, (at 61 deg.C falling to 55 deg.C, each circulation reducing by 0.6 deg.C); cycling 30 times at 94 ℃ for 20sec and 58 ℃ for 1 min. Scanning a fluorescence signal of the PCR product by adopting BMG Pheastar (LGC, Middlesex, UK) to obtain original data; genotype statistics were performed using Kraken software (LGC, Middlesex, UK).
9. Determining a primer for identifying the specific locus of the male sterile cytoplasm type of the corn: based on the test data of 65 parts of the above materials, 20 pairs of S-type sterility specific primers, 11 pairs of C-type sterility specific primers, and 7 pairs of T-type sterility specific primers were evaluated in the following four aspects. First, whether primer amplification was successful; second, whether maternal genetic characteristics are reflected in the triple sample; thirdly, whether the genotype data is consistent with the sequencing result; fourthly, verifying whether the primer is a specific primer for identifying the sterility type. The validation results showed that only the S-sterile 1 pair of InDel site primers and the C-sterile 1 pair of InDel site primers did not meet the above requirements. Finally obtaining 19 pairs of specific primer combinations for identifying the maize male sterility S-type cytoplasm, wherein the specific primer combinations comprise 17 SNP primers and 2 InDel site primers; c-type cytoplasm identification specific primer combination 10 pairs, including primers of 9 SNPs and 1 InDel site; t-type cytoplasm identification specific primer combination 7 pairs of SNP primers.
Example 2 high throughput identification of maize male sterile cytoplasm type based on KASP technique
Purpose of the experiment: and identifying the male sterile cytoplasm types of the 96 maize inbred line germplasm materials.
1. Extracting DNA of a maize inbred line sample to be identified: and randomly selecting 50 seeds from each sample, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a mode of mixed plant extraction DNA, green leaves of 30 individual plants are randomly selected from 50 individual plants in each sample and mixed, and the specific steps of DNA extraction are carried out according to the corn DNA molecular identification standard (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, the agricultural industry standard of the people's republic of China). The DNA was diluted to give a working solution at a concentration of 20 ng/. mu.L.
2. And (3) PCR amplification: from the S, C, T-type sterility specific identification primers provided in tables 4-6 of the present invention, 1 or more pairs were selected for amplification. The PCR reaction system is as follows: DNA template 1.5. mu.L, KASP ROX standard reaction mix (Kbiosciences, Herts UK) 0.5. mu.L, KASP Primer mix 0.014. mu.L, ddH2O0.5 μ L; the concentration of each Primer in the KASP Primer mix is 100 mu M.
The PCR reaction program is: 15min at 94 ℃; the falling PCR mode is adopted, the temperature is 94 ℃ for 20s, the temperature is 61-55 ℃ for 1min (the temperature is reduced by 0.6 ℃ per cycle), and 10 cycles are carried out; 94 ℃ for 20s, 58 ℃ for 1min, 30 cycles.
3. Fingerprint data acquisition: the amplification product was scanned for fluorescence signal using a BMG Pheastar (LGC, Middlesex, UK) instrument to obtain raw data. The raw data was imported into Kraken software (LGC, Middlesex, UK) for analysis to obtain fingerprint data for each data point of each corn variety.
4. And (4) judging a result: the determination is made based on the alleles of each of the loci listed in tables 4-6 in the sterile cytoplasmic type of maize S, C, T, which is the cytoplasmic sterility type to which it corresponds if the alleles in the tables are the same and which is not the cytoplasmic sterility type if the alleles in the tables are the other.
The test results of cytoplasmic type specific identification primers for maize male sterility S, C, T are shown in FIG. 2 (A-C).
The analysis result graphs of identifying whether the maize inbred line is S, C, T-type sterile or not by using the 19 pairs, 10 pairs and 7 pairs of primers provided by the invention are shown in figure 2A, figure 2B and figure 2C. In the results typing map for each pair of primers in FIG. 2, each data point is the result of typing for each sample in each pair of primers; the data points near the origin are negative control samples, and part of the rest data points are close to the X axis and the other part of the data points are close to the Y axis, which respectively represent two homozygous genotype results displayed by each pair of primers; genotype data read by software was checked against the information of each pair of primers in tables 1, 2 and 3, respectively, and it was S, C, T type sterile if it was consistent with "maize S, C, T type cytoplasmic male sterility sample allele" and non S, C, T type sterile if it was another genotype.
Example 3 identification of whether maize hybrid material is male sterile for seed production and which type of sterile cytoplasm belongs to using the specific primers provided by the invention
Purpose of the experiment: the method comprises the following steps of identifying whether 96 hybrid seeds are hybrid seeds produced by cytoplasmic male sterile seed production and which type of male sterile cytoplasm belongs to the hybrid seeds, and specifically comprises the following steps:
1. and (3) extracting DNA of a corn hybrid sample to be identified: and randomly selecting 50 seeds from each packaging bag, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a mode of extracting DNA from mixed plants, and green leaves of 30 individual plants are randomly selected from 50 individual plants in each sample to form 96 samples to be detected. The specific steps of DNA extraction are carried out according to the identification standard of the corn DNA molecules (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, and agricultural industry standard of the people's republic of China), and the DNA is diluted to form working solution with the concentration of 20 ng/mu L.
2. PCR amplification and fingerprint data acquisition were performed as in example 2.
3. And (4) judging a result: the determination is carried out based on the alleles of each locus listed in tables 4-6 in the maize S, C, T type sterile cytoplasm types, if the alleles are the same as the sterile material alleles in the tables, the corresponding cytoplasm sterile types are determined, and if the alleles are different from the sterile types in the tables, the hybrid formed by male sterile seed production is not included.
Example 4 identification of the purity of sterile samples Using the loci provided by the invention
Purpose of the experiment: the purity of 1 package of sterile sample seeds, namely the content of the sterile seeds, is identified by the following specific method:
1. and (3) extracting DNA of a sample to be identified: randomly selecting 120 seeds from a sample package to be identified, sprouting, and giving sufficient light to form green seedlings. The DNA extraction adopts a single-plant extraction mode to extract DNA of 100 single-plant green leaves, and the specific steps of DNA extraction are carried out according to the corn DNA molecular identification standard (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, and the agricultural industry standard of the people's republic of China). The DNA was diluted to give a working solution at a concentration of 20 ng/. mu.L.
2. PCR amplification and fingerprint data acquisition were performed as in example 2.
3. And (4) judging a result: the determination of which type of sterility to belong to is first determined based on the alleles of each of the loci listed in tables 4-6 in the maize S, C, T type sterile cytoplasmic type. And according to all loci of the determined sterile types, performing purity calculation, wherein the calculation formula is as follows:
(sterile single plant number/total single plant number detected) × 100%
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Sequence listing
<110> agriculture and forestry academy of sciences of Beijing City
<120> method for identifying maize male sterile cytoplasm type based on KASP technology
<130> KHP171117992.8
<160> 183
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agaaatcgca actcttttcc gttacacata ataaataaag ggtttcaaaa gtcaattttt 60
<210> 2
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttttcaatat ctttcttttt tttttcagaa ttccattttt gttcttccac ccatgcaata 60
<210> 3
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tggttggttc ttctcgccga gttttggcgt agcagctata tttcgcttca tccttttctt 60
<210> 4
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
caaggatttc ataattggac gttgaaacca tttcatatga tgggagttgc cggagtatta 60
<210> 5
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aatagaaaat gaaacggtcg acccagacat agacggtcga cccagacata gacggtcgac 60
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caggtggata taccctataa aatataggac gtagcaagcg tagttcaatg tagcgagcgt 60
<210> 7
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caaacggttt tgaaaggggg ataggctatg cttttctttc attttttttt tttttttttc 60
<210> 8
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcctgctaaa taaaaaaaaa gggttggata tagccctcta tcatatatat acaaatagaa 60
<210> 9
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cctttttttc ttttatcttg aatctaattc tagttagttt tttagaatct ttttcaaaat 60
<210> 10
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtttctgctt ttttggatcc agtttcgctt attctcctcg atggattcta tcttaaaaca 60
<210> 11
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tattattttt tgaattgcag tagtgaacga ctcttaaatc ggtattcccc cccattattt 60
<210> 12
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gaatggcata ataaaataga ataagaataa atgtttccct aatctgtata tagggaaact 60
<210> 13
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gagaccccgt ttacccctat ttatacgatt taagtataca taaagcaatt ttttttactt 60
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aatttaaact atacttttga ccttagaatg ctaacaggtc tgattttcga ttttgtactt 60
<210> 15
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aaactatact tttgacctta gaatgctaac aggtctgatt ttcgattttg tacttaaatt 60
<210> 16
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aaattgtatt ttaataagta aaagaagtca gttaattcat taaggctatg tttataccgt 60
<210> 17
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tgaatcaaaa gaattccttt tttgaagttc aatttttatc agaggacaat atgaatatta 60
<210> 18
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
accatgttcc attaaaacac tcaaggggtt atatgatata tcgggtgtag aagtagggca 60
<210> 19
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gacagaagga attgttagtt cacctcacct tccccaagcg cgggtttcct ttactaattt 60
<210> 20
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gttctctcta tgcgaaccct ctctctttct cgtaagaatg agatataggt agggctaaaa 60
<210> 21
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tcaagtccct ctatccccaa accctctttt attccctaac catagttgtt atcctttttt 60
<210> 22
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cttttatcaa tgggtttaag attcactagc tttctcattc tactctttca caaaggagtg 60
<210> 23
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
caccatatca taagattcgc gttcttgaaa atcggcactt ctccaaaccc agaaaacgga 60
<210> 24
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gggattctag gattatcctt ttgggcaaag acttttatgc atacctcttc tgggttatct 60
<210> 25
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acagagaatt ttcttagtat ttaggtattt agattcaaaa tatcaaaggg gaagaacttt 60
<210> 26
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaaattgtaa aataaagatt agggtttggg ttgcgctata tctatcaaag agtatacaat 60
<210> 27
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
actttttctt ggttaaagga acagatgatt cgatcgattt ctgtatcgat catgatatac 60
<210> 28
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
taataactcg cacatctatt tcaaacgcat atcccatttt tgcgcagcag ggttatgaaa 60
<210> 29
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ttgtatcgaa gtaagaatca tttttcttct tatttgtttt gtcaaagatt actatttatt 60
<210> 30
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tttattcggg tctgtctttt ggacttcctt tgcttaggtt cgggcgcggt agtggacaaa 60
<210> 31
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cgatcttaac ctgatgattc atcatcatga agtatttcta ttttctatag cataaaaccc 60
<210> 32
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
aatttaggtt tgagataaat ttacaagaaa tccacccact accaatcctt aaacatttct 60
<210> 33
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ggaagataag cattcaatgt attttagagg aaaaagatcc tattttaacg aatcacacgt 60
<210> 34
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gagatattgc tagtacacaa aaagttaatg gtatttcata actaatagat tgagcagccg 60
<210> 35
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tatgaaatga tctactaact catctcagat gcaagtccac tttcaatata tctctgtata 60
<210> 36
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
agcggtaagg aaaaagtttt aataaaaaga agaatcaatg gattcatgat taaacccctc 60
<210> 37
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ctctccccct ttgtataaat atttcacatt tcaaatgcaa gtttgaaaga ttgtactgct 60
<210> 38
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ttttctttct ttttctgcac aaaagaaccc ctcctaattc actaatttgt aggaagatac 60
<210> 39
<211> 83
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
actgtataca cggatacaga atccgctata tccgtttgtg aaataaaggc taaatcccct 60
cccctcaact ccatatctaa ata 83
<210> 40
<211> 5
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tcttt 5
<210> 41
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aataaataaa gggtttcaaa agtcaatttt tc 32
<210> 42
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aataaataaa gggtttcaaa agtcaatttt ta 32
<210> 43
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ggaattctga aaaaaaaaag aaagatattg 30
<210> 44
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tcaacgtcca attatgaaat ccttgg 26
<210> 45
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gttcaacgtc caattatgaa atccttga 28
<210> 46
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gtagcagcta tatttcggtt catccttt 28
<210> 47
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
atattttata gggtatatcc acctgg 26
<210> 48
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
cctatatttt atagggtata tccacctgt 29
<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
acatagacgg tcgacccaga cata 24
<210> 50
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tttctttcat tttttttttt tttttttct 29
<210> 51
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gcttttcttt catttttttt tttttttttt cc 32
<210> 52
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tatccaaccc ttttttttta tttagcaggc 30
<210> 53
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
acttactttt ttagaatctt tttcaaaaaa ta 32
<210> 54
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
acttactttt ttagaatctt tttcaaaaaa tg 32
<210> 55
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
agcgaaactg gatccaaaaa agcagaaat 29
<210> 56
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
atttattctt attctatttt attatgccat tca 33
<210> 57
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tattcttatt ctattttatt atgccattcc 30
<210> 58
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tcttaaatcg gtattccccc ccattattt 29
<210> 59
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ttaagtatac ataaagcaat tttttttact tt 32
<210> 60
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
taagtataca taaagcaatt ttttttactt g 31
<210> 61
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
gttagcattc taaggtcaaa agtatagttt 30
<210> 62
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
actgacttct tttacttatt aaaatacaat tta 33
<210> 63
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
actgacttct tttacttatt aaaatacaat ttc 33
<210> 64
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ctaacaggtc tgattttcga ttttgtactt 30
<210> 65
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
caatttttat cagaggacaa tatgaatatt ac 32
<210> 66
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
caatttttat cagaggacaa tatgaatatt at 32
<210> 67
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
tataacccct tgagtgtttt aatggaacat 30
<210> 68
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
aagcgcgggt ttcctttact aatttt 26
<210> 69
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
agcgcgggtt tcctttacta atttg 25
<210> 70
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
agagagaggg ttcgcataga gagaa 25
<210> 71
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
agtgaatctt aaacccattg ataaaaga 28
<210> 72
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
agtgaatctt aaacccattg ataaaagc 28
<210> 73
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
tttattccct aaccatagtt gttatccttt 30
<210> 74
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ccaaaaggat aatcctagaa tcccg 25
<210> 75
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
cccaaaagga taatcctaga atccca 26
<210> 76
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
atcggcactt ctccaaaccc agaaa 25
<210> 77
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gattcaaaat atcaaagggg aagaacttta 30
<210> 78
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
caaaatatca aaggggaaga actttt 26
<210> 79
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
gcaacccaaa ccctaatctt tattttacaa 30
<210> 80
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
cgatttctgt atcgatcatg atatacg 27
<210> 81
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
atcgatttct gtatcgatca tgatataca 29
<210> 82
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gatatgcgtt tgaaatagat gtgcgagtt 29
<210> 83
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tatttgtttt gtcaaagatt actatttatt c 31
<210> 84
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
cttatttgtt ttgtcaaaga ttactattta ttt 33
<210> 85
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ggaagtccaa aagacagacc cgaat 25
<210> 86
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gtatttctat tttctatagc ataaaacccg 30
<210> 87
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
aagtatttct attttctata gcataaaacc ct 32
<210> 88
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
ggatttcttg taaatttatc tcaaacctaa 30
<210> 89
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
aaaagatcct attttaacga atcacacgta 30
<210> 90
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
agatcctatt ttaacgaatc acacgtg 27
<210> 91
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
taccattaac tttttgtgta ctagcaatat 30
<210> 92
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ttttattaaa actttttcct taccgctttt a 31
<210> 93
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ctttttatta aaactttttc cttaccgctt ttt 33
<210> 94
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
atgcaagtcc actttcaata tatctctgta 30
<210> 95
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ccctcccctc aactccatat ctaaa 25
<210> 96
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
caagtttgaa agattgtact gctctttc 28
<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gcaagtttga aagattgtac tgctctttt 29
<210> 98
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
attaggaggg gttcttttgt gcagaaaaa 29
<210> 99
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
cccatttttt tttttttttg caattttatg acttagttta gtgcgagatg cccacatttt 60
<210> 100
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
ggtctgaaca ctaaacgagc acagacttaa aattacaaaa aaaaatgaaa ttggactctt 60
<210> 101
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
tcaggggggc caagtcaggt tagatctata tctttaatgc ctataagaca gtcatctttt 60
<210> 102
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
tgtggctttc taaagaatga ttttagaatc ggattcaata gaaaatgaga aaataggcaa 60
<210> 103
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
ttcgtctttt attatgttag atgaagggga aaaaatggga actcaaagat atcgaagagt 60
<210> 104
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
aaaaaaagga tggaataaaa gagtgattgg ttgaaaagaa agagaaatag aataatgaga 60
<210> 105
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tagttgtatc gacccagtcg ctcactaatt gatctttacg gtgttttctc tatcaatttc 60
<210> 106
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gctttatcca tagaatagta gtataggctc tactttcttc ctattttgat tctcgtgaag 60
<210> 107
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ccaatcaaat attgagtaat caaatccttc aattcattgt tttcgagatc ttttaatttt 60
<210> 108
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
aaaagtggat taatcggacg aggataaaga gagagtccca ttctacatgt caatactgac 60
<210> 109
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
gcgtttcgat gaatgagcct atggtaatgc ttttatctct attctatggc gcaatcgacc 60
<210> 110
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tcgagtctat aaacaagtac taaataagga aaagaaaact atactaaagg aaacataaga 60
<210> 111
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
cccgggacgc gaagtagtag gattggttct cataattatc acataatttt caaaaaaaaa 60
<210> 112
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
gaatttgtcg aaattttttt tcttgttgaa taatgccaaa tcaaaaaaaa tatccaaaaa 60
<210> 113
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ttttttaatt atagttattc ctatgcgaga gatagaattc ttcgtgacat gacgaaaatt 60
<210> 114
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
cccctttttg aattcttttt tagtatatga agcaaaaaga aagaaaagat ggataaggat 60
<210> 115
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
aaaagcttct ttttcgaaag attacccctg tctttgttta tgcttcggat tggaacaaat 60
<210> 116
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
actctaattc gcccacgcct gcgaatcagt cgacattttg tacaaatttt acgaacggaa 60
<210> 117
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 118
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ctctatccta ttcattggta ccgatcatgg atacttcaaa aattttatta tttgtttgaa 60
c 61
<210> 119
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
gtgctcgttt agtgttcaga cca 23
<210> 120
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
gtgctcgttt agtgttcaga ccc 23
<210> 121
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
cttagtttag tgcgagatgc ccacat 26
<210> 122
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
attctaaaat cattctttag aaagccacac 30
<210> 123
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
ctaaaatcat tctttagaaa gccacat 27
<210> 124
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ggccaagtca ggttagatct atatcttta 29
<210> 125
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
atgggaactc aaagatatcg aagagta 27
<210> 126
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gggaactcaa agatatcgaa gagtc 25
<210> 127
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
caaccaatca ctcttttatt ccatcctttt 30
<210> 128
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gcctatacta ctattctatg gataaagct 29
<210> 129
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
cctatactac tattctatgg ataaagcg 28
<210> 130
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tcgctcacta attgatcttt acggtgttt 29
<210> 131
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
atcctcgtcc gattaatcca ctttta 26
<210> 132
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
atcctcgtcc gattaatcca cttttt 26
<210> 133
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
ccttcaattc attgttttcg agatctttta 30
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
tatttagtac ttgtttatag actcgac 27
<210> 135
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
ccttatttag tacttgttta tagactcgat 30
<210> 136
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aatgctttta tctctattct atggcgcaat 30
<210> 137
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
attcaacaag aaaaaaaatt tcgacaaatt cc 32
<210> 138
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
attcaacaag aaaaaaaatt tcgacaaatt ct 32
<210> 139
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gcgaagtagt aggattggtt ctcataatt 29
<210> 140
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
tcatatacta aaaaagaatt caaaaagggg a 31
<210> 141
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
catatactaa aaaagaattc aaaaaggggg 30
<210> 142
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gagatagaat tcttcgtgac atgacgaaa 29
<210> 143
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
aggcgtgggc gaattagagt c 21
<210> 144
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
caggcgtggg cgaattagag tt 22
<210> 145
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
gtctttgttt atgcttcgga ttggaacaa 29
<210> 146
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cggtaccaat gaataggata gags 24
<210> 147
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
atcggtacca atgaatagga tagaga 26
<210> 148
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ccaaaggaaa ccataaattc catataccat 30
<210> 149
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tttttactta ctttagttct ttagttttgg gaaaataaat agggggtact tcttttcttt 60
<210> 150
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
atattggggg tcaaatggat cctgtaagaa ttcccacttc atagatacgg ggtataaagt 60
<210> 151
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
ttttacttac tttagttctt tagttttggg aaaataaata gggggtactt cttttctttc 60
<210> 152
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tattgggggt caaatggatc ctgtaagaat tcccacttca tagatacggg gtataaagtt 60
<210> 153
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tttctagtgc ttataaattc ttatggcttt atcccgtttc atagaaagga gataaaacga 60
<210> 154
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
aaatctttgc tatccaatct ttttagaata tcataaagtt tcagtggcag aatttttttc 60
<210> 155
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tgccaagaga ttggcatttt catttgatca ttatatacat ttttgagata ttttgttttt 60
<210> 156
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
tatttgttaa taatttaagg ataaatagtt cactaaggag aagatagaat catagcaaat 60
<210> 157
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
cggtagtgga caaacagagg gaaagaaggt atggcgggga cacatttctt gtgagcaaat 60
<210> 158
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
gaattgcttt actttttgaa ttaagttcaa ctttgaactt acagaaattt tgtaaaaaaa 60
<210> 159
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tctctatgtt ttattactta atttacgaat ttcaaaaatt ttgtattcta ttggattgga 60
<210> 160
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
ttgttcgaga attcgaagaa ttacaacaaa atctttagaa atcacatttt tagttaggaa 60
<210> 161
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
tttgtcgttc ccacagcttc tcctttaatg gttaggtttg aatcctgcaa tggagcttcc 60
<210> 162
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
attttttttt ccgagtcaat tttctcagtt ttattaaccc ggctgctctt tatttattgc 60
<210> 163
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
aggatccatt tgacccccaa tatg 24
<210> 164
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
aggatccatt tgacccccaa tatc 24
<210> 165
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
ggaaaataaa tagggggtac ttcttttctt 30
<210> 166
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
aaataaatag ggggtacttc ttttctttca 30
<210> 167
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
aaataggggg tacttctttt ctttcg 26
<210> 168
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
cttacaggat ccatttgacc cccaa 25
<210> 169
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
atattctaaa aagattggat agcaaagatt tc 32
<210> 170
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gatattctaa aaagattgga tagcaaagat tta 33
<210> 171
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
gctttatccc gtttcataga aaggagata 29
<210> 172
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
gaactattta tccttaaatt attaacaaat aa 32
<210> 173
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
gaactattta tccttaaatt attaacaaat ac 32
<210> 174
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
gccaagagat tggcattttc atttgatcat 30
<210> 175
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
agttgaactt aattcaaaaa gtaaagcaat tct 33
<210> 176
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
gttgaactta attcaaaaag taaagcaatt cg 32
<210> 177
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
cggggacaca tttcttgtga gcaaa 25
<210> 178
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
atttcaaaaa ttttgtattc tattggattg gat 33
<210> 179
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
tcaaaaattt tgtattctat tggattggac 30
<210> 180
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
tttgttgtaa ttcttcgaat tctcgaacaa 30
<210> 181
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
ttgaatcctg caatggagct tcca 24
<210> 182
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
gaatcctgca atggagcttc cc 22
<210> 183
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
gcagccgggt taataaaact gagaaaatt 29
Claims (5)
1. The primer combination of the molecular marker for identifying the corn C-type cytoplasmic sterile material, which is developed based on the KASP technology, is characterized in that the molecular marker for identifying the corn C-type cytoplasmic sterile material comprises the following 9 SNP markers and 1 InDel marker, and the information of the SNP markers and the InDel markers is as follows:
2. the identification method of the corn C-type cytoplasmic sterile material based on the KASP technology is characterized by comprising the following steps:
1) extracting DNA of a corn sample to be detected;
2) KASP reaction: adding KASP Primer mix and KASP ROX standard reaction mix into the DNA template extracted in the step 1) for PCR amplification;
3) analyzing the PCR product by a fluorescence detector;
the KASP Primer mix is a mixture of the KASP primers of claim 1 and the downstream universal Primer after the 5' end of the upstream Primer is modified with different fluorescent tag sequences;
wherein the fluorescent tag sequence is: 5 '-FAM-GAAGGTGACCAAGTTCATGCT-3';
5’-HEX-GAAGGTCGGAGTCAACGGATT-3’。
3. the method of claim 2, wherein the PCR reaction system in step 2) is: DNA template 1.5. mu.L, KASP ROX standard reaction mix 0.5. mu.L, KASP Primer mix 0.014. mu.L, ddH2O0.5 μ L; the concentration of each Primer in the KASP Primer mix is 100 mu M;
the PCR reaction program is: 15min at 94 ℃; at 94 ℃ for 20s and 61-55 ℃ for 1min, reducing the temperature by 0.6 ℃ per cycle and performing 10 cycles; 30 cycles of 94 ℃ for 20s and 58 ℃ for 1 min.
4. A detection reagent or kit comprising the primer combination of claim 1.
5. The primer combination of claim 1 or the detection reagent or kit of claim 4, which is used for corn sample detection and corn molecular marker-assisted breeding;
the corn is a corn C-type cytoplasmic male sterile material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810118136.0A CN108486265B (en) | 2018-02-06 | 2018-02-06 | Method for identifying type of male sterile cytoplasm of corn based on KASP technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810118136.0A CN108486265B (en) | 2018-02-06 | 2018-02-06 | Method for identifying type of male sterile cytoplasm of corn based on KASP technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108486265A CN108486265A (en) | 2018-09-04 |
| CN108486265B true CN108486265B (en) | 2022-06-24 |
Family
ID=63344485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810118136.0A Active CN108486265B (en) | 2018-02-06 | 2018-02-06 | Method for identifying type of male sterile cytoplasm of corn based on KASP technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108486265B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129470B (en) * | 2019-04-15 | 2022-05-03 | 北京市农林科学院 | Core SNP locus combination for corn molecular breeding and application thereof |
| CN111154908B (en) * | 2020-01-20 | 2022-09-16 | 中国农业科学院油料作物研究所 | KASP labeled primer for identifying Brassica crop Cam and Pol cytoplasm types and application thereof |
| CN113373256A (en) * | 2021-06-22 | 2021-09-10 | 扬州大学 | Development and application of KASP molecular marker of sugarcane mosaic virus disease resistance gene of corn |
| CN117737296A (en) * | 2024-02-21 | 2024-03-22 | 北京康普森生物技术有限公司 | SNP marker for identifying purity of Qingzao 510 maize hybrid and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088018A (en) * | 2012-12-27 | 2013-05-08 | 河南农业大学 | Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591470B (en) * | 2017-01-11 | 2020-05-08 | 北京市农林科学院 | One set of chloroplast SNP and INDEL molecular marker combination for corn maternal traceability |
-
2018
- 2018-02-06 CN CN201810118136.0A patent/CN108486265B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088018A (en) * | 2012-12-27 | 2013-05-08 | 河南农业大学 | Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108486265A (en) | 2018-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108486265B (en) | Method for identifying type of male sterile cytoplasm of corn based on KASP technology | |
| CN108486266B (en) | Molecular marker of corn chloroplast genome and application of molecular marker in variety identification | |
| CN102747138B (en) | Rice whole genome SNP chip and application thereof | |
| ES2393318T3 (en) | Strategies for the identification and detection of high performance polymorphisms | |
| ES2357549T3 (en) | STRATEGIES FOR THE IDENTIFICATION AND DETECTION OF HIGH PERFORMANCE OF POLYMORPHISMS. | |
| CN106048042B (en) | For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits color traits | |
| CN112195264B (en) | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of tomato hybrid and application | |
| CN112195265B (en) | SNP (Single nucleotide polymorphism) locus and primer set for identifying purity of pepper hybrid and application | |
| CN110904266B (en) | Identification of maize stalk lodging resistance QTL and development and application of molecular marker | |
| CN108220473B (en) | Identification of maize S-type cytoplasmic male sterile material by using chloroplast InDel marker | |
| CN113817867A (en) | SNP molecular marker related to alfalfa quality traits and application thereof | |
| CN108441572B (en) | Method for identifying maize chloroplast cytoplasm type based on KASP technology | |
| CN112538535B (en) | Molecular marker related to hair yield of long-hair rabbits and application of molecular marker | |
| CN109811077B (en) | KASP marker closely linked with wheat dwarf gene and application thereof | |
| CN108192991B (en) | Identification of maize male sterility S-type cytoplasmic types using chloroplast markers | |
| KR102087024B1 (en) | Single nucleotide polymorphism marker set for line purity checking and early fixed line selecting in Brassica rapa and uses thereof | |
| CN108486264B (en) | Identification of maize male sterility type C cytoplasm type by chloroplast marker | |
| CN108411020B (en) | Corn chloroplast InDel molecular marker suitable for capillary electrophoresis detection platform | |
| CN116622877B (en) | SNP molecular marker related to lotus rhizome internode shape and application thereof | |
| CN108486263A (en) | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker | |
| CN114736979B (en) | Molecular marker closely linked with watermelon complete leaf shape gene ClLL and application thereof | |
| CN114606341B (en) | dCAPS molecular marker of aegilops sieboldii based on genome resequencing SNP and application | |
| CN114525362B (en) | Primer combination for identifying Apostichopus japonicus population of Anyuan No. 1 and application thereof | |
| 間瀬誠子 et al. | Analysis of bud sport cultivars of peach (Prunus persica (L.) Batsch) by simple sequence repeats (SSR) and restriction landmark genomic scanning (RLGS) | |
| KR101892461B1 (en) | Molecular marker for discrimination genetic male sterility gene and detection method using the same |
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 |