CN108441572B - Method for identifying maize chloroplast cytoplasm type based on KASP technology - Google Patents
Method for identifying maize chloroplast cytoplasm type based on KASP technology Download PDFInfo
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Abstract
The invention provides a method for identifying the cytoplasmic type of corn chloroplast based on the KASP technology. Five specific primers for identifying chloroplast cytoplasm types are B type and 12 pairs of SNP primers respectively; type C, 10 pairs of primers including 1 pair InDel and 9 pairs of SNP primers; d type, 3 pairs of primers comprise 1 pair of InDel and 2 pairs of SNP primers; HS type, 19 pairs of primers including 2 pairs of InDel and 17 pairs of SNP primers; t type, 7 pairs of SNP primers. The method can be used for identifying the cytoplasm type of the maize inbred line or the hybrid, identifying the cytoplasm type of the maize inbred line or the hybrid female parent, and classifying and dividing the maize material to reflect the maternal evolution process. The application of the method in genome level expands the analysis methods of corn material group division and maternal line tracking, and provides a new thought and means for the research of the cytoplasmic hereditary property of 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 chloroplast cytoplasm type based on a KASP (Kasp technology).
Background
Corn is the first crop in the world and has an important position in agricultural production in China; is the crop which is the first most popular in the application of hybrid fine variety and is the crop with the largest market share in the species industry of the whole world and China. The screening of parents is a key factor for breeding excellent corn hybrid, so that research reports aiming at the division of parents inbred line groups are many, but the research reports are all based on the analysis of nuclear genome molecular markers. That is, the genetic variation is mainly caused by artificial combination and breeding of excellent materials.
Chloroplasts are organelles of green plants that perform photosynthesis and possess an intact set of genomes known as the chloroplast genome. The chloroplast genome structure is well conserved, DNA is generally a double-stranded circular molecule, and the chloroplast genome size in higher plants is generally 120-160 kb. Unlike nuclear genomes, chloroplast genomes have the characteristic of cytoplasmic inheritance, and plant chloroplast genomes are uniparental inheritance (mostly maternal inheritance) and generally do not recombine; the chloroplast has smaller and relatively conservative genome, the molecular evolution rate is slow, and the genetic variation of the genome is easily influenced by the group genetic effect; therefore, the chloroplast molecular marker is an effective means for studying population differentiation. The chloroplast genome of the gramineous plant corn belongs to strict maternal inheritance, and several chloroplast cytoplasm types formed by the differentiation of the corn can be identified by analyzing a corn material based on the molecular marker of the chloroplast genome.
The maturity of high-throughput sequencing and genome bioinformatics analysis technology effectively promotes the research on chloroplast genomes. It becomes possible to excavate molecular marker sites such as Single Nucleotide Polymorphism (SNP), insertion deletion polymorphism (InDel) and the like from the chloroplast genome level. KASP (Kompetitive Allele Specific PCR) is a competitive Allele Specific PCR technology, has the characteristics of simple test flow, high efficiency, flexibility, easiness in operation and the like, and is suitable for genotyping detection of SNP/InDel and other binary marker types.
The existing maize inbred line and the classification and grouping of germplasm resource materials are all based on nuclear genome information, and plant cells have cytoplasmic inheritance, namely chloroplast and mitochondrial genome information, besides nuclear inheritance. The cytoplasmic genome information, particularly the chloroplast genome, has the advantages, and is more suitable for classifying and dividing the corn material from the aspect of early differentiation and reflecting the maternal uniline evolution process. Therefore, the invention utilizes the re-sequencing data to develop the specific locus of the cytoplasmic type of the corn chloroplast, designs the primer based on the KASP genotyping platform and carries out verification and evaluation, thereby establishing a method for identifying the cytoplasmic type of the corn chloroplast.
Disclosure of Invention
The invention aims to provide a method for identifying the cytoplasmic type of corn chloroplast based on KASP technology.
The invention concept of the invention is as follows: (1) and selecting 170 parts of corn representative materials, including all heterosis groups in China, samples such as sweet glutinous, local varieties, cytoplasmic 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 and polymorphic site determination, namely, annotating the chloroplast genome by using DOGMA software, comparing 170 parts of material chloroplast genome sequences, and screening 100 SNP/InDel chloroplast genome variation sites. (6) The method comprises the steps of determining five types of chloroplast cytoplasm of corn, respectively establishing a phylogenetic clustering diagram by utilizing an MP (maximum simplicity) maximum likelihood method and an ML (maximum likehood) maximum likelihood method based on 170 parts of material chloroplast genome sequences, and displaying that all samples are divided into five types. The five types are respectively: type B is modified Ruide, modified blue card and American backbone inbred line material; the type C is a type C cytoplasmic sterile material; d type is Luodan red bone miscellaneous excellent group, local variety and tropical material; the HS type is S type cytoplasm sterile material and Tangsichun Heipitou Heteroyou group material; t type is T type cytoplasm sterile material. (7) And (3) determining the specific identification sites of the five cytoplasmic types, and analyzing Fst values (genetic differentiation coefficients) among the five types of groups, wherein the Fst value of more than 0.9 is the specific site of the group. Five types of cytoplasm of corn B, C, D, HS and T are respectively analyzed to obtain 12B type specific sites, 11C type specific sites, 4D type specific sites, 20 HS type specific sites and 7T type specific sites. (8) Designing, evaluating and verifying primers, designing the primers of the specific sites based on a KASP technical system, and utilizing 94 samples representing five types to carry out primer evaluation on the following four aspects, namely whether the first amplification is successful, whether the second amplification reflects maternal inheritance, whether the third and sequencing results are consistent, and whether the fourth is the specific primers of five chloroplast cytoplasm types. The five chloroplast cytoplasm types of identification specific primers of corn B, C, D, HS and T are respectively 12, 10, 3, 19 and 7 pairs. The site details are shown in tables 1-5, and the primer details are shown in Table 6. The technical scheme obtained by identifying the specific primers based on the five chloroplast cytoplasm types of the corn of the KASP technical system is shown in a figure 1.
In order to achieve the object, the invention provides a molecular marker for identifying maize B-type cytoplasmic material, which is developed based on chloroplast genome, and is selected from at least one of the following 12 SNP markers, and the site information of the molecular marker is shown in Table 1:
TABLE 1
The invention provides a molecular marker developed based on chloroplast genome and used for identifying corn C-type cytoplasmic sterile material, wherein the molecular marker is selected from at least one of the following 9 SNP markers and 1 InDel marker, and the information of the markers is shown in Table 2:
TABLE 2
The invention provides a molecular marker developed based on chloroplast genome and used for identifying D-type cytoplasmic material of corn, wherein the molecular marker is selected from at least one of the following 2 SNP markers and 1 InDel marker, and the site information of the markers is shown in Table 3:
TABLE 3
The invention provides a molecular marker which is developed based on chloroplast genome and used for identifying corn HS-type cytoplasmic sterile material, the molecular marker is selected from at least one of the following 17 SNP markers and 2 InDel markers, and the site information of the markers is shown in Table 4:
TABLE 4
Wherein the nucleotide sequences of the InDel markers CPMIDP01 and CPMIDP02 are respectively shown as SEQ ID NO: 103-104.
The invention provides a molecular marker for identifying T-type cytoplasmic sterile material of corn, which is developed based on chloroplast genome, and is selected from at least one of the following 7 SNP markers, and the site information of the molecular marker is shown in Table 5:
TABLE 5
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 present invention also provides primers developed based on the KASP technique for detecting the molecular markers described in tables 1-5.
Preferably, the KASP primer sequences for detecting the markers CPMSNP 01-CPMSNP 93 in Table 1 are shown as SEQ ID NO:105-140, respectively, wherein the KASP primer for detecting the marker CPMSNP01 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer with the sequences of SEQ ID NO:105-107, the KASP primer for detecting the marker CPMSNP02 is SEQ ID NO:108-110, and so on.
Preferably, the KASP primer sequences for detecting the markers CPMIDP 10-CPMSNP 81 in Table 2 are shown as SEQ ID NO 141-170, respectively, wherein the KASP primer for detecting the marker CPMIDP10 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer with the sequences of SEQ ID NO 141-143, the KASP primer for detecting the marker CPMSNP18 is SEQ ID NO 144-146, and so on.
Preferably, the KASP primer sequences for detecting the markers CPMIDP 07-CPMSNP 19 in Table 3 are shown as SEQ ID NO:171-179, respectively, wherein the KASP primer for detecting the marker CPMIDP07 comprises an upstream primer 1, an upstream primer 2 and a downstream universal primer with the sequences of SEQ ID NO:171-173, the KASP primer for detecting the marker CPMSNP07 is SEQ ID NO:174-176, and so on.
Preferably, the KASP primer sequences for detecting the markers CPMIDP 01-CPMSNP 96 in Table 4 are respectively shown as SEQ ID NO:180-237, wherein 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 with the sequence of SEQ ID NO:180-183, the KASP primer for detecting the marker CPMIDP02 comprises the upstream primer 1, the upstream primer 2 and the downstream universal primer with the sequence of SEQ ID NO:184-186, the KASP primer for detecting the marker CPMSNP08 comprises the upstream primer 1, the upstream primer 2 and the downstream universal primer with the sequence of SEQ ID NO:187-189, and so on.
Preferably, the KASP primer sequences for detecting the markers CPMSNP 03-CPMSNP 86 in Table 5 are shown as SEQ ID NO 238-258, respectively, 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 sequences of SEQ ID NO 238-240, the KASP primer for detecting the marker CPMSNP04 is SEQ ID NO 241-243, and so on.
The invention also provides a detection reagent or a kit containing the KASP primer.
The 51 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; buying MasterMix PCR amplification system matched with KASP technique; 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 cytoplasmic type of the corn chloroplast. The method specifically comprises the following steps: (1) extracting the total DNA of the detected inbred line or hybrid material; (2) the SNP/IDNEL primer provided by the invention is utilized to carry out PCR amplification and fluorescence scanning based on a KASP genotyping platform to obtain genotype data, and determine which chloroplast cytoplasm type belongs to. The primer provided by the invention can also be used for identifying the cytoplasmic type of the female parent of corn or hybrid, and can be used for classifying and dividing corn materials to reflect the maternal evolution process.
The invention also provides a method for identifying five types of chloroplast cytoplasm of corn based on 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 KASP Primer mix is a mixture formed by the upstream Primer and the downstream universal Primer (downstream Primer) after the 5' end of the upstream Primer in each KASP Primer is respectively modified with different fluorescent label sequences.
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 μ 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. Based on the fingerprint data for each locus, a determination is made that each locus listed in Table 6 represents an allele of that type, which is the corresponding cytoplasmic type if the allele is identical to the allele in the table and which is not the cytoplasmic type if it is another allele.
The invention also provides application of the KASP primer, or a detection reagent or a kit containing the primer in identifying the five chloroplast cytoplasm types 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 sample 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 scheme, coupled with alignment with a maize chloroplast reference genome. The key steps for obtaining the chloroplast genome data of the corn are that a Blast program is utilized to screen contigs of chloroplast genomes, Sequencher software is used to assemble the chloroplast genome contigs, and the assembled sequences are compared with a corn chloroplast reference genome (corn variety B73, AGPv3) for verification and confirmation, so that guarantee is provided for obtaining accurate and reliable chloroplast genome sequences. (2) Determination of five chloroplast cytoplasmic types in maize: based on chloroplast genome sequence information of 170 materials with wide sources and abundant phenotypes and genotypes, a phylogenetic clustering graph is respectively established by utilizing PAUP4.0 and RAxML7.04 software, an MP (maximum Parsimony) maximum reduction method and an ML (maximum likelihood) maximum likelihood method. The results of the cluster maps established by both methods show that all maize samples are classified into five chloroplast cytoplasmic types. (3) Based on KASP technology, five chloroplast cytoplasm types of corn are specifically determined: analyzing the variation sites of the inbred lines of all types of corns, screening and determining five specific sites of the cytoplasmic types, and designing and evaluating primers based on a KASP technical system. The primer screening standard is whether the amplification is successful or not, whether the maternal inheritance is followed or not, the accuracy of genotype data and the like are followed, and finally the specific primer for identifying the cytoplasm type is determined.
170 parts of maize inbred line materials with wide sources, rich phenotypes and genotypes and strong representativeness are collected to obtain a chloroplast genome sequence, and the cytoplasm types of the 170 parts of materials are analyzed. Comparing nucleotide polymorphism of chloroplast genomes, developing a specific site for identifying the cytoplasmic type of the corn chloroplast, designing a primer based on a KASP technical system and evaluating to establish the method for identifying the cytoplasmic type of the corn chloroplast. The method can be used for identifying the cytoplasm type of a corn inbred line or a hybrid, identifying the cytoplasm type of a corn or a hybrid female parent, and classifying and dividing corn materials to reflect the maternal evolution process. The application of the method expands the analysis methods of corn material group division and maternal line tracking on the genome level, and provides a new thought and technical means for the research of the cytoplasmic hereditary property of corn and the like.
Drawings
FIG. 1 is a technical scheme diagram obtained by identifying specific primers for five chloroplast cytoplasm types of corn based on KASP technical system.
FIGS. 2A-2E show the results of the experiments of the primers for identifying maize cytoplasm types B, C, D, HS and T according to the present invention. The circles in the figure represent the genotype of the corn material. FIG. 2A is a B-type specific identification primer, FIG. 2B is a C-type specific identification primer, FIG. 2C is a D-type specific identification primer, FIG. 2D is an H/S-type specific identification primer, and FIG. 2E is a T-type specific identification primer.
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 identification of specific primers based on the KaSP technical System corn five chloroplast cytoplasmic types
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, cracked 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 five chloroplast cytoplasmic types in maize: based on chloroplast genome sequence information of 170 materials with wide sources and abundant phenotypes and genotypes, a phylogenetic clustering map is respectively established by using PAUP4.0 and RAxML7.04 software, MP (maximum Parsimony) maximum reduction method and ML (maximum likelihood) maximum likelihood method. The results of the cluster maps established by both methods show that all maize samples are classified into five chloroplast cytoplasmic types. The five types are respectively: type B is modified Ruide, modified blue card and American backbone inbred line material; the type C is a type C cytoplasmic sterile material; d type is Luda red bone miscellaneous excellent group, local variety and heat belt material; HS type is S type cytoplasm sterile material and Tangsijie Heteroyou group material; t type is T type cytoplasm sterile material.
7. Determination of specific sites of five chloroplast cytoplasmic types in maize: 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. Five types of cytoplasm of corn B, C, D, HS and T are respectively analyzed to obtain 12B type specific sites, 11C type specific sites, 4D type specific sites, 20 HS type specific sites and 7T type specific sites.
8. Designing, evaluating and verifying a primer based on a KASP platform: the primers of the specific sites are designed based on the KASP technical system. SNP and InDel (insertion deletion less than 26 bp) sites need to design 3 primers, two are allele specific primers, and one is a universal reverse primer. For InDel sites with inserts larger than 26bp, two pairs of primers were designed for insertions and deletions, respectively. 94 samples representing five types were selected and the designed primers were verified based on the evaluation of the KASP technology system. The material mainly verifies the amplification effect of the primers, whether the marker loci strictly follow maternal inheritance or not, the consistency of genotype data and sequencing results or not and whether the marker loci are five types of specific primers or not.
9. 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).
10. Specific primers for identifying five chloroplast cytoplasmic types of corn are determined: based on the above test data for 94 materials, the primers designed were evaluated in the following four aspects. First, whether primer amplification was successful; second, whether maternal genetic characteristics are reflected; thirdly, whether the genotype data is consistent with the sequencing result; fourth, it was verified whether there were five specific primers for chloroplast cytoplasmic types. The five chloroplast cytoplasm types of identification specific primers of corn B, C, D, HS and T are respectively 12, 10, 3, 19 and 7 pairs.
The technical scheme obtained by identifying the specific primers based on the five chloroplast cytoplasm types of the corn of the KASP technical system is shown in a figure 1. FIGS. 2A-2E show the results of experiments with primers specific for the five cytoplasmic types of maize B, C, D, HS, T.
The analysis result graphs of whether the maize inbred line is B, C, D, HS and T sterile or not are respectively identified by using the 12 pairs, 10 pairs, 3 pairs, 19 pairs and 7 pairs of primers provided by the invention are shown in the figures 2A-2E. In the results typing plots for each pair of primers in FIGS. 2A-2E, each data point is the result of typing 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 the software was checked against the information for each pair of primers in tables 1, 2, 3, 4, 5, respectively, for the corresponding B, C, D, HS, T cytoplasmic types if they are consistent with the "maize B, C, D, HS, T five cytoplasmic type alleles" and for the non-B, C, D, HS, T cytoplasmic types if they are of another genotype.
Example 2 high throughput identification of five chloroplast cytoplasmic types in maize based on KASP technology
Purpose of the experiment: and identifying five chloroplast cytoplasm types of 96 maize inbred line germplasm materials.
1. Extracting DNA of a maize inbred line sample to be identified: 50 seeds are randomly selected from each sample, and then sprouting is carried out, and sufficient light is given 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: 1 pair or more pairs of primers for identifying the specificity of the chloroplast cytoplasm types B, C, D, HS and T are selected from the primers provided in the table 6 of the invention respectively 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 was 100. mu.M. The PCR reaction program is: 15min at 94 ℃; adopting a touchdown PCR mode, at 94 ℃ for 20s, at 61-55 ℃ for 1min (each cycle is reduced by 0.6 ℃),10 cycles; 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 sample.
4. And (4) judging a result: the determination was made based on the alleles in five chloroplast cytoplasmic types of maize for each pair of primers listed in table 6.
Example 3 identification of chloroplast cytoplasmic types in inbred lines or hybrid parents of maize using specific primers provided by the invention
The method for extracting the DNA of the maize inbred line or hybrid sample to be identified, PCR amplification and fingerprint data acquisition is the same as the example 2.
And (4) judging a result: because the corn chloroplast genome is inherited as a maternal line, the chloroplast cytoplasm type of the inbred line or hybrid of the corn to be detected is completely the same as that of the maternal line. The determination was made based on the alleles in five chloroplast cytoplasmic types of maize for each pair of primers listed in table 6.
Example 4 group marking of corn material by using the specific primer provided by the invention to reflect maternal evolution relationship
Purpose of the experiment: 100 parts of corn materials (including inbred lines and hybrid species) with different genetic backgrounds are subjected to grouping based on chloroplast specific primers, and maternal evolution relations of the corn materials are analyzed.
The method for extracting the DNA of the corn material to be detected, PCR amplification and fingerprint data acquisition is the same as the example 2.
Constructing a cluster map: using the Power-Marker ver.3.25 software, Rogers (1972) genetic distance calculation method was used to analyze and establish NJ clustering dendrograms (neighbor-join tree). 100 samples are divided into different groups, and the groups are five groups at most, namely five groups of B \ C \ D \ HS \ T determined in the invention. And analyzing the cluster to which each sample belongs and the correlation relationship among different clusters based on the topological structure of the cluster map.
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, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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<400> 4
ctccttttct atctaattag tttatgtttg ttctaattat aaaacaaaca agaacaaatc 60
<210> 5
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttcctagtgt tcggaaacgg aaaaccctga acctggagga gttaggtatg taggatatgc 60
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttttattttt tgttggacag gcagtagtat aattttgata ctctgcagta taaattcgac 60
<210> 7
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggttcgaatc cgataaagta cttttctact aaattcattc atttcttttt tgaaaatgtc 60
<210> 8
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccattttttt ttttttttgc aattttatga cttagtttag tgcgagatgc ccacattttt 60
<210> 9
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tagtagtgaa ttgggaacaa gaagaaaaag agggagctcg tgcttctctt gttgaggtaa 60
<210> 10
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aacaaatgat ctaattcgcg atttcctaag aattgagtta gtcaagtcta ctattttgta 60
<210> 11
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ttggatttga agaaaaaata aaaggaattc tatcaatttt tattttccat ttatttagtt 60
<210> 12
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gtttttctta atgaaattga aattattaac taacagagca aacacaaata aagaaacaac 60
<210> 13
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttcgaatcca tgaagtaaga cattgatttt gcaacaaggt caattatgtt cattgcataa 60
<210> 14
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aagttccctt gaaaagcatt ggcgcacgtg taaacgagtt gctctaccga actgagctat 60
<210> 15
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tgaatccact tttgttgggg ttcaaaaaac gaataaaaat aaataaaaaa aagtaaattt 60
<210> 16
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aggaatagtt ccctttttga gggggccctc gggggtcgtg gaatgctttt cttctcctct 60
<210> 17
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tttattactt aatttacgaa tttcaaaaat tttgtattct attggattgg atttgttcga 60
<210> 18
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aattcgaaga attacaacaa aatctttaga aatcacattt ttagttagga acttctatgg 60
<210> 19
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggttcctcct aatttttcta taatcaacat gttttccctt ttctttaaat ttaggatttt 60
<210> 20
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aagcatatac gtgagacaaa atctactaat tttttctttg atctatatct cgtctactag 60
<210> 21
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ataaaaacga ggattctatt ataaaaagta gactattctt gcaataggac ttacaacctc 60
<210> 22
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acctaatctt atccaaattc ccatataaat gggtttagga ctggtacatc atattaataa 60
<210> 23
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctcccagtct cgacgattca cgataaaata actattattc ttttaagtta actattattt 60
<210> 24
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aaattagccg ccatggtgaa attggtagac acgctgctct taggaagcag tgctcaagca 60
<210> 25
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 26
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ctctatccta ttcattggta ccgatcatgg atacttcaaa aattttatta tttgtttgaa 60
c 61
<210> 27
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cccatttttt tttttttttg caattttatg acttagttta gtgcgagatg cccacatttt 60
<210> 28
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggtctgaaca ctaaacgagc acagacttaa aattacaaaa aaaaatgaaa ttggactctt 60
<210> 29
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcaggggggc caagtcaggt tagatctata tctttaatgc ctataagaca gtcatctttt 60
<210> 30
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgtggctttc taaagaatga ttttagaatc ggattcaata gaaaatgaga aaataggcaa 60
<210> 31
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttcgtctttt attatgttag atgaagggga aaaaatggga actcaaagat atcgaagagt 60
<210> 32
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
aaaaaaagga tggaataaaa gagtgattgg ttgaaaagaa agagaaatag aataatgaga 60
<210> 33
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tagttgtatc gacccagtcg ctcactaatt gatctttacg gtgttttctc tatcaatttc 60
<210> 34
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gctttatcca tagaatagta gtataggctc tactttcttc ctattttgat tctcgtgaag 60
<210> 35
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ccaatcaaat attgagtaat caaatccttc aattcattgt tttcgagatc ttttaatttt 60
<210> 36
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aaaagtggat taatcggacg aggataaaga gagagtccca ttctacatgt caatactgac 60
<210> 37
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gcgtttcgat gaatgagcct atggtaatgc ttttatctct attctatggc gcaatcgacc 60
<210> 38
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tcgagtctat aaacaagtac taaataagga aaagaaaact atactaaagg aaacataaga 60
<210> 39
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cccgggacgc gaagtagtag gattggttct cataattatc acataatttt caaaaaaaaa 60
<210> 40
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gaatttgtcg aaattttttt tcttgttgaa taatgccaaa tcaaaaaaaa tatccaaaaa 60
<210> 41
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttttttaatt atagttattc ctatgcgaga gatagaattc ttcgtgacat gacgaaaatt 60
<210> 42
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cccctttttg aattcttttt tagtatatga agcaaaaaga aagaaaagat ggataaggat 60
<210> 43
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
aaaagcttct ttttcgaaag attacccctg tctttgttta tgcttcggat tggaacaaat 60
<210> 44
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
actctaattc gcccacgcct gcgaatcagt cgacattttg tacaaatttt acgaacggaa 60
<210> 45
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ctagagttgt aggagagaga atagactata ttatggaaga ggtaaagtat atatgcattc 60
a 61
<210> 46
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gggggccaag tcaggttaga tctatatctt taatgcctat aagacagtca tcttttatgt 60
g 61
<210> 47
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ccttcgattc taaaatgaaa attcttctac attgaatgta tagctgcagc aataaatttt 60
<210> 48
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
atcagccttt ctaccccctg cgcctacgtt gaacaggtac ctttaggtac ccacacaata 60
<210> 49
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gtattctata ccctttaaac gaatttcctt aaccttaaaa agcttaaaaa gtaggggatg 60
<210> 50
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tccgtgaatt aacctaacca tcaactaaaa aaaatcctat gaaagcataa cagaaaagta 60
<210> 51
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tatgaaatga tctactaact catctcagat gcaagtccac tttcaatata tctctgtata 60
<210> 52
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
agcggtaagg aaaaagtttt aataaaaaga agaatcaatg gattcatgat taaacccctc 60
<210> 53
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ctctccccct ttgtataaat atttcacatt tcaaatgcaa gtttgaaaga ttgtactgct 60
<210> 54
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ttttctttct ttttctgcac aaaagaaccc ctcctaattc actaatttgt aggaagatac 60
<210> 55
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
agaaatcgca actcttttcc gttacacata ataaataaag ggtttcaaaa gtcaattttt 60
<210> 56
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ttttcaatat ctttcttttt tttttcagaa ttccattttt gttcttccac ccatgcaata 60
<210> 57
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tggttggttc ttctcgccga gttttggcgt agcagctata tttcgcttca tccttttctt 60
<210> 58
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
caaggatttc ataattggac gttgaaacca tttcatatga tgggagttgc cggagtatta 60
<210> 59
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
aatagaaaat gaaacggtcg acccagacat agacggtcga cccagacata gacggtcgac 60
<210> 60
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
caggtggata taccctataa aatataggac gtagcaagcg tagttcaatg tagcgagcgt 60
<210> 61
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
caaacggttt tgaaaggggg ataggctatg cttttctttc attttttttt tttttttttc 60
<210> 62
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gcctgctaaa taaaaaaaaa gggttggata tagccctcta tcatatatat acaaatagaa 60
<210> 63
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
cctttttttc ttttatcttg aatctaattc tagttagttt tttagaatct ttttcaaaat 60
<210> 64
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gtttctgctt ttttggatcc agtttcgctt attctcctcg atggattcta tcttaaaaca 60
<210> 65
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tattattttt tgaattgcag tagtgaacga ctcttaaatc ggtattcccc cccattattt 60
<210> 66
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gaatggcata ataaaataga ataagaataa atgtttccct aatctgtata tagggaaact 60
<210> 67
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gagaccccgt ttacccctat ttatacgatt taagtataca taaagcaatt ttttttactt 60
<210> 68
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
aatttaaact atacttttga ccttagaatg ctaacaggtc tgattttcga ttttgtactt 60
<210> 69
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
aaactatact tttgacctta gaatgctaac aggtctgatt ttcgattttg tacttaaatt 60
<210> 70
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
aaattgtatt ttaataagta aaagaagtca gttaattcat taaggctatg tttataccgt 60
<210> 71
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
tgaatcaaaa gaattccttt tttgaagttc aatttttatc agaggacaat atgaatatta 60
<210> 72
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
accatgttcc attaaaacac tcaaggggtt atatgatata tcgggtgtag aagtagggca 60
<210> 73
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gacagaagga attgttagtt cacctcacct tccccaagcg cgggtttcct ttactaattt 60
<210> 74
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gttctctcta tgcgaaccct ctctctttct cgtaagaatg agatataggt agggctaaaa 60
<210> 75
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tcaagtccct ctatccccaa accctctttt attccctaac catagttgtt atcctttttt 60
<210> 76
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
cttttatcaa tgggtttaag attcactagc tttctcattc tactctttca caaaggagtg 60
<210> 77
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
caccatatca taagattcgc gttcttgaaa atcggcactt ctccaaaccc agaaaacgga 60
<210> 78
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gggattctag gattatcctt ttgggcaaag acttttatgc atacctcttc tgggttatct 60
<210> 79
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
acagagaatt ttcttagtat ttaggtattt agattcaaaa tatcaaaggg gaagaacttt 60
<210> 80
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
aaaattgtaa aataaagatt agggtttggg ttgcgctata tctatcaaag agtatacaat 60
<210> 81
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
actttttctt ggttaaagga acagatgatt cgatcgattt ctgtatcgat catgatatac 60
<210> 82
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
taataactcg cacatctatt tcaaacgcat atcccatttt tgcgcagcag ggttatgaaa 60
<210> 83
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ttgtatcgaa gtaagaatca tttttcttct tatttgtttt gtcaaagatt actatttatt 60
<210> 84
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
tttattcggg tctgtctttt ggacttcctt tgcttaggtt cgggcgcggt agtggacaaa 60
<210> 85
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
cgatcttaac ctgatgattc atcatcatga agtatttcta ttttctatag cataaaaccc 60
<210> 86
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
aatttaggtt tgagataaat ttacaagaaa tccacccact accaatcctt aaacatttct 60
<210> 87
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
ggaagataag cattcaatgt attttagagg aaaaagatcc tattttaacg aatcacacgt 60
<210> 88
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gagatattgc tagtacacaa aaagttaatg gtatttcata actaatagat tgagcagccg 60
<210> 89
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tttttactta ctttagttct ttagttttgg gaaaataaat agggggtact tcttttcttt 60
<210> 90
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
atattggggg tcaaatggat cctgtaagaa ttcccacttc atagatacgg ggtataaagt 60
<210> 91
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
ttttacttac tttagttctt tagttttggg aaaataaata gggggtactt cttttctttc 60
<210> 92
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
tattgggggt caaatggatc ctgtaagaat tcccacttca tagatacggg gtataaagtt 60
<210> 93
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
tttctagtgc ttataaattc ttatggcttt atcccgtttc atagaaagga gataaaacga 60
<210> 94
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
aaatctttgc tatccaatct ttttagaata tcataaagtt tcagtggcag aatttttttc 60
<210> 95
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
tgccaagaga ttggcatttt catttgatca ttatatacat ttttgagata ttttgttttt 60
<210> 96
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
tatttgttaa taatttaagg ataaatagtt cactaaggag aagatagaat catagcaaat 60
<210> 97
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cggtagtgga caaacagagg gaaagaaggt atggcgggga cacatttctt gtgagcaaat 60
<210> 98
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gaattgcttt actttttgaa ttaagttcaa ctttgaactt acagaaattt tgtaaaaaaa 60
<210> 99
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tctctatgtt ttattactta atttacgaat ttcaaaaatt ttgtattcta ttggattgga 60
<210> 100
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
ttgttcgaga attcgaagaa ttacaacaaa atctttagaa atcacatttt tagttaggaa 60
<210> 101
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
tttgtcgttc ccacagcttc tcctttaatg gttaggtttg aatcctgcaa tggagcttcc 60
<210> 102
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
attttttttt ccgagtcaat tttctcagtt ttattaaccc ggctgctctt tatttattgc 60
<210> 103
<211> 83
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
actgtataca cggatacaga atccgctata tccgtttgtg aaataaaggc taaatcccct 60
cccctcaact ccatatctaa ata 83
<210> 104
<211> 5
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
tcttt 5
<210> 105
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
agcaatctga gtttttcatt tttactaact ta 32
<210> 106
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gcaatctgag tttttcattt ttactaactt c 31
<210> 107
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
cttcatttac caaatccaaa aatttgggaa 30
<210> 108
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
aacaaacata aactaattag atagaaaagg agt 33
<210> 109
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
caaacataaa ctaattagat agaaaaggag c 31
<210> 110
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gaaagaaagg gagtctaatc catagaactt 30
<210> 111
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
atgtaggata tgctttttat tttttgttgg a 31
<210> 112
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
gtaggatatg ctttttattt tttgttggg 29
<210> 113
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ctgcagagta tcaaaattat actactgcct 30
<210> 114
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
aaattcattc atttcttttt tgaaaatgtc c 31
<210> 115
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ctaaattcat tcatttcttt tttgaaaatg tct 33
<210> 116
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
ggcatctcgc actaaactaa gtcataaa 28
<210> 117
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
taggaaatcg cgaattagat catttgttt 29
<210> 118
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ggaaatcgcg aattagatca tttgttc 27
<210> 119
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
gctcgtgctt ctcttgttga ggtaa 25
<210> 120
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
ctatcaattt ttattttcca tttatttagt ta 32
<210> 121
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
ctatcaattt ttattttcca tttatttagt tt 32
<210> 122
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
gtttctttat ttgtgtttgc tctgttagtt 30
<210> 123
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
caacaaggtc aattatgttc attgcataaa 30
<210> 124
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
caacaaggtc aattatgttc attgcataat 30
<210> 125
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
gcgccaatgc ttttcaaggg aactt 25
<210> 126
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
cccctcaaaa agggaactat tccta 25
<210> 127
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
cccctcaaaa agggaactat tcctt 25
<210> 128
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
ccacttttgt tggggttcaa aaaacgaat 29
<210> 129
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
tgtattctat tggattggat ttgttcgat 29
<210> 130
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gtattctatt ggattggatt tgttcgag 28
<210> 131
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
tctaaagatt ttgttgtaat tcttcgaatt 30
<210> 132
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tagtagattt tgtctcacgt atatgctta 29
<210> 133
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
agtagatttt gtctcacgta tatgcttt 28
<210> 134
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
catgttttcc cttttcttta aatttaggat 30
<210> 135
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
cttgcaatag gacttacaac ctcc 24
<210> 136
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
cttgcaatag gacttacaac ctct 24
<210> 137
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
cccatttata tgggaatttt ggataagatt 30
<210> 138
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
ccaatttcac catggcggct aattta 26
<210> 139
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
ccaatttcac catggcggct aatttt 26
<210> 140
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
cccagtctcg acgattcacg ataaa 25
<210> 141
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
cggtaccaat gaataggata gags 24
<210> 142
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
atcggtacca atgaatagga tagaga 26
<210> 143
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
ccaaaggaaa ccataaattc catataccat 30
<210> 144
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gtgctcgttt agtgttcaga cca 23
<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
gtgctcgttt agtgttcaga ccc 23
<210> 146
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cttagtttag tgcgagatgc ccacat 26
<210> 147
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
attctaaaat cattctttag aaagccacac 30
<210> 148
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ctaaaatcat tctttagaaa gccacat 27
<210> 149
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
ggccaagtca ggttagatct atatcttta 29
<210> 150
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
atgggaactc aaagatatcg aagagta 27
<210> 151
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
gggaactcaa agatatcgaa gagtc 25
<210> 152
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
caaccaatca ctcttttatt ccatcctttt 30
<210> 153
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
gcctatacta ctattctatg gataaagct 29
<210> 154
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
cctatactac tattctatgg ataaagcg 28
<210> 155
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tcgctcacta attgatcttt acggtgttt 29
<210> 156
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
atcctcgtcc gattaatcca ctttta 26
<210> 157
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
atcctcgtcc gattaatcca cttttt 26
<210> 158
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
ccttcaattc attgttttcg agatctttta 30
<210> 159
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tatttagtac ttgtttatag actcgac 27
<210> 160
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
ccttatttag tacttgttta tagactcgat 30
<210> 161
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
aatgctttta tctctattct atggcgcaat 30
<210> 162
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
attcaacaag aaaaaaaatt tcgacaaatt cc 32
<210> 163
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
attcaacaag aaaaaaaatt tcgacaaatt ct 32
<210> 164
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
gcgaagtagt aggattggtt ctcataatt 29
<210> 165
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tcatatacta aaaaagaatt caaaaagggg a 31
<210> 166
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
catatactaa aaaagaattc aaaaaggggg 30
<210> 167
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
gagatagaat tcttcgtgac atgacgaaa 29
<210> 168
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
aggcgtgggc gaattagagt c 21
<210> 169
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
caggcgtggg cgaattagag tt 22
<210> 170
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gtctttgttt atgcttcgga ttggaacaa 29
<210> 171
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
atctaacctg acttggcccc ct 22
<210> 172
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
ctaacctgac ttggcccccc 20
<210> 173
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
gagagaatag actatattat ggaagaggta 30
<210> 174
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
gcagggggta gaaaggctga ta 22
<210> 175
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
cagggggtag aaaggctgat c 21
<210> 176
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
ctacattgaa tgtatagctg cagcaataaa 30
<210> 177
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
agttgatggt taggttaatt cacggat 27
<210> 178
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
gttgatggtt aggttaattc acggag 26
<210> 179
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
taaccttaaa aagcttaaaa agtaggggat 30
<210> 180
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
ttttattaaa actttttcct taccgctttt a 31
<210> 181
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
ctttttatta aaactttttc cttaccgctt ttt 33
<210> 182
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
atgcaagtcc actttcaata tatctctgta 30
<210> 183
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
ccctcccctc aactccatat ctaaa 25
<210> 184
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
caagtttgaa agattgtact gctctttc 28
<210> 185
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
gcaagtttga aagattgtac tgctctttt 29
<210> 186
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
attaggaggg gttcttttgt gcagaaaaa 29
<210> 187
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
aataaataaa gggtttcaaa agtcaatttt tc 32
<210> 188
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
aataaataaa gggtttcaaa agtcaatttt ta 32
<210> 189
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
ggaattctga aaaaaaaaag aaagatattg 30
<210> 190
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
tcaacgtcca attatgaaat ccttgg 26
<210> 191
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
gttcaacgtc caattatgaa atccttga 28
<210> 192
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
gtagcagcta tatttcggtt catccttt 28
<210> 193
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
atattttata gggtatatcc acctgg 26
<210> 194
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
cctatatttt atagggtata tccacctgt 29
<210> 195
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
acatagacgg tcgacccaga cata 24
<210> 196
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
tttctttcat tttttttttt tttttttct 29
<210> 197
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
gcttttcttt catttttttt tttttttttt cc 32
<210> 198
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
tatccaaccc ttttttttta tttagcaggc 30
<210> 199
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
acttactttt ttagaatctt tttcaaaaaa ta 32
<210> 200
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
acttactttt ttagaatctt tttcaaaaaa tg 32
<210> 201
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 201
agcgaaactg gatccaaaaa agcagaaat 29
<210> 202
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 202
atttattctt attctatttt attatgccat tca 33
<210> 203
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 203
tattcttatt ctattttatt atgccattcc 30
<210> 204
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 204
tcttaaatcg gtattccccc ccattattt 29
<210> 205
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 205
ttaagtatac ataaagcaat tttttttact tt 32
<210> 206
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 206
taagtataca taaagcaatt ttttttactt g 31
<210> 207
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 207
gttagcattc taaggtcaaa agtatagttt 30
<210> 208
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 208
actgacttct tttacttatt aaaatacaat tta 33
<210> 209
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 209
actgacttct tttacttatt aaaatacaat ttc 33
<210> 210
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 210
ctaacaggtc tgattttcga ttttgtactt 30
<210> 211
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 211
caatttttat cagaggacaa tatgaatatt ac 32
<210> 212
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 212
caatttttat cagaggacaa tatgaatatt at 32
<210> 213
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 213
tataacccct tgagtgtttt aatggaacat 30
<210> 214
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 214
aagcgcgggt ttcctttact aatttt 26
<210> 215
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 215
agcgcgggtt tcctttacta atttg 25
<210> 216
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 216
agagagaggg ttcgcataga gagaa 25
<210> 217
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 217
agtgaatctt aaacccattg ataaaaga 28
<210> 218
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 218
agtgaatctt aaacccattg ataaaagc 28
<210> 219
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 219
tttattccct aaccatagtt gttatccttt 30
<210> 220
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 220
ccaaaaggat aatcctagaa tcccg 25
<210> 221
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 221
cccaaaagga taatcctaga atccca 26
<210> 222
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 222
atcggcactt ctccaaaccc agaaa 25
<210> 223
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 223
gattcaaaat atcaaagggg aagaacttta 30
<210> 224
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 224
caaaatatca aaggggaaga actttt 26
<210> 225
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 225
gcaacccaaa ccctaatctt tattttacaa 30
<210> 226
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 226
cgatttctgt atcgatcatg atatacg 27
<210> 227
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 227
atcgatttct gtatcgatca tgatataca 29
<210> 228
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 228
gatatgcgtt tgaaatagat gtgcgagtt 29
<210> 229
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 229
tatttgtttt gtcaaagatt actatttatt c 31
<210> 230
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 230
cttatttgtt ttgtcaaaga ttactattta ttt 33
<210> 231
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 231
ggaagtccaa aagacagacc cgaat 25
<210> 232
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 232
gtatttctat tttctatagc ataaaacccg 30
<210> 233
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 233
aagtatttct attttctata gcataaaacc ct 32
<210> 234
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 234
ggatttcttg taaatttatc tcaaacctaa 30
<210> 235
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 235
aaaagatcct attttaacga atcacacgta 30
<210> 236
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 236
agatcctatt ttaacgaatc acacgtg 27
<210> 237
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 237
taccattaac tttttgtgta ctagcaatat 30
<210> 238
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 238
aggatccatt tgacccccaa tatg 24
<210> 239
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 239
aggatccatt tgacccccaa tatc 24
<210> 240
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 240
ggaaaataaa tagggggtac ttcttttctt 30
<210> 241
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 241
aaataaatag ggggtacttc ttttctttca 30
<210> 242
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 242
aaataggggg tacttctttt ctttcg 26
<210> 243
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 243
cttacaggat ccatttgacc cccaa 25
<210> 244
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 244
atattctaaa aagattggat agcaaagatt tc 32
<210> 245
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 245
gatattctaa aaagattgga tagcaaagat tta 33
<210> 246
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 246
gctttatccc gtttcataga aaggagata 29
<210> 247
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 247
gaactattta tccttaaatt attaacaaat aa 32
<210> 248
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 248
gaactattta tccttaaatt attaacaaat ac 32
<210> 249
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 249
gccaagagat tggcattttc atttgatcat 30
<210> 250
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 250
agttgaactt aattcaaaaa gtaaagcaat tct 33
<210> 251
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 251
gttgaactta attcaaaaag taaagcaatt cg 32
<210> 252
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 252
cggggacaca tttcttgtga gcaaa 25
<210> 253
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 253
atttcaaaaa ttttgtattc tattggattg gat 33
<210> 254
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 254
tcaaaaattt tgtattctat tggattggac 30
<210> 255
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 255
tttgttgtaa ttcttcgaat tctcgaacaa 30
<210> 256
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 256
ttgaatcctg caatggagct tcca 24
<210> 257
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 257
gaatcctgca atggagcttc cc 22
<210> 258
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 258
gcagccgggt taataaaact gagaaaatt 29
Claims (5)
1. A primer combination of molecular markers for identifying maize D-type cytoplasmic material developed based on KASP technology, characterized in that,
the molecular markers for identifying the maize D-type cytoplasmic material include the following 2 SNP markers and 1 InDel marker, whose information is as follows:
wherein, the KASP primer for detecting the marker CPMIDP07 comprises an upstream primer 1, an upstream primer 2 and a downstream general primer with the sequence of SEQ ID NO. 171-.
2. A detection reagent or kit comprising the primer combination according to claim 1.
3. The method for identifying the D-type cytoplasmic material of the corn based on the KASP technology is characterized by comprising the following steps of:
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’。
4. the method as claimed in claim 3, 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; 94 ℃ for 20s, 58 ℃ for 1min, 30 cycles.
5. The primer combination of claim 1 or the detection reagent or kit of claim 2, which is used for corn sample detection and corn molecular marker-assisted breeding;
the maize is maize D-type cytosolic material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810119094.2A CN108441572B (en) | 2018-02-06 | 2018-02-06 | Method for identifying maize chloroplast cytoplasm type based on KASP technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810119094.2A CN108441572B (en) | 2018-02-06 | 2018-02-06 | Method for identifying maize chloroplast cytoplasm type based on KASP technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108441572A CN108441572A (en) | 2018-08-24 |
| CN108441572B true CN108441572B (en) | 2022-06-24 |
Family
ID=63192004
Family Applications (1)
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