CN108441572A - The identification method of DCIPThe chloroplast of maize cytoplasm type based on KASP technologies - Google Patents

Abstract

The present invention provides the identification method of the DCIPThe chloroplast of maize cytoplasm type based on KASP technologies.Five kinds of chloroplaset cytoplasm type identification special primers, respectively Type B, 12 pairs of SNP primers；C-type, 10 pairs of primers include 1 pair of InDel and 9 pair of SNP primer；D types, 3 pairs of primers include 1 pair of InDel and 2 pair of SNP primer；HS types, 19 pairs of primers include 2 pairs of InDel and 17 pair of SNP primers；T-type, 7 pairs of SNP primers.This method can be used for identifying the cytoplasm type of corn inbred line or cenospecies, identify the cytoplasm type of corn inbred line or cenospecies female parent, and carrying out classification to corn material draws the maternal evolution process of group's reflection.The applying of this method expanded in genomic level corn material guild division, maternal tracking analysis method, provide new approaches and means for maize cell cytoplasmic inheritance characteristic research.

Description

The identification method of DCIPThe chloroplast of maize cytoplasm type based on KASP technologies

Technical field

The invention belongs to crops technical field of molecular biology, specifically, being related to the maize leaves based on KASP technologies The identification method of green body cytoplasm type.

Background technology

Corn is global first big crop, has critical role in China's agricultural production；It is that hybridization breeding application is earliest The maximum crop of the industry market share is planted in most popular crop, and the whole world and China.The screening of Parent parents is that cultivation is excellent The key factor of corn hybrid seed, therefore the research report for being directed to parents' self-mating system guild division is a lot of, but it is all based on core The analysis of genome molecules label.It is mainly analyzed in terms of Matrix attachment region Chromosome recombination, the hereditary variation is main It is due to manually assembling caused by selection and breeding excellent material.

Chloroplaset is that green plants carries out photosynthetic organelle, possesses itself complete a set of genome, referred to as leaf Green body genome.Chloroplast gene structure is very conservative, and DNA is generally double-stranded circular molecule, higher plant Chloroplast base Because a group size is generally 120-160kb.Different from Matrix attachment region, Chloroplast gene has the characteristics that cytoplasmic inheritance, plant Chloroplast gene is monolepsis (majority is matrilinear inheritance), is not recombinated generally；Chloroplaset have genome it is smaller and Relatively conservative, molecular evolutionary rate is slow, and the hereditary variation of genome is easy to be influenced by population genetic effect；Therefore chloroplaset Molecular labeling is the effective means for studying Population Differentiation.Grass DCIPThe chloroplast of maize genome belongs to stringent matrilinear inheritance, Molecular labeling based on Chloroplast gene analyzes corn material, and it is green can to identify several leaves that corn is differentiated to form Body cytoplasm type.

The maturation of high-flux sequence and genome bioinformatic analysis technology, has effectively facilitated to Chloroplast gene Research.Single nucleotide polymorphism (SNP), polymorphic (InDel) the equimolecular mark of insertion and deletion are excavated from Chloroplast gene level Note site is possibly realized.KASP (Kompetitive Allele Specific PCR) is competitive ApoE gene Technology has the features such as experiment process is simple, efficient, flexible, easy to operate, is suitble to the dimorphisms types such as SNP/InDel Genotyping detects.

Existing corn inbred line, the classification of Germplasms and a stroke group are all based on cell nucleus gene group information, plant In cell in addition to Mesoplast heredity, also there is cytoplasmic inheritance, i.e. chloroplaset and chondriogen group information.Cytoplasmic skeleton Information especially Chloroplast gene has the advantages that above-mentioned, is more suitable for carrying out corn material from early differentiation classification and draws Group reflects maternal monosystem evolution process.Therefore, the present invention develops the special of DCIPThe chloroplast of maize cytoplasm type using weight sequencing data Site based on KASP Genotyping Platform Designing primers and carries out verification assessment, establishes DCIPThe chloroplast of maize cytoplasm type identification A kind of method.

Invention content

The object of the present invention is to provide the identification methods of the DCIPThe chloroplast of maize cytoplasm type based on KASP technologies.

The inventive concept of the present invention is as follows：The selection of (1) 170 part of corn representative materials, including all hybrids in China are excellent Gesture group, the samples such as sweet tea is glutinous, local varieties and cytoplasm CMS infertility types.(2) prepared by high concentration, high quality total DNA.(3) Based on the high-flux sequence of two generation microarray datasets, structure library size is 500bp, PE140, sequencing depth is 5 times.(4) full base Because of data unit sequence processing, Chloroplast gene splicing, is independently spliced using two softwares, be based on corn B73 chloroplast genes Group sequence belongs to the contig of Chloroplast gene using blast program screening, is assembled, and sequence accuracy is verified.(5) leaf Green body genome annotation, polymorphic site determine, using DOGMA software annotation Chloroplast genes, by 170 parts of material chloroplaset bases Because a group sequence is compared, 100 SNP/InDel Chloroplast gene variant sites are filtered out.(6) five kinds of chloroplaset born of the same parents of corn The determination of matter type utilizes MP (Maximum Parsimony) maximum parsimony method based on 170 parts of material Chloroplast gene sequences Phyletic evolution dendrogram is established respectively with ML (Maximum Likelihood) maximum likelihood method, as a result shows that all samples are drawn It is divided into five types.Five types are respectively：Type B is to improve auspicious moral, the blue card of improvement and U.S.'s Inbred Lines material；C-type For c-type cytoplasmic sterility material；D types are the miscellaneous excellent group of Lucia Red Cob, local varieties and tropical material；HS types are S type cytoplasm Sterile material and the miscellaneous excellent group's material of tangsipingtou；T-type is T-type cytoplasmic sterility material.(7) five kinds of cytoplasm type specifics identify position The determination of point, analyzes the Fst values (genetic differentiation coefficient) between five kinds of monoids, and Fst values are more than 0.9 for the special position of the monoid Point.Five kinds of cytoplasm types of corn B, C, D, HS, T are analyzed respectively obtains Type B specific site 12, c-type specific site 11, D types Specific site 4, HS types specific site 20, T-type specific site 7.(8) design of primers, assessment and verification are based on KASP skills The primer of the above-mentioned specific site of art System Design, the sample for representing five types using 94 parts carry out primer in terms of following four Assessment, first amplification whether succeed, whether second reflect matrilinear inheritance, and whether third consistent with sequencing result, the 4th whether For the special primer of five kinds of chloroplaset cytoplasm types.It is final to obtain five kinds of chloroplaset cytoplasm type identifications spies of corn B, C, D, HS, T Different primer is 12,10,3,19,7 pairs respectively.Site specifying information is shown in Table 1- tables 5, and primer specifying information is shown in Table 6.Base in the present invention See Fig. 1 in the Technology Roadmap that five kinds of chloroplaset cytoplasm type identification special primers of corn of KASP technical systems obtain.

It is provided by the invention to be used to identify corn Type B based on Chloroplast gene exploitation in order to realize the object of the invention The molecular labeling of cytosolic material, the molecular labeling are selected from least one of following 12 SNP markers, their site information As shown in table 1：

Table 1

Molecule mark for identifying corn C type cytoplasmic sterility material provided by the invention based on Chloroplast gene exploitation Note, the molecular labeling are selected from least one of following 9 SNP markers and 1 InDel label, their information such as 2 institute of table Show：

Table 2

Molecular labeling for identifying corn D type cytosolic materials provided by the invention based on Chloroplast gene exploitation, The molecular labeling is selected from least one of following 2 SNP markers and 1 InDel label, their site information such as table 3 It is shown：

Table 3

Molecule for identifying corn HS type cytoplasmic sterility materials provided by the invention based on Chloroplast gene exploitation Label, the molecular labeling are selected from least one of following 17 SNP markers and 2 InDel labels, their site information As shown in table 4：

Table 4

Wherein, the nucleotide sequence of the InDel labels CPMIDP01 and CPMIDP02 is respectively such as SEQ ID NO:103- Shown in 104.

Molecule mark for identifying corn T-type cytoplasmic sterility material provided by the invention based on Chloroplast gene exploitation Note, the molecular labeling are selected from least one of following 7 SNP markers, their site information is as shown in table 5：

Table 5

The physical location of the molecular labeling is the determination based on corn variety B73 Chloroplast genes sequence, described The version number of corn variety B73 Chloroplast genes is AGPv3.

The present invention also provides the primers for detecting molecular labeling described in table 1- tables 5 based on KASP technological development.

Preferably, for detecting the KASP primer sequences of label CPMSNP01~CPMSNP93 in table 1 respectively such as SEQ ID NO:Shown in 105-140, wherein for detect mark CPMSNP01 KASP primers include sense primer 1, sense primer 2 and under Universal primer is swum, sequence is SEQ ID NO:105-107, the KASP primers for detecting label CPMSNP02 are SEQ ID NO: 108-110, and so on.

Preferably, for detecting the KASP primer sequences of label CPMIDP10~CPMSNP81 in table 2 respectively such as SEQ ID NO:Shown in 141-170, wherein for detect mark CPMIDP10 KASP primers include sense primer 1, sense primer 2 and under Universal primer is swum, sequence is SEQ ID NO:141-143, the KASP primers for detecting label CPMSNP18 are SEQ ID NO: 144-146, and so on.

Preferably, for detecting the KASP primer sequences of label CPMIDP07~CPMSNP19 in table 3 respectively such as SEQ ID NO:Shown in 171-179, wherein for detect mark CPMIDP07 KASP primers include sense primer 1, sense primer 2 and under Universal primer is swum, sequence is SEQ ID NO:171-173, the KASP primers for detecting label CPMSNP07 are SEQ ID NO: 174-176, and so on.

Preferably, for detecting the KASP primer sequences of label CPMIDP01~CPMSNP96 in table 4 respectively such as SEQ ID NO:Shown in 180-237, wherein for detect mark CPMIDP01 KASP primers include sense primer 1, sense primer 2 and under Primer 1, downstream primer 2 are swum, sequence is SEQ ID NO:180-183, the KASP primers for detecting label CPMIDP02 include Sense primer 1, sense primer 2 and downstream universal primer, sequence are SEQ ID NO:184-186, for detecting label The KASP primers of CPMSNP08 include sense primer 1, sense primer 2 and downstream universal primer, and sequence is SEQ ID NO:187- 189, and so on.

Preferably, for detecting the KASP primer sequences of label CPMSNP03~CPMSNP86 in table 5 respectively such as SEQ ID NO:Shown in 238-258, wherein for detect mark CPMSNP03 KASP primers include sense primer 1, sense primer 2 and under Universal primer is swum, sequence is SEQ ID NO:238-240, the KASP primers for detecting label CPMSNP04 are SEQ ID NO: 241-243, and so on.

The present invention also provides detection reagents or kit containing the KASP primers.

51 pairs of SNP/InDel primers provided by the invention can realize the acquisition of genotype data based on KASP platforms. Concrete scheme is to be required to be directed to site design primer provided by the invention according to KASP technologies, and primer is free of for general primer Fluorophor；Purchase the PCR amplification system MasterMix of KASP complete sets of Techniques；Configure reaction system be added DNA, primer and MasterMix；Run response procedures；In-situ scanning fluorescence signal；Data analysis obtains genotype data.

The present invention also provides a kind of identification methods of DCIPThe chloroplast of maize cytoplasm type.Specifically comprise the following steps：(1) it extracts It is detected the total DNA of self-mating system or cenospecies material；(2) SNP/IDNEL primers provided by the invention are utilized, KASP genes are based on Parting platform carries out PCR amplification, fluorescent scanning obtains genotype data, and judges which kind of chloroplaset cytoplasm type belonged to.This hair The primer of bright offer can be additionally used in the cytoplasm type for identifying corn or cenospecies female parent, can be used for carrying out classification to corn material drawing Group reflects maternal evolution process.

The present invention also provides a kind of identification methods of five kinds of chloroplaset cytoplasm types of corn based on KASP technologies, including with Lower step：

1) DNA of corn sample to be measured is extracted；

2) KASP reacts：KASP Primer mix and KASP ROX is added in the DNA profiling extracted to step 1) Standard reaction mix carry out PCR amplification；

3) fluorescence detector is used to analyze PCR product.

The KASP Primer mix are that different fluorescence labels sequences are modified at each KASP primers middle and upper reaches primer 5 ' end respectively Afterwards, the mixture formed with downstream universal primer (downstream primer).

Preferably, the fluorescence labels sequence is：5’-FAM-GAAGGTGACCAAGTTCATGCT-3’；

5’-HEX-GAAGGTCGGAGTCAACGGATT-3’。

Preferably, PCR reaction systems are in step 2)：1.5 μ L, KASP ROX standard reaction of DNA profiling 0.5 μ L, KASP Primer mix of mix (Kbiosciences, Herts UK) 0.014 μ L, ddH₂O 0.5μL；The KASP A concentration of 100 μM of each primer in Primer mix.

Preferably, PCR response procedures are：94℃15min；By the way of touchdown PCR, 94 DEG C of 20s, 61-55 DEG C of 1min (each cycle reduces by 0.6 DEG C), 10 cycles；94 DEG C of 20s, 58 DEG C of 1min, 30 cycles.

Preferably, amplified production is swept using BMG Pherastar (LGC, Middlesex, UK) instrument progress fluorescence signal It retouches, obtains initial data.Initial data imports Kraken softwares (LGC, Middlesex, UK) and carries out each sample of analysis acquisition The finger print data of each data point.According to the finger print data in each site, the type is represented according to each site listed in table 6 Allele judged, if identical as allele described in table be its corresponding cytoplasm type, if it is another A allele is then not belonging to the cytoplasm type.

The present invention also provides the KASP primers, or the detection reagent containing the primer or kit are in identification corn five Application in kind chloroplaset cytoplasm type or selection and breeding sterile material.

The present invention further provides the molecular labeling or the KASP primers, or the detection reagent containing the primer or Application of the kit in corn sample detection, corn molecular mark.

The inventive point of the present invention is：(1) the isolated Chloroplast gene data from full-length genome data：Due to leaf Green body genome sequence is relatively conservative, and sequencing quality and length are enough, therefore Chloroplast gene data in the present invention Be obtained by connection scheme, in combination with DCIPThe chloroplast of maize reference gene group alignments.DCIPThe chloroplast of maize genome number It is to screen the contig of Chloroplast gene using Blast programs, use Sequencher software combinations according to committed step is obtained Chloroplast gene contig, assembled sequence are carried out with DCIPThe chloroplast of maize reference gene group (corn variety B73, AGPv3) Comparison confirms, provides safeguard to obtain accurately and reliably Chloroplast gene sequence.(2) five kinds of chloroplaset cytoplasm classes of corn The determination of type：Based on derive from a wealth of sources, the Chloroplast gene sequence information for 170 parts of materials that phenotype and genotype are abundant, utilize PAUP4.0 and RAxML7.04 softwares, MP (Maximum Parsimony) maximum parsimony methods and ML (Maximum Likelihood) maximum likelihood method establishes phyletic evolution dendrogram respectively.The dendrogram result that two methods are established shows all Corn sample is divided into five kinds of chloroplaset cytoplasm types.(3) five kinds of chloroplaset cytoplasm type specifics of KASP technologies corn are based on Primer determines：The variant sites of all types corn inbred line are analyzed, screens and determines five kinds of cytoplasm type specific sites, Based on KASP technical system design evaluation primers.Primer screening standard is whether to expand success, if follows matrilinear inheritance, gene Type data accuracy etc., the final special primer for determining cytoplasm type identification.

The present invention is derived from a wealth of sources by collection, phenotype and genotype are abundant, representative 170 parts of strong corn inbred line materials Material obtains Chloroplast gene sequence, and analyzes the cytoplasm type of 170 parts of materials.Compare Chloroplast gene nucleotide polymorphism Property, the specific site of exploitation identification DCIPThe chloroplast of maize cytoplasm type based on KASP technical systems design primer and is assessed, is built Found a kind of identification method of DCIPThe chloroplast of maize cytoplasm type.This method can be used for identifying the cytoplasm class of corn inbred line or cenospecies Type identifies the cytoplasm type of corn or cenospecies female parent, and carrying out classification to corn material draws the maternal evolution process of group's reflection.The party The applying of method expanded in genomic level corn material guild division, maternal tracking analysis method, for for maize cell The researchs such as cytoplasmic inheritance characteristic provide new approaches and technological means.

Description of the drawings

Fig. 1 is that the present invention is based on five kinds of chloroplaset cytoplasm type identification special primers of corn of KASP technical systems to obtain Technology Roadmap.

Fig. 2A-Fig. 2 E are the experiment knot that in the present invention five kinds of cytoplasm type specifics of corn B, C, D, HS, T are identified with primer Fruit.Figure orbicular spot represents the genotype of corn material.Fig. 2A is that Type B cytoplasm type specific identifies that primer, Fig. 2 B are c-type cytoplasm class Type unique identification primer, Fig. 2 C are that D type cytoplasm type specifics identify that primer, Fig. 2 D are that H/S type cytoplasm type specifics identify primer, Fig. 2 E are that T-type cytoplasm type specific identifies primer.

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Acquisition of the embodiment 1 based on five kinds of chloroplaset cytoplasm type identification special primers of KASP technical systems corn

1, sample is chosen：Choosing 170 parts has representative corn inbred line progress genome sequencing extensively.This 170 Part sample includes the Types of Maize such as conventional corn, waxy corn, corn, pop corn；Include all Heterotic Groups in China, Tangsipingtou, Lucia Red Cob, Rui De, Lan Ka, the auspicious moral of improvement, the blue card of improvement, tri- kinds of P groups, local varieties and T, C, S cells Matter infertility types of material.

2, sample preparation：The seed of 170 parts of corn samples sends out seedling 5 days in incubator, first 3 days be non-illuminated conditions, rear 2 It is illumination condition (giving substantial light photograph after being unearthed).Each sample is therefrom chosen 30 plants of green seedling leafs and is mixed, liquid nitrogen Under the conditions of be fully ground.Total DNA is extracted using CTAB methods, and removes RNA.Distinguished with ultraviolet specrophotometer and agarose electrophoresis The quality of the extracted DNA of detection, agarose electrophoresis show that DNA bands are single, do not degrade；UV spectrophotometer measuring A260/280 (DNA does not have protein contamination, rna content low) between 1.8-2.0；A260/230 is between 1.8-2.0 (DNA salt ion contents are low)；DNA concentration is more than 1000ng/ μ L.

3, total DNA high-flux sequence：170 parts of corn sample DNAs and PCR product are interrupted using ultrasound, gel extraction The DNA fragmentation of 400-600bp utilizesThe library for building library kit structure 500bp sizes, utilize Hiseq The microarray dataset of 4000PE150 is sequenced, and sequencing depth is 5 times, and average each sample obtains about 10GB data.

4, high-flux sequence data processing, Chloroplast gene splicing：High-flux sequence data independently use Two softwares of SPAdes (Bankevich et al., 2012) and SOAPdenovo2 (Luo et al., 2012) are spliced. Using Blast programs filter out the contig (Altschul of Chloroplast gene for the contig of each software splicing et al.,1997)；The Chloroplast gene contig filtered out is assembled using Sequencher.Then it uses Geneious 8.1 (Kearse et al., 2012) is all reads map to the Chloroplast gene sequence spliced On, whether correct verify the contig sequences being spliced into.

5, Chloroplast gene annotation, polymorphic site determine：Chloroplast gene annotation DOGMA (Dual Organellar Geno Me Annotator) (Wyman et al., 2004) carry out, with BLASTX and BLASTN search identifications The position of encoding gene.170 DCIPThe chloroplast of maize genomes be compared with MAFFT softwares (Katoh and Standley, 2013), then manual setting comparison result is carried out with Se-al softwares.The principle compared for the inversion occurred inside sequence is It is pulled open, in order to avoid cause the multimodality of mistake.The change dystopy in two Chloroplast genes is counted using DnaSp 5.0 Point and sequence polymorphism (Librado and Rozas, 2009), exploitation obtain 100 SNP/InDel polymorphic sites.

6, the determination of five kinds of chloroplaset cytoplasm types of corn：Based on the 170 parts of materials derived from a wealth of sources, phenotype and genotype are abundant The Chloroplast gene sequence information of material, using PAUP4.0 and RAxML7.04 softwares, MP (Maximum Parsimony) is maximum Parsimony principle and ML (Maximum Likelihood) maximum likelihood method establish phyletic evolution dendrogram respectively.What two methods were established Dendrogram result shows that all corn samples are divided into five kinds of chloroplaset cytoplasm types.Five types are respectively：Type B is improvement Rui De, the blue card of improvement and U.S.'s Inbred Lines material；C-type is c-type cytoplasmic sterility material；D types are that Lucia Red Cob is miscellaneous excellent Group, local varieties and tropical material；HS types are S type cytoplasmic sterility materials and the miscellaneous excellent group's material of tangsipingtou；T-type is T-type Cytoplasmic sterility material.

7, the determination in five kinds of chloroplaset cytoplasm type specific sites of corn：100 based on 170 parts of corn representative materials SNP/InDel polymorphic gene type data, using between the weir-fst-pop functional analysis Different groups in VCFtools softwares Fst values, i.e. genetic differentiation coefficient between Different groups, Fst values think that the site is specific site more than 0.9.To corn B, five kinds of cytoplasm types of C, D, HS, T are analyzed respectively obtains Type B specific site 12, c-type specific site 11, D type specific sites 4, HS types specific site 20, T-type specific site 7.

8, KASP platforms are based on and carry out design of primers, assessment and verification：Above-mentioned special position is designed based on KASP technical systems The primer of point.SNP and InDel (insertion and deletion for being less than 26bp) site, need to design 3 primers, two are allele specific Primer, one be general reverse primer.For Insert Fragment be more than 26bp the sites InDel, be directed to respectively insertion and Missing two pairs of primers of design.94 parts of samples for representing five types are chosen, designed based on the assessment verification of KASP technical systems Primer.Above-mentioned material mainly verifies primer amplification effect, and whether marker site follows strictly matrilinear inheritance, genotype data and survey Sequence result consistency, if be the special primer of five types.

9, primer verification test process：The mode of mixed strain extraction DNA is taken in DNA extractions, is mixed with the greenery of 30 single plants, DNA extract specific steps according to maize dna Molecular Identification standard execute (Wang Fengge etc., 2014, corn variety identification technology regulation SSR marker method, People's Republic of China's agricultural industry criteria).DNA mass and concentration 2000 (Thermo of NanoDrop Scientific) ultraviolet specrophotometer is measured, and a concentration of 20ng/ μ L of working solution are adjusted according to measured value.PCR reactants System is 1 μ L, including 1.5 μ L total DNAs (drying), 0.5 μ L KASP ROX standard reaction mix (Kbiosciences, Herts UK), 0.014 μ L primer mixtures, 0.5 μ L deionized waters.Response procedures are in water-bath PCR instrument It is executed in (Hydrocycler HC-64), response procedures are 94 DEG C of 15min；Cycle 10 times, takes touchdown PCR mode, 94 DEG C 20s, 61 DEG C of 1min, (61 DEG C drop to 55 DEG C, and each cycle reduces by 0.6 DEG C)；94 DEG C of 20sec, 58 DEG C of 1min are recycled 30 times. Fluorescence signal scanning is carried out to PCR product using BMG Pherastar (LGC, Middlesex, UK), obtains initial data；Profit Genotype statistics (LGC, Middlesex, UK) is carried out with Kraken softwares.

10, the special primer of identification five kinds of chloroplaset cytoplasm types of corn is determined：Experiment number based on above-mentioned 94 parts of materials According to the primer for being directed to design is assessed in terms of following four.First, whether primer amplification succeeds；Second, if reflection Maternal inheritance characteristics；Whether third, genotype data are consistent with sequencing result；4th, it verifies whether as five kinds of chloroplaset born of the same parents The special primer of matter type.Finally obtaining corn five kinds of chloroplaset cytoplasm type identification special primers of B, C, D, HS, T is respectively 12,10,3,19,7 pairs.

The skill that five kinds of chloroplaset cytoplasm type identification special primers of corn in the present invention based on KASP technical systems obtain Art route map is shown in Fig. 1.Fig. 2A-Fig. 2 E illustrate the experiment knot of five kinds of cytoplasm type specific identification primers of corn B, C, D, HS, T Fruit.

Using 12 pairs provided by the invention, 10 pairs, 3 pairs, 19 pairs, 7 pairs of primers differentiate respectively corn inbred line whether be B, C, D, HS, T-type infertility analysis result figure see Fig. 2A-Fig. 2 E.In Fig. 2A-Fig. 2 E in each pair of primer result parting figure, per number Strong point is each sample genotyping result in each pair of primer；Data point is negative control sample, remainder data near origin A point part has respectively represented two kinds of homozygous genotype results that each pair of primer is shown close to X-axis, a part close to Y-axis；It utilizes Information of the genotype data data that software is read respectively with each pair of primer in table 1, table 2, table 3, table 4, table 5 is checked, It is corresponding B, C, D, HS, T cytoplasm type if consistent with " five kinds of cytoplasm type allele of corn B, C, D, HS, T ", such as Fruit is that another genotype is then non-B, C, D, HS, T cytoplasm type.

The high throughput identification method of the five kinds of chloroplaset cytoplasm types of the corn based on KASP technologies of embodiment 2

Experiment purpose：Its five kinds of chloroplaset cytoplasm types are identified to 96 corn inbred line germplasm materials.

1, corn inbred line sample DNA extraction to be identified：Each sample randomly selects 50 seeds, carries out hair seedling, gives Substantial light is shone, and green seedling is formed.DNA extractions take the mode of mixed strain extraction DNA, each sample to be randomly selected from 50 single plants The greenery of 30 single plants and mixing, DNA extract specific steps execute according to maize dna Molecular Identification standard (Wang Fengge etc., 2014, corn variety identification technology regulation SSR marker method, People's Republic of China's agricultural industry criteria).Dilution DNA forms work Make liquid, a concentration of 20ng/ μ L.

2, PCR amplification：Five kinds of chloroplaset cytoplasm type specifics of B, C, D, HS, the T provided from table 6 of the present invention identify primer In, 1 pair is chosen respectively or multipair is expanded.PCR reaction systems are：1.5 μ L, KASP ROX standard of DNA profiling 0.5 μ L, KASP Primer mix of reaction mix (Kbiosciences, Herts UK) 0.014 μ L, ddH₂O 0.5μL； A concentration of 100 μM of each primer in the KASP Primer mix.PCR response procedures are：94℃15min；Using touchdown PCR Mode, 94 DEG C of 20s, 61-55 DEG C of 1min (each cycle reduces by 0.6 DEG C), 10 cycles；94 DEG C of 20s, 58 DEG C of 1min, 30 Cycle.

3, finger print data obtains：Amplified production is carried out using BMG Pherastar (LGC, Middlesex, UK) instrument Fluorescence signal scans, and obtains initial data.Initial data importing Kraken softwares (LGC, Middlesex, UK) carry out analysis and obtain Obtain the finger print data of each each data point of corn sample.

4, result judgement：Equipotential based on each pair of primer listed in table 6 in five kinds of chloroplaset cytoplasm types of corn Gene is judged.

Embodiment 3 utilizes special primer provided by the invention identification corn inbred line or the chloroplaset cytoplasm of cenospecies female parent Type

Corn inbred line to be identified or the extraction of cenospecies sample DNA, PCR amplification, the same embodiment of finger print data preparation method 2。

Result judgement：Since DCIPThe chloroplast of maize genome is matrilinear inheritance, so corn inbred line to be detected or cenospecies Chloroplaset cytoplasm type it is identical with its female parent.Based on each pair of primer listed in table 6 in five kinds of chloroplaset born of the same parents of corn Allele in matter type is judged.

Embodiment 4 carries out corn material using special primer provided by the invention to draw the maternal Evolvement of group's reflection

Experiment purpose：The corn material (including self-mating system and cenospecies) of 100 parts of different genetic backgrounds, it is special based on chloroplaset Different primer carries out drawing group and analyzes its maternal Evolvement.

Corn material DNA extractions to be measured, PCR amplification, finger print data preparation method is the same as embodiment 2.

Dendrogram is built：Using Power-Marker ver.3.25 softwares, Rogers (1972) genetic distance is selected to calculate NJ Dendrograms (neighbor-joining trree) are established in method, analysis.100 parts of samples are divided into different monoids It is interior, up to five big monoids, i.e., five major class of B C D HS T determined in the present invention.Topological structure based on dendrogram, analysis The correlativity between monoid and Different groups belonging to each sample.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

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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 (10)

1. the molecular labeling for identifying corn Type B cytosolic material based on Chloroplast gene exploitation, which is characterized in that described Molecular labeling is selected from least one of following 12 SNP markers, their information is as shown in table 1：

Table 1

2. the molecular labeling for identifying corn C type cytoplasmic sterility material based on Chloroplast gene exploitation, which is characterized in that The molecular labeling is selected from least one of following 9 SNP markers and 1 InDel label, their information is as shown in table 2：

Table 2

。

3. the molecular labeling for identifying corn D type cytosolic materials based on Chloroplast gene exploitation, which is characterized in that described Molecular labeling is selected from least one of following 2 SNP markers and 1 InDel label, their information is as shown in table 3：

Table 3

。

4. based on the molecular labeling for identifying corn HS type cytoplasmic sterility materials of Chloroplast gene exploitation, feature exists In the molecular labeling is selected from least one of following 17 SNP markers and 2 InDel labels, their information such as table 4 It is shown：

Table 4

5. the molecular labeling for identifying corn T-type cytoplasmic sterility material based on Chloroplast gene exploitation, which is characterized in that It is described.Molecular labeling is selected from least one of following 7 SNP markers, their information is as shown in table 5：

Table 5

。

6. the primer for requiring any one of the 1-5 molecular labelings for test right based on KASP technological development, feature exist In for detecting the KASP primer sequences of label CPMSNP01~CPMSNP93 in table 1 respectively such as SEQ ID NO:105-140 institutes Show, wherein the KASP primers for detecting label CPMSNP01 include sense primer 1, sense primer 2 and downstream universal primer, sequence It is classified as SEQ ID NO:105-107, the KASP primers for detecting label CPMSNP02 are SEQ ID NO:108-110, with this Analogize；

For detecting the KASP primer sequences of label CPMIDP10~CPMSNP81 in table 2 respectively such as SEQ ID NO:141-170 It is shown, wherein the KASP primers for detecting label CPMIDP10 include sense primer 1, sense primer 2 and downstream universal primer, Sequence is SEQ ID NO:141-143, the KASP primers for detecting label CPMSNP18 are SEQ ID NO:144-146, with This analogizes；

For detecting the KASP primer sequences of label CPMIDP07~CPMSNP19 in table 3 respectively such as SEQ ID NO:171-179 It is shown, wherein the KASP primers for detecting label CPMIDP07 include sense primer 1, sense primer 2 and downstream universal primer, Sequence is SEQ ID NO:171-173, the KASP primers for detecting label CPMSNP07 are SEQ ID NO:174-176, with This analogizes；

For detecting the KASP primer sequences of label CPMIDP01~CPMSNP96 in table 4 respectively such as SEQ ID NO:180-237 It is shown, wherein for detect mark CPMIDP01 KASP primers include sense primer 1, sense primer 2 and downstream primer 1, under Primer 2 is swum, sequence is SEQ ID NO:180-183, for detect label CPMIDP02 KASP primers include sense primer 1, Sense primer 2 and downstream universal primer, sequence are SEQ ID NO:184-186, the KASP for detecting label CPMSNP08 draw Object includes sense primer 1, sense primer 2 and downstream universal primer, and sequence is SEQ ID NO:187-189, and so on；

For detecting the KASP primer sequences of label CPMSNP03~CPMSNP86 in table 5 respectively such as SEQ ID NO:238-258 It is shown, wherein the KASP primers for detecting label CPMSNP03 include sense primer 1, sense primer 2 and downstream universal primer, Sequence is SEQ ID NO:238-240, the KASP primers for detecting label CPMSNP04 are SEQ ID NO:241-243, with This analogizes.

7. the detection reagent containing primer described in claim 6 or kit.

8. the identification method of five kinds of chloroplaset cytoplasm types of corn based on KASP technologies, which is characterized in that include the following steps：

1) DNA of corn sample to be measured is extracted；

2) KASP reacts：KASP Primer mix and KASP ROX standard are added in the DNA profiling extracted to step 1) Reaction mix carry out PCR amplification；

3) fluorescence detector is used to analyze PCR product；

The KASP Primer mix are that difference is modified at each KASP primers middle and upper reaches primer 5 ' end described in claim 6 respectively After fluorescence labels sequence, the mixture with the formation of downstream universal primer；

Preferably, the fluorescence labels sequence is：5’-FAM-GAAGGTGACCAAGTTCATGCT-3’；

5’-HEX-GAAGGTCGGAGTCAACGGATT-3’。

9. according to the method described in claim 8, it is characterized in that, PCR reaction systems are in step 2)：1.5 μ L of DNA profiling, 0.5 μ L, KASP Primer mix of KASP ROX standard reaction mix 0.014 μ L, ddH₂O 0.5μL；It is described A concentration of 100 μM of each primer in KASP Primer mix.

PCR response procedures are：94℃15min；94 DEG C of 20s, 61-55 DEG C of 1min (each cycle reduces by 0.6 DEG C), 10 cycles； 94 DEG C of 20s, 58 DEG C of 1min, 30 cycles.

10. primer described in any one of the claim 1-5 molecular labelings or claim 6 is in corn sample detection, corn dividing Application in sub- marker-assisted breeding.