CN108486263A - Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker - Google Patents
Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker Download PDFInfo
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Abstract
The present invention provides corn male sterility T-type cytoplasm type is differentiated using chloroplastic marker, chloroplastic marker of the invention is to develop to obtain based on Chloroplast gene, shares 7 SNP markers.The molecular labeling or its any one or more molecular labeling combination can be used for identifying the germplasm materials of corn T-type male cytoplasmic sterility and normal cytoplasm, can be used for identifying the cenospecies of the production of hybrid seeds of T-type infertility and the conventional production of hybrid seeds, it may also be used for the purity of identification T-type sterile material.Molecular labeling provided by the invention is combined as the production of hybrid seeds of corn T-type cytoplasmic sterility, sterile material selection and breeding provide identification technology support, and strong guarantee is provided for the mating seed production of corn three-line；The range that corn serviceable indicia site has been expanded in genomic level provides new idea and method for researchs such as maize cell cytoplasmic inheritance characteristics.
Description
Technical field
The invention belongs to crops technical field of molecular biology, specifically, relating to the use of chloroplastic marker differentiates jade
Rice male sterility T-type cytoplasm type.
Background technology
Corn is the earliest crop for realizing heterosis utilization, and the selection and breeding and utilization of cenospecies are known as in crop production
Revolution.Hybrid maize seed production method has two methods of routine and male sterility, and it is miscellaneous to prepare corn using male sterility
Kind is handed over, a large amount of labours is not only saved, reduces cost, but also purity of hybrid can be improved, improves yield.
Corn male sterility (male sterile, MS) refer to corn stamen development extremely generate nonfunctional pollen and it is female
Stamen develops the solid phenomenon that normally can normally pollinate.Corn male sterility can substantially be divided into cytoplasmic male sterility
(cytoplasmic male sterility, CMS) and nuclear male sterility (genetic male sterility, GMS)
Two classes.Cytoplasmic male sterility (CMS) is the nucleo-cytoplasmic interaction male sterility system by nucleus and cytogene co- controlling,
Sterile line easy to implement, maintainer and restorer it is mating, be the main male sterility utilized in corn breeding and breeding production
Type.So far it has been found that the CMS materials in more than 200 kinds of different cytoplasms source will according to the special validity response of male flower fertility restorer
CMS materials are divided into T-type, c-type and S type three types, and different types of material male flower Fertility reaction and cytoplasmic effect performance are not
Together.
Need to solve the problems, such as three using the CMS production of hybrid seeds, the first male cytoplasmic sterility type identification, the second male sterile material
Purity, third utilize the identification of sterile line preparing hybrid kind.Therefore a set of Sites Combination for being suitable for identifying CMS materials is obtained
It has a very important significance.Chloroplast gene group information is widely used in plant variety, kind due to having the advantage that
Matter resource identifies that affiliation is evaluated, phyletic evolution, in the research such as cytoplasmic inheritance characteristic and application.(1) Chloroplast gene
Smaller and relatively conservative, complete sequence is easy to get；(2) chloroplast gene is matrilinear inheritance, the exchange of gene between Different Individual
Seldom occur with fusion, has good synteny between each gene of chloroplaset；(3) Chloroplast gene is in addition to inverted repeat area
Single copy gene there's almost no paralog gene interference；(4) chloroplaset code area and noncoding region evolutionary rate difference are aobvious
It writes, there are some high saltation zones, can solve the problems, such as kind or less taxon.Since CMS is closely related with cytoplasm, adopt
It is a kind of stabilization, reliable, innovation method with marker site identification Sterile cytoplasm classification on Chloroplast gene.Exploitation is suitable for
Chloroplaset SNP or the InDel marker site of CMS materials identification, multiple Sites Combinations whiles, use, and it is reliable can to enhance identification
Property reduce it is uncertain.
Report that corn male sterility cytoplasm type identification method has crop field Fertility observation, cytology comparison in difference, fertility
Restore specially effect property identification, the methods of marker site PCR amplification.First three methods are long qualification cycle, and expert evidence range is limited；Base
In PCR method currently without report for the corn male sterility cytoplasm type in the sites Chloroplast gene exploitation SNP or InDel
Identification method, can not achieve the identification of high-throughput automation.It is reflected for corn C MS types based on Chloroplast gene exploitation
Fixed marker site and method has not been reported.Therefore, it is suitable for corn male not using the exploitation of the heavy sequencing data of high quality is a set of
The chloroplaset molecular labeling site for educating T-type cytoplasm type is very necessary.
Invention content
The object of the present invention is to provide the methods for differentiating corn male sterility T-type cytoplasm type using chloroplastic marker.
In order to realize the object of the invention, the present invention by collect derive from a wealth of sources, phenotype and genotype are abundant, representative strong
170 parts of inbreds (including three kinds of CMS types of material) obtain Chloroplast gene sequence, it is more to compare nucleotide
State property, the sites SNP and InDel of exploitation identification corn male sterility T-type cytoplasm type.It has main steps that：(1) 170 parts are chosen
Corn representative test material, type include all Heterotic Groups in China, the samples such as sweet tea is glutinous, local varieties, CMS infertility types
Product.(2) prepared by high concentration, high quality total DNA.(3) high-flux sequence based on two generation microarray datasets, structure library size are
500bp, PE140, sequencing depth are 5 times.(4) whole genome sequence data processing, Chloroplast gene splicing, it is soft using two
Part independently splices, and belongs to Chloroplast gene using blast program screening based on corn B73 Chloroplast genes sequence
Contig is assembled, and sequence accuracy is verified.(5) Chloroplast gene annotation, polymorphic site determine, utilize DOGMA softwares
Annotate Chloroplast gene.(6) 170 parts of material Chloroplast gene sequences are compared, and filter out 100 SNP/InDel leaves
Green body genome mutation site.(7) corn T-type Sterile cytoplasm classification identification specific site determination, analyze Different groups between
Fst values (genetic differentiation coefficient), Fst values be more than 0.9 be monoid specific site, corn T-type infertility types of material analysis obtain
Obtain 7 SNP sites.Site specifying information is shown in Table 1, and the physical location in site is to be based on corn variety B73 Chloroplast gene sequences
Row compare determining.It is suitable for differentiating the technology of 7 chloroplaset sites exploitation of corn male sterility T-type cytoplasm type in the present invention
Route map is shown in Fig. 1.
The present invention provides the molecular labeling for differentiating corn male sterility T-type cytoplasm, including 7 SNP markers,
Their information is shown in Table 1.
Table 1
Since above-mentioned 7 SNP sites provided by the invention are two condition label, so the molecular labeling is any one or more
Molecular labeling combination can realize the acquisition of genotype data based on KASP platforms.Concrete scheme is to be wanted according to KASP technologies
It asks and is directed to site design primer provided by the invention, primer is that general primer is free of fluorescence group；Purchase KASP complete sets of Techniques
PCR amplification system MasterMix；It configures reaction system and DNA, primer and MasterMix is added；Run response procedures；It is in situ
Scan fluorescence signal；Data analysis obtains genotype data.
The molecular labeling of the present invention can be obtained by following primer amplification respectively：CPMSNP03, SEQ ID NO.15-17；
CPMSNP04, SEQ ID NO.18-20；CPMSNP29, SEQ ID NO.21-23；CPMSNP47, SEQ ID NO.24-26；
CPMSNP67, SEQ ID NO.27-29；CPMSNP73, SEQ ID NO.30-32；CPMSNP86, SEQ ID NO.33-35.
Further, the present invention provides the specific primer sets for detecting molecular labeling of the present invention, specifically
It is following any one group：CPMSNP03, SEQ ID NO.15-17；CPMSNP04, SEQ ID NO.18-20；CPMSNP29, SEQ
ID NO.21-23；CPMSNP47, SEQ ID NO.24-26；CPMSNP67, SEQ ID NO.27-29；CPMSNP73, SEQ ID
NO.30-32；CPMSNP86, SEQ ID NO.33-35.
Further, the answering in identifying corn male sterility T-type cytoplasm type the present invention provides above-mentioned molecular labeling
With.
The present invention provides above-mentioned molecular labeling answering in the cenospecies that identification corn T-type cytoplasm male sterility line is prepared
With.
The present invention provides application of the above-mentioned molecular labeling in identifying T-type cytoplasmic male sterilty corn material.
The present invention provides application of the above-mentioned molecular labeling in corn molecular mark.
The present invention provides application of the above-mentioned molecular labeling in Identification of Corn Germplasms.
Above-mentioned application, specifically includes following steps：
1) DNA of corn sample to be measured is extracted；
2) it using the DNA of step 1) extraction as template, according to the molecular labeling, is set respectively based on KASP detection platforms technology
Primer is counted, PCR amplification is carried out；
3) fluorescence detector is used to analyze PCR product.
Wherein, step 2) PCR amplification program is：94℃15min；94 DEG C of 20s, 61 DEG C of 1min are recycled 10 times, each to recycle
Reduce by 0.6 DEG C；94 DEG C of 20s, 58 DEG C of 1min are recycled 30 times.
Above-mentioned steps 3) in, if PCR product is T-type infertility single plant if identical as allele described in table 1, if it is
Another allele is then not belonging to T-type infertility single plant.
The present invention provides application of the above-mentioned molecular labeling in identifying T-type cytoplasmic male sterilty corn material purity.
In the detailed process of identification T-type cytoplasmic male sterilty corn material purity, include the following steps：
1) DNA of corn sample to be measured is extracted；
2) using the DNA of step 1) extraction as template, according to the above-mentioned molecular labeling of the present invention, KASP detection platform technologies are based on
Primer is separately designed, PCR amplification is carried out；
3) fluorescence detector is used to analyze PCR product；
4) if PCR product is T-type infertility single plant if identical as the allele, if it is another allele
Then it is not belonging to T-type infertility single plant；Each site carries out purity calculating, and computational methods are：(T-type infertility single plant number/detection
Total single plant number) × 100%.
The key point of the present invention is：
(1) DCIPThe chloroplast of maize genomic data detaches：The isolated Chloroplast gene data from full-length genome data
It is the difficult point and key point of the present invention.Since Chloroplast gene sequence is relatively conservative, and sequencing quality and length are enough, because
Chloroplast gene data are obtained by the scheme of splicing in the present invention for this, in combination with DCIPThe chloroplast of maize reference gene
Group alignments.DCIPThe chloroplast of maize genomic data is mainly comprised the following steps, and Chloroplast gene is screened using Blast programs
Contig, using Sequencher software combination Chloroplast gene contig, assembled sequence is referred to DCIPThe chloroplast of maize
Genome (corn variety B73, AGPv3) is compared verification and confirms, is carried to obtain accurately and reliably Chloroplast gene sequence
For ensureing.
(2) chloroplaset variant sites are excavated：Pass through representative sample selection, high quality sequencing data, accurate data analysis
Ensure the Accuracy and high efficiency of acquisition chloroplaset polymorphic site.Selection in the present invention derives from a wealth of sources, phenotype and genotype are rich
170 parts of rich materials, 170 Chloroplast gene sequences based on splicing utilize the 5.0 statistical variation or dispersion sites DnaSp and sequence
Polymorphic, final to determine chloroplaset polymorphic site, wherein different genetic background materials, high quality gene order is to obtain accurately may be used
An important factor for by chloroplaset polymorphic site.
(3) corn T-type Sterile cytoplasm classification identification site determines：By by all types corn inbred line (including T, C, S
Three classes CMS materials) the variant sites of Chloroplast gene analyzed, filter out the specific site of T-type sterile material, specifically
Site show as T-type infertility and it is other it is all types of selfing based materials genotype it is different, belong to T-type sterile material
Peculiar site.
The present invention has the following advantages：The present invention resurveys ordinal number in view of the feature of Chloroplast gene based on high quality
According to one group of DCIPThe chloroplast of maize genome SNP marker of exploitation, can be used for identifying corn T-type male cytoplasmic sterility and normal cytoplasm
Germplasm materials, can be used for identifying the cenospecies of the production of hybrid seeds of T-type infertility and the conventional production of hybrid seeds, it may also be used for identification T-type sterile material
Purity.Molecular labeling provided by the invention provides identification technology branch for the production of hybrid seeds of corn T-type cytoplasmic sterility, sterile material selection and breeding
It holds, strong guarantee is provided for the mating seed production of corn three-line.
Description of the drawings
Fig. 1 is to be suitable for identifying that 7 chloroplaset sites of corn male sterility T-type cytoplasm type are obtained in the embodiment of the present invention 1
The Technology Roadmap obtained.
Fig. 2 is whether to differentiate corn inbred line using 7 pairs of primers provided by the invention for being directed to the design of 7 chloroplaset sites
For the analysis result figure of T-type infertility.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.The content not being described in detail in present specification belongs to professional and technical personnel in the field
The well known prior art.
The acquisition of embodiment 1 and the relevant DCIPThe chloroplast of maize genome molecules label of corn male sterility T-type cytoplasm type
(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, and first 3 days are non-illuminated conditions, after
2 days are 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
Geneious8.1 (Kearse et al., 2012) on all reads map to the Chloroplast gene sequence spliced,
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) it is suitable for differentiating the determination in corn T-type Sterile cytoplasm classification site：100 based on 170 parts of corn representative materials
A SNP/INDEL polymorphic genes type data, using the weir-fst-pop functional analyses Different groups in VCFtools softwares it
Between Fst values, i.e. genetic differentiation coefficient between Different groups, Fst values think that the site is monoid specific site more than 0.9.
The analysis of corn T-type infertility types of material obtains 7 SNP specific sites.Site specifying information is shown in Table 1, and the physical location in site is
It is determined based on corn variety B73 Chloroplast gene sequence alignments.For differentiating corn male sterility T-type cytoplasm in the present invention
The Technology Roadmap of 7 chloroplaset sites exploitation of type is shown in Fig. 1.
It finally screens to obtain 7 SNP markers for differentiating corn male sterility T-type cytoplasm, information is shown in Table 1.
The present embodiment devises the primer for above-mentioned 7 SNP markers, is respectively：CPMSNP03, SEQ ID
NO.15-17；CPMSNP04, SEQ ID NO.18-20；CPMSNP29, SEQ ID NO.21-23；CPMSNP47, SEQ ID
NO.24-26；CPMSNP67, SEQ ID NO.27-29；CPMSNP73, SEQ ID NO.30-32；CPMSNP86, SEQ ID
NO.33-35。
The molecular markers for identification corn T-type cytoplasmic sterility self-mating system that embodiment 2 is obtained using the present invention
T-type cytoplasmic sterility self-mating system sample is identified from 96 corn inbred line samples, the specific method is as follows.
Corn inbred line sample DNA extraction to be identified：Each sample randomly selects 50 seeds, carries out hair seedling, gives and fill
Sufficient illumination forms green seedling.DNA extractions take the mode of mixed strain extraction DNA, each sample to randomly select 30 from 50 single plants
The greenery of a single plant 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 working solution,
A concentration of 20ng/ μ L.
The design of primers synthesis of 7 T-type infertility specific sites：The polymorphic site provided in the present invention is two allele
SNP and InDel label, therefore can by 7 sites be based on KASP technology platform design primers, synthetic primer be without fluorescence
General primer.
PCR amplification：It is arbitrary to choose 1 group or multigroup expanded from 7 groups of primers that embodiment 1 provides.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.PCR response procedures are 94 DEG C
15min；Cycle 10 times takes touchdown PCR mode, and 94 DEG C of 20s, 61 DEG C of 1min, (61 DEG C drop to 55 DEG C, and each cycle reduces
0.6℃)；94 DEG C of 20sec, 58 DEG C of 1min are recycled 30 times.
Finger print data obtains：Amplified production is glimmering using the progress of BMG Pherastar (LGC, Middlesex, UK) instrument
Optical signal scans, and obtains initial data.Initial data imports Kraken softwares (LGC, Middlesex, UK) and carries out analysis acquisition
The finger print data of each data point of each corn variety, and import data management system.
Result judgement：Allele based on each site listed in table 1 in corn T-type Sterile cytoplasm classification into
Row judgement is T-type sterile material if identical as allele described in table, is not belonging to if if it is another allele
T-type sterile material.The results show that 7 groups of primers that embodiment 1 designs all have good specificity, can realize respectively to institute
For the specific detection in site, to complete the appraisal of corn T-type cytoplasmic sterility self-mating system.
For 7 chloroplaset sites design 7 pairs of primers differentiate corn inbred line whether be T-type infertility analysis result figure
See Fig. 2.In Fig. 2 in each pair of primer result parting figure, each data point is each sample genotyping result in each pair of primer；Data
Point is negative control sample near origin, and a remaining data point part is respectively represented close to X-axis, a part close to Y-axis
Two kinds of homozygous genotype results that each pair of primer is shown；The genotype data read using software is drawn with each pair of in table 1 respectively
The information of object is checked, and is T-type infertility if consistent with " corn T-type cytoplasmic male sterility sample allele ", separately
One type is then non-T-type infertility.
Whether embodiment 3 is the T-type cytoplasmic sterility production of hybrid seeds using the molecular markers for identification corn hybrid seed that the present invention obtains
Identify 96 Different Package bags or separate sources capital section 968 whether be T-type cytoplasmic sterility production of hybrid seeds production hybridization
Kind, the specific method is as follows.
Corn hybrid seed sample DNA extraction to be identified：Each packaging bag 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 form 96 parts of detected samples.DNA extracts specific steps and is executed 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 working solution, a concentration of 20ng/ μ L.
Design of primers synthesizes, and PCR amplification, finger print data preparation method is referring to embodiment 2.
Result judgement：Allele based on each site listed in table 1 in corn T-type Sterile cytoplasm classification into
Row judgement, the cenospecies generated for T-type infertility mode if certain sample to be tested is identical as allele described in table, if it is
Another allele is then not belonging to the cenospecies that T-type infertility mode produces.
Embodiment 4 identifies the purity of T-type infertility sample using the site that the present invention obtains
Identify the purity of 1 packet corn T-type infertility sample seeds, the i.e. content of T-type sterile seed, the specific method is as follows.
Sample DNA extraction to be identified：120 seeds are randomly selected from sample packaging to be identified, are carried out hair seedling, are given and fill
Sufficient illumination forms green seedling.The mode that single plant extracts is taken in DNA extractions, extracts the DNA of 100 single plant greenery, and DNA extractions are specific
Step 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).Dilution DNA forms working solution, a concentration of 20ng/ μ L.
The design of primers synthetic method of 7 T-type infertility specific sites is the same as embodiment 2.
PCR amplification：Embodiment 1 is selected to design 7 obtained group-specific primers, every group of primer carries out PCR amplification.PCR
Reaction 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 KASP Primer mix mixtures, 0.5 μ L deionized waters.PCR reaction intervals
Sequence is 94 DEG C of 15min；Cycle 10 times, takes touchdown PCR mode, 94 DEG C of 20s, 61 DEG C of 1min, (61 DEG C drop to 55 DEG C, each
Cycle reduces by 0.6 DEG C)；94 DEG C of 20sec, 58 DEG C of 1min are recycled 30 times.
Finger print data obtains：Amplified production is glimmering using the progress of BMG Pherastar (LGC, Middlesex, UK) instrument
Optical signal scans, and obtains initial data.Initial data imports Kraken softwares (LGC, Middlesex, UK) and carries out analysis acquisition
The finger print data of each data point of each single plant, and import data management system.
Result judgement：Allele based on each site listed in table 1 in corn T-type Sterile cytoplasm classification into
Row judgement is T-type infertility single plant if identical as allele described in table, is not belonging to if if it is another allele
T-type infertility single plant.Each site carries out purity calculating, and computational methods are (total single plant number of T-type infertility single plant number/detection
Mesh) × 100%.It can be realized respectively to corn T-type cytoplasm not the results show that embodiment 1 designs 7 obtained group-specific primers
Educate the appraisal of sample purity.
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 made some modifications or improvements, 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.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker
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<213>Artificial sequence (Artificial Sequence)
<400> 8
tatttgttaa taatttaagg ataaatagtt cactaaggag aagatagaat catagcaaat 60
<210> 9
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
cggtagtgga caaacagagg gaaagaaggt atggcgggga cacatttctt gtgagcaaat 60
<210> 10
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gaattgcttt actttttgaa ttaagttcaa ctttgaactt acagaaattt tgtaaaaaaa 60
<210> 11
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
tctctatgtt ttattactta atttacgaat ttcaaaaatt ttgtattcta ttggattgga 60
<210> 12
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
ttgttcgaga attcgaagaa ttacaacaaa atctttagaa atcacatttt tagttaggaa 60
<210> 13
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tttgtcgttc ccacagcttc tcctttaatg gttaggtttg aatcctgcaa tggagcttcc 60
<210> 14
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
attttttttt ccgagtcaat tttctcagtt ttattaaccc ggctgctctt tatttattgc 60
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
aggatccatt tgacccccaa tatg 24
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
aggatccatt tgacccccaa tatc 24
<210> 17
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
ggaaaataaa tagggggtac ttcttttctt 30
<210> 18
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
aaataaatag ggggtacttc ttttctttca 30
<210> 19
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
aaataggggg tacttctttt ctttcg 26
<210> 20
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
cttacaggat ccatttgacc cccaa 25
<210> 21
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
atattctaaa aagattggat agcaaagatt tc 32
<210> 22
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
gatattctaa aaagattgga tagcaaagat tta 33
<210> 23
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
gctttatccc gtttcataga aaggagata 29
<210> 24
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
gaactattta tccttaaatt attaacaaat aa 32
<210> 25
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
gaactattta tccttaaatt attaacaaat ac 32
<210> 26
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
gccaagagat tggcattttc atttgatcat 30
<210> 27
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
agttgaactt aattcaaaaa gtaaagcaat tct 33
<210> 28
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
gttgaactta attcaaaaag taaagcaatt cg 32
<210> 29
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
cggggacaca tttcttgtga gcaaa 25
<210> 30
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
atttcaaaaa ttttgtattc tattggattg gat 33
<210> 31
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
tcaaaaattt tgtattctat tggattggac 30
<210> 32
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
tttgttgtaa ttcttcgaat tctcgaacaa 30
<210> 33
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
ttgaatcctg caatggagct tcca 24
<210> 34
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
gaatcctgca atggagcttc cc 22
<210> 35
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
gcagccgggt taataaaact gagaaaatt 29
Claims (10)
1. the molecular labeling for differentiating corn male sterility T-type cytoplasm type, which is characterized in that it includes 7 SNP molecule marks
Any one or more in note, information is as follows：
2. molecular labeling as described in claim 1, which is characterized in that the molecular labeling is obtained by following primer amplification respectively
It arrives：CPMSNP03, SEQ ID NO.15-17；CPMSNP04, SEQ ID NO.18-20；CPMSNP29, SEQ ID NO.21-
23；CPMSNP47, SEQ ID NO.24-26；CPMSNP67, SEQ ID NO.27-29；CPMSNP73, SEQ ID NO.30-
32；CPMSNP86, SEQ ID NO.33-35.
3. the answering in the cenospecies that identification corn T-type cytoplasm male sterility line is prepared of molecular labeling described in claims 1 or 2
With.
4. application of the molecular labeling described in claims 1 or 2 in identifying T-type cytoplasmic male sterilty corn material.
5. application of the molecular labeling described in claims 1 or 2 in corn molecular mark.
6. application of the molecular labeling described in claims 1 or 2 in Identification of Corn Germplasms.
7. according to any applications of claim 3-6, which is characterized in that include the following steps：
1) DNA of corn sample to be measured is extracted；
2) using the DNA of step 1) extraction as template, molecular labeling according to claim 1 or claim 2 is based on KASP detection platform skills
Art separately designs primer, carries out PCR amplification；
3) fluorescence detector is used to analyze PCR product.
8. application according to claim 7, which is characterized in that step 2) PCR amplification program is：94℃15min；94℃
20s, 61 DEG C of 1min are recycled 10 times, and each cycle reduces by 0.6 DEG C；94 DEG C of 20s, 58 DEG C of 1min are recycled 30 times.
9. application of the molecular labeling described in claim 1 in identifying T-type cytoplasmic male sterilty corn material purity.
10. application as claimed in claim 9, which is characterized in that include the following steps：
1) DNA of corn sample to be measured is extracted；
2) using the DNA of step 1) extraction as template, molecular labeling according to claim 1 or claim 2 is based on KASP detection platform skills
Art separately designs primer, carries out PCR amplification；
3) fluorescence detector is used to analyze PCR product；
4) if PCR product is T-type infertility single plant if identical as the allele, not if it is another allele
Belong to T-type infertility single plant；Each site carries out purity calculating, and computational methods are：(total list of T-type infertility single plant number/detection
Strain number mesh) × 100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810117191.8A CN108486263A (en) | 2018-02-06 | 2018-02-06 | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker |
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| CN201810117191.8A CN108486263A (en) | 2018-02-06 | 2018-02-06 | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker |
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| CN108486263A true CN108486263A (en) | 2018-09-04 |
Family
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|---|---|---|---|
| CN201810117191.8A Pending CN108486263A (en) | 2018-02-06 | 2018-02-06 | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157834A (en) * | 2019-07-01 | 2019-08-23 | 北京市农林科学院 | SNP site OE2-5 relevant to corn high-lysine character and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591470A (en) * | 2017-01-11 | 2017-04-26 | 北京市农林科学院 | Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize |
| CN107217101A (en) * | 2017-06-30 | 2017-09-29 | 北京市农林科学院 | Differentiate and really weigh the detection method of identification suitable for variety of crops molecular identity |
-
2018
- 2018-02-06 CN CN201810117191.8A patent/CN108486263A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591470A (en) * | 2017-01-11 | 2017-04-26 | 北京市农林科学院 | Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize |
| CN107217101A (en) * | 2017-06-30 | 2017-09-29 | 北京市农林科学院 | Differentiate and really weigh the detection method of identification suitable for variety of crops molecular identity |
Non-Patent Citations (1)
| Title |
|---|
| MASSIMO BOSACCHI 等: "Plastid Genotyping Reveals the Uniformity of Cytoplasmic Male Sterile-T Maize Cytoplasms", 《PLANT PHYSIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157834A (en) * | 2019-07-01 | 2019-08-23 | 北京市农林科学院 | SNP site OE2-5 relevant to corn high-lysine character and its application |
| CN110157834B (en) * | 2019-07-01 | 2022-04-19 | 北京市农林科学院 | SNP site OE2-5 related to high lysine character of corn and application thereof |
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