CN108486264A - Differentiate corn male sterility c-type cytoplasm type using chloroplastic marker - Google Patents
Differentiate corn male sterility c-type cytoplasm type using chloroplastic marker Download PDFInfo
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Abstract
The present invention provides corn male sterility c-type cytoplasm type is differentiated using chloroplastic marker, chloroplastic marker of the invention is to develop to obtain based on Chloroplast gene, shares 11 molecular labelings, including 9 SNP and 2 InDel labels.Molecular labeling combination can be used for identifying the germplasm materials of corn C type male cytoplasmic sterility and normal cytoplasm, can be used for identifying the cenospecies of the production of hybrid seeds of c-type infertility and the conventional production of hybrid seeds, it may also be used for the purity of identification c-type sterile material.Molecular labeling provided by the invention is combined as the production of hybrid seeds of corn C 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 c-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 S types, c-type and T-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 c-type cytoplasm type is very necessary.
Invention content
The object of the present invention is to provide differentiate corn male sterility c-type cytoplasm type using chloroplastic marker.
It is a further object of the present invention to provide SNP the and INDEL molecules for differentiating corn male sterility c-type cytoplasm type
Label combination.
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 c-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 C 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 C type infertility types of material analysis obtain
Obtain 11 specific sites, including 9 SNP sites and 2 sites InDel.Site specifying information is shown in Table 1, the physical location in site
It is to be determined based on corn variety B73 Chloroplast gene sequence alignments.It is suitable for differentiating corn male sterility c-type born of the same parents in the present invention
The Technology Roadmap of 11 chloroplaset sites exploitation of matter type is shown in Fig. 1.
The present invention provides the molecular labelings for differentiating corn male sterility c-type cytoplasm, including following 11 molecule marks
Any one or more molecular labeling in note, 11 molecular labelings be 9 SNP markers and 2 InDel molecular labelings,
Their information is shown in Table 1.
Table 1
Due to above-mentioned 11 sites SNP/InDel provided by the invention be two condition label, so the molecular labeling or its
Any multiple molecular labeling combination can realize the acquisition of genotype data based on KASP platforms.Concrete scheme is, according to
KASP technologies require to be directed to site design primer provided by the invention, and primer is that general primer is free of fluorescence group；It purchases
The PCR amplification system MasterMix of KASP complete sets of Techniques；It configures reaction system and DNA, primer and MasterMix is added；Operation is anti-
Answer program；In-situ scanning fluorescence signal；Data analysis obtains genotype data.
In addition to the molecular labeling that site number is CPMIDP09, other molecular labelings are expanded by following primer the present invention respectively
Increasing obtains：CPMSNP18, SEQ ID NO.23-25；CPMSNP38, SEQ ID NO.26-28；CPMSNP39, SEQ ID
NO.29-31；CPMSNP44, SEQ ID NO.32-34；CPMSNP48, SEQ ID NO.35-37；CPMSNP57, SEQ ID
NO.38-40；CPMSNP59, SEQ ID NO.41-43；CPMSNP72, SEQ ID NO.44-46；CPMSNP81, SEQ ID
NO.47-49；CPMIDP10, SEQ ID NO.50-52.
Further, the present invention provides the specific primer sets for detecting molecular labeling of the present invention, specifically
It is following any one group：CPMSNP18, SEQ ID NO.23-25；CPMSNP38, SEQ ID NO.26-28；CPMSNP39, SEQ
ID NO.29-31；CPMSNP44, SEQ ID NO.32-34；CPMSNP48, SEQ ID NO.35-37；CPMSNP57, SEQ ID
NO.38-40；CPMSNP59, SEQ ID NO.41-43；CPMSNP72, SEQ ID NO.44-46；CPMSNP81, SEQ ID
NO.47-49；CPMIDP10, SEQ ID NO.50-52.
Although the embodiment of the present invention does not provide the amplimer in the sites CPMIDP09, those skilled in the art can be directed to
Site number designs suitable specific primer, and the site information of the CPMIDP09 provided based on table 1 for CPMIDP09, can
Realize identification to corn C type male cytoplasmic sterility type, thus site number be CPMIDP09 molecular labeling and its can
For differentiating that this purposes of corn C type male cytoplasmic sterility type belongs to the scope of protection of the invention.
Further, the present invention provides above-mentioned molecular labelings or specific primer in identification corn male sterility c-type
Application in cytoplasm type.
The present invention provides above-mentioned molecular labelings or specific primer to being prepared in identification corn C type cytoplasm male sterility line
Cenospecies in application.
The present invention provides above-mentioned molecular labelings or specific primer to the application in corn molecular mark.
The present invention provides above-mentioned molecular labelings or specific primer to the application in being identified in corn material.
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 c-type infertility single plant if identical as allele described in table 1, if it is
Another allele is then not belonging to c-type infertility single plant.
The present invention provides application of the above-mentioned molecular labeling in identifying c-type cytoplasmic male sterilty corn material purity.
In the detailed process of identification c-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 c-type infertility single plant if identical as allele described in table 1, if it is another equipotential
Gene is then not belonging to c-type infertility single plant；Each site carries out purity calculating, and computational methods are：(c-type infertility single plant number/inspection
The total single plant number surveyed) × 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 C 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 c-type sterile material, specifically
Site show as c-type infertility and it is other it is all types of selfing based materials genotype it is different, belong to c-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 the combination of one group of DCIPThe chloroplast of maize genome SNP marker and InDel molecular labelings and these molecular labelings of exploitation, can use
In the germplasm materials of identification corn C type male cytoplasmic sterility and normal cytoplasm, it can be used for identifying the production of hybrid seeds of c-type infertility and the conventional production of hybrid seeds
Cenospecies, it may also be used for identification c-type sterile material purity.Molecular labeling provided by the invention be corn C type cytoplasm not
Educate the production of hybrid seeds, sterile material selection and breeding provide identification technology support, produced for the mating seed of corn three-line and provide strong guarantee.
Description of the drawings
Fig. 1 is to be suitable for identifying that 11 chloroplaset sites of corn male sterility c-type cytoplasm type are obtained in the embodiment of the present invention 1
The Technology Roadmap obtained.
Fig. 2 be 10 pairs of primers provided by the invention differentiate corn inbred line or cenospecies whether be c-type infertility analysis knot
Fruit is schemed, and in addition to the molecular labeling that site number is CPMIDP09, the primer of other 10 molecular labelings can be realized to corn
Self-mating system or cenospecies whether be male sterility c-type cytoplasm type identification.
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 c-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
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) it is suitable for differentiating the determination in corn C 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 C type infertility types of material obtains 11 specific sites, including 9 SNP sites and 2 sites IDNEL.
The technology path of 11 chloroplaset sites exploitation in the present invention for differentiating corn male sterility c-type cytoplasm type
Figure is shown in Fig. 1.It finally screens to obtain 11 molecular labelings for differentiating corn male sterility c-type cytoplasm, including 9 SNP molecules
Label and 2 InDel molecular labelings, their information are shown in Table 1.
In addition to the molecular labeling that site number is CPMIDP09, other molecular labelings are obtained by following primer amplification respectively
It arrives：CPMSNP18, SEQ ID NO.23-25；CPMSNP38, SEQ ID NO.26-28；CPMSNP39, SEQ ID NO.29-
31；CPMSNP44, SEQ ID NO.32-34；CPMSNP48, SEQ ID NO.35-37；CPMSNP57, SEQ ID NO.38-
40；CPMSNP59, SEQ ID NO.41-43；CPMSNP72, SEQ ID NO.44-46；CPMSNP81, SEQ ID NO.47-
49；CPMIDP10, SEQ ID NO.50-52.
The molecular markers for identification corn C type cytoplasmic sterility self-mating system that embodiment 2 is obtained using the present invention
C-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 11 c-type infertility specific sites：The polymorphic site provided in the present invention is two equipotential bases
The SNP and InDel of cause are marked, therefore 11 sites can be based on to KASP technology platform design primers, synthetic primer be without
The general primer of fluorescence.
PCR amplification：From 10 pairs of primers that embodiment 1 determines, expanded for every pair of primers.PCR reaction systems
For 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 of 15min；Cycle 10 times, is adopted
Take touchdown PCR mode, 94 DEG C of 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.
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 C type Sterile cytoplasm classification into
Row judgement is c-type sterile material if identical as allele described in table, is not belonging to if if it is another allele
C-type sterile material.The results show that other molecules in addition to the molecular labeling for being CPMIDP09 except site number that embodiment 1 determines
The primer of label design all has good specificity, can realize the specific detection to targeted site respectively, to complete
At the appraisal of corn C type cytoplasmic sterility self-mating system.
Differentiate whether corn inbred line is that the analysis result figure of c-type infertility is shown in Fig. 2 using 10 pairs of primers provided by the invention.
In addition to the molecular labeling that site number is CPMIDP09, the primer of other 10 molecular labelings can be realized to corn inbred line
Or cenospecies whether be male sterility c-type cytoplasm type identification.In Fig. 2 in each pair of primer result parting figure, each data point
For each sample in each pair of primer genotyping result；Data point is negative control sample, remaining data point one near origin
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；Utilize software
Information of the genotype data of reading respectively with each pair of primer in table 1 is checked, if with " corn C type cytoplasmatic male
Consistent sterile sample allele " is then c-type infertility, and another type is then non-c-type infertility.
Whether embodiment 3 is the c-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 c-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 C type Sterile cytoplasm classification into
Row judgement, the cenospecies generated for c-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 c-type infertility mode produces.
Embodiment 4 identifies the purity of c-type infertility sample using the site that the present invention obtains
Identify the purity of 1 packet corn C type infertility sample seeds, the i.e. content of c-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, DNA extractions at random
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).Dilution DNA forms working solution, a concentration of 20ng/ μ L.
One or more sites, design primer are randomly selected with 11 c-type infertility specific sites that embodiment 1 determines.PCR
Amplification：From 10 groups of primers that embodiment 1 determines, every group of primer carries out PCR amplification.PCR reaction systems are 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 response procedures are 94 DEG C of 15min；Cycle 10 times,
Take touchdown PCR mode, 94 DEG C of 20s, 61 DEG C of 1min, (61 DEG C drop to 55 DEG C, and each cycle reduces by 0.6 DEG C)；94℃
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 C type Sterile cytoplasm classification into
Row judgement is c-type infertility single plant if identical as allele described in table, is not belonging to if if it is another allele
C-type infertility single plant.Each site carries out purity calculating, and computational methods are (total single plant number of c-type infertility single plant number/detection
Mesh) × 100%.
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 c-type cytoplasm type using chloroplastic marker
<130> KHP171117986.1
<160> 52
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cccatttttt tttttttttg caattttatg acttagttta gtgcgagatg cccacatttt 60
<210> 2
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ggtctgaaca ctaaacgagc acagacttaa aattacaaaa aaaaatgaaa ttggactctt 60
<210> 3
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tcaggggggc caagtcaggt tagatctata tctttaatgc ctataagaca gtcatctttt 60
<210> 4
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tgtggctttc taaagaatga ttttagaatc ggattcaata gaaaatgaga aaataggcaa 60
<210> 5
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ttcgtctttt attatgttag atgaagggga aaaaatggga actcaaagat atcgaagagt 60
<210> 6
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
aaaaaaagga tggaataaaa gagtgattgg ttgaaaagaa agagaaatag aataatgaga 60
<210> 7
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
tagttgtatc gacccagtcg ctcactaatt gatctttacg gtgttttctc tatcaatttc 60
<210> 8
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gctttatcca tagaatagta gtataggctc tactttcttc ctattttgat tctcgtgaag 60
<210> 9
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ccaatcaaat attgagtaat caaatccttc aattcattgt tttcgagatc ttttaatttt 60
<210> 10
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
aaaagtggat taatcggacg aggataaaga gagagtccca ttctacatgt caatactgac 60
<210> 11
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gcgtttcgat gaatgagcct atggtaatgc ttttatctct attctatggc gcaatcgacc 60
<210> 12
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
tcgagtctat aaacaagtac taaataagga aaagaaaact atactaaagg aaacataaga 60
<210> 13
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
cccgggacgc gaagtagtag gattggttct cataattatc acataatttt caaaaaaaaa 60
<210> 14
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
gaatttgtcg aaattttttt tcttgttgaa taatgccaaa tcaaaaaaaa tatccaaaaa 60
<210> 15
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
ttttttaatt atagttattc ctatgcgaga gatagaattc ttcgtgacat gacgaaaatt 60
<210> 16
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
cccctttttg aattcttttt tagtatatga agcaaaaaga aagaaaagat ggataaggat 60
<210> 17
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
aaaagcttct ttttcgaaag attacccctg tctttgttta tgcttcggat tggaacaaat 60
<210> 18
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
actctaattc gcccacgcct gcgaatcagt cgacattttg tacaaatttt acgaacggaa 60
<210> 19
<211> 61
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 20
<211> 61
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
cttctctatc ctattcattg gtaccgatca tggatacttc aaaaatttta ttatttgttt 60
g 61
<210> 21
<211> 61
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
gattggattt gcaccaaagg aaaccataaa ttccatatac catagaaatc ttaggataga 60
g 61
<210> 22
<211> 61
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
ctctatccta ttcattggta ccgatcatgg atacttcaaa aattttatta tttgtttgaa 60
c 61
<210> 23
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
gtgctcgttt agtgttcaga cca 23
<210> 24
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
gtgctcgttt agtgttcaga ccc 23
<210> 25
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
cttagtttag tgcgagatgc ccacat 26
<210> 26
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
attctaaaat cattctttag aaagccacac 30
<210> 27
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
ctaaaatcat tctttagaaa gccacat 27
<210> 28
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
ggccaagtca ggttagatct atatcttta 29
<210> 29
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
atgggaactc aaagatatcg aagagta 27
<210> 30
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
gggaactcaa agatatcgaa gagtc 25
<210> 31
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
caaccaatca ctcttttatt ccatcctttt 30
<210> 32
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
gcctatacta ctattctatg gataaagct 29
<210> 33
<211> 28
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
cctatactac tattctatgg ataaagcg 28
<210> 34
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
tcgctcacta attgatcttt acggtgttt 29
<210> 35
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
atcctcgtcc gattaatcca ctttta 26
<210> 36
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
atcctcgtcc gattaatcca cttttt 26
<210> 37
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
ccttcaattc attgttttcg agatctttta 30
<210> 38
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
tatttagtac ttgtttatag actcgac 27
<210> 39
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
ccttatttag tacttgttta tagactcgat 30
<210> 40
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
aatgctttta tctctattct atggcgcaat 30
<210> 41
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
attcaacaag aaaaaaaatt tcgacaaatt cc 32
<210> 42
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
attcaacaag aaaaaaaatt tcgacaaatt ct 32
<210> 43
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 43
gcgaagtagt aggattggtt ctcataatt 29
<210> 44
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 44
tcatatacta aaaaagaatt caaaaagggg a 31
<210> 45
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 45
catatactaa aaaagaattc aaaaaggggg 30
<210> 46
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 46
gagatagaat tcttcgtgac atgacgaaa 29
<210> 47
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 47
aggcgtgggc gaattagagt c 21
<210> 48
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 48
caggcgtggg cgaattagag tt 22
<210> 49
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 49
gtctttgttt atgcttcgga ttggaacaa 29
<210> 50
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 50
cggtaccaat gaataggata gags 24
<210> 51
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 51
atcggtacca atgaatagga tagaga 26
<210> 52
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 52
ccaaaggaaa ccataaattc catataccat 30
Claims (10)
1. the molecular labeling for differentiating corn male sterility c-type cytoplasm type, which is characterized in that it includes following 11 molecules
Any one or more molecular labeling combination in label, 11 molecular labelings are 9 SNP markers and 2 InDel molecules
Label, their information are as follows：
2. molecular labeling as described in claim 1, which is characterized in that in addition to the molecular labeling that site number is CPMIDP09,
He is obtained by following primer amplification molecular labeling respectively：CPMSNP18, SEQ ID NO.23-25；CPMSNP38, SEQ ID
NO.26-28；CPMSNP39, SEQ ID NO.29-31；CPMSNP44, SEQ ID NO.32-34；CPMSNP48, SEQ ID
NO.35-37；CPMSNP57, SEQ ID NO.38-40；CPMSNP59, SEQ ID NO.41-43；CPMSNP72, SEQ ID
NO.44-46；CPMSNP81, SEQ ID NO.47-49；CPMIDP10, SEQ ID NO.50-52.
3. application of the molecular labeling described in claims 1 or 2 in identifying corn male sterility c-type cytoplasm type.
4. the answering in the cenospecies that identification corn C type cytoplasm male sterility line is prepared of molecular labeling described in claims 1 or 2
With.
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 corn material identification.
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 claims 1 or 2 in identifying c-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, is based on KASP detection platform technologies according to claim 1
Primer is separately designed, PCR amplification is carried out；
3) fluorescence detector is used to analyze PCR product；
4) if PCR product is c-type infertility single plant if identical as the allele, not if it is another allele
Belong to c-type infertility single plant；Each site carries out purity calculating, and computational methods are：(total list of c-type infertility single plant number/detection
Strain number mesh) × 100%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088018A (en) * | 2012-12-27 | 2013-05-08 | 河南农业大学 | Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn |
| 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 |
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2018
- 2018-02-06 CN CN201810117639.6A patent/CN108486264B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088018A (en) * | 2012-12-27 | 2013-05-08 | 河南农业大学 | Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn |
| 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 |
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