CN109486989A - Sterile stability analysis method and application of the corn Male sterile gene ms1-alb under extensive genetic background - Google Patents
Sterile stability analysis method and application of the corn Male sterile gene ms1-alb under extensive genetic background Download PDFInfo
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Abstract
The present invention relates to a kind of corn recessive nucleus male sterility mutated genesms1‑albLead to method for analyzing stability and its application of male-sterility under extensive genetic background.The present invention will by way of hybridizationms1‑albMutated gene imports 300 parts of different genetic background corn inbred lines, to F2It is counted for plant Fertility segregation ratio, and utilizes mutant genems1‑albThe functional label that isolates carry out genotyping, sterile mutated gene as the result is shownms1‑albComplete male-sterility is shown as under all 300 genetic backgrounds, it was demonstrated that it causes stable male flower thoroughly sterile, can be used for initiative maternal in corn hybridization breeding and the sterilization production of hybrid seeds.Infertility mutated gene provided by the invention leads to the method for analyzing stability of the thorough infertility of male flower, is equally applicable to detect male flower thorough Sterile stability of other corn recessive nucleus male sterility genes under different genetic backgrounds.
Description
Technical field
Generally, the invention belongs to Crop Genetic Breeding, molecular breeding and seed engineering fields.More particularly to a kind of corn
Recessive nucleus male sterility genems1-albSterile stability analysis method and application under extensive genetic background.
Background technique
Plants male sterility (male sterility, MS) is phenomenon generally existing in higher plant, arrenotoky
Allelotaxis is abnormal, can not generate functional andro gamete (pollen), but female reproductive organ's development is normal, can receive normal hero
Gamete and fertilization, and the infertility character can be hereditary to offspring.
Corn is important grain and industrial crops, male-sterile mutation material anther development mechanism is parsed and
Cenospecies production all has significance.According to the difference of mode of inheritance, existing corn male sterility material can be divided into two classes:
Nucleo-cytoplasmic interreaction male sterility (Cytoplasmic male sterility, CMS) and nuclear male sterility (Genic male
sterility, GMS).The abortion of CMS is generally caused by mitochondrial toxicity albumen, is existed in the nucleus of part germplasm and is restored
Gene can release influence of the toxic protein to fertility；Because restoring gene exists only in the germ plasm resource of part, and sterile degree is easy
It is influenced by genetic background, the category feature of CMS greatly reduces germ plasm resource utilization efficiency；In addition, CMS improved variety cytoplasm
Unification is widely applied that there are risks vulnerable to infecting for single-minded pathogeny microspecies.GMS is mutated by single cell nucleus gene
Caused male sterility, theoretically all fertile germplasm materials can be used as restorer and restore its offspring's fertility, this feature pole
The earth increases germplasm resource utilization efficiency；And the non-matrocliny of the genoid, it not will lead to cytoplasm unification, reduce miscellaneous
Kind offspring's potential risks, can be used as the ideal female parent material of hybrid seeding.
Have many advantages, such as that restoring gene is widely present, will not cause cytoplasm unification, but such base compared to CMS, GMS
It is imported not because the stability of the thorough abortion of caused by male flower under different genetic backgrounds is not apparent, therefore by the mutated gene
With under nucleus and cytoplasmic inheritance background, and the genotype of offspring's segregating population is identified, to judge such mutation base
The stability of sterile phenotype is of great significance caused by because under different genetic background.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of detection maize cell kernel male sterile mutated genes in difference
Genetic background under caused male sterility phenotype method for analyzing stability.Provided method is specifically, pass through hybridization skill
Art and molecular marker assisted selection, by the different nucleus of corn recessive nucleus male sterility channel genes and cytoplasmic inheritance background
Under, by fertility analysis and genotype detection, judge maize cell Male sterile gene being led under different genetic backgrounds
The stability for causing male-sterility phenotype, provides reference for subsequent crossbreeding and the selection of production of hybrid seeds infertility female parent material.
An object of the present invention is to provide a kind of identification corn recessive nucleus male sterility gene in different heredity back
Lead to the method for analyzing stability of male-sterility under scape.
The second object of the present invention is to provide under 300 parts of different genetic backgroundsms1-albThe germplasm of homozygous mutation
Material is used for corn male sterility crossbreeding and the production of hybrid seeds.
Detailed description of the invention
Fig. 1 be maize wild-type (A, C, E) andms1-albTassel, anther and the iodo- iodine of pollen grain of mutant (B, D, F)
Change the comparison diagram of potassium dyeing.Scheming scale in E, F is 200 μm.
Fig. 2 is the corn inbred line phylogenetic analysis result of 300 parts of extensive genetic background.Based on 3072 SNP markers
Chip analysis, it is found that this 300 parts of corn inbred lines are divided into four big monoids, genetic diversity is abundant, in hybrid maize breeding and
There is good utility value in the production of hybrid seeds；Also illustrate the group for testing simultaneouslyms1-albThe Sterile stability of sterile gene
Representative and reliability.
Fig. 3 isms1-albMutated gene caused male-sterility character under 300 parts of different corn germplasm genetic backgrounds is steady
The operational flowchart of qualitative analysis.
Fig. 4 is F1For plant genotype identification the primer schematic diagram and qualification result electrophoretogram.Utilize mutant genems1-albFunction phenotypic marker ms1-IN1 and ms1-IN2 to F1Genotype identification is carried out for plant.Ms1-IN1 and ms1-IN2
It can specific detection mutated genems1-albSite expands obtain the band of 602 bp and 945 bp sizes respectively, andZmMs1/ ZmMs1In homozygous material, it can not expand to obtain band.
Fig. 5 is representativeness F2The distribution of the statistic and segregation ratio of fertile plant and sterile plant segregation ratio in group.
According to the actual ratio of fertile single plant and sterile single plant, 300 parts of materials are divided into three classes, the first kind includes 29 and is, fertile
The ratio of plant and sterile plant is between 0.7 and 2.0, and P value is between 0.01 and 0.56；Second class includes 207,
Its fertile plant and the ratio of sterile plant are between 2.0 and 4.6, and P value is between 0.27 and 1.00；Third class includes 64
The ratio of a system, fertile single plant and sterile single plant is greater than 4.6, P value between 0.06 and 0.64.
Fig. 6 is 3 representativeness F2Separation strain genotype identification electrophoretogram partially.It utilizesms1-albFunctional label Ms1-1 and
Ms1-IN1 is to 3 F2Separation strain single plant partially carries out genotype identification, and Ms1-1 can specific detection wild type geneZmMs1Position
Point, therefore can beZmMS1/ZmMs1Homozygous wildtype andZmMs1/ms1-albAmplification obtains 763 bp sizes in heterozygous
Band；Ms1-IN1 can specific detection mutated genems1-albSite, therefore can beZmMS1/ms1-albHeterozygous with
Andms1-alb/ms1-albAmplification obtains the band of 602 bp sizes in homozygous mutant.Testing result shows, 3 F2Strain
In all fertile single plant genotype beZmMS1/ZmMs1OrZmMs1/ms1-alb, the genotype of sterile single plant isms1- alb/ms1-alb, genotype is completely the same with phenotype.
Fig. 7 is wild type, mutant and 2 F2Sterile single plant tassel phenotype and pollen grain iodate in representative strain
Potassium coloration result comparison diagram.Scale is 200 μm in figure.
Specific embodiment
Following embodiments are used to illustrate the present invention, but do not limit the scope of the invention.Without departing substantially from spirit of that invention and reality
In the case where matter, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.Such as without spy
Different to illustrate, the synthesis and sequencing of the primer and gene are complete by Sangon Biotech (Shanghai) Co., Ltd. in embodiment
At.It is conventional commercial reagent that other biochemical reagents, which non-specifically indicate outer, and technological means used in embodiment is this field skill
Conventional means known to art personnel.
Embodiment 1: corn male sterility mutant material and 300 parts of corn inbred lines are obtained
Material to be tested of the invention is corn normally fertile self-mating systemZmMs1The sterile plant that gene mutation obtains, has shown as
Full male sterility, as shown in Figure 1, it is showed specifically: loose powder phase anther is not exposed, and anther becomes smaller, generates without pollen grain.This is not
Based material is educated to be named asms1-alb。
300 parts randomly select in the corn inbred line of 1400 parts of this laboratory for trying maternal corn inbred line, material
Title still continues to use original number.
2:300 parts of corn inbred line group structure identifications of embodiment
First with the Illumina GoldenGate SNP Genotyping detection chip comprising 3072 sites to 300 parts
Selfing based material is analyzed, and the genetic distance (0- between different materials is then calculated by PHYLIP DNADIST program
1.15), 300 parts are selfed using PHYLIP NEIGHBOR software building phylogenetic tree according to genetic distance calculated result
Based material is classified.As shown in Fig. 2, 300 parts of corn inbred lines can be divided into 4 monoids, preferable polymorphism is represented.
Embodiment 3:ms1-albHeterozygous plant is mutated as male parent and 300 parts of hybridizations between selfed lines and obtains F1For seed
Corn genic male sterile mutant can only be handed over by sisters, i.e., Mutants homozygous hybridizes with Heterozygous mutants
Mode saves, the offspring's fertile plant theoretically obtained be heterozygote (ZmMs1/ms1-alb), sterile plant is homozygote
(ms1-alb/ms1-alb), thus genotype can be judged by phenotype.The present invention utilizesms1-albSisters hand over group in can
Educate single plant (ZmMs1/ms1-alb) to 300 parts of self-mating system pollinations, obtain F1For seed.
Embodiment 4:F1For heterozygous genotypes in groupZmMs1/ms1-albThe screening and selfing of plant
In all 300 parts of self-mating systemsZmMs1Gene loci isZmMs1/ZmMs1, therefore the F hybridized in embodiment 31Generation
In, wild typeZmMs1/ZmMs1With heterozygousZmMs1/ms1-albGenotypic segregation ratio theoretically should be 1:1.In seedling stage, it takes
F1DNA is extracted for the blade of plant, and is utilizedms1-albFunctional label ms1-IN1 and ms1-IN2 carry out genotype identification, Fig. 4
Part strain genotype identification is illustrated as a result, the standard that heterozygous genotypes judge expands respectively as ms1-IN1 and ms1-IN2
To the band of 602 bp, 945 bp；Pollination self is carried out to the loose powder phase to obtained heterozygote plant listing mark.
Wherein, maize leaf DNA extraction method is as follows: 1) the appropriate blade of clip and shredding, be put into and finish writing number in advance
In 2.0 ml centrifuge tubes, a steel ball is added.2) that the centrifuge tube for being put into blade and steel ball is placed in proofing instrument in order is special
On centrifuge tube shelf (8 × 5), being integrally immersed in the crisper for fill liquid nitrogen 1-2 minutes, (note: liquid nitrogen was not to have centrifugation just
Pipe support is advisable a little).3) centrifuge tube shelf freezed is put into proofing instrument (Thmorgan cell killer ck-1000)
It in card slot, tightens, closes lid, 1200 revs/min of speed is drawn a design 20 seconds.4) steel ball in centrifuge tube is sucked out with magnet.5) add
Enter 700 μ L CTAB extracting solutions (65 DEG C preheating), 65 DEG C water-bath 30 minutes, centre is taken out 1-2 times reverse.6) 700 μ L chlorine are added
Imitative: isoamyl alcohol (24:1) extract liquor covers tightly lid, mixings of turning upside down, and careful label not be rubbed off that (note: chloroform is to corrode
Property reagent, need band PE disposable glove operated in draught cupboard).7) 5 minutes are centrifuged to clear split-phase for 12000 revs/min.It inhales
400 μ L of supernatant is taken, (dehydrated alcohol for being previously added 800 μ l pre-cooling) is transferred in 1.5 new mL microcentrifugal tubes, abandons rifle
Head covers tightly lid, finishes writing number and checks errorless, mixing of turning upside down.It is placed 30 minutes in -20 DEG C of refrigerators.8) 12000 turns/
Centrifugation bottom of the tube is invested to precipitating within minute centrifugation 10 minutes, abandon supernatant.9) 70% ethanol washing precipitates 2 times, and 1.5 mL are micro-
Amount centrifuge tube is inverted on the paper being laid on table, is spontaneously dried.10) 1 × TE buffer or ddH of 100-200 μ L is added2O
Dissolution precipitating.11) sample is placed in -20 DEG C of refrigerators and saves backup.
PCR amplification is carried out using the primer pair of design DNA obtained, methods and procedures is as follows: 1) first opening ice machine
Water source switch, then open the power switch of ice machine, make ice.2) following reagent is taken out to defrosting: PCR from -20 DEG C of refrigerators
Buffer, dNTP solution, forward and reverse primer solution and template DNA (note: when a certain reagent thaws completely, that is, are placed on ice.
Through common ddH2O, primer working solution, template DNA and a small amount of PCR buffer, dNTP solution can be placed in 4 DEG C of refrigerators).
3) after all reagents thaw, 8000 revs/min of centrifugation several seconds are put back to stand-by on ice.4) mixed liquor of PCR reaction is prepared,
Sequentially add each reagent according to following table (reaction system is 10 μ L).The following table 1 has listed file names with 1 reaction (1R '), 10 reactions
(10R '), 50 reaction (50R '), 100 reaction (100R ') mixture formula.After preparing by all reagents put back to 4 DEG C or-
(note: Taq polymerase needs careful operation to 20 DEG C of refrigerators, takes out from -20 DEG C of refrigerators before use, and the used time need to be placed on ice, after being finished
It is put back in -20 DEG C of refrigerators immediately).5) it mixes, 8000 revs/min of centrifugation several seconds.6) mixed liquor is dispensed anti-to 200 μ L PCR
Ying Guanzhong adds 1 μ L template DNA, and marking (note: if without using hot lid function, to prevent sample to be evaporated, needs last
About 20 μ L paraffin oils are added or cover PCR pipe lid).7) PCR reaction plate (pipe) is inserted into 200 holes μ L of PCR amplification instrument,
Hot lid is closed, is screwed.8) PCR amplification instrument power switch is opened, preset response procedures (such as table 2) is selected, starts to expand.9) expand
After having increased, response procedures are exited, return to main menu, are turned off the power switch.Heat lid is opened, PCR reaction tube is taken out, is placed in PCR pipe
On frame, it is put into 4 DEG C of refrigerators, for use.
Embodiment 5:F2It is counted for fertile plant in group and sterile plant segregation ratio
The F of different female parents will be derived from2Filial generation branch plants, and unites in the loose powder phase to fertile plant and sterile plant segregation ratio
Meter, and reduced value carries out Chi-square statistic, calculatesPValue, to judge whether fertile plant and sterile plant segregation ratio meet 3:1.Knot
Fruit shows, except A141 filial generation (P=0.01) other than, 299 parts of F2Meet 3:1 for the Fertility segregation ratio of plant,PValue between
Between 0.06 and 1.00.Fig. 5 (under) it is the Fertility segregation of some materials than statistic and Chi-square Test result.To verify F2Group
The consistency of genotype and phenotype, utilizes wild type gene in bodyZmMs1Functional label Ms1-1 and mutated genems1- albFunctional label ms1-IN1, to A141 hybridize F2Offspring and other 2 F separated partially2The institute of group (A160 and A440)
There is single plant to carry out genotype detection, as a result as shown in fig. 6, the genotype of all fertile single plants is allZmMs1/ZmMs1OrZmMs1/ms1-alb, the genotype of sterile single plant is allms1-alb/ms1-alb, genotype is consistent with phenotype.
Embodiment 6:F2For group's sterile plant fringe type and pollen grain dyeing detection
F in different maternal sources2For in sterile plant tassel, anther exposing phenomenon is not observed, it means thatms1-alb
Male-sterility phenomenon caused by mutated gene is stable under different genetic background；For the stabilization for further verifying infertility
Property, in the F of each parent23 sterile single plants are randomly selected in offspring, are taken its anther in the loose powder phase, are utilized the iodo- iodine of 1% concentration
Change potassium solution dyeing, all F2The generation pollen grain of sterile single plant can not colour, withms1-albMutant pollen grain embodies phase
Same morphological feature；Two representativeness F2Generation sterile single plant tassel and pollen grain coloration result are as shown in Figure 7.
Under 7:300 parts of embodiment different genetic backgroundsms1-albMutant answering in initiative male sterility breeding female parent
With
Under 300 parts of different genetic backgroundsms1-albMutant can be used as basic material, carry out 4- with corresponding female parent material
5 wheel backcrossings, are had using ms1-IN1 or ms1-IN2 selection before each round backcrossingms1-albThe plant of mutated gene is returned
It hands over, final can get both hadms1-albMutated gene while the again new material with recurrent parent genetic background (ZmMs1/ms1-alb), it is selfed, is can be obtained under new genetic background using a wheelms1-albMaize cell kernel male sterile green wood
Material, the female parent material as hybrid seeding are produced for cenospecies.
In short, the present invention by hybridization and selfing in the way of, assist with molecular marker assisted selection, establish a set of detection
The method of corn genic male sterile gene caused male-sterility Detection of Stability under different genetic background；The
One step crossing performance Heterozygous mutants are male parent, and 300 parts of self-mating systems ensure that cytoplasmic diversity after hybridization as female parent,
Therefore the influence that different cytoplasms acts on mutated gene is had detected simultaneously.This method is prominent for corn genic male sterile
Become gene and provides more fully theoretical foundation for the sterile production of hybrid seeds.
Pertinent literature
1, Wu Suowei, just now minister, Deng Lianwu, universal member (2012) corn recessive nucleus male sterility progression and its breeding
Application approach analysis, Molecular Plant Breeding (network edition), 10: 1001-1011
2, Liu Shuanshuan, Wu Suowei, Rao Liqun, the male sterile Study on Molecular Mechanism of universal member (2018) maize kernel and application point
Analysis, Chinese biological engineering magazine, 38(1): 100-107
3, universal member, Wu Suowei, Zhou Yan, Xie Ke, Li Jinping, An Xueli, Plant Pollen Development controlling geneMs1And its coding egg
It is white, 2017.6.16, Chinese invention patent, ZL201410381072.5
4, universal member, Wu Suowei, Xie Ke, An Xueli, Li Jinping, Zhang Danfeng, Xiao Zhonghua, Liu Shensi are based onMs1Gene constructed
More control infertility carriers of mediation corn male fertility and its application, 2017.2.22, Chinese invention patent,
ZL201510298173.0
5, universal member, Wu Suowei, An Xueli, Xie Ke, Li Jinping, Liu Xinjie, Liu Shuanshuan, Zhang Chunxia, Dong Zhenying, Tian Youhui, it is beautiful
Rice recessive nucleus male sterility mutated genems1Functional label and its application, Chinese invention patent application number:
201810868535.9。
Claims (4)
1. a kind of corn recessive nucleus male sterility mutated genems1-albSterile stability analysis side under different genetic backgrounds
Method, which is characterized in that with 300 parts of corn inbred lines (ZmMs1/ZmMs1) be female parent, with Heterozygous mutants (ZmMs1/ms1- alb) it is male parent, carry out artificial pollination；300 parts of F of harvest1It is sowed for seed in next plantation season, in seedling stage, utilizes mutation base
Causems1-albIsolate functional indicia ms1-IN1 (1F/1.2R) and ms1-IN2 (2F/2.2R) screen heterozygote single plant
(ZmMs1/ms1-alb), and be selfed in pollination period；To 300 parts of F of acquisition2It is carried out for the Fertility segregation ratio of group plant
Statistics, Chi-square Test result deviate the group of 3: 1 segregation ratio, utilize wild type geneZmMs1-1Isolate label Ms1-1
(1F/2.2R) and mutated genems1-albIsolate functional indicia ms1-IN1 to its all single plantZmMs1Equipotential position
The genotype of point is identified, the consistency between phenotype and genotype is investigated, to verify mutated genems1-albDifferent
Sterile stability under nucleus and cytoplasmic inheritance background.
2. carrying out corn recessive nucleus male sterility gene using analysis method described in claim 1ms1-albOther equipotentials are prominent
Become Sterile stability analysis and application of the sterile gene under different genetic backgrounds.
3. the corn recessiveness core male under the 300 parts of different genetic backgrounds obtained using analysis method described in claim 1 is not
Educate material, which is characterized in that all material existsZmMs1Gene loci isms1-albHomozygous mutant type (ms1-alb/ms1- alb), and all homozygous mutation materials show as complete male-sterility.
4. the 300 parts including but not limited to as claimed in claim 3 different heredity back obtained using method described in claim 1
Corn male recessive cytoblast sterile material under scape, the application in the female parent of initiative male sterility crossbreeding and the production of hybrid seeds.
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| CN112680460A (en) * | 2021-03-12 | 2021-04-20 | 北京科技大学 | Male sterile gene ZmTGA9 and application thereof in creating male sterile line of corn |
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