CN1514017A - Method of discriminating corn cytoplasm male sterality material cytoplasm kind - Google Patents

Abstract

A method for discriminating the cytoplasmic type T, C, or S of the cytoplasmic male steril (CMS) material for corn features that the genotype determined selfing line of corn and the material to be tested take part in conventional sexual hybridization and the phynotype of restoring or maintaining the fertility of male flower of hybridized descendant is used to discriminate the sterile cytoplasmic type, or the molecular hybridization of mitochondrial DNA and the resultant RFLP fingerprint are used to discriminate the sterile cytoplasmic type, or the PCR of mitochondrial DNA by the designed primer and the resultanjt PCR fingerprint to discriminate the sterile cytoplasmic type.

Description

Differentiate the method for maize cell matter male sterile material kytoplasm type

Technical field

The invention belongs to field of plant variety breeding technology, be specifically related to differentiate maize cell matter male sterile (Cytoplasmic Male Sterility, CMS) method of material kytoplasm, be specifically designed to the corn C MS breeder and the corn hybrid seed sterilization production of hybrid seeds producer to kytoplasm type (normal kytoplasm (N) and T, C, S type sterile cytoplasm) identify and laboratory qualification in field.

Background technology

Maize cell matter male sterile material is found in 1931 by Rhoades.M.M the earliest.So far, found the CMS material in the different kytoplasms of kind more than 100 source in the world, corn is to utilize the CMS germplasm to produce the important food crop of cross-fertilize seed in the world the earliest.Utilize CMS genetic germplasm preparing hybrid kind, can not only guarantee the quality of cross-fertilize seed seed, and can reduce the labour and drop into, reduce production costs, reduce labor intensity, thereby obtain huge economic benefit.Therefore receive the concern of various countries breeding man and seed producers, by 1970, utilization was selected from Texas always " Mexico June " the CMS cross-fertilize seed of corn sterile line production accounted for 85% of entire United States Maize Production area.Yet the huge gain of CMS cross-fertilize seed on producing ignored people fully and found in Philippines that specialization infected Texas in 1961 " Mexico June " report of corn southern leaf blight T type physiological strain of sterile cytoplasm.Thereby cause these cause of disease microspecies to spread insanely in the U.S. large-area corn sterile cytoplasm host colony, cause the whole America serious underproduction of Maize Production in 1970, loss is up to 3,000,000,000 US dollars.Painful historical lessons are recognized scientist and are sought novel cytoplasmic sterility germplasm, and avoiding the widely-used of single cell matter sterile material is the important prerequisite of effectively utilizing crop CMS.The evaluation of CMS kytoplasm is the important assurance that realizes this prerequisite with classification.Beckett J.B. (Crop Science in 1971,1971,11, p724) take the lead in selecting for use 18 corns test kind, the CMS material in 30 kinds of different kytoplasms sources is carried out widely the fertility of test cross and filial generation identify, extensive according to fertility, the reductive analysis of the relation of protecting is T with the kytoplasm classification of type of corn C MS, C, three kinds of S, and proof S type sterile cytoplasm is maximum one class in the corn C MS germplasm.Hua Zhong Agriculture University's corn group has successively been bred beautiful No. 2 of China, China beautiful No. 3 and beautiful No. 4 sterile hybrids of China of S group kytoplasm since the seventies in 20th century.China Agricultural University, Agricultural University Of He'nan successively breed the cross-fertilize seed of C type cytoplasmic male sterility, have all received good economic benefit, still have the trend that enlarges development in recent years.

Since corn T, C, and the CMS of three kinds of kytoplasm types of S has different genetic developments and phenotypic effect separately.When the breeder makes up at the assembly triple crossing, need at first to understand the kytoplasm type of the CMS material that is utilized; The investigator need study the ownership of its sterile cytoplasm type to newfound corn C MS material; The cross-fertilize seed producer needs the kytoplasm quality of CMS seed is carried out strict detection when processing and packing in results.And the huge test cross identification system that 18 tests that Beckett J.B is adopted are planted uses in almost can't and producing in research.He also is difficult to seek in China at employed test kind.

Summary of the invention

Purpose of the present invention is intended to set up a kind of accurate, identify the method for corn C MS kytoplasm type fast, it both can carry out kytoplasm to newfound CMS material and sort out evaluation, can be used for again the seed specimen after plant sample between seed farm and the results is carried out quality examination, therefore the dna fingerprinting technology that has comprised the CMS kytoplasm among the present invention simultaneously also can be the dispute of solution seed quality the judicial evidence with law benefit is provided.

The present invention is achieved through the following technical solutions:

1, the method for a kind of discriminating maize cell matter male sterile (CMS) material T, C, S kytoplasm type, it is characterized in that, utilize definite genotypic corn inbred line and detected materials to carry out conventional sexual hybridization, be resumed or maintained phenotype according to filial generation male flower fertility, differentiate the sterile cytoplasm type (the male flower fertility restorer of abbreviation kytoplasm type discriminating is effect property mensuration specially) of detected materials; Or utilize the molecular hybridization of Mitochondrial DNA, according to polytypism (RFLP) finger printing of restriction fragment length, differentiate the sterile cytoplasm type (the RFLP fingerprint pattern technology that is called for short the Mitochondrial DNA that the kytoplasm type differentiates) of detected materials; Or utilize the primer that designs, and Mitochondrial DNA is carried out polymerase chain reaction (PCR) reaction, according to the PCR finger printing, differentiate the sterile cytoplasm type (the PCR fingerprint pattern technology of the Mitochondrial DNA that abbreviation kytoplasm type is differentiated) of detected materials.

2, the male flower fertility restorer differentiated of described kytoplasm type specially effect property mensuration according to the following step;

1) to determine genotypic self-mating system test kind extensive 313, that differentiate as corn C MS material kytoplasm type from phoenix 1;

2) with detected materials respectively with extensive 313, from phoenix 1 hybridization between selfed lines, the preparing hybrid kind, the land for growing field crops mode is planted routinely;

3) flowering period sterile fully, highly sterile to the test cross offspring by male flower, half can educate, height can educate and can educate fully 5 grades of fertility phenotypes and carry out fertility and identify, determines the phenotype that sterility is held or is resumed;

4) differentiate the sterile cytoplasm type of corn C MS according to the phenotype of fertility.

3, the RFLP finger printing of the Mitochondrial DNA of described kytoplasm type discriminating is according to the following step;

1) detected materials is sowed under 30 ℃ of dark conditions, the back of emerging is dark cultivated 7 days;

2) extract the total DNA of etiolated seedling;

3) cut total DNA with BamHI or HindIII restriction endonuclease enzyme;

4) clone B376 and the pH2-7-1 from Mitochondrial DNA respectively, separate the cob cloned sequence of 5.6kb and the coxII cloned sequence of 1.7kb, the preparation probe;

5) hybridize by probe/enzyme combination carrying out Southern of cob/BamH I or coxII/HindIII

6) differentiate corn C MS sterile cytoplasm type according to the RFLP finger printing.

In the Southern hybridization collection of illustrative plates of cob/HindIII probe/enzyme combination (Fig. 2)

The experimental result of N, T, C material is just the same, and hybrid belt is 6.4kb and 1.8kb

The S kytoplasm has the hybrid belt line of 4.4kb and 1.8kb,

The CMS kytoplasm of all 4.4kb of having band lines is confirmed as the S kytoplasm.

In the Southern hybridization collection of illustrative plates of coxII/BamH I probe/enzyme combination (Fig. 1)

T kytoplasm material has the specific cross band line of 2.5kb

The CMS kytoplasm of all 2.5kb of having band lines is confirmed as the T kytoplasm

C kytoplasm material has the specific cross band line of 5.0kb

The CMS kytoplasm of all 5.0kb of having band lines is confirmed as the C kytoplasm

S kytoplasm material has the specific cross band line of 2.0kb

The CMS kytoplasm of all 2.0kb of having band lines is confirmed as the S kytoplasm

N kytoplasm material has the specific cross band line of 6.0kb,

The kytoplasm of all 6.0kb of having band lines is confirmed as (N) normal kytoplasm

4, the PCR finger printing of the Mitochondrial DNA of described kytoplasm type discriminating is according to the following step;

1) gets detected materials seed or blade, extract total DNA with small-sample method;

2) add the special primer of differentiating corn T, C, S material sterile cytoplasm;

3) carry out a polymerase chain reaction (PCR), amplification, electrophoresis;

4) differentiate corn C MS sterile cytoplasm type according to the PCR finger printing..

5, described primer is shown in sequence table SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5 and SEQ ID NO:6.

Below the PCR measuring method of Mitochondrial DNA that CMS material kytoplasm type is differentiated do to describe further:

(1) gets detected materials seed or blade, extract total DNA with small-sample method

(2) add corn T, the C of the present invention's design, three couple (shown in sequence table SEQ ID NO:1-6) special primer of S material cytoplasmic sterility genes involved

Below be the designed primer of registration sequence Z11843 according to GENEBANK:

Design primer C-1 5 '-TGAAAGGGTGGTGGAATA-3 (being numbered in the sequence table SEQ ID NO:1)

Primer C-2 5 '-GAGCCAAAGTAATGAGAAAA-3 (being numbered in the sequence table SEQ ID NO:2)

The target segment of amplification is 176Nt to the 854Nt totally 698 bases of Z11843.

Below be primer according to the registration sequence A F542203 design of GENEBANK:

Design primer S-1 5 '-GATGCTATGCTAAGCGAGAT-3 ' ((being numbered in the sequence table SEQ ID NO:3))

The target segment of primer S-2 5 '-CCGCTAACCCACTCTTCT-3 ' (being numbered in the sequence table SEQ ID NO:4) amplification is 3766Nt to the 4633Nt totally 885 bases of AF542203.Below be primer according to the registration sequence M12582 design of GENEBANK:

Design primer T-1 5 '-GTCGTGTCCTGGTAGCCT-3 ' (being numbered in the sequence table SEQ ID NO:5)

The target segment of being numbered in the primer T-2 5-CCTCCTTCATTCCGTTGT-3 sequence table (SEQ ID NO:6) amplification is 1052Nt to the 1469Nt totally 435 bases of M12582.

(3) carry out a polymerase chain reaction (PCR), amplification, electrophoresis

The program of pcr amplification is:

94 ℃/1min (sex change)--55 ℃/1min (annealing)---72 ℃/1min (extension), 30 circulations,

Amplified production on Agarose gel (ethidium bromide staining), 90V electrophoresis 1 hour 30 minutes.

(4) differentiate corn C MS sterile cytoplasm type according to PCR finger printing (as shown in Figure 3).

Electrophoretogram shows that pcr amplification product is the examination material of 689 base band lines, is confirmed as the C kytoplasm

Electrophoretogram shows that pcr amplification product is the examination material of 885 base band lines, is confirmed as the S kytoplasm

Electrophoretogram shows that pcr amplification product is the examination material of 435 base band lines, is confirmed as the T kytoplasm

Electrophoretogram shows the examination material that does not have pcr amplification product, is confirmed as normal (N) kytoplasm

The user can select for use a certain authentication method among the present invention to carry out the work according to the actual conditions and the purpose of constituent parts; The male flower fertility restorer that the kytoplasm type is differentiated is effect property measuring method specially;

Method economical and effective of the present invention does not need special plant and instrument, but needs just can draw reliable conclusion through two seasons of growth, is suitable for the breeder and uses.

The RFLP fingerprint spectrum method of the Mitochondrial DNA that kytoplasm type of the present invention is differentiated needs the special plant and instrument of being engaged in molecular biology research, can obtain the kytoplasm classification results in about 15 days, is suitable for applied basic research person and uses.

The PCR fingerprint spectrum method of the Mitochondrial DNA that kytoplasm type of the present invention is differentiated is simple and practical, and it does not only need a little maize leaf or simple grain corn, and just can obtain qualification result accurately and reliably in 6 hours.Thereby be more suitable for seed producers and administrative authority carries out quality examination to field large sample and seed large sample.

The RFLP finger printing of Mitochondrial DNA of the present invention and the biotechnology of PCR finger printing, have and be not subjected to environmental influence, not limited by the season of growth, the advantage that the banding pattern feature is stable, therefore also be applicable to the seed quality dispute and need mediate, accept the test analysis task of the trust of relevant judicial expertise mechanism.

Accompanying drawing and explanation thereof

Fig. 1: the finger printing of corn C MS Mitochondrial DNA coxII/BamHI probe/enzyme combination RFLP;

M represents molecular weight marker among the figure, and digital 1-18 represents the sample of material sequence number in the table 1, N, and T, C, S represent the kytoplasm type respectively

Fig. 2: the finger printing of corn C MS Mitochondrial DNA cob/HindIII probe/enzyme combination RFLP;

M represents molecular weight marker among the figure, and digital 1-18 represents the sample of material sequence number in the table 1, N, and T, C, S represent the kytoplasm type respectively

Fig. 3: the finger printing of corn C MS Mitochondrial DNA PCR;

M represents molecular weight marker among the figure, and digital 1-10 represents the sample of material sequence number in the table 3, N, and T, C, S represent the kytoplasm type respectively

Embodiment

Embodiment 1: the evaluation of corn 77CMS-X kytoplasm classification

1, for the examination material

This test adopts 18 kind of two cover with dyskaryosis and homogeneity heteronuclear material altogether, is provided by the applicant Hua Zhong Agriculture University corn chamber.Every kind of material is extensive 313 with corn inbred line respectively, from phoenix 1 hybridization, carry out the kytoplasm classification according to the F1 fertility.

Table 1 is for the examination material

Sequence number title material sequence number title material

1 Mo17 10 77CMS-T

2 Mo17CMS-T 11 77CMS-EL

3 Mo17CMS-RB 12 77CMS-C

Xu of 4 Mo17CMS-EL, 13 77CMS-Tang

Xu 14 77CMS-Vg of 5 Mo17CMS-Tang

6 Mo17CMS-C, 15 77CMS-X (unknown kytoplasm)

The two 16 77CMS-rivers of 7 Mo17CMS-

8 Mo17CMS-J 17 77CMS-S

9 77 18 77CMS-R gold

2, experimental technique

2.1 specially effect property evaluation of fertility restorer

18 kinds extensive 313 with the corn inbred line that has the specific gene type respectively for the examination material, from phoenix 1 hybridization, preparing hybrid kind; The land for growing field crops mode is planted routinely; Carrying out fertility by 5 grades of standards of perfection of male flower fertility flowering period identifies.

The grade scale of table 2 corn male flower fertility phenotype

The grade scale of table 2 corn male flower fertility phenotype

1 complete sterile flower pesticide is shrivelled, does not expose clever shell, WUHUAFEN or pollen abortion

2 highly sterile only a few flower pesticide expose grain husk visitor, WUHUAFEN or pollen abortion

3 half fertile flower medicines are half-full full, and most flower pesticide expose, the part loose powder

4 height fertile flower medicines are full, and pollen is normal, and most of flower pesticide exposes and loose powder

5 can educate normally fully and bloom, a large amount of loose powder

2.2 corn etiolated seedling total DNA extraction and purifying:

The etiolated seedling DNA extraction of all examination materials adopts CTAB method (as described below)

1. getting maize leaf 5.0g grinds in liquid nitrogen, change in the 50ml centrifuge tube, add 2ml by every gram sample and be preheated to 65 ℃ CTAB extracting solution (1.17MNaCL, 0.1016M EDTA-8.0,0.835M Ttis-7.5,1.6%CTAB, 1% β-thin basic ethanol), mixing, 65 ℃ water-bath 60-90 minute, every 15min shakes up once.

2. after water-bath finishes, be cooled to room temperature (25 ℃), add the equal-volume chloroform: different alcohol (24: 1), carefully shook test tube 10 minutes, make lower floor's organic phase by colourless → green → black (deep green).

3. (〉=20 ℃) under the room temperature, centrifugal 10 minutes of 3.2Krpm shifts supernatant in another centrifuge tube with the 5ml suction nozzle that cuts off head, carries once if supernatant is still then used 24: 1 for green again.

4. add 2/3 volume Virahol, softly put upside down twice, leave standstill behind the 20min gently fully mixing, make DNA agglomerating.Choose DNA with hook, in the 1.5ml centrifuge tube, embathed 24 hours, change primary wash liquor midway with 70% ethanol.

5. DNA purifying: DNA is dried up, add an amount of TE-8.0 damping fluid, place 65 ℃ of water-baths 15 minutes, with dissolving DNA; After being chilled to room temperature, add 10 μ l 10mg/ml RNaseA, mixing; Under 37 ℃, incubation 2 hours, use equal-volume phenol again: chloroform (1: 1) and chloroform: each extracting of primary isoamyl alcohol (24: 1) is once, under the room temperature, centrifugal 10 minutes of 8Krpm, the 3M NaAC (pH5.2) of adding 1/10 volume adds 2 times of volume dehydrated alcohol deposit D NA in supernatant liquor; Hook goes out cotton-shaped DNA, puts into 1.5ml Eppendorf test tube, and is with 70% washing with alcohol once of short duration centrifugal, makes DNA adherent, removes ethanol, dries up the DNA precipitation.Every pipe adds an amount of T.E-8.0 damping fluid, and fully the dissolving DNA precipitation exists in-20 ℃ of refrigerators standby.

2.3 the polytypism of restriction fragment length (RFLP) is analyzed

1. with the total DNA of an amount of TE damping fluid dissolving blade, measure light absorption value under the wavelength of 260nm and 280nm, determine DNA concentration, and with the quality of 0.8% agarose electrophoresis inspection DNA extraction, master tape sample clearly can be used for next step experiment.

2. get total DNA about 10 μ g in the Eppendorf test tube of 1.5 μ l, add the restriction enzyme of 30 units, 5 μ l10 * damping fluids, 2 μ lBSA add ddH ₂O makes final volume to 50 μ l, cuts 16-20hrs at 37 ℃ of following enzymes behind the mixing.Sample after enzyme is cut is got 4 μ l electrophoresis detection on 0.8% agarose, and 100V electrophoresis 1.5hrs, DNA are the disperse shape and show that the DNA enzyme digestion reaction is complete.After confirming that enzyme is cut effect, the sepharose of preparation 0.8%, per 50 μ lDNA endonuclease reaction liquid add 5 μ l point samples buffering and stop endonuclease reaction, and each swimming lane is clicked and entered all the DNA enzyme of (55 μ l) and cut sample liquid, finishes electrophoresis process.

3. gel is cut into suitable size, with 0.2mol/L HCl liquid sex change 8 minutes until the blob of viscose flavescence, promptly use 0.4mol/L NaOH solution as shifting liquid, by capillary theory the DNA on the glue is transferred on N '-nylon membrane, after 24 hours with nylon membrane with 2 * SSC liquid rinsing 2 times, each 5 minutes, dry the back and clip with filter paper, baking is 2-3 hour in 80 ℃ of-120 ℃ of vacuum drying ovens, and DNA is fixed on the film.

4. the mark of probe

Every Hybond membrane uses probe liquid 25 μ l

ddH ₂O 7.7μl

Dna probe (100ng/ μ l) 2.0 μ l

λ/HindIII(1ng/μl) 0.8μl

Random primer+damping fluid 7.5 μ l

dNTP(-dCTP)(2mM) 3.0μl

Klenow enzyme (2unit/ μ l) 2.0 μ l

α ³²P-dCTP 20μci

Cumulative volume: 25 μ l

Behind the mixing, 37 ℃ are incubated 4-8 hour, add equal-volume TE damping fluid again, the probe prehybridization solution 250 μ l (0.05Mtris-8.0 of 10 times of volumes, 0.01M EDTA-8.0,5XSSC, 1 times of denhardts, 0.2%SDS), 100 ℃ of water-bath 10min, ice bath 10min is standby again.

5. molecular hybridization and radioautograph

Nylon membrane after the transfer is in prehybridization solution, after carrying out 4 ~ 6 hours prehybridization under 65 ℃, to falling prehybridization solution, add the good probe of mark, be preheated to 65 ℃ hybridization solution (10%Dextran Sulfate according to quantity (by every the film 5 ~ 10ml hybridization solution) adding of film, 0.05Mtris-8.0,0.01M EDTA-8.0,5 * SSC, 1 * denhardts, 0.2%SDS), slowly rotate hybridization about 16 ~ 20 hours in 65 ℃.

Discard hybridization solution, add 2 * SSC, the film damping fluid of washing of the low rigorous degree of 0.5%SDS is washed film 2 times under room temperature, and each 20 minutes, with 0.2 * * SSC, the high rigorous film damping fluid of washing of 0.1%SDS is washed film 2 times, each 30min in 65 ℃.The taking-up film blots the film surface with thieving paper and swims, and preservative film is wrapped Hybond membrane, detects radioactive intensity on the film, and record data are opened and shielded before camera obscura is put sensitizing successively well, the X-ray sheet, and Hybond membrane shields behind the intensifying screen.Shut camera obscura and be stored in-70 ℃ of refrigerators, according to the radioactive intensity size, autography 10 ~ 15 days.

Hybond membrane goes probe to reuse: 0.1 * SSC, 0.1%SDS washes film 30min under 90 ℃ ~ 95 ℃ conditions, determine the probe wash-out after, recross is used.

3, experimental result

3.1 fertility restorer is effect property qualification result specially:

According to the special method of effect property classification of fertility restorer, all by extensive 313 (test cross offspring's fertility can be educated or can educate fully for height) of recovering, detected materials is confirmed as having S type kytoplasm, all by extensive 313 maintenances (test cross offspring's fertility is for fully sterile or highly sterile), recovered from phoenix 1 simultaneously, detected materials is confirmed as having C type kytoplasm, is allly kept by extensive 313, again by what keep from phoenix 1, detected materials is confirmed as having T type kytoplasm simultaneously.

Except that 77 for normally educating the kytoplasm, in other 17 kinds of CMS materials, be accredited as

Having of T kytoplasm: Mo17CMS-T, 77CMS-T;

Having of C kytoplasm: Mo17CMS-C, Mo17CMS-RB, Mo17CMS-EL, 77CMS-EL, 77CMS-C;

Having of S kytoplasm: Mo17CMS-Tang Xu, Mo17-are two, Mo17CMS-J, Xu of 77CMS-Tang, 77CMS-Vg

77CMS-river, 77CMS-R gold, 77CMS-S.

Unknown kytoplasm 77CMS-X is accredited as the C kytoplasm.

3.2 RFLP technical measurement result

3.21 the probe of cob/HindIII/enzyme combination

For examination material RFLP result such as Fig. 2 in probe/enzyme combination of cob/HindIII.

The result shows: No. 15 the unknown kytoplasm 77CMS-X of material is accredited as and does not belong to the S kytoplasm.Supply examination material RFLP result such as Fig. 1 in probe/enzyme combination of cox II/BamH I: the result shows: No. 15 the unknown kytoplasm 77CMS-X of material is accredited as the C kytoplasm.

Embodiment 2: the PCR fingerprint identification of corn kytoplasm

1, for the examination material

The PCR fingerprint identification of table 3 corn kytoplasm is for the examination material

Sequence number title material sequence number title material

1 Mo17CMS-C 6 Mo17CMS-S

2 77CMS-C, 7 Mo17CMS-are two

3 Mo17CMS-EL 8 77CMS-T

4 Mo17 9 Mo17CMS-T

5 77CMS-S 10 W23-T

2, experimental technique:

2.1 DNA extraction is used " small sample total DNA extraction method

1. get the 100mg sample in the 1.5ml centrifuge tube, add 500 μ lCTAB extracting solution (1M tris-8.0 100mM; 0.5MEDTA-8.0 150mM; 5M NaCl 2100mM; PVP 2%; CTAB 2%; 14M β ME 140mM),

With the Glass drill head that self-control and centrifuge tube match, electric drill was milled one minute;

2. will grind good sample and place 65 ℃ of water-bath 20min; In the water-bath process, at interval the mixing sample is once gently for 5-10min;

3. in the process sample of water bath processing, add 24: 1 chloroform of 600 μ l: primary isoamyl alcohol solution, mixing 3min gently

4. centrifugal 8 minutes of mixing is good sample 12000rpm in desk centrifuge;

5. supernatant liquor is changed in another new 1.5ml centrifuge tube, add the dehydrated alcohol of 400 μ l-20 ℃ precoolings, centrifugal 3 minutes of 10000rpm;

6. outwell supernatant liquor, the ethanol with 75% cleans the DNA throw out once, carefully blots ethanol, dries the DNA throw out under the room temperature;

7. add the went out distilled water dissolving DNA precipitation of bacterium of 500 μ l.Get 5 μ l and be used for the PCR reaction.

2.2 polymerase chain reaction (PCR) reaction system:

DNA sample liquid 5ul

10 * damping fluid (Mg ²⁺) 2ul

dNTPs(2mM) 2ul

Taq enzyme (5u/ul) 0.2ul

Primer (50uM) 0.2ul

ddH ₂O 10.6ul

Tatal?V 20ul

Annotate: C1, C2, S1, S2, each 0.2ul of six kinds of primers of T1, T2 are added in the reaction system

2.3 polymerase chain reaction (PCR) program:

Step 1 94 ℃/3 minutes

Step 2 { 94 ℃/1 minute; 55 ℃/1 minute; 72 ℃/1 minute } 30 circulations

Step 3 72 ℃/5 minutes, 1 circulation

4 ℃ of steps 4 are preserved

3, experimental result:

3, experimental result:

Experimental result as shown in Figure 3;

Sequence number is that (Mo17CMS-EL) the amplified band size is accredited as the C kytoplasm for 698bp to the 1-3 test material for Mo17CMS-C, 77CMS-C;

Sequence number is that No. 4 test materials (Mo17) do not have the band line, is accredited as normal (N) kytoplasm;

Sequence number is that 5-7 test material (Mo17CMS-is two for 77CMS-S, Mo17CMS-S) amplified band size is accredited as the S kytoplasm for 885bp;

Sequence number is that (W23-T) the amplified band size is accredited as the T kytoplasm for 435bp to the 8-10 test material for 77CMS-T, Mo17CMS-T.

Embodiment 3; The present invention is used for the administration of justice of seed quality dispute and entrusts evaluation and test

The inventor in 2000 accepts the trust of certain judicial expertise mechanism the normal kytoplasm of a certain corn hybridization combination and the kytoplasm type of two kinds of cross-fertilize seed of sterile cytoplasm is identified, the inventor adopts RFLP fingerprint of the present invention that sample has been carried out check and analysis (described test procedure as specification sheets of the present invention as described in).Test shows with analytical results: the tested kind of part label has the sample conclusive evidence that normally can educate the cybridization kind to be S type sterility cytoplasmic hybrid kind in the sample, and the tested kind of part label has the sample conclusive evidence of sterility cytoplasmic hybrid kind for normally educating kytoplasm (N) cross-fertilize seed.The inventor's analyzing and testing is reported as the correct ruling of judicial department, and this plays the scientific basis that the seed quality dispute provides acquire full legal force.

SEQUENCE?LISTING

＜110〉Hua Zhong Agriculture University

＜120〉method of discriminating maize cell matter male sterile material kytoplasm type

<130>

<141>2003-03-10

<160>6

<170>PatentIn?version?3.1

<210>1

<211>18

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(18)

<223>

<400>1

tga?aag?ggt?ggt?gga?ata 18

Lys?Gly?Gly?Gly?Ile

1 5

<210>2

<211>20

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(20)

<223>

<400>2

gag?cca?aag?taa?tga?gaa?aa 20

Glu?Pro?Lys Glu

1

<210>3

<211>20

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(20)

<223>

<400>3

gat?gct?atg?cta?agc?gag?at 20

Asp?Ala?Met?Leu?Ser?Glu

1 5

<210>4

<211>18

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(18)

<223>

<400>4

ccg?cta?acc?cac?tct?tct 18

Pro?Leu?Thr?His?Ser?Ser

1 5

<210>5

<211>18

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(18)

<223>

<400>5

gtc?gtg?tcc?tgg?tag?cct 18

Val?Val?Ser?Trp Pro

1 5

<210>6

<211>18

<212>DNA

＜213〉corn (maize)

<220>

<221>exon

<222>(1)..(18)

<223>

<400>6

cct?cct?tca?ttc?cgt?tgt 18

Pro?Pro?Ser?Phe?Arg?Cys

1 5

Claims (5)

1, the method for a kind of discriminating maize cell matter male sterile (CMS) material T, C, S kytoplasm type, it is characterized in that, utilize definite genotypic corn inbred line and detected materials to carry out conventional sexual hybridization, be resumed or maintained phenotype according to filial generation male flower fertility, differentiate the sterile cytoplasm type of detected materials, i.e. the male flower fertility restorer of kytoplasm type discriminating is effect property mensuration specially; Or utilize the molecular hybridization of Mitochondrial DNA, according to polytypism (RFLP) finger printing of restriction fragment length, differentiate the sterile cytoplasm type of detected materials; Or utilize the primer that designs, and Mitochondrial DNA is carried out polymerase chain reaction (PCR) reaction, according to the PCR finger printing, differentiate the sterile cytoplasm type of detected materials.

2, require 1 described method according to profit, it is characterized in that, the male flower fertility restorer that described kytoplasm type is differentiated specially effect property mensuration according to the following step;

1) to determine genotypic corn inbred line test kind extensive 313, that differentiate as CMS material kytoplasm type from phoenix 1;

2) with detected materials respectively with extensive 313, from phoenix 1 hybridization between selfed lines, the preparing hybrid kind, the land for growing field crops mode is planted routinely;

3) flowering period sterile fully, highly sterile to the test cross offspring by male flower, half can educate, height can educate and can educate fully 5 grades of fertility phenotypes and carry out fertility and identify, determines the phenotype that sterility is held or is resumed;

4) differentiate the sterile cytoplasm type of corn C MS according to the phenotype of fertility.

3, method according to claim 1 is characterized in that, the RFLP finger printing of the Mitochondrial DNA that described kytoplasm type is differentiated is according to the following step;

1) detected materials is sowed under 30 ℃ of dark conditions, the back of emerging is dark cultivated 7 days;

2) extract the total DNA of etiolated seedling;

3) cut total DNA with BamHI or HindIII restriction endonuclease enzyme;

4) clone B376 and the pH2-7-1 from Mitochondrial DNA respectively, separate the cob cloned sequence of 5.6kb and the coxII cloned sequence of 1.7kb, the preparation probe;

5) hybridize by probe/enzyme combination carrying out Southern of cob/BamH I or coxII/HindIII;

6) differentiate corn C MS sterile cytoplasm type according to the RFLP finger printing.

4, method according to claim 1 is characterized in that, the PCR finger printing of the Mitochondrial DNA that described kytoplasm type is differentiated follows these steps to;

1) gets detected materials seed or blade, extract total DNA with small-sample method;

2) add the special primer of differentiating corn T, C, S material sterile cytoplasm;

3) carry out a polymerase chain reaction (PCR), amplification, electrophoresis;

4) differentiate corn C MS sterile cytoplasm type according to the PCR finger printing..

5, method according to claim 1 is characterized in that, described primer is shown in sequence table SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

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