CN108812298A - A kind of quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea - Google Patents
A kind of quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea Download PDFInfo
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- CN108812298A CN108812298A CN201810775751.9A CN201810775751A CN108812298A CN 108812298 A CN108812298 A CN 108812298A CN 201810775751 A CN201810775751 A CN 201810775751A CN 108812298 A CN108812298 A CN 108812298A
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Abstract
The invention discloses one kind, quick and precisely the glutinous double recessive corn inbred line method of breeding sweet tea, step are：Screeningsh2sh2Type super-sweet corn meets breeding objective as donor parents, selectionwxwxCorn constructs BC as receptor parent1F1, to BC1F1Seed shows that light seed screens using iodine stainingwxwxGermplasm, at the same it is rightwxwxGermplasm is had using DNA marker detectionSH2sh2For middle seed selection matter, each backcross generations use seed selection grain in iodine staining and DNA marker acquisition, are returned to BC3F1, selfing acquisition BC3F2, wherein fold seed is the glutinous double recessive corn inbred line of sweet tea.The glutinous double recessive corn inbred line method of quick and precisely breeding sweet tea of the invention passes through the primer detection of specific primer T1, T2, can accurately and efficiently screen containing recessivenesssh2The individual of gene significantly improves the Breeding Efficiency of the glutinous double recessive corn inbred line of sweet tea by the M8003 line in backcross generations.
Description
Technical field
The present invention relates to breed of crop technical fields, especially design a kind of quick and precisely glutinous double recessive corn of breeding sweet tea from
Hand over system, method.
Background technique
Fresh edible maize is divided into Sweety and waxy corn.It is popular with consumers again because of its peculiar flavour and nutritional ingredient.With diet
Structure changes, and people increase fresh edible maize demand year by year.Especially China's the Course of Urbanization is accelerated and agricultural industry knot
Structure optimization, causes to fresh edible maize increase in demand, thus the initiative of fresh edible maize new germ plasm has become fresh edible maize breeding and fresh
Important research content and the direction of corn breeding scholar are eaten, important foundation will be established for sweet-waxy maizes breed breeding.
Breeding of Sweet-waxy Corn dependent on double recessive (such as wxwxsh2sh2) self-mating system as parent, but existing conventional herd breeding sweet tea
Glutinous double recessive self-mating system only relies on phenotypic evaluation, thus Breeding Process is more time-consuming, increases the new germ plasm initiative time.
Summary of the invention
Object of the present invention is to solve the above problems, a kind of quick and precisely glutinous double recessive corn inbred line side of breeding sweet tea is provided
Method is able to achieve quickly accurate breeding sweet-waxy maizes new germ plasm.The present invention is based on modern molecular labelling techniques, to recessive sh2 gene
It carries out the initiative that Rapid identification is quickly accurate sweet-waxy maizes new germ plasm and has established technology and material foundation.
To achieve the goals above, the technical scheme is that：
It is a kind of that quick and precisely the glutinous double recessive corn inbred line method of breeding sweet tea, this method step are：
S1, the DNA for extracting corn；
S2, using specific primer T1, T2 according to the mutational site characteristic Design of sh2 sequence, detect corn sh2 base
Cause screens candidate donor parent；T1 and T2 sequence is that the sh2 gene based on existing super-sweet corn sh2sh2 material tests is distinctive
DNA marker；
S3, select the wxwx genotype waxy corn for meeting breeding objective as receptor parent；
S4, auxiliary backcross breeding, utilize waxy corn receptor parent (wxwxSH2SH2) and donor parents (WXWXsh2sh2)
Hybridization obtains BC until being most selfed afterwards through a generation3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive of sweet tea
(wxwxsh2sh2) self-mating system.
Further, the T1 is ACTTACAGAGGTGCTCCAAAGGTAC, and T2 is
TCCTGAATTGTTATTTAATGGCATG。
Further, donor parents are to contain sh2sh2 genotype super-sweet corn.
Further, the step of step S1 is：
S11,65 DEG C of preheating CTAB buffers；
S12, it takes the fresh blade 0.2g of corn to grind in 1mL CTAB buffer, is transferred in 2mL centrifuge tube；
S13, lapping liquid is placed in 30min in 65 DEG C of water-baths, frequently jog centrifuge tube；
S14, after being cooled to room temperature, isometric chloroform and isoamyl alcohol (v is added:V=24:1), jog test tube 10min；
S15, centrifugation 10min is carried out with the revolving speed of 10000rpm, later goes to supernatant in another 1.5mL centrifuge tube；
S16, finally, plus 500 μ L ice cold ethanols, jog mix to generate flocculent deposit (can be in -20 DEG C of standings 20min effects
Fruit is more preferably), 10000rpm is centrifuged 10min, abandons supernatant, is precipitated 2-3 times with 75% ethanol washing, 75% ethyl alcohol is outwelled, by DNA
It dries, 50 μ L ddH is added2O dissolution；
S17, after DNA is completely dissolved, be diluted to 50ng/ μ L, carry out PCR detection, obtain the DNA of corn.
Further, band length 636bp.
Further, the composition of CTAB buffer is 1.17mM NaCl, 0.0016mM EDTA pH=8.0,0.835mM
Ttis-HCl pH=7.5,1.6%CTAB, 1% β-dredge base ethyl alcohol.
Further, in the step S2, the method for screening candidate donor parent is PCR detection method, PCR detection inspection
The step of survey method is：
S21, select PCR reaction total system be 20 μ L, wherein：2μL 10×Taq Buffer(Mg2+), 1.6μL dNTP
Primer after primer, 1.0 μ L before (2.5mM), 1.0 μ L, 0.2 μ L Taq enzyme (2.5 U/L), 1.0 μ L DNA profilings (50ng/ μ L),
13.2μL ddH2O；Response procedures are：95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 52 DEG C of annealing 45s, 72 DEG C of extension 1min,
35 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of heat preservations；
S22,10 μ L pcr amplification products are taken to be detected with 1% agarose gel electrophoresis, selection has purpose band (length
Corn 636bp) is as candidate donor parent.
Further, in the step S4, the step of auxiliary backcross breeding, is：
S41, hybridized using waxy corn receptor parent (wxwxSH2SH2) with donor parents (WXWXsh2sh2), F1(glutinous jade
Meter Shou Ti parent (wxwxSH2sh2)) it is returned with waxy corn receptor parent；
S42, backcross progeny BC is screened using iodine staining1F1In glutinous matter seed, by 3g potassium iodide be dissolved in 80mL steam
It is made into liquor kalii iodide in distilled water, 1g iodine is dissolved in the liquor kalii iodide prepared, that is, is made into iodine staining reagent；It will when use
10 times of iodine staining dilution agent, point 1~2 is dripped in the corn embryosperm scraped off, observation chromogenic reaction, glutinous matter seed displaing amaranth, commonly
The aobvious blue of seed；
S43, glutinous matter seed is sowed, seedling stage screens the plant containing recessive sh2 gene using PCR, and DNA is extracted and the side PCR
Method is the same as step S1 and step S2.
S44, the crlutinous maize containing recessive sh2 gene in backcross generations is screened using the screening technique until BC3F1, finally
It is selfed through a generation and obtains BC3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive of sweet tea (wxwxsh2sh2) from
Hand over system.
Compared with prior art, the invention has the advantages and positive effects that：
Compared with traditional breeding technology, the glutinous double recessive corn inbred line method of quick and precisely breeding sweet tea of the invention passes through spy
The primer detection of specific primer T1, T2 can accurately and efficiently screen the individual containing recessiveness sh2 gene, by backcross generations
M8003 line significantly improve the Breeding Efficiency of the glutinous double recessive corn inbred line of sweet tea.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is the flow diagram of breeding sweet glutinous maize self-bred line of the invention；
Fig. 2 is BCnF1Sh2sh2 genotype detection schematic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
As shown in Figure 1 to Figure 2, the glutinous double recessive corn inbred line method of quick and precisely breeding sweet tea of the invention, step are：
S1, the DNA for extracting corn；
S2, using specific primer T1, T2 according to the mutational site characteristic Design of sh2 sequence, detect corn sh2 base
Cause screens candidate donor parent；T1 and T2 sequence is that the sh2 gene based on existing super-sweet corn sh2sh2 material tests is distinctive
DNA marker；
S3, select the wxwx genotype waxy corn for meeting breeding objective as receptor parent；
S4, auxiliary backcross breeding, utilize waxy corn receptor parent (wxwxSH2SH2) and donor parents (WXWXsh2sh2)
Hybridization obtains BC until being most selfed afterwards through a generation3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive of sweet tea
(wxwxsh2sh2) self-mating system.
The T1 is ACTTACAGAGGTGCTCCAAAGGTAC, T2 TCCTGAATTGTTATTTAATGGCATG.Donor
Parent is to contain sh2sh2 genotype super-sweet corn.
Wherein the step of step S1 is：
S11,65 DEG C of preheating CTAB buffers；
S12, it takes the fresh blade 0.2g of corn to grind in 1mL CTAB buffer, is transferred in 2mL centrifuge tube；
S13, lapping liquid is placed in 30min in 65 DEG C of water-baths, frequently jog centrifuge tube；
S14, after being cooled to room temperature, isometric chloroform and isoamyl alcohol (v is added:V=24:1), jog test tube 10min；
S15, centrifugation 10min is carried out with the revolving speed of 10000rpm, later goes to supernatant in another 1.5mL centrifuge tube；
S16, finally, plus 500 μ L ice cold ethanols, jog mix to generate flocculent deposit (can be in -20 DEG C of standings 20min effects
Fruit is more preferably), 10000rpm is centrifuged 10min, abandons supernatant, is precipitated 2-3 times with 75% ethanol washing, 75% ethyl alcohol is outwelled, by DNA
It dries, 50 μ L ddH is added2O dissolution；
S17, after DNA is completely dissolved, be diluted to 50ng/ μ L, carry out PCR detection, obtain the DNA of corn.
The composition of CTAB buffer is 1.17mM NaCl, 0.0016mM EDTA pH=8.0,0.835mM Ttis-HCl
PH=7.5,1.6%CTAB, 1% β-dredge base ethyl alcohol.
In the step S2, the method for screening candidate donor parent is PCR detection method, which detects the step of detection method
Suddenly it is：
S21, select PCR reaction total system be 20 μ L, wherein：2μL 10×Taq Buffer(Mg2+), 1.6μL dNTP
Primer after primer, 1.0 μ L before (2.5mM), 1.0 μ L, 0.2 μ L Taq enzyme (2.5 U/L), 1.0 μ L DNA profilings (50ng/ μ L),
13.2μL ddH2O；Response procedures are：95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 52 DEG C of annealing 45s, 72 DEG C of extension 1min,
35 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of heat preservations；
Sh2sh2 genotype detection Primer and sequence table
S22,10 μ L pcr amplification products are taken to be detected with 1% agarose gel electrophoresis, selection has purpose band (length
Corn 636bp) is as candidate donor parent.
Wherein in the step S4, the step of auxiliary backcross breeding, is：
S41, hybridized using waxy corn receptor parent (wxwxSH2SH2) with donor parents (WXWXsh2sh2), F1(glutinous jade
Meter Shou Ti parent (wxwxSH2sh2)) it is returned with waxy corn receptor parent；
S42, backcross progeny BC is screened using iodine staining1F1In glutinous matter seed, by 3g potassium iodide be dissolved in 80mL steam
It is made into liquor kalii iodide in distilled water, 1g iodine is dissolved in the liquor kalii iodide prepared, that is, is made into iodine staining reagent；It will when use
10 times of iodine staining dilution agent, point 1~2 is dripped in the corn embryosperm scraped off, observation chromogenic reaction, glutinous matter seed displaing amaranth, commonly
The aobvious blue of seed；
S43, glutinous matter seed is sowed, seedling stage screens the plant containing recessive sh2 gene using PCR, and DNA is extracted and the side PCR
Method is the same as step S1 and step S2；
S44, the crlutinous maize containing recessive sh2 gene in backcross generations is screened using the screening technique until BC3F1, finally
It is selfed through a generation and obtains BC3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive of sweet tea (wxwxsh2sh2) from
Hand over system.
The glutinous double recessive corn inbred line method of quick and precisely breeding sweet tea of the invention is drawn by specific primer T1, T2's
Analyte detection can accurately and efficiently screen the individual containing recessiveness sh2 gene, be significantly improved by the M8003 line in backcross generations
The Breeding Efficiency of the glutinous double recessive corn inbred line of sweet tea.
Claims (8)
1. a kind of quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea, it is characterised in that：This method step is：
S1, the DNA for extracting corn；
S2, using specific primer T1, T2 according to the mutational site characteristic Design of sh2 sequence, detect corn sh2 gene,
Screen candidate donor parent；T1 and T2 sequence is the distinctive DNA of sh2 gene based on existing super-sweet corn sh2sh2 material tests
Label；
S3, select the wxwx genotype waxy corn for meeting breeding objective as receptor parent；
S4, auxiliary backcross breeding, are hybridized with donor parents using waxy corn receptor parent, are obtained until being most selfed afterwards through a generation
BC3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive self-mating system of sweet tea.
2. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as described in claim 1, it is characterised in that：T1 is
ACTTACAGAGGTGCTCCAAAGGTAC, T2 TCCTGAATTGTTATTTAATGGCATG.
3. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as described in claim 1, it is characterised in that：Donor parent
This is to contain sh2sh2 genotype super-sweet corn.
4. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as described in claim 1, it is characterised in that：The step
The step of rapid S1 is：
S11,65 DEG C of preheating CTAB buffers；
S12, it takes the fresh blade 0.2g of corn to grind in 1mL CTAB buffer, is transferred in 2mL centrifuge tube；
S13, lapping liquid is placed in 30min in 65 DEG C of water-baths, frequently jog centrifuge tube；
S14, after being cooled to room temperature, isometric chloroform and isoamyl alcohol, jog test tube 10min is added；
S15, centrifugation 10min is carried out with the revolving speed of 10000rpm, later goes to supernatant in another 1.5mL centrifuge tube；
S16, finally, plus 500 μ L ice cold ethanols, jog mix to generate flocculent deposit, 10000rpm be centrifuged 10min, abandon supernatant
Liquid is precipitated 2-3 times with 75% ethanol washing, outwells 75% ethyl alcohol, DNA is dried, and 50 μ L ddH are added2O dissolution；
S17, after DNA is completely dissolved, be diluted to 50ng/ μ L, carry out PCR detection, obtain the DNA of corn.
5. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as claimed in claim 4, it is characterised in that：CTAB is slow
The composition of fliud flushing is 1.17mM NaCl, 0.0016mM EDTA pH=8.0,0.835mM Ttis-HCl pH=7.5,1.6%
CTAB, 1% β-dredge base ethyl alcohol.
6. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as described in claim 1, it is characterised in that：The step
In rapid S2, the step of method of screening candidate donor parent is PCR detection method, PCR detection detection method, is：
S21, select PCR reaction total system be 20 μ L, wherein：2 μ L 10 × Taq Buffer, 1.6 μ L dNTP, 1.0 μ L leadings
Primer, 0.2 μ L Taq enzyme, 1.0 μ L DNA profilings, 13.2 μ L ddH2O after object, 1.0 μ L；Response procedures are：95 DEG C of initial denaturations
5min；95 DEG C of denaturation 30s, 52 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 recycle；Last 72 DEG C of extensions 10min, 4 DEG C of heat preservations；
S22, it takes 10 μ L pcr amplification products to be detected with 1% agarose gel electrophoresis, chooses the corn conduct for having purpose band
Candidate donor parent.
7. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as claimed in claim 6, it is characterised in that：Belt length
Spend 636bp.
8. the quick and precisely glutinous double recessive corn inbred line method of breeding sweet tea as described in claim 1, it is characterised in that：Step S4
In, the step of auxiliary backcross breeding is：
S41, hybridized using waxy corn receptor parent with donor parents, waxy corn receptor parent and waxy corn receptor parent are returned；
S42, backcross progeny BC is screened using iodine staining1F1In glutinous matter seed, 3g potassium iodide is dissolved in 80mL distilled water
It is made into liquor kalii iodide, 1g iodine is dissolved in the liquor kalii iodide prepared, that is, is made into iodine staining reagent；By iodine staining when use
10 times of dilution agent, 1~2 drop of point observes chromogenic reaction, glutinous matter seed displaing amaranth in the corn embryosperm scraped off, and common seed is shown
Blue；
S43, glutinous matter seed is sowed, seedling stage screens the plant containing recessive sh2 gene using PCR, and DNA is extracted and PCR method is same
Step S1 and step S2.
S44, the crlutinous maize containing recessive sh2 gene in backcross generations is screened using the screening technique until BC3F1, most afterwards through one
Generation selfing obtains BC3F2Group, in BC3F2In select fold seed to get to the glutinous double recessive self-mating system of sweet tea.
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|---|---|---|---|---|
| CN110106282A (en) * | 2019-06-20 | 2019-08-09 | 河北农业大学 | A kind of primer of quick detection sh2sh2 genotype corn and its application |
| CN110132963A (en) * | 2019-04-26 | 2019-08-16 | 安徽省农业科学院烟草研究所 | A kind of method of Rapid identification waxy corn germplasm |
| CN111560462A (en) * | 2020-06-10 | 2020-08-21 | 忻州师范学院 | Molecule detection method for co-separation of waxy genes of millet |
| CN111690643A (en) * | 2020-07-14 | 2020-09-22 | 吉林省农业科学院 | DNA extraction reagent, kit and method for detecting corn kernel transgenosis |
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| CN111690643A (en) * | 2020-07-14 | 2020-09-22 | 吉林省农业科学院 | DNA extraction reagent, kit and method for detecting corn kernel transgenosis |
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