Method for quickly distinguishing bud mutation of grape

Abstract

The invention discloses a method for quickly distinguishing bud mutation of grape. The method comprises the following steps in sequence: extracting DNA (Deoxyribose Nucleic Acid) of parent grape plant and corresponding suspected grate plant with bud mutation; amplifying the DNA by iPBS (inter Primer Binding Site) primers; distinguishing the bud mutation of the amplified products by agarose gel electrophoresis; and if difference is generated between the electrophoretic bands of the parent and the suspected variety with bud mutation, determining that the suspected variety with bud mutation is subjected to bud mutation. According to the method, three groups of precocious grape varieties with bud mutation are adopted as the raw materials, and the grape varieties are effectively distinguished by iPBS molecular markers; and by adopting the method, relative sequences of retrotransposon do not need to be predicted, so that high generality is ensured, and the theoretical basis is provided for effective selection mutation. The method is simple and convenient to operate and wide in application, saves much manpower and material resources, and is convenient to popularize.

Description

technical field

[0001] The invention relates to a method for quickly distinguishing grape bud mutations, belonging to the field of biological molecular markers. Background technique

[0002] Bud mutation (bud sport, bud mutation) is a kind of somatic mutation. As a method of grape breeding, bud mutation selection has unique advantages compared with other breeding methods (seed selection, cross breeding, radiation mutation breeding, protoplast fusion breeding, etc.). Its variable traits are easy to be found in the field, and can be preserved through grafting and reproduction without childhood interference, and can be quickly identified and popularized.

[0003] Budding is a mutation of genetic material in cells, and only when cells in the meristem of the terminal tissue undergo mutations can it become a bud. The histogenetic layer theory is currently the main theory for explaining bud change. Angiosperm apical meristems have several distinct cell layers called histogenetic ...

Examples

Embodiment 1

[0029] The extraction of embodiment 1, DNA

[0030] 1. After pre-cooling the mortar, add a small amount of liquid nitrogen and pre-cool it sufficiently;

[0031] 2. Take 3g of fresh, intact and clean leaves pre-cooled at -20°C and put them into a mortar, add half a medicine spoon of polyvinylpyrrolidone (PVP) and appropriate amount of liquid nitrogen, grind to fine powder, put into a centrifuge tube, and refrigerate at -20°C 30min;

[0032] 3. Prepare CTAB extraction buffer: weigh CTAB 2g, add distilled water 40ml, 1M Tris-cl (pH8.0) 10ml, 0.5M EDTA (pH 8.0) 4ml and 5M NaCl 28ml, after CTAB is dissolved, dilute to 100ml with distilled water , heat, and then add 2% β-mercaptoethanol to boiling;

[0033] 4. Put the hot CTAB extraction buffer into the centrifuge tube of step 2), 5ml per tube, and evenly disperse the leaf tissue in the buffer, and then put it in a constant temperature water bath at 65°C for 90 minutes, gently light every 5 minutes Gently invert and shake once;

...

Embodiment 2

[0040] Embodiment 2, iPBS-PCR amplification

[0041] Refer to Kalendar et al. for the PCR amplification system and procedures, as shown in Table 2 and Table 3, and the primers were synthesized by Shanghai Bioengineering Technology Co., Ltd., as shown in Table 4.

[0042] Table 2 iPBS-PCR amplification reaction system

[0043] Reagent name

[0044] Table 3 iPBS-PCR amplification reaction program

[0045] program name

[0046] Note: The three steps of denaturation, annealing and extension were cycled 30 times.

[0047] Table 4 iPBS-PCR amplification primers

[0048] serial number

[0049] Wherein, the nucleotide sequence of primer 2224 is the sequence listing SEQ ID NO: 1; the nucleotide sequence of the primer 2229 is the sequence listing SEQ ID NO: 2; the nucleotide sequence of the primer 2298 is the sequence listing SEQ ID NO: 3; The nucleotide sequence of primer 2373 is SEQ ID NO: 4 in the sequence listing.

Embodiment 3

[0050] Embodiment 3, agarose gel electrophoresis detection

[0051] 1) Preparation of buffer solution: Dilute 5×TBE to 0.5×TBE.

[0052] 2) Preparation of glue solution: Weigh 0.70g of agarose, put it in a conical flask, add 70mL of 0.5×TBE dilution buffer, put it in a microwave oven and heat and boil three times until the agarose is completely melted, take it out and shake well, this is 1.0% agarose gel solution.

[0053] 3) Preparation of the gel plate: Cool the boiled agarose gel to 50°C, slowly pour it into the gel tank placed on a horizontal table, let it cool for 60 minutes, and gently pull out the comb.

[0054] 4) Adding samples: Take 5 μL of DNA amplification product, add 1 μL of staining solution, 2.5 μL of bromophenol blue, flick to mix well, and add 5 μL of sample to the well.

[0055] 5) Electrophoresis: After adding the sample, close the cover of the electrophoresis tank, turn on the power immediately, and perform electrophoresis for 40 minutes.

[0056] 6) Ob...

technical field

[0001] The invention relates to a method for quickly distinguishing grape bud mutations, belonging to the field of biological molecular markers. Background technique

[0002] Bud mutation (bud sport, bud mutation) is a kind of somatic mutation. As a method of grape breeding, bud mutation selection has unique advantages compared with other breeding methods (seed selection, cross breeding, radiation mutation breeding, protoplast fusion breeding, etc.). Its variable traits are easy to be found in the field, and can be preserved through grafting and reproduction without childhood interference, and can be quickly identified and popularized.

[0003] Budding is a mutation of genetic material in cells, and only when cells in the meristem of the terminal tissue undergo mutations can it become a bud. The histogenetic layer theory is currently the main theory for explaining bud change. Angiosperm apical meristems have several distinct cell layers called histogenetic ...

Examples

Embodiment 1

[0029] The extraction of embodiment 1, DNA

[0030] 1. After pre-cooling the mortar, add a small amount of liquid nitrogen and pre-cool it sufficiently;

[0031] 2. Take 3g of fresh, intact and clean leaves pre-cooled at -20°C and put them into a mortar, add half a medicine spoon of polyvinylpyrrolidone (PVP) and appropriate amount of liquid nitrogen, grind to fine powder, put into a centrifuge tube, and refrigerate at -20°C 30min;

[0032] 3. Prepare CTAB extraction buffer: weigh CTAB 2g, add distilled water 40ml, 1M Tris-cl (pH8.0) 10ml, 0.5M EDTA (pH 8.0) 4ml and 5M NaCl 28ml, after CTAB is dissolved, dilute to 100ml with distilled water , heat, and then add 2% β-mercaptoethanol to boiling;

[0033] 4. Put the hot CTAB extraction buffer into the centrifuge tube of step 2), 5ml per tube, and evenly disperse the leaf tissue in the buffer, and then put it in a constant temperature water bath at 65°C for 90 minutes, gently light every 5 minutes Gently invert and shake once;

...

Embodiment 2

[0040] Embodiment 2, iPBS-PCR amplification

[0041] Refer to Kalendar et al. for the PCR amplification system and procedures, as shown in Table 2 and Table 3, and the primers were synthesized by Shanghai Bioengineering Technology Co., Ltd., as shown in Table 4.

[0042] Table 2 iPBS-PCR amplification reaction system

[0043] Reagent name

[0044] Table 3 iPBS-PCR amplification reaction program

[0045] program name

[0046] Note: The three steps of denaturation, annealing and extension were cycled 30 times.

[0047] Table 4 iPBS-PCR amplification primers

[0048] serial number

[0049] Wherein, the nucleotide sequence of primer 2224 is the sequence listing SEQ ID NO: 1; the nucleotide sequence of the primer 2229 is the sequence listing SEQ ID NO: 2; the nucleotide sequence of the primer 2298 is the sequence listing SEQ ID NO: 3; The nucleotide sequence of primer 2373 is SEQ ID NO: 4 in the sequence listing.

Embodiment 3

[0050] Embodiment 3, agarose gel electrophoresis detection

[0051] 1) Preparation of buffer solution: Dilute 5×TBE to 0.5×TBE.

[0052] 2) Preparation of glue solution: Weigh 0.70g of agarose, put it in a conical flask, add 70mL of 0.5×TBE dilution buffer, put it in a microwave oven and heat and boil three times until the agarose is completely melted, take it out and shake well, this is 1.0% agarose gel solution.

[0053] 3) Preparation of the gel plate: Cool the boiled agarose gel to 50°C, slowly pour it into the gel tank placed on a horizontal table, let it cool for 60 minutes, and gently pull out the comb.

[0054] 4) Adding samples: Take 5 μL of DNA amplification product, add 1 μL of staining solution, 2.5 μL of bromophenol blue, flick to mix well, and add 5 μL of sample to the well.

[0055] 5) Electrophoresis: After adding the sample, close the cover of the electrophoresis tank, turn on the power immediately, and perform electrophoresis for 40 minutes.

[0056] 6) Ob...

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