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Method for quickly detecting ploidy of new brassica hexaploid germplasm

A Brassica and new germplasm technology, applied in the biological field, can solve the problems of poor effect of very young leaves, unsuitable for large-scale detection, blurred description, etc., to achieve easy judgment, obvious peak bands, and high detection efficiency Effect

Active Publication Date: 2016-12-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Predecessors' descriptions of sample preparation are too vague, generally expressed as taking young leaves, but the specific stage of how tender the leaves are is not clearly expressed, and the effect of very young leaves (such as heart leaves) is not good; at the same time Predecessors often used blades to chop tissue samples to prepare single-cell suspensions, but this method was very time-consuming and not suitable for large-scale detection

Method used

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  • Method for quickly detecting ploidy of new brassica hexaploid germplasm
  • Method for quickly detecting ploidy of new brassica hexaploid germplasm
  • Method for quickly detecting ploidy of new brassica hexaploid germplasm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] (1) Treat the tender leaf (from the heart leaf to the third tender leaf) sample with a mill to prepare a single-cell suspension:

[0085] 1) At the five-leaf stage of the plant, take about 50 mg of the third tender leaf (the third tender leaf from the heart leaf to the outside) and place it in a centrifuge tube (1.5 mL) containing 3 mm steel balls and 500 μL buffer solution. Store at ℃ for 10min;

[0086]2) Put the tissue sample into the sample grinder, grind the sample at 25Hz for 1 min, then change the direction, and grind the sample for 1 min again.

[0087] 3) Filter the suspension into a clean centrifuge tube with a 300-mesh nylon mesh, add 4 μL RNase to 120 μL of the filtrate, mix well, and incubate at 37°C for 20-30 minutes;

[0088] 4) Before loading the sample, add 120 μL of staining solution to the mixture and store in the dark.

[0089] (2) Determination and analysis of the ploidy of the samples by using a flow cytometer.

[0090] Test results: The number ...

Embodiment 2

[0092] (1) Treat the tender leaf (from the heart leaf to the third tender leaf) sample with a mill to prepare a single-cell suspension:

[0093] 1) At the four-leaf stage of the plant, take about 50 mg of the third young leaf (the third young leaf from the heart leaf to the outside) and place it in a centrifuge tube (1.5 mL) containing 3 mm steel beads and 500 μL buffer solution. Store at ℃ for 10min;

[0094] 2) Put the tissue sample into the sample grinder, grind the sample at 25Hz for 1 min, then change the direction, and grind the sample for 1 min again.

[0095] 3) Filter the suspension into a clean centrifuge tube with a 300-mesh nylon mesh, add 4 μL RNase to 120 μL of the filtrate, mix well, and incubate at 37°C for 20-30 minutes;

[0096] 4) Before loading the sample, add 120 μL of staining solution to the mixture and store in the dark.

[0097] (2) Determination and analysis of the ploidy of the samples by using a flow cytometer.

[0098] Test results: The number o...

Embodiment 3

[0100] (1) Treat the tender leaf (from the heart leaf to the third tender leaf) sample with a mill to prepare a single-cell suspension:

[0101] 1) At the five-leaf stage of the plant, take about 50 mg of the third tender leaf (the third tender leaf from the heart leaf to the outside) and place it in a centrifuge tube (1.5 mL) containing 3 mm steel balls and 500 μL buffer solution. Store at ℃ for 10min;

[0102] 2) Put the tissue sample into the sample grinder, grind the sample at 25Hz for 1.5min, then change the direction, and grind the sample for another 1.5min.

[0103] 3) Filter the suspension into a clean centrifuge tube with a 300-mesh nylon mesh, add 4 μL RNase to 120 μL of the filtrate, mix well, and incubate at 37°C for 20-30 minutes;

[0104] 4) Before loading the sample, add 120 μL of staining solution to the mixture and store in the dark.

[0105] (2) Determination and analysis of the ploidy of the samples by using a flow cytometer.

[0106] Test results: The nu...

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Abstract

The invention discloses a method for quickly detecting ploidy of new brassica hexaploid germplasm. The method includes: (1) taking a third leaf of a new brassica germplasm from an interior leaf to the outside, putting in a steel ball containing centrifugal tube, adding extraction buffer liquor into the centrifugal tube, standing for 5-20min at a temperature ranging from -20 to -10 DEG C, and grinding in a grinder; (2) filtering the ground leaf through a filter screen to obtain filtrate, adding RNAse solution into the filtrate, and incubating for 20-30min at 37 DEG C; (3) adding staining fluid into the incubated filtrate, and performing lightproof staining to obtain a unicellular suspension sample; (4) using a flow cytometer for detecting the unicellular suspension sample. By adoption of the grinder for grinding leaf tissues, a great quantity of unicells are generated in the unicellular suspension sample, and broken cells are few. In addition, the method is high in detection efficiency and suitable for large-scale detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly detecting the ploidy of new Brassica hexaploid germplasm. Background technique [0002] Brassica spp. is the most important genus in the Brassicaceae (formerly known as Cruciferae) plants. In my country, Brassica crops are widely planted, including many important vegetables, oil plants, medicinal and feed crops. After polyploidization, many plants show many new phenotypes due to the cumulative effect of multiple chromosome groups, which are more suitable for the needs of agricultural production, such as wheat, cotton, tobacco, apple, etc. The three genomes of Brassica A, B, and C are considered to originate from an ancient hexaploid ancestor (reference: Lysak M A, et al. Chromosome triplication found across the tribe Brassiceae[J]. Genome Research, 2005, 15: 516-525). However, in nature, there is no hexaploid Brassica plant (AABBCC) containing three genomes of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N15/14
CPCG01N1/28G01N15/14
Inventor 杨素周伟军张康妮许玲王尖葛常青徐建祥
Owner ZHEJIANG UNIV
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