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Rapid identification method for present generation genotype of gramineous seed

A kind of undergraduate and seed technology, applied in the fields of molecular biology and genetic breeding, can solve the problems of increasing screening costs, complicated operation procedures, and long time consumption, and achieve the effects of saving manpower, safe operation, and simple operation

Active Publication Date: 2016-12-21
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The premise of molecular marker-assisted selection is to prepare the plant genome DNA first. The traditional method of extracting the genome DNA of a single plant is to first cultivate the plant seeds to seedlings in the greenhouse or in the field, and then collect the leaves of the plant seedlings for DNA extraction. There are many problems in this method, such as complicated operation procedures, long time-consuming, troublesome sampling, and heavy workload, which greatly increases the screening cost
At present, although there are reports on the use of seeds to extract plant genomic DNA, most of them are destructive methods. The seeds are completely destroyed and cannot be used for subsequent planting, induction, etc.

Method used

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  • Rapid identification method for present generation genotype of gramineous seed
  • Rapid identification method for present generation genotype of gramineous seed
  • Rapid identification method for present generation genotype of gramineous seed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 seed treatment

[0039] The specific method is as follows:

[0040] 1. Hull removal: remove the glume of a single seed;

[0041]2. Cut the endosperm: After shelling the single seed, cut the endosperm horizontally at 1 / 3-1 / 2 of the seed with a knife, put the endosperm in a 2mL centrifuge tube, and put the part containing the complete embryo in another centrifuge tube, corresponding to serial number, store dry at room temperature;

[0042] 3. Soaking: add 0.5-1mL distilled water to the endosperm in step 2), soak at room temperature for 3 days.

[0043] Comparative example 1 seed treatment

[0044] The specific method is as follows:

[0045] 1. Hull removal: remove the glume of a single seed;

[0046] 2. Cut the endosperm: After shelling the single seed, cut the endosperm horizontally at 1 / 3-1 / 2 of the seed with a knife, put the endosperm in a 2mL centrifuge tube, and put the part containing the complete embryo in another centrifuge tube, corresponding to...

Embodiment 2

[0058] Example 2 Seed Endosperm DNA Extraction

[0059] The specific method is as follows:

[0060] 1. Crushing: Drain the differently treated endosperms with distilled water, add 1 mL of 2×CTAB solution, add a small steel ball with a diameter of 5-6 mm, and then crush them on a fast sample preparation machine (FastPrep-24 from MP Company). Start 30sec each time, break 2-4 times in total;

[0061] 2. Water bath: put the crushed endosperm in a water bath for water bath, 65°C, 30-60min, and mix several times during the period;

[0062] 3. Centrifugation: Centrifuge the extract after the water bath, 12000rpm, 15min, take 0.8mL supernatant into a new centrifuge tube;

[0063] 4. Precipitation: Add 0.8 times the volume of isopropanol to the supernatant, mix it upside down, store at low temperature (-20°C) for 10 minutes; centrifuge at 12,000 rpm for 10 minutes at 4°C, discard the supernatant, and keep the precipitate;

[0064] 5. Washing: add 0.5mL of 75% ethanol to the precipit...

Embodiment 3

[0067] Example 3 Genomic DNA Quality Identification

[0068] Pipette 5 μL of the extracted single-grain endosperm DNA solution and 1 μL of loading buffer (China Keruitai) to mix, and then pipette 5 μL of the mixed sample and Maker (China Keruitai) to 1% agarose gel to make Electrophoresis at 350 mA for 25 min. see results figure 1 , the DNA band after treatment 3d is clearly visible, and there are few impurities, which shows that the single-grain endosperm DNA extracted by the method of the present invention has good quality, high concentration and high purity. The DNA of treatment 0-2d had no obvious bands. The DNA concentration of measuring treatment 3d is 110-130ng / μL, and its OD260 / OD280 value is between 1.9-2.0, shows to contain a little RNA sample in the DNA sample extracted by the present invention, and this is relevant with undigested RNA, but does not affect PCR amplification effect.

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Abstract

The invention relates to a rapid identification method for a present generation genotype of a gramineous seed. The rapid identification method comprises the steps of seed processing, seed genome DNA extracting and identifying, target gene amplifying and detecting and the like. According to the method, genome DNA of the single seed can be rapidly obtained in the seed period, and operation is easy and safe; the genotype of the seed can be identified in the seed period, the seed of the needed genotype is obtained and used for follow-up study, the field planting workload in the seeding period is reduced, the seed breeding efficiency is improved, follow-up growth and development of an embryo-containing seed are not damaged, and the seed can be preserved for a long time and also can be used for tissue culturing.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic breeding, in particular to a method for rapidly identifying contemporary genotypes of Poaceae seeds. Background technique [0002] Molecular marker-assisted selection (MAS for short) is an auxiliary method for applying molecular markers in the process of crop improvement (Ribaut J M, Hoisington D (1988) Marker-assisted selection: new tools and strategies[ J]. Trends in Plant Sci. 3:236-239). The basic principle is to use molecular markers that are closely linked to the target gene or show a co-segregation relationship to screen the target region and the whole genome of the selected individuals, so as to reduce linkage redundancy, obtain desired individuals, and achieve the purpose of improving breeding efficiency (Lee M (1995) DNA markers in plant breeding programs [J]. Adv Agron. 55:265-344). Compared with individual selection by phenotypic traits and isoenzyme markers, molecular m...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1003C12Q1/6895C12Q2600/156C12Q2565/113
Inventor 黄培劲安保光陈思兰李新鹏吴永忠
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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