A method for rapid identification of chromosomal ploidy in Avena plants and its application

A chromosome ploidy, plant technology, applied in the field of plant biology, can solve the problems of high cytology operation technology, difficult large-scale quantification, time-consuming and labor-intensive

Inactive Publication Date: 2016-05-18
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0008] In summary, as a direct method for ploidy identification, although the chromosome preparation method is intuitive and credible, the operation process is cumbersome, the workload is heavy, time-consuming and labor-intensive, and requires high cytological operation techniques, and it is difficult to quantify on a large scale. People have been exploring simple, rapid and accurate methods to identify chromosome ploidy
The development of modern biotechnology can use molecular markers to identify these differences according to the differences in genomic DNA between different ploidy species, so as to achieve the purpose of ploidy identification, but there is no such research report in oats, so the development of a can The method of rapid identification of ploidy of Avena species is of great significance for the identification and utilization of Avena germplasm resources, molecular breeding, variety purity detection and research on the origin and evolution of Avena species

Method used

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  • A method for rapid identification of chromosomal ploidy in Avena plants and its application
  • A method for rapid identification of chromosomal ploidy in Avena plants and its application
  • A method for rapid identification of chromosomal ploidy in Avena plants and its application

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Experimental program
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Embodiment 1

[0032] The extraction of embodiment 1 Avena genome DNA

[0033] In this embodiment, A.strigosa (sand oat), A.hispanica (Spanish oat), A.brevis (short oat), A.nudabrevis (small grain naked oat), A.maroccana (Maloca oat), A.murphy (Murphy oats), A.magna (large oats), A.sativa (common cultivated oats), A.fatua (common wild oats), A.sterilis (wild red oats) and A.chinensis (naked oats ) as the experimental object.

[0034] Using the young leaves of the above-mentioned plants of the genus Avena as materials, oat DNA was extracted by using the CTAB method. The specific method is as follows: take 0.1 g of the material, add liquid nitrogen and grind it quickly. Transfer the ground material into a 1.5ml centrifuge tube, immediately add 600μl of preheated 2×CTAB buffer solution, mix by inverting, keep warm at 65°C for 10-20min, and shake it from time to time. Add an equal volume of chloroform / isoamyl alcohol (volume ratio 1 / 1, the same below), mix gently by inversion, and centrifuge a...

Embodiment 2

[0035] Embodiment 2 to the PCR amplification of Avena plant genomic DNA

[0036] Various oat genome DNAs extracted in embodiment 1 are carried out PCR amplification, and method is as follows:

[0037] Take a clean and sterile 0.2ml centrifuge tube, add the following reagents to it: ddH 2 O, 36 μl; 10×PCRBuffer, 5 μl; dNTP (10 mMeach), 1 μl; upstream primer (SP1) / downstream primer (SP2) of a specific primer pair, 2 μl; oat genomic DNA, 0.1-1 μg; Taq enzyme, 1 μl.

[0038] The nucleotide sequence of the specific primer pair is:

[0039] Upstream primer (SP1): 5'-AGCGTCTGCTTCAAAATCTGTT-3' (as shown in SEQ ID NO.1)

[0040] Downstream primer (SP2): 5'-TTTCTTCCTGCCGCGTTAAGTT-3' (shown in SEQ ID NO.2).

[0041] Amplification was carried out according to the following reaction conditions: 94°C for 5 min; 94°C for 20 sec, 55°C for 30 sec, 72°C for 1 min, 35 cycles; 72°C for 7 min.

[0042] The polyacrylamide gel electrophoresis detection of embodiment 3 amplification result

[00...

Embodiment 4

[0049] Example 4 Observation and Identification of Chromosomal Ploidy in Avena Plants

[0050] With reference to methods such as Liu Wei [Liu Wei, Zhang Zongwen, Wu Bin. Karyotype identification of the diploid oat germplasm introduced in Canada. Journal of Plant Genetic Resources. 2013,14(1):141-145], for example 1- 3 The chromosome ploidy of the Avena genus involved is observed and identified, and the specific methods are as follows:

[0051] Take oat seeds with full grains and put them in a clean petri dish, add water to submerge the seeds, and place them in a constant temperature incubator at 23°C to cultivate until they are white, then put the white seeds in a clean petri dish lined with sterilized water-soaked filter paper, and place Put it in the refrigerator at 4°C for 48 hours, and then put it in a constant temperature incubator at 23°C to grow roots. When the roots grow to 1.0-2.0cm, cut the new and strong root tips and pretreat them in ice water for 48 hours, and the...

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Abstract

The invention belongs to the technical field of plant biotechnology, and specifically discloses a method for rapidly identifying chromosome ploidy of an avena plant. An SSR (Simple Sequence Repeats) molecule is utilized to mark characteristics that avena plants with different ploidy have different sizes and copy numbers, and the chromosome ploidy of the avena plants can be identified rapidly by combining a PCR amplification technology and a gel electrophoresis technology by virtue of different numbers of amplified product segments. The method for rapidly identifying chromosome ploidy of the avena plant has the advantage that ploidy of the plant can be identified rapidly under the condition that the ploidy of the avena plan is not known in advance, and the method is applicable to avena plants.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a method for rapidly identifying ploidy of chromosomes in plants of the genus Avena and an application thereof. Background technique [0002] Oats are widely distributed all over the world and are important food and forage crops. Oats are rich in nutrients. Compared with wheat, rice, corn and other crops, the nutritional indicators such as protein, unsaturated fatty acids, vitamins, and mineral elements in oat grains are all in the forefront. In addition, oats are also rich in soluble dietary fiber. A large number of studies at home and abroad have proved that oats can lower blood fat and cholesterol without side effects, and have the functions of regulating human immune function, enhancing resistance and inhibiting diabetes [RipsinCM, KeenanJM, JacobsDR, ElmerPJ, WelchRR, VanHornL, LiuK, TurnbullWH, ThyeFW, KestinM, HegstedM, DavidsonDM, DavidsonMH, DuganLD, Demar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 吴斌张宗文
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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