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Simple sequence repeats (SSR) primer and method for identifying purity of hybrid seeds of Yunnan raphanus sativus L. No. 2

A hybrid seed purity, cloud radish technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as heavy workload, long cycle, false hybrids, etc., and achieve high efficiency and accurate quality. The effect of control and labeling with good repeatability and fast detection method

Active Publication Date: 2020-06-30
云南省农业科学院园艺作物研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fertile plants in the female parent are not completely removed during seed production, the field isolation measures are not perfect, the male parent is not pulled out in time before harvest, and other varieties are mixed in after harvest, all of which will lead to the generation of false hybrids
At present, planting identification in field plots is the main method for identifying the purity of dried processed radish seeds. It has disadvantages such as heavy workload, long cycle, and easy to be affected by experience and environment, and may miss the best sales and sowing time of hybrids.

Method used

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  • Simple sequence repeats (SSR) primer and method for identifying purity of hybrid seeds of Yunnan raphanus sativus L. No. 2
  • Simple sequence repeats (SSR) primer and method for identifying purity of hybrid seeds of Yunnan raphanus sativus L. No. 2
  • Simple sequence repeats (SSR) primer and method for identifying purity of hybrid seeds of Yunnan raphanus sativus L. No. 2

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Experimental program
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Effect test

Embodiment 1

[0046] This embodiment provides a SSR primer SSR06 for the purity identification of Yunluobo No. 2 hybrid seeds. The screening process is as follows:

[0047] According to the genome information of radish, 200 pairs of primers were designed to screen among the parents of Yunluobo No. 2, and the primer pair SSR06( figure 1 ), the marker has good repeatability and clear bands, and the sequence is as follows:

[0048] Upstream primer: GAAGCAGATGATTGCAAAACC (SEQ ID NO: 1)

[0049] Downstream primer: CATCCGTTTTGAAGATGGAAA (SEQ ID NO: 2).

Embodiment 2

[0051] This embodiment provides a method for identifying the purity of Yunluobo No. 2 hybrid seeds, comprising the following steps:

[0052] 1) Extraction of genomic DNA:

[0053] Soak the hybrid seeds of Yunluobo No. 2 for 12 hours, sow them in plug trays after being white, and place them in a light incubator. After germination, remove the hypocotyls and place them in a 1.5mL centrifuge tube, add liquid nitrogen and grind them with a grinding rod, add 200 μL 2% CTAB extraction buffer, grind, make up to 700 μL, and bathe in water at 65°C for 40 minutes;

[0054] Add 700 μL of chloroform and isoamyl alcohol in total, where V chloroform: V isoamyl alcohol = 24:1, shake gently for 5 min, and then centrifuge at 12000 rpm for 10 min;

[0055] Take 250 μL of supernatant, add 250 μL of pre-cooled isopropanol and mix well, place at -20°C for 30 min; centrifuge at 12,000 rpm for 10 min; discard the supernatant, add 150 μL of pre-cooled ethanol, mix gently and wash, centrifuge at 12,00...

Embodiment 3

[0071] The purity detection of Yunluobo No. 2 hybrid produced in different seed production bases

[0072] The SSR primer SSR06 was used to perform PCR detection on 100 Yunradish No. 2 radish hybrids produced by farmers in three seed production bases.

[0073] The results are shown in Table 1. The purity of No. 1 and No. 3 seed production base Yunluobo No. 2 hybrids is 100% ( image 3 , Figure 5 ); No. 2 seed production base Yunluobo No. 2 hybrid has a purity of 99%, such as Figure 4 The figure shows that one of the seeds with the paternal band type (the band shown in the triangle) is a non-hybrid, and the rest of the seeds are Yunluobo No. 2 hybrids.

[0074] Table 1 The purity detection of Yunluobo No. 2 hybrid produced in different seed production bases

[0075]

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Abstract

The invention provides a simple sequence repeats (SSR) primer SSR06 for identifying the purity of hybrid seeds of Yunnan raphanus sativus L. No. 2, and further provides a method for identifying the purity of the hybrid seeds of the Yunnan raphanus sativus L. No. 2. The method comprises the following steps: Step 1, extracting genome DNA of to-be-detected samples, the hybrid seeds of the Yunnan raphanus sativus L. No. 2; Step 2, performing polymerase chain reaction (PCR) amplification by employing the SSR primer SSR06 by taking the genome DNA, extracted in the step 1, of the hybrid seeds of theYunnan raphanus sativus L. No. 2 as a template; Step 3, separating an amplification product in the step 2 by polyacrylamide gel electrophoresis, performing staining by a silver staining method, performing photographing and making statistics on results; and Step 4, calculating the purity of the hybrid seeds of the Yunnan raphanus sativus L. No. 2 by utilizing the statistical results in the step 3.The SSR primer SSR06 provided by the invention has high specificity, and accurate identification results, can quickly, accurately and conveniently identify the purity of hybrid seeds, and overcomes the shortcoming that time and vigor are consumed by using field plot planting identification to identify the purity of the hybrid seeds.

Description

technical field [0001] The invention relates to the technical field of purity identification of hybrid seeds, in particular to an SSR primer and a method for identifying the purity of Yunluobo No. 2 hybrid seeds. Background technique [0002] Radish (Raphanus sativus L.) is a vegetable of the Brassicaceae (Brassi-caceae) genus Raphanus (Raphanus). Its perennial cultivation area in China is second only to Chinese cabbage, and it occupies an important position in agricultural production. Radishes are planted in a wide range of areas and there are many varieties. Dried and processed radishes are an important characteristic agricultural pillar industry in the mountainous areas of Yunnan in autumn and winter. Relying on the unique climate advantages of Yunnan, they can develop rapidly. According to statistics, in 2018, the radish planting area in Yunnan Province was about 1.1 million mu, mainly in Qujing, Zhaotong, Chuxiong, Honghe and other areas. In recent years, with the cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2600/13C12Q2531/113C12Q2565/125
Inventor 陶婧李石开孙一丁汪骞袁艺钟秋月
Owner 云南省农业科学院园艺作物研究所
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