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A kind of ssr primer and its application for the purity identification of Mobao wax gourd hybrid seeds

A hybrid seed purity, wax gourd technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of short identification period, broad application prospect and low cost

Active Publication Date: 2021-03-02
INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Routine identification includes field planting identification, grain and seedling morphological identification, and physical and chemical identification. These methods have disadvantages such as time-consuming, easily affected by environmental factors, large land use, and few marker traits that can be used for identification.
[0005] Biochemical identification includes methods such as isoenzyme electrophoresis, protein electrophoresis and high-performance liquid chromatography, and the techniques are cumbersome and complicated

Method used

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  • A kind of ssr primer and its application for the purity identification of Mobao wax gourd hybrid seeds
  • A kind of ssr primer and its application for the purity identification of Mobao wax gourd hybrid seeds
  • A kind of ssr primer and its application for the purity identification of Mobao wax gourd hybrid seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The screening process of the SSR primer SSR11c160 of the Mobao wax gourd hybrid seed purity identification provided by the present embodiment is as follows:

[0039] Several pairs of primers designed according to the genome sequencing results of wax gourd were found in the parents (male and female parents of Mobao wax gourd) and F 1 The primer SSR11c160 for the co-dominant differential marker band was selected, and the marker band was clear and reproducible. The sequence is as follows:

[0040] SSR11c160-F: 5'-GCCCACACTCACCTACACA-3' (SEQ ID NO.1);

[0041] SSR11c160-R: 5'-AGCCCCACCGAATTAATGCT-3' (SEQ ID NO. 2).

[0042] Using wax gourd genomic DNA as a template, use the above SSR primer SSR11c160 for PCR amplification—PAGE for polyacrylamide gel electrophoresis of the amplified product—recover DNA from specific fragments (polyacrylamide non-denaturing gel)—PCR amplification—agarose gel Recovery—After TA clone sequencing, the analysis results showed that the primer SSR...

Embodiment 2

[0044] The Mobao wax gourd hybrid seed purity identification method provided by the present embodiment comprises the following steps:

[0045] (1) Extraction of wax gourd DNA

[0046] The experimental material is the cotyledon of Mobao wax gourd hybrid and its male and female parents. The steps for extracting cotyledon DNA are as follows:

[0047] ①Put a piece of cotyledon into a 2mL centrifuge tube, add liquid nitrogen and grind it with a grinding pestle, quickly add 800 μL 2% CTAB extract when the liquid nitrogen is quickly evaporated to dryness, mix well and place in a 65°C water bath for 45 minutes (shake well once every 10 minutes) );

[0048] ② After standing at room temperature, add 800 μL of chloroform:isoamyl alcohol (24:1), mix gently, let stand for 2 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant (about 510 μL) to a new 1.5 mL centrifuge tube ;

[0049]③ Add 1 / 3 volume of NaAc (3mol / l) to the supernatant, add 1.5 times the volume of sup...

Embodiment 3

[0067] The method of embodiment 2 is adopted to carry out field planting and sampling to Mobao wax gourd hybrid, the sample is 80 in total, its single plant is numbered, and the single plant DNA is extracted and detected (see figure 2 ); Simultaneously, the purity identification was carried out by morphological detection method. Test results: 74 hybrid seeds, 6 female plants (the one in the red box is the female parent plant), the seed purity is 92.5%, which is consistent with the field test results.

[0068] The above examples show that the method of the present invention can effectively distinguish Mobao wax gourd seeds from their parent seeds, and accurately and quickly detect the purity of the seeds.

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Abstract

The invention discloses an SSR primer for the purity identification of Mobao winter melon hybrid seeds, including the upstream primer SSR11c160-F and the downstream primer SSR11c160-R of the primer SSR11c160, and the nucleotide sequence of the upstream primer SSR11c160-F is as shown in SEQ ID NO.1 As shown, the nucleotide sequence of the downstream primer SSR11c160‑R is shown in SEQ ID NO.2. Also disclosed is a method for identifying the purity of Mobao wax gourd hybrid seeds, the primer can produce a 193bp female parent specific marker and a 225bp male parent specific marker, and the primer has good repeatability and strong specificity. The method utilizes the above-mentioned SSR primers, and can accurately, rapidly and effectively identify the purity of the Mobao wax gourd hybrid seeds. It also discloses the application of the primers in Mobao wax gourd hybrid seeds and the purity identification of Mobao wax gourd hybrid seeds.

Description

technical field [0001] The invention belongs to the technical field of purity identification of wax gourd hybrid seeds, and in particular relates to an SSR primer used for purity identification of Mobao wax gourd hybrid seeds and an application thereof. [0002] technical background [0003] Seed purity is an important indicator of seed quality, which affects crop yield and product quality. It is entirely possible that yield reductions due to low purity could negate the yield potential of a new variety, and low purity seeds can cause significant losses in agricultural production. Seed purity identification mainly includes conventional identification, biochemical identification and molecular marker identification. [0004] Routine identification includes field planting identification, grain and seedling morphological identification, and physical and chemical identification. These methods have disadvantages such as time-consuming, easily affected by environmental factors, larg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘文睿谢大森江彪王敏闫晋强彭庆务何晓明林毓娥梁肇均陈林
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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