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A kind of SSR primer and identification method for the purity identification of Tiezhu wax gourd hybrid seeds

A technology of hybrid seed purity and identification method, which is applied in the field of hybrid seed purity identification, can solve the problems of delayed sales of newly produced seeds, impossibility of purity identification, long identification time, etc., and achieve short identification period, low cost and easy operation Effect

Active Publication Date: 2019-09-20
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

The disadvantage of this method is that the identification time is long, and it usually takes about 3 months to get the identification result after the wax gourd plants bear fruit, and because the growth of melons is limited by seasons and climatic conditions, it is usually impossible to carry out the purity identification immediately after the seeds are harvested , leading to a delay in the sale of newly produced seeds
Molecular markers are more and more widely used in the identification of hybrid seed purity, and tend to gradually replace the traditional morphological identification method. At present, they have been used in the identification of hybrids of rice, cucumber, squash, zucchini and other crops, but in wax gourd Not yet seen in the application

Method used

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  • A kind of SSR primer and identification method for the purity identification of Tiezhu wax gourd hybrid seeds
  • A kind of SSR primer and identification method for the purity identification of Tiezhu wax gourd hybrid seeds
  • A kind of SSR primer and identification method for the purity identification of Tiezhu wax gourd hybrid seeds

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The screening process of the SSR primer SSR327 of the Tiezhu wax gourd hybrid seed purity identification provided by the present embodiment is as follows:

[0036] Several pairs of primers designed according to the sequencing results of the wax gourd transcriptome were screened between the parents (paternal and female Tiezhu wax gourd) and F1 (Tiezhu wax gourd hybrid), and the primer SSR327 with a co-dominant differential marker band was selected. The marking band is clear and repeatable, and the sequence is as follows:

[0037] SSR327-F: 5'-AAACAACACCGTTTCTTCCG-3' (SEQ ID NO.1);

[0038] SSR327-R: 5'-TCAGCGACATCAGAAACACC-3' (SEQ ID NO. 2).

[0039] Using the wax gourd genomic DNA as a template, use the above SSR primer SSR327 for PCR amplification—PAGE the amplified product by polyacrylamide gel electrophoresis—recover DNA from specific fragments (polyacrylamide non-denaturing gel)—PCR amplification—agarose gel Recovery—After TA clone sequencing, the analysis results...

Embodiment 2

[0045] The method for identifying the purity of "Tie Zhu" wax gourd hybrid seeds provided in this embodiment comprises the following steps:

[0046] (1) Extraction of wax gourd DNA

[0047] The experimental material is the cotyledon of Tiezhu wax gourd hybrid and its male parent and female parent. The steps for extracting cotyledon DNA are as follows:

[0048] ①Put a 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 every 10 minutes) once);

[0049] 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 ;

[0050] ③ Add 1 / 3 volume of NaAc (3mol / l) to the supernatant, add 1.5 times the volume of...

Embodiment 3

[0067] The method of embodiment 2 is adopted to carry out field planting and sampling to the Tiezhu wax gourd hybrid planted in Suixi, Guangdong, with a total of 57 samples, numbering its individual plants, and extracting individual plant DNA for detection (see figure 2 , image 3 ); Simultaneously, the purity identification was carried out by morphological detection method. Test results: 54 hybrid seeds, 3 female plants, the seed purity is 95%, consistent with the field test results.

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Abstract

The invention discloses an SSR primer, namely SSR327, for identifying the purity of an iron pillar white gourd hybrid seed. The primer SSR327 comprises an upstream primer SSR327-F and a downstream primer SSR327-R, wherein the nucleotide sequence of the upstream primer SSR327-F is as shown in SEQ ID NO.1; and the nucleotide sequence of the downstream primer SSR327-R is as shown in SEQ ID NO.2. The invention further discloses a method for identifying the purity of the iron pillar white gourd hybrid seed. The primer is capable of generating a female parent specific mark SSR327184 of 184bp and a male parent specific mark SSR327199 of 199bp, and is good in repeatability and good in specificity. With the SSR primer, the purity of the iron pillar white gourd hybrid seed can be accurately, rapidly, conveniently and effectively identified by using the method.

Description

technical field [0001] The invention belongs to the technical field of purity identification of hybrid seeds, and in particular relates to an SSR primer SSR327 used for identification of the purity of Tiezhu wax gourd hybrid seeds and an identification method. [0002] technical background [0003] Seed purity is an important indicator of seed quality. Rapid and accurate identification of seed purity has important application value for seed producers, sellers and users. [0004] Tiezhu winter melon is a hybrid first-generation winter melon variety bred by the Vegetable Research Institute of Guangdong Academy of Agricultural Sciences. Because of its high yield, disease resistance, and storage and transportation characteristics, it is very popular among growers, and the supply of seeds is often in short supply. The traditional method of identifying the purity of wax gourd seeds is the morphological identification method. Through the identification of the morphological charact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 何晓明谢大森江彪马金露彭庆务刘文睿罗少波林毓娥郭汉权刘洪彪孙保娟王瑞梁肇均
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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