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A kind of SNP molecular marker of wax gourd seed type gene and its application

A technology of molecular markers and wax gourd seeds, which is applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., to achieve the effects of strong specificity, easy operation, and short identification cycle

Active Publication Date: 2019-06-21
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

Since the invention of dCAPS technology, it has been widely used in the research of molecular genetics and identification of germplasm resource varieties and strains, but no one has used it for wax gourd seed type.

Method used

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  • A kind of SNP molecular marker of wax gourd seed type gene and its application
  • A kind of SNP molecular marker of wax gourd seed type gene and its application
  • A kind of SNP molecular marker of wax gourd seed type gene and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The development and verification process of the SNP molecular marker of the wax gourd seed type gene provided in this example and the primer pair (dCAPS primer pair) for amplifying the SNP molecular marker is as follows:

[0045] Materials: Sources of parents: B227: Unilateral seed parent, B214: Bilateral seed parent.

[0046] A six-generation segregation population was constructed with unilateral-seeded wax gourd B227 and bilateral-seeded wax gourd B214 as parents, and the genetic law of wax gourd seed type was studied. The results showed that bilateral and unilateral are the quality traits controlled by a pair of nuclear genes, and bilateral to unilateral is dominant. . Subsequently, using the F 2The high-density genetic map of wax gourd was constructed by the population, and the seed type gene was located on chromosome 4, and the genetic distances between it and the markers on both sides were 0.4cM and 0.8cM, respectively.

[0047] According to the genome resequenc...

Embodiment 2

[0057] The identification method of the wax gourd seed type that the present embodiment provides, comprises the following steps:

[0058] (1) Extraction of wax gourd DNA

[0059] The experimental materials are fresh leaves of B227 and B214, and the steps of extracting genomic DNA are as follows:

[0060] ①Put a small amount of fresh leaves into a 2mL centrifuge tube, add liquid nitrogen and grind with a grinding pestle, quickly add 800 μL 2% CTAB extract when the liquid nitrogen is quickly evaporated, mix well and place in a 65°C water bath for 45 minutes (shaking every 10 minutes) uniform once);

[0061] 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 ;

[0062] ③ Add 1 / 3 volume of NaAc (3mol / L) to the supernatant, add 1.5 times the volume of supernatant to absolute ethanol (pre-cooled at...

Embodiment 3

[0081] The method of Example 2 was used to verify 96 individual plants of the F4:5 family population of B227 and B214 (the first generation of F2 population selfing is F2:3 family, and the second generation of selfing is F3:4, and then F4:5) . Each individual plant was numbered separately, and the DNA of the individual plant was extracted for detection (see image 3 ); if two bands are heterozygous, the band type is bilateral, and for one band, the large segment is bilateral, and the small segment is unilateral.

[0082] According to the results of marker detection, there were 60 individual plants with 199bp band type (bilateral homozygosity), 8 individual plants with 173bp band type (unilateral homozygous), 20 heterozygous band type (bilateral heterozygous), and the remaining 8 unamplified bands.

[0083] The field trait identification results were consistent with the marker detection results, and the accuracy rate reached 100%.

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Abstract

The invention discloses an SNP molecular marker of a wax gourd seed type gene. The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1. The nucleotide sequence shown in SEQ ID NO. 1 is a SNP site from the 5 'end, and the nucleotide of the nucleotide sequence is T or C. Primer pairs for amplifying the SNP molecular markers described above are also disclosed, the pair of primers comprising an upstream primer and a downstream primer, The nucleotide sequence of the upstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3. The primer has good repeatability and high specificity for producing unilateral seed material specific marker and bilateral seed material specific marker; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. Also disclosed is a method for identifying wax gourd seed type, which is accurate, rapid, low in cost, short in identification period and simple in operation. Further disclosed are the application of the SNP molecular marker and the primer pair in identifying the seed type of winter gourd.

Description

technical field [0001] The invention belongs to the technical field of molecular markers, and in particular relates to a SNP molecular marker of a wax gourd seed type gene and an application thereof. Background technique [0002] According to the shape and structure of the wax gourd seed coat, the mature seeds can be divided into non-prismatic seeds (commonly known as single-sided seeds) and ribbed flat seeds (commonly known as double-sided seeds). Unilateral seeds usually have different degrees of dormancy, and their germination rate is generally below 60%, and they germinate slowly and irregularly, while bilateral seeds germinate quickly and neatly, and their germination rate exceeds 90%. According to the national seed standard, the germination rate of winter melon commercial seeds is only 60%, while the germination rate standard of other cucurbit crops is above 90%. Therefore, seed type is an important trait of winter melon, and double-seed varieties are favored by seed ...

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 江彪谢大森刘文睿王敏何晓明彭庆务林毓娥梁肇均
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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