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Muskmelon SSR molecular marker system and application thereof

A technology of molecular markers and melons, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems that the melon varieties cannot be quickly and accurately identified, and achieve rapid and accurate identification and high polymorphism , Repeatable and stable effect

Active Publication Date: 2018-11-20
新疆维吾尔自治区葡萄瓜果研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the embodiment of the present invention provides a muskmelon SSR molecular marker system and its application, the main purpose is to solve the problem that the variety of SSR molecular markers used to identify muskmelon varieties cannot be quickly and accurately identified

Method used

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  • Muskmelon SSR molecular marker system and application thereof
  • Muskmelon SSR molecular marker system and application thereof
  • Muskmelon SSR molecular marker system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 (adopting CTAB method to extract muskmelon DNA)

[0027] a. Take 0.2-0.5g of young leaves, put them in a mortar, add enough liquid nitrogen to grind until it becomes powder;

[0028] b. Quickly transfer the powder into a 2ml centrifuge tube; 760μl CTAB extraction buffer 100mmol / LTris (pH8.0), 1.4mol / LNaCl, 50mmol / L EDTA, 2%CTAB, 2%PVP, 20μlβ-mercapto have been added to the tube Mix well with ethanol;

[0029] c. Water bath at 65°C for 30 minutes, open and close the tube cap and shake it 2 to 3 times during the water bath;

[0030] d. Cool to room temperature, add an equal volume of 24:1 (V / V) chloroform:isoamyl alcohol, about 900 μl, and mix well;

[0031] e. Centrifuge at room temperature, 12000rpm, 15min;

[0032] f. Take a 2ml centrifuge tube and transfer the supernatant (about 750μl) into it;

[0033] g. Add the same volume of chloroform as the supernatant: isoamyl alcohol (V / V is 24:1), invert the centrifuge tube several times, and mix well;

[00...

Embodiment 2

[0046] Embodiment 2 (PCR amplification)

[0047]The melon DNA (such as Xizhou Mi 1, Xizhou Mi 3, Xizhou Mi 25, Xizhou Mi 17, Xizhou Mi 21 or Xizhou Mi 29) extracted in Example 1 was used as an amplification template, Use primer 73 (C30), primer 47 (MU5499), primer 14 (MU5554-1), primer 53 (SSR04219) or primer 5 (SSR12083) to amplify the DNA of the above six melon varieties; the primer pair information is shown in the table 1, the PCR reaction system is as shown in Table 2;

[0048] Table 1. Melon SSR primer system

[0049]

[0050] Table 2. PCR reaction system

[0051] reaction system

Volume (20μL)

wxya 2 o

7.9 μL

DNA template

1.5μL

Primers P1, P2

0.3μL each

10×Buffer

10.0 μL

[0052] PCR reaction program: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 15 s, appropriate annealing temperature for 15 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 4 min;

[0053] After th...

Embodiment 3

[0054] Embodiment 3 (construction of melon SSR fingerprints)

[0055] Utilize 5 pairs of SSR primers designed in Example 2 to amplify the genomic DNA (total DNA) of 18 muskmelon materials shown in Table 3, and the amplification result of each primer will obtain different band patterns (different numbers represent different belt type, such as Image 6 The band patterns appearing sequentially in the primer 73 are represented by 1, 2, 3, 4, and 5 respectively, that is, 1, 2, 3, 4, and 5 respectively represent 5 different band patterns), so the primer pair 73, 47 , 14, 53, and 5 can get the fingerprints of 18 materials, among which the new varieties Xizhoumi No. 1, Xizhoumi No. 3, Xizhoumi No. 25, Xizhoumi No. 17, Xizhoumi No. 21 and Xizhoumi The fingerprint of secret 29 is as follows Image 6 as shown, Image 6 The numbers 1-18 represent the female parent of Xizhoumi No. 1, Xizhoumi No. 1, and male parent of Xizhoumi No. 1; the female parent of Xizhoumi No. 3, Xizhoumi No. 3, ...

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Abstract

The invention discloses a muskmelon SSR molecular marker system and application thereof, and relates to the technical field of molecular markers. The names of primer pairs for identifying muskmelon varieties are C30, MU5499, MU5554-1, SSR12083 and SSR04219 respectively. The researched SSR molecular marker primer pairs have high polymorphism, clear stripes and stable repeatability, can be used forconstructing muskmelon DNA fingerprints and identifying the muskmelon varieties, especially identifying XizhouMi 1, XizhouMi 3, XizhouMi 25, XizhouMi 17, XizhouMi 21 or XizhouMi 29, and have a more obvious effect.

Description

technical field [0001] The invention relates to the technical field of molecular markers, in particular to a muskmelon SSR molecular marker system and its application. Background technique [0002] SSR (Simple Sequence Repeats) marking is a molecular marking technology based on specific primer PCR developed in recent years. 6) is a tandem repeat sequence of dozens of nucleotides consisting of repeating units. The sequences flanking each SSR are generally relatively conserved single-copy sequences. [0003] The genomes of organisms, especially the genomes of higher organisms, contain a large number of repetitive sequences. Studies have found that the number of repeat units in microsatellites is highly variable, and these variations are manifested as euploid variations in the number of microsatellites or repeat unit sequences. The sequences may not be identical, resulting in polymorphisms at multiple sites. If these variations can be revealed, the polymorphisms of different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 李超孙玉萍廖新福杨英陈伟郑贺云杨军张翠环杨咪
Owner 新疆维吾尔自治区葡萄瓜果研究所
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