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Chloroplast molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties

A technology of molecular markers and chloroplasts, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems affecting the double control policy of tobacco production, and achieve the effect of strict control

Pending Publication Date: 2022-02-11
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when tobacco farmers plant, they may harvest the conventional seeds planted for the next year’s tobacco planting, pretending to be sterile line seeds, and before the tobacco plants bloom, the fertility of the plants cannot be discerned with the naked eye, so , which seriously affects the double-control policy of tobacco production and poses a challenge to the sustainable development of tobacco production

Method used

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  • Chloroplast molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties
  • Chloroplast molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties
  • Chloroplast molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] See figure 1 Shown:

[0031] 1. Primers:

[0032] trnH1 / psbA1, trnH2 / psbA2, trnH3 / psbA3, trnH4 / psbA4

[0033] 2. Template DNA:

[0034] Conventional varieties: Honghua Dajinyuan (HD), K326,

[0035] CMS 1 (Sua-CMS): MS20 (Sua-CMS Honghua Dajinyuan, MS26 (Sua-CMSK326), CMS 2 (Gla-CMS): MS28 (Gla-CMSK326, MS29 (Gla-CMS Red Huada Jinyuan 3. (1) Reaction system: (25μL)

[0036] 2×Phanta max Buffer: 12.5 μL; trnH1 / psbA1, trnH2 / psbA2, trnH3 / psbA3, rnH4 / psbA4: 1 μL each; ddH2O: 8.5 μL; Template: 2 μL

[0037] (2) Gradient PCR reaction program:

[0038] 94°C, 2min; 94°C, 45s; 59°C, 30s; 72°C, 45s; 72°C, 4min;

[0039] Annealing: 59 degrees

[0040] figure 1 In the description:

[0041] The sample order is: HD, K326, MS20, MS26, MS28, MS29, water.

[0042] The primer sequence is: trnH1 / psbA1, trnH2 / psbA2, trnH3 / psbA3, trnH4 / psbA4 (the left and right arrows indicate the sequence of the primer pair).

[0043] Among them, the control of the last primer has no water.

...

Embodiment 2

[0047] See figure 2 Shown:

[0048] figure 2 In the description:

[0049] Sample order: HD, K326, MS28, MS29, MS20, MS26, water

[0050] Primer sequence: trnH1 / psbA1, trnH2 / psbA2, trnH3 / psbA3, trnH4 / psbA4

[0051] Enzyme: 2XPhanta max Buffer

[0052] Annealing: 59 degrees (35cyl), extension 72 degrees

[0053] Marker:100bp DNA ladder

[0054] Conclusion: The conventional species and the sterile line can be clearly distinguished by using the primers of the present invention, and there are obvious differences between them.

Embodiment 3

[0055] Embodiment three (the case of partial failure):

[0056] See image 3 Shown:

[0057] image 3 In the description:

[0058] Primer order:

[0059]

[0060] Sample order: Sua-CMS Yunyan 87, Sua-CMSK326, MS28, MS29, Honghua Dajinyuan, K326, Yunyan 87, Yunyan 97, Yunyan 116, China Tobacco 100.

[0061] Conclusion: Using the primer pair No. 1-8 can not clearly distinguish the conventional species and the sterile line, and the difference is not obvious.

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Abstract

The invention relates to molecular marker primers, and application thereof in early identification of seed sources of tobacco varieties. The molecular marker primers are trnH1 / psbA1; trnH2 / psbA2; trnH3 / psbA3; and trnH4 / psbA4. The molecular marker primers are applied to early identification of seed sources of tobacco varieties. The markers have the beneficial effects that any one pair of the four pairs of primers can be used for distinguishing and identifying conventional tobacco seeds and sterile line seeds in the prior art, that is, molecular marking of the conventional tobacco seeds and the sterile line seeds is completed, so that strict management and control of the tobacco seeds are realized. By using the method disclosed by the invention, distinguishing can be carried out by naked eyes, so that the sterile line tobacco species can be identified.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of chloroplast molecular marker primers and application technology for early identification of tobacco variety seed sources. Background technique [0002] The control of tobacco seeds is very strict, and the seeds used in tobacco production include conventional seeds and sterile line seeds. Conventional species can self-pollinate, producing seeds that the next generation can continue to plant. Tobacco male sterile line seeds are cytoplasmic male sterile type. The stamens controlled by cytoplasmic genes are abnormally developed or pollen is aborted, unable to self-pollination, and no seeds are produced. However, when tobacco farmers plant, they may harvest the conventional seeds planted for the next year’s tobacco planting, pretending to be sterile line seeds, and before the tobacco plants bloom, the fertility of the plants cannot be discerned with the naked eye, so , seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 曾建敏刘勇黄昌军袁诚童治军于海芹方敦煌肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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