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A method for screening tmv-resistant tobacco varieties

A variety, tobacco technology, applied in the field of plant variety resistance detection, can solve problems such as difficult breeding

Active Publication Date: 2018-08-31
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Ambalema variety in common tobacco shows disease resistance to TMV, and its resistance is controlled by the recessive alleles rm1 and rm2, and the disease resistance gene is linked to the adverse trait gene, which is difficult to use its breeding

Method used

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  • A method for screening tmv-resistant tobacco varieties
  • A method for screening tmv-resistant tobacco varieties
  • A method for screening tmv-resistant tobacco varieties

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: A preferred embodiment of the kit for screening anti-TMV tobacco varieties of the present invention

[0029] This embodiment is a preferred embodiment of a kit of the present invention for screening anti-TMV tobacco varieties. This kit contains upstream and downstream primers, 2×PCR buffer and ddH 2 O; the concentration of the lower and upper primers is 10 μM, the sequence of the primers is shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the buffer is composed of 20 mM Tris-HCl (pH8.3), 100 μM dNTP, 100 mM KCl, 3mM MgCl 2 and 0.1U Taq enzyme / μl.

[0030] The sequence of the upstream primer N5309U is as SEQ ID NO: 1: 5`-GATGCCAGTGATACTCTAA-3`,

[0031] The sequence of the downstream primer N5921L is SEQ ID NO: 2: 5'-ATACACTACTATCCCAACC-3'.

Embodiment 2

[0032] Embodiment 2: A preferred embodiment of screening the resistance of 23 tobacco varieties to be tested using the kit of Embodiment 1.

[0033] Using the kit described in Example 1, 23 tobacco varieties to be tested were screened for resistance. Sow 23 tobacco varieties to be tested in seedling trays, and when the tobacco seedlings grow to 2-3 true leaves, cut 0.1g leaves and put them into 1.5mL EP tubes for DNA extraction. The names and TMV resistance of the 23 tested tobacco varieties are shown in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] DNA extraction:

[0038] 1. Immerse EP in liquid nitrogen for 1 minute, take it out, and immediately use a pre-cooled grinding pestle to quickly grind the leaves into powder.

[0039] 2. Immediately add an equal volume (w / v) of 2×CTAB extraction buffer and incubate at 65°C for 30 minutes, shaking from time to time.

[0040] 3. Add an equal volume of chloroform / isoamyl alcohol, gently invert the centrifuge tube to m...

Embodiment 3

[0056] Embodiment 3: Resistance source identification analysis test

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Abstract

The invention provides primers, a method and a kit for screening of an anti-TMV tobacco variety. The sequences of the primers are shown in SEQ ID No. 1 and SEQ ID No. 2. Two resistant genes consisting of an N gene and a CN gene are detected through PCR amplified reaction; if a 613 bp band exists, a detected variety has TMV resistance; and if the 613 bp band does not exist, the detected variety does not have TMV resistance. The kit contains the upstream and downstream primers, 2*PCR buffer and ddH2O, wherein the the sequences of the primers are shown in SEQ ID No. 1 and SEQ ID No. 2. Compared with single gene detection, the method for detection of two genes in the invention has the advantages of rapidness and low cost due to a few operation steps and saving of materials and reagents; moreover, the problem of missing of other resistant genes due to single gene detection can be overcome.

Description

technical field [0001] The invention belongs to the field of plant variety resistance detection, and in particular relates to a method for screening TMV-resistant tobacco varieties. Background technique [0002] Tobacco mosaic virus (TMV, tobacco mosaic virus) is one of the main diseases in tobacco production. The occurrence of TMV seriously affects the yield and quality of tobacco leaves, and causes serious losses to farmers. At present, there is no effective pesticide for the treatment of TMV, and the breeding of disease-resistant varieties is still the most economical and effective means to prevent common mosaic disease. An important part of breeding disease-resistant varieties is resistance identification. [0003] Traditional resistance identification methods are mainly field inoculation identification and greenhouse inoculation identification. These two methods are cumbersome to operate and require a lot of work, and the identification cycle takes 2-3 months. Due to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
Inventor 谢锐鸿袁清华李集勤马柱文吕锦津陈俊标李淑玲张振臣
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI