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Molecular marker for identifying resistance gene of tomato mosaic virus (TOMV) and application thereof

A tomato mosaic virus and resistance gene technology, applied in the field of molecular biology, can solve the problems of prone to human errors, extremely high requirements for PCR amplification conditions, and high experimental costs, so as to achieve accurate identification of disease resistance and improve breeding efficiency , The effect of speeding up the breeding process

Inactive Publication Date: 2018-07-27
CHINA NAT SEED GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently in Tm-2 2 In terms of marker development, there are currently some markers available, such as Tetra-primer ARMSPCR (four-primer amplification hindered mutant system Ye. PCR amplification of primers requires extremely high conditions for PCR amplification, and it is necessary to constantly try and explore a suitable experimental system. The latter requires enzyme digestion of PCR products, which is complicated and expensive to operate, and is prone to human errors. The degree of automation in the process of using this marker is poor, and it is not suitable for high-throughput and large-scale operations

Method used

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  • Molecular marker for identifying resistance gene of tomato mosaic virus (TOMV) and application thereof
  • Molecular marker for identifying resistance gene of tomato mosaic virus (TOMV) and application thereof
  • Molecular marker for identifying resistance gene of tomato mosaic virus (TOMV) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Development of KASP markers

[0053] Download tm-2, Tm-2 and Tm-2 respectively from NCBI (https: / / www.ncbi.nlm.nih.gov / ) 2 The sequences of the three alleles have accession numbers AF536199, AF536200 and AF536201. Among them, tm-2 is the non-resistant allele, Tm-2 and Tm-2 2 is the allele for disease resistance.

[0054] Using Sequencher5.1 software to compare the sequences of the three, after analysis, it was found that tm-2 was related to Tm-2 and Tm-2 2 There are multiple SNP loci.

[0055] The SNP sites that can be used for the development of KASP markers need to meet the following requirements: the genotype is between Tm-2 and Tm-2 2 The same as in tm-2, different from tm-2; there are no other SNP sites nearby, and it is located in a non-SNP dense area; avoid continuous AT, GC content and other complex sequence areas.

[0056] Finally, the 516th C / T, the 1043rd C / T and the 837th C / T were selected; the genotypes of tm-2 in the above three sites wer...

Embodiment 2

[0070] Example 2 Amplification of molecular markers

[0071] The materials used in this example are the known tomato mosaic virus-resistant material LA1791 and the tomato mosaic virus-susceptible material moneymaker (both from the American Tomato Genetic Resource Center).

[0072] Using the CTAB method to extract the genomic DNA in the tomato leaves, the concentration of the extracted DNA solution is 10-50ng / μl, and it is stored at -20°C.

[0073] Use the KASP V4.0 Master mix (low ROX) kit (LGC Company) to configure the PCR reaction system with a total reaction volume of 5 μl, including: 2xLGC Master mix, 2.5 μl; DNA extraction solution (10-50ng / μl), 1 μl; Primer KTm2-F1, 0.06 μl (10 pmol / μl); Primer KTm2-F2, 0.06 μl (10 pmol / μl); Primer KTm2S01-R or KTm2S02-R or KTm2S03-R, 0.24 μl (10 pmol / μl); and ddH 2 O, 1.14 μl. Set up three technical replicates.

[0074] The PCR reaction program was: 94°C for 15 min; 94°C for 20 sec, 61-55°C (0.6°C drop per cycle) for 1 min, 10 cycl...

Embodiment 3

[0084] Example 3 Using KTm2S02, KTm2S02-2 and KTm2S02-3 to identify disease-resistant and susceptible tomatoes

[0085] The high-generation homozygous disease-resistant material ZZ01 selected by the applicant was used as the male parent, and the susceptible material moneymaker was used as the female parent, and the two were crossed to prepare F 1 , F 1 180 strains of F 2 Separate groups.

[0086] to F 2 The plants of the colony carry out inoculation virus identification, friction inoculation of tobacco mosaic virus (Fang Zhongda, "Plant Pathology Research Methods", China Agricultural Press, 2007) at the 7-8 true leaf stage, after two weeks, investigate the incidence, statistics f 2 Resistant and susceptible individual plants in populations. Disease investigation is divided into 6 grades: Grade 0 without any symptoms; grade 1 acute small scabs on heart leaves or inoculated leaves; grade 3 necrotic spots on mosaic leaves or stems; grade 5 heavy mosaic leaves, deformity or...

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Abstract

The invention provides a molecular marker for identifying resistance gene of tomato mosaic virus (TOMV) and an application thereof, and includes a KASP primer and a kit for identifying the resistancegene of TOMV, a method for identifying whether a tomato contains the resistance gene of TOMV or not, and a method of distinguishing anti-TOMV tomatoes and TOMV-infected tomatoes. In the invention, themolecular marker can be used for quick and accurate resistance identification in a large throughput during seedling period of tomato plants, thus increasing seed breeding efficiency, saving seed breeding cost, and accelerating seed breeding procedure.

Description

technical field [0001] The application belongs to the field of molecular biology, and in particular relates to the molecular marker of tomato mosaic virus resistance gene and its application. Background technique [0002] Tomato is a very widely cultivated vegetable all over the world, and it is also one of the main cultivated vegetables in my country. However, in the process of tomato production, viral diseases are becoming more and more serious, resulting in a decline in yield and quality, so it is very important to breed disease-resistant varieties. [0003] Tomato mosaic virus (ToMV) is the most common pathogenic bacteria in tomato virus diseases. (The virus is spread on hands or tools), or spreads by sucking juice from aphids. It can not only cause mosaic or stripe spots on tomato leaves, but also cause internal necrosis of fruits, which can lead to a 65% reduction in tomato yield. The use of chemical pesticides is not only ineffective, but also likely to cause excess...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 孔会利井立军何宗顺张龙雨夏琴芳张文丽孙吉庆韦懿喻辉辉周发松
Owner CHINA NAT SEED GRP
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