Co-dominant SSR markers closely linked to tobacco TMV resistant gene N and application of co-dominant SSR markers

A resistance gene and co-dominant technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial measurement / inspection, etc., can solve the problems of cumbersome detection system, PCR amplification failure, and inability to distinguish all genotypes, etc. Achieve the effect of low cost and fast cost

Active Publication Date: 2017-03-15
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all the above-mentioned molecular markers developed based on the N gene are dominant markers, which have serious defects in the actual tobacco breeding practice: 1) cannot distinguish all genotypes of the N gene in the material, that is, the above-mentioned dominant markers only It can tell whether the material contains the resistance gene N, but cannot further distinguish homozygous resistance (NN) and heterozygous resistance (Nn); In the N gene detection of N gene markers, if no characteristic (target) band appears, it cannot be determined that the test material is susceptible, and there is a high possibility of PCR amplification failure caused by experimental factors; 3) The detection system is cumbersome and has Some markers are designed based on the full-length cDNA of the N gene, so the actual detection involves the extraction of RNA, the synthesis of cDNA, and the PCR amplification of long fragment DNA.

Method used

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  • Co-dominant SSR markers closely linked to tobacco TMV resistant gene N and application of co-dominant SSR markers
  • Co-dominant SSR markers closely linked to tobacco TMV resistant gene N and application of co-dominant SSR markers
  • Co-dominant SSR markers closely linked to tobacco TMV resistant gene N and application of co-dominant SSR markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Screening of co-dominant SSR markers N-linked to tobacco anti-TMV gene by Bulked Segregation Analysis (BSA)

[0031] 1. Experimental materials

[0032] The flue-cured tobacco Y3 with excellent comprehensive traits but susceptible to TMV was used as the female parent, and the TMV-resistant flue-cured tobacco material Coker176 (whose resistance was controlled by the N gene) was used as the male parent. In 2014, the TMV-resistant and susceptible parent materials were planted, and F1 was obtained by crossing. In 2015, F1 and two parents were planted, and Y3 was used as the recurrent parent, and the backcross generation (BC1F1) population was obtained by crossing. In 2016, two parents, F1 and BC1F1 generation materials were planted.

[0033] 2. Identification of TMV resistance in parents and BC1F1 isolates

[0034] The test materials were transplanted to the field after they became seedlings, with a row-to-plant spacing of 100cm × 50cm; 3 weeks after transplanting, artific...

Embodiment 2

[0051] Map Distance of Codominant Linkage Markers and Its Validation in Individual Plants of BC1F1 Population

[0052] 1. Data analysis

[0053] First, extract and purify tobacco genome DNA, identify TMV resistance of individual plants of BC1F1 population and analyze SSR markers according to the method described in Example 1. Secondly, 123 individual plants in the BC1F1 population were genotyped using the markers TM508-007 and TM508-118 obtained by BSA screening. Finally, carry out data statistics on the band type of each individual plant. The resistant heterozygous band is marked as "H", the susceptible band is marked as "A", and the bands with unclear or no amplification bands are marked as "U". ".

[0054] 2. Calculation of genetic distance of co-dominant linked markers

[0055] Using JoinMap 4.0 software combined with the TMV resistance identification data of individual plants in BC1F1 population, genetic linkage analysis was carried out on the genotype data of co-domin...

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Abstract

The invention discloses co-dominant SSR markers closely linked to tobacco TMV resistant gene N and application of the co-dominant SSR markers. Serial numbers of the co-dominant SSR markers are TM508-007 and TM508-118, and nucleotide sequences of amplified products of the co-dominant SSR markers are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. The application refers to application of the co-dominant SSR markers in detecting whether tobacco genome DNA contains the tobacco TMV resistance gene N or not. The co-dominant SSR markers have the advantages of stability, reliability, simplicity, convenience, quickness and low cost, thereby being applicable to N gene molecular marker assisted selection in tobacco TMV disease resistant breeding.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a co-dominant SSR marker closely linked with tobacco TMV resistance gene N and application thereof. Background technique [0002] Tobacco mosaic is caused by Tobacco Mosaic Virus (TMV), the first single-stranded positive-sense virus identified (Beijerinck MW, Uebereincontagiumvivumfluidumalsursachederfleckenkrankheit der tabaksblatter.VerhKonAkadWetensch, 1898, 5: 3-21) , belonging to the mosaic virus family (Dawson WO and Lehto KM, Regulation of tobamovirus gene expression, Adv. Virus. Res., 1990, 8: 307-342), with infectious activity (Erickson FL, Holzberg S, Calderon-Urrea A, Handley V , Axtell M, Corr C, and Baker B, Thehelicase domain of the TMV replicase proteins induces the N-mediated defense response in tobacco. Plant J., 1999, 18: 67-75). Because the host is mainly Solanaceae crops including tobacco, it seriously affects the yield and quality of tobacco leaves, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 童治军焦芳婵肖炳光陈学军李梅云吴兴富李永平
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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