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Molecular marker related with cotton fertility restoration, and application thereof

A cotton and restorer line technology, applied in the field of molecular biology genetics and breeding, can solve the problems of speeding up the field selection and backcross improvement process of excellent restorer lines, easy loss of restorer genes, time-consuming and labor-intensive, etc., and achieves good commercial application prospects, Improve selection efficiency and accuracy, low cost effect

Active Publication Date: 2019-10-15
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a molecular marker identification method related to cotton fertility restoration, aiming to solve the problem of time-consuming and labor-intensive field identification and detection methods in the breeding of cotton restorer lines containing Harknessy cotton cytoplasmic male sterile cytoplasm in the prior art. Poor accuracy and easy loss of restorer genes in the process of backcross improvement of restorer lines, etc., so as to speed up the process of field breeding and backcross improvement of excellent restorer lines, while ensuring the seed production purity of restorer lines and "three-line" hybrid cotton

Method used

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  • Molecular marker related with cotton fertility restoration, and application thereof
  • Molecular marker related with cotton fertility restoration, and application thereof
  • Molecular marker related with cotton fertility restoration, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1, the extraction of genomic DNA

[0082] Four restorer line materials were extracted by the improved CTAB method: Zhonghui 46 (H46), Zhonghui 80 (H80), DR and ZR, two transgenic insect-resistant three-line hybrids Zhongmiansuo 83 and Zhongmiansuo 99, And 4 conventional cotton varieties: Zhongmian 45, Zhongmian 69, Zhongmian 100 and Zhongmian 110, a total of 10 materials (or leaves) genomic DNA, the specific method is as follows:

[0083] (1) First peel off the shell of the cotton seeds, grind each seed into powder separately, transfer to a 2ml centrifuge tube, and quickly add 800μl of preheated CTAB lysate (65°C), mix it upside down and place in 65°C water bath for 30-40 minutes, and slowly invert and mix once every 10 minutes.

[0084] (2) After the water bath, take out the centrifuge tube, add 800 μl of chloroform:isoamyl alcohol (24:1), invert for about 30-50 times, and mix until no layering occurs.

[0085] (3) Centrifuge at 12000rpm for 10min at 4°C. ...

Embodiment 2

[0093] Example 2, Gene Mapping and Molecular Marker Development

[0094] (1) Population construction and fertility survey

[0095] In this experiment, the "three lines" of upland cotton: Hackney cotton cytoplasmic male sterile line ZBA, maintainer line ZB and restorer line Zhonghui 46 (H46) were used as parents to construct restorer gene near-isogenic lines and F 2 Separate groups. The test materials were planted in the East Field Test Base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences (East of Baibi Town, Anyang County, Henan Province; 36°10'N, 114°35'E), and the field was managed according to conventional methods. Fertility investigation method: During the flowering period from early July to mid-to-late August, try to choose sunny weather, and start investigating the near-isogenic line of the restorer gene and the F 2 Fertility of individual plants in a population. In order to improve the accuracy of group physical survey, a total of 5 s...

Embodiment 3

[0103] Example 3, Fertility recovery correlation verification of molecular markers

[0104] Using the above primers, the polymorphism and amplification stability of the InDel molecular marker were verified by PCR amplification and agarose gel electrophoresis detection. Specifically, using the genomic DNA of 10 cotton material seeds extracted in Example 1 as a template, the above-mentioned amplification primers were used to perform PCR amplification, wherein:

[0105] (1) PCR reaction system and conditions:

[0106] 20 μl reaction system: 1 μl of DNA template and 19 μl of PCR reaction solution (10 μl of Kangwei Century Company 2×Es TaqMasterMix (Dye), 0.8 μl of each 10 μM InDel primer pair, 7.4 μl of ddH2O), and mix well.

[0107] The amplification program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 15 sec, 30 cycles; storage at 4°C.

[0108] (2) Analysis of results:

[0109] After the PCR ampl...

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Abstract

The present invention discloses a molecular marker related with cotton fertility restoration, and an application thereof. A provided primer pair for identification or assistant identification of cotton restoration genes to be tested consists of a primer 1 and a primer 2; the primer 1 is as follows (a) or (b): (a) a single-stranded DNA molecule shown in a sequence 2 in a sequence listing; and (b) asingle-stranded DNA molecule with the sequence 2 in the sequence listing substituted and / or deleted and / or added by one or several nucleotides and having a same function as the sequence 2 in the sequence listing; and the primer 2 is as follows (c) or (d): (c) a single-stranded DNA molecule shown in a sequence 3 in the sequence listing; and (d) a single-stranded DNA molecule with the sequence 3 inthe sequence listing substituted and / or deleted and / or added by one or several nucleotides and having a same function as the sequence 3 in the sequence listing. The molecular marker is closely related to cotton fertility restoration, and can be effectively used for molecular marker-assisted selection breeding of restoration lines and "three-line" hybrid cotton.

Description

technical field [0001] The invention relates to the technical field of genetic breeding of molecular biology, in particular to a molecular marker related to cotton fertility recovery and its application. Background technique [0002] Field breeding practice shows that the use of cotton heterosis can greatly increase fiber yield, improve fiber quality and enhance stress resistance. At present, most of the cotton hybrids widely promoted in production are obtained by artificial emasculation and pollination. However, labor costs in major cotton producing areas and surrounding areas are increasing year by year, making it difficult for many seed companies engaged in hybrid cotton production to survive. Therefore, it is the general trend to vigorously develop simplified, efficient, and low-cost cotton production. The "three-line method" hybrid seed production procedure is simple, and the cost of seed production is only 30-40% of that of manual emasculation hybridization, which is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11A01H1/02A01H1/04
CPCA01H1/02A01H1/04C12Q1/686C12Q1/6895C12Q2600/13C12Q2600/156C12Q2565/125
Inventor 邢朝柱张梦吴建勇冯娟娟郭立平戚廷香张学贤王海林唐会妮乔秀琴
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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