Molecular Identification of Cytoplasmic Male Sterility Restorer Gene in Cotton Trifida

A technology for male sterility and gene restoration, which is applied in biochemical equipment and methods, microbiological measurement/inspection, DNA/RNA fragments, etc., can solve the problem of long time required for trait investigation, high consumption of time, material and financial resources, and accelerated recovery system. Breeding and other issues to achieve the effect of reducing backcross tests, promoting breeding and improvement, and reducing risks

Active Publication Date: 2022-03-15
INST OF COTTON RES CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problems of traditional breeding methods such as large field workload, long time required for character investigation and poor accuracy, and large consumption of time, material and financial resources in the process of breeding three-lobed cotton cytoplasmic male sterile restorer lines. InDel molecular markers, molecular marker primers and molecular identification methods closely linked to the three-lobed cotton cytoplasmic male sterility restorer gene can speed up the process of restorer line breeding and improvement, shorten the breeding cycle, and at the same time ensure that restorer line seeds and three-line hybridization purity of the seeds

Method used

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  • Molecular Identification of Cytoplasmic Male Sterility Restorer Gene in Cotton Trifida
  • Molecular Identification of Cytoplasmic Male Sterility Restorer Gene in Cotton Trifida
  • Molecular Identification of Cytoplasmic Male Sterility Restorer Gene in Cotton Trifida

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Embodiment 1

[0030] The InDel molecular marker of the present invention is a deletion sequence 5'-TATTTGCACCCTAAGCAATAGTTTTAGAACAATTAA-3' (SEQ ID NO.1) with a deletion of 36 bp. The deletion site of the InDel marker is in upland cotton chromosome ChrD05: 54213468-54213503 (refer to the upland cotton sequencing results of Nanjing Agricultural University, the download URL is http: / / mascotton.njau.edu.cn / info / 1054 / 1118.htm ).

[0031] In the upstream and downstream of the marker, according to the InDel site, sequence and PCR primer design principles, design primers, successfully convert it into InDel marker primers, and amplify the InDel molecular marker primer pair as shown in Table 1:

[0032] Table 1: InDel Molecular Labeling Primers

[0033]

[0034] In the genome of the homozygous restorer line of the three-lobed cotton cytoplasmic male sterile restorer line, the 239bp band was specifically amplified and deleted, and 275bp and A 239bp band, a 275bp band was specifically amplifie...

Embodiment 2

[0037] 1. The CTAB method was used to extract the seed DNA of the first-generation backcross population (BCF1) constructed by three lines of cotton male sterile (CMS-D8).

[0038] 2. Using the InDel molecular marker primers, the extracted DNA is amplified by PCR. The PCR reaction system is shown in Table 2.

[0039] Table 2 20μl PCR reaction system

[0040]

[0041] The PCR program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 15 s, and 35 cycles; then final extension at 72°C for 2 min, and storage at 4°C.

[0042] Perform agarose gel (2.5%) electrophoresis on the PCR product, electrophoresis detection in 1×TAE electrophoresis buffer, voltage 80-90V, current 100mA, 40min-60min, take pictures with a gel imaging system and save, and electrophoresis results Perform band analysis.

[0043] 3. Test results

[0044] Judgment criteria: In the statistics, if only 239bp band appears, it contains homozygo...

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Abstract

The invention provides a method for molecular identification of the three-lobed cotton cytoplasmic male sterility restoration gene, the InDel molecular marker is a 36bp nucleotide sequence deletion, and the InDel molecular marker primer is shown in SEQ ID NO.2‑3. Using InDel molecular marker primers for PCR amplification, if the fragment length is a 275bp band, it is a material that does not contain the three-fied cotton cytoplasmic male sterility restoration gene; if the fragment length is a 239bp band, it is a three-fied cotton cytoplasmic material Restorer line material that is homozygous for the male sterility restorer gene locus; if two bands with fragment lengths of 275bp and 239bp appear in the results of electrophoresis of the PCR product at the same time, it is the material for the heterozygous locus of the restorer gene. The invention can speed up the breeding and improvement process of restorer lines, shorten the breeding cycle, ensure the purity of restorer line seeds and three-line hybrid seeds, and significantly reduce the workload of field material property investigation.

Description

technical field [0001] The invention belongs to the application field of molecular marker development and breeding, and specifically relates to the application of an InDel marker and its corresponding primers to the identification of the CMS restoration gene of trifida cotton and the molecular marker assisted breeding. Background technique [0002] Cotton is an important economic crop, an important fiber energy crop, and an important industrial raw material in the world. Cotton has an important position and significance in world agricultural production. Cotton is a crop with frequent cross-pollination, and frequent cross-pollination provides a biological basis for the research and utilization of heterosis in cotton. The research and utilization of cotton heterosis have significantly increased cotton yield, improved cotton quality, improved cotton multiple resistance, etc. Cytoplasmic male sterility, which widely exists in plants, is an important tool for hybrid breeding and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 邢朝柱冯娟娟吴建勇张梦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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