Primer pair for identifying carrot petal cytoplasmic male sterility and application thereof

A technology for male sterility and male sterility, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of complex genetic background of male sterility in carrots, etc. The effect of reliable technical means

Active Publication Date: 2022-03-08
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional conventional method of cultivating male sterile lines of carrots is to use the method of backcrossing and transferring, using the backcrossing of high-quality fertile single plants to carry out fertility identification on the backcross offspring, focusing on examining the method of fertility identification with maintainer lines and no restorer genes. It needs to be in the flowering stage of the plants, but due to the complex genetic background of carrot male sterility, fertile plants often appear in the pre-selected sterile lines after several generations in breeding

Method used

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  • Primer pair for identifying carrot petal cytoplasmic male sterility and application thereof
  • Primer pair for identifying carrot petal cytoplasmic male sterility and application thereof
  • Primer pair for identifying carrot petal cytoplasmic male sterility and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A group of inflorescence RNAs from carrot petal cytoplasmic male sterile lines and their maintainers (HYs698 and HYd698) of the Hongyue strain bred by the Vegetable Research Institute of Tianjin Academy of Agricultural Sciences were extracted, a cDNA library was established, and the transcriptome was sequenced. , compared with the carrot reference mitochondrial genome, the URL of the reference mitochondrial genome is as follows: http: / / ftp.ensemblgenomes.org / pub / plants / release-51 / fasta / daucus_carota / dna / ; Screen out 32 positions with base number differences (Indel≥4bp) between fertile and sterile, as shown in Table 1 for schematic examples.

[0053] Table 1 Nine differential positions screened by transcriptome analysis

[0054]

[0055] On the basis of including this difference site, the sequence was extended up and down by about 100 bp each, and 9 pairs of primers were designed in order to amplify the bands, corresponding to the difference positions 1-9 respective...

Embodiment 2

[0061] (1) Sample group 1: WMA-477 and WMB-477 are derived from Japanese Kuroda-type carrot petal-type cytoplasmic male sterile lines and their maintainers; LSsA-128 and LSsB-128 are derived from American cut-type carrots The petalized cytoplasmic male sterile line and its maintainer line; SKs35-027 and SKd35-027 are derived from the carrot petalized cytoplasmic male sterile line and its maintainer line with the combination of Nantes and Kuroda; HYs698 and HYd698 are derived from Carrot petal-type cytoplasmic male sterile line and its maintainer line of Hongyue strain bred by Vegetable Research Institute of Tianjin Academy of Agricultural Sciences.

[0062] (2) Reagents: 2×Taq Master Mix (for PAGE); specific amplification primers synthesized by Jinweizhi.

[0063] The forward primer sequence is SEQ ID NO.1: 5'-TTGTGCGGTTAAGGTTGTTGT-3'

[0064] The reverse primer sequence is SEQ ID NO.2: 5'-CCTGTTCTTGTGGATCAATGGA-3'.

[0065] (3) Polymorphism detection: extract the genomic DN...

Embodiment 3

[0075] (1) Sample group 2: 8 commercial varieties available on the market (Mendel No. 3, SK316, Chunao 709, Nobel No. 2, Rose Red, Laxian, Chunao 705, Hongbao Six-inch Ginseng).

[0076] (2) Reagents: 2×Taq Master Mix (for PAGE); specific amplification primers synthesized by Jinweizhi.

[0077] The forward primer sequence is SEQ ID NO.1: 5'-TTGTGCGGTTAAGGTTGTTGT-3'

[0078] The reverse primer sequence is SEQ ID NO.2: 5'-CCTGTTCTTGTGGATCAATGGA-3'.

[0079] (3) Polymorphism detection: extract the genomic DNA of young carrot leaves to be tested, and use it as a template to perform PCR amplification using the specific primer pair provided by the present invention.

[0080] (4) PCR reaction system: a total volume of 10 μL, including 5 μL of 2×Taq Master Mix (for PAGE), 0.5 μL of upstream and downstream primers (10 μmol / L), 1 μL of DNA template (25ng / μL), supplemented with sterile water to 10 μL.

[0081] (5) PCR amplification program: pre-denaturation at 95°C for 5 min, 1 cycle;...

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Abstract

The invention discloses a primer pair for identifying carrot petal cytoplasmic male sterility and application thereof, the primer pair is designed according to an Indel molecular marker screened by transcriptome analysis, and the application is that the primer pair is adopted to identify carrot petal cytoplasmic male sterility. According to the application, transcriptome sequencing analysis is carried out on total RNA (Ribonucleic Acid) of a group of stable carrot petal cytoplasmic male sterile lines and maintainer lines thereof, comparison with a published carrot reference mitochondrial genome is carried out, Indel loci with differences between the sterile lines and the maintainer lines are screened out, and primers are designed. Specific amplification verification is carried out between fertile plants and sterile plants of different varieties of carrots, the result shows that the primer pair can completely distinguish the fertility of the variety to be detected, the carrot variety with cytoplasmic male sterility can be rapidly screened out in the seedling stage, the accuracy rate is high, and the method can be used for removing abnormal plants in the breeding process. The method has important practical application value and market prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology breeding, and in particular relates to a pair of primers for identifying carrot petal-type cytoplasmic male sterility and an application thereof. Background technique [0002] Carrot (Daucus carota L.) belongs to a class of important root vegetables belonging to the genus Carota of Umbelliferae. Its fleshy root is rich in carotenoids and has high nutritional value. It is widely planted all over the world and is a global One of the top ten vegetable crops. The rapid increase of carrot planting area not only greatly increases the demand for seeds, but also requires higher seed quality and production efficiency. [0003] Carrot is a biennial crop with high degree of cross-pollination, and the flower organs are extremely small, so it is difficult to artificially detassell, and it is difficult to carry out conventional artificial cross-breeding. Due to the difficulty in carrying out conventional artifici...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 孙海波王一衡付任胜岳东霞陈磊杨迎霞
Owner 天津市农业科学院
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