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Application of molecular marker MtD4 in identifying carrot petal male sterility

A technique for male sterility and carrots, applied in the field of breeding, can solve the problems of verification, inability to use multiple genotypes of petal flower type sterile lines and maintainer lines, complex structure, etc., and achieve the effect of accelerating the pace of breeding.

Active Publication Date: 2019-04-16
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Nakajima et al. (Nakajima Y., Yamamoto T, Muranaka T and Oeda K, 1999. Genetic variation of petaloid male-sterile cytoplasm of carrot revealed by sequencetagged sites (STSs). Theor Appl Genet 99:837–843.) using RAPD and STS markers Qualitative research was carried out on the mitochondrial DNA of 13 sterile lines and fertile lines, and two specific markers, STS1 and STS4, which can distinguish between sterile lines and fertile lines were obtained. The STS1 marker contains a sequence homologous to the orfB gene. The structure of the STS4 marker is relatively complex, including a retrotransposon-like sequence and a small fragment of chloroplast DNA sequence, but these two markers cannot be used for the identification of petal-flowered sterile lines and maintainer lines of various genotypes
Yu Chunxia et al. (Yu Chunxia, ​​Liang Yi, Li Longnian. 2007. Establishment of the CAPS marker of the petalized carrot male sterility gene atp6. China Agricultural Science Bulletin, 23(12): 68-72.) Development of CAPS marker identification based on the mitochondrial gene atp6 The petalized male sterile line H05A and its maintainer line H05B can produce a 60bp specific fragment in the sterile line, but this marker has not been verified in other materials

Method used

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  • Application of molecular marker MtD4 in identifying carrot petal male sterility
  • Application of molecular marker MtD4 in identifying carrot petal male sterility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1, Development of mitochondrial haplotype-specific (Mitotype-specific sequence, MSS) molecular markers and polymorphism detection

[0068] 1. Development of MSS molecular markers

[0069] 1. Extract the mitochondrial DNA of carrot 50119 and carrot OK respectively, and then perform mitochondrial genome sequencing.

[0070] 2. After completing step 1, compare and analyze the mitochondrial genome sequencing results of carrot 50119 and carrot O K, and obtain 3 MSS sequences (named MSS sequence 2-MSS sequence 4 in turn). The nucleotide sequence of MSS sequence 2 is shown in sequence 1 in the sequence listing. The nucleotide sequence of MSS sequence 3 is shown as sequence 2 in the sequence listing. The nucleotide sequence of MSS sequence 4 is shown as sequence 3 in the sequence listing.

[0071] 3. Based on the two variant forms of MSS sequence 2 in the carrot mitochondrial genome, the molecular marker MtD2 was developed.

[0072] The nucleotide sequence of the am...

Embodiment 2

[0113] Embodiment 2, the application of MSS molecular marker

[0114] The PCR reaction system in this embodiment is all 10 μ L, consists of 1 μ L genomic DNA (concentration is 50-100 ng / μ L) of young carrot leaves to be tested, 1 μ L 10 × PCR reaction buffer (containing Mg 2 +), 1 μL dNTPs (2.5 mM concentration), 0.2 μL upstream primer (10 μM concentration), 0.2 μL downstream primer (10 μM concentration), 0.2 μL Taq DNA polymerase (2.5 U / μL concentration), and 6.4 μL ddHO composition.

[0115] 10× PCR reaction buffer (containing Mg 2 +) is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0116] The PCR reaction procedures in this example are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and 35 cycles; extension at 72°C for 5 min.

[0117] 1. Application 1

[0118] The carrots to be tested were 16 carrot materials imported and preserved from the Vegetable Research Center of the Un...

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Abstract

The invention discloses application of a molecular marker MtD4 in identifying carrot petal male sterility. Genomic DNA of a carrot to be tested is used as a template, and a primer pair consisting of aprimer F and a primer R is used for PCR amplification to obtain a DNA fragment. The DNA fragment is the molecular marker MtD4 related to the carrot petal male sterility. The nucleotide sequences of the primer F and the primer R are sequentially shown as the sequence 8 and the sequence 9 in the sequence listing. The molecular marker MtD4 may specifically be a DNA fragment 1 or a DNA fragment 2. The nucleotide sequences of the DNA fragment 1 and the DNA fragment 2 are sequentially shown as the sequence 14 and the sequence 15 in the sequence listing. The carrot variety carrying the DNA fragment1 and not carrying the DNA fragment 2 has the petal male sterility. By using the molecular marker MtD4, the carrot variety with the petal male sterility can be rapidly screened out. The application has important application value.

Description

technical field [0001] The invention belongs to the field of breeding, and in particular relates to the application of the molecular marker MtD4 in identifying carrot petal male sterility. Background technique [0002] Since carrot is a cross-pollinated crop with small flower organs and artificial pollination is difficult, the self-incompatibility of carrot is still unclear, so the use of carrot male sterile lines has become the key to hybrid breeding, without complicated procedures such as artificial pollination and emasculation , the production cost is lower, and the seed purity of the first generation of hybrids can be ensured. [0003] The sources of male sterility of existing carrot cultivars mainly come from wild species, all of which are cytoplasmic male sterility (CMS) (Fang Zhiyuan et al. 2017. Chinese Vegetable Breeding, Beijing: China Agricultural Press. 268-269 .). There are two types of male sterility in carrot: brown anther type and andropetalized type. The ...

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 庄飞云欧承刚曹琼文陈姝敏赵志伟
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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