Insertion/deletion (InDel) molecular marker AAD closely-linked to tomato anthocyanin absent character and application thereof

Abstract

The invention relates to the field of molecular breeding, and in particular to an insertion / deletion (InDel) molecular marker AAD closely-linked to the tomato anthocyanin absent character and application thereof. The physical position of the InDel molecular marker closely-linked to the tomato anthocyanin absent character is No. 45339693 base to No. 45343097 base on chromosome 2 of tomato. The invention can replace a traditional method of selecting a green plant to ensure that a breeding material of tomato contains an anthocyanin absence (AA) mutation site. An AA site hybrid plant in the breeding material with normal phenotype is assist-selected by mean of the molecular marker, the breeding period can be shortened, and the breeding efficiency can be improved.

Description

technical field

[0001] The invention relates to the field of molecular breeding, in particular to an insertion-deletion molecular marker AAD closely linked to tomato anthocyanin deficiency traits and an application thereof. Background technique

[0002] Tomato is one of the most important vegetables in China and even in the world. The heterosis of tomato is relatively obvious, and most of the varieties currently planted are hybrids. However, at present, manual detasseling and pollination are mostly used for tomato hybrid seed production, which is time-consuming and labor-intensive, and the cost of seed production is high; and the purity of hybrids may be affected due to untimely or incomplete detasseling. In addition, the purity detection of hybrids is time-consuming and expensive, whether using traditional phenotypic identification methods or molecular marker detection methods.

[0003] The surface of each part of the tomato anthocyanin absent (aa) plant is green due to t...

Examples

Embodiment 1

[0021] Example 1 Determination of tomato anthocyanin deficiency gene locus InDelAAD

[0022] The tomato material containing anthocyanin-deficient mutation site aa is used as the female parent, and the gooseberry tomato material with purple plant surface is used as the male parent, and the two are crossed to obtain F 1 . F1 single plant is self-crossed, and its seeds are propagated into F 2 There are nearly 6,000 plants isolated from generation to generation, and conventional water and fertilizer management. Observe the color (purple or green) of the hypocotyls of the seedlings, and pick out the plants containing anthocyanins and lacking homozygous mutation sites whose hypocotyls are green. For the seedlings with green hypocotyls in the female parent, male parent, F1, and F2 populations, the genomic DNA was extracted by conventional CTAB method in individual plants.

[0023] Comparing the sequence of tomato variety Heinz1706 sequenced by Tomato International Genome Project a...

Embodiment 2I

[0027] Example 2 Development and Verification of InDel Molecular Marker AAD

[0028] Primer Design of InDel Molecular Marker AAD

[0029] According to AAD and its flanking sequences, 3 primers were designed. The forward primer (AAD-F) was 5-GGGACAAAATACAACGACGAC-3, the reverse primer 1 (AAD-R1) was 5-GCTGATGTTGATGTGAAGGAA-3, and the reverse primer 2 (AAD-R2) was 5-TAAAAGGGCTAGTGACTTGGTGT-3.

[0030] Polymorphism Detection of InDel Molecular Marker AAD

[0031] PCR amplification.

[0032] Based on female parent, male parent and its F 1 The genomic DNA of AAD was used as a template, and three specific primers for detecting InDel-labeled AAD were used for PCR amplification. A 363bp band can be detected in the female parent plant containing tomato anthocyanin deficiency (aa) homozygous mutation site; a 487bp band can be detected in the male parent plant whose aa site is normal homozygous (wild type) ; The aa site is F in a heterozygous state 1 Plants can detect the above two...

technical field

[0001] The invention relates to the field of molecular breeding, in particular to an insertion-deletion molecular marker AAD closely linked to tomato anthocyanin deficiency traits and an application thereof. Background technique

[0002] Tomato is one of the most important vegetables in China and even in the world. The heterosis of tomato is relatively obvious, and most of the varieties currently planted are hybrids. However, at present, manual detasseling and pollination are mostly used for tomato hybrid seed production, which is time-consuming and labor-intensive, and the cost of seed production is high; and the purity of hybrids may be affected due to untimely or incomplete detasseling. In addition, the purity detection of hybrids is time-consuming and expensive, whether using traditional phenotypic identification methods or molecular marker detection methods.

[0003] The surface of each part of the tomato anthocyanin absent (aa) plant is green due to t...

Examples

Embodiment 1

[0021] Example 1 Determination of tomato anthocyanin deficiency gene locus InDelAAD

[0022] The tomato material containing anthocyanin-deficient mutation site aa is used as the female parent, and the gooseberry tomato material with purple plant surface is used as the male parent, and the two are crossed to obtain F 1 . F1 single plant is self-crossed, and its seeds are propagated into F 2 There are nearly 6,000 plants isolated from generation to generation, and conventional water and fertilizer management. Observe the color (purple or green) of the hypocotyls of the seedlings, and pick out the plants containing anthocyanins and lacking homozygous mutation sites whose hypocotyls are green. For the seedlings with green hypocotyls in the female parent, male parent, F1, and F2 populations, the genomic DNA was extracted by conventional CTAB method in individual plants.

[0023] Comparing the sequence of tomato variety Heinz1706 sequenced by Tomato International Genome Project a...

Embodiment 2I

[0027] Example 2 Development and Verification of InDel Molecular Marker AAD

[0028] Primer Design of InDel Molecular Marker AAD

[0029] According to AAD and its flanking sequences, 3 primers were designed. The forward primer (AAD-F) was 5-GGGACAAAATACAACGACGAC-3, the reverse primer 1 (AAD-R1) was 5-GCTGATGTTGATGTGAAGGAA-3, and the reverse primer 2 (AAD-R2) was 5-TAAAAGGGCTAGTGACTTGGTGT-3.

[0030] Polymorphism Detection of InDel Molecular Marker AAD

[0031] PCR amplification.

[0032] Based on female parent, male parent and its F 1 The genomic DNA of AAD was used as a template, and three specific primers for detecting InDel-labeled AAD were used for PCR amplification. A 363bp band can be detected in the female parent plant containing tomato anthocyanin deficiency (aa) homozygous mutation site; a 487bp band can be detected in the male parent plant whose aa site is normal homozygous (wild type) ; The aa site is F in a heterozygous state 1 Plants can detect the above two...

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