Breeding method of tomato recessive genetic male sterility maintainer line

A nuclear male and maintainer line technology, applied in the field of genetic breeding, can solve problems such as difficult application, ineffective application of hybrid seeds, difficulty in self-pollination, etc., and achieve the effect of important application value

Inactive Publication Date: 2019-11-05
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various tomato male sterile materials available in natural resources have their own advantages and disadvantages, which limit their application in hybrid seed production
Although the type with exposed stigma has difficulty in self-pollination, in the case of group planting, pollination between adjacent plants and adjacent flowers is prone to occur, so the natural self-pollination rate is relatively high. From the perspective of sterile lines, self-pollination cannot be completely avoided. possibility of delivery
The non-dehiscing type can basically avoid the possibility of self-fertilization, but because the style is shorter than the stamen anther tube, artificial pollination is still required
Stamenless or stamen-degenerated types can also completely avoid selfing, but the agronomic traits in such materials are generally poor and difficult to apply (Susan et al., 1997; Chen Yuhui et al., 2004; Xing Hucheng et al., 2004)
At present, more than 40 pollen abortion types found in natural resources have great application potential in tomato hybrid seed production, but such materials are generally controlled by a pair of recessive nuclear genes, most of which have not yet been cloned (Susan et al., 1997; Jeong et al., 2014), also limits the application
To sum up, the various male sterile materials currently available in tomato cannot be effectively used in the production of hybrid seeds, and tomato hybrid seed production is still mainly carried out by artificial emasculation.

Method used

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  • Breeding method of tomato recessive genetic male sterility maintainer line
  • Breeding method of tomato recessive genetic male sterility maintainer line
  • Breeding method of tomato recessive genetic male sterility maintainer line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the acquisition of Solyc03g053130 homozygous mutant plant

[0069] 1. Construction of CRISPR / Cas9 gene editing vector containing Solyc03g053130 gene-specific sgRNA target

[0070] 1. Sequence 1 is the nucleotide sequence of Solyc03g053130 gene, its structure is as follows figure 1 As shown, 1-1528bp is the promoter sequence, 1529-1920bp is the first exon sequence, 2089-2374bp is the second exon sequence, 2461-2628bp is the third exon sequence, 2737-3137bp is the fourth exon sequence exon sequence. Submit the Solyc03g053130 gene sequence shown in Sequence 1 to the CRISPRdirect online target analysis database (http: / / crispr.dbcls.jp / ), set the PAM sequence to NGG, and set the species data to Tomato (Solanum lycopersicum) str.Heinz 1706genome SL2.50, CRIPSR / Cas9 target design. Finally, the reverse complementary sequence of positions 1601-1619 (the first exon) of sequence 1 was selected as the sgRNA target sequence for editing the Solyc03g053130 gene.

[0...

Embodiment 2

[0110] Example 2, Pollen Vitality and Fertility Detection of Solyc03g053130 Homozygous Mutant Plants

[0111] 1. Detection of pollen viability of Solyc03g053130 homozygous mutant plants

[0112] 1. Reagent configuration

[0113] Fluorescein diacetate (FDA) stock solution: Dissolve 10 mg FDA in 5 mL acetone, aliquot into 1.5 mL centrifuge tubes, and store at -20°C in the dark.

[0114] BK buffer S15MOPS (pH7.5) buffer solution: mix 5mL MOPS (100mM, pH7.5), 7.5g sucrose, 6.35μL Ca(NO 3 ) 2 (1M), 4.05 μL MgSO 4 (1M) and 5 μL KNO 3 (1M) was dissolved in water, then dilute to 50mL with water. Aliquot into 1.5mL centrifuge tubes and store at -20°C in the dark.

[0115] 2. Staining and observation of pollen with fluorescein diacetate

[0116] (1) Add 1 μL of FDA stock solution to 1 mL of BK buffer S15MOPS buffer, mix well, and take 1 drop onto a clean glass slide.

[0117] (2) Take a small amount of pollen from the anther of the tomato plant to be tested (tomato Moneymaker plan...

Embodiment 3

[0121] Example 3, T 0 Obtaining the Purple Homozygous Mutant Plants

[0122] 1. Construction of a recombinant vector (i.e. recombinant plasmid pCAMBIA2300-p35S-ANT1-pLAP3-LAP3) containing both the fertility restoration gene expression cassette and the purple marker gene expression cassette

[0123] The schematic diagram of the structure of the recombinant plasmid pCAMBIA2300-p35S-ANT1-pLAP3-LAP3 is shown in Figure 8 (p35S is the 35S promoter, ANT1 is the purple marker gene, and the plants (such as leaves and stems) of plants introduced with the ANT1 gene appear purple; pLAP3-LAP3 is the Solyc03g053130 gene and its promoter).

[0124] 1. Using the total RNA of the leaves of the tomato line LA1996 as a template, reverse transcription to obtain the cDNA of the tomato line LA1996.

[0125] 2. Using the cDNA of tomato line LA1996 as a template, using 2300-ANT1-F: 5'-CGG GGTACC ATGAACAGTACATCTATGTCTTCA-3' (the underline is the recognition site of restriction endonuclease KpnI) ...

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Abstract

The invention discloses a breeding method of a tomato recessive genetic male sterility maintainer line. The method comprises the following steps that a first expression cassette for expressing a pollen fertility protein and a second expression cassette for expressing a labeled protein are introduced into a tomato recessive genetic male sterility line to obtain transgenic tomatoes, wherein sterility of the tomato recessive genetic male sterility line is caused by the function loss of the pollen fertility protein or inhibition of the expression of the pollen fertility protein, meanwhile, the transgenic tomatoes meeting the conditions A1), A2) and A3) are the tomato recessive genetic male sterility maintainer line, A1) refers to male fertility, A2) refers to expression of the labeled protein,and A3) refers to insertion of the first expression cassette and the second expression cassette in the same locus of the same chromosome of the tomato recessive genetic male sterility line; the maintainer line serves as a male parent, and the sterility line serves as a female parent for hybridization; a strain capable of expressing the labeled protein in hybrid offspring is a male fertility maintainer line, and a strain incapable of expressing the labeled protein is a sterility line. The method has important application value.

Description

technical field [0001] The invention belongs to the field of genetic breeding, and in particular relates to a breeding method for tomato recessive male sterility maintainer lines. Background technique [0002] Tomato (Solanum lycopersicum) belongs to Solanaceae tomato genus, is an important economic crop and model plant, and also the solanaceous vegetable crop with the largest planting area and high output value in Beijing. Strengthening tomato-related basic research and applied research is of great significance for promoting the construction of urban modern agriculture in Beijing. [0003] As a strictly self-pollinated crop, tomato has obvious heterosis, high hybrid uniformity and strong stress resistance, so hybrids are mainly used in production. At present, tomato hybrid seed production is mainly carried out by artificial castration. Using the male sterile line as the female parent to produce hybrid seeds can save the process of manual detasseling, reduce costs, improve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/02A01H1/04
CPCA01H1/02A01H1/04
Inventor 李常保杜敏敏李传友周明邓磊周科刘圆圆
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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