The method and application of screening non-heading Chinese cabbage recessive genital male sterile lines for fertility-related molecular markers by using snp and ssr technology
A technology related to molecular markers and recessive genital sterility, applied in biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of saving investment and shortening the breeding period
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Embodiment 1
[0055] Example 1: Molecular marker screening of non-heading Chinese cabbage recessive male sterile line fertility gene linkage
[0056] 1. DNA extraction
[0057] The numbers of each individual plant in the F2 generation segregation population were listed, and 0.5g of fresh leaves were selected at the seedling stage, packed in a 2.0ml centrifuge tube, and stored at -20°C or -70°C for later use. Genomic DNA was extracted using the CTAB method (Lu et al, 2004). The specific steps are: pour the leaves into a mortar, add 700-800 μl of 2% CTAB buffer solution (2% CTAB; EDTA: 20mmol / L; pH=8.0Tris-HCl: 100mmol / L; NaCl: 1.4mol / L ; PVP1%), ground into a homogenate. Water bath at 65°C for 60 minutes, with gentle shaking every 15 minutes. Leave it at room temperature for 10 min, add an equal volume of chloroform:isoamyl alcohol (24:1), shake gently, and mix well. Centrifuge at 12,000r / min for 15min, take it out gently, pipette the supernatant into a new 2ml centrifuge tube, add 1 / 10 ...
Embodiment 2
[0099] Example 2: Using molecular markers linked to the fertility gene of Banana A of non-heading Chinese cabbage to assist in the selection of excellent sterile lines and transfection, the steps are as follows:
[0100] a) The non-heading Chinese cabbage recessive male sterile line Bianjiaohuang A was used as the female parent, and the fertile inbred line of non-heading Chinese cabbage with excellent agronomic traits was selected as the male parent, and the F1 generation was produced by crossing.
[0101] b) The F1 generation is used as the female parent, and the above-mentioned excellent fertile inbred line is used as the male parent to backcross to produce the BC1F1 generation.
[0102] c) Plant BC1F1 generation plants, take samples from individual plants, extract DNA samples from individual plants, use labeled SSR60-1, SSR60-2 and SSR8-2 primers to perform single-plant PCR amplification, 6% polyacrylamide gel electrophoresis, and silver staining After developing, the ampli...
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