Preparation method and application of molecular marker of rape male sterile restoring gene

Abstract

The invention discloses a preparation method and an application of a molecular marker of a rape male sterility restoring gene. The preparation method comprises the following steps of A, designing degenerate primers according to PPR gene conservative amino acid sites, B, extracting a lamina DNA of a rape cytoplasm male sterility restoring line through a cetyltrimethyl ammonium bromide (CTAB) method and magnifying the lamina DNA through a touchdown polymerase chain reaction (PCR) process to obtain a specific restoring gene candidate segment, C, carrying out recovery, conversion, sequencing and sequence analysis processes on PCR products, D, carrying out 5 ' RACE and 3 ' RACE processes and splicing a sequencing result to obtain a full-length cDNA sequence, and E, designing specific primers in 5 ' UTR and 3 ' UTR zones of the full-length cDNA sequence according to the full-length cDNA sequence to obtain a molecular marker. The invention also discloses an application of a restoring gene molecular marker in hybrid rape parent breeding. The preparation method has the advantages of improvements of a speed of restorer seeding and an efficiency of verification, simple and easy method, good operability, rapid identification speed of restoring genes, great reduction of necessary workload of test cross and progeny planting observation in a restorative verification process, and improvement of an efficiency of hybrid seeding.

Description

technical field [0001] The invention belongs to the fields of rapeseed breeding and molecular biology, and more specifically relates to a method for preparing a molecular marker of a rapeseed heterologous cytoplasmic male sterility restoration gene, and also relates to a molecular marker of a rapeseed heterologous cytoplasmic male sterility restoration gene Application in parental selection of rapeseed hybrids. Background technique [0002] At present, the promotion area of ​​hybrid rapeseed has accounted for more than 60% of the total production area of ​​rapeseed. Cytoplasmic male sterility is the main pollination control system for the production of rapeseed hybrids. About 70% of the miscellaneous rapeseed used in my country's production are cytoplasmic male sterile hybrids, which are basically formulated from Pol CMS, which was discovered by Huazhong Agricultural University in 1972 from the imported variety "Polima". . The fertility of Pol CMS is easily affected by tem...

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Problems solved by technology

The fertility of Pol CMS is easily affected by temperature. Micro pollen is produced at low temperature, and the sterile plants can reproduce by themselves, resulting in the existence of a certain proportion of sterile plants when preparing hybrids, which reduces the purity of hybrids, and the sterile plants have a low seed setting rate. Affect the yield potential of hybrids and increase the risk of hybrid rapeseed utilization

Other male sterile cytoplasms currently researched and applied in my country include Shan 2A CMS, MI CMS, Nca CMS, etc. These sterile cytoplasms are all identified by natural variation or interspecific hybridization in Brassica cultivars, and belong to homologous Male sterile cytoplasm has the disadvantage of insufficient stability of fertility, and there is a problem that the purity of hybrids is difficult to guarantee in application, and its complete maintainer and restorer lines are not easy to screen, and most rapeseed varieties are neither restored nor preserved Type (Li Dianrong. Preliminary report on three-line breeding of Brassica napus. Shaanxi Agricultural Sciences, 1980, (1): 26-29; Fu Shouzhong, Qi Cunkou, Tang Jihong. Breeding of the cytoplasmic male sterile line MI CMS of Brassica napus. Acta Crops , 1989, 17(2): 151-156. Liu Guihua, Cai Ming, Sheng Xiaoyan. Breeding of NCa male sterile material in Brassica napus and its restoration relationship in Brassica napus. Chinese Agricultural Sciences, 1997, 30(3 ):61-65)

Radish male sterile material with relatively stable fertility obtained by transferring radish sterile cytoplasm abroad (Bannerot H, Boulidard L, Cauderon Y and Tempe J. Transfer of cytoplasmic male sterility from Raphanus sativus to Brassicaoleracea. Proc. Eucarpia Meeting Cruciferae: 1974 , 52-54), but in the process of utilization, it is difficult to find the restorer line, and the problems of linkage between the restorer gene and the bad quality traits make the breeding and utilization of the sterile line slow (Delourme R., Foisset N., Horvais R., Barret P., Champagne G., Cheung W.Y., Landry B.S., Renard M. Characterization of the radish introgression carrying the Rforestorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassicanapus L.). 1998, Theor Appl Genet 97:129 -134)

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