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...

Examples

Embodiment 1

[0036] A method for preparing a rapeseed male sterility restorer gene molecular marker, the specific steps are:

[0037] 1. Acquisition of relevant fragments of the cytoplasmic male sterility restoration gene of Brassica japonica

[0038] A. Experimental materials and population construction: CMS lines of wild mustard cytoplasmic male sterile (Nsa CMS) (both selfed or backcrossed progenies with Brassica napus, no wild mustard chromosomes in the nucleus), Zhongshuang 4 (for The maintainer line of Nsa CMS is also the original parent of Brassica napus for somatic cell hybridization), Yeyou 18 (the original wild mustard parent for somatic cell hybridization) and the male sterile line of Nsa CMS crossed with No. 1 restorer line (Huai 1) After the obtained fertile F1 generation single plant was crossed with the maintainer line, fertile plants and sterile plants in the fertile segregation population obtained after continuous backcrossing of the fertile plant and the maintainer line f...

Embodiment 2

[0066] The molecular markers of wild mustard cytoplasmic male sterility restoration gene and linkage detection of restoration traits, the specific steps are:

[0067] A. Construction of wild mustard cytoplasmic male sterile segregation population: the wild mustard cytoplasmic male sterile line (Nsa CMS) was pollinated with bagging self-bred Nsa restorer line for multiple generations, and F1 generation seeds were obtained. Bagging selfing of F1 to obtain a fertilely separated F2 population;

[0068] B. Fertility identification: During the full flowering stage of rapeseed, the fertility identification of each individual plant of the F2 population is carried out in the field. Since the Nsa CMS is completely sterile, it is easy to determine whether each individual plant is viable according to whether the petals are open or not and the amount of pollen. Fertile or sterile;

[0069] C. Sampling and extracting individual plant DNA: Sampling the sterile plants and fertile plants as i...

Embodiment 3

[0072] The application of the molecular marker of the wild mustard cytoplasmic male sterility restorer gene in parental selection of rapeseed hybrids, the specific steps are as follows:

[0073] A. The original restorer line Hui 1 obtained from the initial screening was used to cross with rapeseed lines 141 and TR008 with excellent agronomic properties and early maturity, and fertile plants were selected from the F2 generation for bagging and selfing to produce F3-F6 generation high-generation plants Tie;

[0074] B, from the F3 generation, according to the performance of agronomic traits, the field selects the excellent lines with neat growth, strong, many branches, and suitable flowering period, and gets 10 individual plant mixed samples to extract genomic DNA, and adopts the developed method of implementation example 2 The specific primers for the cytoplasmic male sterility restorer gene of Brassica japonica were amplified by PCR ( Figure 8), keep the strain that can ampl...

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...

Examples

Embodiment 1

[0036] A method for preparing a rapeseed male sterility restorer gene molecular marker, the specific steps are:

[0037] 1. Acquisition of relevant fragments of the cytoplasmic male sterility restoration gene of Brassica japonica

[0038] A. Experimental materials and population construction: CMS lines of wild mustard cytoplasmic male sterile (Nsa CMS) (both selfed or backcrossed progenies with Brassica napus, no wild mustard chromosomes in the nucleus), Zhongshuang 4 (for The maintainer line of Nsa CMS is also the original parent of Brassica napus for somatic cell hybridization), Yeyou 18 (the original wild mustard parent for somatic cell hybridization) and the male sterile line of Nsa CMS crossed with No. 1 restorer line (Huai 1) After the obtained fertile F1 generation single plant was crossed with the maintainer line, fertile plants and sterile plants in the fertile segregation population obtained after continuous backcrossing of the fertile plant and the maintainer line f...

Embodiment 2

[0066] The molecular markers of wild mustard cytoplasmic male sterility restoration gene and linkage detection of restoration traits, the specific steps are:

[0067] A. Construction of wild mustard cytoplasmic male sterile segregation population: the wild mustard cytoplasmic male sterile line (Nsa CMS) was pollinated with bagging self-bred Nsa restorer line for multiple generations, and F1 generation seeds were obtained. Bagging selfing of F1 to obtain a fertilely separated F2 population;

[0068] B. Fertility identification: During the full flowering stage of rapeseed, the fertility identification of each individual plant of the F2 population is carried out in the field. Since the Nsa CMS is completely sterile, it is easy to determine whether each individual plant is viable according to whether the petals are open or not and the amount of pollen. Fertile or sterile;

[0069] C. Sampling and extracting individual plant DNA: Sampling the sterile plants and fertile plants as i...

Embodiment 3

[0072] The application of the molecular marker of the wild mustard cytoplasmic male sterility restorer gene in parental selection of rapeseed hybrids, the specific steps are as follows:

[0073] A. The original restorer line Hui 1 obtained from the initial screening was used to cross with rapeseed lines 141 and TR008 with excellent agronomic properties and early maturity, and fertile plants were selected from the F2 generation for bagging and selfing to produce F3-F6 generation high-generation plants Tie;

[0074] B, from the F3 generation, according to the performance of agronomic traits, the field selects the excellent lines with neat growth, strong, many branches, and suitable flowering period, and gets 10 individual plant mixed samples to extract genomic DNA, and adopts the developed method of implementation example 2 The specific primers for the cytoplasmic male sterility restorer gene of Brassica japonica were amplified by PCR ( Figure 8), keep the strain that can ampl...

Images