Genetic Analysis And Improvement Of A Restore Line For The Ogura Cytoplasmic Male Sterility (CMS) In Brassica Napus L.

Cytoplasmic male sterility（CMS）system is an important approaches for heterosis application in Brassica napus.Using this system,complete sterile population could easily be obtained,and the cost of producing hybrid seeds could be reduced,therefore,it could apply widely for hybrid seed production.Ogura CMS（Ogu CMS）derived from radish（Raphanus sativus）has stable and complete sterility,and has been transferred into B.napus,being considered as the most promising CMS type for heterosis utilization in rapeseed.However,restorer resources for Ogu CMS only exist in radish,making it quite difficult to directly transfer the restorer gene from radish to B.napus.This research utilized a Ogu-CMS restorer line CLR650 as study material,which was created by Hunan Crop Research Institute through bridging species Raphanobrassica（AACCRR）.After multiple generations of single plant selection,a novel restorer line CLR6430 with stable phenotype and fertility was obtained.The Rfo gene and its nearby exogenous radish DNA sequences were applied BAC library and DNA re-sequencing technologies for analysis.As a result,specific molecular markers for the restorer gene and its exogenous DNA were developed,achieving an updated Ogu-CMS restorer with significantly improved quality and agronomic traits.Major results are listed as follows:1.The results of chromosome counting of somatic cell through root tips showed that:The chromosome number of somatic cells in CLR6430 was 38;genomic library of CLR6430 were created using Copy Control TM p CC1BACTM（Hind III Cloning-Ready）Vector as the carrier,and made the BAC probe for restorer gene（Rfo）.The anthers were used for BAC-FISH experiments and the results demonstrated that there were no hybrid signals with complete radish chromosome and a pair of Rfo signals were detected in A genome,indicating different locations of exogenous radish fragments in CLR6430 from that of in R2000.2.Two Ogu CMS sterility lines and their corresponding maintainers 20QA（maintainer line 20B）and SC3QA（maintainer line SC3）were crossed[S（rfrf）×S（Rfrf）]and back-crossed with CLR6430[S（Rfrf）×N（rfrf）],respectively,and selfing for F₂ and F₃to generate segregating populations and back-cross（BC₁F₁,BC₂F₁）populations.The fertility versus sterility plants did not fitted 3:1 segregating ratio in F₂ and F₃ segregating populations,while expected 1:1 ratio were not found among reciprocal crosses（BC₁F₁,BC₂F₁）,implying segregating distortion while the probabilities of pass Rfo to offsprings through female or male gametes were the same.Meanwhile,earlier published Rfo-specific markers were applied for analyzing segregating populations of F₂ and BC₁F₁,showing co-segregating for Rfo-specific markers and fertility in plants.3.The re-sequencing data of CLR6430 was matched with the 127Kb sequence of Rfo gene（Gene Bank:AJ550021.2）with 99.83%coverage,indicating the recover gene in CLR6430 was Rfo.The results were further matched with radish genome at scaffold level,and found a highly homologous region in linkage group Rs＿scaf131 in R9.Furthermore,assembled contigs from resequencing results of CLR6430 were matched with a combined a reference genome combining B.napus and Rs＿scaf131,and a region that homologous to Rs＿scaf131 was found at a contig homologous to Chr A6 of B.napus after filtering the identity,direction and location,indicating the possibility of partial sequences of radish fragment that containing the restorer gene.4.A total of 100 pairs of specific primers were designed covering R9 linkage group based on scaffold sequences from radish genome.After primer screening and verification on segregating populations,32 specific markers of exogenous radish fragment were obtained,locating along the regions of 66.13～138.23c M in R9 linkage group,while no amplification product or the same as B.napus were found from the primers designed from other genomic regions,implying the length of exogenous radish fragment was about72.4c M comparable to R9 in radish.5.Thirty two specific markers of exogenous radish fragment were applied for PCR amplification and product sequencing for both CLR6430 and R2000,and the results showed differences between CLR6430 and R2000 in both size and sequence for amplified fragments.6.Through MAS back-crosses and mass selection,a novel Ogu-CMS restorer line of B.napus CLR095 were bred using a"double-low"inbred line as reciprocal parent.The cytoplasm was Ogu sterility type,with all fertile offsprings for selfing and all recovered for test-crossing.The traits of silique length and seed number per silique were significantly improved than CLR6430 and CLR650,as well as reduced glucosinolate content in seeds to 68.7μmol/g（meal）,but still significantly higher than its reciprocal parent which was 19.3μmol/g（meal）.The glucosinolate-linked marker was used to analyze CLR6430,CLR650 and CLR095 which amplified the same size PCR products in all three restorer lines,indicating the linkage between Rfo and the glucosinolate gene did not break through during backcrossing and selfing recombination.Ogu CMS restore resources in B.napus through cell fusion and embryo rescue were applied long-term patent protection,utilization of the Ogu CMS in B.napus has not been achieved due to the lack of restorer in China.In this research,the novelty and originality of CLR650 were clarified and the characteristics of restorer were significantly improved.The results provide a new restorer in Ogu CMS for China.