Creation And Genetic Dissection Of Ogura CMS Fertility-restored Materials In Brassica Oleracea L.

Male sterile breeding system is a maior way in the production of commercial hybrids of Brassica oleracea vegetable.Ogura cytoplasmic male sterility（Ogura CMS）,with stable sterility and easy to transfer,is the most widely used cytoplasmic sterility in B.oleracea vegetable.However,all the offsprings produced by Ogura CMS lines are male sterile.So the excellent germplasm resources with Ogura cytoplasmic type cannot be transferred and utilized.Development of B.oleracea Ogura CMS restorer lines is of great importance for the innovation and utilization of Ogura germplasm resources.Previously,the Ogura CMS fertility-restored interspecific hybrids and BC₁ progenies between rapeseed and Chinese kale have been successfully produced.However,these progenies,which still contained a large proportion of rapeseed genomic components,showed poor fertility and a low seed setting rate under natural pollination,and can not be used in breeding.In this study,the triploid interspecific hybrids between Chinese kale and rapeseed and its doubled hexaploid individuals were used as male parent to backcross with Ogura CMS Chinese kale,respectively.With marker-assisted selection（MAS）,morphology,fertility,cytological observations and genetic component analysis,the Ogura CMS fertility-restored materials of Chinese kale type（2n=18）were developed.In addition,we tried to transfer the Rfo gene to some Ogura CMS B.oleracea germplasm resources with excellent agronomic traits using this fertility-restored materials.The main results are as follows:1.The BC₂ and BC₃ fertility-restored polyploid individuals,with stable fertility performance（pollen viability>85%）and a closer morphological characteristics and genetic background to the parent Chinese kale,were obtained.And the seed setting of these individuals was significantly improved and could produce seeds under natural pollination（seed setting rate>0.2 seeds per pod）.The Ogura CMS Chinese kale,used as backcrossed female parent,was backcrossed with hexaploid Chinese kale-rapaseed interspecific individual（AACCCC）and the BC₂,BC₃ and BC₄ were generated.MAS,evaluation of agronomic traits,fertility identification and genetic background analysis were performed.One BC₂ Rfo-positive individual（code:15Q23）,with a higher pollen viability（87.07）,closer morphological characteristics and genetic background（genetic similarity coefficient=0.76）with the parent Chinese kale,was identified.And it could produce seeds under natural pollination（305seeds/4295 pods）.The individual 15Q23 was further backcrossed with Chinese kale to produce the BC₃ and BC₄ generations.The results indicated the Rfo gene can be stably transferred.Two individuals,16Q1-4 and 16Q1-10,morphologically similar to parent Chinese kale 15Y102,with closer genetic background to Chinese kale compared with the BC₂pollen donor 15Q23,and significantly improved seed-setting（seed setting rate>0.2 seeds per pod）.have been successfully created.Ploidy identification results showed that most backcross progenies between Chinese kale and hexaploid individual（AACCCC）were polyploid individuals.Moreover,individuals with ploidy o that of parent Chinese kale were identified in the BC₄ generation.Cytological observations showed that the meiosis behavior of all backcross polyploid progenies was irregular.2.The Ogura CMS fertility-restored materials,with 18 chromosomes,normal seed setting,stable fertility and closer genetic background to the parent Chinese kale were successfully developed.In order to accelerate the transfer process of Ogura CMS fertility-restored materials in B.oleracea,the successive backcross between triploid Chinese kale-rapeseed interspecific hybrids and Ogura CMS Chinese kale were peRformed.Using embryo rescue,MAS and ploidy identification,one BC₁ Rfo-positive individual（code:15CMSF-Y1）,with morphology near to the parent Chinese kale and pollen viability between15%and 65%,was obtained.The ploidy of 15CMSF-Y1 was nearly the mean of Chinese kale and interspecific triploid,and the chromosome number of 15CMSF-Y1 was 22.A total of 167BC₂ progenies was produced from the continue backcross with Chinese kale using15CMSF-Y1 as male parent.Two Rfo-positive individuals（16Q2-11 and 16Q2-58）were identified.The individual 16Q2-11,with 18 chromosomes,was morphologically identical to parent Chinese kale.And it showed stable fertility performance,which pollen viability between 60%and 90%,and could produce seeds normally under natural pollination（4¹0seeds per pod）.Cytological observations showed that the meiosis behavior of 16Q2-11 was still irregular.The further backcross was performed and 9 BC₃ Rfo-positive individuals were identified,all of which have 18 chromosomes.The 9 BC₃ Rfo-positive individuals showed stable fertility performance（pollen viability above 85%）and the pairing and segregating of chromosomes have become normal in some pollen mother cell of BC₃ Rfo-positive individuals.3.The restored gene Rfo and clubroot resistant loci（CRb）were integrated in Ogura CMS Brassica materials.The fertility of Ogura CMS materials,which contained clubroot resistant loci（CRb）,was successfully restored in B.oleracea for the first time The Ogura CMS germplasm resources,which contained clubroot-resistant loci（CRb）,were crossed with Chinese kale Ogura CMS fertility-restored individual,16Q2-11.A total of eleven fertility-restored F₁ individuals,with pollen viability above 85%,were obtained,which showed intermediate morphological characteristics between Chinese kale and cabbage.Among them,four individuals harbored CRb,and showed clubroot resistance growed in Plasmodiophora brassicae hypnospore soil.4.Genetic component of BC₁ and BC₂ progenies,derived from triploid and hexaploid strategy,was analyzed by high-throughput sequencing technology and molecular marker.The results showed that the BC offsprings of hexaploid contained higher proportion of B.napus genomic background and more difficult to eliminate,compared with BC offsprings of triploid.Two Rfo-positive individuals,16Q2-11 and 16Q2-58,have almost recovered to the C genome genetic constitution.Most chromosome segments of them were Chinese kale homozygous type（C^o/C^o）,whereas the proportion of heterozygous type segment（C^o/Cⁿ）in 16Q2-11 and16Q2-58 was 0.36 and 0.36,respectively;most Aⁿ genomes segment have been lost in16Q2-11 and 16Q2-58.