Development And Cytological Analysis Of The Complete Set Of Nullisomics And Monosomics Lines Of C Chromosome In B. Napus

Brassica napus(A_nA_nC_nC_n,2n=38)is an alloteraploid species,originating about 7,500 years ago by interspecific hybridization between B.rapa(A_nA_n,2n=20)and B.oleracea(C_nC_n,2n=18)under the natural conditions and the whole genome duplication,which also is the most important oil crops in the world.The A and C subgenomes originated from the same ancestor,and there is a very high homology between the two genomes.In addition,the genome become more complicated because of chromosome exchange were occurred during the process of diploidization,which bring great challenges to analysis of the genome and the location of target genes in B.napus.The nullisomics materials effectively reduce the complexity of B.napus genome,which can be used to map the target traits.However,there are few reports of nullisomics materials in B.napus.In this study,an aneuploidy population of more than 6000 plants was constructed by hybridization,backcrossing and multiple self-crossing between B.napus“Oro”(recurrent parent)and restituted B.rapa“Oro”(RBR Oro)for many years.The seven nullisomics(2n=36)and two deletion lines of C chromosome were screened by molecular marker,cytological detection,fliorescence in situ hybridization.The five monosomics(C2,C5,C7,C8 and C9)were obtained by hybridization between the parental B.napus and nullisomics.Subsequently,the phenotype and cytology of these nullisomics and monosomics were systematically analyzed,and the important characters of B.napus were analyzed based on these genetic materials.The main results of this study as follows:(1)In this study,a large number of aneuploidy population was constructed by hybridization,backcrossing and multiple self-crossing between B.napus“Oro”(recurrent parent)and restituted B.rapa“Oro”(RBR Oro).The C genome nullisomic materials were obtained through the C genome chromosome specific molecular marker,a total of 93 plants,including nine type of NC1-NC9.The number of NC2 and NC5 plants was the most,with 18 and 17 plants respectively,while the number of other types was 5-13 plants.The count of chromosome results that there were 7types(NC1,NC2,NC5,NC6,NC7,NC8 and NC9)of the candidate materials with the expected chromosome number(2n=36),a total of 46plants.The 9 C-deletion lines in B.napus were obtained,the number of chromosomes in C3,C4 deldetion lines was between 24 and 29,28 and 32respectively.(2)Fluorescence in situ hybridization analysis of the candidate materials results indicate that there were 38 chromosomes with DAPI and18 red hybridization signals in parent B.napus“Oro”,and there were 36chromosomes with DAPI and 16 red hybridization signals in 5 nullisomics candidate materials(NC1,NC5,NC6,NC7 and NC9),was the consistent with the count of somatic chromosome.The above materials were confirmed to be nullisomics of C chromosome set of B.napus again.(3)The 7 nullisomic lines had 18 bivalents at diakinesis in the pollen mother cells,but monovalent and trivalent were appeared with low frequency,indicating that may exist the genes of inhibit and affect homologous pairing.Some lagging chromosomes,chromosome bridge unequal separation of chromosomes were observed at meiosis anaphase I(MA I)and meiosis anaphase II(MA II)in the pollen mother.Compared with their parental B.napus,the pollen fertility of the NC5,NC7 and NC9was significantly different,which may be caused by the production of sterile gametes during the relatively disordered meiosis process,the unequal segregation of chromosomes.(4)Phenotypic observation of 9 types of aneuploid materials showed that they all showed different phenotypic characteristics from their parents“Oro”.The leaves and stems of NC1 plants covered with white burrs,and the white burrs were more obvious in the back stems particularly.NC2showed significantly early flowering time of two months earlier than its parental B.napus,smaller plant size.The leaves of C3 deficient plants were folded and had multiple lateral branches.The C4 deletion plants lack the main stem,multiple base branching.The branches of NC5 are small.The flowering time of NC6 was delayed about one month.NC7 showed significantly early flowering time.NC8 were greatly dwarfed and full of muti-branched.The leaves’types of NC9 were similar to cabbage.(5)The five monosomics materials(C2,C5,C7,C8 and C9)were obtained by hybridization between the nullisomics(NC2,NC5,NC7,NC8and NC9)and B.napus.The C2 monosomic plants exhibited similar morphology to NC2,which showed significantly early flowering time of two months earlier than its parental B.napus,smaller plant size.The C5monosomic showed significantly early flowering time.The height of C7,C8 and C9 monosomics were higher than“Oro”,and the pollen fertility also greater than their parental.