Extraction And Interaction Of The Constituent Subgenomes Of The Natural Allotetraploid Rapeseed(Brassica Napus L.)

Allopolyploid derived from the interspecific hybridizations between related diploid ancestors is a vital way for speciation in nature,and they outcompete their progenitors to a wide range of climatic conditions.To unravel the origin and structure of the component genomes in these allopolyploids has been the goal of the extensive genetic researches over the long period.The extraction of the constituent subgenome(s)from a natural allopolyploid species of long history and then restitution of the progenitor(s)provides the unique opportunity to study the genome evolution and interplay.The allotetraploid oilseed rape(Brassica napus L.,AACC)has been taken as a model to decipher the allopolyploidization.In the present study,A-subgenome from the allotetraploid oilseed rape was extracted through inducing the preferential elimination of C-subgenome chromosomes in intertribal crosses and the progenitor B.rapa was restituted(RBR).Meanwhile,to dissect the C sub-genome in B.napus,one RBR(RBR Oro)obtained previously in our lab as pollen donor was crossed and backcrossed with parental B.napus(Oro)to generate a battery of aneuploids of A sub-genome added single C sub-genome chromosome.Finally,one complete set of monosomic alien additional lines(MAALs)of A sub-genome added one of nine C sub-genome chromosome was determined by PCR amplification of C chromosome specific gene makers and FISH analysis.Establishment of RBR and MAALs provide a unique avenue to explore the component sub-genome evolution and interplay in B.napus.The main results of this study are as follows:1.Restitution of RBR ZS11 induced by intertribal crossB.napus cultivar “Zhongshuang 11”,the first genotype which was selected for genome sequencing in China,was extensively pollinated by the crucifer Crambe abyssinica(2n=90)and four F1 hybrids were identified to have 2n=29.FISH with C genome specific probe and PCR amplification of C chromosome specific gene makers manifested that one copy of C sub-genome was eliminated and the complete A sub-genome was inherited in F1(2n=29)hybrid,moreover,no chromosome chromosomal fragments from of C.abyssinica was detected by GISH in these plants.Then,a plant with 2n=22 was obtained from the backcross between hybrid and C.abyssinica.Among the assisted self-pollination progenies from one BC1 plant with 2n=22,some plants with 2n=20 were harvested.These plants showed same chromosomes pairing with nature B.rapa as 10 bivalents and 10:10 segregation in meiotic cells,and FISH analysis furtherly confirmed no introgression of C chromosomal fragments,indicating extraction of A sub-genome(RBR ZS11)in B.napus “Zhuangshuang 11”.Comparing with those natural B.rapa,RBR ZS11 showed some specific features,such as decreased vigor,leaves with dense hairs,more susceptible to diseases,barrier of selfing seed-sets,small flowers with ridgy petals,and in particular,procumbent leaves in cold winter etc.By AFLP analysis with randomly selected 57 pairs of primers,the four hybrids and RBR ZS11 showed some deleted and novel bands for B.napus-specific fragments,as well as a bit of specific fragments for C.abyssinica.Phylogenetic analysis of RBR ZS11 and other 17 accessions by AFLP with randomly selected 32 pairs of primers demonstrated RBR ZS11 clustered with B.rapa “Baiyou 1 hao”.2.Dissection of C sub-genome in B.napusTo dissect the C-subgenome of natural allotetraploid B.napus,the parental B.napus cv.Oro was crossed and backcrossed with RBR Oro as pollen donor.Out of 646 BC1 and BC2 plants(2n=20-29)with chromosome numbers determined in the young ovary cells by cytological method,129(20.0%)had 2n=21 for putative monosomic alien addition lines(MAALs).Based on the genome sequences of B.napus genotype “Darmor”,49 pairs of chromosome-specific genes makers of C-subgenome were designed and screened.A complete set of MAALs of RBR Oro added one of nine C sub-genome chromosome was determined by PCR amplification of these specific gene makers.And then,BAC-FISH with several repeated sequences as probes were performed to confirm the reliablity of the gene markers in MAALs.MAALs expressed some peculiar features likely associated with each additional C-subgenome chromosome in comparison with parental RBR Oro,such as leaves and stems covered with much waxen powder in AAC2.These MAALs exhibited obviously different frequencies in homoeologous pairing and transmission of additional C-subgenome chromosomes,which were associated with the distinct degrees of their relatedness,and even with the possible genetic regulation for meiotic pairing evolved in B.napus.AAC1 and AAC2 showed the two highest percentages of PMCs with the allosyndetic trivalent and transmission rate of additional chromosome through male and female gametes,but AAC9 showed lowest allosyndetic trivalent and transmission rate,likely owing to the regulation of Pr Bn located on C9.Finally,22 large scaffolds undetermined for sequence assembly of B.napus were anchored to specific C-subgenome chromosomes using MAALs by PCR amplification of specific gene markers,indicating a large role of MAALs for improvement of B.napus genome information.