Applicaiont Of Method Of Anchor Mapping Of Alien Chromosome Fragment In Intergeneric Hybrid Progenies

Oilseed rape（Brassica napus L.）is an important oilseed crop extensively grown around the world,which also is a relatively new species with limited history of evolution,domestication and cultivation.In order to broaden gene pools and create new germplasm suitable for modern rapeseed breeding,valuable chromosome fragments or genes from related species have been transferred to Brassica napus using wide hybridization technology and a series of elite germplasm containing alien chromosome fragments have been obtained.However,identifying these fragments from individual plants generated by distant hybridization and developing molecular markers for further marker-assisted breeding poses a challenge.This study proposed a method called anchor mapping of alien chromosome fragment（AMAC）for identifying events of exotic introgressions resulting from distant hybridization.Radish（Raphanus sativus L.）is one of the important closely related species for germplasm improvement and genetic base expansion of Brassica napus.A number of elite chromosome fragments or genes have been transferred to Brassica napus genome through distant hybridization between Raphanus sativusand Brassica napus.In this study,the Ogura-CMS restore line 16 C which donor parent is radish,and red-purple flower line 2021 RP which donor parent is unknown,were used as materials for AMAC analysis.The results proved the effectiveness and feasibility of AMAC method on identifying events of exotic introgressions.The main results of this study are as follows:1.The AMAC analysis process involves four steps（1）developing genome-wide molecular markers for donor species of distant hybridization and predictive analysis of specific-single-locus markers（hereafter referred as SSL markers）;（2）constructing of SSL markers map after verification of PCR and electrophoresis experiments;（3）screening SSL markers for alien chromosome fragments in individual plant and obtaining anchor-SSL marker sites;（4）further screening and validating new SSL markers near the anchor-SSL marker and mapping for the alien chromosome fragments.2.Based on AMAC method,radish Rsa10 was used to as reference genome for molecular markers development.A total of 20,3625 markers,including 123,861 IP markers and 76,764 SSR markers,were developed.Among them,87,911 markers,including 61,757 IP markers and 26,154 SSR makers,were predictively amplifying single locus in radish,and 44,176 markers,including 23,816 IP markers and 20,360 SSR makers,were predictively amplifying single locus only in radish using e PCR software.3.24 varieties of Raphanus sativus,18 varieties of Brassica napus,2 varieties of Brassica rapa ssp.chinensis,2 varieties of Brassica oleracea and 2 varieties of Brassica juncea were used as materials for PCR and agarose electrophoresis experiment.215 radish SSL markers evenly distributed on 9 radish chromosomes were verified and employed to construct a radish SSL markers map.4.The 215 radish SSL markers were used to identify alien chromosome segment in Ogura-CMS restore line 16 C,Ogura-CMS sterile line 81 A and their hybrid Yunyouza 15.One SSL marker,Rsa10025823_intron₃,only had PCR product in16 C and Yunyouza15,recorded as anchor-SSL marker.Then four new SSL markers near Rsa10025823_intron₃ were obtained and only had PCR product in 16 C and Yunyouza15.Finally,we confirmed the alien chromosome fragment of 16 C were originated from radish R9:8.4807-11.7798 Mb by further integrating previous study.These results domonstrate the effectiveness and feasibility of the AMAC method in identifying events of exotic introgressions.5.The 215 radish SSL markers were also employed to detect alien chromosome segments in red-purple petal line 2021 RP,whose donor parent was unknown.Three radish chromosome fragments were identified in 2021 RP,originated from R5,R7 and R9 chromosome,respectively,and subsequently,marker maps were constructed for these alien chromosome fragments.It is reasonable to assume that the unknown donor parent of 2021 RP is a radish.6.We compared the transcriptome data of buds from 2021 RP and normal yellow flower line,and analyzed differentially expressed genes（DEGs）base on ZS11 and Rsa10 reference genome,respectively.4588 and 2966 genes up-regulated in red-purple buds were identified,respectively.By integrating AMAC results,we identified three radish genes（Rsa10015104,Rsa10008592,and Rsa10033481）located in the alien R7 and R9 chromosome fragments,were up-regulated in red-purple buds of 2021 RP.These genes are potential candidate genes controlling trait of red-purple petal,and further experiments are needed to validate their function in future.7.In this study,we also constructed a marker map of radish using 735 SSR markers,which could be used to develop fingerprint and facilitate marker-assisted breeding in radish.The expected sequences amplified by these 735 SSR markers contain SSR motif and derive from genes.In summary,this study proposed a novel method,Anchor Mapping of Alien Chromosome fragment（AMAC）,for detecting events of exotic introgressions and domenstrated its effectiveness and accuracy in intergeneric hybrids lines between Brassica napus and Raphanus sativus.By combining the results of AMAC and transcriptome analysis,we identified three candidate genes from radish genome that controlling the trait of red-purple petal in 2021 RP.Additionally,we developed a large number of IP and SSR marker from radish genome and constructed two marker maps of radish,which would facilitate marker-assisted breeding in Brassica napus and Raphanus sativus in the future.