Comparative Study The Response Of PbBa8.1 And CRb Pyramided Brassica Napus Lines Against Genetically Diverse Clubroot Pathogen Plasmodiophora Brassicae Isolates

Brassica napus is one of the most important oilseed crops in the world as well as in China,and clubroot disease caused by Plasmodiophora brassicae is a serious threat causing yield reduction.Even though a number of clubroot resistant(CR)cultivars have been developed,but resistance breakdown has been reported in single resistance genes containing homozygous and heterozygous lines.Pathogen type 4 is a more prevailing isolate found in China with variance in virulence.A broad genetic diversity among pathogen isolates were recorded in Yunnan and Liaoning Provinces,and low genetic polymorphisms were observed in Hubei,Sichuan and Anhui provinces.On the other hand,Pb Ba8.1 and CRb are the two main resistant sources are present in China.Here,we carried out a series of experiments to knockout the comparative physical and genetical analyses through genome sequencing using molecular markers and RNA-Seq techniques of combining of these two resistant genes into one line,to analyze the genetic polymorphism among clubroot field isolates,and then to reveal the physical and genetical potentials in fortifying the resistance of rapeseed oil crops to Plasmodiophora brassicae.The main outcomes of these studies are summarized below.1-In this study,we analyzed the genetic variation among P.brassicae isolate 4 through simple sequence repeats(SSR)molecular marker technique.Genetic diversity of 9field isolates revealed a diverse divergence — the phylogenic analysis clustered P.brassicae into two distinct clades.Based on the results obtained from SSR markers,it is inferred that the clubroot disease pathogen P.brassicae isolates were genetically closely related.The environmental factors of the geographical regions of China and cultivation of resistant varieties were the major cause of genetic variations.These results are in consistence with greenhouse and field experiments.Further,our results suggest that extensive study with more pathogen types should be conducted with the combination of other systems can elaborate on the genetic variations among clubroot field populations.2-In this work,we combined two clubroot resistance(CR)genes(Pb Ba8.1 and CRb)through marker-assisted selection(MAS)and developed CR homozygous lines.The CR parental and pyramided lines were crossed with genetically male sterile(GMS)plants to generate heterozygous lines.In order to confirm their differences on resistance,the parental and generated lines were inoculated with 9 P.brassicae field isolates in the greenhouse and resistant test also evaluated in different naturally infested fields.The CR pyramiding lines exhibited high levels of resistance to the most isolates than line containing any single resistance gene;comparatively,the homozygous lines demonstrated a higher resistance than the heterozygous.However,the pathogen isolate Pb Xm displayed a drastic effect on all lines,including pyramiding lines.This study will certainly provide a novel molecular strategy for the breeding of the durable CR B.napus varieties in future breeding programs.3-Here,we conducted a comparative RNA-Seq analysis between 618 R and its parental lines(305R and 409R)to uncover the transcriptomic response of superior defense mechanisms of 618 R,and how these two different resistant genes coordinated to each other.We showed here,the number and expression of differentially expressed genes(DEGs)in 618 R were significantly higher than parental lines and Pb Ba8.1 shared more DEGs and played a dominant role.The DEGs common among the lines largely exhibited non-additive expression patterns and enrichment in resistance pathways.Among enriched pathways,plant-pathogen interaction,plant-hormone signaling transduction,and secondary metabolites were the key observation.However,the expressions of salicylic acid signaling pathway and reactive oxygen species(ROS)appear to be crucial regulatory components.Our findings will provide a comprehensive transcriptome insight to understand the interaction of pyramiding of resistance genes in single line and to facilitate in breeding of improved resistance in B.napus.Conclusively,Plasmodiophora brassicae field isolates had a significant adverse effect on B.napus.However,pyramiding of resistant genes strongly increased the resistance by activating the ROS activity and SA pathway.These studies will provide a novel molecular and genetic strategy to understand the genetic mechanism and facilitate for further understanding of these mechanisms.