Genetic Improvement Of Clubroot Resistance Of Huayouza 62 Restorer Line And Analysis Of Molecular Mechanism Of MiRNA Response

Clubroot disease caused by the soil-borne pathogen Plasmodiophora brassicae War(P.brassicae),is one of the world's oldest and most destructive disease of Brassica species and cruciferous crops.The rapeseed clubroot disease incidence in China is about 0.67 million hectares,accounting for 10%of the canola production area.In addition,with the continuous improvement of mechanization,there is a trend of large area outbreak of rape clubroot in China,especially at present,there is an extreme shortage of rape varieties resistant to clubroot,which considered a serious threat for the safety of Brassica napus(B.napus)industry.Based on this,a new clubroot-resistant restorer line Bing409R(409R for short)was established in this study,and a new clubroot-resistant hybrid variety Huayouza 62R was formulated on this basis,which provided a strong guarantee for the resistance to clubroot disease,for the sustainable and healthy development of canola industry in China.At the same time,this work systematically investigated the roles of miRNAs and their targets during P.brassicae infection in two B.napus near-isogenic lines(NILs)of clubroot-resistant line(409R)and clubroot-susceptible line(409S)by means of small RNA sequencing and degradome sequencing,combined with published transcriptome data.The main results were described as follows:1.Genetic improvement of clubroot resistance of B.napus restorers Bing409We used CR Shinki,a Chinese cabbage material containing CRb clubroot disease resistance locus,as the donor parent,and Pol.CMS restorer line Bing409,the parent of Brassica napus national approved varieties Huayouza 62,as the recipient parent,and the CRb resistance locus was introduced into Bing409 by breeding programs such as crossing and backcrossing.The clubroot disease resistance identification of F₁ and BC₁ indicated that CRb disease resistance locus appeared as a dominant single-gene inheritance in B.napus background.Five pairs of foreground markers and 124 pairs of background markers uniform in the A genome of B.napus were used to screen the foreground and background of each generation of BC₁F₁ to BC₃F₁.In the BC₃F₂generation,a new restorer line Bing409R with a genetic background close to Bing409containing CRb resistance locus was obtained.Bing409R were showed immune-resistance to physiological races of P.brassicae in Sichuan,Hubei,and Anhui provinces in China.The quality test of the improved resistant lines indicated that the oil content of all tested lines was similar to that of un improved parent 409S,and the contents of erucic acid and glucosinolate were in line with the production standard of“double low rapeseed”in China.2.Breeding of a novel clubroot disease-resistant B.napus variety Huayouza 62RIn the summer of 2016,Huayouza 62 male sterile line firstly crossed with Bing409R,and then a novel clubroot disease-resistant B.napus variety Huayouza62R was successfully developed in China.The results of clubroot disease resistance identification indicated that Huayouza 62R showed total resistance to P.brassicae from Zhijiang,Hubei Province and Huangshan,Anhui Province,and its field growth strength was significantly better than that of the local control variety.The yield and the main agronomic characters of Huayouza 62R were investigated in non-clubrootpathic areas.Compared with the control cultivar Huayouza12,the genetic improvement of clubroot resistance had no adverse effects on the main yield traits of Huayouza 62R,such as plant height,effective branches number,silique number per plant,seeds per horn,thousand seed weight and yield per plant.This research will provide valuable resources for the breeding of rapeseed in China,and provide an important guarantee for our country to resist the threat of rapeseed clubroot disease.3.Identification of miRNAs during P.brassicae infection in two B.napus NILs of clubroot-resistant line and clubroot-susceptible lineTwelve small RNA libraries generated from 20 days post P.brassicae inoculated(DPI)roots(Int409R,Int409S)and mock roots(Mock409R,Mock409S)were sequenced and 2.03 million small RNA sequences on average from each library were identified.The length of these s RNA ranged from 18-to 30-nt,mainly 21-nt and 24-nt.We totally identified 48 known miRNAs and 72 novel miRNAs,among which 5 miRNAs were specifically conserved in brassica species.The overall expression level of these miRNAs indicated that about 20%miRNAs in B.napus genome had a high expression level(reads>200).18 miRNAs were significantly differentially expressed in the resistant materials in response to P.brassicae infection,while only 1 miRNA was differentially expressed in the susceptible materials.The expression of 4 miRNAs validated by q RT-PCR was consistent with the up-or down-regulation results of high-throughput sequencing.These differentially expressed miRNAs may be directly or indirectly involved in the resistance process to P.brassicae.4.Analysis of regulation mechanism of miRNA and its target in response to P.brassicae infection in clubroot-resistant line and clubroot-susceptible lineFour degradome libraries generated from post P.brassicae inoculated roots and mock roots were sequenced.Analysis of the degradome libraries identified 938 target transcripts of 84 miRNAs,and functional annotation showed that most of the targets are transcription factors,enzymes and proteins,which involved in multiple biological processes.Targets of 409R and 409S materials after P.brassicae infection were subsequently grouped into 12 and 18 significant KEGG pathway,respectively,which most significantly enriched in plant hormone signal transduction pathway,and 8 different degradation pathways were identified.We also constructed miRNA-target regulatory network modules.Finally,the expression profiles of both P.brassicae responsive miRNAs and their target transcripts were integrated through combining with transcriptome data.We totally identified 8 pairs of antagonistic miRNA-target in two B.napus NILs in response to P.brassicae infection,including miR395d-APS4,novel?147-NAC076,and novel?147-PNSL2,etc.,which were involved in root development,hypersensitive(HR)cell death and chloroplast metabolic synthesis related-pathways.Our data reveals that P.brassicae infection result in fundamental changes on miRNA pool and target transcripts between the clubroot-resistant versus-susceptible lines.This research will shed light on possible molecular mechanisms of resistance response to P.brassicae infection in B.napus.