Mining Of Genes Associated With Resistances To Triticeae Fusarium Head Blight And Flax Pasmo

The thesis mainly included two parts.First part was the results of research carried out during the post-graduated studying and training in China.In previous study,the fast neutron induced susceptible mutant of high Fusarium head blight（FHB）resistance wheat landrace Wangshuibai was obtained and characterized in our lab.According to the results from previous transcriptome analysis of Wangshuibai and its mutant pre-and post-inoculation of Fusarium graminearum（Fg）,resistance-related genes were identified.Meanwhile,small RNA and degradome sequencing of Wangshuibai and its mutant were performed to unravel the epigenetic regulatory mechanism of the FHB resistance in Wangshuibai.The second work was supported by China scholarship council（CSC）,and all the results were obtained in the lab of Dr.Frank M You in Agriculture and Agri-Food Canada（AAFC）.Genome-wide association studies（GWAS）were performed using the multi-location and five years data of pasmo resistance assessment of flax core collection,.Pasmo resitance quantitative trait loci（QTL）were identified by optimization of GWAS method.Part 1.Identification of miRNAs and genes related to FHB resistance in wheat landrace Wangshuibai.Fusarium head blight（FHB）,mainly caused by Fusarium graminearum（Fg）is one of the most devastating diseases threatening wheat production.Wangshuibai is a wheat landrace from Jiangsu province with high resistance to FHB.It is important for disease resistance breeding to mine and identify FHB resistance related genes and unravel the genetic and epigenetic regulatory mechanism of FHB resistance in Wangshuibai.Previous transriptome sequencing study using Wangshuibai and its FHB susceptible mutant in our lab showed that multiple genes and pathways in response to Fg infection.Based on this,the function of previous identified genes was further validated.Furthermore,using spikes of pre-inoculated and 24 h post Fg inoculation,small RNA and degradome sequencing libraries of Wangshuibai（with high FHB resistance carrying Fhb1）and its mutant NAUH117（with increased FHB susceptibility due to the fragment deletion in the Fhb1 region）were constructed and sequenced to identify Fg-responsive miRNAs and their targets and reveal the molecular mechanisms of post-transcriptional regulatory roles of miRNAs in wheat and Fg interaction.Based on the work of reported TaUGT3 gene in Wangshuibai,a FHB resistance related gene HvUGT-10W1 encoding UDP-glucosyltransferase was cloned from barely.Functional analyses hold the promise to dispher the molecular mechanism of it on FHB resistance.The main results of this study are as following:1.Identification of wheat miRNAs in reponse to Fg infection（1）Identification and analyses of miRNAs pre-and post-Fg inoculation in Wangshuibai and NAUH117Genome sequence of wheat cultivar Chinese spring（released by IWGSC,version RefSeq v1.0）was used as the reference sequence for miRNA annotation.Four small RNA sequencing libraries including Fg pre-and post-inoculation of Wangshuibai and NAUH117 were constructed and sequenced.A total of 166 miRNAs were identified from four small RNA sequencing libraries.The number of miRNAs identified from them was 114（w0,Wangshuibai pre-inoculated）,112（w24,Wangshuibai 24 h post-inoculated,hpi）,109（h0,NAH117 pre-inoculated）and 114（h24,NAUH117 24 hpi）,respectively.Of 166 miRNAs,the number of known miRNAs and novel miRNAs was 67 and 99.All miRNAs were originated from 460 precursor miRNAs（MIRNAs）with 59-227 nt in length.Post-Fg inoculation,the expression of 78 miRNAs（including 25 known and 53 novel miRNAs）were significantly different in the two genotypes.Among them,27 and 29 miRNA were specically expressed in Wangshuibai and NAUH117,respectively.In the FHB resistant Wangshuibai,pre-and post-Fg inoculation,the expression of 82 miRNAs（including 29 known and 53 novel miRNAs）were significantly different.Further comparisons of the 82 miRNAs differentially expressed pre-and post-inoculation and the 78 miRNAs differentially expressed in the two genotypes post-inoculation showed that,54 miRNAs were commonly present.These are considered as key Fg-responsive miRNAs in Wangshuibai.（2）Prediction of miRNA target genes and analysis of the regulatory relationshipTo identify the miRNA targets,degradome sequencing was performed using the above RNA samples for small RNA sequencing library construction.A total of 2987 genes were predicted as the target genes for the above 166 miRNAs with high confidence.In average,each miRNA regulated 18 target genes.GO enrichment and classification of target genes showed that target genes were involved in hormone pathways,signal transduction and stress responses.These inferred that wheat miRNAs in response to Fg infection via regulating the pathways above.Based on the results obtained from degradome sequencing,seven target genes were selected to verify the regulatory relationship between miRNA and target by qRT-PCR assay.Five target genes showed negatively correlated expression pattern with corresponding miRNAs in Wangshuibai pre-and post-inoculation of Fg.The five target genes differentially expressed in Wangshuibai post-Fg inoculation were involved in five defense-related pathways,such as auxin,ROS（reactive oxygen species）,cell wall degradation,PTI（PAMP-triggered immunity）and ETI（Effector-triggered inmunity）.Briefly,the five miRNA-target modules are novel₅4₂ and GH3,tae-miR9658-3p₂1 and NADP-dependent oxidoreductase,miR528a and PG（Polygalacturonase）,novel₁6₂1 and CERK1,ata-miR9863a-3p₂2 and RPM1.According to the results of transcriptome obtained in our previous study,NADP-dependent oxidoreductase was induced post-Fg inoculation which may positively regulate the FHB resistance in wheat.（3）Analysis of miR528a and PG regulatory module and functional analysis of miR528aIn this study,miR528a was induced by Fg infection may enhance FHB resistance by targeting PG and further resulting in the suppression of cell wall degradation.Expression pattern of miR528a in Wangshuibai was investigated using Northern blot and qRT-PCR assay.Compared to pre-inoculated Wangshuibai,miR528a was significntly induced postFg inoculation.Correspondingly,decreased expression of PG was observed by qRT-PCR.Using transient expression in tobacco,qRT-PCR and western blot assay,expression of PG was investigated to validate the interactions between miRNA and target in vivo.It was found that expression of PG on transcriptional and translational level was reduced when it was coexpressed with the miR528a.These indicated the regulatory relationship between miR528a and PG was on transcriptional and translational level.Cis-regulatory elements analysis in MIR528 promoter region showed that defense and stress responsive elements and hormone responsive elements were generally present in MIR528 promoter region.It indicated the key role of miR528 involved in stress response and hormone signaling in plants.In order to better understand the function of miR528a in wheat,miR528a was transformed into susceptible cultivar Alondra’s by bombardment.One positive T0 plant was identified by PCR and qRT-PCR.FHB resistance evaluation showed the positive T0 plant could increase the resistance to FHB in Alondra’s,which preliminarily confirmed the role of miR528a participating in defense response to FHB.2.Cloning and functional analysis of HvUGT-10W1Using trancriptome sequencing and transgenic technology,the results showed that TaUGT3 in Wangshuibai was induced post-Fg inoculation,the FHB resistance of susceptible cultivar Alondra’s was enhanced by the over-expression of TaUGT3 from Wangshuibai.Phylogenetic analysis showed that HvUGT14077 in barley was relatively close to Ta UGT3 in Wangshuibai,and it may contribute to FHB resistance in barley.In this study,seven alleles of barley Hv UGT14077（GenBank No.GU 170356.1）were cloned using RT-PCR.Among them,HvUGT-10W1,which was isolated from a FHB resistant barley variety 10W1,was significantly up-regulated in young spikes post-Fg inoculation.HvUGT-10W1::GFP was subcellularly located in the plasma membrane and cytoplasm of the wheat protoplasts.In vitro antifungal activity assay showed that the HvUGT-10W1 protein exerted obvious inhibition against the growth of Fg.The silencing of the HvUGT-10W1 by virus-induced gene silencing（VIGS）resulted in compromised FHB resistance of 10W1,which was shown by the increased infected colonies on the leaves.These indicated that the barley HvUGT-10W1 may also contribute to FHB resistance in barley and provided a potential candidate gene to develop transgenic barley with enhanced FHB resistance.Part 2.Genome-wide association studies for pasmo resistance in flaxPasmo,caused by Septoria linicola,is one of destructive diseases threatening flax production.Despite of chemical control approaches applied on flax,yield loss increased up to 75%when susceptible plants were infected at flowering stage.Genetic improvement on pasmo resistance is one of the most effective and ecofriendly strategies to prevent yield losses caused by the disease.Resistance to pasmo in flax is controlled by polygenes which is quantitatively inherited.In this study,genotypic data of 370 accessions from 39 countries and representing 11 geographical regions in flax core collection were obtained using genotyping-by-sequencing（GBS）technology.Phenotypic data from field assessments of pasmo resistance was collected in five years.Genome-wide association study（GWAS）analyses were performed using ten statistical models.Quantitative trait loci（QTL）underlying pasmo resistance were identified and used for candidate gene prediction.The main results of this study are as following:（1）Analyses of the phenotypic and genotypic data of flax core collection on pasmo resistanceEvaluation of pasmo severity was performed in the field from 2012 to 2016 in Morden,MB,Canada.Analyses of pasmo severity datasets showed different distribution among the evaluation stages,a peak of pasmo severity was observed at the last stage in each dataset.It also indicated a large variation on pasmo severity of the 370 accessions.Significant correlations and gene and environment interaction were found among pasmo severity datasets（r=0.44-0.70）.24.23%of phenotypic variation explained by gene and environment interaction were obtained by ANOVA.Thus,it is necessary to carry out the GWAS using six phenotypic datasets,which are five individual years and average across years of pasmo severity.GBS has identified 258873 single nucleotide polymorphisms（SNPs）distributed on all 15 flax chromosomes for GWAS hereafter.（2）Identification of QTL associated with pasmo resistance in flaxMarker-trait associations were identified using ten different statistical models,which contained three single-locus methods（GLM,MLM and GEMMA）and seven multi-locus methods（mrMLM,FASTmrEMMA,ISIS EM-BLASSO,pLARmEB,pKWmEB,FASTmrMLM and FarmCPU）.A total of 692 unique QTNs associated with 500 putative QTL were detected from six phenotypic PR datasets（five individual years and average across years）.GLM identified the largest number of QTL（209）or QTNs（346）of all methods and these had relatively large effects with an average R2 of 5.57%,ranging from 0.48 to 15.02%.QTL differed depending on the statistical methods.QTL detected by at least two methods accounted for a small proportion of overall QTL.The mrMLM methods detected more common QTL than the other methods.Multi-locus methods detected more small-effect QTL than the single-locus methods.Generally,QTL with large effects were identified by both GLM and mrMLM methods（mrMLM,FASTmrEMMA,ISIS EM-BLASSO,pLARmEB,pKWmEB and FASTmrMLM）.QTL also differed across individual year datasets,but most（240）were identified from the mean dataset which comprised two to four times more QTL than the individual year datasets.This is indicative of strong gene × environment interactions and reinforces the representability of the mean dataset for QTL identification.（3）Validation of QTL associated with pasmo resistance in flaxOf the 500 QTL,134 were detected in all six PS datasets and explained 27.4-60.9%of the total variation;they are considered stable.By construction of forward stepwise multiple regression models,67 out of the 134 stable QTL were detected;they explained 31.5-64.2%of the total variation in individual datasets.The 67 QTL subset represents the non-redundant and large effect QTL as each of them could explain 3.3-23.4%of the total variation.Based on the information of 67 QTL,statistics of QTL with positive-effect alleles（PQTL）showed the tally of the PQTL in the 370 accessions ranged from 3 to 60 per accession.Number of PQTL（NPQTL）were compared between two extreme subsets of 23 resistant（R）and 23 susceptible（S）,respectively.Notably,all accessions of the R group belong to the fiber type and those of the S group were oilseed type.The R group,with an average pasmo severity rating of 3.2,contained an average of 42.5 PQTL per accession ranging from 14 to 60;the S group,with an average pasmo severity rating of 8.3,averaged only 9.4 PQTL per accession.Significant negative correlations between NPQTL and pasmo severity were observed in all six datasets（r=-0.45 to-0.74）.（4）Evaluation of flax core collection by pasmo resistance associated QTLA positive correlation between pasmo severity and morphotype（r=0.49）was observed,showing that fiber accessions were more resistant to pasmo.NPQTL were negatively correlated with morphotypes（r=-0.65）.The chi-square test results indicated that most PQTL alleles were significantly associated with fiber type accessions.Bidimensional cluster analyses were conducted based on the 67 core QTL of the flax collection.The 370 accessions grouped into four clusters.Cluster 1 with 269 accessions and Cluster 2 with 35 were mostly susceptible to pasmo.Most accessions（243）of Cluster 1 and all accessions of Cluster 2 were linseed type.Cluster 3 comprised 40 moderately susceptible accessions including 11 of linseed.Cluster 4 contained 26 accessions,of which,25 were fiber type and only one was a linseed;they were resistant to pasmo,respectively.The 26 resistant accessions ofCluster 4 represent an important germplasm for PR breeding.The 67 QTL were clustered into four sub-groups.Group 1 included 13 QTL widely distributed across the germplasm（68.27%of the accessions）but with relatively low QTL effects（R2 ranging from 3.32 to 10.85%）.Groups 2 and 3 contained 7 and 11 QTL,respectively.Present in 31.08%of the accessions,these QTL had an R2 ranging from 4.64 to 16.17%.The 36 QTL of Group 4 had an R2 ranging from 6.61 to 23.39%and contributing to the majority of the PR.These QTL were mostly found in the resistant accessions of Cluster 4 which amounts to a mere 9.70%of the germplasm.（5）Resistance gene analogs co-localized with QTLAmong the 67 stable and large-effect QTL,45 co-localized with 85 RGAs within the pre-defined 200Kb QTL flanking window.Four types of RGAs were harbored at these loci:receptor like protein（RLP）,receptor like kinase（RLK）,nucleotide-binding site（NBS）coding genes（including TNL,TX,CNL,NL,TN,NBS,and others）,and transmembranecoiled coil protein（TM-CC）.The majority of the RGAs were RLKs with 36.47%.A TNL cluster associated with QTL 37 and 38 was co-localized on chromosome 8.Additional TNL-type RGA clusters co-localized with QTL 13 and 14 on chromosome 4 and QTL 15 and 16 on chromosome 5.An RLP gene（Lus10039958）located only 56 bp downstream of QTN Lu10-8700793（QTL 49）exemplifies close linkage between the RGA and the QTL identified in this study.