XU Wen,ZHOU Tengsheng,LU Dandan,AN Sufang,HOU Jinna,LI Genyi,

(1.Crop Designing Center,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;2.Department of Plant Science,University of Manitoba,Winnipeg,MB R3T2N2,Canada)

Abstract: Blackleg and sclerotinia stem rot caused by Leptosphaeria maculans and Sclerotinia sclerotiorum respectively are two major diseases in rapeseed worldwide,which cause serious yield losses.Chitinases are pathogenesis-related proteins and play important roles in host resistance to various pathogens and abiotic-stress responses.However,a systematic investigation of the chitinase gene family and its expression profile against L.maculans and S.sclerotiorum infection in rapeseed remains elusive.In this study,68 chitinase genes were identified in Brassica napus genome.These genes were divided into five different classes and distributed among 15 chromosomes.Evolutionary analysis indicated that the expansion of the chitinase gene family was mainly attributed to segmental and tandem duplication.Moreover,the expression profile of the chitinase gene family was investigated using RNA sequencing (RNA-Seq) and the results revealed that some chitinase genes were both induced while the other members exhibited distinct expression in response to L.maculans and S.sclerotiorum infection.Thus,the functions of different chitinase gene family members have diverged during long-term evolution and might exhibit different roles against different disease stresses in Brassica napus.

Key words: Brassica napus; chitinase gene family; expression pattern; Leptosphaeria maculans; Sclerotinia sclerotiorum

Introduction

Plant chitinases (EC 3.2.1.14) are enzymes that hydrolyze the N-acetyl glucosamine polymer chitin,a major component of fungal cell walls and exoskeleton of insects[1]and are considered as one group of pathogenesis-related (PR) proteins[2],which can be induced in response to the infection of various pathogenic micro-organisms.In the light of classification of glycosyl hydrolases based on amino acid sequence similarities,plant chitinases have been put in glycoside hydrolasis family 18(GH-18) and 19 (GH-19)[3].According to the CAZy database (http://www.cazy.org/Glycoside-Hydrolases.html)[4],plant chitinases have been grouped into five different classes ranging from Ⅰ to Ⅴ. Of these,classes Ⅰ,Ⅱ and Ⅳ belong to the GH-19 family whereas the GH-18 family is composed of classes Ⅲ and Ⅴ chitinases[3].The details of plant chitinase classification are described as follows.Class Ⅰ chitinases have an N-terminal chitin-binding domain and a GH-19 catalytic domain.Class Ⅱ chitinases consist of only a catalytic domain with a high level of sequence and structure similarity to class Ⅰ chitinases but lack the chitin-binding domain and linker regions.Class Ⅳ chitinases show high homology with class Ⅰ chitinases but are smaller due to one deletion in the chitin-binding domain and three deletions in the catalytic domain[5].Both class Ⅲ and Ⅴ chitinases have a GH-18 catalytic domain and a consensus sequence DXDXE,but there is no homology for other amino acids[6].GH-18 chitinases are widely distributed in plants,animals,fungi,bacteria and viruses whereas GH-19 members almost exclusively exist in higher plants[7].

In higher plants,the expression of chitinase genes is involved in defense against biotic and abiotic stress as well as in growth and developmental processes[1,8]. For instance,a class Ⅲ chitinase gene,MtchitinaseIII-3,had been found to be induced upon the infection of fungiGlomusmosseaeandGlomusintraradicesin cortical root[9].PSCHI4,a putative extracellular class Ⅱ chitinase,was up-regulated in pine seedlings infected with the necrotrophic pathogenFusariumsubglutinansf.sp.Pini[10].InArabidopsisthaliana,a class Ⅳ chitinase geneAtchitIVaccumulated very rapidly in leaves after inoculation withXanthomonascampestrisand reached maximum mRNA accumulation after one hour infection[11].In addition,several transgenic studies showed that enhanced levels of chitinase genes in transgenic plants could indeed improve resistance against pathogens and reduce the damage caused by fungi and some insect pests[12-17].There were several reports of induced expression of plant chitinases when plants were exposed to abiotic stresses such as heavy-metal stress[18],drought[19-20],salt[19],cold[21],heat[22],UV light and wounding[23].Furthermore,some chitinases are essential in physiological processes such as somatic embryo development[24]and formation of root nodules[25].In conclusion,chitinases play important roles in plant defense and plant health.

Rapeseed (Brassicanapus) is an important oilseed crop worldwide.This crop is affected by various fungal diseases,especially blackleg caused byLeptosphaeriamaculansand sclerotinia stem rot bySclerotiniasclerotiorum,which are the most destructive rapeseed diseases in Canada,Australia,Europe and many other regions around the world[26].Recently,a few studies have been conducted on chitinase genes responding to some pathogens infection inB.napus[17,27-30]. For example,constitutive overexpression of a chimeric chitinase gene in rapeseed had been shown to exhibit an increased resistance to three fungal pathogens compared with their nontransgenic parental plants[27].Co-expression of defensin geneRs-AFP1fromR.sativusand chimeric chitinase genechit42fromT.atroviridein rapeseed viaAgrobacterium-mediated transformation demonstrated enhanced resistance against sclerotinia stem rot disease[31].In addition,global studies of transcriptome dynamics of defense responses toL.maculansandS.sclerotioruminB.napuspresented that pathogen responsive genes including chitinases were rapidly induced during early infection[32-35].However,to date,the chitinase genes in rapeseed have not been systematically identified and thus the genetic resistance toL.maculansandS.sclerotiorumhas been not yet studied.Recently,the availability of the whole genome sequence and RNA-Seq enable further investigations into chitinase genes and their response toL.maculansandS.sclerotioruminfection on a genome-wide scale[36-37].

To further extend the understanding of the chitinase gene family,a global analysis,including identification,sequence features,physical location,the evolutionary relationship and expression pattern of the chitinase gene family in response toL.maculansandS.sclerotioruminfection inB.napususing the RNA-Seq data collected by LI’s lab in University of Manitoba and some transcriptome data from NCBI database was performed.Expression analysis revealed that some chitinase genes were induced by both pathogens while others displayed differential expression pattern in response toL.maculansandS.sclerotioruminfection,suggesting that they may have distinct roles in different pathogens stress response.Together,our findings will be helpful for further understanding of the functions of the chitinase gene family against different stress in rapeseed.

1 Materials and methods

1.1 Identification of chitinase genes in B.napus

The v4.1 genome sequences and annotations ofB.napuswere downloaded from the FTP site of theBrassicadatabase (ftp://brassicadb.org/Brassica_napus/)[38].To identify chitinase genes inB.napus,Glyco_hydro_18 (PF00704) and Glyco_hydro_19(PF00182) domains were obtained from the Pfam website (http://pfam.xfam.org/).The HMMER software version 3.0 was employed to identify chitinase genes from all known protein sequences[39].All candidate genes were further submitted to Pfam analysis (http://pfam.xfam.org/) to confirm the presence of one of the above two domains with E-value 0.000 1.For annotation,the identified protein sequences were aligned with NCBI nr database using BLAST alignment (E-value cut-off of 1e-5)[40].The identification of signal peptide was performed in the website (http://www.cbs.dtu.dk/services/SignalP/)[41].

1.2 Phylogenetic tree construction and sequence analysis

All identified chitinase genes were aligned using the MUSCLE program within MEGA 7.0 software[42].Subsequently,a neighbor-joining (NJ) method was applied to construct a phylogeny of chitinase genes with a 1 000 bootstrap replication.Motifs of chitinase proteins inB.napuswere investigated statistically using online MEME software (http://meme-suite.org/tools/meme),which set the maximum number of motifs at 10.Subsequently,InterProScan (http://www.ebi.ac.uk/interpro/search/sequence-search) was employed to annotate all the identified motifs.In addition,the exon-intron structures of genes were analyzed with the gene structure display server program(http://gsds.cbi.pku.edu.cn/).

1.3 Analysis of chromosomal distribution and evolution patterns of chitinase genes

The chromosomal locations of chitinase genes were determined based on annotation data obtained from theB.napusdatabase.The orthologous relationships between the chitinase genes inB.napusandA.thaliana,B.rapa,andB.oleraceagenes were evaluated following the criteria:We used program BLAST to identify putative orthologs between chitinase genes inB.napusand one ofA.thaliana,B.rapa,andB.oleraceaspecies with both coverage over 70% and identity more than 70% and all chitinase sequences fromB.napuswere searched against all gene sequences from one ofA.thaliana,B.rapa,andB.oleraceaspecies.Tandem duplication was characterized as multiple genes of one family located within the same or neighboring intergenic region[43].

1.4 Analysis of transcriptome sequencing data

The sequence data responsive toL.maculansinfection was deposited in the BioProject database of NCBI under accession number PRJNA378851.Transcriptome data under accession number PRJNA274853 publicly available on the NCBI SRA database were mined and analyzed for expression patterns of the rapeseed chitinase genes in response toS.sclerotioruminfection.Sequencing reads were then aligned to theB.napusreference genome sequence(ftp://brassicadb.org/Brassica_napus/) using TopHat,v2.1.1[44].Mapping data was used to estimate expression values for annotated genes using htseq-count tool[45].Differential gene expression analyses were performed using the R/Bioconductor package,DESeq2[46].An absolute value of log2 fold change>1.5 and the false discovery rate(FDR)<0.05 was set to declare differentially expressed genes.

2 Results and analysis

2.1 Identification and phylogenetic analysis of the chitinase gene family in B.napus

The complete genome sequence and gene annotation were used for the genome-wide identification of the chitinase gene family and a total of 68 putative chitinase genes were identified in theB.napusgenome(Tab.1).All these identified proteins had at least one typical “Glyco_hydro_19” or “Glyco_hydro_18” domain which is responsible for catalyzing the degradation of chitin.Of these,GH-18 family and GH-19 family included 12 and 56 putative chitinase genes,respectively.These chitinase genes inB.napusencoded proteins ranging from 130 to 1 005 amino acids in length with an average of 294.The average number of exons among these chitinase genes was 3.04,a value that was smaller than the average number of exons among all predictedB.napusgenes(4.9).BLAST search of these 68 proteins against NCBI non-redundant database showed that the top matched hits were endochitinases,chitinases,chitinase-like proteins,which further confirmed the reliability of the identified chitinase genes.Furthermore,the signal peptides in 54 predicted chitinase sequences were also identified.To examine the evolutionary relationships among the chitinase genes inB.napus,sequence alignment was performed with amino acid sequences and an unrooted phylogenetic tree of the 68 chitinase genes using neighbor-joining method was constructed (Fig.1).

The phylogenetic tree highlighted that the 68 chitinase genes could be divided into five well-supported subfamilies,which were consistent with Class Ⅰ,Ⅱ,Ⅲ,Ⅳ,and Ⅴ.As expected,the chitinase genes of Glyco_hydro_19 and Glyco_hydro_18 families were clustered into two relatively distinct branches.Chitinase genes from subfamilies Class Ⅲ and Class Ⅳ were in the Glyco_hydro_18 clade,whereas subfamilies Class Ⅰ,Ⅱ and Ⅳ that belonged to Glyco_hydro_19 clade were clustered together and showed close relationships (Fig.1).According to the phylogenetic tree,chitinases in different subfamilies had various characteristics.Among the five subfamilies,Class Ⅳ was found to be the biggest group with 36 members,accounting for almost a half of the chitinase gene family,whereas there were 11,9,4,8 chitinase genes in Class Ⅰ,Ⅱ,Ⅲ,Ⅴ subfamilies,respectively.

Tab.1 Chitinase genes in the B.napus genome and their sequence characteristics

Tab.1(Continued) Chitinase genes in the B.napus genome and their sequence characteristics

Note:+ means forward strand of genome reference sequence; - means reverse strand of genome reference sequence.

Fig.1 Phylogenetic relationships and motif compositions of chitinase genes

2.2 Conserved motifs and gene structures of the chitinase gene family in B.napus

To gain further insights into the structural diversity and functional evolution of chitinase genes,10 motifs of all 68 chitinase genes were captured by MEME software and displayed schematically in Fig.1,2 and 3.As shown in Fig.1,most chitinases in the same class shared common motif compositions.Among the GH-19 family,motifs 1,3,4,9 were annotated as glycoside hydrolase catalytic domain and motif 7 was annotated as chitin-binding domain.Each member of chitinases in classes Ⅰ,Ⅱ and Ⅳ had at least one glycoside hydrolase catalytic domain and motif 4 was only detected in classes Ⅰ and Ⅱ.Most chitinase genes from subfamilies Ⅰ and Ⅳ also harbored a chitin-binding domain whereas motif 7 did not exist in the subfamily Ⅱ.In the GH-18 family,except motifs 8 and 9,other 8 motifs were annotated as glycoside hydrolase catalytic domains.Similarly,in the GH-18,at least one glycoside hydrolase catalytic domain was detected in every chitinase gene of class Ⅲ and Ⅴ.Interestingly,motif 7 and motif 10 were unique in class Ⅲ whereas motif 2 could be only detected in class Ⅴ.In addition,we analyzed the coding sequences with corresponding genome sequences of each chitinase gene inB.napus.A detailed illustration of the chitinase gene structure was shown in Fig.4.Most chitinase genes contained two or three exons,whereas three chitinase genes(BnaA01g30550D,BnaA0626330D and BnaUnng03570D)had no introns.In general,most chitinase genes in the same class showed similar conserved motifs and exon-intron structures.These findings revealed that motif compositions and gene structures of each class in the chitinase gene family were relatively conserved.The similar features of chitinase genes in the same class may fulfill similar functions and this claim needs to be supported by their expression and related data.

Fig.2 Details of the ten conserved motifs of chitinase GH-18 family as derived by MEME analysis

Fig.3 Details of the ten conserved motifs of chitinase GH-19 family as derived by MEME analysis

Fig.4 Exon-intron structures of all chitinase genes in B.napus

2.3 Chromosomal distribution and evolution patterns of chitinase genes in B.napus

The chromosomal distribution of 68 chitinase genes was analyzed based on the available gene annotation and genome sequence assembly.The results revealed that all 68 chitinase genes were distributed among 15 out of 19 chromosomes with the exception of chromosomes A02,A07,C02 and C06 in theB.napusgenome(Fig.5a).There were 32 genes mapped in the A genome,and 35 genes located in the C genome while one gene BnaUnng03570D was not assigned to a chromosome.GH-19 family presented on 13 chromosomes except chromosomes A06 and C07 while GH-18 family was absent from chromosomes A04,A05,A10,C03,C04,C05,C08.The number of chitinase genes varied considerably among different chromosomes and a large number of chitinase genes were found on chromosomes A03 and C03,harbouring 11 and 14 genes,respectively (Fig.5a).Furthermore,the classes of the chitinase gene family were distributed in different chromosomes and up to three classes in A03 and C03 chromosomes were identified (Fig.5b).

To understand the evolution of the chitinase gene family,twenty-six pairs of paralogs were detected in 68 chitinase genes based on criteria of both coverage≥70% and identity≥70%(Tab.2).The phylogenetic relationship analysis of chitinase genes also showed that most pairs of paralogs could be clustered together (Fig.1).For example,the four members in two pairs of paralogs (BnaA09g05050D and BnaC09g04600D,BnaA06g26630D and BnaC07g30330D) in Class Ⅲ subfamily were clustered into two parts in a single clade.As genome duplication was considered,one member in the A subgenome would correspond to one homologous gene in the C subgenome inB.napus.In fact,50 members of 68 chitinase genes showed such a one-to-one correspondence.

a.Gene distribution of GH-18 family and GH-19 family on B.napus chromosomes; b.Distribution of five subfamilies of chitinase genes on B.napus chromosomesFig.5 Distribution of the chitinase gene family on B.napus chromosomes

The distribution of chitinase genes indicated a relatively deep evolutionary origin of these chitinase genes as well as gene duplication.The allotetraploidB.napusis a spontaneous hybridisation ofB.rapa(A genome) andB.olearecea(C genome).To understand the origin and duplication patterns of these chitinase genes,putative orthologs of chitinase genes inB.napuswere also identified inA.thaliana,B.rapaandB.olearecea(Tab.2).The results demonstrated that 30 orthologs of 32 chitinase genes in the A subgenome inB.rapaand 32 orthologs of 35 chitinase genes in the C subgenome were identified inB.olearecea.The gene BnaUnng03570D had both orthologs (Bo1g021980.1 and Bra020951.1) inB.oleareceaandB.rapa,respectively,but it had much higher identities with Bo1g021980.1.Furthermore,the order and synteny of chitinase genes BnaA03g20290D,BnaA03g20300D,BnaA03g20310D,BnaA03g20320D,BnaA03g20330D,BnaA03g20340D in chromosome A03 and BnaC03g24270D,BnaC03g24280D,BnaC03g24290D,BnaC03g24300D,BnaC03g24340D,BnaC03g24360D in chromosome C03,as well as those genes for both subgenomes of the allopolyploid (the A and C subgenomes inB.napusvs the A and C genomes inB.rapaandB.olearecea) revealed that there were a few structural rearrangements and genomic collinearity inB.napuswith regard to the chitinase gene family evolution and expansion.These findings showed that most chitinase genes (63/68) of allotetraploidB.napuswere inherited from their diploid ancestors by recombination or segmental duplication.It was interesting to observe that the best hit of chitinase gene BnaA09g15430D was BnaA09g15440D,which were tandem repeats on the A09 chromosome.It was also observed that five chitinase genes inB.napushad no orthologs in their diploid ancestors.Of these,two genes (BnaC04g09720D and BnaC03g37600D) and one paralog of two genes (BnaA03g32270D and BnaC03g37570D) did not have orthologs inA.thaliana,B.rapaandB.olearecea,which might result from incomplete and error-filled genome assemblies and gene annotation errors or gene structure rearrangement in the evolutionary process.Perhaps these five genes might be the new members of the chitinase gene family during the evolution inB.napus.

2.4 Transcriptomic profiles of the chitinase gene family in response to L.maculans and S.sclerotiorum during early infection in B.napus

Blackleg,also known as stem canker caused byL.maculansand sclerotinia stem rot caused byS.sclerotiorumare two major rapeseed diseases in most major rapeseed growing areas.The expression profiles of all chitinase genes in response toL.maculansandS.sclerotioruminfection in resistant and susceptibleB.napusaccessions were investigated using whole-transcriptome sequencing data to understand the roles of chitinase genes at early stages of infection.The results showed that these two biostresses caused a significant expression induction of some members in the chitinase gene family.The differentially expressed genes (DEGs) of chitinases in response toL.maculanswere identified.Among all 68 chitinase genes in the genome,31 and 25 chitinase genes were up-regulated in the resistant lines and susceptible lines inoculated with the pathogen compared with their water control,respectively.Of these up-regulated chitinase genes,24 were overlapped in the resistant lines and susceptible accessions.Combined data of all resistant and susceptible lines revealed that 15 chitinase genes were upregulated and no chitinase gene was dramatically downregulated during the infection ofL.maculans(Fig.6).

Relative fold change in control as compared to resistant and susceptible B.napus lines is used to generate heatmap; R and S represent resistant and susceptible lines,respectively; The colored scale for the relative expression levels is shownFig.6 Expression profiles of chitinase genes in response to L.maculans (lma) and S.sclerotiorum (scl) infection

In a previous report,the abundance of transcripts in resistant and susceptibleB.napusaccessions under the 4 day post-inoculation treatments was analyzed to understand the differential defense response toS.sclerotiorum[34].Using these data and all the 68 chitinase genes identified in this study,23 and 20 chitinase genes were up-regulated while 6 and 11 members were downregulated in the resistant accession and susceptible accession compared with water control,respectively.Compared with the expression of chitinase genes in response to blackleg pathogen infection,16 up-regulated and 4 down-regulated chitinase genes were overlapped respectively.Compared with the susceptible accession,the analyses showed that 13 and 7 chitinase genes were the same as those upregulated and downregulated ones in the resistant accession againstS.sclerotiorumattack,respectively.The distinctive expression pattern of the chitinase gene family suggested that the functions of different members in the chitinase gene family had diverged during long-term evolution and might exhibit different roles against different biotic and abiotic stresses.

3 Conclusion and discussion

Chitinases are believed to play important roles in plant-pathogen interactions and catalyze the hydrolysis of the β-1-4-linkage in the N-acetyl-D-glucosamine polymer of chitin,which is a major component of many fungal cell walls,but absent in higher plants[1-2].In this study,a total of 68 chitinase genes were identified inB.napusgenome.Of these,GH-18 family and GH-19 family had 12 and 56 chitinase genes,respectively,which was further supported by analysis of gene structures and conserved motifs.GH-18 family was divided into Class Ⅲ (4 genes) and Class Ⅴ (8 genes).GH-19 family was composed of Class Ⅰ (11 genes),Class Ⅱ (9 genes) and Class Ⅳ(36 genes).However,there were 13 and 26 chitinase genes in GH-19 family and GH-18 family in para rubber tree,respectively[47]. Class Ⅳ had the most members inB.napuswhereas Class Ⅲ posed the most genes of chitinase in para rubber tree,which may reveal that there is evolutionary divergence of specific classes of chitinases in different species.The chitinase gene family has 24,32,35 and 68 members inA.thaliana,B.rapa,B.oleraceaandB.napus,respectively[48],which suggested that chitinase genes inB.napushad expanded in comparison to its ancestors.Gene duplication events,such as tandem duplication and segmental duplication play important roles in the rapid expansion and evolution of gene families[49].The AACC genome ofB.napuswas formed through recent allopolyploidy between the ancestors ofB.rapa(AA) andB.oleracea(CC)[36].From our analysis,30 orthologs of 32 chitinase genes in the A genome inB.rapaand 32 orthologs of 35 chitinase genes in the C genome ofB.oleareceawere identified.Most orthologous gene pairs inB.rapaandB.oleraceawere still homeologous pairs inB.napus.Most chitinase genes inB.napusshowed a close relationship to their ancestor chitinase genes,which suggested that segmental duplication or polyploidy events contributed to the expansion of the chitinase gene family inB.napus.Only one pair of tandemly duplicated genes (BnaA09g15430D and BnaA09g15440D) was identified.These findings suggest that segmental duplication and tandem duplication likely play an important role in the expansion of the chitinase gene family inB.napus.Moreover,four genes (BnaC04g09720D,BnaC03g37600D,BnaA03g32270D and BnaC03g37570D) did not have orthologs inA.thaliana,B.rapaandB.olearecea,suggesting that they may be the new members of the chitinase gene family and coevolve with fungi in response to variation in pathogen defenses.

Chitinases play a major role in host defense by directly attacking fungal pathogens inA.thaliana[11],rice[12],grapevine[13],tobacco[14-15,50],peanut[16]and pepper[19].Some chitinase members can be induced by fungal pathogens,such asCylindrosporiumconcentricum,Phomalingam,andS.sclerotiorum.The role of chitinase gene also had been studied inB.napusand overexpression of chitinase genes could increase tolerance in transgenic plants previously[27,31].Transgenic plants ofB.napuscv.ZS 758 carrying sporamin and chitinase PjChi-1 genes exhibited increased levels of resistance toS.sclerotiorumand reduced the size of leaf spot in transformants compared to untransformed wild-type plants[51].However,constitutive expression of pea chitinase gene showed little or no enhancement of resistance toL.maculansin transgenic rapeseed compared with non-expressing transgenic lines[52].Some chitinases such as pineapple leaf chitinase-A do not have any antifungal activity[53].Although many studies about the individual member of the chitinase gene family have been published,there is little information about analysis of expression divergence of the chitinase gene family at a genome-wide level in rapeseed,especially under different fungal pathogen stresses.In this study,detailed expression pattern of the chitinase gene family againstL.maculansandS.sclerotioruminfection in rapeseed was analyzed using RNA-Seq data.The results showed that many chitinase genes could transcriptionally respond toL.maculansandS.sclerotioruminfection in rapeseed,implying possible function of chitinase genes in response to these two fugal pathogens inB.napus.The results revealed that the resistant accesions differentiated from the susceptible ones in pathogen defense,so we hypothesize that the function of different chitinase genes has been diverged against pathogens in resistant and susceptibleB.napusaccessions.Previously,the expression of chitinase genes could be induced in response to all kinds of pathogens,such asG.mosseae,F.subglutinansf.sp.Pini,X.campestris,L.maculansandS.sclerotiorum[9-11,33-34]. Next,to identify genes critically responsible forL.maculansandS.sclerotiorumresistance in resistant accessions,we compared theL.maculansandS.sclerotiorumresponsive chitinase genes in both resistant and susceptible accessions,respectively. Furthermore,we compared the 18 up-regulated chitinase genes against the pathogenL.maculansaggression with 18 up-regulated chitinase genes againstS.sclerotiorumattack.Interestingly,the upregulation after infection withL.maculanswas stronger in resistant accessions than in susceptible accessions.In contrast,the upregulation afterS.sclerotiorumattack showed higher levels in both resistant and susceptible accessions whereas there were much less differences in both resistant and susceptible accessions.In addition,there were some chitinase members with no expression changes detected,which may account for that some chitinases show little or no enhancement of resistance and do not have any antifungal activity[52-53].The above results indicate that some members of the chitinase gene family have developed as powerful basal defence against various pathogens attack and other individual members of the chitinase gene family have evolved different roles in response to different environmental stresses inB.napus.

In summary,our study provides a comprehensive analysis of the chitinase gene family in rapeseed,including gene identification,sequence features,physical location,evolutionary relationship,and expression patterns of chitinase genes responding toL.maculansandS.sclerotioruminfection,which could facilitate further dissection of the function of the chitinase gene family in rapeseed.