Evolution analysis and expression divergence of the chitinase gene family against Leptosphaeria maculans and Sclerotinia sclerotiorum infection in Brassica napus

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. The recent release of assembled genome sequence of rapeseed allowed us to perform a genome-wide identification of the chitinase gene family. 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 profiling 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 exhibit distinct expression in response to L. maculans and S. sclerotiorum infection. This study presents a comprehensive survey of the chitinase gene family in B. napus and provides valuable information for further understanding the functions of the chitinase gene family.


36
In higher plants, the expression of chitinase genes is involved in defense against biotic and abiotic stress as well as in growth and 37 developmental processes (COLLINGE et al. 1993;PUNJA and ZHANG 1993). For instance, a class III chitinase gene, Mtchitinase III-3, 38 has been found to be induced upon the infection of fungi Glomus mosseae and Glomus intraradices in cortical root (BONANOMI et al.

65
To further extend the understanding of the chitinase gene family, a global analysis, including identification, sequence features, 66 physical location, the evolutionary relationship and expression pattern of the chitinase gene family in response to L. maculans and S. 67 sclerotiorum infection in B. napus using the RNA-seq sequencing data collected in our lab and some transcriptome data from NCBI 68 database was performed. Expression analysis revealed that some chitinase genes were induced by both pathogens while others 69 displayed differential expression pattern in response to L. maculans and S. sclerotiorum infection, suggesting that they may have 70 distinct roles in different pathogens stress response. Together, our findings will be helpful for further understanding of the functions of

74
The complete genome sequence and gene annotation was used for the genome-wide identification of the chitinase gene family 75 and a total of 68 putative chitinase genes were identified in the B. napus genome (Table 1). All these identified proteins have at least 76 one typical "Glyco_hydro_19" or "Glyco_hydro_18" domain which is responsible for catalyzing the degradation of chitin. Of these, 77 GH-18 family and GH-19 family include 12 and 56 putative chitinase genes, respectively. These chitinase genes in B. napus encode 78 proteins ranging from 130 to 1005 amino acids in length with an average of 294. The average number of exons among these chitinase 79 genes was 3.04, a value that is smaller than the average number of exons among all predicted B. napus genes (4.9). BLAST search of 80 these 68 proteins against NCBI non-redundant database showed that the top matched hits were endochitinases, chitinases, chitinase-81 like proteins, which further confirm the reliability of the identified chitinase genes. Furthermore, the signal peptides in 54 predicted 82 chitinase sequences were also identified. To examine the evolutionary relationships among the chitinase genes in B. napus, sequence 83 alignment was performed with amino acid sequences (Supplementary Table 1) and an unrooted phylogenetic tree of the 68 chitinase 84 genes using neighbor-joining method was constructed (Figure 1).

140
To understand the evolution of the chitinase gene family, twenty-six pairs of paralogs were detected in 68 chitinase genes based 141 on criteria for both coverage ≥70% and identity ≥70% ( Table 2). The phylogenetic relationship analysis of chitinase genes also 142 showed that most pairs of paralogs could be clustered together (Figure 1). For example, the four members in two pairs of paralogs 143 (BnaA09g05050D and BnaC09g04600D, BnaA06g26630D and BnaC07g30330D) in Class III subfamily were clustered into two parts 144 in a single clade. As genome duplication was considered, one member in the A subgenome would correspond to one homologous gene 145 in the C subgenome in B. napus. In fact, 50 members of 68 chitinase genes showed such a one-to-one correspondence.

221
In this study, a total of 68 chitinase genes were identified in B. napus genome. Of these, GH-18 family and GH-19 family have 222 12 and 56 chitinase genes, respectively, which was further supported by analysis of gene structure and conserved motifs (Figure 2, 3).

253
The results showed that many chitinase genes could transcriptionally respond to L. maculans and S. sclerotiorum infection in rapeseed

309
Supplementary Figure 2 Details of the ten conserved motifs of chitinase GH-19 family as derived by MEME analysis.

322
The authors declare no conflict of interest.