Identification of NBS gene family and its effect on fruit disease resistance in apricot

Abstract:【Objective】Apricot (Prunus armeniaca L.) is one of the characteristic fruit trees. However, the apricot fruits are often infected by various pathogens, ranging from powdery mildew, anthracnose and brown-rot fungi to scab and soft-rot bacteria. NBS (Nucleotide-Binding Site, NBS) gene family in-cluding a large amount of resistance (R) genes is usually necessary for plants to recognize toxic effec-tors secreted by pathogens and further activate defense responses. Therefore, this study aimed to system-atically investigate the NBS genes in the apricot genome and analyze their expression patterns at 4 fruit developmental stages (G1, G2, CT, FR) in 2 apricot cultivars (Chuanzhihong and Dabaixing), which would provide information on the mechanism of disease-resistant of apricot fruits.【Methods】NB-ARC hmm (hidden Markov model) profile was used as a query to search the longest transcripts of NBS cod-ing genes (E-value < 1e-50). Based on these sequences obtained above, multiple sequence alignment was performed using a MAFFT program (maximum iterations = 1000). Then, an apricot NBS hmm profile was built and used as a query to identify NBS candidates (E-value < 1e-50) again. Finally, NBS candidate genes were further confirmed by NCBI Conserved Domains Database and Pfam Database, and the genes without NB-ARC and PF03210 superfamily domains were deleted. The raw data of RNA-seq were processed by fastp, hista2, feature Counts, and DESeq2 programs. All analyses were performed us- ing default parameters.【Results】A total of 155 NBS genes were identified in the apricot reference ge-nome, which were classed into 2 groups, including 126 CNL (coiled-coil NBS leucine-rich-repeat) and 29 NL (NBS-LRR), respectively. CNL group was divided into 5 subgroups, including CNL1, CNL2, CNL3, CNL4 and CNL5. Phylogenetic tree showed that NL genes were more ancient than CNL genes. The NBS genes were mainly located in linkage groups 1 (LG1) and LG2 (45%). Functional annotation suggested that NBS genes were homologous with previously-studied resistance (R) protein, including RPP13-like, RPP8, RGA, RPM1, RPS2, SUMM2, RPS5 and RQ11. We found 61 genes were associated with 4 pathogens, including Peronospora parasitica, Pseudomonas syringae, and Phytophthora infes- tans and Xanthomonas spp., which would cause apricot disease such as bacterial canker and downy mil-dew. We identified 10 conserved motifs in the apricot NBS genes, of which motif CNBS-1 was a specif-ic one discovered in the apricot NBS genes. A large number of cis-elements were found in the upstream 2000 bp of NBS genes, including salicylic acid (SA), gibberellin (GA), auxin, ethylene and a variety of environmental stress, which indicated that the signals of hormones and stress might regulate NBS genes expression. The distribution of Ka/Ks indicated that the NBS gene might undergo genome-wide replica-tion events during its evolution. 4 genes pairs were subjected to positive selection, of which PARG16921-PARG16918 might confer resistance to downy mildew, PARG08758- PARG08765 might confer resis-tance to Phytophthora root rot and PARG03801-PARG03800 might be related to the resistance to diseas-es including fruit green crinkle, russet ring and powdery mildew. Transcriptome analysis between two apricot cultivars Chuanzhihong and Dabaixing showed that 86 genes in Chuanzhihong and 84 genes in Dabaixing were constantly expressed during the 4 fruit developmental stages. The stage-specific expres-sion occurred in 5 genes (PARG15350 and PARG15400 in G1 stage, PARG02388, PARG21703 and PARG01953 in G2, CT and FR stage, respectively) in Dabaixing, while there was only 1 gene (PARG15350) in Chuanzhihong. The expression of NBS genes showed that 8 genes (PARG04668, PARG09017, PARG15825, PARG15832, PARG16406, PARG17721, PARG22597, and PARG30352) in Chuanzhihong were significantly higher than those of Dabaixing at different fruit developmental stages of apricot fruit. In addition, the expression level of some other genes were significantly higher in Chuan-zhihong than that in Dabaixing at each developmental stages. There were 4 in the G1 stage (PARG03817, PARG11106, PARG15350 and PARG29976), 3 in the G2 stage (PARG03716, PARG21218 and PARG22609), 2 in the CT stage (PARG21684 and PARG22582) and 7 in the FR stage (PARG05521,PARG08801, PARG08803, PARG16630, PARG17764, PARG25364 and PARG30351). The difference in fruit disease resistance between Chuanzhihong and Dabaixing was mainly related to the genes resistant to Phytophthora infestans and downy mildew pathogens, and especially the 8 NBS genes that were sig-nificantly differentially expressed at all stages might be more closely related to the disease resistance of apricot fruit.【Conclusion】We identified 155 NBS genes from apricot genome and found that 8 genes showed a higher expression in Chuanzhihong compared with Dabaixing, which were mainly related to the resistance to Phytophthora infestans and downy mildew pathogens. These results would provide the-oretical guidance for disease-resistant breeding in apricot.