c Indian Academy of Sciences RESEARCH NOTE Genomewide analysis of NBS-encoding genes in kiwi fruit (Actinidia chinensis) YINGJUN LI, YAN ZHONG, KAIHUI HUANG and ZONG-MING CHENG∗ College of Horticulture, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China [Li Y., Zhong Y., Huang K. and Cheng Z.-M. 2016 Genomewide analysis of NBS-encoding genes in kiwi fruit (Actinidia chinensis). J. Genet. 95, 997–1001] Introduction (Huang et al. 2013). Although NBS-encoding genes were just identified by Huang et al. (2013) no evolutionary his- In plants, there are many layers of defense system against tory of NBS-encoding genes was detected. In this study, we pathogens in the environment. The first is structural bar- identified NBS-encoding genes in kiwi fruit genome and then rier and second is pathogen-associated molecular pattern divided them into families based on three criteria. Duplica- (PAMP) recognition receptors. The third is resistance genes tion time, phylogenetic relationship and selection pressure (R genes) against specific pathogens, which work in the trig- were also examined to obtain insight into evolutionary pat- gering effector immunity (ETI) that produces a hypersensi- terns of NBS-encoding genes. As a result, a total of 96 NBS- tive response (HR) (Jones and Dangl 2006). R genes confer encoding genes were identified, include 74 NBS–LRR genes. resistance to a diverse range of pathogens, including bacte- The recent duplication mainly contributed to the existing ria, fungi, oomycetes, viruses, insects and nematodes (Martin NBS-encoding genes. Further, purifying selection played an et al. 2003). important role in evolution process of NBS-encoding genes. Kiwi fruit (Actinidia chinensis) is a commercially valu- The analysis will help us deeply understand the evolution of able and nutritionally important fruit, which is well known as NBS-encoding genes in Actinidia. ‘the king of fruits’ for remarkably high vitamin C content. However, pathogen infections have lowered the yield and quality of kiwi fruit (Ferrante and Scortichini 2010; Biondi Methods and materials et al. 2013; Li et al. 2013). Therefore, better understanding Identification of NBS-encodings and gene family classification of resistance (R) genes in kiwi fruit could provide the strategy of kiwi fruit for improving resistance to pathogens. The class of NBS– LRR resistance genes, which encode nucleotide-binding sites Kiwi fruit (A. chinensis) assembly and annotation were (NBS) and leucine-rich repeat (LRR) domains, is one of the downloaded from kiwi fruit genome database (http://bioinfo. largest R genes families (McHale et al. 2006). bti.cornell.edu/cgi-bin/kiwi/download.cgi). The amino acid NBS-encoding genes are categorized as NBS-only genes sequence of NB-ARC domain was downloaded from Pfam and NBS–LRR genes. Based on an N-terminal domain of database (http://pfam.xfam.org/) by using Pfam ID (PF00931), toll and interleukin-1 receptors (TIR) NBS-encoding genes which was employed as a query in BLASTP searches, with are divided into two subclasses, TIR type genes and non- the threshold expectation set to one, for searching candidate TIR type genes. Some nonTIR NBS-encoding genes have NBS-encoding genes in kiwi fruit. Further, all hits were ver- a coil–coil motif in N-terminus (Dangl and Jones 2001), ified for the presence of NB-ARC domain by Pfam ver. 28.0 therefore, they are subdivided into CC-NBS genes (CNs), (http://pfam.xfam.org/). All NBS-encoding genes were fur- X-NBS genes (XNs), CC-NBS–LRR genes (CNLs) and ther analysed to detect the LRR, TIR and RPW8 domain X-NBS–LRR genes (XNLs). by Pfam ver. 28.0 and SMART analysis. The CC domain Recently, the genome of a heterozygous kiwi fruit cultivar was predicted by COILS server (http://www.ch.embnet.org/ ‘Hongyang’ (A. chinensis) was sequenced (616.1 Mb), which software/COILS_form.html) with a threshold of 0.9 (Lupas provides an opportunity for the study of NBS-encoding genes et al. 1991). There were three criteria to classify gene family. Both the coverage (aligned sequence/gene lengths) and iden- tity between sequences were not less than 70%. The stricter ∗ For correspondence. E-mail: [email protected]. criteria were not less than 80 and 90%. Keywords. R genes; NBS-encoding genes; NBS–LRR genes; Actinidia chinensis. Journal of Genetics, DOI 10.1007/s12041-016-0700-8, Vol. 95, No. 4, December 2016 997 Yingjun Li et al. Sequence alignment and phylogenetic analysis Table 1. Number of identified NBS-encoding genes in kiwi fruit. The NB-ARC domain sequences of 96 NBS-encoding genes Predicted protein domain Letter code A. chinensis were aligned by using MUSCLE program in MEGA 5.0 (Tamura et al. 2011). The phylogenetic tree was constructed NBS-encoding genes 96 based on the neighbour-joining (NJ) method with the default NBS–LRR type 74 options and 1000 bootstraps by ClustalW 2.0 (Larkin et al. TIR-NBS–LRR TNL 9 nonTIR-NBS–LRR non-TNL 65 2007). CC-NBS–LRR CNL 17 X-NBS–LRR XNL 48 Calculation of Ks and Ka/Ks NBS 22 TIR-NBS TN 2 The ratios of nonsynonymous substitution (Ka) to synony- nonTIR-NBS non-TN 20 mous substitution (Ks) were computed in the gene fami- CC-NBS CN 3 lies and divided according to the criterion of the coverage X-NBS XN 17 and the identity between sequences not less than 70%. The Whole genome genes 39040 Proportion of NBS-encoding genes 0.246% nucleotide coding sequences (CDSs) in each gene family Proportion of NBS–LRR genes 0.190% were aligned by ClustalW 2.0 and the values of Ka, Ks and Proportion of TIR-NBS–LRR genes 0.023% Ka/Ks were calculated by MEGA ver. 5.0. Proportion of nonTIR-NBS–LRR genes 0.166% Average exon of all genes 4.63 Average exon of TIR-NBS–LRR 3.33 Test for positive pressures Average exon of nonTIR-NBS–LRR 2.34 The phylogenetic analysis by maximum likehood 4 (PAML4) Average exon of CC-NBS–LRR 2.24 Average exon of NBS-encoding genes 2.35 package was used to test selection pressures on NBS- Average exon of NBS–LRR genes 2.46 encoding genes in gene families with three or more mem- bers using the site model and branch model (Yang 2007). For the site model, one single dN/dS ratio (model = 0) and models M7 (beta) and M8 (beta-ω) (NS site = 7, 8) were of it, which may be the reason that the number of NBS- set to identify the positive selection sites. Moreover, the LR encoding genes was larger in papaya and cucumber genomes test between model M7 and M8 was performed by the criti- (Huang et al. 2009). cal criterion of chi-square 5.991 (P < 0.05, df = 2) and 9.210 The average number of exons of NBS-encoding genes and (P < 0.01, df = 2), respectively. For the branch model, one NBS–LRR genes were 2.35 and 2.46, respectively which single dN/dS ratio (model = 0) and models 0 (NS site = 0) were less than the average number of whole-genome pre- were used to detect the dN/dS in gene families. dicted genes (4.63) (table 1). The phenomenon was also observed in other species, such as Arabidopsis, rice, poplar Results and discussion and strawberry. Identification of NBS-encodings in kiwi fruit Recent duplications of NBS-encoding genes were detected in the kiwi fruit genome We compared our results with the NBS-encoding genes identified in Huang et al.’s (2013), two sequences (Achn Gene duplication provides new genes for different mecha- 047351 and Achn 064331) were found as the same gene, and nisms of evolution and creates genetic novelty in organisms Achn 088471 was also detected encoding NB-ARC domain, (Magadum et al. 2013). We divided 96 NBS-encoding genes which were not in Huang’s results. As a result, 96 NBS- into gene families with three criteria to detect the duplica- encoding genes were identified containing 74 NBS–LRR tion events. For the criterion of 70%, 50% of NBS-encoding genes (table 1), which were more than that in papaya (36) genes (48) were classified into 13 multigene families, which (Ming et al. 2008), and cucumber (52) (Wan et al. 2013), suggested that half of the NBS-encoding genes could be but less in strawberry (144) (Zhong et al. 2015), and Ara- detected under duplication events. Moreover, the average bidopsis (147) (Meyers et al. 2003). Further, the propor- number of each family was significantly greater than that in tions of NBS–LRR genes in whole genome genes in papaya Arabidopsis (t-test, P < 0.01), and smaller than that in straw- (0.145%), cucumber (0.0821%), strawberry (0.439%) and berry (t-test, P < 0.01). To detect more recent duplication, we Arabidopsis (0.544%), were 0.76-, 0.33-, 1.78- and 2.86- applied the criteria of 80%. As a result, there are 40 NBS- fold to that of kiwi fruit (0.246%), which indicated that encoding genes (41.67%) belonging to 13 multigene families the number of NBS–LRR genes did not evolve proportion- and the proportion of NBS-encoding genes was significantly ally with the genome. Further, a relatively fewer number of larger than that of Arabidopsis (t-test, P < 0.01). When the third NBS–LRR disease-resistant genes in kiwi fruit, ‘Hongyang’ criterion of 90% was applied, the proportion of multigenes may be related to its disease susceptibility. In addition, kiwi (21.88%) in all NBS-encoding genes reduced significantly fruit genome underwent the recent whole-genome duplica- (t-test, P < 0.01).