Genomewide analysis of NBS-encoding genes in kiwi fruit (Actinidia chinensis)

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1 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 , People s Republic of China [Li Y., Zhong Y., Huang K. and Cheng Z.-M Genomewide analysis of NBS-encoding genes in kiwi fruit (Actinidia chinensis). J. Genet. 95, ] Introduction In plants, there are many layers of defense system against pathogens in the environment. The first is structural barrier and second is pathogen-associated molecular pattern (PAMP) recognition receptors. The third is resistance genes (R genes) against specific pathogens, which work in the triggering effector immunity (ETI) that produces a hypersensitive response (HR) (Jones and Dangl 2006). R genes confer resistance to a diverse range of pathogens, including bacteria, fungi, oomycetes, viruses, insects and nematodes (Martin et al. 2003). Kiwi fruit (Actinidia chinensis) is a commercially valuable and nutritionally important fruit, which is well known as 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 et al. 2013; Li et al. 2013). Therefore, better understanding of resistance (R) genes in kiwi fruit could provide the strategy for improving resistance to pathogens. The class of NBS LRR resistance genes, which encode nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) domains, is one of the largest R genes families (McHale et al. 2006). NBS-encoding genes are categorized as NBS-only genes and NBS LRR genes. Based on an N-terminal domain of toll and interleukin-1 receptors (TIR) NBS-encoding genes are divided into two subclasses, TIR type genes and non- TIR type genes. Some nontir NBS-encoding genes have a coil coil motif in N-terminus (Dangl and Jones 2001), therefore, they are subdivided into CC-NBS genes (CNs), X-NBS genes (XNs), CC-NBS LRR genes (CNLs) and X-NBS LRR genes (XNLs). Recently, the genome of a heterozygous kiwi fruit cultivar Hongyang (A. chinensis) was sequenced (616.1 Mb), which provides an opportunity for the study of NBS-encoding genes For correspondence. zmc@njau.edu.cn. Keywords. R genes; NBS-encoding genes; NBS LRR genes; Actinidia chinensis. (Huang et al. 2013). Although NBS-encoding genes were just identified by Huang et al. (2013) no evolutionary history of NBS-encoding genes was detected. In this study, we identified NBS-encoding genes in kiwi fruit genome and then divided them into families based on three criteria. Duplication time, phylogenetic relationship and selection pressure were also examined to obtain insight into evolutionary patterns of NBS-encoding genes. As a result, a total of 96 NBSencoding genes were identified, include 74 NBS LRR genes. The recent duplication mainly contributed to the existing NBS-encoding genes. Further, purifying selection played an important role in evolution process of NBS-encoding genes. The analysis will help us deeply understand the evolution of NBS-encoding genes in Actinidia. Methods and materials Identification of NBS-encodings and gene family classification of kiwi fruit Kiwi fruit (A. chinensis) assembly and annotation were downloaded from kiwi fruit genome database ( bti.cornell.edu/cgi-bin/kiwi/download.cgi). The amino acid sequence of NB-ARC domain was downloaded from Pfam database ( by using Pfam ID (PF00931), which was employed as a query in BLASTP searches, with the threshold expectation set to one, for searching candidate NBS-encoding genes in kiwi fruit. Further, all hits were verified for the presence of NB-ARC domain by Pfam ver ( All NBS-encoding genes were further analysed to detect the LRR, TIR and RPW8 domain by Pfam ver and SMART analysis. The CC domain was predicted by COILS server ( software/coils_form.html) with a threshold of 0.9 (Lupas et al. 1991). There were three criteria to classify gene family. Both the coverage (aligned sequence/gene lengths) and identity between sequences were not less than 70%. The stricter criteria were not less than 80 and 90%. Journal of Genetics, DOI /s , Vol. 95, No. 4, December

2 Yingjun Li et al. Sequence alignment and phylogenetic analysis The NB-ARC domain sequences of 96 NBS-encoding genes were aligned by using MUSCLE program in MEGA 5.0 (Tamura et al. 2011). The phylogenetic tree was constructed based on the neighbour-joining (NJ) method with the default options and bootstraps by ClustalW 2.0 (Larkin et al. 2007). Calculation of K s and K a /K s The ratios of nonsynonymous substitution (K a ) to synonymous substitution (K s ) were computed in the gene families and divided according to the criterion of the coverage and the identity between sequences not less than 70%. The nucleotide coding sequences (CDSs) in each gene family were aligned by ClustalW 2.0 and the values of K a, K s and K a /K s were calculated by MEGA ver Test for positive pressures The phylogenetic analysis by maximum likehood 4 (PAML4) package was used to test selection pressures on NBSencoding genes in gene families with three or more members 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 set to identify the positive selection sites. Moreover, the LR test between model M7 and M8 was performed by the critical criterion of chi-square (P < 0.05, df = 2) and (P < 0.01, df = 2), respectively. For the branch model, one single dn/ds ratio (model = 0) and models 0 (NS site = 0) were used to detect the dn/ds in gene families. Results and discussion Identification of NBS-encodings in kiwi fruit We compared our results with the NBS-encoding genes identified in Huang et al. s (2013), two sequences (Achn and Achn ) were found as the same gene, and Achn was also detected encoding NB-ARC domain, which were not in Huang s results. As a result, 96 NBSencoding genes were identified containing 74 NBS LRR genes (table 1), which were more than that in papaya (36) (Ming et al. 2008), and cucumber (52) (Wan et al. 2013), but less in strawberry (144) (Zhong et al. 2015), and Arabidopsis (147) (Meyers et al. 2003). Further, the proportions of NBS LRR genes in whole genome genes in papaya (0.145%), cucumber (0.0821%), strawberry (0.439%) and Arabidopsis (0.544%), were 0.76-, 0.33-, and fold to that of kiwi fruit (0.246%), which indicated that the number of NBS LRR genes did not evolve proportionally with the genome. Further, a relatively fewer number of NBS LRR disease-resistant genes in kiwi fruit, Hongyang may be related to its disease susceptibility. In addition, kiwi fruit genome underwent the recent whole-genome duplication (WGD), but papaya and cucumber genomes were absent Table 1. Number of identified NBS-encoding genes in kiwi fruit. Predicted protein domain Letter code A. chinensis NBS-encoding genes 96 NBS LRR type 74 TIR-NBS LRR TNL 9 nontir-nbs LRR non-tnl 65 CC-NBS LRR CNL 17 X-NBS LRR XNL 48 NBS 22 TIR-NBS TN 2 nontir-nbs non-tn 20 CC-NBS CN 3 X-NBS XN 17 Whole genome genes Proportion of NBS-encoding genes 0.246% Proportion of NBS LRR genes 0.190% Proportion of TIR-NBS LRR genes 0.023% Proportion of nontir-nbs LRR genes 0.166% Average exon of all genes 4.63 Average exon of TIR-NBS LRR 3.33 Average exon of nontir-nbs LRR 2.34 Average exon of CC-NBS LRR 2.24 Average exon of NBS-encoding genes 2.35 Average exon of NBS LRR genes 2.46 of it, which may be the reason that the number of NBSencoding genes was larger in papaya and cucumber genomes (Huang et al. 2009). The average number of exons of NBS-encoding genes and NBS LRR genes were 2.35 and 2.46, respectively which were less than the average number of whole-genome predicted genes (4.63) (table 1). The phenomenon was also observed in other species, such as Arabidopsis, rice, poplar and strawberry. Recent duplications of NBS-encoding genes were detected in the kiwi fruit genome Gene duplication provides new genes for different mechanisms of evolution and creates genetic novelty in organisms (Magadum et al. 2013). We divided 96 NBS-encoding genes into gene families with three criteria to detect the duplication events. For the criterion of 70%, 50% of NBS-encoding genes (48) were classified into 13 multigene families, which suggested that half of the NBS-encoding genes could be detected under duplication events. Moreover, the average number of each family was significantly greater than that in Arabidopsis (t-test, P < 0.01), and smaller than that in strawberry (t-test, P < 0.01). To detect more recent duplication, we applied the criteria of 80%. As a result, there are 40 NBSencoding genes (41.67%) belonging to 13 multigene families and the proportion of NBS-encoding genes was significantly larger than that of Arabidopsis (t-test, P < 0.01). When the third criterion of 90% was applied, the proportion of multigenes (21.88%) in all NBS-encoding genes reduced significantly (t-test, P < 0.01). Thus, we could find out that recent duplication mainly resulted in the existing NBS-encoding genes 998 Journal of Genetics, Vol. 95, No. 4, December 2016

3 NBS-encoding genes in kiwi fruit in kiwi fruit genome. In addition, K s peaked in the range of , and the proportion of the frequency of K s in the range of was 45.92% (figure 1), demonstrating that the recent duplication played an important role in the existing NBS-encoding genes in kiwi fruit. In the evolutionary history, kiwi fruit underwent an ancient WGD shared by core eudicots, and two recent WGD events (Huang et al. 2013). Moreover, the recent WGD events occurred after the kiwi fruit tomato or kiwi fruit potato divergences (Huang et al. 2013). It was assumed that the duplications of NBS-encoding genes might appear in recent WGD events. Besides, the K s peaked at about 0.2 in kiwi fruit whole genome, close to K s peak of in NBS-encoding genes, which further demonstrated our speculation that NBS-encoding genes duplication event occurred approximately at recent WGD. Phylogenetic analysis of NBS-encoding genes In general, the NBS region has high conservative and is usually used to construct phylogenetic tree, while 5 region preceding the NBS and 3 region following the NBS are various and not included for phylogenetic analysis. To study the evolutionary relationships of the NBS-encoding genes in kiwi fruit genome, a phylogenetic tree was constructed based on the nucleotide sequences of NBS domain using the neighbour-joining (NJ) method. All 96 gene types are marked in the phylogenetic tree (figure 2). In addition, five NBS-encoding genes encoding RPW8 domain were marked with solid squares (figure 2). RPW8 domains had a wide range of resistance to powdery mildew pathogens in Arabidopsis (Xiao et al. 2001). Among the five genes, four clustered in the evolutionary analysis at the basal position with longer length branches. Table 2. Selective pressures of NBS-encoding genes in kiwi fruit. Family no. 2 ln a K a /K s ω b Family Family ** Family ** Family Family ** Family ** Family ** Family Family Family Family Family Family ** a The result of the LR test for the site model; ** highly significant (2 ln > 9.210, P < 0.01) tests for positive selection between model M7 and M8. b The dn/ds ratio for each gene family using the branch model., Families with only two members not in PAML analysis. Selection pressure on NBS-encoding genes of kiwi fruit Positive selection drives the host pathogen coevolution and selection for new resistance specificities (Mondragon- Palomino et al. 2002). To detect and measure the direction and intensity of selection, we estimated the ratios of the nonsynonymous substitution to the synonymous substitution (K a /K s or dn/ds) using MEGA 5.0 in all families and using PAML analysis in families with more than two members. Firstly, by using MEGA 5.0, K a /K s in 84.62% (11/13) of the gene families were less than one, showing the majority of the duplicated genes underwent purifying selection (table 2). Besides, there were also two families under positive selection. The relationship of K a /K s and K s in figure 1 implied that younger genes had less selective pressure. Secondly, in PAML analysis, the values of ω (dn/ds) calculated in families with more than two members were all less than one, meaning the purifying selection functioned on the evolution of NBS-encoding genes (table 2). Besides, the average of K a /K s ratio (0.7571) was significantly higher than that in strawberry (0.6638) (t-test, P < 0.01). Finally, combined results of two showed that the purifying selection Figure 1. The frequency distribution of K s (bar chart) and the relationship between K s and K a /K s (line chart). The x-axis denotes average K s per unit of 0.1; y-axis denotes frequency and average K a /K s ratios, respectively. played a main role in the evolution of NBS-encoding genes in kiwi fruit. In addition, amino acid sites under positive selection pressure were identified in PAML analyses in gene families with more than two members. A total of 125 amino acid sites were subjected to positive selection, containing 42 sites under significant positive selection and 83 sites under highly significant positive selection. Therefore, positive selections played a certain role in the NBS-encoding genes. Meanwhile, many amino acid sites were under positive selection pressure, which might be an evolutionary profile to identify NBS-encoding genes that possibly played a role in disease resistance (Mondragon-Palomino et al. 2002). Journal of Genetics, Vol. 95, No. 4, December

4 Yingjun Li et al RNL Achn RNL Achn RNL Achn RNL Achn TNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XNL Achn CN Achn XN Achn XN Achn XNL Achn XNL Achn XNL Achn XNL Achn XN Achn XNL Achn CN Achn XNL Achn TNL Achn TNL Achn XNL Achn XN Achn XNL Achn CNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XNL Achn XN Achn XN Achn TNL Achn XNL Achn XNL Achn XNL Achn XNL Achn TNL Achn XNL Achn XN Achn XN Achn XNL Achn TNL Achn CNL Achn RNL Achn CNL Achn XN Achn XN Achn XN Achn TN Achn XN Achn XNL Achn XNL Achn XNL Achn CNL Achn XNL Achn CNL Achn CNL Achn CNL Achn CNL Achn CNL Achn XNL Achn XN Achn XN Achn XN Achn CNL Achn XN Achn CNL Achn TNL Achn CNL Achn XNL Achn XNL Achn CNL Achn CNL Achn CN Achn XNL Achn CNL Achn XNL Achn CNL Achn XNL Achn XNL Achn XNL Achn TNL Achn TNL Achn XNL Achn CNL Achn XNL Achn XNL Achn TN Achn XNL Achn Figure 2. Phylogenetic tree of NBS-encoding genes in kiwi fruit. Solid squares represent NBS-encoding genes encoding the RPW8 domain. Journal of Genetics, Vol. 95, No. 4, December 2016

5 NBS-encoding genes in kiwi fruit Acknowledgements This project was funded by National Natural Science Foundation of China ( ), the National Agriculture Ministry 948 Project (#2011-G21), China, and by the Priority Academic Programme Development of Modern Horticultural Science in Jiangsu province, China. References Biondi E., Galeone A., Kuzmanović N., Ardizzi S., Lucchese C. and Bertaccini A Pseudomonas syringae pv. actinidiae detection in kiwi fruit plant tissue and bleeding sap. Ann. Appl. Biol. 162, Dangl J. L. and Jones J. D Plant pathogens and integrated defence responses to infection. Nature 411, Ferrante P. and Scortichini M Molecular and phenotypic features of Pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker on yellow kiwi fruit (Actinidia chinensis) in central Italy. Plant Pathol. 59, Huang S. W., Li R. Q., Zhang Z. H., Li L., Gu X. F., Fan W. et al The genome of the cucumber, Cucumis sativus L. Nat. Genet. 41, U1275 U1281. Huang S. X., Ding J., Deng D. J., Tang W., Sun H. H., Liu D. Y. et al Draft genome of the kiwi fruit Actinidia chinensis. Nat. Commun. 4, 2640, doi: /ncomms3640. Jones J. D. G. and Dangl J. L The plant immune system. Nature 444, Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H. et al Clustal W and clustal X version 2.0. Bioinformatics 23, Li C., Jiang J. X., Liu L. P., Cui C. Y., Huang T. and Liu D. Q Fruit stem blight on kiwi fruit (Actinidia chinensis) in China. Canadian J. Plant Pathol. 35, Lupas A., Van Dyke M. and Stock J Predicting coiled coils from protein sequences. Science 252, Magadum S., Banerjee U., Murugan P., Gangapur D. and Ravikesavan R Gene duplication as a major force in evolution. J. Genet. 92, Martin G. B., Bogdanove A. J. and Sessa G Understanding the functions of plant disease resistance proteins. Annu. Rev. Plant Biol. 54, McHale L., Tan X., Koehl P. and Michelmore R. W Plant NBS LRR proteins: adaptable guards. Genome Biol. 7, 212. Meyers B. C., Kozik A., Griego A., Kuang H. and Michelmore R. W Genome-wide analysis of NBS LRR-encoding genes in Arabidopsis. Plant Cell 15, Ming R., Hou S. B., Feng Y., Yu Q. Y., Dionne-Laporte A., Saw J. H. et al The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).Nature 452, U991 U997. Mondragon-Palomino M., Meyers B. C., Michelmore R. W. and Gaut B. S Patterns of positive selection in the complete NBS LRR gene family of Arabidopsis thaliana. Genome Res. 12, Tamura K., Peterson D., Peterson N., Stecher G., Nei M. and Kumar S MEGA 5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, Wan H., Yuan W., Bo K., Shen J., Pang X. and Chen J Genome-wide analysis of NBS-encoding disease resistance genes in Cucumis sativus and phylogenetic study of NBS-encoding genes in Cucurbitaceae crops. BMC Genomics 14, 109. Xiao S. Y., Ellwood S., Calis O., Patrick E., Li T. X., Coleman M. et al Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8. Science 291, Yang Z. H PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, Zhong Y., Yin H., Sargent D. J., Malnoy M. and Cheng Z. M Species-specific duplications driving the recent expansion of NBS LRR genes in five Rosaceae species. BMC Genomics 16, 77. Received 5 November 2015, in final revised form 16 March 2016; accepted 17 March 2016 Unedited version published online: 21 March 2016 Final version published online: 24 November 2016 Corresponding editor: UMESH C. LAVANIA Journal of Genetics, Vol. 95, No. 4, December