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). Thus, we could find out that recent dupli- tion (WGD), but papaya and cucumber genomes were absent cation mainly resulted in the existing NBS-encoding genes

998 Journal of Genetics, Vol. 95, No. 4, December 2016 NBS-encoding genes in kiwi fruit in kiwi fruit genome. In addition, Ks peaked in the range of Table 2. Selective pressures of NBS-encoding genes in kiwi fruit. 0.1 − 0.2, and the proportion of the frequency of Ks in the a b range of 0.1 − 0.2 was 45.92% (figure 1), demonstrating that Family no. 2 ln Ka/Ks ω the recent duplication played an important role in the exist- Family 0 – 0.220 – ing NBS-encoding genes in kiwi fruit. In the evolutionary Family 1 57.365** 0.732 0.671 history, kiwi fruit underwent an ancient WGD shared by core Family 2 191.015** 0.670 0.770 eudicots, and two recent WGD events (Huang et al. 2013). Family 3 – 0.461 – Moreover, the recent WGD events occurred after the kiwi Family 4 12.976** 0.747 0.937 fruit–tomato or kiwi fruit–potato divergences (Huang et al. Family 5 173.359** 0.494 0.655 Family 6 231.024** 0.829 0.834 2013). It was assumed that the duplications of NBS-encoding Family 7 – 0.671 – genes might appear in recent WGD events. Besides, the Family 8 – 0.603 – Ks peaked at about 0.2 in kiwi fruit whole genome, close Family 9 2.420 0.425 0.477 to Ks peak of 0.1–0.2 in NBS-encoding genes, which fur- Family 10 – 1.175 – ther demonstrated our speculation that NBS-encoding genes Family 11 – 1.004 – Family 12 104.619** 0.826 0.932 duplication event occurred approximately at recent WGD. aThe result of the LR test for the site model; ** highly significant (2 ln > 9.210, P < 0.01) tests for positive selection between Phylogenetic analysis of NBS-encoding genes model M7 and M8. bThe dN/dS ratio for each gene family using In general, the NBS region has high conservative and is the branch model. –, Families with only two members not in PAML usually used to construct phylogenetic tree, while 5 region analysis. preceding the NBS and 3 region following the NBS are var- ious 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.

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 direc- Figure 1. The frequency distribution of Ks (bar chart) and the rela- tionship between K and K /K (line chart). The x-axis denotes tion and intensity of selection, we estimated the ratios of s a s average Ks per unit of 0.1; y-axis denotes frequency and average the nonsynonymous substitution to the synonymous substi- Ka/Ks ratios, respectively. tution (Ka/Ks or dN/dS) using MEGA 5.0 in all families and using PAML analysis in families with more than two mem- bers. Firstly, by using MEGA 5.0, Ka/Ks in 84.62% (11/13) played a main role in the evolution of NBS-encoding genes of the gene families were less than one, showing the major- in kiwi fruit. ity of the duplicated genes underwent purifying selection In addition, amino acid sites under positive selection pres- (table 2). Besides, there were also two families under posi- sure were identified in PAML analyses in gene families tive selection. The relationship of Ka/Ks and Ks in figure 1 with more than two members. A total of 125 amino acid implied that younger genes had less selective pressure. Sec- sites were subjected to positive selection, containing 42 sites ondly, in PAML analysis, the values of ω (dN/dS) calculated under significant positive selection and 83 sites under highly in families with more than two members were all less than significant positive selection. Therefore, positive selections one, meaning the purifying selection functioned on the evo- played a certain role in the NBS-encoding genes. Mean- lution of NBS-encoding genes (table 2). Besides, the aver- while, many amino acid sites were under positive selection age of Ka/Ks ratio (0.7571) was significantly higher than pressure, which might be an evolutionary profile to identify that in strawberry (0.6638) (t-test, P < 0.01). Finally, com- NBS-encoding genes that possibly played a role in disease bined results of two showed that the purifying selection resistance (Mondragon-Palomino et al. 2002).

Journal of Genetics, Vol. 95, No. 4, December 2016 999 Yingjun Li et al.

1000 RNL Achn014741 977 RNL Achn280441 850 RNL Achn028111 1000 RNL Achn136851 TNL Achn208841 882 XNL Achn022151 XNL Achn034191 679 961 XNL Achn034201 XNL Achn034181 736 XNL Achn192091 227 394 XNL Achn192101 788 XNL Achn163791 298 XNL Achn163791 257 CN Achn328231 XN Achn204661 351 XN Achn334461 512 917 XNL Achn047351 XNL Achn238081 XNL Achn047361 943 XNL Achn047371 835 339 XN Achn151011 606 569 XNL Achn334451 521 969 CN Achn334431 828 XNL Achn334411 462 TNL Achn334471 TNL Achn369911 180 XNL Achn067721 973 944 960 XN Achn237541 XNL Achn370391 999 769 CNL Achn369921 XNL Achn369901 738 XNL Achn066161 860 XNL Achn096341 835 XNL Achn228931 XNL Achn098511 1000 XN Achn074481 XN Achn074501 1000 TNL Achn388651 177 515 1000 627 XNL Achn388661 XNL Achn340061 645 XNL Achn230571 598 567 XNL Achn078231 TNL Achn337321 510 XNL Achn294261 974 XN Achn373371 1000 XN Achn075521 357 XNL Achn255261 325 TNL Achn378361 1000 CNL Achn112411 662 RNL Achn112661 444 CNL Achn178051 1000 XN Achn178071 XN Achn238071 629 XN Achn111221 1000 TN Achn345031 250 XN Achn025391 XNL Achn025411 781 250 XNL Achn122641 XNL Achn027941 608 275 CNL Achn356511 XNL Achn025421 992 CNL Achn037891 997 590 942 CNL Achn356001 CNL Achn320751 CNL Achn095991 531 1000 CNL Achn070111 742 XNL Achn231791 XN Achn128441 1000 XN Achn260981 881 XN Achn180711 882 CNL Achn180731 983 XN Achn153631 1000 CNL Achn173061 1000 TNL Achn174311 CNL Achn348041 313 1000 XNL Achn348061 XNL Achn155461 371 997 953 CNL Achn328251 CNL Achn328301 1000 CN Achn328271 444 996 XNL Achn227881 474 CNL Achn376721 1000 XNL Achn376751 736 901 CNL Achn377051 1000 XNL Achn376741 XNL Achn276081 XNL Achn165411 951 TNL Achn275231 917 TNL Achn030861 1000 XNL Achn356971 CNL Achn099851 1000 XNL Achn075601 263 XNL Achn231191 959 TN Achn079101 996 XNL Achn180641

0.05

Figure 2. Phylogenetic tree of NBS-encoding genes in kiwi fruit. Solid squares represent NBS-encoding genes encoding the RPW8 domain.

1000 Journal of Genetics, Vol. 95, No. 4, December 2016 NBS-encoding genes in kiwi fruit

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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

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