Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula

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1 Chin. Sci. Bull. (2014) 59(11): DOI /s Article csb.scichina.com Bioinformatics Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula Hui Song Zhibiao Nan Received: 6 May 2013 / Accepted: 13 November 2013 / Published online: 23 February 2014 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2014 Abstract The class of nucleotide-binding site (NBS)- Leucine-rich repeat (LRR) disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Consequently, many NBS-LRR genes have been identified in various plant species. In this study, we identified 617 NBS-encoding genes in the Medicago truncatula genome (Mt3.5v5) and divided them into two groups, regular (490) and non-regular (127) NBS- LRR genes. The regular NBS-LRR genes were characterized on the bases of structural diversity, chromosomal location, gene duplication, conserved protein motifs, and EST expression profiling. According to N-terminal motifs and LRR motifs, the 490 regular NBS-LRR genes were then classified into 10 types: CC-NBS (4), CC-NBS-LRR (212), TIR-NBS (20), TIR-NBS-LRR (160), TIR-NBS-TIR (1), TIR-NBS-TIR-LRR (2), NBS-TIR (7), NBS-TIR-LRR (1), NBS (10), and NBS-LRR (73). Analysis of the physical location and duplications of the regular NBS-LRR genes revealed that the M. truncatula genome is similar to rice. Interestingly, we found that TIR-type genes are more frequently expressed than non-tir-type genes in M. truncatula, whereas the number of non-tir-type regular NBS- LRR genes was greater than TIR-type genes, suggesting the gene expression was not associated with the total number of NBS-LRR genes. Moreover, we found that the phylogenetic tree supported our division of the regular Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. H. Song Z. Nan (&) Key Laboratory of Grassland Agro-Ecosystems of Ministry of Agriculture, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou , China zhibiao@lzu.edu.cn NBS-LRR genes into two distinct clades (TIR-type and non-tir-type), but some of the non-tir-type lineages contain TIR-type genes. These analyses provide a robust database of NBS-LRR genes in M. truncatula that will facilitate the isolation of new resistance genes and breeding strategies to engineer disease resistance in leguminous crop. Keywords Medicago truncatula Disease resistance genes Nucleotide-binding sites Phylogenetic tree 1 Introduction Diseases are one of plant s major threats and may be caused by diverse microorganisms, including bacteria, fungi, viruses, nematodes, and insects. Over evolutionary time plants have developed various sophisticated mechanisms to protect themselves. One of the roles of the plant immune response is characterized by gene-for-gene interactions between a host disease resistance (R) gene and a pathogen avirulence (Avr) gene [1]. The R genes may be divided into at least five classes based on the structure of the encoded proteins [2 5] and the biggest category is NBS-LRR proteins [6]. Although how NBS-LRR proteins function in disease resistance is not completely clear, previous studies show that plants contain a large number of NBS-LRR proteins to confer resistance to diverse pathogens [7]. The NBS-LRR protein contains two major domains, the nucleotide-binding site (NBS) and the leucine-rich repeat (LRR) [8]. However, many plant NBS-LRR proteins also contain coiled-coil (CC) or a Toll/mammalian interleukin-1 receptor (TIR) domain at their N-terminal end [8, 9].

2 1130 Chin. Sci. Bull. (2014) 59(11): Therefore, NBS-LRR proteins are classified into TIR-NBS- LRR (TNL) and CC-NBS-LRR (CNL) groups [10]. In general, CNL genes are found in both monocot and dicot genomes, whereas TNL genes have been identified only in dicots [11]. Generally, the NBS domain contains several conserved motifs, which have been confirmed to play a role in binding and hydrolysis of ATP and GTP [2, 12, 13]. In addition, over-expression of the TIR or CC domain of some NBS-LRR proteins can induce hypersensitive response [14 16]. Recently, Sayar-Turet et al. [17] used NBS conserved motifs as a molecular marker to assess variation associated with potentially functional regions of the genome underlying specific phenotypes. Previous studies found that the LRR domain mediated direct or indirect interactions with pathogen molecules [18]. In addition, the LRR domain seems to act as a negative regulator to block inappropriate NBS activation [6]. Medicago truncatula (2n = 16) is an annual diploid in the tribe Trifolieae, cultivated as a forage crop and closely related to tetraploid alfalfa (Medicago sativa). However, M. truncatula is susceptible to a variety of pathogens. As an important economic crop, it is essential that we find ways to improve the disease resistance in M. truncatula. In recent years, more and more draft sequences of M. truncatula have been described [19], providing a unique opportunity for analysis of NBS-LRR genes in M. truncatula. NBS-LRR genes belong to one of the largest gene families in plant genomes and have been detected in a wide variety of plants, including nonvascular plants and angiosperms (i.e., Physcomitrella patens and Arabidopsis thaliana) [20, 21]. Although NBS-LRR genes have been identified in the M. truncatula draft genome (Mt1.0) and analyzed according to their phylogenetic pattern and genomic organization [22], the M. truncatula genome was resequenced in 2011 and this draft have better coverage of the genome [23]. Therefore, in this study, we identified 617 NBS-LRR genes in the recently sequenced genome (Mt3.5v5), including 490 regular and 127 non-regular NBS-LRR genes. We analyzed structural diversity, phylogenetic relationships, gene duplication, chromosomal location, conserved motif, and EST expression profiling in the NBS-LRR gene family. 2 Materials and methods 2.1 Database search and sequence retrieval The M. truncatula genome sequence (Mt3.5v5) was downloaded from The complete set of NBS-LRR gene sequences was identified using a deliberative process. First, a Hidden Markov Model (HMM) profile of NBS domain (PF00931) was downloaded from the Pfam database ( ac.uk/) [24]. We employed the NBS domain as a query to identify all possible NBS-LRR sequences in the M. truncatula genome database using the BLASTP program (P = 0.001), according to the method from previous studies [10, 25]. To insure that there were no additional related genes missing from the NBS-LRR gene prediction, another NBS domain was generated from alignments of A. thaliana NBS-LRR sequences downloaded from niblrrs.ucdavis.edu. Subsequently, searches on the Pfam database and COILS ( were used to confirm and classify each predicted NBS-LRR sequence. We located overlapping genes by aligning all of the candidate NBS-LRR sequences using Clustal W [27] and manually checking the alignment. Only the nonoverlapping NBS-LRR sequences were used for further analysis. 2.2 Identification of conserved motifs The program MEME (version 4.8, [28] was used to elucidate motifs in M. truncatula NBS- LRR protein sequences with the following parameters as described in previous methods [29]: (1) optimum motif widths were constrained to residues; (2) the number of repetitions was set to any, and (3) the maximum number of motifs was set to 20. Structural motif annotation was performed using the Pfam and COILS databases, as well as the SMART program ( de) [30]. 2.3 Phylogenetic analysis It is well known that amino acid sequences of NBS resistance genes are highly conservative [10]. Multiple sequence alignments of the highly conserved NBS domain were performed by Clustal X (version 1.83) [31]. The tree was analyzed and displayed in MEGA (version 4.0) software [32] with the neighbor-joining method and 1,000 bootstrap replicates. NBS-LRR genes are known to be subdivided into two clades: TIR-type and non-tir-type. Therefore, to perform phylogenetic analyses, we constructed TIR-type and non-tir-type phylogenetic trees between M. truncatula and Glycine max using the whole protein sequences by the same method. 2.4 Chromosomal locations of NBS-LRR genes and gene duplication The chromosomal locations of NBS-LRR genes were identified by methods described by Meyers [10]. Genome

3 Chin. Sci. Bull. (2014) 59(11): Pixelizer software was used to draw the location images of the M. truncatula NBS genes ( edu/genomepixelizer/genomepixelizer_w-elcome.html). To detect potential gene duplication, we aligned and calculated all of the relevant genes identified in M. truncatula genomes. We defined gene duplication such that between any two loci [25]: (1) the alignable nucleotide sequence covered [70 % of the longer sequence; (2) the amino acid identity between the sequences was [70 % identical. 2.5 Digital expression analysis: EST expression profiling Medicago truncatula EST sequences were downloaded from GenBank ( The NBS-LRR genes were used as query to perform a local BLASTN search against all of the ESTs. The following parameters were employed [29]: maximum sequence similarity [96 %, length [ 200 bp, and E-value \ Results 3.1 Identification and classification of NBS-LRR genes In this study, we identified 617 NBS-LRR genes with the 375 Mb Mt3.5v5 draft genome [19], including 490 regular and 127 non-regular NBS-LRR genes (Table 1, Table S1). The length of non-regular NBS domain is shorter (B2/3 of normal NBS domain) than that of normal NBS domain [25, 33, 34]. TIR-only and TIR-X sequences were excluded in this work. The 617 regular and non-regular NBS-LRR genes were analyzed against the Pfam database. In 490 out of 617 NBS-LRR genes, all of the characteristic NBS domain motifs are conserved. The non-regular genes are totally different in their structures and lack specific motifs and/or domains. In the regular NBS-LRR genes, we detected 191 TIRtype resistance genes, including NBS-TIR (7), TIR-NBS (20), NBS-TIR-LRR (1), TIR-NBS-LRR (160), TIR-NBS- TIR (1), and TIR-NBS-TIR-LRR (2). We found 216 non- TIR-type genes, including CC-NBS (4) and CC-NBS-LRR (212). Eighty-three NBS-LRR genes lacked CC and TIR motif regions (10 NBS and 73 NBS-LRR; Table 1). In addition, we found 22 potential pseudogenes among the 127 non-regular NBS-LRR genes with either a premature stop codon or a frameshift mutation (Table S2). The non-regular NBS-LRR genes include CC (25), TIR (31), and LRR (70) motif regions and 37 non-regular NBS-LRR genes contained only a conserved NBS motif (Table 1). Noticeably, the TIR motif was not detected in regular Table 1 Numbers of NBS-LRR gene in M. truncatula and rice genomes Predicted Letter code Medicago Rice a Regular NBS genes NBS N NBS-LRR NL CC-NBS CN 4 7 CC-NBS-LRR CNL NBS-TIR NT 7 0 TIR-NBS TN 20 0 NBS-TIR-LRR NTL 1 0 TIR-NBS-LRR TNL TIR-NBS-TIR TNT 1 0 TIR-NBS-TIR-LRR TNTL 2 0 Total regular NBS genes Non-regular NBS genes NBS NBS-LRR CC-NBS 3 0 CC-NBS-LRR 22 0 TIR-NBS 15 3 NBS-TIR 2 0 TIR-NBS-LRR 14 0 Total non-regular NBS genes a Data from Zhou et al. [25] NBS-LRR genes of the rice genome, except in non-regular NBS-LRR genes (3TIR-NBS; Table 1). 3.2 Identification of conserved motifs To analyze the conserved motifs of NBS-LRR genes in M. truncatula, we divided CNL (212) and TNL (160) groups into three parts defined by their N-terminal, NBS, and LRR domains (Table 1). The detailed motif sequences are shown in Table S3. The N-terminal domain in NBS-LRR genes covers amino acids. The N-terminal CC domain has been identified as a representative motif in the amino terminal of CNL proteins [35], and the presence or absence of a CC motif can be anticipated on the basis of characteristic motifs present in the NBS-LRR [21, 36]. In this study, we identified 20 motifs in the N-terminal region from CNL genes (Table S3). Compared with the CNL gene, motifs found in A. thaliana and rice [10, 25], the CNL genes in M. truncatula are highly variable and show many different patterns. The CNL genes all contained a Q (L/I/V) RD motif at the N-terminal [37] and we found similar motifs in the N-terminal domain (Table S3, motif 10).

4 1132 Chin. Sci. Bull. (2014) 59(11): Several conserved motifs were confirmed as present in the TIR domain of plant NBS-LRR genes and related proteins (Table S3). Sequence analysis showed high similarity between TIR motifs of M. truncatula and counterparts in A. thaliana [10]. A previous study in A. thaliana identified eight major NBS motifs [10], including P-loop, RNBS-A, Kinase 2, RNBS-B, RNBS-C, GLPL, RNBS-D, and MHDV. The sequences of these motifs differ in CNL and TNL proteins. In this study, MEME analyses identified seven consensus sequence motifs, but lack MHDV motif in CNL and TNL genes of M. truncatula (Table 2). The MHDV motif in different plant species has been classified as MHDL, MHSL, QHDV, or QHDL [11, 25, 34, 38]. The MHDV motif was not detected in the M. truncatula draft genome (Mt3.5v5) using MEME software, prompting us to consider several possibilities. (1) MEME may not have been able to recognize MHDV motif in M. truncatula if the MHDV motif possesses high levels of diversity in different plants. (2) MHDV motif in M. truncatula may have originated through a different origin of CNL and TNL genes. (3) An ancestral MHDV motif may have been lost during the evolution of M. truncatula or evolved into other structures through mutation. The LRR regions were characterized and found to be variable in sequence, size, and occurrence per gene [11]. High sequence variability in LRR is consistent with their role in determining the recognition specificity of pathogen encoded Avr proteins [38]. To investigate structural features in the LRR domain, we analyzed those domains using the COILS program, excluding some of the CNL and TNL genes that had a short LRR domain (\300 amino acids). 3.3 Phylogenetic analysis A phylogenetic analysis of 490 regular NBS-LRR sequences was performed to classify genes into groups and identify relationships between genes. One hundred and twenty-seven genes (non-regular genes) were excluded due to their excessively short length (B2/3 the normal length). Since a previous study showed that phylogenetic analyses of NBS domains allow groups of R genes to be distinguished [21], we constructed a composite phylogenetic tree for all candidate genes using the NBS domain sequence. There was a clear separation between the TIR-type and non-tir-type clades with well-supported bootstrap values, reflecting ancient differentiation of NBS-LRR genes into two major groups (Figs. 1, S1). However, in some cases, the distribution of M. truncatula NBS-LRR proteins reveals a high level of sequence divergence within TIRtype and non-tir-type clades. For example, a clade of non- TIR-type sequences resolves within eight TIR-type proteins (Fig. 1). Moreover, NBS/NBS-LRR genes were distributed in both TIR and non-tir groups. This topology indicates a paraphyletic origin of NBS/NBS-LRR genes and suggests that they may be distinguished by a loss of a domain [38]. Based on the major division evident in the NBS domain tree, two neighbor-joining trees were constructed from the aligned whole protein sequences of G. max and M. Table 2 Consensus sequences of major NBS domain motif in predicted M. truncatula CNL and TNL proteins Class Motif Consensus sequence a TIR-NBS-LRR P-loop IWGMSGIGKTTIAKQLFAKLF RNBS-A LVHLQEQLLSKLLGEK Kinase 2 RLxRKKVLIVLDDVD RNBS-B WFGPGSRIIVTTRDKHLLxSH RNBS-C YEVKELNETEALELFCWKAF GLPL YAGGLPLALKVLGSNLFGKSIEEWKSALDKLEKIPN RNBS-D EKSIFLDIACFFNGx CC-NBS-LRR P-loop VISIVGMGGLGKTTLAQLVYN RNBS-A QKHFDLKIWVCVSED Kinase 2 KRFLLVLDDVW RNBS-B DGSKGSKVLVTTRSEKVAxIM RNBS-C HxLxGLSEEEAWSLFKKHAFG GLPL LExIGKEIAKKCGGLPLAIKTLGGLLRSK RNBS-D CFLYCALFPEDYEIxKKxLIRLWIAEGFIxS a Consensus amino acid sequence derived from MEME. Related motifs in the NBS domains of CNL and TNL proteins are aligned. X indicates a non-conserved residue. Underlined residues indicate consensus sequences identical to the motifs detected in Arabidopsis NBS domains by Meyers et al. [10]

5 Chin. Sci. Bull. (2014) 59(11): Fig. 1 Phylogenetic relationship of the TIR-type and non-tir-type NBS-LRR proteins in M. truncatula. The neighbor-joining tree was constructed using the NBS domains. Black circle, red circle, and diamond indicate the TIR-type, non-tir-type, and NBS/NBS-LRR genes, respectively, truncatula, resulting in trees of TIR-type and non-tir-type NBS-LRR genes (Figs. 2, S2, and S3). In some cases, one clade includes many genes from a single species, indicating that some gene clades are species-specific, and these sequences were highly conserved by the gene duplication. However, in some cases, clades contain genes from G. max and M. truncatula, indicating the same origin or evolution of NBS-LRR genes from G. max and M. truncatula. The phylogeny-based method was often used to identify orthologs or paralogs [39]. In this study, we applied this method to identify 10 possible orthologs pair between G. max and M. truncatula (Fig. 2). The research of the NBS- LRR orthologs of G. max and M. truncatula contributes to the study on genetic transformation between G. max and M. truncatula. 3.4 Chromosomal location of NBS-LRR genes and gene duplication Figure 3 shows the locations of the NBS-LRR genes on the chromosomes of M. truncatula. The chromosomal distribution of the genes is unevenly (Fig. 3). Chromosome 3 contains the largest number of NBS-LRR genes (129), while chromosome 1 contains 15 NBS-LRR genes. There was no obvious difference between the distributions of the TIR-type and non-tir-type genes on the chromosomes (Fig. 3). However, chromosome 3 contributes roughly 89 (44.5 %) of all non-tir-type genes, while chromosome 6 contributes roughly 44 (27.5 %) of all TIR-type genes. Studies in A. thaliana and rice have shown the most NBS-LRR genes are found clustered on certain chromosomes [10, 25]. Holub [40] defined a gene cluster as a chromosome region with four or more genes within 200 kb. Using this window size, we found that 309 NBS- LRR genes reside in 78 gene clusters, and that the average number of NBS-LRR genes in a cluster was The ratio is more than that in A. thaliana (3.21), rice (3.48), maize (2.77), respectively (Table 3). The largest cluster contains 10 NBS-LRR genes located Chromosome 6 and 8, respectively (Table S4). In addition, 181 NBS-LRR singletons were dispersed over all of the chromosomes (Table 3). In this study, we confirmed previously hypothesized genome duplications by BLAST comparison [37]. A total

6 1134 Chin. Sci. Bull. (2014) 59(11): Fig. 2 Phylogenetic comparison of G. max (blue triangle) and M. truncatula (red circle) NBS-LRR genes. Each NBS-LRR predicted orthologous gene was indicated in green color. a Phylogenetic tree base on the whole protein sequence from G. max (blue triangle) and M. truncatula (red circle) CC-NBS-LRR genes. b Phylogenetic tree base on the whole protein sequence from G. max (blue triangle) and M. truncatula (red circle) TIR-NBS-LRR genes of 250 out of the 490 regular NBS-LRR genes with duplication events were detected in 86 gene families in the M. truncatula genome (Table 3). The percentage of NBS-LRR genes in M. truncatula multigene genes (51.0 %; n = 86) and the total number of genes was similar in rice (53.7 %; n = 93), while the number in M. truncatula was greater than A. thaliana (46.6 %; n = 25), maize (37.6 %; n = 17), or sorghum (47.3 %; n = 20). The largest number of genes in a family in M. truncatula was 14 and the average number of family members was Moreover, the average number of genes in each NBS-LRR gene family was 3.24, 3.00, 2.41, and 6.10 in A. thaliana, rice, maize, and sorghum, respectively. In addition, these results suggest that the number of NBS- LRR gene duplications in M. truncatula is similar to those found in rice. 3.5 Digital expression analysis: EST expression profiling Publicly available Expressed Sequence Tags (ESTs) were considered a useful means of studying gene expression profiles (a type of digital northern) [41]. A total of 269,501 M. truncatula EST sequences were downloaded from GenBank. The digital expression profiles of 255 regular NBS-LRR genes were acquired from the EST database at NCBI, which resulted in the assignment of NBS-LRR genes of M. truncatula to eight groups on the basis of tissue and organ types, including shoot, stem, leaf, root, flower, seed, glandular trichome, and multiple tissues. The EST database did not contain sequences for the rest of the 235 NBS-LRR genes (Table S5). EST expression profiling revealed that the maximum number of NBS-LRR genes expressed in multiple tissues was 205 (80.4 %), but only 12 (4.7 %) of the NBS-LRR genes were expressed in seeds. In addition, we discovered that the number of NBS-LRR genes expressed in shoots, stems, leaves, roots, flowers, and glandular trichomes was 34 (13.3 %), 47 (18.4 %), 25 (9.8 %), 77 (30.2 %), 14 (5.5 %), and 23 (9.0 %), respectively. Furthermore, we detected 112 TIR-type, 103 non-tir-type, and 40 NBS/ NBS-LRR genes in the expression database (Table S5). TIR-type genes are more prevalent in the expression data than non-tir-type genes in M. truncatula. 4 Discussion Many aspects of the NBS-LRR disease resistance gene family have been studied extensively and this gene family has been described in monocotyledonous and dicotyledonous plants, including A. thaliana [10, 42], Oryza sativa [25, 43], Vitis vinifera [44], Brassica rapa [11], G. max [45], Sorghum bicolor [46], Brachypodium distachyon [37], Zea mays [34], and Solanum tuberosum [47]. Although the complete genome of M. truncatula had not

7 Chin. Sci. Bull. (2014) 59(11): Fig. 3 Chromosomal locations of M. truncatula NBS-LRR genes. The sizes of chromosome can be estimated using the scale at the top. Boxes above and below each M. truncatula chromosome (chrm; gray bars) designate the approximate locations of each gene. Unmapped NBS-LRR genes are show on the chromosome 0. The arrow on MtChr 5 indicates the TIR-type proteins distributed in non-tir-type clade. Color code: CN (yellow), CNL (orange), N (pink), NL (red), NT (brown), NTL (gray), TN (cyan), TNL (violet), TNT (blue), and TNTL (green) Table 3 Organization of NBS-LRR genes in the five plant genomes Organization Medicago Arabidopsis a Rice a Maize b Sorghum b Single-genes Multi-genes Gene family number Maximal member of a family Average members of a family Percentage of multi-genes Singleton genes Cluster genes Cluster number Maximal members of a cluster Mean members per cluster a Data from Yang et al. [44] Data from Cheng et al. [34]

8 1136 Chin. Sci. Bull. (2014) 59(11): been fully sequenced, approximately 94 % of the genome is assembled. In this study, we identified 617 NBS-LRR genes in the Mt3.5v5 draft genome, which represents 0.99 % of all the predicted proteins. The number of NBS- LRR genes was similar to NBS-LRR genes found in rice (1.00 %) [43] and Populus trichocarpa (1.00 %) [38], while the number of NBS-LRR genes in M. truncatula was higher compared to A. thaliana (0.43 %) [10], B. distachyon (0.77 %) [37], and Carica papaya (0.22 %) [48]. Interestingly, the ratio of NBS-LRR genes in the Mt3.5v5 draft genome was lower than that (1.33 %) of the Mt1.0 draft genome [22], suggesting that the number of NBS- LRR genes would no longer increase with the increase of genome size. In addition, our results show that in M. truncatula there are more non-tir-type genes (241) than TIR-type genes (222), suggesting that the gene duplication of non-tir-type experienced more duplications and more recent duplications than that of TIR-type. This result is similar to that found based on the Mt1.0 genome (152 non-tir-type genes and 122 TIRtype genes) [22]. A previous study analyzed A. thaliana NBS domain regions and estimated a tree that formed two clades [49], corresponding to TIR and non-tir-nbs-lrr genes. However, NBS-LRR genes from rice did not cluster into two clades and generally lacked topological resolution [25]. In this study, the phylogenetic analysis found two distinct clades of 490 NBS-LRR genes. The tree showed multigene genes clustered together (Fig. 1; Table S4), but also recovered clades of a mixture of cluster gene types (Fig. 3, the arrow), suggesting chromosomal rearrangement, to transposition, or to large-scale genomic duplications have occurred in the Medicago lineage. We note that eight TIR-type genes (Medtr5g , Medtr5g , Medtr5g , Medtr5g , Medtr5g , Medtr5g , Medtr5g , and Medtr5g ) were resolved within a clade of non-tir-type genes, suggesting non-tir-type NBS-LRR genes were more conservative than that of the TIR-type. Similarly, the interesting results were also found in grapevine, but the two CNL genes nested in TIR-NBS clade [44]. McHale et al. [7] and Yang et al. [44] confirmed that the CNL group originated before the divergence of the monocots and dicots, while the TNL group appeared more recently in the eudicots, after the divergence of the monocots. Recently, however, a study suggested that TNL genes actually originated earlier than CNL genes [50]. In this study we identified both TNL and CNL genes, and we predicted that the loss of CC domain and fusion of TIR domain could lead to the composition of eight TIR-type genes based on the analysis of phylogentic tree. Therefore, we support an earlier origin of CNL genes by phylogenetic tree. Further study of these eight TIR-type genes may give us additional insights in NBS-LRR evolution. For example, phylogenetic analysis showed that seven TIR-type genes formed a clade, to the exclusion of Medtr5g Chromosomal localization results showed that the seven closely related TIR-type genes are arranged in a cluster on chromosome 5 (Fig. 3, the arrow). This suggests that Medtr5g has a different origin from the other seven TIR-type genes. We attribute above two different conclusions to the fact that the studies had different research objectives. McHale et al. [7] and Yang et al. [44] sampled flowering plants, whereas Yue et al. [50] sampled fungi and non-vascular plants. Although some of the TNL genes were identified in bryophytes through degenerate primer-based PCR and BLAST-based homologous searches, these genes were not obtained as full-length sequences [20, 36]. Therefore, we can not verify the function of TNL genes in bryophytes by Yue et al. s experimental methods [50]. In addition, bryophytes (e.g., P. patens) may be not susceptible to diseases during their relatively short life span, suggesting this may result in rapid divergence of NBS-LRR genes due to natural selection. NBS-LRR contains three main domains, including an N-terminal variable domain, a central nucleotide-binding site (NBS) domain, and a C-terminal leucine-rich repeat (LRR) domain. Depending on whether they also encoded an N-terminal TIR domain, NBS-LRR genes could be further divided into two groups, the TNL group and the CNL group [21]. Previous studies have shown that the TIR, NBS, and LRR domains originated before the split of prokaryotes and eukaryotes [50]. Moreover, some studies have revealed that the TIR domain is more conserved across prokaryotes and eukaryotes than the CC domain [10, 25]. Our results are consistent with that generalization. The M. truncatula LRR regions were highly variable in sequence and size, which we deemed to be evidence that LRR plays an important role in the pathogen recognition process and activation of signal transduction in response to pathogen attack [8, 51, 52]. Although three primary LRR motif patterns were detected in A. thaliana [10], the highly diverse LRR repeats in M. truncatula could not classified. Table S3 demonstrates how difficult it is to identify the motif. According to previous studies, most plant NBS-LRR genes are unevenly distributed and clustered in certain chromosomes [22, 43]. Gene duplication, including tandem and whole-genome duplication (WGD), play a crucial role in increasing gene copy numbers. Nobuta et al. [53] and Yu et al. [54] found that the ratio of NBS-LRR genes in WGD blocks in A. thaliana and O. sativa was 10 % and 0 %, respectively. Yang et al. [44] compared four plants of A. thaliana, O. sativa, P. trichocarpa, and V. vinifera and found that in plant genomes tandem duplication played a primary role in NBS-LRR gene family expansion, but

9 Chin. Sci. Bull. (2014) 59(11): WGD does not. The same conclusions were found in B. distachyon [37]. This study classified 78 gene clusters, including 309 NBS-LRR genes and found that they were randomly distributed on chromosomes 0 8. The results indicted NBS-LRR genes in M. truncatula genome were formed by tandem duplication. We used an EST database to test the expression levels of the regular NBS-LRR from different tissues and organs of M. truncatula. We found approximately 52 % of the regular NBS-LRR genes were expressed in various tissues and organs. The result is similar to that found by Ameline- Torregrosa et al. [22] study. Interestingly, Cheng et al. [34] and Kohler et al. [38] showed that most of the R genes present in a plant genome are not expressed in the transcriptome during a pathogen infection [34, 38]. On the contrary, NBS-LRR genes were only expressed under specific conditions in specific tissues and not necessarily during an infection [55]. 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