All relevant data are within the paper and its Supporting Information files.

Introduction {#sec001}
============

Plant growth and crop production are adversely affected by environmental stresses, such as drought, low temperature, high salinity, pathogen infection, microbial elicitors and wounding. To adapt to these environmental conditions, plants have developed a mechanism that includes the perception of stress signals, subsequent signal transduction, and the activation of various physiological and metabolic responses \[[@pone.0155590.ref001]\]. Calcium ions (Ca^2+^) play a central role as a second messenger in the signal transduction pathways \[[@pone.0155590.ref002]\]. In plants, the perturbation of cytosolic Ca^2+^ concentration responds to diverse endogenous and external signals, including phytohormone status, abiotic stress and biotic stress, as well as mechanical wounding \[[@pone.0155590.ref003]--[@pone.0155590.ref006]\]. These calcium signatures can be recognized by different Ca^2+^ sensor molecules which subsequently transduce the signal to downstream signaling cascades such as phosphorylation of target proteins.

In plants, four main classes of Ca^2+^ sensors have been identified, including calmodulin (CaM), calmodulin-like proteins (CML), calcineurin B-like proteins (CBL) and calcium-dependent protein kinases (CPKs) \[[@pone.0155590.ref007]--[@pone.0155590.ref009]\]. Among them, CPKs are best characterized and constitute a large multigene family, which widely exist in plant kingdom from algae to angiosperms. Structurally, CPK proteins contain four domains, a variable N-terminal domain, a conserved kinase domain, an autoinhibitory region, and a calmodulin-like domain \[[@pone.0155590.ref010]\]. The highly variable N-terminal domain contains myristoylation or palmotylation sites, which facilitate membrane association \[[@pone.0155590.ref011]\]. And the calmodulin-like domain contains EF-hands for binding calcium ions through interactions with alpha-helices \[[@pone.0155590.ref012]\].

CPK proteins have been implicated to participate in immune and stress signaling, growth and development, and hormone responses. Ectopic expression of Arabidopsis *CPK* gene *AtCPK1* enhanced NADPH oxidase activity and oxidative burst\[[@pone.0155590.ref013]\]. AtCPK1 also activates the accumulation of salicylic acid (SA) \[[@pone.0155590.ref014]\]. AtCPK5 was reported to activate respiratory burst oxidase homolog D (RBOHD) to induce a reactive oxygen species (ROS) burst \[[@pone.0155590.ref015]\]. CPKs help to enhance drought tolerance by regulating abscisic acid (ABA) signal transduction (e.g. AtCPK4/11) \[[@pone.0155590.ref016]\], and via stomatal closure (e.g. AtCPK3/6) \[[@pone.0155590.ref017]\]. AtCPK23 helps to enhance plant salt tolerance through controlling of K^+^ channels \[[@pone.0155590.ref018]\]. CPKs also function in hormone signaling, such as ABA \[[@pone.0155590.ref019]\], MeJA\[[@pone.0155590.ref020]\] and ethylene\[[@pone.0155590.ref021], [@pone.0155590.ref022]\]. In addition, some *CPK* genes also participate in root development \[[@pone.0155590.ref023]\] and pollen tube growth \[[@pone.0155590.ref024]--[@pone.0155590.ref026]\].

Because of the critical regulatory functions of *CPK* genes in plant responses to different stresses and developmental processes, *CPK* gene family have been extensively studied in Arabidopsis (*Arabidopsis thaliana*)\[[@pone.0155590.ref027]\], rice (*Oryza sativa*) \[[@pone.0155590.ref028]\], poplar (*Populus trichocarpa*) \[[@pone.0155590.ref029]\], maize (*Zea mays*) \[[@pone.0155590.ref030]\], etc. However, compared to the extensive studies in other species, the *CPK* gene family in the Rosaceae has not been widely surveyed. The most economically important fruit, such as apple (*Malus × domestica*), pear (*Pyrus communis*), peach (*Prunus persica*), plum (*Prunus mume*) and strawberry (*Fragaria vesca*), all belong to the Rosaceae family \[[@pone.0155590.ref031]\]. *M*. *× domestica* is one of the most economically important fruit cultivated worldwide, and 30 *CPK* genes have been identified by bioinformatics \[[@pone.0155590.ref032]\]. Recently, the whole genome of apple, peach, strawberry, plum, and pear has been fully sequenced. This resource provides an opportunity to further analyze the *CPK* gene family in Rosaceae species. In this study, we aim to analyze the *CPK* genes in apple and other Rosaceae fruit species at whole genome scale, elucidate their evolutionary history, and provide a relatively complete overview of the *CPK* gene family in Rosaceae. Using a HMMER-BLASTP-InterProScan strategy, we identified 37 *CPK* genes from apple, and other 86 *CPK* genes from pear, peach, strawberry and plum. Then, their evolution event were surveyed by phylogenetic and synteny analysis. Furthermore, the expression profiles of *CPK* genes in response to pathogen infection were investigated. The identification and comprehensive study for *CPK* genes from Rosaceae will provide valuable information for breeding biotic stress-resistant fruit tree and further studying of the biological function and evolutionary relationship of this family in Rosaceae.

Results {#sec002}
=======

Identification and classification of *CPK* genes in the Rosaceae {#sec003}
----------------------------------------------------------------

A HMMER-BLASTP-InterProScan strategy was used to search for genes encoding CPK proteins. The HMM (Hidden Markov Model) profiles of protein kinase domain (PF00069) and EF-hand domain (PF13499 and PF13202) were downloaded from the Pfam protein family database (<http://pfam.sanger.ac.uk/>). These HMM profiles were used to search proteomes of apple, pear, peach, plum and strawberry by hmmsearch with the threshold set of the Pfam GA gathering cutoff. Then, the HMMER selected proteins were used for a BLASTP query of the original proteomes. Finally, the BLASTP hits were scanned for kinase and EF-hand domains using InterProScan\[[@pone.0155590.ref033]\], then truncated sequences and pseudogenes were removed. Initially, 129 nonredundant putative genes were identified. After manually checking the sequences of these genes, 6 genes were removed for having diverse sequences.

In comparison with a previous study\[[@pone.0155590.ref032]\], in which 30 *MdCPK* genes have been identified, 37 *MdCPK* genes were found in this work, although the apple genome data used is the same version deposited in Phytozome. After carefully checking, we found that the discrepancy in number of *CPK*genes lies in the methods used by these two studies. The previous work used BLASTP to search apple genome using *CPK* genes from *Arabidopsis thaliana*, then all the candidate *CPKs* of apple were scanned using SCAN PROSITE software to confirm the presence of the EF-hands signature motif. We adopted a HMMER-BLASTP-InterProScan strategy that mined the *CPK* genes based on the existence of the HMM profiles of protein kinase domain and EF-hand domain. The advantage of our method is that HMM profile was constructed from some representative *CPKs* from various species, which covered more domain information than that only from Arabidopsis *CPKs*. Our search strategy was tested against Arabidopsis and detected all previously reported 34 *CPKs*, with *AtCPK25* removing for having truncated conserved domain. Using this strategy, we detected 37 *CPK* genes in apple genome, which include all the genes reported by\[[@pone.0155590.ref032]\] except one gene, MDP0000169895, in which no EF-hand was detected. The existence of protein kinase domain and EF-hand domain were further confirmed by SMART (<http://smart.embl-heidelberg.de/>), PfAM (<http://pfam.xfam.org/>), and SUPERFAMILY (<http://supfam.org/SUPERFAMILY/>). Therefore, additional eight *CPK* genes were identified by this study.

Finally, a total of 123 *CPK* genes were determined as *CPK* genes in the five species. These *CPK* genes in each species were named based on the similarity with Arabidopsis *CPKs* as the nomenclature in \[[@pone.0155590.ref032]\]. A total of 37 apple *CPK* (*MdCPK*) genes, 37 pear *CPK* (*PbCPK)* genes, 17 peach CPK (*PpCPK*) genes, 16 strawberry *CPK* (*FvCPK*) genes, and 16 plum *CPK* (*PmCPK*) genes were identified ([Table 1](#pone.0155590.t001){ref-type="table"}, [S1](#pone.0155590.s001){ref-type="supplementary-material"}--[S4](#pone.0155590.s004){ref-type="supplementary-material"} Tables). At the time of analysis, nearly complete genomes were available for apple, peach, strawberry and plum, while pear only had scaffold assemblies data available. In apple genome, chromosome 16 did not contain any *CPK* genes. The highest number of *MdCPK* genes (5, or 13.6% of the total) was found on chromosome 8 ([Fig 1](#pone.0155590.g001){ref-type="fig"}). Only one *CPK* gene was located on chromosome 1, 3, 4, 5, 6, and 13, respectively. Similar to that of *MdCPK*s, the distribution of the *CPK* genes in peach, strawberry and plum genomes is random ([Fig 1](#pone.0155590.g001){ref-type="fig"}). *CPK* genes in pear can only be anchored to scaffold assemblies now ([S1 Table](#pone.0155590.s001){ref-type="supplementary-material"}).

![Localization and duplication of the *CPK* genes in the apple, peach, plum and strawberry genome.\
Circular visualization of the *CPK* genes mapped on the different chromosomes in the genome using the Circos software. Chromosome number is indicated on the chromosome. The synteny relationship between each pair of *CPK* genes were detected by using the MicroSyn software. The genes have synteny relationship are linked by lines. Red link: \>30 anchors in a synteny block, blue link: 20--30anchors, green link: 10--20 anchors, gray link: 5--10 anchors.](pone.0155590.g001){#pone.0155590.g001}

10.1371/journal.pone.0155590.t001

###### *CPK* genes and related information in apple.

![](pone.0155590.t001){#pone.0155590.t001g}

  Gene name[\*](#t001fn002){ref-type="table-fn"}   Gene ID         Chr          Start      End        Str   Len    MW       pI
  ------------------------------------------------ --------------- ------------ ---------- ---------- ----- ------ -------- ------
  MdCPK1c                                          MDP0000128057   chr7         5521968    5529057    \-    660    73.99    6.40
  MdCPK1b                                          MDP0000142687   chr7         5521790    5528992    \-    618    69.01    5.45
  MdCPK1a                                          MDP0000153100   chr2         29570514   29576411   \+    566    62.87    4.94
  MdCPK2                                           MDP0000232344   chr12        4101197    4105359    \+    775    86.67    6.44
  MdCPK20a                                         MDP0000318339   chr14        5713952    5723780    \-    1023   116.65   6.01
  MdCPK20b                                         MDP0000513005   chr12        4295808    4308804    \+    679    76.21    5.63
  MdCPK5a[\*](#t001fn002){ref-type="table-fn"}     MDP0000162676   chr8         9964949    9969366    \-    756    85.71    5.46
  MdCPK26b                                         MDP0000457940   chr8         9964776    9971063    \+    1403   158.29   5.59
  MdCPK6c[\*](#t001fn002){ref-type="table-fn"}     MDP0000554360   chr8         9981452    9988787    \+    581    64.76    6.51
  MdCPK5b[\*](#t001fn002){ref-type="table-fn"}     MDP0000306850   chr10        13769805   13772951   \+    588    65.74    6.36
  MdCPK26a                                         MDP0000297184   chr8         9975429    9978531    \-    571    64.04    6.34
  MdCPK4b                                          MDP0000232885   chr10        5888120    5891732    \+    518    58.15    5.74
  MdCPK4a                                          MDP0000260834   chr9         22196543   22200169   \-    517    58.17    5.50
  MdCPK11                                          MDP0000494270   chr9         17647795   17649291   \+    498    55.59    5.00
  MdCPK11a[\*](#t001fn002){ref-type="table-fn"}    MDP0000203275   unanchored   13999639   14000609   \-    323    36.74    5.32
  MdCPK11c[\*](#t001fn002){ref-type="table-fn"}    MDP0000895339   unanchored   73579661   73580722   \-    353    40.10    4.91
  MdCPK17b                                         MDP0000138436   unanchored   20561522   20564258   \+    534    59.75    5.92
  MdCPK17a                                         MDP0000802997   chr8         16451427   16454495   \+    533    59.69    5.79
  MdCPK3[\*](#t001fn002){ref-type="table-fn"}      MDP0000920355   chr1         1097749    1101688    \+    501    56.12    6.33
  MdCPK29                                          MDP0000142398   chr12        20910182   20913063   \-    527    59.86    6.17
  MdCPK21                                          MDP0000232001   chr5         1385740    1389468    \+    554    62.12    6.44
  MdCPK9a[\*](#t001fn002){ref-type="table-fn"}     MDP0000320888   chr3         24125216   24129169   \-    607    68.35    7.10
  MdCPK19                                          MDP0000180811   chr11        23439921   23443151   \-    504    57.10    7.19
  MdCPK9b[\*](#t001fn002){ref-type="table-fn"}     MDP0000629000   chr11        23437472   23439940   \+    293    33.23    5.67
  MdCPK24a                                         MDP0000262701   chr17        6495752    6498333    \+    541    61.70    5.25
  MdCPK24b                                         MDP0000282003   chr9         5964148    5972280    \+    954    108.28   5.83
  MdCPK8b                                          MDP0000119457   chr15        40625083   40627959   \+    476    52.88    6.39
  MdCPK32a                                         MDP0000649508   chr15        40603145   40610990   \-    709    79.03    8.96
  MdCPK8a                                          MDP0000269423   chr2         26382762   26389283   \+    553    62.14    7.06
  MdCPK8c                                          MDP0000260857   chr12        6533214    6538860    \-    664    75.23    5.86
  MdCPK32b                                         MDP0000179069   chr14        9875913    9880717    \-    676    76.66    8.24
  MdCPK13a                                         MDP0000164868   chr4         15515595   15520798   \-    585    65.77    6.60
  MdCPK13b                                         MDP0000649496   chr13        15942217   15944688   \+    345    39.18    4.97
  MdCPK10a                                         MDP0000218522   chr6         20540708   20544197   \+    570    64.33    6.86
  MdCPK10b                                         MDP0000301254   unanchored   24840430   24843853   \+    548    62.22    7.32
  MdCPK10c                                         MDP0000308706   unanchored   24860733   24864154   \+    548    62.22    7.32
  MdCPK28                                          MDP0000208913   chr2         11285557   11290409   \-    626    70.28    9.14

**Note:**Chr: Chromosome; Str: Strand; MW: molecular weight; Len: Amino acid length; pI: Isoelectric point. Gene name in a previous study \[[@pone.0155590.ref032]\], and

\* indicates addition *CPK* genes detected in our study.

Phylogeny of *CPK* genes {#sec004}
------------------------

To detect the evolutionary relationships of *CPK* genes in Rosaceae, we performed phylogenetic analyses on the 123 *CPK* genes from apple, pear, peach, plum and strawberry using Neighbor-Joining (NJ), Minimal Evolution (ME) and Maximum Parsimony (MP) algorithms, respectively. The tree topologies produced by the three methods are largely consistent, with only minor modifications (data not shown). Therefore, the NJ tree was selected for further analysis ([Fig 2](#pone.0155590.g002){ref-type="fig"}). Based on the phylogenetic tree topology and previous studies, we divided the *CPK* gene family into 4 distinct groups (subfamilies): Group I, Group II, Group III, and Group IV. Group I contains 16 *CPK* genes from apple, 14 from pear, and 6 from each of strawberry, peach and plum. Group II consists of 8 *MdCPKs*, 7 *PbCPKs*, 5 *FvCPKs*, 5 *PpCPKs* and 5 *PmCPKs*. In Group III, 12 *CPK* genes are from apple, 14 form pear, 4 from strawberry, 5 form peach and 4 from plum. Group IV genes constitute the smallest subfamilies in all of the five species, which contain two *CPK* genes from pear, one from each of other species. The number of *CPK* genes in Group I and Group III from apple and pear were greater than that from strawberry, peach and plum, mainly due to the expansion of *CPK* genes in apple and pear. And the size of Group II in apple and pear were slightly bigger than other three species. These results implied that the variance in the number of *CPK* genes was mainly due to the occurrence of gene gain or loss in subfamilies independently among the different species.

![Phylogenetic trees of *CPK* genes in apple, pear, peach, plum, strawberry and Arabidopsis.\
The phylogenetic tree of *CPK* full length protein sequences was constructed with MEGA6 program with the neighbor-joining method. A *CPK* gene from *Ostreococcus lucimarinus* was used as the outgroup. The numbers beside the branches represent bootstrap values based on 1000 replications.](pone.0155590.g002){#pone.0155590.g002}

Using a *CPK* gene from *Ostreococcus lucimarinus* as the outgroup for the five Rosaceae species and Arabidopsis *CPKs*, the general topology of the resulting Neighbor--Joining tree appeared similar to that of a previous study \[[@pone.0155590.ref027]\]: the Group IV lineage appeared to have split first from the last common ancestor. Group III formed a clade close to Groups I and II, while the divergence between Groups I and II occured at the most recent time.

Gene structure and conserved domains in *MdCPK* genes {#sec005}
-----------------------------------------------------

Most of the apple *CPK* genes have six or seven introns, with clear intron phase patterns ([Fig 3](#pone.0155590.g003){ref-type="fig"}). For the members in each subfamily, the intron number and phase pattern on the full length protein sequences are variable, but conserved on the protein kinase domain. Of the 16 members in Group I, 11 genes contain 3 introns with the same phase pattern in kinase domain. Interestingly, three intronless *CPK* genes were found in Group I. These *CPK* genes without introns were also observed in other *Rosaceae* species(pear, peach, plum and strawberry; data not shown), while they have not been reported previously outside *Rosaceae* species, indicating introns may be lost during the evolution of these *CPK* genes in *Rosaceae*. All of the Group II members share the same intron-exon organization as most of the Group I, except *MdCPK9b* and *MdCPK19*. Group III members have 4 introns with the same phase pattern, except *MdCPK13a* and *MdCPK13b*, which contain 3 introns. Compared with the other three groups, Group IV only contains one *MdCPK* gene, which has 6 introns on kinase domain.

![Schematic representations of the exon--intron compositions of *CPK* genes in apple.\
Phylogenetic tree of Md*CPK* genes are placed on the left. Exons, represented by boxes, were drawn to scale. Dashed lines connecting two exons represent an intron. Protein kinase domain and EF-hand domain is marked in red and blue, respectively.](pone.0155590.g003){#pone.0155590.g003}

The most prominent feature of the proteins in *CPK* gene family is the typical signature domains. We surveyed top 10 motifs in the 37 MdCPK proteins using The Multiple EM for Motif Elicitation (MEME) motif search tool. Protein sequences were also compared with well-characterized signature domains in databases, such as SMART, SUPERFAMILY, Pfam, ProSite and Profiles (data not shown). As showed in [Fig 4](#pone.0155590.g004){ref-type="fig"}, motifs 1, 2, 3, 6 and 7 correspond to protein kinase domain; motifs 4, 5, 8 and 9 correspond to calmodulin-like domain; motif 10 corresponds to the junction domain ([S5 Table](#pone.0155590.s005){ref-type="supplementary-material"}).

![Schematic representations of the conserved motifs of *CPK* genes in apple.\
Names of genes are indicated on the left. Different motifs are highlighted with different colored boxes with numbers 1 to 10. Lines represent protein regions without detected motif.](pone.0155590.g004){#pone.0155590.g004}

Expansion of the *CPK* gene family in Rosaceae {#sec006}
----------------------------------------------

It is thought that genes in a family usually evolved from multiple gene duplication events. Gene duplication mechanisms mainly include tandem duplication and large segmental/whole-genome duplication (WGD). To examine the relative contribution of these kinds of mechanisms in the expansion of the *CPK* gene family in Rosaceae, we detected the origins of duplicate genes for the *CPK* gene family using the MicroSyn package\[[@pone.0155590.ref034]\]. Different patterns of gene duplication contributed differentially to the expansion of the *CPK* gene family in the investigated species ([Table 2](#pone.0155590.t002){ref-type="table"}). Remarkably, 15*CPK* genes in apple were duplicated and retained from WGD events, compared to 10 in peach and 6 in plum. No WGD events can be detected between *CPK* genes in strawberry. We did not find tandem duplication events for *CPK* genes in strawberry, peach and plum, however, 10 *CPK* genes in apple genome were found to form 4 tandem duplicated clusters. These results showed that WGD or segmental duplication played vital roles in the expansion of the *CPK* genes in apple, peach and plum. However, for the *CPK* genes in strawberry, other duplication events, such as dispersed gene duplication, is the main source of expansion.

10.1371/journal.pone.0155590.t002

###### Duplication genes in apple, peach and plum.

![](pone.0155590.t002){#pone.0155590.t002g}

  Species               Duplicated pair   Anchors    E value   Mean Ks     SD Ks   Ka/Ks   
  --------------------- ----------------- ---------- --------- ----------- ------- ------- ------
  *Malus × domestica*   MdCPK24b          MdCPK24a   79        1.57E-142   0.28    0.28    0.77
                        MdCPK20a          MdCPK20b   11        3.81E-20    0.38    0.36    0.63
                        MdCPK26b          MdCPK5b    10        6.41E-16    0.28    0.3     0.56
                        MdCPK4a           MdCPK4b    9         2.41E-17    0.26    0.34    0.17
                        MdCPK9b           MdCPK9a    8         2.74E-11    0.19    0.1     0.72
                        MdCPK32b          MdCPK8c    7         8.04E-07    0.55    0.75    0.54
                        MdCPK1b           MdCPK1a    6         6.94E-09    0.13    0.09    0.27
                        MdCPK9b           MdCPK21    5         8.78E-05    1.15    0.67    0.26
  *Prunuspersica*       PpCPK24           PpCPK8b    41        3.32E-113   0.6     0.67    0.17
                        PpCPK8b           PpCPK1     39        3.38E-178   0.79    0.62    0.25
                        PpCPK21           PpCPK9     37        3.56E-48    1.29    0.38    0.1
                        PpCPK8b           PpCPK4     27        8.44E-24    0.69    0.74    0.16
                        PpCPK17           PpCPK4     21        2.54E-18    0.55    0.76    0.14
                        PpCPK8b           PpCPK8a    9         5.82E-10    1.07    1.16    0.12
                        PpCPK10           PpCPK17    7         2.13E-04    1.14    0.75    0.18
                        PpCPK21           PpCPK6     6         5.35E-05    1.32    0.92    0.22
                        PpCPK8b           PpCPK17    6         3.48E-04    1.19    0.98    0.15
                        PpCPK8a           PpCPK4     6         3.94E-05    1.37    0.47    0.2
                        PpCPK8b           PpCPK6     5         4.43E-04    0.95    0.44    0.31
  *Prunusmume*          PmCPK21           PmCPK9     7         8.01E-10    1.21    0.37    0.1
                        PmCPK8a           PmCPK8b    5         2.09E-05    1.02    0.31    0.13
                        PmCPK28           PmCPK3     5         6.22E-04    0.63    0.2     0.74

**Note:** Mean Ks: mean value of each pair of genes within a duplicated block. Ka/Ks: ratio of no-synonymous substitution (Ka) and synonymous substitution (Ks) value of a pair of duplicated genes.

With the availability of genome sequence, studies of *CPK* gene family duplication history become possible in part by detecting synteny or clustering and ordering of neighboring matching gene pairs \[[@pone.0155590.ref035]\]. Using the information of neighboring matching gene pairs between two interested genes, synteny can usually detect duplication event of two chromosome fragments containing interested genes. For the five Rosaceae species investigated here, apple and pear belong to the *Maloideae*, peach and plum belong to the *Prunoideae*, and strawberry belongs to the *Rosoideae*. Owing to the lack of complete genome sequence for pear and computational limit, we select apple from the *Maloideae*, peach from the *Prunoideae* and strawberry from the *Rosoideae* to further survey the origin and evolution of *CPK* genes in Rosaceae. The synteny analysis showed that synteny relationship was mainly detected within subgroups, indicating that if the synteny between two members of a gene family is more significant, these two members evolved from a duplication event more recently ([Fig 5](#pone.0155590.g005){ref-type="fig"}, [S6 Table](#pone.0155590.s006){ref-type="supplementary-material"}). Two syntenies were also detected between subgroups, which indicates that some duplication history can be traced between subgroup. As illustrated in [Fig 5](#pone.0155590.g005){ref-type="fig"}, one synteny relationship was detected between subgroup I and II, and subgroup II and III, respectively, which support the relationship of 4 subgroups inferred by the feature of phylogenetic tree \[[@pone.0155590.ref027]\].

![Phylogenetic tree of the *CPK* gene family in apple, peach and strawberry annotated with collinear and tandem relationships.\
Curves connecting pairs of genes suggest either the collinear relationship (red) or tandem relationship (blue).](pone.0155590.g005){#pone.0155590.g005}

The synteny analysis showed that *CPK* genes can be divided into two types in Rosaceae. The first type of syntenic genes has a single strawberry and plum gene that corresponds to two apple gene, such as *FvCPK1*/*PpCPK1*/*MdCPK1a*/*MdCPK1b*, *FvCPK20*/*PpCPK20*/*MdCPK20a*/*MdCPK20b* ([Fig 5](#pone.0155590.g005){ref-type="fig"}). The second type has a single strawberry and peach gene that corresponds to a single apple gene, such as *MdCPK29*/*PpCPK29*/*FvCPK29* and *FvCPK28*/*MdCPK28*/*PpCPK28*. These results provide insights that will assist in understanding of orthologous relationship among *CPK* genes in Rosaceae.

Ks value and Ka/Ks ratio of *CPK* genes {#sec007}
---------------------------------------

To estimate the evolutionary dates of the segmental duplication events among the *CPK* gene family, we calculated the synonymous substitution (Ks) values between each pair of duplicated genes. The mean Ks of the duplicated *CPK* gene pairs in the syntenic region are shown in [Table 2](#pone.0155590.t002){ref-type="table"}. The Ks values for the *CPK* gene pairs ranged from 0.13 to 1.15. We found that most of segmental duplication pairs in peach and plum and one pair in apple (*MdCPK13a*/*MdCPK2*; Ks = 1.15) may have arisen from the γ triplication (\~140 million years ago \[MYA\]). While in apple genome, many duplicated gene pairs had lower Ks values (0.19--0.55), suggesting that these duplications may have been derived from the recent WGD (30\~45 MYA).

To further detect which selective force has been acted on the evolution of the *CPK* gene family, we also calculated the ratio of non-synonymous to synonymous substitution ratio (Ka/Ks) for 8, 11 and 3 pair of duplicated *CPK* genes in apple, peach and plum, respectively. All the Ka/Ks ratios of gene pairs were less than one, implying that purifying selection was the primary influence on the functional evolution of *CPK* family genes.

Expression profiles of the *MdCPK* genes in response to *Alternaria alternata* {#sec008}
------------------------------------------------------------------------------

Recent studies have provided compelling evidence for the involvement of *CPK*s in most of the immune signaling events. Alternaria blotch disease of apple is one of the most serious fungal diseases, which is caused by the apple pathotype of *Alternaria alternata*. To examine the expression pattern of *MdCPK* genes responding to this pathogen, we examined their expression information using quantitative real-time RT-PCR (qPCR) analysis with RNA from apple leaves sampled at 18 h, 36 h, and 72 h after *A*. *alternata* inoculation (hai). Among the 37 *MdCPK* genes, the qPCR products have been confirmed by sequencing except 9 genes for their unspecific amplification or undetectable expressions, providing a measure of the reliability of the qPCR results of *MdCPK* expression. The sequences of primers were listed in [S7 Table](#pone.0155590.s007){ref-type="supplementary-material"}. As shown in [Fig 6](#pone.0155590.g006){ref-type="fig"}, the expression pattern of *MdCPK* genes in response to *A*. *alternata* can be divided into 4 clusters. Cluster A contains 12 (42.9%) members of detectable genes, which were significantly up-regulated at 18, 36 and 72 hai, except *MdCPK32a*, which showed slight down-regulation at 18 hai. Cluster B contains 3 genes, which exhibited lower abundance of expression at 18 hai, while were highly induced at 36 and 72 hai compared with control. In Cluster C (3 genes), all the *MdCPK* genes were down-regulated after pathogen infection throughout the three time points. Genes in Cluster D are up-regulated at 36 hai, whereas down-regulated at18 and 72 hai, except two genes (*MdCPK1b* and *MdCPK17b*), which displayed no significant changes of expression at 36 hai.

![Relative changes in expression of *MdCPK* genes in response to *A*. *alternata* infection using quantitative real-time RT-PCR.\
The expression of *MdCPK* genes were normalized to tubulin expression. The fold changes of *MdCPK* genes were obtained by calculating the log2 ratio between treated (18, 36 and 72 hours after inoculation (hai) of *A*. *alternata*) and untreated samples. **(A)** The colour scheme in the heat map is blue/red: white indicate a low variation in expression, blue indicate a decrease and red indicate an increase. *MdCPK* gene names are displayed to the right of each row and are clustered in different clusters A, B, C and D using Hierarchical Clustering method. **(B)** Y axis represents the log2 value of fold change of *MdCPK* genes. For example, \> 0 means up-regulation and \< 0 indicates down-regulation of expression, and "1" represents genes up-regulated with 2 fold change in treated sample relative to control. Bars indicate standard error (SE) in three biological replicates.](pone.0155590.g006){#pone.0155590.g006}

Discussion {#sec009}
==========

CPKs are encoded by a multigene family, which have been identified and analyzed in many land plant species. The number and composition of *CPK* family members differ in various plants. Ancient polyploidy events (hexaploidization) and additional recent lineage-specific WGDs have presumably resulted in varying numbers of gene family within flowering plants. In this study, the number of the *CPK* gene families identified from the five Rosaceae genomes is diverse. The number of *CPK* genes in apple (37) and pear (37) are greater than that in peach (17), strawberry (16), and plum (16). Apple and pear were suggested to have undergone a recent WGD, while peach, strawberry, and plum did not undergo this event \[[@pone.0155590.ref036]\]. Therefore, this recent WGD event likely led to the different size of *CPK* genes in these Rosaceae species.

For the five Rosaceae species investigated here, apple and pear belong to the *Maloideae*, peach and plum belong to the *Prunoideae*, and strawberry belongs to the *Rosoideae*. The specification of the *Rosoideae* occurred prior to the split of the *Maloideae* and *Prunoideae*. The phylogenetic analysis showed that *MdCPKs* were closest to *PbCPKs* in the phylogenetic tree, while *PpCPKs* and *PmCPKs* had a closer relationship, which was consistent with the evolutionary history among the three subfamilies.

Different expansion mechanisms, such as genome-wide, tandem, and dispersed duplications, were thought to play a significant role in the expansion of specific gene families in plant genomes\[[@pone.0155590.ref037]\]. It has been shown that the three whole-genome duplications in Arabidopsis were responsible for more than 90% of the increase in transcription factors, signal transducers, and developmental genes \[[@pone.0155590.ref038]\]. Recently, genome-wide studies have revealed that the apple and pear genomes have experienced at least two genome duplications, one ancient and one before the apple-pear divergence \[[@pone.0155590.ref039]\]. In this study, the size of apple and pear *CPK* gene family is as twice as that of peach, strawberry and plum. The results of the synteny analysis verified that the expansion of the *CPK* gene family in apple, peach and plum was mainly derived from WGD or segmental duplications and tandem duplication. In strawberry, only a few or no significant segmental duplications were detected. However, the result of synteny analysis across apple, strawberry and peach revealed that almost all *CPK* genes in strawberry have orthologous genes in apple and peach genome. In strawberry, other duplication events, such as dispersed duplications might be the major drivers for *CPK* gene family expansion. The mean Ks value of the duplicated blocks of *CPK* genes in peach and plum are greater than that of apple, indicating that expansion of *PpCPK* and *PmCPK* genes may have arisen from the γ triplication (\~140 MYA). It is worth noticing that the synteny detection algorithm determines the relationship between two members in a gene family by searching for conserved, flanking collinear homologous gene pairs between two genomic fragments. For the ancient duplication events, the original flanking homologous gene pairs were hard to determine, leading to uncertain result of synteny analysis. Therefore, it might be that *CPK* genes in strawberry were evolved from the ancient WGD or segment duplications, but they are difficult to be detected now. Other mechanisms, such as genome rearrangement, gene loss, gene transposition and retrotransposition after the ancient polyploidy event, may also have affected the evolution of the *CPK* gene family in strawberry.

The genes duplicated through WGD might experience three alternative fates during the process of evolution, including (i) one copy may become silenced and lost original functions (nonfunctionalization), (ii) one copy may acquire a novel, beneficial function, while the other copy retained the original function (neofunctionalization) and (iii) both copies may become partition of original functions (subfunctionalization) \[[@pone.0155590.ref040]\]. It has been shown that the retention of genes duplicated through WGD is biased in plant genomes \[[@pone.0155590.ref041]\]. In Arabidopsis, genes encoding transcription factors, protein kinases, and ribosomal proteins have been preferentially retained after WGD \[[@pone.0155590.ref042]\]. It is hypothesized that the overretention of duplicated genes through WGD is strongly correlated with greater structural complexity, highly conserved domains, and lower evolutionary rates in the plant genome. Multiple models may simultaneously drive the evolution of genes duplicated after WGDs. In this study, the *CPK* gene family has undergone specific expansion and been preferentially retained in Rosaceae. Rosaceae *CPK* family genes contain several highly conserved functional domains, and present lower Ka/Ks ratios, corresponding to a slower evolutionary rate. These results implied that functions of the duplicated *CPK* genes in Rosaceae did not diverge much during subsequent evolution. These stable function of *CPK* genes over recent years may serve as good targets for dosage balance selection\[[@pone.0155590.ref043]\].

To resist pathogen, plants have evolved two defense mechanisms to sense pathogens invasion \[[@pone.0155590.ref044]\]. On the cell-surface of host, pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs), which are subsequently stimulated to trigger PAMP-triggered immunity (PTI).In response, pathogens have evolved the means to suppress PTI by secreting effectors inside the plant cell. These pathogen effectors are recognized by intracellular nucleotide-binding leucine-rich repeat (NB-LRR) immune sensors, which activate the second type of immune defense mechanism, effector-triggered immunity (ETI). PAMPs initiate an influx of calcium ions and an oxidative burst, followed by activation of MAPK and calcium-dependent protein kinase \[[@pone.0155590.ref045], [@pone.0155590.ref046]\]. In these studies, the expression pattern of *MdCPK* genes after *A*. *alternata* infection was surveyed by qPCR. These genes included 10 *MdCPKs* from Group I, 8 from Group II, 9 from Group III and 1 from Group IV. In Arabidopsis, the expression of *AtCPK1* from Group I is rapidly induced by fungal elicitors. Over-expression of *AtCPK1* confers broad-spectrum resistance to bacteria and fungi. Long-term *AtCPK1* over-expression triggered salicylic acid (SA) accumulation and constitutive expression of SA-regulated defense and resistance genes \[[@pone.0155590.ref014]\]. *MdCPK2*, a closest homolog of *AtCPK1*, displayed continuously up-regulation during the three time points (18, 36 and 72 hai). A functional genomic screen identified four related *AtCPKs* in Group I (*AtCPK4*, *AtCPK5*, *AtCPK6* and *AtCPK11*), as early transcriptional regulators in MAMP signaling \[[@pone.0155590.ref047]\]. The closest homolog of *AtCPK4* and *AtCPK11* are *MdCPK4b* and *MdCPK4a*. The transcript accumulation of *MdCPK4b* reduced at early stage of infection, and subsequently increased 36 hai, and then declined 72 hai, whereas *MdCPK4a* showed continuously up-regulation. These results indicate that some *CPKs* from Group I exhibittransient and sustained transcriptional modifications upon pathogen infection. Interestingly, in this study, most of the members (77.8%) in Group III exhibited continuously up-regulation at the three time points, suggesting their conserved function in plant defense response. By contrast, several *MdCPKs* (*MdCPK17b*, *MdCPK1b*, *MdCPK28*, *MdCPK5b*, and *MdCPK20b*) showed down-regulation throughout all the treatments. These results suggest that *CPK* genes in apple might have evolved independently in different biological contexts.

Conclusions {#sec010}
===========

A total of 123 *CPK* genes were identified in the five Rosaceae genomes. Based on the phylogenetic tree topology and structural characteristics, these *CPK* genes were divided into 4 distinct subfamilies (Group I, II, III and IV). Collinearity analysis showed that many duplicated genes in apple genome may have been evolved from a recent WGD (30\~45 MYA), whereas, most of segmental duplication genes in peach and plum may have arisen from the γ triplication (\~140 MYA). Purifying selection is the major force driving the function evolution of *CPK* family genes. qPCR evidence showed that *MdCPKs* genes might have evolved independently in different biological contexts. These results in this study laid a foundation for further examining the function and complexity of the *CPK* gene family in the Rosaceae.

Methods {#sec011}
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Gene Identification {#sec012}
-------------------

The genome sequences of apple, peach, and strawberry were downloaded from Phytozome (<http://phytozome.jgi.doe.gov/pz/portal.html#>). The pear genome sequence was downloaded from the pear genome project(<http://peargenome.njau.edu.cn/>), and the plum genome sequence was downloaded from the *Prunus mume* Genome Project (<http://prunusmumegenome.bjfu.edu.cn/index.jsp>).The complete genome, proteome sequences of Arabidopsis was obtained from The Arabidopsis Information Resource (version 10; [http://www.arabidopsis.org](http://www.arabidopsis.org/)). In proteome datasets, if two or more protein sequences at the same locus were identical where they overlapped, we selected the longest sequence. The HMM profiles of protein kinase domain Pkinase (PF00069) and EF-hand domain (PF13499 and PF13202) were downloaded from the Pfam protein family database (<http://pfam.sanger.ac.uk/>). HMMER was used to search a customized database containing the proteome with the threshold set of the Pfam GA gathering cutoff. The HMMER selected proteins were used for a BLASTP query of the original protein database. Finally, the BLASTP hits were scanned for protein kinase domain Pkinase and EF-hand domains using InterProScan.

Phylogenetic tree building and protein motif prediction {#sec013}
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The amino acid sequences of full length sequences were aligned using the MUSCLE \[[@pone.0155590.ref048]\] with default parameters. Phylogenetic trees for the aligned sequences were constructed using NJ, ME and MP methods. The statistical support of the retrieved topology was assessed using a bootstrap analysis with 1000 replicates for trees. The conserved motifs in the proteins were detected by MEME (<http://meme.nbcr.net/meme/cgibin/meme.cgi>), with the following parameters: number of repetitions: any; maximum number of motifs: 20; the optimum motif widths: 6--200 amino acid residues.

Chromosomal distribution and gene duplication {#sec014}
---------------------------------------------

The genes were plotted separately onto the chromosomes according to their locations on the chromosomes in the GFF file. Genes with a maximum of 5 genes distance were considered to be tandem duplicates. The syntenies between each pair of members were detected by using the MicroSyn software. The parameters were set as follows: window size of 100 genes, tandem gap value of 2, expected threshold value cut off of 1e-10, and 8 homologous pairs to define a syntenic segment. The mean Ks values of orthologous gene pairs in the same synteny block, the Ka and Ks were calculated by Microsyn.

Plant materials and pathogen treatments {#sec015}
---------------------------------------

To examine the expression of *MdCPK* genes, the leaves of cultivar, 'Red Delicious', inoculated with *A*.*alternata* were used. *A*. *alternata* was expanded on potato dextrose agar (PDA; 200 g potato extract, 20 g dextrose, 20 g agar, 1L water) medium for 5 d at 26°C under dark conditions. The inoculation method was carried out according to the protocol described previously\[[@pone.0155590.ref049]\]. The mycelia were punched using a hole puncher (DIA. = 5 mm). Six pieces of mycelium 'cake' were put on each side of midrib of the abaxial leaf surface, and then incubated at 25°C under a 14 h light/10 h dark cycle in sterilized plastic chambers. Leaves were sampled at 0, 18, 36, and 72 hours after inoculation.

Gene expression analysis by quantitative real-time PCR {#sec016}
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Quantitative real-time PCR was carried out on three independent biological replicates of each sample, as well as three technical replicates for each reaction. Total RNA was extracted using the CTAB method. The qualities and quantities of RNA were determined by 1.5% agarose gel electrophoresis and Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc.; USA), respectively. A SMART™ PCR cDNA Synthesis kit was used to synthesize the cDNA from 1μg total RNA. Primers of *MdCDPK* genes for qPCR were designed using Beacon Designer v7.0 (Premier Biosoft International, California, USA). The real-time quantification was performed using an ABI7300 Detection System using SYBR Green qRT PCR kits (TaKaRa, Toyoto, Japan). PCR mixtures (final volume, 20 μL) contained 10 μL of 2×SYBR Premix, 2 μL of cDNA template, 200 nM of each primer. The PCR reaction was performed as the following conditions: for 3 min denaturation at 94°C followed by 40 cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 40 s. Following amplification, melting curves were determined with the following program: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. For the accuracy of results, each reaction was conducted in triplicate. Expression levels of the selected genes were normalized to tubulin expression. 2^-ΔΔCt^ method \[ΔΔCt = (Ct~target\ gene~−Ct~tubulin\ gene~) ~treatment~−(Ct~target\ gene~--Ct~tubulin\ gene~) ~control~\] was used to calculate the relative expression of *MdCDPK* genes. The gene expression levels were visualized as the heat map and histogram based on the value of log~2~(2^-ΔΔCt^). Heatmaps were generated using R package 'gplots' (<http://www.bioconductor.org/>). Clustering in the heatmap was carried out using Hierarchical Clustering by hclust function in R (<http://www.r-project.org/>).

Supporting Information {#sec017}
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###### *CPK* genes and related information in pear.

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###### *CPK* genes and related information in strawberry.

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###### *CPK* genes and related information in peach.

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Click here for additional data file.

###### *CPK* genes and related information in plum.

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Click here for additional data file.

###### Consensus sequences of motifs in *CPK* genes in apple detected by MEME.

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Click here for additional data file.

###### Synteny relationship between apple, strawberry and peach.

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Click here for additional data file.

###### Primer sequences used for real-time PCR analysis.

(XLS)

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Click here for additional data file.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: SHW. Performed the experiments: MHW HD. Analyzed the data: MHW SHW. Contributed reagents/materials/analysis tools: BHC JMT SHW. Wrote the paper: SHW MHW.
