Genome-wide identification and expression analysis of the cytokinin gene family in Brassica oleracea L. reveals its role in clubroot resistance

Background: cytokinins have cytokinin family genes have been described in several plant species, but a comprehensive analysis of the cytokinin family genes in Brassica oleracea has not been reported to date, especially their roles in dealing with the invasion of P. brassicae. Results: Cytokinins are a class of phytohormones that promote cell division and differentiation and are thought to affect plant immunity to multiple pathogens. To reveal the mechanisms of the Brassica oleracea cytokinin family genes in response to clubroot disease, a total of 36 cytokinin genes were identified using a genome-wide search method. Phylogenetic analysis classified these genes into three groups. They were distributed unevenly across nine chromosomes in B. oleracea, and 15 of them did not contain introns. The results of colinear analysis showed that each cytokinin gene in the B. oleracea genome had at least one homologous gene in the Arabidopsis genome. A cis-element analysis indicated that these genes possessed several stress response cis-elements. The heatmap of the cytokinin gene family showed that these genes were expressed in various tissues and organs. Five and eight genes were up- and downregulated, respectively, in the susceptible material after inoculation. In addition, two and one genes were up- and downregulated, respectively, in resistant material. This may indicate that these cytokinin genes play important roles in the host plant response to clubroot disease. In addition, the results provide insights for better understanding the role of cytokinin in the B. oleracea–P. brassicae interaction. Conclusions: Our results are helpful to elucidate the role of cytokinin family genes in cabbage response to infection by P. brassicae, and lay a foundation for further study on the function of these genes.

where auxins and cytokinins are synthesized in large amounts (Dekhuijzen andOvereem 1971 Ando et al. 2005). Similar to clubroot disease, leguminous plants form tumors in the roots when they are infected by rhizobia (Miri et al. 2015). As a key endogenous plant signal, the role of cytokinin in this process is to induce the formation of a mass. Many phenomena indicate that cytokinin must play an essential role in the plant response to the invasion of P. brassicae. Cytokinin and auxin synergistically induce hypertrophy and hyperplasia by increasing the cell division rate and cell enlargement during the formation of a mass in the roots (Ludwig-Müller and Schuller 2008). The detection and analysis of five cytokinin synthase genes from Brassica rapa indicated that the expression of cytokinin synthase genes was increased during the primary developmental stage of clubroot disease (Ando et al. 2005;Schuller et al. 2014). Similarly, throughout the process of gall formation, cytokinin biosynthesis is upregulated, with downregulation of adenosine kinase (Ludwig-Müller et al. 2009).
RNA-seq is considered a powerful approach for detecting differentially expressed genes and is widely used to study the interactions between host plants and P. brassicae. Siemens et al. (2006) identified more than 1000 DEGs in infected individuals compared with the normal roots of Arabidopsis. A transcriptome study of Brassica oleracea var. italica and Brassica macrocarpa Guss. in different infection periods of P. brassicae showed that genes associated with glucosinolate biosynthesis, cell wall biosynthesis, and plant hormone signal transduction were activated much earlier in resistant lines (Zhang et al. 2016). In addition, comparative transcriptome analysis between susceptible and resistant Chinese cabbage varieties revealed that genes associated with pathogen-associated molecular patterns (PAMPs), pathogenesis-related (PR), calcium ion influx, hormone signaling, transcription factors, and cell-wall modification played important roles in the interactions between B. rapa and P. brassicae during the primary infection stages (Chen et al. 2016).
Although the function of cytokinin has been analyzed in various plant cultivars, such as Arabidopsis (Riou-Khamlichi et al. 1999), Spirodela polyrhiza (Kurepa et al. 2018), Phaseolus lunatus (Martin et al. 1999), rice (Ashikari et al. 2005), tomato (Chen et al. 2016), apple (Feng et al. 2017), potato (Lomin et al. 2018) and maize (Schmülling et al. 2003;Chettoor et al. 2017), the roles of the cytokinin gene family in cabbage with clubroot disease are currently unknown. In this study, we identified 36 cytokinin genes in B. oleracea. The phylogenetic relationships, chromosome locations, colinearity relationships, and gene structures of all detected genes as well as the cis-acting regulatory elements in promoters were then systematically analyzed. The transcription and expression patterns of cytokinin genes during different periods following inoculation with P. brassicae were also investigated.
Our results will promote the understanding of the molecular mechanism of the cytokinin response to the development of clubroot disease in B. oleracea.

Genome-wide identification and phylogenetic analysis of cytokinin genes in B. oleracea
A total of 36 cytokinin genes were identified, and the detailed information for each cytokinin gene is shown in Table 1. The proteins encoded by the cytokinin genes varied from 355 to 594 amino acids (aa) in length. Among these proteins, the Bol024927 protein sequence was the longest, with 594 amino acids, and the Bol006929 protein sequence was the shortest, with 355 amino acids. Among all of the proteins, 18 members shared similar localization to cytoplasm, three to the extracellular region, four to the vacuole, and eleven members were located in more than one compartment.
To study the evolutionary relationships of the cytokinin genes in B. oleracea, Arabidopsis, and B. rapa, 135 cytokinin genes were analyzed to construct an unrooted phylogenetic tree. The cytokinin genes were classified into three groups ( Fig. 1), which contained 34 (class I), 32 (class II) and 56 (class III) members. The remaining 10 members could not be clustered into any group. The numbers of cytokinins from B. oleracea, A. thaliana and B. rapa in each class are represented in Supplemental Table S1 (Additional file 1).

Chromosomal distribution and differential retention of cytokinin genes in B. oleracea
The 36 cytokinin genes were assigned to nine chromosomes of B. oleracea (Fig. 2). The distribution of the cytokinin genes on each chromosome was uneven. Chromosome C07 contained the largest number (8) of cytokinin genes, followed by chromosomes C01, which contained six genes. Both chromosome C03 and C04 contained five genes. Three genes were located on C05. Chromosome C02, C06 and C08 each contained only two genes.
To obtain a better understanding of the evolutionary history of the cytokinin gene family in B. oleracea and Arabidopsis, duplications of putative cytokinin genes in the B. oleracea and Arabidopsis genomes were analyzed. Every cytokinin gene in the B. oleracea genome has at least one homologous gene in the Arabidopsis genome. In addition, segmentally duplicated gene pairs were observed among the 36 identified cytokinin genes in B. oleracea ( Fig. 3; Additional file 2: Table S3).
The B. oleracea genome contained 1-7 copies of each cytokinin gene found in Arabidopsis, while 13 of the A. thaliana cytokinin genes had no homologs in B. oleracea ( Fig. 3; Additional file 3:

Structure and conserved motif analysis of cytokinin genes
The structures and phases of introns/exons were determined to further study the cytokinin genes. In general, genes with closer phylogenetic relationships exhibited similar genetic structures, such as The intron pattern is also related to the phylogenetic classification ( Fig. 1; Fig. 4). Fifteen intron-free genes were classified into two subclasses. Bol018140, Bol031036 and Bol010168 all contained only one intron and showed a very close relationship. The same phenomenon occurred between Bol035751 and Bol036047. Taken together, the distribution patterns of the introns in cytokinin genes with similar relationships were roughly the same.

Cis-elements in the promoters of B. oleracea cytokinin genes
To further elucidate the regulatory mechanisms of B. oleracea cytokinin genes in response to clubroot disease, the promoter sequences were submitted to the PlantCARE database to identify cis-elements.
Seventeen types of cis-acting regulatory elements were detected (Fig. 6). All 36 cytokinin genes contained 2-16 cis-elements related to light-responsiveness. Twelve cytokinin genes contained defense and stress-response-related cis-elements. MeJA-responsiveness cis-elements were detected in 28 chitinase genes, while cis-elements related to endosperm expression and anoxia-specific inducibility were only detected in five and one cytokinin genes, respectively. Anaerobic induction ciselements was detected in 28 cytokinin genes, as were drought induction cis-elements. The numbers of cis-elements related to circadian control, flavonoid biosynthesis and seed-specific regulation were relatively small, being detected in 2, 2 and 3 cytokinin genes, respectively.

Expression patterns of cytokinin genes in different organs and treatments
The RNA-Seq dataset (GSE42891) was examined to determine the expression levels of cytokinin genes in the leaves, stem, flowers, siliques, buds, calli and roots of B. oleracea. Most of the cytokinin genes exhibited different expression patterns (Fig. 7a). Fifteen were expressed in all organs, while the expression of two was not detected. Some genes were expressed in only one or two organs. For example, Bol024949, Bol024952, Bol028359 and Bol028360 were only expressed in siliques, whereas To explore the relationship between cytokinin and clubroot disease, we tried to inoculate two cabbage of cytokinin genes between Jingfeng No.1 and Xiangan 336 were quite different under the same treatment, possibly because of the difference in resistance between them.
To further examine these findings, quantitative reverse-transcription PCR (qRT-PCR) was performed to analyze the expression patterns of eight cytokinin genes under different treatments. The results showed that the expression levels of the eight genes were basically consistent with those in the heat map ( Fig. 7b; Fig. 8). Generally, the expression levels of Bol028363 and Bol006383 were relatively low in each treatment. Bol011927 and Bol005172 showed similar expression patterns because of their higher expression levels in X28C and X28I and low expression levels in J28C and J28I. Overall, the expression levels of the 8 genes in X7I were relatively low.
families such as the Hsp20 family (Peng et al. 2018), the leucine-rich repeat family (Zhou et al. 2016) and the GRF family (Sang et al. 2018) also contain few introns. Among the genes examined in this study, 15 genes were intronless, and 5 genes contained only 1 intron, potentially supporting the above hypothesis. In other words, these genes will be able to respond faster when plants are invaded by P. brassicae. . In this study, we found that each homologous gene in Arabidopsis presented several copies in the B. oleracea genome; for example, AT1G26380.1 presented 3 copies, and AT5G44400.1 presented 5 copies ( Fig. 3; Additional file 2: Table S3). This may suggest that the cytokinin genes were replicated during the evolution of B. oleracea, resulting in more diverse functions. Conserved genes are more likely to give rise to functional and persistent duplicates, thus contributing more to their long-term survival ability in eukaryotic genomes. Moreover, genes with many cis-regulatory regions, which are expressed in numerous tissues or encode multidomain proteins, will be preferentially preserved (Davis et al. 2004). The promoter region of each cytokinin gene examined in this study contained a large number of cis-elements, which may indicate that they have more diverse functions during gene evolution and thus have a greater chance to be retained.

Many plants increase
According to the RNA-seq data of B. oleracea, a certain number of cytokinin genes, such as Bol036074 may be play an important role in the process of cell division after inoculation because they were upregulated in J7I compared to J7C. Bol014258 and Bol024330 showed the same expression characteristics between J28I and J28C; however, the opposite expression pattern was found for Bol018140, Bol027388, Bol033608, and Bol006389, which may indicate the different roles of these genes. Additionally, Bol024952, Bol1009917 and Bol1036948 were considered to have no effect on the response to pathogen invasion because they exhibited the same expression pattern under each treatment.

P. brassicae infects the root hairs of oleracea at 7 dai and the cortex cells at 28 dai (Ning et al. 2018).
Therefore, genes with significant differential expression between Jinfeng No.1 and Xiangan 336 after inoculation may be crucial genes for the plant response to invasion by P. brassicae. Bol022730 was upregulated in Jinfeng No.1 after inoculation but downregulated in Xiangan 336, possibly because its expression is inhibited by P. brassicae in resistant materials. In contrast, Bol020547 and Bol045724 were downregulated in Jinfeng No.1 after inoculation but upregulated in Xiangan 336. This may mean that these two genes play a key role in responding to infection by P. brassicae. Further analysis showed that the promoter regions of the two genes contained MeJA-responsiveness-related ciselements, and Bol020547 also contained a salicylic acid responsiveness-related cis-element.
Therefore, Bol020547 and Bol045724 may be key genes in the B. oleracea response to clubroot disease, and their specific functions need to be further studied.

Conclusions
Here, a genome-wide analysis of B. oleracea cytokinin gene family was performed, and 36 cytokinin genes were confirmed. Subsequently, analyses of cytokinin genes on gene structures, phylogeny, chromosomal location, gene duplication and gene expression patterns were conducted based on bioinformatics and qRT-PCR methods. Five and eight genes were up-and downregulated, respectively, in the susceptible material after inoculation. In addition, two and one genes were upand downregulated, respectively, in resistant material. This may indicating that cytokinin genes play important roles in the interaction between B. oleracea and P. brassicae. The study provides comprehensive information on the cytokinin gene family in B. oleracea and will aid in determining the cytokinin gene functions.

Identification of cytokinin family genes in Brassica oleracea
The cabbage whole-genome protein sequences were downloaded from the Brassica oleracea Genomics Database (www.ocri-genomics.org/bolbase/blast/blast.html). The HMM profile of the

Phylogenetic analyses of cytokinin genes
The amino acid sequences of cytokinin proteins derived from B. oleracea, Arabidopsis, and B. rapa were used for phylogenetic analysis. An unrooted neighbor-joining phylogenetic tree was constructed using MEGA 6.0 software (Tamura et al. 2013) with 1000 bootstrap test replicates.     Distribution of conserved motifs in cytokinin genes. Ten putative motifs are indicated in different colored boxes. The lengths of the motifs in each protein are shown proportionally.

Figure 6
Predicted cis-acting elements in the promoter regions of cytokinin genes.  Supplementary Files