Genome-wide identification of chickpea WRKY gene family
The
WRKY domain (PF03106) obtained from the Pfam protein family database (
http://pfam.sanger.ac.uk/)
(Finn et al., 2008) was used for HMM profile. SMART tools were used for testing of each
WRKY TFs matching sequence with less than 0.01 e-value, this resulted in to 67 putative genes that could be annotated as
WRKY TFs. The six
WRKY genes (Ca_01713, Ca_03832, Ca_5782, Ca_06124, Ca_16393 and Ca_21883) were removed due to the lack of
WRKY domain. Remaining 61 putative genes were named as per their location on chromosome as
CarWRKY01 to
CarWRKY61. Five genes were not mapped to any chromosomes were names as
CarWRKY57 to
CarWRKY61 (Table 1).
As WRKY transcription family is one of the most important family for regulation of genes in plants, it primarily involved in modulation of stress and not in developmental processes in plants
(Rushtom et al., 2000). Thus, based on the publicly available sequence information total 61 CarWRKY genes were identified. Comparatively chickpea has a large number of transcription factors as compared to grapes 59 (WRKYs)
(Guo et al., 2014), castor bean (58 WRKYs)
(Zou et al., 2016), physic nut (58 WRKYs)
(Xiong et al., 2013), peach (58 WRKYs)
(Chen et al., 2016), cucumber (57 WRKYs)
(Ling et al., 2011), tea (50 WRKY)
(Wu et al., 2016), barley (45 WRKYs),
(Mangelsen et al., 2008) and hemp (40 WRKYs)
(Xin et al., 2016). However, maize (136 WRKYs)
(Wei et al., 2012), soybean (131 WRKYs)
(Yu et al., 2016), apple (127 WRKYs)
(Meng et al., 2016), poplar (104 WRKYs)
(He et al., 2012), rice (100 WRKYs)
(Ross et al., 2007), kiwi fruit (97 WRKYs), (
Jing and Liu, 2018), tomato (81 WRKYs)
(Huang et al., 2012), Arabidopsis (72 WRKYs)
(Eulgem et al., 2000) reported more number of transcription factors in comparison with chickpea.
More number of WRKY transcription factors may be due increased plant genome size during evolution
(Meng et al., 2016). The genome size of some plant species can be given as: apple (927 and 724.3 Mb)
(Han et al., 2007), soybean (978 Mb)
(Song et al., 2016), chickpea (738.09 Mb)
(Varshney et al., 2013), kiwifruit (616.1 Mb)
(Huang et al., 2013), grape (490 Mb)
(Jaillon et al., 2007) and finally cucumber (367 Mb)
(Huang et al., 2009). However, some scientists interpreted that there is not any correlation between number of
WRKY TFs and the genome size of respective plant
(Song et al., 2016). There is variation in
WRKY TFs of apple and soybean although their genome size is approximately similar.
Physiochemical property analysis
ProtParam tool ExPASy revealed longest amino acid CarWRKY33 (724 aa) and CarWRKY41 is the shortest (105 aa) found in chickpea however protein size ranged between 12.63 kDa (
CarWRKY41) to 78.62 kDa (
CarWRKY33) and theoretical isoelectric point (pI) ranged 4.77 (
CarWRKY01) to 9.91 (
CarWRKY51). This indicates the physiological traits of
WRKY proteins extraordinarily varied for PI and MW, exhibiting a high degree of complexity within the
CarWRKY genes. The GRAVY value for all studied genes was below zero and ranged from -1.157 to -487 (Table 1), which suggests that all 61
CarWRKY proteins were hydrophilic. As per
Drews et al., (2004) grand average of hydropathicity (GRAVY) value of protein above zero shows hydrophobic characteristics, while the value below zero shows its hydrophilic nature.
Chromosomal localization
Sixty-one
CarWRKY genes distributed unevenly on among chromosomes. Fifty-six out sixty-one
CarWRKY genes were distributed on seven chromosomes (Fig 1). Another five genes (
CarWRKY57-
CarWRKY61) are not assigned to any chromosome, because they reported on scaffold region. Chromosome 1 located largest number of genes (10
CarWRKY genes) and lowest number of gens were present on chromosome 4 (6
CarWRKY genes). The chromosome number 3 and 6 contain 9
CarWRKY genes while chromosome 7 contain 8
CarWRKY genes. Chromosome 2 and 5 contained equal number of 7 genes. In common bean
Wang et al., (2016) reported similar results, they reported 90
PvWRKY genes distributed across all 11 chromosomes and named as PvWRKY1 to PvWRKY90. Chromosome 8 contained largest 18 WRKY genes, followed by chromosome 2 and 9 with 16 and 12 genes respectively.
Gene structure analysis
As per Gene Structure Display Server (GSDS 2.0) we determined the structure of all 61
CarWRKY genes
i.e. the intron/exon distribution pattern to get information about the evaluations of these genes in chickpea. Most of the introns reported to be located near the 5’ end. The diversity exhibited in terms of number of introns ranged between 1 to 5. Five genes reported presence of one intron, however, two introns were reported in 33
CarWRKY genes, seven genes reported three introns, 4 Introns in 9 genes and 5 introns in 6 genes. Larger introns were reported in 33 genes whereas
CarWRKY56 did not contain any intron.
Li et al., (2015) reported similar structural features in rubber plant. They reported introns ranged 1-5 introns in
WRKY gene. Some rice species didn’t report any introns in their
WRKY genes
(Xie et al., 2005; Ross et al., 2007) and supposed that during the evolution there might be intron loss events. Phylogenetic grouping of genes, there diversity in structure between exons and introns is helpful tool for the evolution, diversification and neofunctionalization of gene families (
Shiu and Bleecher 2003;
Wang et al., 2014).
Conserved domain analysis
MEME suite tools showed diversity among
CarWRKY proteins and predicted motifs in
CarWRKY genes family. From the candidate
CarWRKY protein, we reported 15 conserved motifs (Fig 3 and 4) each indicating its conserved
CarWRKY domain.
Few
WRKY proteins have showed that motifs such as leu-zipper motif, a putative new class of DNA binding protein also exist besides the 60 conserved amino residues
(Eulgem et al., 2000; Cormack et al., 2002). The complexity and diversity study of these
WRKY motifs shows importance of
WRKY genes in growth regulation and stress responses. The WRKY proteins harbor a characteristic WRKY domain of around 60 amino acids with conserved oligopeptide sequence (WRKYGQK) at N-terminal along with Cys2His2/Cys2HisCys zinc finger motif (CX4-7CX22-23HXH/C) at C-terminal
(Rushton et al., 2010; Schluttenhofer and Yuan 2015). The conservation of cysteine and histidine in the WRKY domain is responsible for the formation of unique zinc finger-like motif and sequence of WRKY amino acids can directly bind with W box (TTG ACT /C)
cis-regulatory element, which are found in upstream regions of target genes
(Zhang et al., 2018).
Multiple sequence alignment and phylogenetic analysis
The ClustalW and Neighbor-Joining (NJ) method criteria was used for construction of unrooted phylogenetic tree with the passion correlation, pairwise detection and bootstrap 1000 replicates parameters. On the basis of classification done by
Sakuma et al., (2002) we generated phylogenetic tree, it was classified into three different groups designated as group I, II and III group. Group II was subdivided into three subgroups such as group IIa, IIb and IIc. Group I consisted six genes, group III consisted 15 genes, however, group II reported total 40 genes which was largest among all other groups. Subdivisions of group II consisted 12 genes in subgroup IIa and 14 genes in each sub group IIb and IIc (Fig 5). The phylogenetic tree-based classification of chickpea followed the same trend as in other crop species. The characterization of WRKYs with respect to intron/exon distribution and conserved domains revealed the conservation of gene structure as well as domains among the members of a same group in the phylogenetic tree. These three major groups were subdivided into seven sub classes
i.e. I, II, IIa, IIb, IIc and III as reported by
Eulgem et al., (2000).
