Genome-wide Identification And Expression Analysis Of WRKY Gene Family In Saccharum Spontaneum

WRKY transcription factors are one of the largest transcription factor families in plants,and were revealed to play important roles in the regulation of developmental and physiological processes.To date,although many studies have performed on the identification of WRKY proteins in various plant species,the WRKY gene family has not been identified in Saccharum species because of its complex polyploid genome.In this study,we used comparative genomics and bioinformatics to identify the WRKY genes of S.spontaneum based on our recent assembled S.spontaneum genome.Phylogenetic trees were constructed to explore their evolutionary relationship,and the expression patterns of different WRKY genes were analyzed by transcriptome sequencing.These results provided the valuable basis for the further understand of the function of Ss WRKY genes.The main results are as follows:(1)Based on the genomic database of S.spontaneum and HMMserch program,a total of 154 Ss WRKY genes were identified and named on the basis of their chromosome locations,including 13(8.4%)genes with four alleles,29(18.8%)with three and 41(26.6%)with two.According to the classification of OsWRKYs and primary amino acid structure feature of Ss WRKYs,these 154 Ss WRKY genes were classified into group ?(17 genes),group ?(81 genes),group ?(51 genes)and group IV(5 genes).Group ? consists of two subgroups,subgroup la contained 13 Ss WRKYs,and subgroup Ib with 4Ss WRKYs.Group ? was further divided into five subgroups,Ila,?b,?c,?d and IIe,which contained 6,12,40,11 and 12 genes,respectively.The SsWRKYs of group IV were observed to contain an incomplete domain and only possessed a WRKYGQK motif.Except for SsWRKY2,SsWRKY52 and Ss WRKY131,the Ka/Ks ratios of the rest Ss WRKY alleles were under 1,indicating that these SsWRKY genes were under purifying selection.(2)As a proximal species of sugarcane,the number of WRKY TFs in Sorghum bicolor was less than that in S.spontaneum.Collinearity analysis showed that 217(73.8%)and 47(16.0%)SsWRKYs were originated from segmental duplication and tandem duplication,respectively,indicating that both tandem duplication and segmental duplication contributed to the expansion of SsWRKY family,but segmental duplication may play a more critical role.Nineteen genes,including SsWRKY1,SsWRKY26,SsWRKY32,SsWRKY83,SsWRKY89,SsWRKY95,SsWRKY110,and SsWRKY115,were observed to lack of the orthologous SbWRKY genes,suggesting that these WRKYs may be produced after the differentiation of S.spontaneum and Sorghum,(3)Consistent with previous study,in S.spontaneum,the phylogenetic analysis showed that the WRKY genes of subgroup ?e was derived from the group la WRKY genes by losing a N-terminal domain or a C-terminal domain,while the genes within group ?b were evolved by a duplication of group ? WRKY domains.In addition,subgroup ?a and?b merge into one clade,and subgroup ?d and He merge into another clade,which demonstrates a close evolutionary relationship between the subgroup ?a and Ilb,as well as between the subgroup ?d and ?e.(4)Expression profile of SsWRKYs showed that SsWRKY76 and SsWRKY93 were highly expressed in all tissues at different developmental stages of S.spontaneum,suggesting that these two genes are the main functional genes.The expression levels of SsWRKY67,SsWRKY68,SsWRKY71,SsWRKY78 and SsWRKY79 increased with the maturity of the leaves,indicating that they may play an important role in leaves growth and development SsWRKY39 and SsWRKY104 exhibited predominant expression in leaves and leaves roll of maturing plant of S.spontaneum,while decreased from top to bottom in stem during the period of sugar accumulation,indicating that these two genes may play important role in the regulation of sugar metabolism in the stem of S.spontaneum and the development of the their leaves.In addition,the expression patterns of SsWRKY genes in continuous gradient leaf segments of the developmental S.spontaneum SES208 showed that SsWRKY21/22/-39/-56/-93/-109/-124 were expressed at the high levels near the base of the leaves,indicating the involvement of those genes in basic cellular activities such as DNA synthesis,cell wall synthesis and hormone signaling.The expression of SsWRKY71 and SsWRKY145 was dominant in the region of transition from sink to source tissues,suggesting that these two genes are associated with the photosynthetic machinery.SsWRKY68,Ss WKY122 and SsWRKY129 exhibited peak expression in the distal region of the leaves,indicating these three WRKY genes may be highly correlated with the photosynthetic reactions of leaves.Finally,SsWRKY38,SsWRKY40,SsWRKY45,SsWRKY56,SsWRKY67,SsWRKY71,SsWRKY1 45 and other SsWRKY genes expression fluctuated in the mature leaves of S.spontaneum SES208 during diurnal cycle,suggesting that the transcriptional levels of those SsWRKY genes may be subject to the diurnal control.