The rapid alkalinization factor (RALF) gene family is essential for the plant growth and development. However, there is little known about these genes among Rosaceae species. Here, we identify 124 RALF-like genes from seven Rosaceae species, and 39 genes from Arabidopsis, totally 163 genes, divided into four clades according to the phylogenetic analysis, which includes 45 mature RALF genes from Rosaceae species. The YISY motif and RRXL cleavage site are typical features of true RALF genes, but some variants were detected in our study, such as YISP, YIST, NISY, YINY, YIGY, YVGY, FIGY, YIAY, and RRVM. Motif1 is widely distributed among all the clades. According to screening of cis-regulatory elements, GO annotation, expression sequence tags (EST), RNA-seq, and RT-qPCR, we reported that 24 RALF genes coding mature proteins related to tissue development, fungal infection, and hormone response. Purifying selection may play an important role in the evolutionary process of RALF-like genes among Rosaceae species according to the result from ka/ks. The tandem duplication event just occurs in four gene pairs (Fv-RALF9 and Fv-RALF10, Md-RALF7 and Md-RALF8, Pm-RALF2 and Pm-RALF8, and Pp-RALF11 and Pp-RALF14) from four Rosaceae species. Our research provides a wide overview of RALF-like genes in seven Rosaceae species involved in identification, classification, structure, expression, and evolution analysis.
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