Genome-wide identiﬁcation and analysis of cystatin family genes in Sorghum ( Sorghum bicolor L.)

To set a systematic study of the Sorghum cystatins ( SbCys ) gene family, a comprehensive genome-wide analysis of the SbCys family genes was performed by bioinformatics-based methods. In total, 18 SbCys genes were identiﬁed in Sorghum, which were distributed unevenly on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes had similar exon/intron structure and motifs, indicating their high evolutionary conservation. Transcriptome analysis showed that 16 SbCys genes were expressed in diﬀerent tissues, and most genes displayed higher expression levels in reproductive tissues than in vegetative tissues, indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the expression proﬁles of the SbCys genes revealed that 7 cystatin family genes were induced during Bipolaris sorghicola infection and only 2 genes were responsive to aphid infestation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) conﬁrmed that 17 SbCys genes were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed development and responses to diﬀerent stress conditions. ABSTRACT 25 To set a systematic study of the Sorghum cystatins ( SbCys ) gene family, a comprehensive 26 genome-wide analysis of the SbCys family genes was performed by bioinformatics-based 27 methods. In total, 18 SbCys genes were identified in Sorghum, which were distributed unevenly 28 on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes 29 had similar exon/intron structure and motifs, indicating their high evolutionary conservation. 30 Transcriptome analysis showed that 16 SbCys genes were expressed in different tissues, and most 31 genes displayed higher expression levels in reproductive tissues than in vegetative tissues, 32 indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the 33 expression profiles of the SbCys genes revealed that 7 cystatin family genes were induced during 34 Bipolaris sorghicola infection and only 2 genes were responsive to aphid infestation. In addition, 35 quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 17 SbCys genes 36 were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the 41 structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed 43 development and responses to different stress conditions. expression profile in different tissues. We also explored the expression patterns of SbCys genes in response to biotic and abiotic stresses. The results may lay a foundation for further functional analyses of cystatin genes.