Comprehensive Evaluation Of Salt Tolerance And Leaf Transcriptome Analysis Under Salt Stress Of Willow At Seedling Stage

Willow is widely distributed in China,and has fast growth rate and strong saline-alkali stress tolerance.Willow is the main afforestation tree species for coastal saline and alkaline soil treatment.The research on its salt-tolerance and salt-tolerance molecular mechanism can better play its important role in saline and alkaline soil improvement.In this study,the salt tolerance of 5 willow species was comprehensively evaluated and the identification indexes of salt tolerance were screened.On this basis,the transcriptome sequencing were performed on the leaves of Salix psammophila ‘Yanliu NO.1’ and Salix matsudana ‘Bohailiu NO.1’ to explore the molecular mechanism of willow response to salt stress from transcriptome level.With the hope of providing a reference for salt tolerance evaluation of willow germplasm resources and laying a foundation for further research on salt tolerance mechanism of willow.The main results are as follows:(1)16 growth and physiological indexes of 5 willow species at seedling stage were measured under different salinity treatments,the comprehensive salt tolerance evaluation of 5willow species were screened by principal component analysis and membership function method.The salt tolerance of 5 willow species was Salix psammophila ‘Yanliu NO.1’ > Salix×aureopendula CL.‘Gaokang’ > Salix matsudana ‘Bohailiu NO.3’> Salix matsudana ‘Bohailiu NO.2’>Salix matsudana ‘Bohailiu NO.1’.The optimal regression equation of salt tolerance prediction was established by stepwise regression analysis,peroxidase activity,transpiration rate and malondialdehyde content were selected as the salt tolerance identification indexes of willow seedlings.(2)The transcriptome sequencing of the leaves in Salix psammophila ‘Yanliu NO.1’and Salix matsudana ‘Bohailiu NO.1’ before and after salt stress treatment stress were conducted by Illumina sequencing technology.A total of 588 832 108 clean reads were obtained.After De Novo assembly,183 987 Unigenes were obtained with an average length of 1 080.18 bp and N50 of 1 690 bp,among them,14 9864(81.45%)Unigenes successfully annotated in each database.33 684 SSRs were found with a frequency of 15.26%.And a total of 6 125 transcription factors were identified,which were from 58 transcription factor families.(3)The differentially expressed genes of two willow species were screened.After salt stress,5698 differentially expressed genes were selected from Salix psammophila ‘Yanliu NO.1’,including 1428 up-regulated genes and 4270 down-regulated genes.and 536 differentially expressed genes were selected from Salix matsudana ‘Bohailiu NO.1’ including 232 upregulated and 304 down-regulated genes.There are 218 genes were both differentially expressed in Salix psammophila‘Yanliu NO.1’ and Salix matsudana ‘Bohailiu NO.1’.(4)GO enrichment analysis was performed on the differentially expressed genes of two willows.The differentially expressed genes in Salix psammophila‘Yanliu NO.1’ were mostly enriched in the GO term such as protein phosphorylation(BP),regulation of transcription,DNAtemplated(BP),metabolic process(BP),ATP binding(MF),DNA binding(MF),protein serine/threonine kinase(MF),integral component of membrane(CC)and nucleus(CC).The differentially expressed genes of Salix matsudana ‘Bohailiu NO.1’ were mostly enriched in the GO term such as oxidation-reduction process(BP),metabolic process(BP),cysteine biosynthetic process(BP),metal ion binding(MF),chlorophyll binding(MF),integral component of membrane(CC),photosystem I(CC)and photosystem II(CC).(5)KEGG pathway enrichment analysis of the differentially expressed genes showed that the differentially expressed genes of Salix psammophila‘Yanliu NO.1’ were significantly enriched in 14 pathways including plant-pathogen interaction,plant hormone signal transduction,amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,isoflavonoid biosynthesis,regulation of autophagy,phosphatidylinositol signaling system.The differentially expressed genes of Salix matsudana ‘Bohailiu NO.1’ were significantly enriched in 17 pathways including photosynthesis-antenna proteins,photosynthesis,metabolic pathway,porphyrin and chlorophyll metabolism,pyruvate metabolism,starch and sucrose metabolism,and biosynthesis of secondary metabolites.(6)q RT-PCR validation of selected 7 DEGs was used to evaluate the rseults of transcriptome sequencing,the expression trend of q RT-PCR and transcriptome sequencing results were consistent to each other indicated the reliability of the results of transcriptome sequencing.