Distant Somatic Hybridization Alters The Structure And Function Of Wheat Root Bacterial Microbiota And Its Molecular Mechanism

The root microbiota is an important regulator of plant growth and stress resistance,and it is also a treasure trove of agricultural production resources to be developed.Plants are exposed to a variety of biotic and abiotic stresses in the environment.These biotic and abiotic stresses can induce changes in the plant transcriptome and metabolome,leading to changes in root exudates,which in turn alter plant-associated microbial communities.Root microbiota improve plant stress resistance by promoting plant uptake of nutrients,while plants regulate the composition and function of root microbiota through various root metabolites.In this study,the saline-alkali-tolerant wheat Shanrong 4(SR4)and its parent Jinan 177(JN177),which were selected from the wheat somatic hybrid introgression line,were used as experimental materials.By combining amplicon sequencing technology with RNA-seq technology Combined,the root bacteria and fungi genera related to the resistance to saline-alkali stress of wheat were identified,the possible regulatory genes of the wheat root microbiome were found,and the influence of distant hybridization on the structure and function of the wheat root microbiome and its molecular mechanism were revealed.The results of this study are as follows:1.In this study,the differences in dry weight,growth vigor,and aboveground Na⁺content of SR4 and JN177 were compared between ordinary field and saline-alkali soil.The results showed that the resistance of SR4 to saline-alkali stress was stronger than that of JN177.We then measured the content of O₂^-and sodium ion in SR4 and JN177 under three conditions:saline-alkali soil,sterilized saline-alkali soil,and adding soil to sterilized saline-alkali soil to extract microorganisms.The results showed that SR4 could recruit soil microorganisms better than JN177 to help resist saline-alkali stress.2.In this study,16S r RNA amplicon sequencing technology was used to investigate the effects of distant hybridization on the structure and function of bacterial communities in wheat roots.The results are as follows:A total of 946 operational taxonomic units(OTUs)were obtained for JN177 and SR4 root samples in normal and saline soils.Alpha diversity analysis showed that distant hybridization had no significant effect on the?-diversity of wheat root bacterial communities.The root bacterial communities of JN177 and SR4 were mainly composed of Proteobacteria,Bacteroidetes,Actinobacteria,Firmicutes and other phyla.Proteobacteria was the dominant bacteria.At the genus taxonomic level,the root bacterial communities of JN177 and SR4 were composed of Pseudomonas,Pantoea,Flavobacterium and other genera,with Pseudomonas dominant.PCo A analysis showed that wheat genotype was the most important factor affecting the bacterial community structure in wheat roots,followed by soil type.Lefse found that the characteristic taxa significantly enriched in SR4-C belonged to Proteobacteria,and the characteristic taxa significantly enriched in SR4-SL belonged to Actinobacteria;the parental JN177(JN177-SL and JN177-C)Significantly enriched characteristic taxa belonged to Proteobacteria.The difference test between groups showed that there were statistical differences between Pseudomonas and Variovorax in the four groups of JN177-C,SR4-C,SR4-SL and JN177-SL.The functional prediction analysis of wheat root bacteria by PICRUSt found that amino acid transport and metabolism,energy production and conversion,transcription,carbohydrate transport and metabolism,cell wall/membrane/envelope biosynthesis,and inorganic ion transport and metabolism are important for wheat root bacterial communities Function.Carbon metabolism,Biosynthesis of amino acids metabolism,Pyruvate metabolism,Glyoxylate and dicarboxylate metabolism,ABC transporters,and Two-component system are related to the response of wheat root bacterial community to saline-alkali stress.3.In this study,ITS amplicon sequencing technology was used to investigate the effects of distant hybridization on the structure and function of wheat root fungal communities.The results are as follows:For JN177 and SR4 root samples in normal soil and saline-alkali soil,a total of 899 fungal operational taxonomic units(OTUs)were obtained.Alpha diversity analysis showed that distant hybridization had no significant effect on the?-diversity of wheat root fungal communities.The root fungal community of wheat is composed of Ascomycota,Basidiomycota,Chytridiomycota and other phyla.Ascomycota is the dominant fungus.At the genus taxonomic level,the root fungal communities of JN177 and SR4 are composed of Pyrenochaetopsis,Alternaria,Holtermanniella,and Preussia,Fusarium,Mortierella,Cyphellophora and other genera,Pyrenochaetopsis is dominant.PCo A analysis showed that soil type was the most important factor affecting the fungal community structure in wheat roots,followed by plant genotype.Lefse found that the characteristic taxa significantly enriched in SR4 in the distant hybrid introgressed line belonged to Basidiomycota,and the characteristic taxa significantly enriched in parental JN177 belonged to Ascomycoat.The difference test between groups showed that Pyrenochaetopsis had statistical differences among the four groups of JN177-C,SR4-C,SR4-SL and JN177-SL;Preussia had extremely significant differences among the four groups.PICRUSt2 function prediction heat map analysis found that the categories with higher fungal functional abundance were all related to basal metabolic or physiological functions.Adenosinetriphosphatase,Glucan 1,4-alpha-glucosidase,L-arabinose isomerase and Protein-tyrosine-phosphatase were involved in the response of wheat root fungal community to saline-alkali stress.4.This study used a transcriptomic approach to investigate the key genes and signaling pathways regulated by the root microbiom e of JN177 and SR4.19,600 differentially expressed genes between JN177 and SR4 were obtained.In saline-alkali soil,JN177 and SR4 had a total of 2854 differentially expressed genes,of which 1428were up-regulated and 1426 were down-regulated.There were 8126 differentially expressed genes in JN177 and SR4 in common soil,4999 were up-regulated and 3127 were down-regulated.The GO f unctional annotations of JN177 and SR4 differential genes are mainl y divided into:Molecular function,Biological process and Cellular components.WGCNA analysis identified 27 expression modules.Thr ough the correlation analysis between modules and phenotypes,four modules of ME turquoise,ME pink,ME salmon and ME brown were excavated.The hub genes of these four modules are key gene s regulating the root microbial communities of JN177 and SR4 gro wing in saline-alkali soil.These hub genes(eg Traes CS1D02G295800,Traes CS1A02G296300,Traes CS1B02G224900 and Traes CS6A02G102400)are key regulatory genes.The response of the wheat root microbiome to saline-alkali stress mainly involves:nitrogen uptake,transport,primary nitrate response,signal transduction pathways,anti-oxidative damage,regulation of channel proteins to maintain Na⁺-K⁺ion balance,and epigenetic mechanisms.This suggests that SR4 u nder salinity stress activates these genes regulating root microbiota and recruits specific root bacteria and fungi to enhance their toleran ce to salinity stress.In conclusion,root microbiota is the key factor to maintain the better balance of sodium and potassium ions in SR4 than JN177 under saline-alkali stress.Through systematic comparison,we identified specific bacterial and fungal taxa of SR4 under saline-alkali stress.Through WGCNA analysis,key functional genes(eg:Traes CS1B02G224900,Traes CS1A02G210900)and pathways were identified.These results suggest that somatic hybridization alters key genes regulating the wheat root microbiome,further enhancing the salinity tolerance of SR4.This study lays a foundation for further revealing the regulation mechanism of wheat root microbial community,and points out the direction for the development of microbial inoculants that can improve the salinity and alkali tolerance of wheat,which has important practical significance for the sustainable development of agriculture.