| Maize is an important crop for food,feed and industrial raw materials.Maize is rich in endophytic microorganisms which includes both beneficial and pathogenic bacteria.During the whole growth cycle of maize,endophytic microorganisms may not only promote maize growth and resist biotic and abiotic stresses,but also may cause maize diseases.Maize kernel is an important reproductive organ,the endophytic microorganisms can be transmitted to offspring with vertical inheritance,which has an important impact on the quality of maize kernel,yield and plant growth and development.The differences in the composition of endophytic microorganisms in maize germplasm are often related to the origin of germplasm,growth location and the host genes,and the microbial secondary metabolites produced by them also have different physiological activities,which is one of the influencing factors that causes differences in growth characteristics of different maize germplasm.Amplicon sequencing is currently the main method for the study of endophytic microorganisms in animals and plants,but it can only be used to obtain species composition of bacteria and fungi by amplifying bacterial and fungal marker sequences in the sample,respectively,which can not obtain all the microorganism composition in samples,nor to compare the relative content between endophytic microorganisms and plants and endophytes,and it is easy to overlook the protozoa that have received attention in recent years,which has already contrained the faster development of endophytic microbial research.At present,limited by the research methods and costs of endophytic microorganisms in animals and plants,the changes of endophytic microorganisms during the differentiation of different germplasm maize populations and the mechanism of endophytic microbial communities regulated by host genes have not been reported.Therefore,it is of great significance for plant production and germplasm innovation to develop a new method for mining plant endophytic microorganisms to study the differences in endophytic microbial communities of different germplasms and the association with plants.In this study,a new method for mining information from the endophytic microbiome of animals and plants was developed,and endophytic microorganisms of368 maize inbred line germplasm kernels from tropical/subtropical(TST)and temperate(NSS)regions were used to study the characteristics,community differences and association factors of endophytic microbiome in different maize germplasms kernel from the perspectives of community composition,interaction network and genome-wide association analysis,the main research and results were as follows:(1)A new method to mine endophytic microbiome information from animal and plant transcriptome data was developed,which can directly obtain microbial community composition and relative content information between endophytic microorganisms and plants and endophytes from the transcriptome sequencing data of animals and plants.Compared with the amplicon sequencing method,more types of endophytic microorganism information can be obtained with the new method,and it can solve the defect that amplicons cannot analyze the relative content of bacteria and fungi and save experimental costs.The effectiveness of the method was verified by various experimental data.(2)The endophytic microbiome information in the kernels of 368 maize germplasm inbred lines was mined based on the new method,and the microbial species annotation,classification and abundance information were obtained,and the contents of different classifications were compared.The results showed that the content of plant genome generally accounted for more than 99.5%,and the majority content of microbial genome was less than 0.5%.Among the endophytic microorganisms of developing maize kernels,bacteria and fungi had the highest content,followed by protozoa,and less virus.(3)The composition of the endophytic microbial community in the kernels of 368 maize inbred lines was analyzed.The results showed that there were great differences in the composition and relative abundance of endophytic microorganisms in the kernels of different maize inbred lines.A total of 9 phyla,12 classes,22 orders,30 families,84 genera,and 166 species of archaea were detected;A total of 52 phyla,73 classes,169 orders,390 families,1425 genera,and 6194 species of bacteria were detected;A total of 9 phyla,57 classes,200 orders,588 families,1905 genera and4815 species were detected in fungi.(4)Diversity analysis of endophytic microorganisms was carried out according to tropical/subtropical(TST),temperate(NSS)and other maize germplasm groups.The results showed that the community composition structure of the groups were generally similar,but the number and relative abundance of the community were different.In the tropical/subtropical group,1 order,2 families,1 genus and 4 species of bacteria were significantly enriched,and 1 phylum,3 orders,3 families,3 genera and 5 species of fungi were significantly enriched.In the temperate group,3 phyla,4 classes,3orders,6 families,9 genera and 9 species of bacteria were significantly enriched,1phylum,4 orders,3 families,1 genus and 1 species of fungi were significantly enriched.(5)A co-occurrence network of endophytic microorganisms in tropical/subtropical and temperate maize kernel at the genus level was constructed.The interactions of microorganisms in the network were mainly positive and the endophytic microbial network in tropical/subtropical maize kernels was more closely connected and more stable than that in temperate maize kernels.The species of high degree in networks were mainly bacteria,and different core species of the network were microbial phylum specificity.Although some species were low abundance,the stability of the network as core species was mantained.Most of the core species of endophytic microbial network in tropical/subtropical maize kernels were beneficial endophytes,while most of the core species of endophytic microbial network in temperate maize kernels were pathogens.21 modules were detected in the TST group network and 15 modules were detected in the NSS group network,and the species composition of each module showed a high degree of microbial phylum specificity.(6)With genome-wide association analysis between richness,diversity,dominant species abundance,network core species abundance of 368 maize endophytic microbial community and maize genome,72 significantly associated SNP loci were obtained and 142 association candidate genes were identified.Seventeen of these genes were found in previous studies that may be associated with microbial diversity and abundance.GO and KEGG analysis revealed that these candidate genes were involved in different functions and metabolic pathways,which may be the genetic basis for maize to influence and regulate the endophytic microbial community.The results of the above research provided a new method and theoretical basis for the efficient exploration of the maize endophytic microbiome,the isolation and utilization of beneficial endophytes in the kernels,and the in-depth study of the differential formation and regulation mechanism of endophytic microbial community. |
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