Automated Rna Extraction And Discovering Candidate Gene Of Wood Property Traits In Eucalyptus Urophylla×Eucalyptus Tereticonis By RNA-seq

Eucalyptus as one of the three planted forest trees in the world,because of their fast growth and high-quality wood,is an ideal materials for forest genetic study.The genetic improvement to the properties of wood is one of the main research contents in Eucalyptus genetic breeding.It is critical to find the key regulatory genes related to wood traits.With the fast development of genomics,developing one quick,efficient and automatic RNA extraction method to accelerate the research of Eucalyptus molecular biology research is getting more and more important.Based on the established method of RNA extraction,the extreme phenotypic traits of different wood including wood basic density,cellulose content,hemicellulose content and lignin content were analyzed.Seedlings and clones in Eucalyptus urophylla×E.tereticornis were selected for RNA sequencing and the expression of genes and transcription factors related to wood properties synthesis were analyzed.In order to analyze gene network changes and expression patterns of wood properties in eucalyptus,key functional candidate genes and transcription factors of wood properties traits were discovered.Our results will provide a solid genetic basis for the tree breeding and speed up the process of molecular assisted breeding in eucalyptus.The main conclusion are as follows:（1）Based on Kingfisher Flex automatic magnetic bead extraction and purification system,the quality and purity of RNA extracted from Eucalyptus tereticornis Smith leaves were evaluated for five magnetic RNA isolation kits（A/B/C/D/E,with the manufacturers’default programs）,and the optimal magnetic RNA isolation kit C was selected.In order to improve the RNA concentration and purity,orthogonal experimental design L₉（3⁴）was applied to test the washing speed,elution speed and time on the Kingfisher Flex system.Orthogonal test results indicated that the washing speed was the most important factor affecting the concentration and purity of RNA extracted.Although the purity of RNA extracted with the optimal program of L9（3⁴）was slightly better than the original program of kit C,the RNA yield was only one third of that of its default program.So the default program of kit C was selected as the optimum program for Eucalyptus tereticornis Smith leaf RNA extraction.（2）The four groups wood properties in seedling/clones that include basic density,hemicellulose content,cellulose content and lignin content were analyzed by RNA-seq data,and the differentially expressed genes（DEGs）were obtained.The number of DEGs of basic density,hemicellulose content,cellulose content and lignin content were individually 396/831,139/3543,172/1814,800/831;UP regulation DEGs were 262/361,43/910,82/524,513/1306;and DOWN regulation DEGs were 134/470,96/2633,90/392,287/508.And the enrichment analysis of Gene ontology and Kegg pathway were analyzed by using those DEGs.The GO terms enriched by the DEGs in basic density,hemicellulose contern,cellulose content and lignin content were individually 86/78,46/105,65/123 and 179/326;the number of signinficant enriched GO terms were 8/1,1/27,0/26 and 0/46.The number of the KEGG pathway enriched by the DEGs in four traits of wood properties（basic density,hemicellulose contern,cellulose content and lignin content）were 42/78,20/115,52/85 and 83/115 individually,the number of significant enriched pathway were 0/0,0/9,0/1 and 0/11 separately.（3）42,32,25 and 37 candidate functional genes were separately related to wood basic density,hemicellulose,cellulose and lignin.Some of genes are the homologous genes of Arabidopsis thaliana L.which related to secondary wall synthesis,such as cellulose synthetase gene Eucgr.H00185（CLSG3）,Cinnamyl alcohol dehydrogenase gene Eucgr.D01622（CAD9）,methyltransferase gene Eucgr.I01392（CCOMT9）.（4）Using the DEGs transcription factor enrichment analysis of seedling and clone in 4groups of wood traits,33 regulatory transcription factors possibly related to the regulation of secondary wall synthesis were obtained.And 8 regulatory transcription factors both in seedling and clone samples,13 regulatory transcription factors in seedling samples and 13 transcription factors in clone samples.Combining the results of 33 regulatory transcription factors and DEGs,8 differentially expressed regulatory transcription factors were found,which were mainly involved in the biological process such as regulation of phloem or xylem histogenesis and the biological process of differentiation of vascular component cells in xylem.