Genetic Analysis Of Ability When Leaf To Regenerate Adventitious Shoots In Populus Euphratica

Plant regeneration is a widespread phenomenon in nature which is applied in tissue culture,cutting grafting and other agricultural practices.Injured plants can regenerate adventitious roots or shoots by means of de novo organogenesis.The de novo pathway of shoots has been attracted attention and research has gradually shifted from the physiological to genetic mechanism.In this study,the leaves of Populus euphratica from natural populations were used as experimental materials to collect dynamic growth data of regeneration shoots by establishing a leaf differentiation system.The methods which were combined with QTL mapping and transcriptome sequencing,were explored candidate genes related to the regeneration process.Subsequently,gene expression of different genotypes were conducted by qRT-PCR to reveal the genetic pattern.The main research results are as follows:1.Establishment a differentiation system of Populus euphratica leaf and collection the growth data of adventitious shoots: 164 Populus euphratica were cultured in dark for 10 days and light for 30 days and recorded the amounts of regenerated shoots,proliferation coefficient and regeneration rate at an interval of 5 days.It was found that 139 genotypes had the ability to differentiate regenerative shoots,while 25 genotypes did not.It showed a trend with fast at first and then slow to the growth trend of regenerated shoots.The regeneration rate was high before 15 days and the regenerated shoots developed quickly.From 20 to 30 days,the proliferation coefficient increased,and the amounts of regenerated shoots increased slowly after 25 days.Finally,we obtained SNP markers of Populus euphratica by Illumina platform and a total of 202,573 SNP markers were screened by the vcftools tool for subsequent model studies.2.The QTL mapping of leaf regeneration was carried out,including case-control model,function mapping of multi-time points and growth parameters were located 18,24,73 significant QTLs respectively,intend to explore the opening time,the differentiated degree,and the growth process of regenerative shoots.The GO enrichment of these genes was most significant in biological functions such as nucleotide transferase,nuclear activity,and nuclear fusion.Screen the genes related to the regeneration process and draw the genotype curves,each QTL showed significant differences.The functions of these genes mainly involved plant organ growth and senescence,stress response,plant immunity,amino acid and protein metabolism,the process which affected the growth of plant stems,flowers,shoots and so on.3.The semi-thin section experiment obtained the key growth points of all genotypes.The p0,p1,and p3 of adventitious shoots were cultured for 0 days,dark culture for 7 days and light culture for 30 days,but in all genotypes,p2 time were 13 days for light culture except for 1274 and T056,which need cluture 15 days.This indicated that there showed early or late phenomenon when various genotypes regenerated shoot.Meanwhile,it was observed that all genotypes with strong differentiation ability could complete callus initiation and adventitious shoot initiation,while the genotypes with weak differentiation ability could only complete callus initiation but not shoot initiation,thus affecting the development of adventitious shoot.4.In transcriptome sequencing,163,711 transcripts was obtained through Trinity assembly,at the same time,46,196 annotations were obtained after functional annotation of the transcripts in Nr,Nt,Pfam,KOG/COG,Swiss-Prot,KEGG and GO databases and the functions involve in signal transduction,cell growth and death,environmental adaptation,plant development and so on.At the same time,a total of 19,349 differential genes were obtained and 107 genes related to regeneration process were screened,which contained wound or other stress pathway,plant hormone behavior,callus,or root formation pathway,adventitious shoots formation pathway and leaf morphology,epidermal development and cell proliferation,division,and apoptosis.5.The longest transcripts in transcriptome sequencing were locally constructed to compare candidate genes obtained by QTL mapping,and finally to screen out 9 genes related to regeneration process,which were mainly involved in plant hormone behavior,plant morphogenesis,plant defense and other biological development processes.Among them,HAT3 and CYP40 genes appeared simultaneously in QTL mapping and the new genes by library construction,and LOG7 gene appeared simultaneously in transcriptome and the new genes by library construction.6.Combined with QTL mapping and transcriptome sequencing,we performed 12 genes at specific growth stages by qRT-PCR.The expression of regeneration-related genes at specific growth stages showed differences among the genotypes with strong and poor differentiation ability.Among them,the genes which were consistent with previous studies include ERF109,SAUR21WOX11 and WOX9 genes,the genes with weak differentiation ability that are not expressed on time include SHR,CLE1,ATHB8 and WOX1,and genes with early and late gene expression differences included CUC3,YUCCA4 and CLE19.This study revealed the key points in regeneration of adventitious shoots under different genotypes and obtained a vast amount of genes associated with the regeneration process in Populus euphratica.The genes functions of specific stages in growth development were analyzed,which was beneficial to the genetic analysis of leaf regeneration ability in Populus euphratica and laid a theoretical foundation for other tree species in regeneration research.