Protocol For Automated DNA Extraction And Detection Of QTLs Related With Lignin In Eucalyptus

Plantation forestry supplies high-quality woody biomass for several industrial applications while acting as a carbon storage.Recently,with the completion of genome sequencing of several tree taxa including Populus and Eucalyptus and the development of high-throughput sequencing technology,molecular biology of trees has entered a high-throughput genomics era and some progresses have been achieved.The experiments for such tree studies as population geneomics and genome-wide association genetics need a large scale of samples,of which automated and high-throughput experiment operation is indispensable.Therefore,this study aimed to establish a protocol for automated DNA extraction in trees.The protocol was successfully applied to DNA extraction of a set of samples used for paternity identification of Eualyptus hybrids.Moreover,as Eualyptus species are currently a widely planted woody species in tropical and subtropical regions in the word,we focused on detecting QTLs related with lignin content and Gyringyl-to-suaiacyl ratio in Eucalyptus.The main contents and results are as follows:(1)Optimization and establishment of an automated protocol for DNA extraction from tree leaves.Five magnetic DNA kits were tested for yield and purity of DNA isolated from leaves of four Eucalyptus species/hybrid using KingFisher Flex purification system,and two superior kits were preliminarily selected out.The washing speed ‘Medium' was determined from the comparison of four settings on KingFisher Flex.DNA samples isolated with the two superior kits could result in effective PCR amplification,indicating acceptable DNA purity,and the highest yielding kit was then the choice of our method.As compared with manually performing CTAB method(DNA yield 19.0 ?g/g,OD260/OD280 1.97 and OD260/OD230 2.03),our method could increase considerably the DNA yield(103.6 ?g/g)while retaining similar DNA purity(OD260/OD280 1.93 and OD260/OD230 1.89).Our method was effectively applied to isolate DNA from leaves of Acacia mangium,Populus alba × Po.berolinensis,Dalbergia odorifera,Pinus kesiya var.langbianensis,Pi.massoniana and Casuarina equisetifolia.This is valuable for high throughput DNA isolation in tree molecular biology studies and would also provide potential method for isolating DNA from non-woody plants.(2)Application of this automated protocol for DNA extraction in paternity identification of Eualyptus hybrids.A total of 96 samples,including an maternal parent,two unidentified paternal parents and 93 putative hybrid sibs,were analysed using 16 polymorphic EST-SSR markers.The paternity of 92 putative sibs was successfully identified at 95% confidence level.Among them,the paternal tree of 87 sibs was the pollen parent in the seed orchard.,and the paternal parent of 5 sibs was a parent identified outside the seed orchard.Hence,it proved that the automated DNA extraction protocol established can be successfully applied to fast DNA extraction of a relatively large number of samples.This may lay a foundation for further application of this method to other tree species.(3)Detection of QTLs related with lignin in Eucalyptus.QTLs related with lignin and G/S ratio were detected in a cloned E.urophylla × E.tereticornis cross.A total of four QTLs were identified for the two traits on both parental linkage maps constructed earlier,with LOD scores 3.8-20.6 and explaining 5.1-23.1% of the total phenotypic variation in the respective traits.