Identification,evaluation And Genetic Study On Dwarf Traits In Litchi Chinensis Sonn.

Litchi(Litchi chinensis Sonn.)is the most important fruit tree widely cultivated in South China.Under the traditional management,the litchi tree is very tall and can grow to more than ten meters,which causes inconvenience to management.In recent years,dwarf cultivation has become an important cultivation mode for the modern management of orchards,and the innovation and utilization of dwarf varieties or rootstocks is an important way to achieve dwarf cultivation.But in most litchi orchards,heavy pruning techniques are usually adopted to control plant height to achieve dwarf cultivation purposes,and dwarf germplasm resources are not effectively cultivated and utilized.The inheritance and mechanism of dwarf-related traits in litchi are still unclear.This study carried out the identification and genetic laws of dwarf traits,and the screening for dwarf-related genes and QTLs,which would provide a theoretical reference for the exploration and innovation of dwarf resources in litchi.The main results are as follows:1.Using all or part of 120 litchi germplasm resources as materials,dwarf-related phenotypes such as length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),diameter of common petiole(DCP),length of internode(LI),length of single leaf(LSL),width of single leaf(WSL)and bark percentage(BP)were measured and analyzed.According to the data,except the bark percentage,there is more or less correlation between any of the two traits.Through the factor analysis,the concept and calculation formula of the dwarf index(D value)of litchi was proposed.A standard for determining the dwarf classification of litchi based on D value was established,and the dwarfing characteristics of 120 litchi resources were evaluated and graded by five levels.The results of the classification were basically consistent with the judgment of practical experience.In addition,through the comparative analysis of the bark percentage,it is obvious that the bark percentage has a significant negative correlation with the diameter of new branch,and the correlation coefficient is-0.69.Even in different varieties,there would be also no significant difference in the bark percentage if there is no significant difference in diameter of new branch.Therefore,the bark percentage is not suitable as a key indicator for evaluation of dwarf degree in litchi.2.High-throughput transcriptome sequencing and gene expression analysis were performed on Litchi chinensis cv.‘Feizixiao'(FZX,vigorous varieties)and‘Ziniangxi'(ZNX,dwarf varieties).Results indicated that a total of 55,810 representative transcripts(unigenes)were obtained,45,740(81.96%of 55,810)unigenes were matched to one or more of the databases,9190 DEGs were differentially expressed in the transcriptome data.A total of 3,248 DEGs were detected between FZX-apical-buds and ZNX-apical-buds and1,750 DEGs were detected between FZX-leaves and ZNX-leaves.These DEGs mainly focused on‘phytohormone signal transduction',‘biosynthesis of other secondary metabolites',‘genetic information process',‘oxidative phosphorylation'and other related biological processes.42 key genes involved in plant hormone signal transduction pathways and 69 key genes involved in energy metabolism pathways were analyzed.Some candidate genes and gibberellin biosynthesis and metabolic genes were confirmed by RT-q PCR.In addition,three Lc GA2ox genes,Lc GA2ox1(MSTRG.54748),Lc GA2ox2(MSTRG.12960),and Lc GA2ox3(MSTRG.14947),under the expression of the 35S promoter were transformed into tobacco.The result indicated that the 35S::Lc GA2ox transgenic tobacco plants were dwarf and had smaller leaves or branches than wild type plants.These results showed that ectopic overexpression of Lc GA2ox genes in tobacco leads to the dwarf phenotype in transgenic plants,which will be helpful for the litchi dwarf mechanism studies.3.The dwarf cultivar‘Ziniangxi'was used as the female parent,and the extremely vigorous variety‘Feizixiao'was used as the male parent for hybridization.The SRAP molecular markers were used to identify the true or false hybrids of the offspring,and then,the genetic laws and characteristics of dwarf-related traits in hybrids were investigated and analyzed.The results showed that 266 strains of progeny were obtained by hybridization,and 194 true hybrids were screened by SRAP molecular markers,and the true hybrid rate was 72.9%.Finally,178 surviving true hybrids were used to construct the F₁populations.The dwarf traits of the hybrid population were investigated,and the genetic characteristics of dwarf-related traits such as length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),et al.,and also the vigorous index(F value),were identified.The coefficient of variation of each phenotype is from 11.50%to 23.23%,and the mid-parent heterosis is from-7.37%to 18.68%.Overall,the F₁hybrid population contains suitable individuals,and the target traits of the offsprings are significantly separated,which is consistent with the basic requirements for mapping population.4.We applied a genotyping-by-sequencing(GBS)protocol to discover and genotype single nucleotide polymorphism(SNP)markers in the‘Ziniangxi'בFeizixiao'F₁hybrid populations.A total of 8,654,911 single nucleotide polymorphism locus were identified,and5,100,050 of them which marker-typed as‘hk×hk',‘lm×ll',‘ef×eg',‘ab×cd',‘nn×np'were represented in the F₁population.After the examination and screening for genetic markers,3,027 markers were comprised of the genetic map and spanned the cumulative length of1711.97 c M.Compared with the map constructed by traditional molecular marker technique,this GBS-based genetic map showed a substantial improvement in marker density,with one SNP marker in every 0.57 c M.Thirty-seven quantitative trait loci(QTL)for eight dwarf-related traits were detected,which could explain 8.0%to 14.7%(mean=9.7%)of the phenotypic variation.126 candidate genes were identified within the QTL regions,50 of which are differentially expressed genes by RNA-seq analysis between‘Ziniangxi'and‘Feizixiao'.These DEGs were found to participate in regulation of cell development,material transportation,signal transduction,and morphogenesis of plants,which might play important roles in regulating plant dwarf-related traits.The high-density genetic map newly constructed,and the QTL and candidate genes identified would provide a valuable genetic resource and a basis for marker-assisted selection and genomic studies for litchi.