Chromosome Doubling Effects In Lilium Davidii Var.unicolor Based On The Phenotype And Genetics Analysis

Lanzhou lily(Lilium davidii var.unicolor)is the only edible sweet lily species in China with its bulbs edible.Currently,the largest bottlenecks in the production of bulbs involve the decrease in quality and the degeneration in yield.Chromosome doubling has been proved to be an effective way to breed new varieties with higher yield,better quality and improved stress tolerance in horticultural plants.The effects of chromosome doubling in L.davidii var.unicolor are not clear although some studies have been reported.Therefore,in order to elucidate its chromosome doubling effects,the technology including tissue culture,molecular marker,gene cloning and bioinformatics analysis were employed to optimize the chromosome doubling technique,and analyze the effect of chromosome doubling on genomic sequence variation,DNA methylation,and phenotypic.The main findings are as follows:1.Autotetraploid plants were induced by colchicine,using scale,seed or tube-bulb as the explants.The factors that affected the autotetraploid induction rate,including colchicine concentration and explant status,were examined.The results showed that scale was the ideal explant,and the highest autotetraploid induction rate(33.33%)resulted from a 15-day-preculture treatment followed by soaking in 0.025%colchicine solution for 24 h.2.The ploidy stability and the genetic integrity of the subcultured plantlets were studied.The results showed stable ploidy level and slight genomic sequence variation(7.69%)between the subcultured plants and their mother plants.The DNA content of both diploid and tetraploid of L.davidii var.unicolor were tested by flow cytometry,using Triticum aestivum “Chinese spring” as an internal standard.The results showed the genome size of L.davidii var.unicolor was 64.9 Gbp,four times greater than that of “Chinese spring”.3.The autotetraploids showed some differences from the donor diploid.In morphology,the leaves of the autotetraploids were fewer in number per plant,darker in color,and wider and deeper in shape and size.Their roots were greater in diameter.The overall plants showed a lower growth rate,and had greater chlorophyll content.In cytology,the autotetraploid had lower stomata density,and longer guard cells,greater depth of the overall leaf and mesophyll tissue,and greater cell size in the upper epidermal cells and mesophyll.In quality,the starch content in 40% of the tested autotetraploid lines was higher than that of the donor,and the soluble sugar and crude fiber content in 20% of autotetraploids were higher than that of the donor,but these values were lower in another 20% of the lines.Fifty percent of autotetraploid lines showed better drought stress tolerance than the donor.4.The wide genetic variation was produced by chrosome doubling.The polymorphism between 15 autopolyploid lines and the donor was assayed using ISSR,and the results showed wide genetic variation not only between the autopolyploid lines and the donor,but also within the autotetraploid lines.Five primers were screened from 50 ISSR primers.They showed 75% polymorphism.There were 40 amplified bands and 30 polymorphic bands.The genetic identity of autopolyploid lines to the donor was 0.4250?0.8750,and their genetic distance was 0.1335?0.8557.Accordingly,the genetic identity and genetic distance within the autopolyploid lines were 0.4000?0.9750 and 0.0253?0.9163.Cluster analysis based on ISSR data showed 11 autotetraploid lines were categorized into three major groups,indicating the dramatic difference between different autotetraploid lines.Bioinformatic analysis of polymorphism fragments of ISSR showed considerable homology to lexA,dinF,lacZ,tdcC,and mobA.We here speculate that these genes may be related to the yield and quality as well as the response to drought stresses.5.The total methylation rate in L.davidii var.unicolor was decreased by chrosome doubling.We developed and optimized a methylation-sensitive amplification polymorphism(MSAP)protocol of L.davidii var.unicolor.The study of MSAP analysis between 13 autopolyploid lines and the donor showed that there was much variation of methylation modification between them,with 43 selective primer pairs out of 45(about 95.56%)polymorphic.The total methylation rate was decreased by 5.68% in the process of diploid doubled to the autotetraploid,including demethylation of CCGG site as well as hypermethylation.The bioinformatic analysis of the polymorphism fragment of MSAP showed the high homology to the transposons SINE,pre-m RNA cleavage factor Im 25 kDa subunit 2-like,t RNA-Ile and t RNA-Val genes,and so on.These genes may affect the related characteristics including quality,the response to biotic and abiotic stresses,and reproduction.The results aboved provide a theoretical and technical basis for genetic improvement and germplasm innovation in L.davidii var.unicolor.