| Lily is a commercially important bulb flower. Although lily cytogenetic and breeding was studied extensively, its molecular genetic studies are limited. The very complex and huge genome (36Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed. In this research, Illumina HiSeq2000sequencer and bioinformatics tools were implemented to construct a lily organ transcriptome and gene expression profiles. SSR markers were developed base on EST database and applied for genetic studies. Main results are as follows:1. A total of49,991transcripts with an average length of673bp were produced from a4.6Gb transcriptome data. Approximately72%of the transcripts were annotated against public protein databases and39,658coding sequences were predicted. The most common repeat motifs were trinucleotide and dinucleotide, with GA/CT and GGC/CCG the most common repeats. A comprehensive lily hybrid assembly, L.-Unigene-All with69,615transcripts of an average length of1600bp was generated by merging reported lily transcripts. The distribution of gene ontology was very similar for different transcripts. The transcrtiptome will be beneficial to expression profiles studies and functional gene mining.2. Lily gene expression profiling of11Mb for flower, leaf and bulbs were constructed respectively. A total of2685differentially expressed genes were found for leaf-flower,2296for leaf-bulb and1709for flower-bulb. There were74commonly differentially expressed genes among three organs. They participated into pathways like Photosynthesis, biosynthesis of secondary metabolites, signal transduction, nitrogen metabolism, plant-pathogen interaction, metabolic pathway. Totally,17%those differentially expressed genes are new transcripts and more than one hundred pathways were found for each pair of the three groups. Based on functional enrichment analysis, differentially expressed genes for flower mainly participated into flavone and flavonol biosynthesis; those for leaf chiefly took part into photosynthesis and those for bulbs primarily were of starch and sucrose metabolism. Particular attentions were focused on genes like DFR, LcTPS, psbA and SBE with functions for flower color, scent, photosynthesis and starch analyses. This gene expression profiles are available for candidate gene mining and functional researches for Lilium.3. An optimal two-step PCR amplification system with M13as universal fluorescent-label at the5’end of each primer pairs was established based on adjusting of the concentration of DNA, the concentration and kind of polymerase, the kind of adjuvant and anneling temperature. Mining EST database from GenBank and transcriptome we gained,61SSR markers were validated, with a total of153alleles, average alleles of5.7and average polymorphism information content of0.55for32lily accessions. The sequence discrepancy of transcripts from sequences before and after hybrid assembly may be one reason for less SSR developed and the other may due to the huge and complicated lily genome. Briefly, the newly developed SSR markers are of useful for genetic diversity, linkage mapping, and parentage analysis.4. The34SSR markers (selected from51previously reported markers) and61newly developed markers in our lab were used in the analysis of transferability and genetic diversity in69lily accessions and Hemerocallis fulva. A total of611polymorphism alleles were amplified in69lily genotypes with the average alleles of6.43, an average polymorphism information content of0.55, an average observed heterozygosity of0.50and an average expected heterozygosity of0.60. Among those polymorphic loci,75of them were rare alleles that maninly existed in wild lily. The analyzed lilies were divided into two groups:Oriental and non-Oriental, with L. brownii var. giganteum clustered with L. browni and L. pumilum from Beijing clustered with that from Shanxi. The candidate fathers,’Cocar D’or’,’Cold Play’’Ariosto’,’Souvenir’and’Love Story’ for11-45,11-93,11-94,11-150, and11-173respectively were identified based on SSR markers of non-null alleles and zero-mismatch by CERVUS software. The information we obtained here on the selected95SSR markers can be valuable in phylogenetic studies and parentage analysis for further breeding programs. Of the95markers,29were transferable in Hemerocallis genus... |
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