Construction On Full-length CDNA Library And EST-SSR Markers Of Lilium Regale Wilson

There are various species of Liliumspp.plants in our country,which have rich variations in colors and shapes of flower.Lilium regale Wilson originating in China's Sichuan Minjiang River area,80-120cm high, strong and cold,horn-shaped flowers,scented flowers,alkaline limestone soil,happyr half shade environment,but not afraid of strong light insolates,white color from light red to deep rich yellow mutation, History from her parents was a lot of growth potential and strong bright colors,flowers and beautiful series of lily hybrids.At present,Lilium regale Wilson is a rejuvenation of China's food stocks as well as anti-virus,resistant to drought,salinity Watch of New Varieties of lily germplasm important innovation of this excellent parent.At the same time, Lilium regale Wilson Wild-type also is important material of international study of the lily's genome.The generation of expressed sequence tags(ESTs)has been proven to be a rapid and efficient approach by which to identify the novel genes and analyze the function of genes.In order to identify and isolate correlative genes of colors,shapes and pollen growth of flower,a flower full-length cDNA library of Lilium regale Wilson was constructed,and it was subjected to 5'EST sequencing and bioinformatical analysis.Expressed sequence tags,increased rapidly in number recently,are important resoures for development of molecular markers.Compared with common makers derived from genomic DNA,the EST-derived maker is a novel type of molecular maker and has remarkable advantages.The study analyzed SSR recouse of ESTs data in Lilium L.rape,and preliminarily developed some EST-SSR markers, providing some referecence on rape breeding.The results were obtained as follows:1.Construction of full-length cDNA Librarycompatible reaction has been established based on Lilium regale Wilson Flower.Total RNA was extracted,equal amount of which then were mixed as reverse transcription template.Compatible cDNA library was constructed utilizing SMART^TM cDNA Library Construction Kit.9.8×10⁵pfu/mL for primary library titer,5.6×10⁹pfu/mL for amplified library titer,and 95.1%recombinants were obtained.The inserted cDNA lengths ranged from 0.5 kb to 2.0 kb,majority at about 0.8 kb.2.Acquisition and analysis of EST sequencesTotally 45 clones were randomly selected from cDNA library of Lilium regale Wilson Flower and partially sequenced.After removing the vector,repeat sequences,clustering and putting together,37 ESTs were acquired.And the ESTs similarity analysis based on BlastN software was carried on by comparing sequences in non-redundant database of GenBank.13 ESTs had the higher homology with sequences in database of GenBank;the ESTs similarity analysis based on BlastX software,22 ESTs had the higher homology with sequences in database of GenBank;the ESTs were further annotated with Geneontology(GO).The Go annotaton showed that the 51.4%ESTs significant homology to genes at the translation level.3.Development of EST-SSR markers101 SSR-ESTs from 1688 ESTs of Lilium L.in the National Center for Biotechnology Information (NCBI) database,representing 5.98%of the total number of ESTs were identified.Among them,the trinucleotide repeat is the dominant type with repeat motifs being the most common,accounting for 2.84% of ESTs.47 kinds of repeat motifs were mined out from all SSRs.23 SSR primers were designed to sequence flanking SSRs,the primer pairs designed were screened against genomic DNA of 'Snow Queen'from which most EST-SSRs were derived,and 18 primer pairs showed the amplification, accounting for 78.26%of total primers.Then the primers showing amplification were subjected to PCR for DNA from 13 Lilium L.cultivars of 5 hybridism series and 12 primer pairs showed polymorphisms, accounting for 66.7%of primers available.