Development Of SSR Markers Based On Transcriptome And Their Application In Germplasm Identification Of Lilium

Lilium regale, also called ‘Regal lily’, originated from Minjiang area of Sichuan province in China,which is very hardy and tolerants lime soil. Its strong resistance to virus had rescued European lilycultivars which were on the verge of extinction. It is an important material in lily breeding. SSR markerswere developed from flowers’ transcriptome sequences of L. regale. The developed markers wereapplied to germplasm identification of Lilium. Molecular fingerprint and molecular identity of22lilyspecies and74cultivars were constructed. Phylogenetic relationships of some species and cultivars wereanalysed and principal coordinate analysis was performed to heterotic grouping of96accessions. Themain results are as follows:(1) EST-SSR markers were developed based on transcriptome of flower of L. regale.Non-redundant59046EST sequences obtained from transcriptome sequences were used to develop SSRmarkers.1072pairs of primers were designed using Primer Premier5.0. Among the1072primers,526(49%) showed amplification products in L. regale, and among the526primer pairs,494(46.1%)generated products in accord with the expected sizes,32pairs larger than expected.(2)20primer pairs were selected as core primers which were polymorphic, robust and easy torecognize. The20core primers were used for germplasm identification research on Lilium.96accessions were fingerprinted using the20core primer pairs.24out of96accessions were selected asstandard accessions, in which amplified all the alleles the20primers amplified in the96accessions.20primers were modified with6-FAM, HEX, TAMRA, ROX respectively. Fragments sizes were analysedusing ABI3130xl genetic analyzer. Fingerprint data were transformed to number code. Molecularidentity of every accession was constructed.(3) Phylogenetic relationships of the96accessions were constructed and principal coordinateanalysis was performed based on fingerprint data. From the phylogenetic diagram it can be inferred thatspecies originated from China were first divided into two groups: GroupⅠincluded L. lancifolium, L.concolor, L. dauricum, L. davidii, L. leichtlinii var. maximowiczii, et al, from which derived Asiatichybrids, Longiflorum hybrids and LA hybrids; GroupⅡ included L. leucanthum, L. henryi, L. regale,L. bakerianum et al, some of them have a close relationship with Trumpet hybrids and Oriental hybrids.The diagram also showed that the Longiflorum hybrids have a close relationship with the Asiatichybrids. Principal coordinate analysis divided the96accessions into5main groups: Asiatic hybrids,Oriental hybrids, OT, LA and Species.Plenty of EST-SSR markers were developed in this study. Research on germplasm identification ofLilium would provide useful reference for further study. Analysis of phylogenetic relationships of Liliumwas helpful for parents’ selection in breeding. The developed EST-SSR markers would have broadapplication prospects in genetic mapping, gene location and clone, comparative genomic, markerassisted selection and so on.