Abstract
BackgroundSimple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low.Methodology/Principal FindingsIllumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity.Conclusions/SignificanceSSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera.
Highlights
SSRs represent an informative class of genetic markers
One approach taken to enhance the efficiency of the process has been to target expressed sequence tag (EST) sequence, which is acquired by sequencing a reverse-transcribed preparation of mRNA
For the RNA required for the transcriptome sequencing, stems and leaves were harvested from three 30 day old C. nankingense cuttings rooted on MS media and grown at a constant temperature of 25uC and a 16 h photoperiod
Summary
SSRs (simple sequence repeats) represent an informative class of genetic markers. They are distributed throughout the coding and non-coding regions of all eukaryotic genomes [1] and have been widely use to characterize genetic diversity, identify germplasm, construct linkage maps and tag genes for the purpose of markerassisted breeding [2,3,4]. SSR markers derived from EST sequence (‘‘EST-SSRs’’) are attractive because their location within coding sequence enhances the probability of successful cross-species transferability [7,8,9]. Few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low
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