De novo sequencing of the Freesia hybrida petal transcriptome to discover putative anthocyanin biosynthetic genes and develop EST–SSR markers

Freesia is an important bulb flower. Based on Illumina platform, the transcriptome profiling of Freesia hybrida ‘Pink Passion’ was conducted by de novo sequencing method in this study. The goal of this study is to reveal basic information and provide data on regulatory mechanism of flower color formation in freesia. Totally, 49,503,460 short reads, corresponding to total 4.46 GB nucleotides, were yielded. These short reads were then classified into 74,192 unigenes, of which 42,934 were annotated in several databases, including Nr, Nt, Swiss-Prot, KEGG, COG, and GO. A total of 43,594 coding sequences were obtained and 25,409 unigenes were allocated to 128 KEGG pathways. The “metabolic pathways” (6642 counts, 26.14%) were present as the largest category. The freesia transcriptome results revealed 205 unigenes involved in the flavonoid biosynthesis pathway and 18 unigenes in anthocyanin biosynthesis pathway. Then, 13 genes related to anthocyanin biosynthesis were identified, including 8 up-stream genes and 5 down-stream genes. MISA software identified 10,249 simple sequence repeats (SSR) as putative molecular markers, from which 4996 primer pairs were designed. Then, over 10,249 motifs were identified, and the most common motif was AG/CT (31.18%), followed by A/T and AAG/CTT. One hundred and fifty SSR primer pairs for loci were further synthesized and tested. The primers for 62 SSR loci amplified reproducible amplicons. Thirty-six polymorphic EST–SSR markers were then chosen to screen the polymorphisms among 16 freesia accessions. The genetic relationships among the 16 accessions were then assessed by the cluster analysis based on these markers. Surprisingly, the 16 freesia accessions cannot be grouped simply by an individual characteristic, indicating a potential complex genetic relationship of the tested freesias. In conclusion, this study is the first freesia transcriptome characterization by large-scale sequencing. The findings provide valuable information for germplasm characterization, genetic diversity and relationship analysis, and marker-assisted breeding in Freesia.