Single Molecule Real Time Transcript Sequencing Unveiled Flowering Regulatory Genes in Crocus Sativus
Annual Conference of ASHS
Crocus sativus (saffron) is prized for purple flower that is well-known for producing spice saffron, a most valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Single molecule real time (SMRT) sequencing is a newly developed technology that can generate kilobase-sized sequence reads. Unveiling flowering regulatory genes using SMRT sequencing is important to breed superior cultivars with increase flower numbers, thus resulting in high saffron yield. Two full length transcriptomes of flowering saffron and non-flowering saffron were established using SMRT sequencing separately. The bulb buds, pistils, stamens, petals and leaves of flowering saffron and terminal buds, lateral buds, leaves of non-flowering saffron were used. Meanwhile, higher-accuracy short-read sequencing of terminal and lateral buds of both flowering and non-flowering saffron and small terminal buds (< 6 g) of non-flowering saffron was obtained separately using next generation sequencing (NGS) method. Sixteen SMRT cells were detected, and 22.85 G data was acquired from the PacBio RS II panel. Flowering and non-flowering saffron had 394,653 and 252,850 high quality full-length transcripts, respectively. A total of 75,351 full-length saffron unigenes were generated. Function annotation showed that 64,562 (85.7%) full-length unigenes were annotated against databases of Cluster of Orthologous Group(COG), Swiss Prot, Non redundant(NR), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and 50,197 coding DNA sequences (CDS) were predicted with an average length of 1081 bp. Alternative splicing (AS) was detected in 72,148 unigenes with 1 to 217 AS events, among which 42 and 28 AS events existed in flowering and non-flowering saffron gene pool, respectively. In addition to long noncoding RNAs (lncRNAs), single-nucleotide polymorphism (SNP) and simple sequence repeats (SSR) in flowering or non-flowering saffron were obtained. A total of 478 full-length differentially expressed genes between flowering terminal buds and non-flowering terminal buds, 74 genes between flowering lateral buds and non-flowering lateral buds, and 1,247 genes between non-flowering small terminal buds and flowering terminal buds was acquired. At least 14 genes were closely related with flowering phenotype using real-time RT-PCR method. Further validation of AS, lncRNAs, SNP, and SSR is needed for flowering and non-flowering saffron.
Single Molecule Real Time Transcript Sequencing Unveiled Flowering Regulatory Genes in Crocus Sativus, Annual Conference of ASHS, Washington, D.C., 2 August 2018.