Analysis of microsatellite markers related to monoterpenoid biosynthesis in medicinal plant ajowan using RNA-Seq

Microsatellite markers play a key role in the genome and gene expression, especially in secondary metabolite biosynthesis in medicinal plants. Trachyspermum ammi, which known as ajowan belonging to family Apiaceae is a highly valued medicinally important seed spice. Ajowan ecotypes ‘Arak’ and ‘Shiraz’ were grown at the experimental station of College of Abouraihan, University of Tehran, Pakdasht, Tehran, Iran. RNA was isolated from T. ammi inflorescences (5 days after anthesis) using TRIzol reagent (Invitrogen, USA) according to the manufacturer s instruction. RNA samples were treated with DNase I (TURBO DNase; Ambion,TX, USA). Quality and quantity of the extracted RNA were checked on 1% agarose gel and by nano-drop, respectively. Subsequent RNA quality control was carried out on a QC Bioanalyzer and each sample received an RNA integrity numbers (RIN) of greater than 8. The RNA sequencing was done on an Illumina HiSeq 2000 instrument for a paired-end run of 2x100 reads at the Beijing Genomes Institute. In average 25 millions of HQ paired-end reads per library were obtained from T. ammi inflorescent and assembled into contigs using Trinity software. In this study, 11,468 unitranscripts (7913 unigenes) were found to contain 13593 potential microsatellites. The frequency of SSRs found to be approximately one per 10.1 kB of assembled sequence. More than 57 percent of unigenes containing SSRs were blasted with carrot genome. A total of 3437 unigenes (43%) were functional categoried, which among them 2,219 unigenes (64.6%) belonged to the metabolic process category and 71 unigenes (1.2%) were assigned to the secondary metabolic process . In this study, 12 genes were detected in the terpenoid backbone biosynthesis pathway, that their transcripts were containing a microsatellite. The development of these markers can be used for future studies of marker-assisted selection, genetic diversity and construct genetic maps in this medicinal plant.

Keywords:

Apiaceae , Secondary metabolic , Transcriptome , Terpenoids