Isolation and characterization of microsatellite markers for Jasminum sambac (Oleaceae) using Illumina shotgun sequencing1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Jasminum sambac (L.) Aiton (Oleaceae) is an evergreen vine or shrub that is native to Pakistan and India; this species is cultivated as an ornamental plant worldwide because of its attractive and sweet fragrance (Ruan, 2014). Previous studies on this plant have mainly focused on its aromatic com-Only one study has reported the genetic diversity of J. sambac using inter-simple sequence repeat (ISSR) markers (Qiu et al., 2008). However, ISSR loci are dominant markers that are diffi cult to use in the calculation of heterozygosity and paternity analysis. As an important ornamental plant, it is necessary to develop a set of powerful markers for the assessment of wild germplasm resources and the development of molecular marker– assisted breeding. Microsatellites or simple sequence repeats (SSRs) are powerful markers used in population genetics and molecular marker– assisted breeding because of their high level of polymorphism, ease of genotyping, and codominant inheritance (Li et al., 2002 ; Oliveira et al., 2006). Emerging high-throughput sequencing platforms make it possible to discover a large number of micro-satellite markers in a short time (Suresh et al., 2013). In the present work, transcript-based microsatellite markers were developed for J. sambac by using Illumina sequencing. METHODS AND RESULTS Because of the temporal and spatial specifi city of gene expression, RNA was isolated from a fl ower from a single individual of J. sambac to fi nd molecular markers associated with the most important ornamental organs. The extraction was performed using a Quick RNA isolation kit (BioTeke Corporation, Beijing, China) following the manufacturer's protocol. RNA concentration was measured using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA). The construction of cDNA libraries and RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). Sequencing was conducted using an Illumina HiSeq 2500 system (Il-lumina, San Diego, California, USA). The obtained raw reads were cleaned by removing adapter sequences and then assembled de novo using Trinity (Grabherr et al., 2011). Microsatellite searching was performed using MISA (Thiel et al., 2003), and searching parameters were set as di-, tri-, tetra-, penta-, and hexanu-cleotide motifs with a minimum of fi ve repeats. Primer pairs were designed with Primer3 (Rozen and Skaletsky, 1999). The product size range was set at 100–400 bp, and the other primer design parameters were set at default values. Fresh leaves of J. sambac were collected from …