Development and validation of EST-SSR markers of Magnolia wufengensis using de novo transcriptome sequencing

Abstract

Key message An EST-SSR database of Magnolia wufengensis was first generated by transcriptome sequencing, the SSRs related to flower pigment synthesis were validated, and the transferability of the EST-SSR markers to other species was additionally investigated. Magnolia (M.) wufengensis is a distinct ornamental species endemic to China and is famous for its gorgeous flower color. However, the limitation of understanding genetic resources has restricted its conservation and breeding. This study used RNA-Seq to generate thousands of expressed sequence tags (ESTs) with which thousands of functional markers were developed. Of the 9567 simple sequence repeats (SSRs) identified, 8306 contained unigene sequences. Among the different SSR motif types, dinucleotide repeats (50.8%) were the most abundant, followed by trinucleotide repeats (32.4%). AG/CT (37.8%) and AAG/CTT (11.8%) were the most enriched repeat motifs. A total of 5203 EST-SSR novel primer pairs were designed to construct a reference database, from which 33 related to floral pigment formation were PCR-validated by testing them on the five cultivars of M. wufengensis. Twenty-two primer pairs displayed clear PCR products in the verification. Moreover, the number of alleles per marker ranged from 1 to 6 with an average of 3.41, and the polymorphic bands ranged from 0 to 6 with an average of 2.77. Additionally, the genetic distances were positively correlated with certain phenotypic indexes, including anthocyanin and flavonoid contents. Furthermore, applying these EST-SSR markers to other related species showed that 50.0–68.1% of the markers were transferable. The identified EST-SSRs will not only inspire future molecular-assisted breeding in Magnoliaceae but also facilitate targeted research in marker-trait association for floral pigment formation and genetic diversity analysis.