Transcriptome Assembly and Identification of EST-SSR Markers in Crocus Sativus

Crocus sativus is a triploide plant and propagating by vegetative propagation. Therefore, trait segregation and genetic diversity are limited in this plant. EST-SSR markers have some priority, for example co-dominant inheritance, locus specific and highly polymorphic against all other markers. Due to the availability of transcriptome data, it is possible to develop EST-SSR markers and polymorphism studies in saffron. Development of EST-SSR markers in C. sativus make it possible to study genetic diversity and molecular polymorphism in different genotypes. In order to develop EST-SSR marker for C. sativus, we downloaded public available C. sativus RNA-seq data. Quality control and preprocessing of raw reads were done using FastQC and Trimmomatic tools, respectively. We performed de novo transcriptome assembly using RNA-Bloom. CD-HIT-EST was used in order to reduce redundancy in transcriptome assembly. The assembly quality was evaluated using the BUSCO software and completeness of transcriptome assembly was 90%. After achieving to high quality transcriptome assembly of C. sativus, EST-SSRs were identified by MISA tool. The EST-SSRs primers were designed using Primer3. 35459 SSR-containing sequences were detected and primer pairs were designed for them. Ten EST-SSR primer pairs were randomly selected to amplify C. sativus DNA. Seven pairs of the primers (70%) generated clear and reproducible bands with the expected size. These EST-SSR markers can be functional and useful for C. sativus genetic studies.