A rapid and cost-effective approach for the development of polymorphic microsatellites in non-model species using paired-end RAD sequencing

Abstract

As one of the most informative and versatile DNA-based markers, microsatellites have been widely used in population and conservation genetic studies. However, the development of microsatellites has traditionally been laborious, time-consuming, and expensive. In the present study, a rapid and cost-effective "RAD-seq-Assembly-Microsatellite" approach was developed to identify abundant microsatellite markers in non-model species using the roughskin sculpin Trachidermus fasciatus as a representative. Overlapping paired-end Illumina reads generated by restriction-site-associated DNA sequencing (RAD-seq) were clustered based on the similarity of reads containing the restriction enzyme recognition site and then assembled into contigs, which were used for microsatellite discovery and primer design. A total of 121,750 RAD contigs were generated with a mean length of 522 bp, and 19,782 contigs contained microsatellite motifs. A total of 156,150 primer pairs were successfully designed based on 16,497 contigs containing priming sites. Experimental validation of 52 randomly selected microsatellite loci demonstrated that 45 (86.54%) loci were successfully amplified and polymorphic in two geographically isolated populations of T. fasciatus. Compared with traditional approaches based on DNA cloning and other approaches based on next-generation sequencing, our newly developed approach could yield thousands of microsatellite loci with much higher successful amplification rate and lower costs, especially for non-model species with shallow background of genomic information. The "RAD-seq-Assembly-Microsatellite" approach holds great promise for microsatellite development in future ecological and evolutionary studies of non-model species.

Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers

“…The high throughput and low cost of next-generation sequencing (NGS) technologies enable the generation of large amounts of genomic sequences, from which putative microsatellite markers can be identified rapidly. NGS sequencing has been used for developing markers in plants [3,[6][7][8], fungi [3,9], arthropods [10][11][12] as well as in vertebrates like fish [13], snakes [14,15], birds [3,14,16] and mammals [2,15]. Furthermore, previously assembled genomes can be exploited [9,17,18].…”

Temporal Genetic Stability Despite Decades of Overexploitation for Large Yellow Croaker in the East China Sea

“…Finally, 7 polymophic microsatellite loci were selected for subsequent analysis (Table S1). The amplification of the 7 microsatellite loci was performed by the PCR protocol described in Xue et al (2017). All the amplified loci were analysed through an automated capillary sequencer ABI (Applied Biosystems) and allele sizes were determined with the GS500-ROX size standard GeneMarker 2.2.0 (SoftGenetics, State College, USA).…”

BigFiRSt: A Software Program Using Big Data Technique for Mining Simple Sequence Repeats From Large-Scale Sequencing Data

“…Several paired-end read merging algorithms have been proposed in recent years, which include FLASH (Magoc and Salzberg, 2011), leeHom (Renaud et al, 2014), PEAR (Zhang et al, 2013), BBMerge (Bushnell et al, 2017) and Konnector (Vandervalk et al, 2014), OverlapPER (Oliveira et al, 2018), Cope (Liu et al, 2012), and XORRO (Dickson and Gloor, 2013). There also exist approaches and tools such as SSRs-pipeline (Miller et al, 2013) and RAD-seq-Assembly-Microsatellite (Xue et al, 2017), which integrate paired-end reads merging and SSRs mining into a single pipeline. These computational methods and tools have been used for merging paired-end reads and identifying novel SSRs.…”