Genotyping-by-Sequencing (GBS) in Tomato using Ion AmpliSeq™ Technology

Genotyping by sequencing (GBS) using next-generation sequencing technologies offers a high-throughput and cost-effective way to study plant genetics, discover and screen molecular markers for genomics-assisted plant breeding. Here, we present a simple, cost effective and flexible single-day workflow for tomato variant validation and discovery using Ion AmpliSeq™ technology. We first designed an Ion AmpliSeq DNA custom panel that targets about 400 SNPs based on the tomato genome. We prepared multiplexed amplicon libraries for 192 tomato leaf samples using only 10 ng of DNA per primer pool, and sequenced the libraries on Ion 318™ chips. The sequencing reads were analyzed for the variants using Torrent Variant Caller of Torrent Suite™ Software. Over 10% of the variants identified are de novo. Additionally, we demonstrated the workflow could be further simplified by using crude cell extract instead of purified DNA to prepare sequencing libraries. For 192 tomato leaf samples, the concordance of over 73,000 variants called from the crude extract and the purified DNA is 99%.