Comprehensive SSR study of 14 Zingiberaceae species based on Microsatellite Capture Sequencing (MiCAPs)

Miao SHI^1*Keisuke Tanaka²Marlon P Rivera^1,3Min San Thein⁴Godfrey M. Ngure¹Kazuo N Watanabe¹

• ¹University of Tsukuba, Tsukuba, Japan
• ²Tokyo Joho Daigaku, Chiba, Japan
• ³University of the Philippines Los Banos, Laguna, Philippines
• ⁴Republic of the Union of Myanmar Department of Agricultural Research Industrial Crops and Horticulture Division, Yezin, Myanmar

The Zingiberaceae family encompasses numerous species renowned for their significant pharmacological properties and culinary importance. Despite this value, many species are considered under-utilized, largely because the absence of basic molecular information hinders effective conservation, sustainable utilization and further exploitation. Simple Sequence Repeat (SSR) markers are particularly suitable for genetic studies in such species, as they are highly polymorphic and do not necessitate a reference genome. However, traditional SSR development can be resource intensive. Microsatellite capture sequencing (MiCAPs) presents a cost-effective solution by enriching libraries for SSR-containing fragments prior to sequencing, thereby substantially reducing data requirements and associated costs for marker discovery, especially crucial for species lacking prior genetic information. MiCAPs was applied to 160 accessions, including 148 samples from 14 Zingiberaceae species and 12 samples from outgroup (Musaceae family), generating 21.78 million raw reads. From this dataset, 612 SSR marker candidates were developed and evaluated via electronic-PCR (ePCR) for seven target species. A genus-level phylogenetic tree reconstructing the relationships among the 14 Zingiberaceae species was successfully built using the consensus sequence from MiCAPs data. Furthermore, genetic similarity patterns of species-specific and transferable SSR loci were assessed across the Curcuma and Zingiber species using Polymorphic SSR Retrieval (PSR) analysis. Comparative genetic diversity studies revealed that Zingiber exhibits a relatively more conserved genetic background compared to Curcuma. This integrated workflow combining MiCAPs, ePCR, and PSR demonstrates a practical approach for marker development and diversity assessment in polyploid species lacking reference genomes. Despite the genetic complexities inherent in Zingiberaceae, especially potential polyploidy, our integrated approach using SSR-enriched sequencing (MiCAPs) and in silico marker validation (PSR) proved highly effective. This methodology successfully established a robust phylogenetic framework and enabled comprehensive genetic diversity assessment at the genus level. Importantly, this study generated a novel set of SSR marker candidates. These markers represent a significant resource, poised to greatly facilitate future genetic studies focused on the diversity, evolutionary relationships, conservation, and sustainable utilization of valuable, yet often under-utilized, Zingiberaceae species.