Location: Vegetable Crops ResearchTitle: Using next generation sequencing approaches for the isolation of simple sequence repeats (SSF) in the plant sciences) Author
Submitted to: American Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2011
Publication Date: 2/1/2012
Citation: Zalapa, J.E., Cuevas, H.E., Steffan, S.A., Simon, P.W., Senalik, D.A., Zhu, H., Mccown, B., Zeldin, E., Harbut, R. 2012. Using next generation sequencing approaches for the isolation of simple sequence repeats (SSR) in the plant sciences. American Journal of Botany. 99(2):193-208. Interpretive Summary: An important application of new sequencing technologies in plants is the large-scale development of genetic markers. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. New sequencing technologies, referred to as next generation sequencing (NGS), allow the efficient identification of large numbers of microsatellites at a fraction of the cost compared to traditional approaches. A review is provided of several recent studies demonstrating the efficient use of NGS technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high throughput methods. Moreover, in cranberry, the application of NGS technologies is demonstrated as a cost-effective and efficient way to identify large numbers of microsatellite-containing sequences for genetic marker development. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied, but economically important crop such as cranberry.
Technical Abstract: The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. The major advantage of using NGS methods to isolate SSR loci is their ability to quickly and cost-effectively produce large amounts of sequence data. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. Compared to traditional SSR discovery methods both 454 and Illumina technologies are strikingly efficient. In fact, one full 454 or Illumina shotgun run can yield thousands of genome- or transcriptome-wide SSR loci that can be used to develop large numbers of microsatellite markers. The previous requirement of SSR enrichment may not be necessary for SSR isolation using either 454 or Illumina due to the large amounts of data produced by both technologies. However, the large sequence data sets can pose a challenge during raw read assembly and SSR isolation. Thus, due to the long and high quality reads obtained and the comparatively minor effort required, genomic shotgun sequencing using 454 technology is currently the easiest and most widely used NGS method to isolate SSRs loci in plants. A working framework for the application of NGS technologies for the isolation of SSR markers is provided, particularly outlining the use of 454 and Illumina sequencing data sets. Additionally, computational methods and high-throughput SSR development strategies are discussed to produce large numbers of SSRs markers. A data set of plastome and mitochondrome microsatellite loci in cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In cranberry, a genetically understudied crop species, whole genome shotgun 454 sequencing was a highly efficient way to identify SSR-containing sequences for marker development. In the future, NGS technologies will massively increase the number of SSR and other genetic markers available to conduct genetic research in a plethora of non-model and understudied plant species such as cranberry.