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Development of robust genomic simple sequence repeat markers for estimation of genetic diversity within and among bulb onion (Allium cepa L.) populations

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Abstract

Improved understanding of genetic diversity in onion and shallot (Allium cepa L.) is required to inform breeding and genetic resource conservation, and to enable development of association genetics and seed quality assurance methods. To develop quantitative estimates of diversity we estimated within- and among-population heterozygosity in a set of onion populations using genomic simple sequence repeat (SSR) markers developed by genomic skim sequencing. Primer sets (166) designed to flank SSR motifs identified were evaluated in a diverse set of lines, with 80 (48 %) being polymorphic. The 20 most robust single copy markers were scored in 12 individuals from 24 populations representing short-day to long-day adapted material from diverse environments. The average genetic diversity estimate (H e) per population was 0.3 (SD 0.08) and the average per marker was 0.49 (SD 0.2). The onion populations assessed in this survey were distinct with moderate to large population differentiation but also had high within-population variation (F st = 0.26). There was evidence of inbreeding (F is = 0.22) with observed heterozygosities lower than the expected. This marker resource will be applicable for DNA fingerprinting, measuring levels of inbreeding in breeding lines, assessing population structure for association mapping and expanding linkage maps that are principally based on expressed sequence tag-based markers. A Galaxy workflow was developed to facilitate bulk SSR marker design from next-generation sequence data. This study provides one of the first quantitative views of population genetic variation in onion and a practical toolset for further genetics.

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Abbreviations

DH:

Doubled haploid

gSSR:

Genomic simple sequence repeat

PCA:

Principal components analysis

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Acknowledgments

We gratefully acknowledge provision of germplasm by the Warwick HRI Genetic Resources Unit (GRU) and Cornell University. We acknowledge Enza Zaden NZ Ltd. and Seminis NZ for field trialling support. We also acknowledge Richard Newcombe and David Chagne for helpful comments on the manuscript. This research was funded by the New Zealand Ministry of Science and Innovation Contract C02X0803. Development of Galaxy tools was carried out under BeSTGRID (Broadband enabled Science and Technology GRID) Summer of eResearch project supervised by Stuart Charters (Lincoln University). We thank Vladimir Mencl (University of Canterbury) for providing Galaxy hosting during development.

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Authors and Affiliations

  1. The New Zealand Institute for Plant and Food Research Limited, Private Bag 4704, Christchurch, 8140, New Zealand

    Samantha Baldwin, Meeghan Pither-Joyce, Kathryn Wright & John McCallum

  2. Department of Applied Computing, Faculty of Environment, Society and Design, Lincoln University, PO Box 84, Lincoln, 7647, New Zealand

    Leshi Chen

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  1. Samantha Baldwin
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  2. Meeghan Pither-Joyce
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  3. Kathryn Wright
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  4. Leshi Chen
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  5. John McCallum
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Corresponding author

Correspondence to John McCallum.

Electronic supplementary material

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11032_2012_9727_MOESM1_ESM.xls

List of Allium germplasm used at the described stages of this study, including the origin (where known), type (OP = open pollinated), description and citations. Where the collection latitude is unknown the day length adaptation is given as short (SD), intermediate (ID) or long day (LD), where known. (XLS 33 kb)

11032_2012_9727_MOESM2_ESM.xls

List of the SSR markers assessed in this study including primer sequences without the Tag or PIG tail sequences and a summary of the amplification results. The product sizes of amplicons from Allium cepa, A. roylei and A. fistulosum are based on sizing of the fluorescent products assessed using the ABI3130xl Genetic Analyser (Applied Biosystems Inc). Zeroes in product sizes denote no amplification and blanks indicate markers that were multi-locus based on agarose gel electrophoresis and were not screened further. (XLS 89 kb)

Screenshot of a simple Galaxy pipeline using MISA tools for SSR identification and flanking primer design. (PNG 188 kb)

11032_2012_9727_MOESM4_ESM.tif

Map showing the origins of each population tested and which of the 11 groups each population was assigned to, based on the clustering by discriminant analysis. (TIFF 947 kb)

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Baldwin, S., Pither-Joyce, M., Wright, K. et al. Development of robust genomic simple sequence repeat markers for estimation of genetic diversity within and among bulb onion (Allium cepa L.) populations. Mol Breeding 30, 1401–1411 (2012). https://doi.org/10.1007/s11032-012-9727-6

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