Genetic Diversity Analysis with 454 Pyrosequencing and Genomic Reduction Confi rmed the Eastern and Western Division in the Cultivated Barley Gene Pool

Next-generation DNA sequencing (NGS) technologies can survey sequence variation on a genome-wide scale, but their utility for crop genetic diversity analysis is poorly known. Many challenges remain in their applications, including sampling complex genomes, identifying single nucleotide polymorphisms (SNPs), and analyzing missing data. This study presented a practical application of the Roche 454 GS FLX Titanium technology in combination with genomic reduction and an advanced bioinformatics tool to analyze the genetic relationships of 16 diverse barley (Hordeum vulgare L.) landraces. A full 454 run generated roughly 1.7 million sequence reads with a total length of 612 Mbp. Application of the computational pipeline called DIAL (de novo identifi cation of alleles) identifi ed 2578 contigs and 3980 SNPs. Sanger sequencing of four barley samples confi rmed 85 of the 100 selected contigs and 288 of the 620 putative SNPs and identifi ed 735 new SNPs and 39 new indels. Several diversity analyses revealed the eastern and western division in the barley samples. The division is compatible with those inferred with 156 microsatellite alleles of the same 16 samples and consistent with our current knowledge about cultivated barley. These results help to illustrate the utility of NGS technologies for crop diversity studies. The NGS application also provides a new informative set of genomic resources for barley research. N DNA sequencing (NGS) technologies can generate unprecedented amounts of genomic data, even in non-model organisms (Nordborg and Weigel, 2008; Bräutigam and Gowik, 2010; Metzker, 2010; Seeb et al., 2011). In recent years, these technologies have become an aff ordable means to survey sequence variation on a genome-wide scale (Seeb et al., 2011; You et al., 2011). In principle, such capability should revolutionize the genetic diversity analysis with high resolution on crop plants with large and complex genomes. However, applications of NGS technologies to assess crop genetic diversity are still full of challenges and feasibility assessments are warranted to inform diversity analysis. Many plants, unlike those model plants such as rice (Oryza sativa L.) and maize (Zea mays L.) with sequenced genomes, have large and complex genomes with variable ploidy and an abundance of repeated sequences (Wicker et al., 2006; Novaes et al., 2008; You et al., 2011). Also, limitations are not lacking in the application of bioinformatics tools to identify single nucleotide polymorphisms (SNPs) without a reference genome (Imelfort et al., 2009; You et al., 2011). Moreover, a diversity analysis of NGS genomic data with sequencing error, assembly error, and missing data may not be always informative (Rokas and Abbot, 2009; Pool et al., 2010). Published in The Plant Genome 4:226–237. Published 2 Nov. 2011. doi: 10.3835/plantgenome2011.08.0022 © Crop Science Society of America 5585 Guilford Rd., Madison, WI 53711 USA An open-access publication All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. Plant Gene Resources of Canada, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Pl., Saskatoon, SK S7N 0X2, Canada. Received 1 Aug. 2011. *Corresponding author ([email protected]). Abbreviations: AMOVA, analysis of molecular variance; DIAL, de novo identifi cation of alleles; dNTP, deoxyribonucleotide triphosphate; EDTA, ethylenediaminetetraacetic acid; MCC, maximum clade credibility; NCBI, National Center for Biotechnology Information; NGS, next-generation DNA sequencing; PCA, principle component analysis; PCR, polymerase chain reaction; RLMID, Rapid Library Multiplex Identifi er; RRL, reduced representation library; SFF, standard fl owgram format; SNP, single nucleotide polymorphism; SSR, simple sequence repeat; Ta, annealing temperature. Published November, 2011