Genome Filtering Using Methylation-Sensitive Restriction Enzymes with Six Base Pair Recognition Sites

The large fraction of repetitive DNA in many plant genomes has complicated all aspects of DNA sequencing and assembly, and thus techniques that enrich for genes and low-copy sequences have been employed to isolate gene space. Methyl-sensitive restriction enzymes, with six base pair recognition sites, were evaluated on genomic DNA of the bread wheat ‘Chinese Spring’ as a different approach to enrich for genes. SacI, SalI, PstI, and AatII were used to digest wheat genomic DNA and fragments ranging from 400 bp to 2.0 kb were cloned and unidirectionally sequenced. All four enzymes provided some level of enrichment for gene space; however, AatII and PstI reduced the number of clones with repeat elements to just 16.2 and 19.1%, respectively. AatII and PstI were also effective in enrichment in corn and tobacco. Corn libraries made with AatII and PstI had 58.7 and 71.2%, respectively, of the clones with signifi cant expressed sequence tag (EST) alignments, while tobacco libraries made with the same enzymes had 51.7 and 65.3%, respectively. With the development of ultra-throughput sequencing technologies, this technique provides an opportunity to rapidly and effi ciently obtain sequencing from gene-rich regions. DURING A SEQUENCING project, repetitive sequences cause signifi cant diffi culties not only in the actual sequencing, but also in the assembly of the genome. Repetitive regions pose even greater problems for the middle and large genome plants. Th e high cost associated with sequencing and the lack of computer algorithms to deal with assembling repeats has led many investigators to focus on the low-copy, gene-rich regions of genomes. Th ese low-copy regions can be obtained by using various gene-enrichment techniques. Th ree main approaches have been developed to preferentially sequence gene-rich regions from repetitive genomes. Expressed sequence tag (EST; Adams et al., 1991) sequencing uses mRNA isolated from one or more tissues which is converted into cDNA, cloned, and sequenced. Th e clones represent the actively expressed coding regions of the genome and are specifi c to the tissue(s) sampled and the environmental conditions they are in (Bonaldo et al., 1996). While ESTs provide details of expressed coding regions of a DNA strand, they do not provide information on promoter sequences or introns (Palmer et al., 2003). Currently there are two predominant methods used for enriching genomic DNA for gene-containing regions including introns and promoter segments. One is methyl fi ltration (MF) which was developed using methyl-sensitive Published in The Plant Genome 1:146–152. Published 1 Oct. 2008. doi: 10.3835/plantgenome2008.05.0245 © Crop Science Society of America 677 S. Segoe 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. J.P. Fellers, USDA-ARS, Plant Science and Entomology Unit, 4008 Throckmorton Hall, Manhattan, KS 66506. Mention of a trademark of a proprietary product does not constitute a guarantee of warranty of the product by the United States Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. Received 13 May 2008. *Corresponding author (john.fellers@ars.usda.gov). Abbreviations: AFLP, amplifi ed fragment length polymorphism; BAC, bacterial artifi cial chromosome; EST, expressed sequence tag; HC, high Cot; kb, kilo base pair; MF, methyl fi ltration; MSLL, methylation-spanning linker libraries; RFLP, restriction fragment length polymorphism; WGS, whole genome shotgun. Published November, 2008