Gene enrichment in maize with hypomethylated partial restriction (HMPR) libraries

Abstract

A new technology was developed to assist gene-enrichment sequencing of any complex plant genome, employing maize as the test organism. Hypomethylated partial restriction (HMPR) libraries were constructed by using independent partial digestions with methylation-sensitive restriction enzymes HpaII (5Ј-CCGG-3Ј) and HpyCH4IV (5Ј-ACGT-3Ј). Fragments of 1-4 kb were purified and cloned, followed by sequence analysis of >2000 clones from 10 separate libraries. Organellar clones comprised ∼10% of each library but were useful in showing that no chimeric clones were generated and that digestion efficiencies were 10%-25% in different libraries. Four separate HMPR libraries, analyzed in detail, exhibited very similar degrees of gene enrichment and repeat depletion. Known gene homologies were found in ∼25% of the HMPR clones, compared with <4% in clones from a fully random set of unfiltered maize shotgun sequences. This six-to sevenfold enrichment for genes compares favorably with the best previous gene enrichment techniques in maize, High Cot analysis and methylation filtration. Compared with High Cot and methylation filtration, HMPR is exceptional in depleting retrotransposons' content to the lowest level yet observed (<5%, compared with >70% for unfiltered maize sequences) and in providing an unmatched enrichment for the "unknown" sequences that contain promoters, introns, and other gene-adjacent regions.[The sequence data from this study have been submitted to GenBank under accession nos. CW539179-CW542054.]Although flowering plant genomes vary enormously in nuclear DNA content, from the <150 Mb of a handful of species to the ∼140,000 Mb of Fritillaria assyriaca, most contain between 1000 and 8000 Mb per haploid genome (with a median of ∼3000 Mb) scattered across anywhere from two to >300 chromosomes (Kenton et al. 1993;Leitch et al. 2005). Gene content is much more constant, for instance varying less than twofold between the large genome of barley (Hordeum vulgare, ∼4900 Mb) and the smaller genome of rice (Oryza sativa, ∼440 Mb) (Van Deynze et al. 1998). Some publications (Goff et al. 2002;Yu et al. 2002;Feng et al. 2003; Rice Chromosome 10 Sequencing Consortium 2003;Sasaki et al. 2002) have concluded that rice might have about twice as many genes as does Arabidopsis thaliana (120-150 Mb), but other studies have indicated that most of these "extra" rice genes are artifacts of annotation, leading to the current conclusion that rice and Arabidopsis contain very similar gene numbers (Bennetzen 2002b;Bennetzen et al. 2004;Jabbari et al. 2004;. Moreover, the average size of genes does not vary >50% between the small genome of Arabidopsis and large genome species such as barley or cotton (Dubcovsky et al. 2001;Wendel et al. 2002). Hence, the space occupied by genes within a plant nuclear genome, the "gene space," is not nearly so variable as is the overall DNA content of the genome.In the maize (Zea mays) genome, ∼2400 Mb of DNA is distributed across the 10 chromosomes in a haploid nucleus. A precise gene number is not known f...

Bifurcation and Enhancement of Autonomous-Nonautonomous Retrotransposon Partnership through LTR Swapping in Soybean

“…Despite extensive proliferation, LTR retrotransposons in plant genomes are nevertheless suppressed by a variety of mechanisms, including DNA methylation (Hamilton et al, 2002;Palmer et al, 2003;Emberton et al, 2005), conversion to heterochromatin (Lippman et al, 2004), formation of solo LTRs by unequal intraelement recombination (Devos et al, 2002;, and accumulation of small deletions by illegitimate recombination (Devos et al, 2002;. It was estimated that >190 Mb of retrotransposon DNA has been removed from the rice (Oryza sativa) genome by unequal recombination and illegitimate recombination within the past 4 million years, leaving a current genome of ;400 Mb that contains <100 Mb of detectable retroelements or fragments .…”

Genome-Wide Distribution of TransposedDissociationElements in Maize

“…Average content for each position(s) (i.e., or for each mono-, di-, or trinucleotide) was calculated as the membership at that position(s) in the alignment. As controls, random, hypomethylated clones were retrieved from GenBank, trimmed of contaminating vector and enzyme recognition site sequences, and their first 248 bases aligned and analyzed as three independent, 248-bp MSAs: 912 Sau3AI sequences, 1360 HpyCH4IV sequences from hypomethylated partial restriction clones, and 1195 HpaII sequences from hypomethylated partial restriction clones (Emberton et al, 2005).…”

Wheat and Barley Genome Sequencing

“…Methylfiltration (MF, Rabinowicz 2003) and hypomethylated partial restriction cloning combined with sequencing (HMPR, Emberton et al 2005) take advantage of the fact that gene-bearing DNA is less methylated (i.e., hypo-methylated) in plants compared to non-coding/repetitive DNA. Another approach to enrich for gene sequences in genomic libraries is based on slower re-association kinetics of low-copy non-repetitive DNA upon complete denaturation of double stranded-genomic DNA (High-C o t, cot filtration, CF, Peterson et al 2002).…”