We have resequenced a group of six elite maize inbred lines, including the parents of the most productive commercial hybrid in China. This effort uncovered more than 1,000,000 SNPs, 30,000 indel polymorphisms and 101 low-sequence-diversity chromosomal intervals in the maize genome. We also identified several hundred complete genes that show presence/absence variation among these resequenced lines. We discuss the potential roles of complementation of presence/absence variations and other deleterious mutations in contributing to heterosis. High-density SNP and indel polymorphism markers reported here are expected to be a valuable resource for future genetic studies and the molecular breeding of this important crop.

Genome-wide patterns of genetic variation among elite maize inbred lines

A massively parallel pyro-sequencing technology commercialized by 454 Life Sciences Corporation was used to sequence the transcriptomes of shoot apical meristems isolated from two inbred lines of maize using laser capture microdissection (LCM). A computational pipeline that uses the POLYBAYES polymorphism detection system was adapted for 454 ESTs and used to detect SNPs (single nucleotide polymorphisms) between the two inbred lines. Putative SNPs were computationally identified using 260 000 and 280 000 454 ESTs from the B73 and Mo17 inbred lines, respectively. Over 36 000 putative SNPs were detected within 9980 unique B73 genomic anchor sequences (MAGIs). Stringent post-processing reduced this number to > 7000 putative SNPs. Over 85% (94/110) of a sample of these putative SNPs were successfully validated by Sanger sequencing. Based on this validation rate, this pilot experiment conservatively identified > 4900 valid SNPs within > 2400 maize genes. These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2007.03193.x

SNP discovery via 454 transcriptome sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with approximately 35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.

/pdf/a-genome-wide-characterization-of-microrna-genes-in-maize-w7rljf0or7.pdf

A genome-wide characterization of microRNA genes in maize.

The plant hormone auxin plays crucial roles in regulating plant growth development, including embryo and root patterning, organ formation, vascular tissue differentiation and growth responses to environmental stimuli. Asymmetric auxin distribution patterns have been observed within tissues, and these so-called auxin gradients change dynamically during different developmental processes. Most auxin is synthesized in the shoot and distributed directionally throughout the plant. This polar auxin transport is mediated by auxin influx and efflux facilitators, whose subcellular polar localizations guide the direction of auxin flow. The polar localization of PIN auxin efflux carriers changes in response to developmental and external cues in order to channel auxin flow in a regulated manner for organized growth. Auxin itself modulates the expression and subcellular localization of PIN proteins, contributing to a complex pattern of feedback regulation. Here we review the available information mainly from studies of a model plant, Arabidopsis thaliana, on the generation of auxin gradients, the regulation of polar auxin transport and further downstream cellular events.

Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development.

Oilseed rape (Brassica napus) was selected as an example of a polyploid crop, and the Solexa sequencing system was used to generate approximately 20 million expressed sequence tags (ESTs) from each of two cultivars: Tapidor and Ningyou 7. A methodology and computational tools were developed to exploit, as a reference sequence, a publicly available set of approximately 94,000 Brassica species unigenes. Sequences transcribed in the leaves of juvenile plants were aligned to approximately 26 Mb of the reference sequences. The aligned sequences enabled the detection of 23,330-41,593 putative single nucleotide polymorphisms (SNPs) between the cultivars, depending on the read depth stringency applied. The majority of the detected polymorphisms (87.5-91.2%) were of a type indicative of transcription from homoeologous genes from the two parental genomes within oilseed rape, and are termed here 'hemi-SNPs'. The overall estimated polymorphism rate (approximately 0.047%-0.084%) is consistent with that previously observed between the cultivars analysed. To demonstrate the heritability of SNPs and to assess their suitability for applications such as linkage map construction and association genetics, approximately nine million ESTs were generated, using the Solexa system, from each of four lines of a doubled haploid mapping population derived from a cross between Tapidor and Ningyou 7. Computational tools were developed to score the alleles present in these lines for each of the potential SNPs identified between their parents. For a specimen region of the genome analysed in detail, segregation of alleles largely, although not entirely, followed the pattern expected for genomic markers.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2008.00396.x

Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C t selection were sequenced to generate ∼200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

Enrichment of gene-coding sequences in maize by genome filtration.

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=2576&context=usdaarsfacpub

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides.

Maize is a staple crop of the grass family and also an excellent model for plant genetics. Owing to the large size and repetitiveness of its genome, we previously investigated two approaches to accelerate gene discovery and genome analysis in maize: methylation filtration and high C0t selection. These techniques allow the construction of gene-enriched genomic libraries by minimizing repeat sequences due to either their methylation status or their copy number, yielding a 7-fold enrichment in genic sequences relative to a random genomic library. Approximately 900 000 gene-enriched reads from maize were generated and clustered into Assembled Zea mays (AZM) sequences. Here we report the current AZM release, which consists of ∼298 Mb representing 243 807 sequence assemblies and singletons. In order to provide a repository of publicly available maize genomic sequences, we have created the TIGR Maize Database (http://maize.tigr.org). In this resource, we have assembled and annotated the AZMs and used available sequenced markers to anchor AZMs to maize chromosomes. We have constructed a maize repeat database and generated draft sequence assemblies of 287 maize bacterial artificial chromosome (BAC) clone sequences, which we annotated along with 172 additional publicly available BAC clones. All sequences, assemblies and annotations are available at the project website via web interfaces and FTP downloads.

The TIGR Maize Database

The large size of the maize genome and the expectation that most of the genome is represented by repetitive elements is a challenge to standard genome sequencing techniques. Genome-filtration sequencing techniques may target gene-rich regions in the genome. Two such approaches, methylation filtration and high Cot selection, may provide rapid and cost-effective alternative to sequencing the maize genome. Approximately 450k sequence reads have been obtained from both methylation filtration (MF) and high Cot (HC) libraries, and these sequences have been clustered and assembled. An analysis was undertaken to examine whether MF and MC enrich for maize genes and target non-identical sequence space. Simple sequence repeat analysis and mapped marker coverage provide gauges to examine whether MF and HC sample identical sequence space. Marker hits also provide evidence that MF and HC enrich for unique genic portions of the genome. Finally, the identification of maize and other protein sequences in the MF and HC sequence sets can indicate the expected fraction and coverage of the maize gene space by MF and HC.

Li Zheng

Papers

Enrichment of gene-coding sequences in maize by genome filtration.

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides.

The TIGR Maize Database

Gene recovery of two genome-filtration sequencing techniques when applied to the maize genome