Kevin A. Fengler

Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize

We report a whole-genome comparison of gene content in allelic BAC contigs from two maize inbred lines. Genic content polymorphisms involve as many as 10,000 sequences and are mainly generated by DNA insertions. The termini of eight of the nine genic insertions that we analyzed shared the structural hallmarks of helitron rolling-circle transposons. DNA segments defined by helitron termini contained multiple gene-derived fragments and had a structure typical of nonautonomous helitron-like transposons. Closely related insertions were found in multiple genomic locations. Some of these produced transcripts containing segments of different genes, supporting the idea that these transposition events have a role in exon shuffling and the evolution of new proteins. We identified putative autonomous helitron elements and found evidence for their transcription. Helitrons in maize seem to continually produce new nonautonomous elements responsible for the duplicative insertion of gene segments into new locations and for the unprecedented genic diversity. The maize genome is in constant flux, as transposable elements continue to change both the genic and nongenic fractions of the genome, profoundly affecting genetic diversity.

Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize

Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an ≈2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize–sorghum–rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution.

Anchoring 9,371 Maize Expressed Sequence Tagged Unigenes to the Bacterial Artificial Chromosome Contig Map by Two-Dimensional Overgo Hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 × 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

A maize wall-associated kinase confers quantitative resistance to head smut

Head smut is a systemic disease in maize caused by the soil-borne fungus Sporisorium reilianum that poses a grave threat to maize production worldwide. A major head smut quantitative resistance locus, qHSR1, has been detected on maize chromosome bin2.09. Here we report the map-based cloning of qHSR1 and the molecular mechanism of qHSR1-mediated resistance. Sequential fine mapping and transgenic complementation demonstrated that ZmWAK is the gene within qHSR1 conferring quantitative resistance to maize head smut. ZmWAK spans the plasma membrane, potentially serving as a receptor-like kinase to perceive and transduce extracellular signals. ZmWAK was highly expressed in the mesocotyl of seedlings where it arrested biotrophic growth of the endophytic S. reilianum. Impaired expression in the mesocotyl compromised ZmWAK-mediated resistance. Deletion of the ZmWAK locus appears to have occurred after domestication and spread among maize germplasm, and the ZmWAK kinase domain underwent functional constraints during maize evolution.

Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens

The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores. While MinION long reads have an error rate substantially higher than the ones produced by short-read sequencing technologies, they can generate de novo assemblies of microbial genomes, after an initial correction step that includes alignment of Illumina sequencing data or detection of overlaps between Oxford Nanopore reads to improve accuracy. In this study, MinION reads were generated from the multi-chromosome genome of Agrobacterium tumefaciens strain LBA4404. Errors in the consensus two-directional (sense and antisense) “2D” sequences were first characterized by way of comparison with an internal reference assembly. Both Illumina-based correction and self-correction were performed and the resulting corrected reads assembled into high-quality hybrid and non-hybrid assemblies. Corrected read datasets and assemblies were subsequently compared. The results shown here indicate that both hybrid and non-hybrid methods can be used to assemble Oxford Nanopore reads into informative multi-chromosome assemblies, each with slightly different outcomes in terms of contiguity and accuracy.