NEW YORK (GenomeWeb News) – In Nature online today, members of the International Barley Genome Sequencing Consortium reported on a newly developed barley resource that brings together physical, genetic, and functional features of the plant's genome.
Using bacterial artificial chromosome sequence and fingerprinting data, the researchers put together a physical genetic map for barley, Hordeum vulgare. From there, they folded in high-throughput shotgun genome sequence data to begin fleshing out this map and to improve on the existing genetic map for barley.
They also sequenced RNA from several barley tissues and cultivars — work that helped in annotating the genome, identifying sequence variants, and exploring ways in which genes are expressed and regulated in the plant.
"This provided us with access to the majority of all genes in the genome of barley that are expressed — so they can be detected also by RNA sequencing," co-corresponding author Nils Stein, a genome diversity researcher at the Leibniz Institute of Plant Genetics and Crop Plant Research, told GenomeWeb Daily News.
"The resulting genomic framework provides a detailed insight into the physical distribution of genes and repetitive DNA and how these features relate to genetic characteristics such as recombination frequency, gene expression, and patterns of genetic variation," he and his co-authors added in the paper.
Going forward, the group hopes to close in on a complete or near complete barley genome within the next year or 16 months by sequencing its way along the plant chromosomes.
For instance, Stein noted that a team in Germany has already received funding to sequence the entire barley chromosome 3H. That work is "pretty far advanced," he said. "And the consortium is currently in the process of directing existing funds into such projects for the other chromosomes."
Like other crop plants in the Triticeae tribe such as wheat and rye, barley has a complex and repeat-rich genome that has proven challenging to completely sequence and assemble. To deal with such complications, the large, international consortium developed what it calls the "barley genome gene space."
This "integrated, multi-layered informational resource … provides access to the majority of barley genes in a highly structured and genetic framework," the team explained. "In association with comparative sequence and transcriptome data, the gene space provides a new molecular and cellular insight into the biology of the species, providing a platform to advance gene discovery and genome-assisted crop improvement."
The team started from a BAC-based physical map of the genome, using sequence generated by Roche 454 GS FLX/FLX Titanium and Sanger sequencing for a barley cultivar called Morex.
"What we did in this study is we developed a high-quality physical map that's tightly anchored to the genetic map," Stein said.
On top of that, researchers added in whole-genome shotgun sequence data from Morex plants and other cultivars that had been produced through paired-end and mate-pair sequencing with Illumina's GAIIx and HiSeq 2000 instruments. To aid in their subsequent annotation and gene expression analysis, they also performed RNA sequencing on eight Morex barley tissues collected over various stages of development.
In an effort to identify as many genetic variants as possible, they generated RNA sequences for plants from four more barley cultivars and from one wild barley representative, focusing on a plant tissue known for extensive gene expression.
Overall, the study authors explained, the resulting physical framework represents an estimated 80 percent of barley's 5.1 billion base genome and roughly 90 percent of the plant's expressed genes.
The sequence available so far covers around 10 percent of that physical map, Stein explained. And members of the barley consortium are in the process of completing the genome sequence by working their way through each of barley's seven chromosomes.
"[T]he barley gene space reported here provides an essential reference for genetic research and breeding," the team concluded. "It represents a hub for trait isolation, understanding and exploiting natural genetic diversity and investigating the unique biology and evolution of one of the world's first domesticated crops."
In the current study, for instance, researchers uncovered 26,159 protein-coding sequences that they classified as high-confidence genes.
Another 53,000 low confidence genes also turned up. Those predicted genes tended to be smaller and less complex, with nearly three-quarters of the low confidence genes being comprised of a single exon.
Such features hint that many of the genes in the low confidence set are actually pseudogenes or fragments of gene sequence that have been carried to new parts of the genome by mobile elements, Stein explained, though the set of low confidence genes is also expected to contain genes that are expressed and translated into proteins.
Consistent with patterns found through previous genetic studies of barley, the team's analyses suggested that barley genes frequently fall closer to the proximal and distal parts of chromosomes, which tend to have higher recombination rates than regions in and around chromosome centromeres.
"The genes are preferentially located in the regions where you have recombination," Stein said. In particular, he noted that genes from certain functional groups — including some pathogen resistance genes — are preferentially located in recombination-rich regions of the barley genome.
"But," he added, "you have also, throughout the entire chromosome, active genes. So it's not like you can omit part of the genome because there are no genes."
Assessment of the barley maps and sequence produced so far are also helping to refine researchers' estimates of the barley genome's repeat content, indicating that annotated repetitive elements make up more than 80 percent of the barley genome.
Using transcript sequence data, meanwhile, the team has started to look at gene expression and alternative splicing patterns in the plant while tracking down new barley SNPs for use in future mapping and genetic diversity studies.