Building upon their success sequencing small RNAs in rice and Arabidopsis, two researchers from the University of Delaware’s Delaware Biotechnology Institute are gearing up to launch a broader effort to sequence small RNAs and target microRNAs in more than 30 different plants and algae.
According to the researchers, Blake Meyers and Pamela Green, the project will take advantage of Solexa’s soon-to-be released Genome Analysis System, which uses the company’s Sequencing by Synthesis technology.
Previously, Meyers and Green had collaborated on sequencing small RNAs in Arabidopsis and rice using Solexa’s now-discontinued massively parallel signature sequencing platform (see RNAi News, 4/1/2005), and later 454 Life Sciences’ sequencing technology.
“454’s introduction was a big thing because it lowered the price barrier substantially [compared with MPSS], although the total throughput is also lowered correspondingly,” Meyers said. Now, “Solexa has this new generation of technology [coming out soon] … which should offer higher throughput and a lower cost than MPSS and 454.”
According to Solexa, Sequencing by Synthesis uses “proprietary fluorescently labeled modified nucleotides to sequence millions of fragments of DNA or cDNA in parallel a single base at a time. These … nucleotides, which also posses a reversible termination property, allow each cycle of the sequencing reaction to occur simultaneously in the presence of all four nucleotides.
“In the presence of all four nucleotides, the polymerase is able to select the correct base to incorporate, with the natural competition between all four alternatives leading to higher accuracy than methods where only one nucleotide is present in the reaction mix at a time which require the enzyme to reject an incorrect nucleotide,” the company explained on its website. “Sequences where a particular base is repeated one after another are dealt with as for any other sequence and with high accuracy.”
Meyers and Green’s latest work is being funded under a two-year National Science Foundation grant worth about $1.1 million. According to Meyers, it was awarded under the NSF’s Plant Genome Research Project, which is designed to support basic research and accelerate development of new technologies in plant genomics.
According to the grant’s abstract, the goal of the project is to “develop extensive sequence resources of plant small RNAs (21 to 24 nucleotides) and target mRNAs from representative species across the plant kingdom. These data will enable functional and evolutionary studies of microRNAs and short interfering RNAs.”
In previous studies, small RNAs have been shown to play roles in plant development, stress responses, and epigenetic regulation,” primarily though transcriptional and post-transcriptional silencing, the abstract adds. “While most studies have been limited to Arabidopsis, the little that is known about small RNA diversity and evolution suggests that miRNAs are a well-conserved gene regulatory system dating back to lower plants and green algae.”
To test this hypothesis, Meyers and Green have selected 32 different plant species, including several green algae, from which samples will be taken and sequenced.
“We looked at the tree of life for plants,” Meyers said. “That includes the angiosperms, which are all of the higher flowering plants, gymnosperms, non-seed plants [including] mosses and ferns, all the way down to the green algae.”
Green noted that they intend to sequence the flowers and leaves from plants that produce them. However, “some of the plants or algae we’re using don’t make the typical flower, so we’d chose a different sample,” she said.
Meyers added that for plants of economic importance, work will focus on the part of the plant with value. “For instance in potatoes, the leaf and flower may be interesting from a small RNA perspective, but economically all the money is in the tuber,” he said. “So in that case, we’d sample the tuber.”
“The project includes sequencing of small RNAs from a diverse and agronomically relevant set of plant species,” the grant abstract states. But Meyers noted that it will also incorporate a focused analysis of five members of the Poaceae and Solanaceae families.
The Poaceae family comprises true grasses including barley, wheat, and maize. Solanaceae is a family of flowering plants that includes the potato and tomato.
The effort will also involve the “sequencing of miRNA target libraries from several species, and development of a small RNA database and web interface for public access and analysis of data,” the grant’s abstract states.
Meyers anticipates that data from the project will
“454’s introduction was a big thing because it lowered the price barrier a substantial amount [compared with MPSS], although the total throughput is also lowered correspondingly. [Now] Solexa has this new generation of technology [coming out soon] … which should offer higher throughput and a lower cost than MPSS and 454.”
allow researchers to “identify microRNAs that are very well-conserved across different clades, whether that’s all flowering plants or gymnosperms or all plants in general. And for some of these species or clades, it may be possible to identify more recently evolved microRNAs that are specific to that particular family,” he added.
Additionally, “within each genome or species we should get an idea of the diversity of the endogenous siRNAs and the types of sequences that they match,” which would be useful to researchers interested in particular plant families, he said.
“To investigators interested both in the specific species and interested in plant functions [such as flowering, for example,] we hope to provide microRNA data that may demonstrate a role in specific pathways, traits, or functions,” Meyers said. A “comparative analysis may show evolutionary differences among plant species in these microRNAs. We really hope that this will be quite a useful data set for a broad range of plant biologists.”
Overall, these data “will allow the experimental characterization of the majority of biologically important small RNAs for a range of plant species, and will be … useful; to a broad set of plant biologists interested in development, stress response, epigenetics, evolution, RNA biology, and other traits impacted by small RNAs,” the grant abstract states.
In the longer term, Meyers said that he hopes to use the data from the project to “look more on the functional side of these things.”
The funds for the project, he noted, were earmarked by the NSF “for the development of resources, not to address biological questions. So our role, therefore, is to provide this as a resource, not necessarily to address any particular questions. But of course as biologists, how can we avoid but to try to make sense of the data and see where that takes us?”
“My lab is quite interested in post-transcriptional control by small RNAs, so this is an area that we’re going to focus on” using the data from the sequencing effort, Green added. “I think the siRNAs that we should see in great abundance, particularly in the grasses, are of great interest to Blake’s lab and some collaborators of his. So I think we will follow up these directions in the future.”