1. Global BYDV/CYDV Genome Sequencing Project Jill Hansen 1, Chris Siguenza 2, Randy Beckett 2, W. Allen Miller 2 1 Bay High School, Panama City Florida, 2 Iowa State University, Ames Iowa ABSTRACT Yellow Dwarf Viruses (YDVs) are plant diseases with a significant effect in determining crop yield and viability. With a global impact, developed and developing countries alike are looking for resources to combat this economically important group of viruses. Members of YDV have a remarkable diversity that is causing taxonomic confusion yet they possess very similar replication genes. For all their diversity, there exist few full- length genome sequences of the multitude of isolates. As a service to the scientific community, plant breeders, and farmers worldwide, we attempted to sequence samples taken from a variety of locations to determine homology and recombination. A database of these and other sequences could provide researchers with avenues to combat new types of infections, better understand genetic resistance, and determine possible phylogenetic relationship. Samples analyzed from California and Iowa showed significant variation that may lead to the a better grasp of viral replication and recombination. Future research may lead to increase in crop yields. RESEARCH QUESTION The goal was to determine and disseminate the complete genomic sequences BYDV and CYDV as a free service to researchers and plant breeders in developed and developing countries. A large database of full-length YDV sequences will be invaluable to help facilitate breeding for resistance, to undertake epidemiological studies, and to guide research on molecular mechanisms that may lead to more sustainable cropping practices and increased yields that benefit the farmers and ultimately the consumer. METHODS Isolates were provided by collaborators from every arable continent. As common sequences appear in all isolates, we used the rapid procedure of cloning and sequencing additional isolates with defined primers. Total RNA was extracted from leaf tissue of oats (Avena sativa) by grinding under liquid nitrogen and homogenized with TRIzol TM. The creation of a full length first strand via a reverse transcriptase (RT) reaction off the viral RNA messenger is often difficult on genomes of this size because the secondary structure can cause RT to terminate prematurely. Creation of a first strand and amplification was achieved using Thermoscript TM reverse transcriptase and Pfx DNA polymerase. PCR products were visualized and extracted on GelRed stained agarose gels, and purified using a QIAquick Gel Extraction Kit (from QIAGEN). For rapid and efficient cloning of the PCR products we used the proven TOPO® blunt cloning method. This allows rapid, efficient (95%) in vitro recombination of any insert with no need for restriction enzyme digestion and ligation. Any pUC derived vector provides convenient restriction sites, high copy number, and Fwd/Rev primer sites. This provided for easy screening of transformants and purification of high quantities of recombinant plasmids to be used for sequencing. Sequencing was performed at the ISU DNA Sequencing and Synthesis Facility, using an ABS 3730xl high throughput capillary system. ACKNOWLEDGEMENTS Thanks to Aurelie Rakotondrafara, Jelena Kraft, Krzysztof Treader, Zhaohui Wang, Betty Chung, Jackie Jackson, Adah Leshem-Ackerman, Eric Hall, Sharon Andrews, Allen Miller, and especially Randy Beckett. RESULTS Sequences obtained from the DNA facility were examined and compared with previously sequenced genomes from common isolates. Tissue sample 104 from California matched BYDV-PAV (IL), one of the most common isolates. Sample 106, also from California, matched the sequence of a previously known CYDV-RPV isolate originally from New York. Subsequent testing failed to reveal the presence of an RMex isolate in the California samples. Samples taken from an oat field near Albia IA (see pictures 1,3) were found to match the sequences of BYDV-PAS. Previously only sequenced in wheat, BYDV-PAS (also known as PAV-129) did not cause striking symptoms. However in oats it has been shown to be lethal and breaks resistance that is effective against other PAV isolates. BYDV-PAS is a PAV isolate based on serology and aphid transmission, but the severe symptoms and nucleotide sequence, especially in the replication genes, reveal a very different virus. BACKGROUND Yellow Dwarf viruses are the most economically important viruses of wheat, barley, oats, corn, and rice. Transferred only by aphids, Barley Yellow Dwarf Virus (BYDV) and Cereal Yellow Dwarf Virus (CYDV) have caused more than $500 million dollars damage in one year and have shown a 100% infection rate in some areas. Reductions in yield considered insignificant in developed countries can be devastating to subsistence farmers trying to provide for their families. In these developed countries, risk of significant loss is high enough that vast quantities or pesticides are sprayed to control the aphid vector. This may cause environmental damage and harm non-target organisms. The epidemiology and ecology of YDV has been referred to as “a study in ecological complexity”. As members of the Luteoviridae family, they share substantial homology to each other in the virion proteins (see Fig.1) The few full-length YDV sequences known reveal amazing variation in different parts of the genome. The differences are so great that BYDV and CYDV have recently been placed in different genera. The few isolates of these viruses that have been sequenced suggest that CYDV and possibly BYDV may be split again into more viruses by the International Committee on the Taxonomy of Viruses. YDV outbreaks are highly variable, being dependent on the time of planting and aphid migrations. Natural host plant resistance has met with limited success since it tends to be isolate/serotype specific, and some viral isolates can break resistance. A great deal of YDV molecular mechanistic biology and resistance breeding has been done using a small number of virus isolates as “lab rats”. Yet, it is clear that the viruses identified as BYDV or CYDV exist as a complex of variants in the field and the predominant populations will change according to host, vector, and environment. DISCUSSION A goal of this project was to sequence resistance breaking isolates towards an understanding of their mechanisms of resistance avoidance. Further research in samples obtained from southern Iowa may yield such data. YDVs offer great potential to understand transmission of circulative viruses due to their unique and extreme vector specificity. Knowing the aphid transmission phenotype and the corresponding virus protein sequence of numerous YDV isolates within each serotype would provide correlative evidence on which amino acid sequences should be targeted for subsequent mutational analysis. A comprehensive database created and supported by Iowa State University could facilitate this effort. Discovery of a polerovirus (CA sample 104) and a luteovirus (CA sample 106) in the same region may help to trace the phylogeny of YDVs (see Fig 3). Luteoviruses undergo striking recombination on an evolutionary time scale and sequencing large numbers of full-length, unbiased genomes may shed light on the evolution of YDVs and similar plant viruses. Future research in other fields may also help to explain the replication and recombination of important human viruses. For example with HIV there is a major variant that exists in just a handful of people in very small geographic regions in Africa. Such a variant might be missed without full-length, unbiased sequencing of large numbers of genomes. REFERENCES Much of this poster was taken from the 2003 United States Department of Agriculture Microbial Genome Sequencing Program Grant Proposal. Other sources: The Aphid: A Virus Vector CD-ROM, APS Press 2004 Invitrogen and Qiagen Corporation Websites From L to R: 1) an Oat farmer with a BYDV-PAS infected crop, 2) the classic symptoms of YDVs, and 3) aphids likely responsible for transmission Fig 3: A possible phylogenetic tree of YDVs Fig 2: the TOPO plasmid Fig 1: Similarities in isolates