BRAGA, D.

FERNANDES, C. D.

JANK, L.

MEIRELES, K. G. X.

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Development of Protein and Transcript DatabasesExpressed During ventcum Maximum X Bipolaris MaydisInteraction, using Data Generated From High-Throughputsequencing MethodsK. Guimarães X. Meireles1, L. Jank-, C. Fernandes3, D. sraga''Plant Biotechnology Laboratory, Researcher of Embrapa Beef Cattle, Campo Grande-MS, Brazil;2Genetic Breeding of Forage, Researcher of Embrapa Beef Cattle, Campo Grande-MS, Brazil,3 Phytopathology Laboratory, Researcher of Embrapa Beef Cattle, Campo Grande-MS, Brazil;'Plant Biotechnology Laboratory, undergraduate Scholarship from Embrapa Beef Cattle.venicum maximum is a C4grass of African origin, widely used in pastures in Brazil,where it is grown on approximately15 million hectares. Cultivars Mombaça, Massai and Tanzânia of P.maximum, released by the Brazilian AgriculturalResearch Corporation - Embrapa, are extensively adopted in the beef and milk production systems in the country,due to their wide adaptation to several types of climates and soils, high productivity and excellent agronomiccharacteristics for use as forage. The Brazilian Market on 2012 for certified seeds of these three cultivars was aroundUS$85 million. Over the past decade, P.maximum pastures have been threatened by the increasing occurrence of leafspot, a disease very well known in the country due to the severity of damage to the corn crop in the 1970s and 1980s.The leaf spot in P.maximum, caused by the fungus Bipolaris maydis, is characterized by lesions on the leaves of thegrass ofvarying sizes and shapes, usually around 0.3-1.0 cm.ln more severe attacks, these lesions coalesce into largernecrotic dark areas, and practically ali aerial parts are affected. Due to the high susceptibility to B.maydis observed incultivar Tanzania,which resulted in significant decrease in area of cultivation, as well as frequent moderate damageto pastures of cultivars Mombaça and Massai, Embrapa's P.maximum breeding program included in its routine, thecharacterization for resistance to B. maydis of its germplasm and promising genotypes for forage use in variousstages of evaluation in the field. With the ongoing reduction in cost and resources required to provide moleculartools to underpin molecular breeding strategies, the breeding program recently incorporated transcriptomics andproteomics approaches aimed at understanding the molecular interaction between P.maximum and B.maydis. TotalRNAextracts were obtained in triplicate from plants of a resistant genotype and of a susceptible one to B.maydis,maintained in interaction with the fungus for 24h, 48h and 72h, as well as their respective controls (plants with nocontact with the pathogen). Twenty-four cDNA libraries were prepared from RNAsamples and sequenced on IIluminaplatform HiSeq2000, resulting in billions of reads of 100bp. For the proteomic analysis, total proteins were extractedfrom 2.0 g of leaf tissue from ali the treatments mentioned above. The samples were desalted and digested withtrypsin, followed by peptide analysis by mass spectrometry, which is underway. In this innovative methodology, asample of peptides is pre-fractionated by three chromatographic columns (nanoACQUITYsystern), followed by massspectrometry analysis model Synapt G2, which allows absolute quantification of proteins and characterization ofthousands of molecules in a single sample, including those of low abundance. The development of a differentiallyexpressed peptide database will assist in the understanding of mechanisms of cellular response of the forage tostress caused by B.maydis. Genetic engineering of plants may in the future benefit from this knowledge through theidentification of gene products that can confer resistance to the pathogen. In this paper, we present the results ofbioinformatics analysis and assembly of de novo transcriptome of P.maximum x B.maydis. As this specie is poorlyrepresented in genome sequence databases, the resulting contigs are being compared to the gene complementfrom the related model grass species. Thus, this study will provide, in an unprecedented manner, the first catalogof transcripts of P.maximum. Bioinformatics analyzes include detection of differential expression of transcripts ineach time of forage x pathogen interaction, aiming at the identification of microsatellite markers for resistance to B.maydis, as well as the selection of candidate genes for this trait. This molecular marker resource will enable futuremolecular breeding efforts in Panicum maximum.Corresponding author: Karem Guimarães Xavier Meireleskarem.meireles@embrapa.br40 14th International Symposium of Forage Breeding

Development of Protein and Transcript Databases Expressed During ventcum Maximum X Bipolaris Maydis Interaction, using Data Generated From High-Throughput sequencing Methods.

http://www.alice.cnptia.embrapa.br/alice/bitstream/doc/973887/1/digitalizar0019PAGINA40.pdf

Development of Protein and Transcript Databases Expressed During ventcum Maximum X Bipolaris Maydis Interaction, using Data Generated From High-Throughput sequencing Methods.

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