Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-12T13:20:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) / Made available in DSpace on 2016-07-12T13:20:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_FlaviaAraujo_2015_VersãoFinal.pdf: 4388867 bytes, checksum: 1ebc175e90318442d9eb1b8a4a688b3c (MD5) Previous issue date: 2015-03-02 / FACEPE / A cultura do feijão-caupi [Vigna unguiculata (L.) Walp.] apresenta importância econômica em nível internacional, entretanto ela é frequentemente acometida por uma diversidade de patógenos. Nesse contexto a família gênica NBS-LRR de genes de Resistência (R) se destaca devido ao seu papel fundamental na defesa das plantas contra o ataque de patógenos, sendo a maior e mais diversificada família desse grupo. O objetivo do presente estudo foi caracterizar genes NBS-LRR em feijão-caupi, desenvolver marcadores moleculares RGA (Resistance Gene Analogs) e validar genes diferencialmente expressos em uma interação planta-vírus. Inicialmente, sequências candidatas para genes NBS-LRR foram obtidas no banco de dados NordEST, procedendo-se com a anotação dos dados, tradução e identificação dos domínios conservados por meio de ferramentas in silico. Um total de 57 sequências NBS-LRR completas foi identificado em feijão-caupi. Como as proteínas codificadas pelos genes R apresentam domínios e motivos conservados, foi possível desenvolver marcadores RGAs usando as sequências de feijão-caupi como sonda contra o banco de Phaseolus vulgaris L. Foram desenhados 16 pares de iniciadores para P. vulgaris, identificando-se um percentual de 87,5% de transferibilidade para o feijão-caupi. Destes, dois foram polimórficos e apresentaram segregação mendeliana em uma população de mapeamento para o vírus do mosaico severo do feijão-caupi (CPSMV). Os 57 candidatos foram ancorados nos 20 pseudocromossomos de Glycine max (L.) Merr., verificando-se repetições in tandem deste grupo gênico. A análise de expressão gênica diferencial in silico foi realizada utilizando dados de RNAseq e SuperSAGE. A validação da expressão gênica via RT-qPCR foi através do desenho de primers, com os dados de SuperSAGE, onde três genes alvo apresentaram indução nos níveis de expressão após 16 horas da inoculação com o patógeno. Esses resultados mostram-se valiosos para o melhoramento genético do feijão-caupi. / The cowpea [Vigna unguiculata (L.) Walp.] culture has an international economic importance, however it is often affected by a diversity of pathogens. In this context the NBS-LRR family of resistance (R) genes stands out because of its key role in plant defense against pathogen attack, being the largest and most diverse family of this group. The present work aimed the characterization of NBS-LRR genes from cowpea, the development of RGA (resistance gene analogs) markers and the validation of differentially expressed genes in plant-virus interaction. Initially, NBS-LRR gene candidate sequences were obtained from NordEST database, proceeding with data annotation, translation and identification of conserved domains through in silico methods. A total of 57 NBS-LRR complete sequences were identified for cowpea. Since R-gene encoded proteins exhibit conserved domains and motifs, it was possible to develop RGA markers using cowpea sequences as probes against Phaseolus vulgaris L. Sixteen primer pairs of P. vulgaris were designed, from which 87.5% were transferable to cowpea. From those, two were polymorphic and showed Mendelian segregation in a mapping population for the cowpea severe mosaic virus (CPSMV). The 57 candidates were anchored in the 20 Glycine max (L.) Merr. pseudo-chromosomes, revealing in tandem repetitions for this group of gene. Differential gene expression analysis in silico was performed using data from RNAseq and SuperSAGE. The validation of gene expression by RT-qPCR was carried out after design of primers using SuperSAGE data, from which three target genes presented induction in expression levels at 16 hours after the pathogen inoculation. These results represent valuable data for the genetic improvement of cowpea.

Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum vulgare) in many regions of the world. Yield losses up to 62% have been reported in susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and 30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants derived from the crosses 'Bowman' (rph5) x 'Magnif 102' (Rph5) and 'Moore' (rph5) x Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman x Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3 was employed to saturate the region within the sub-centimorgan region around Rph5 using sequence-tagged site (STS) markers that were developed based on barley expressed sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 10 EST-based STS markers were incorporated into the 'Bowman' x 'Magnif 102' high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes, represented by these markers, are putative candidates for Rph5. Results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley EST resources for marker saturation of targeted barley genomic region. / Ph. D.