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SummaryTo improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic SNP markers. These markers fail to capture the large amount of haplotype diversity in these species and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6 293 genes implicated in growth and wood properties, pest and disease resistance and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8 915 target regions (at 4 637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13 to 4.52 haplotypes per target region in each species and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.Significance StatementWe developed a haplotype sequencing panel for Eucalyptus targeting 8915 regions at 4637 gene loci associated with growth and wood properties, pest and disease resistance and abiotic stress response providing a genome-wide, multi-allelic, gene centric genotyping resource for eucalypts. We tested the panel in four Eucalyptus species (E. grandis, E. dunnii, E. nitens and E. urophylla) and found an average of 3.65 haplotypes per target region per species, and 9.98 across all four species.

Resistance to fusiform rust disease in loblolly pine (Pinus taeda) is a classic gene-for-gene system. Early resistance gene mapping in the P. taeda family 10-5 identified RAPD markers for a major fusiform rust resistance gene, Fr1. More recently, single nucleotide polymorphism (SNP) markers associated with resistance were mapped to a full-length gene model in the loblolly pine genome encoding for a nucleotide-binding site leucine-rich repeat (NLR) protein. NLR genes are one of the most abundant gene families in plant genomes and are involved in effector-triggered immunity. Inter- and intraspecies studies of NLR gene diversity and expression have resulted in improved disease resistance. To characterize NLR gene diversity and discover potential resistance genes, we assembled de novo transcriptomes from 92 loblolly genotypes from across the natural range of the species. In these transcriptomes, we identified novel NLR transcripts that are not present in the loblolly pine reference genome and found significant geographic diversity of NLR genes providing evidence of gene family evolution. We designed capture probes for these NLRs to identify and map SNPs that stably cosegregate with resistance to the SC20-21 isolate of Cronartium quercuum f.sp. fusiforme (Cqf) in half-sib progeny of the 10-5 family. We identified 10 SNPs and 2 quantitative trait loci associated with resistance to SC20-21 Cqf. The geographic diversity of NLR genes provides evidence of NLR gene family evolution in loblolly pine. The SNPs associated with rust resistance provide a resource to enhance breeding and deployment of resistant pine seedlings.

Strawberry (Fragaria spp.) is a valuable fruit crop as well as an outstanding system for studying functional genomics in plants. The goal of this study was to substantially increase and analyze the available expressed sequence information in the genus by examining the transcriptome of the cultivated strawberry (Fragaria × ananassa Duchesne). To maximize transcript diversity and discovery, plants representing an octoploid strawberry cultivar were subjected to a broad range of treatments. Plant materials were pooled by tissue type. cDNA pools were sequenced by the Roche-454 GS-FLX system and assembled into over 32,000 contigs. Predictions of cellular localization and function were made by associating assembled contigs to annotated homologs, and the tissue pool tags provided a means to assess the overall expression pattern for any given transcript. Contigs comprised of reads originating from only one organ type and those present equally in all plant organs were both identifi ed. Bacterial and fungal sequences found in the strawberry samples provide a metagenomic survey of the microbial community of a greenhouse strawberry plant. This study utilized an innovative assembly strategy on pooled tissues, thus providing a foundation for developing tissue-specifi c tools, an opportunity to identify alleles for marker-assisted selection, a reference of strawberry gene annotations, and a basis for comparative transcriptomics between cultivated strawberry, its diploid ancestors, and the wider Rosaceae family. C ULTIVATED STRAWBERRY is a valuable crop, prized for its fl avorful and nutritious fruits. Strawberry is an important crop for many regional economies worldwide and off ers high levels of healthful compounds. Species within the genus Fragaria occur naturally throughout the northern hemisphere and represent many levels of ploidy, including wild diploids and the wild and cultivated octoploids (reviewed in Folta and Davis, 2006; Hummer and Hancock, 2009). Recent attention has been brought to the diploid strawberry species Fragaria vesca Coville because of its utility as a highly tractable experimental system within the economically important Rosaceae family (Folta and Davis, 2006). Strawberry is emerging as a powerful functional genomics system. Fragaria vesca’s haploid genome size of ~200 Mb is among the smallest of the rosaceous crop plants (Shulaev et al., 2008), and its complete genome sequence will be available in 2010, providing an invaluable resource with reference to its octoploid, cultivated relatives. Most importantly, several genotypes of both diploid and octoploid strawberry are readily transformable, making it an outstanding system for functional studies. Protocols exist for diploid and octoploid lines that allow effi cient regeneration of transgenic materials in a period of weeks to months (Folta and Dhingra, 2006; Mezzetti and

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O gênero Eucalyptus apresenta ampla variabilidade genética, relacionada a características economicamente importantes, passível de ser explorada pela integração de métodos clássicos de melhoramento genético e genômica. Apesar dos avanços obtidos pelo melhoramento genético, espécies de Eucalyptus ainda estão nos estágios iniciais de domesticação apresentando, portanto, ampla oportunidade de ganhos pela seleção direcional. O desenvolvimento de mapas genéticos de alta resolução, enriquecidos com informação de genes, é uma estratégia com possibilidades de impactar futuras aplicações de melhoramento molecular. No entanto, com exceção de organismos para os quais o genoma encontra-se sequenciado, as técnicas atuais para se localizar genes em um mapa-referência têm mostrado eficiência limitada. Recentemente foi demonstrado em organismos modelo que microarranjos de oligonucleotídeos desenvolvidos para estudos de expressão podem ser utilizados para a detecção de polimorfismos de sequência, gerando marcadores denominados polimorfismos de elementos individuais (SFP, do inglês Single Feature Polymorphisms). As sondas no microarranjo detectam sítios polimórficos de SNPs ou -indels- nas regiões expressas fornecendo marcadores específicos de genes que, ao demonstrarem comportamento mendeliano, podem ser mapeados. O objetivo deste trabalho foi aplicar o princípio de descoberta, genotipagem e mapeamento de SFPs em uma população segregante F1 de E. urophylla x E. grandis. SFPs foram detectados com sucesso utilizando microarranjos de oligonucleotídeos contendo sequências derivadas de genes únicos gerados a partir de sequênciasconsenso de ESTs de diferentes espécies de Eucalyptus. Visto que essa classe de marcadores representa regiões gênicas, mapeamento de SPFs se torna uma abordagem eficiente para mapeamento em larga escala de genes em organismos com genoma não sequenciado. O uso da estratégia de pseudo cruzamento teste permitiu a detecção de marcadores dominantes segregando 1:1 e 3:1 em uma amostra da população de mapeamento. Um mapa genético saturado foi gerado com 884 SFPs sobre um mapa de referência pré-existente de 180 microssatélites, atingindo uma densidade média de um marcador a cada 1,2 cM em 11 grupos de ligação. O uso de um delineamento experimental que permite a detecção de SFPs segregando 3:1, além de aumentar a densidade do mapa e o número de genes mapeados, possibilitou o aumento da qualidade do mapa final. Os resultados também demonstraram que aumentando-se o número de sondas testadas por gene aumenta-se a probabilidade de detectar SFPs no gene alvo e consequentemente de mapeálo. Este é o primeiro trabalho de desenvolvimento e mapeamento de SFPs em Eucalyptus. A possibilidade de mapear genes em larga escala de forma rápida e acessível permite a condução de análises de co-localização destes genes com QTLs, abrindo espaço para a descoberta de potenciais genes candidatos que controlam esses QTLs para futuramente serem testados em experimentos de genética de associação. The genus Eucalyptus presents a broad natural genetic diversity for economically important traits that could be explored through an integration of classical breeding and genomic approaches. In despite of the advances obtained by classical breeding, species of Eucalyptus are still in the early stages of domestication and, therefore, present several opportunities of gain through forward selection. The development of high-resolution genetic maps enriched with gene information is a strategy that can possibly impact future molecular breeding applications. However, except for organisms where genome sequence information is available, current techniques to allocate genes on the reference map have shown limited efficiency. Recently, it has been demonstrated in model organisms that oligonucleotide microarrays developed to assay gene expression can be used to detect sequence polymorphisms, generating markers termed Single Feature Polymorphism (SFP). The probes of the microarray detect polymorphic loci containing SNPs or indels on expressed regions, creating gene-specific markers that can be mapped. The objective of this study was to apply the principle of SFP discovery, genotyping and mapping in a segregating F1 population of E. urophylla x E. grandis. SFPs were successfully detected using oligonucleotide microarrays with sequences of unigenes generated by sequencing ESTs from different species of Eucalyptus. Since this class of markers samples gene regions, SFP mapping becomes an efficient approach for large-scale gene mapping in organisms with unsequenced genomes. The use of pseudo-testcross strategy allowed the detection of dominant markers segregating 1:1 and 3:1 in a subset of the mapping population. A saturated gene-rich genetic map was generated with 884 SFPs on a previous reference map of 180 microsatellites, with an average density of one marker every 1.2 cM in 11 linkage groups. The use of an experimental design that enables SFPs segregating 3:1 to be detected not only increased map density and number of mapped genes but, more importantly, improved the overall quality of the final map. The results also demonstrated that increasing the number of probes designed per unigene resulted in a higher probability of SFP detection for the targeted gene and, thus, of ultimately mapping it. This is the first report of SFP detection and genotyping for Eucalyptus. The possibility to map genes in large-scale in a quick and inexpensive way allow for the conduction of co-localization analysis of these genes to QTLs, creating possibilities to discover potential candidate genes for these QTLs to be tested in association genetics experiments.

Breeding for water-deficit tolerance is fundamental to guarantee the sustainability of upland rice production, mainly due to the possibility of an increased frequency of drought episodes due to climate change. This work aimed to identify single nucleotide polymorphism (SNP) markers, derived from RNA sequencing (RNA-Seq), genome-wide association study (GWAS) and candidate genes from Arabidopsis, with potential for use in marker-assisted selection (MAS) for drought tolerance. RNA-Seq and GWAS were efficient in identifying useful SNP markers from the data obtained from three years of field experiments for 175 upland rice accessions, which were sequenced using 32 genes by Capture-Seq. Three genes were equally able to generate SNP markers that discriminated 95% of the 20 most drought susceptible accessions in the joint analysis of the experiments. The elimination of the genotypes with the unfavourable SNP allele reduced the initial number of accessions to one third, and transferring this result in a breeding routine, would enable to conduct smaller experiments per target location, increasing the precision and reducing the cost of the drought phenotyping. Made available in DSpace on 2018-03-10T00:34:10Z (GMT). No. of bitstreams: 1 CNPAF2018gmr.pdf: 333924 bytes, checksum: 202e7a3c0bca406bccdf93cad82d91f8 (MD5) Previous issue date: 2018-03-08