Macaubest: atlas transcriptômico da macaúba (Acrocomia aculeata)

Orientador: Gonçalo Amarante Guimarães PereiraTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A macaúba (Acrocomia aculeata) é uma palmeira nativa de ampla ocorrência, estando presente principalmente em áreas de Cerrado. Sua elevada produtividade de frutos, altos teores e qualidade de óleo, além da possibilidade de plantio em áreas de déficit hídrico a credenciam como uma alternativa de matéria-prima para a produção de óleo para biodiesel e outras demandas. Assim, estudos na área de biologia molecular e diversidade genética formam a base para um programa de melhoramento de sucesso para uma espécie pouco estudada. Diante desse contexto, o trabalho visou construir um banco de genes da macaúba e o desenvolvimento de marcadores moleculares para estudos de caracterização da diversidade genética, seleção assistida e mapeamento das populações. Além disso, o trabalho investigou possíveis diferenças entre a macaúba e dendezeiro, espécies próximas geneticamente, porém com exigências edafoclimáticas distintas. Para isso, o RNA foi extraído de tecidos de raiz, bulbo, folha, flor (feminina e masculina) e mesocarpo de frutos maduros. No primeiro experimento, a montagem De Novo do transcriptoma foi utilizada, permitindo identificar 34.293 transcritos de macaúba em todas as bibliotecas, reunidas em uma única referência. O padrão de expressão gênica mostrou oito transcritos específicos de tecidos de fruto, dois de tecido foliar, oito de tecido de bulbo, cinco de flores femininas, 14 de flores masculinas, 8053 de tecido de raiz e nenhum em tecido de bainha foliar. Quando observados o nível de expressão dos transcritos tecido-específicos, o tecido de raiz apresentou 341 transcritos altamente expressos e específicos. Além disso, a comparação com o dendezeiro mostrou que a macaúba possui 24 famílias gênicas expandidas, 11 famílias possuem pelo menos um transcrito raiz- específico e seis famílias foram exclusivas de macaúba. A maioria dos transcritos expressos foi anotada como proteínas relacionadas a transdução de sinais em resposta a estresses abióticos e bióticos, como quinases, fatores de transcrição, proteínas ligantes de Ca2+. Os dados sugerem uma possível regulação na transdução de sinal em resposta à diversos estresses. Na segunda abordagem foi utilizada a montagem com a referência do genoma do dendê. Neste estudo, um total de 418 EST-SSRs foram identificados e 232 EST-SSR foram selecionados, com repetições trinucleotídicas sendo o motivo mais frequente (380 -90,9%), seguido por compostas (4,5%), di- (3,6%) e hexanucleotídeos (3,6%). Um total de 145 EST-SSR (62,5%) foram validados em dezessete amostras de macaúba, e 100 foram considerados polimórficos com valores de PIC de 0.25 a 0.77. Análise de diversidade genética realizada com os 20 mais informativos EST-SSR mostraram uma separação distinta dos diferentes grupos de macaúba, de acordo com a localização geográfica das amostras. Além disso, 145 marcadores foram transferidos em outras seis espécies de palmeiras resultando em taxas de transferibilidade de 99% (144) em Acrocomia intumescens, 98% (143) em Acrocomia totai, 80,7% (117 EST-EST) em amostras de dendezeiro (Elaeis guineensis) e pupunha (Bactris gasipaes), 70% (102) na palma juçara (Euterpe edulis) e 71,7% (104) em sabal (Sabal causiarum). Análise da distância genética estabeleceu grupos distintos por gênero das palmeiras. Os dados obtidos são exploratórios, mas suportarão as pesquisas em andamento com a plantaAbstract: The macaúba palm (Acrocomia aculeata) is a native and widespread palm tree, being present mainly in Brazilian Cerrado. It is considered an alternative oil-feedstock crop since it has high productivity, high oil content, high-quality oil, as well as it is possible to plant in drought areas. Consequently, molecular biology and genetic diversity studies may provide the basis for a successful breeding program. Therefore, this study aimed to build a macaúba palm gene bank and develop molecular markers for genetic diversity studies, marker-assisted selection, and population mapping. Moreover, the studies investigated possible differences between the macaúba pam and African oil palm, which are genetically close but with different edaphoclimatic requirements. For this, the RNA was extracted from the root, bulb, leaf, flower (female and male), and mesocarp of mature fruits. In the first experiment, De novo transcriptome assembly permitted to identify 34.293 transcripts of macaúba palm in all the libraries, gathered in a single reference. Gene expression analysis showed eight transcripts expressed only in fruit tissues, two in leaf tissue, eight in bulb tissue, five in female flowers, 14 in male flowers, 8053 in root and none in leaf sheath tissue. Furthermore, we observed 341 root-specific transcripts with high expression level. In addition, family turnover analysis between macaúba palm and African oil palm showed that macaúba has 24 expanded gene families, 11 families have at least one root-specific transcript and six families are exclusive of macaúba palm. Most transcripts were similarly to transduction proteins related to abiotic and biotic stresses responses, such as kinases, transcription factors, Ca2+-binding proteins. The data suggest a possible regulation of signal transduction in response to many stresses. In the second experiment, the reference-based transcriptome assembly was performed. We identified 418 EST-SSRs and 232 EST-SSR were selected, with trinucleotide repeats being the most frequent motif (380-90.9%), followed by composited (4.5%), di- (3.6%), and hexanucleotides (3.6%). A total of 145 EST-SSR (62.5%) were validated on seventeen macaúba samples, and 100 were considered polymorphic with PIC values ranging from 0.25 to 0.77. Genetic diversity analysis performed with the 20 most informative EST-SSRs showed a distinct separation of the different macaúba samples, according to the geographical location. Additionally, 145 markers were transferred in six other palm species resulting in 99% transferability rates (144) in Acrocomia intumescens, 98% (143) in Acrocomia totai, 80.7% (117 EST-EST) in samples of African oil palm (Elaeis guineensis) and peach palm (Bactris gasipaes), 70% (102) in juçara palm (Euterpe edulis) and 71.7% (104) in hat palm (Sabal causiarum). Analysis of genetic distance established distinct groups by genera. These are exploratory data but will support ongoing research with macaúba palmDoutoradoBioenergiaDoutora em Ciências2014/07265-2458045/2014-4FAPESPCNP

Development of novel EST-SSR markers in the macaúba palm (Acrocomia aculeata) using transcriptome sequencing and cross-species transferability in Arecaceae species

Abstract Background The macaúba palm is a novel feedstock for oil production suitable for multiple uses, including as biodiesel and in the food and cosmetic industries. As an efficient alternative, the macaúba palm has limited genomic resources, particularly expressed sequence tag (EST) markers. We report a comprehensive set of validated EST-simple sequence repeat (SSR) markers by using transcriptome sequencing, its application in genetic diversity analysis and cross transferability in other palm trees with environmental and economic importance. Results In this study, a total of 418 EST-SSRs were identified to be unique for one transcript and region; 232 EST-SSRs were selected, with trinucleotide repeats being the most frequent motif, representing 380 (90.9%), followed by composited (4.5%), di- (3.6%), and hexanucleotides (3.6%). A total of 145 EST-SSRs (62.5%) were validated for consistent amplification in seventeen macaúba palm samples, and 100 were determined to be polymorphic with PIC values ranging from 0.25 to 0.77. Genetic diversity analysis was performed with the 20 most informative EST-SSR markers showing a distinct separation of the different groups of macaúba palm. Additionally, these 145 markers were transferred in six other palm species resulting in transferability rates of 99% (144) in Acrocomia intumescens, 98% (143) in Acrocomia totai, 80.7% (117 EST-EST) in African oil palm (Elaeis guineensis) and peach palm (Bactris gasipaes) samples, 70% (102) in the juçara palm (Euterpe edulis) and 71.7% (104) in the hat palm (Sabal causiarum). Analysis of genetic distance showed a high separation in accordance with geographic location, establishing distinct groups by genera. Conclusions The EST markers identified in our study are a valuable resource and provide a genomic tool for genetic mapping and further genetic studies, as well as evaluation of co-location between QTLs and functionally associated markers

Staphylococcus aureus Isolated From Traditional Artisanal Raw Milk Cheese from Southern Brazil: Diversity, Virulence, and Antimicrobial Resistance Profile

Staphylococcus aureus is one of the primary pathogenic agents found in cheeses produced with raw milk. Some strains of S. aureus are enterotoxigenic, possessing the ability to produce toxins responsible for staphylococcal food poisoning when present in contaminated foods. This study aimed to genotypically characterize, assess the antimicrobial resistance profile, and examine the enterotoxigenic potential of strains of S. aureus isolated from artisanal colonial cheese. Additionally, a bacterial diversity assessment in the cheeses was conducted by sequencing the 16S rRNA gene. The metataxomic profile revealed the presence of 68 distinct species in the cheese samples. Fifty-seven isolates of S. aureus were identified, with highlighted resistance to penicillin in 33% of the isolates, followed by clindamycin (28%), erythromycin (26%), and tetracycline (23%). The evaluated strains also exhibited inducible resistance to clindamycin, with nine isolates considered multidrug-resistant (MDR). The agr type I was the most prevalent (62%) among the isolates, followed by agr type II (24%). Additionally, ten spa types were identified. Although no enterotoxins and their associated genes were detected in the samples and isolates, respectively, the Panton-Valentine leukocidin gene (lukS-lukF) was found in 39% of the isolates. The presence of MDR pathogens in the artisanal raw milk cheese production chain underscores the need for quality management to prevent the contamination and dissemination of S. aureus strains