Elsa Gonçalves’s research while affiliated with Instituto Superior de Gestão and other places

Métodos de fenotipagem em campo para seleção clonal em videira: contributos para a tolerância às alterações climáticas Fenotipagem de plantas, melhoramento e alterações climáticas A fenotipagem de plantas é uma disciplina emergente que com-bina várias metodologias e protocolos para medir características vegetais, como crescimento, arquitetura, função e composi-ção, em várias escalas de organização biológica. Quando feita manualmente é um processo muito demorado e que exige grande quantidade de mão de obra, pelo que representa um dos princi-pais constrangimentos à fenotipagem de plantas e culturas com objetivos de melhoramento. Atualmente já existem métodos de 'fenotipagem de plantas de alto rendimento' (HTPP), geralmente baseados em captura de imagens, obtenção automatizada de dados e processamento. Mesmo com a automatização da HTTP, ainda existem desafios e nem todos os métodos são adequados ao processo de seleção de plantas em culturas economicamente relevantes, como a videira. As alterações climáticas (menor precipitação, temperaturas mais elevadas, maior frequência de eventos extremos) são um sério risco para a sustentabilidade do setor vitivinícola, diminuindo a produti-vidade e a qualidade, promovendo a senescência e queda precoce das folhas, bem como aumentando a incidência de queimaduras solares em folhas e cachos. O uso de rega tem sido a principal estratégia para enfrentar o desafio climático, mas outras estratégias, mais sustentáveis, tais como a obtenção e seleção de castas e porta enxertos mais resistentes, também podem ser usadas. Fenotipagem da videira A videira é uma espécie com grande diversidade genética que pre-cisa de ser bem caracterizada, pois em cada variedade existem alguns genótipos (clones) que são mais tolerantes do que a média da variedade, ao stresse abiótico. É assim necessário apoiar a sele-ção com a disponibilização de métodos eficazes de identificar este material vegetal mais bem adaptado ao stresse abiótico para uso posterior (material policlonal ou clonal). Este processo envolve a observação de centenas ou milhares de indivíduos no campo. Além de demorados, complexos e dispendiosos, estes ensaios são também afetados por condições de clima e solo heterogéneas e difíceis de controlar. Deste modo, o planeamento cuidadoso do projeto experimental de campo e as metodologias de colheita de dados em campo são cruciais para obter resultados confiáveis. Figura 1. Medição das características morfológicas das folhas de videira com recurso à imagem RGB (A) e a sistemas de análise de imagem "open acess" (ImageJ) (B) (Neves, 2020) Figura 2. Exemplo da variação da temperatura da sebe em função do nível de rega para a casta Aragonez (sin. "Tempranillo") em condições de campo. Imagem feita com câmara térmica de baixa resolução (120 x 120 pixels, 7-14 µm, Emissividade = 0,96) O fenótipo é a parte visível do genótipo. É definido como o resul-tado do genótipo, do ambiente e da interação entre eles (G × E) e é dinâmico, complexo e compreende múltiplos traços quanti-tativos e qualitativos que dificultam o seu estudo. A fenotipagem engloba o conjunto de metodologias e protocolos usados para medir com precisão fenótipos de crescimento, arquitetura, com-posição e função das plantas, e visa fornecer dados para melhorar a gestão dos recursos de biodiversidade, promover a adaptação das culturas ao ambiente, bem como identificar características superiores de, por exemplo, rendimento, qualidade, ou tolerân-cia a stresses bióticos e abióticos. Uma fenotipagem eficiente deve considerar dois aspetos muito importantes: 1) a disponibilidade de ferramentas adequadas para medir as características-alvo e 2) o planeamento das medições. Quanto ao primeiro aspeto, a seleção de plantas necessita de métodos simples, rápidos e fáceis de usar em larga escala (para medição de centenas e mesmo milhares de indivíduos), adaptados às principais características agronómicas, fisiológicas e tecnológi-cas. O segundo aspeto prende-se com as ações tomadas antes e depois da fenotipagem, ou seja, as regras a seguir para garantir que os dados obtidos sejam adequados para efeitos de seleção. Sendo a videira uma espécie perene, com copa e arquitetura com-plexas, a fenotipagem de campo apresenta dificuldades, que o uso de sistemas de imagem com capacidade de identificar situações de stresse tem vindo progressivamente a colmatar. Um exemplo é a análise de imagem por RGB, usada para estimar rendimento, área foliar e vigor (Figura 1), havendo já métodos disponíveis capazes de distinguir uvas com base na forma, o que permite quantificar uvas de castas brancas tão eficientemente como tintas. Já a imagem Figura 3. Exemplo do trabalho de fenotipagem desenvolvido para a caracterização de clones em condições de campo: medição de características relacionadas com a regulação estomática e transpiração e com a capacidade fotossintética através fluorescência da clorofila usando um porómetro-fluorímetro (LI 600, Li-Cor, EUA) A B

The valorisation of genetic variability through the identification suitable genotypes for traits such as yield and must quality is an effective strategy used for grapevine selection. Currently, climate change-driven heat waves and drought affect plant growth and wine quality, but little information is available on intravarietal variability regarding responses to stress. In the current work, the intravarietal genetic variability of the Portuguese variety Arinto was studied for yield, must quality, and tolerance to abiotic stress. An innovative approach using rapid, and nondestructive measurements of surface leaf temperature (SLT), Normalized Difference Vegetation Index (NDVI), Photochemical Reflectance Index (PRI), and chlorophyll content (SPAD), was used in an experimental population of 165 clones of Arinto installed according to a resolvable row-column design with 6 replicates. Also, yield and quality characteristics of the must were quantified. Linear mixed models were fitted to the data, and the empirical best linear unbiased predictors (EBLUPs) of genotypic effects for each trait were obtained as well as the coefficient of genotypic variation (CVG) and broad sense heritability. The results enabled the selection of a group of genotypes with increased tolerance to stress, which maintained the must quality of Arinto.

Since the last decade of the last century, it is known that many old grapevine varieties are descendants of other varieties through natural crossing. Portugal has an important program for the conservation of representative samples of intra-varietal variability of all autochthonous varieties, managed by the Portuguese Association for Grapevine Diversity (PORVID), which makes looking for genotypes with dubious identification an important activity from a perspective of its valorisation. This communication presents the results of the molecular analysis of 5,000 samples (accessions) from the PORVID’s collection, using nine microsatellite loci currently recommended by the International Organization of Vine and Wine (OIV) for genetic grapevine identification. The results obtained confirmed the molecular identity of 4,220 samples corresponding to 214 varieties present in the official list of Portuguese varieties. In 780 samples, 95 profiles with a plural number of accessions revealed not to be listed in the Vitis International Variety Catalogue (VIVC) database, corresponding to possible varieties either descendent from natural crossing from at least one known parental variety, or from undetermined origin. Furthermore, the need for a comprehensive strategy aimed at uncovering other hidden varieties is discussed to prevent their imminent loss, deepen understanding of their origin, and add economic value and sustainability to the vine and wine sector.

The antiquity and diversity of traditional grapevine varieties guarantee a strong historical and natural character and add high economic value to viticulture and wine. A high level of intra-varietal diversity of quantitative traits was naturally created and accumulated over centuries. Nowadays, this diversity allows one to carry out selection within a variety and adapt it to the most diverse environmental, agricultural, and market contexts. However, an unusually intense process of genetic erosion of intra-varietal diversity broke out in the 1980s and threatens to destroy within a few decades the diversity created over centuries and millennia. Therefore, the definition of strategies and methods for conserving and exploring intra-varietal genetic variability is crucial for the sustainability of viticulture rich in history and traditions. Those strategies are presented in this work.

Background and Aims. In ancient grapevine varieties, the experimental design of field trials is crucial to providing a reliable evaluation of quantitative traits. The main purposes of this study are to demonstrate the benefits of the resolvable row-column design (RCD) for quantifying intravarietal variability and performing polyclonal selection and to compare the efficiency of fully and partially replicated designs for quantifying intravarietal variability to implement the latter designs for a preliminary analysis of that variability. Methods and Results. Linear mixed models were fitted to yield data obtained in field trials with fully and partially replicated designs. The results pointed out the importance of the RCD in controlling the spatial variability present in large field trials. Although less precise, a partially replicated design proved to be useful in evaluating intravarietal variability when the average of years was used. Conclusions. The results reinforced the importance of the RCD in increasing the efficiency of intravarietal variability quantification and polyclonal selection. The partially replicated design proved to be useful when the only objective was to perform a preliminary analysis of intravarietal variability. Significance of the Study. Understanding the role of experimental design in grapevine selection field trials will help grapevine breeders enhance their knowledge about variability within ancient varieties and implement more successful polyclonal selection.

There are thousands of ancient grapevine varieties in Europe, each one having a high level of intra-varietal diversity with regard to important economic traits (yield, soluble solids content, acidity, anthocyanins, and others). However, this potential has become exposed to a process of genetic erosion since the middle of the last century. The main objective of this work is to present experimental strategies for conservation and utilization of intra-varietal diversity. A concrete example is given about the actions performed in Portugal since 1978. Two main approaches for the conservation of intra-varietal diversity were performed: (1) strict conservation (in pots and in the field without experimental design) for future generations; and (2) conservation and, simultaneously, evaluation of the intra-varietal variability for selection to fulfil the immediate needs of the grape and wine sector (in the field with experimental design). More than 30,000 accessions of Portuguese autochthonous varieties are conserved. Using the theory of mixed models, intra-varietal diversity of the yield was found for the 59 varieties studied. The conservation and the evaluation of the intra-varietal diversity for quantitative traits will allow to extract high economic value, as well as to ensure its utilization to meet the objectives of the vine and wine sector.

The conservation and evaluation of intravarietal genetic variability, which is under strong erosion pressure, are crucial strategies for preserving traditional viticulture and facing future challenges of adaptation to consumer demands and biotic and abiotic stresses. In this chapter, an overall view concerning the conservation and evaluation of intravarietal grapevine diversity and selection within a variety is drawn. The purpose of this approach is to exploit intravarietal variability for selection and to fulfill the immediate needs of the vine and wine sector. Specifically, different methodologies of conservation are described and compared, and the evaluation of intravarietal genetic diversity with the objective of selection and the practical application in Portugal are discussed.

Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.