Discovery of researchers from Embrapa Grape and Wine paves the way for the development of apple trees that require less cold days to produce. They discovered gene ICE1's action mechanism in the process of plant dormancy induction and release, a fundamental stage to ensure fruit production. According to the scientists' hypothesis, this gene works as a thermal trigger that, once it is stimulated by the cold, sets off a cascade of responses like the induction of dormancy, a fundamental process for plants originating from temperate climates to blossom and produce fruits. "This process is so important that research institutions from the entire world invest resources in studies to try to understand it", emphasizes the Embrapa researcher Luís Fernando Revers, responsible for the team that made the discovery. He explains that the genetic control of dormancy in Rosaceae, the botanical family to which the apple belongs, is a complex process, and the identification of genes that control it is a major challenge. "We can use this knowledge to develop new cultivars with lower chilling requirements and keep producing the fruit despite the warming perceived in recent years", he clarifies. After identifying the responsible gene ICE1, Embrapa Grape and Wine's team created a hypothetical model explaining the process of dormancy induction and release, a fundamental stage to ensure fruit production (see video below). The idea behind the project was to select and study two segregating apple populations from Epagri's Breeding Program, with different late or early bloom and budbreak periods, for seven years. During that period, the research was led in two stages: genotyping and phenotyping. The genotyping stage allowed a genetic map to be assembled. Later on, the integration of the phenotyping data with the genetic map led to the identification of loci associated with the budbreak period and the ICE1 gene. Authors The paper Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus × domestica Borkh.) was authored by Yohanna Evelyn Miotto, Carolina Tessele, Ana Beatriz Costa Czermainski, Diogo Denardi Porto, Vítor da Silveira Falavigna, Tiago Sartor, Amanda Malvessi Cattani, Carla Andrea Delatorre, Sérgio Amorim de Alencar, Orzenil Bonfim da Silva-Junior, Roberto Coiti Togawa, Marcos Mota do Carmo Costa, Georgios Joannis Pappas Jr., Priscila Grynberg, Paulo Ricardo Dias de Oliveira, Marcus Vinícius Kvitschal, Frederico Denardi, Vanessa Buffon and Luís Fernando Revers. Crossing the results allowed the elaboration of the hypothesis of the budbreak model publicized in a scientific article entitled Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus X domestica Borkh.)”, published by the journal Frontiers in Plant Science. The Embrapa scientist reports that the generation of cultivars that are adapted to environments with lower availability of cold requires advances in the basic knowledge of the biological mechanisms to control bud dormancy. "Despite the awareness of the action of some factors on dormancy control, there is no knowledge about how they interrelate and about what can be classified as cause or consequence yet", punctuates the researcher Marcus Vinícius Kvistchal, who coordinates the apple breeding and genetic improvement program of Santa Catarina Agricultural Research and Rural Extension Corporation (Epagri), one of Embrapa Grape and Wine's research partners. Kvistchal's team is going to test the knowledge generated in the study for apple breeding in practice. Researcher explains apple tree's dormancy and cold perception mechanism Accelerated cultivar development The Epagri researcher comments that, if the hypothesis is confirmed, the discovery of ICE1's function could speed up the process of developing a new cultivar in up to ten years. "The discovery will enable more precise and fast breeding activities through molecular marker-assisted selection", he anticipates. The scientist explains that from now on, the selection of a new cultivar with a lower chilling requirement can be made as soon as the seed germinates, regardless of how it develops in the field. Evelyne Costes, a researcher from the Mediterranean and Tropical Plant Genetic Improvement and Adaptation (AGAP) section of the French National Institute of Agricultural Research (INRA), considers the partnership between Embrapa and INRA important to face the challenge imposed by climate change. "It is expected that each group's complementary expertise benefits the partnership, which has been implemented since 2016 through a joint project called Dormap, with funding from Embrapa and from Fundação Agropolis", she recalls. She explains that the work is organized in three main scientific axes: the generation and exchanges of genomic data; the functional analysis of genes related to dormancy and the exploration of gene variability to support plant breeding. Among the future results, Evelyne highlights the elaboration of an agreement for the establishment of an "International Associated Laboratory", to be built through a partnership between the institutions, in order to facilitate mutual staff (students and researchers) visits and the elaboration of new projects to be funded to support the research. Over 600 hours of cold The apple tree, like other fruit trees from temperate climates, is induced to the state of dormancy during the first colds of autumn and spends the winter accumulating hours of chilling to break the dormancy and resume growth in the spring. Cultivars from the Gala and Fuji groups account for over 90% of the Brazilian production and require an average of 600 to 800 hours of cold to break the dormancy and reach sustainable production. To make up for the exposure to less chill hours than ideal, sustainable apple production in Southern Brazil depends on the application of chemical agents to induce budbreak. According to the technical team, the occurrence of production losses attributed to insufficient accumulated chilling is common during the period of hibernal rest, and with the perspectives of climate change, productivity could be affected. According to the Brazilian Association of Apple Growers (ABPM), today, in addition to supplying the national market, Brazilian production is responsible for annually exporting about US$ 52 million in fresh fruit. Hence, since 2007 Embrapa's team, in partnerships with Brazilian and foreign universities and research institutes, has been concentrating efforts on the theme, based on the implementation of several research projects (see box at the end of the text). The research "The choice of the research populations was based on the work developed by now-retired Epagri breeder Frederico Denardi. He had already selected some hybrid populations that has lower chilling requirements in a study he had been leading since 1972, at Epagri", explains Kivtchal, who gave the work some continuity. For this research, two populations were selected based on the phenotype of medium chilling requirement of their genitors. One of the stages of the genetic mapping included the partial sequencing of the genitors' genome to find polymorphisms, that is, to identify differences. "It is only possible to make a genetic map once segregating traits are identified, and this is the first stage", Revers explains. The following stage was to make the genetic map based on DNA chips for genotyping on a large scale (9,000 single-polymorphism markers). Besides the genotyping in the laboratory, the team also performed the phenotyping, that is, they assessed how such populations behaved in the field with regard to cold and budbreak. This activity was under the responsibility of the Embrapa researcher Ana Beatriz Costa Czermainski, who followed up on the crossing populations in the field for seven years. The two populations were respectively cultivated in the city and mountain ranges of Bento Gonçalves, in Serra Gaúcha, and Vacaria, in Campos de Cima da Serra, a region with more rigorous winters, with the purpose of measuring the effect of climate in each region in the budbreak period. "During the experiment, all the plants were assessed two to three times a week, in the months from July to November, to monitor the precise moment of bloom and budbreak", the researcher reports, which was followed by the analysis that resulted in the phenotyping. Discovery will help genetic improvement in the whole world According to Revers, after using a series of complex programs that helped to make the genetic map and after exploring phenotyping to identify loci (DNA regions) associated with bloom/budbreak, the scholarship holder Tiago Sartor conducted a detailed visual inspection in the DNA segment of the tip of chromosome 9 and identified the ICE1 gene in the most significant portion of the locus associated with budbreak. He reports that, in the course of this work, several papers were published on advances related to the dormancy and budbreak mechanism of apple trees, and that the Embrapa group was the only one to identify this gene and its relationship with the process. "Finding the ICE1 gene was crucial to elaborate the hypothesis of how dormancy induction and budbreak happens after the winter period. Now we need to continue to test our hypothesis in practice", discloses the researcher, who will be able to count on contributions from INRA and Epagri at this new stage. Faster genetic improvement Kvistchal explains that if the hypothesis is proven, genetic improvement will be faster, making an interesting advantage possible. "Instead of having to wait until a new apple tree selection presents the traits in the fields, we will be able to make the test as soon as the seed germinates and, through DNA extraction, assess the ICE1 gene. If it tests positive, the selection goes on to further assessments; if it does not, it will be discarded right away", he explains. The researcher ponders that the discovery will be of extreme importance for all of the world's breeding programs, especially those aimed at the development of new cultivars that are more suited to less cold regions, as in Brazil's case, ensuring more expeditiousness and precision in the creation of cultivars. In the 47 years of existence of Epagri's Apple Breeding and Genetic Improvement Program, 19 cultivars were launched, including 15 hybrids and four selections from spontaneous mutations. For Costes, from INRA, the discovery significantly contributed to the study of the dormancy process and genetic control in apple trees. She considers that the team led by Revers confirmed the robustness of the association between the budbreak date and the locus at the tip of chromosome 9, where the genes ICE1, FLC and PRE1 are present. "The discoveries and the budbreak hypothesis reported in the paper open new perspectives for the scientific community and for applications in fruit farming", she assesses. Costes stresses that the discovery will not only help the production sector in the South of Brazil, but also many other places that suffer with reduced exposure to the cold, such as the producing regions by the Mediterranean Sea, for instance. "Some of the genes that had been associated with the budbreak date can be potentially used in breeding programs to obtain new varieties that are more adapted to present and future climate scenarios, not only in Brazil but also in different countries and for different climate conditions", the researcher asserts. Research with apple trees For 12 years, researchers from Embrapa, Epagri and the Federal University of Rio Grande do Sul (UFRGS) have been making a series of experiments, collections and comments on the mechanisms of dormancy control in the areas of molecular genetics, breeding and genetic improvement, modelling and plant physiology. The main concern was the effect of climate change on the planet, which has negatively affected Brazilian apple farming. Database about dormancy: the Apple Bud Dormancy Database (Apple BDDB) is a web app to consult a database of genes related to the dormancy process. It shows sample records for the 57,000 apple tree genes from eight comparative experiments, resulting in more than 450,000 records of levels of expression. Coordination: Embrapa Grape and Wine. Partner institutions: Fundação Agropolis, Associação Brasileira de Produtores de Maçã (ABPM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Embrapa Wheat, Embrapa Genetic Resources and Biotechnology, Embrapa Agricultural Informatics, Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Epagri), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (Fapergs), Financiadora de Inovação e Pesquisa (Finep), LABEX Europe, MUSE (Montpellier Université d’Excellence) and Universidade Federal do Rio Grande do Sul (UFRGS). The project is funded by Embrapa, Epagri, Finep, Capes, CNPq and Fapergs. Translation: Mariana Medeiros