Marker development for the traceability of certified sustainably produced cacao (Theobroma cacao) in the chocolate industry Pedro Lafargue-Molina “A thesis submitted in partial fulfilment of the requirements of the University of the West of England (UWE), Bristol for the degree of Doctor of Philosophy (PhD)” Faculty of Health and Applied Sciences Centre for Research in Biosciences Director of studies Dr Joel Allainguillaume Ph.D. March 2021 Word Count: 47795 Marker development for the traceability of certified sustainably produced cacao (Theobroma cacao) in the chocolate industry Pedro Lafargue-Molina “A thesis submitted in partial fulfilment of the requirements of the University of the West of England (UWE), Bristol for the degree of Doctor of Philosophy (PhD)” Faculty of Health and Applied Sciences Centre for Research in Biosciences Director of studies Dr Joel Allainguillaume Ph.D. March 2021 Word Count: 47795 i PUBLICATION STATEMENT The candidate confirms that the work submitted is his/her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2020 The University of the West of England and Pedro Lafargue ii ABSTRACT Theobroma cacao (cocoa) is one of the most studied commodities around the world and the source of one of the world’s most consumed and familiar products, chocolate. The multibillion-pound industry has changed to a higher demand for sustainably certified cacao (Rainforest Alliance, UTZ, and Fair Trade) and closer attention is being paid to how this such cacao can be traced. The present work describes a new concept, “From Shelf to Farm & Cooperative”, a study to identify the geographical origin of the fermented cacao beans used to manufacture premium and bulk chocolate products. The research sought to assess how DNA based approaches for traceability of food products can be utilised within the supply chain of cacao and chocolate. To identify the factors that influence cacao traceability and the importance of assessing it in different supply chain systems, multi-disciplinary stakeholders from policy makers, small-scale farmers in South and Central America, to the biggest cacao and chocolate manufacturers in Europe were interviewed. Two stages in chocolate production were identified as key to be screened for tracking implementation: The farm (Stage 1) to identify cacao trees genotype composition and the cooperative (Stage 2) where fermentation of cacao beans occur. A reliable modified cacao DNA extraction protocol was developed using the DNeasy mericon Food Kit which enable higher DNA yield from a range of chocolate products including, for the first time, ‘cocoa butter’. DNA markers characterising the chloroplast genome of T. cacao were assessed to trace back the chocolate to Stage 1 (farm). Reference genotypes from the International Cocoa Quarantine Centre at the University of Reading were screened with 25 chloroplast single sequence repeat (cpSSR) markers revealing a level of DNA polymorphism sufficient to reliably identify lineages below the species level to characterise farms. Allelic proportions for nine cpSSR were quantified and compared in DNA extracted from 116 chocolate samples revealing distinct clustering in single-origin chocolate produced from beans harvested in Peru, Ecuador, Venezuela, Trinidad and Madagascar. In contrast, no differentiation was observed for bulk chocolate samples (Mars, Nestle) and beans originating from Ivory Coast farms thus reflecting the lack of allelic diversity found in cultivars in West Africa. To identify unique biomarkers for Stage 2 (cooperative), the fermentation microbiome was assessed by performing amplicon Illumina sequencing on 47 single origin chocolate using the universal 16S v3-v4 ribosomal region and three housekeeping genes from Acetobacter pasteurianus. Variation in microbiome diversity was characterised with unique Amplicon Sequence Variants (ASV) identified per continent, country and fermentation location for which signature bacterial profile was found to be conserved across years. Markers identified in Stage 1 and Stage 2 can be used for tracking cocoa beans origin. To make these biomarkers applicable in industrial scenarios, it will be essential to create a machine learning model that could recognize the specific markers from multiple regions. iii ACKNOWLEDGEMENT Thank you! Gracias! Merci! What a journey! Writing up during the first Pandemic of my life is clearly one thing that I’d have never imagined. It has been ten years since I started working in Cacao, which went from planting the trees as a small farmer, creating a chocolate company from scratch to finally finishing this PhD research thesis. Was it my passion, tenacity or something else that drove me? I certainly don’t know. But what I know is that without the advice and support of my family, farmers, academics, business partners and researchers, this work would have not been accomplished. Firstly I would like to thank my director, Assoc. Prof Joel Allainguillaume for all the guidance, continued support, enthusiasm and advice throughout these three years. I could not have imagined having a better mentor to build this innovative project, who constantly challenged and guided me towards the right direction to connect the dots. I really appreciated how Joel has shared the same passion for the project and taught me all the scientific skills to support and confidently lead the research to a bigger scope that can be applied into the industry. Besides Joel, I would like to thank the rest of my research supervisory team at UWE: Prof Dawn Arnold, Assoc. Prof Emma Weitkamp and Dr Rey Loor from the National Cacao Research Institution in Ecuador, for their insightful comments and encouragement, but also for the hard questions which helped to shape my research from various perspectives and for future projects. A huge thanks to Brigitte Laliberté, who coordinates Cocoa of Excellence Programme (CoEx), Bioversity International and the International Cocoa Awards (ICA) for supplying unique single-origin chocolates from across the globe. To Dr Andrew Daymond for all the DNA samples and metadata from the ICQC at the University of Reading, UK. To Dr Rey Loor, who personally drove me more than 400km to selected farms in the Amazon, rainforest and coastal Ecuador for collecting antique cacao genotypes, and who in addition granted me complete access to the INIAP national research laboratories. A special thanks to Tree of Wisdom Chocolates Ltd (TOW) crew in Ecuador for supplying single-origin chocolates. A further big thank you to Dr Vicente Norero from Latiali and Camino Verde for his initial mentoring, sourcing and fermenting TOW cacao beans under unique conditions. To the Shaman Gilberto from the community Cayapas in Esmeraldas for hosting me and supporting my fieldwork in the tropical rainforest. To Karen and Paul from the NGS sequencing facility at the University of Exeter, UK, who gave to me in-depth feedback on the experimental design of Illumina sequencing and for processing the DNA samples. iv I would like to express my sincere appreciation to all my trainers in metagenomics and bioinformatics from the Harvard T.H Chan School of Public Health from University in Boston Massachusetts, USA Catchen Laboratory from the University of Illinois, USA to the QIIME developers: Dr Antonio Gonzales, Dr Yoshiki Vázquez from Knight labs from University of California San Diego, USA who originally thought I was a bit insane to try to write a program to study a 5 genes multiplexed experiment. A huge thank you to Prof Cherif Guermat from the UWE Faculty of Business and Law, UK who took the time to understand my vision and help me to build a prediction model based on financial algorithms (who nicely constantly complain about the genetic terminology, which lead me to translate my research into business vocabulary to be in the same page). I would like to thank all my interviewees for their time, but especially to Dr Martin Gilmour, Global head of Agronomy at Barry Callebaut who granted me the access to show my research to the RD board of directors in Belgium. I must thank Dr Tim Moss at UWE for awarding me my scholarship. I am extremely grateful to the finance and graduate school departments (Caterina, Neil, Vicky, Annie, Joel and Lyn) who made my self-funding agreements possible and approved all my fieldworks. Without them, I wouldn’t have been able to present the project to multiple audiences in Cambridge, Berlin, Netherland, Belgium and Indonesia. I would like to thank Dr Robin Thorne, Shonna Nelson, Andy Wetten, Gareth Robinson, David Veal and Man-Kim Cheung for making me part of the lecturing teams, which helped me to learn more about research in applied science and gave me feedback in order to accomplish my research goals. To all my friends at the HAS and FBL PGR centre, I salute you and thank you for making me feel at home. Last but not the least, I would like to thanks my mother Sandra, my sister Michelle, my little nephew Elias, my big Molinas and Cucalon family (Diegos, Mireya, Lorena, Edgar, Lucho, Jose, Andres), my father Miguel and my grandmas Fanny and Juana for all the support and encouragement to follow my dreams. To my amazing best friends and second family (Polits, Calderon, Garces, Polanco, Moscoso, Urresta), and my Bristolian community (Tanmay, Leo, Isabel, Goncalo, Ewan, Berrbizne, Chris, Buffy, Cyril) for believing in me and helping me to cope with the stress and difficulties throughout these three years. v “THE PURSUIT OF KNOWLEDGE CAN LEAD TO CHAOS, BUT YOU MUST ACCEPT THE FACT THAT THE JOURNEY WILL NOT BE SMOOTH AND THAT YOU CAN SHAPE CHAOS TO BUILD A MAGNIFICENT PIECE OF ART”.