High-throughput phenotyping of yield parameters for modern grapevine breeding

Weinbau wird auf 1% der deutschen Agrarfläche betrieben. Auf dieser vergleichsweise kleinen Anbaufläche wird jedoch ein Drittel aller in der deutschen Landwirtschaft verwendeten Fungizide appliziert, was auf die Einführung von Schaderregern im 19. Jahrhundert zurück zu führen ist. Für einen nachhaltigen Anbau ist eine Reduktion des Pflanzenschutzmittelaufwands dringend notwendig. Dieses Ziel kann durch die Züchtung und den Anbau neuer, pilzwiderstandsfähiger Rebsorten erreicht werden. Die Rebenzüchtung als solche ist sehr zeitaufwendig, da die Entwicklung neuer Rebsorten 20 bis 25 Jahre dauert. Der Einsatz der markergestützten Selektion (MAS) erhöht die Effizienz der Selektion in der Rebenzüchtung fortwährend. Eine weitere Effizienzsteigerung ist mit der andauernden Verbesserung der Hochdurchsatz Genotypisierung zu erwarten. Im Vergleich zu den Methoden der Genotypisierung ist die Qualität, Objektivität und Präzision der traditionellen Phänotypisierungsmethoden begrenzt. Die Effizienz in der Rebenzüchtung soll mit der Entwicklung von Hochdurchsatz Methoden zur Phänotypisierung durch sensorgestützte Selektion weiter gesteigert werden. Hierfür sind bisher vielfältige Sensortechniken auf dem Markt verfügbar. Das Spektrum erstreckt sich von RGB-Kameras über Multispektral-, Hyperspektral-, Wärmebild- und Fluoreszenz- Kameras bis hin zu 3D-Techniken und Laserscananwendungen. Die Phänotypisierung von Pflanzen kann unter kontrollierten Bedingungen in Klimakammern oder Gewächshäusern beziehungsweise im Freiland stattfinden. Die Möglichkeit einer standardisierten Datenaufnahme nimmt jedoch kontinuierlich ab. Bei der Rebe als Dauerkultur erfolgt die Aufnahme äußerer Merkmale, mit Ausnahme junger Sämlinge, deshalb auch überwiegend im Freiland. Variierende Lichtverhältnisse, Ähnlichkeit von Vorder- und Hintergrund sowie Verdeckung des Merkmals stellen aus methodischer Sicht die wichtigsten Herausforderungen in der sensorgestützen Merkmalserfassung dar. Bis heute erfolgt die Aufnahme phänotypischer Merkmale im Feld durch visuelle Abschätzung. Hierbei werden die BBCH Skala oder die OIV Deskriptoren verwendet. Limitierende Faktoren dieser Methoden sind Zeit, Kosten und die Subjektivität bei der Datenerhebung. Innerhalb des Züchtungsprogramms kann daher nur ein reduziertes Set an Genotypen für ausgewählte Merkmale evaluiert werden. Die Automatisierung, Präzisierung und Objektivierung phänotypischer Daten soll dazu führen, dass (1) der bestehende Engpass an phänotypischen Methoden verringert, (2) die Effizienz der Rebenzüchtung gesteigert, und (3) die Grundlage zukünftiger genetischer Studien verbessert wird, sowie (4) eine Optimierung des weinbaulichen Managements stattfindet. Stabile und über die Jahre gleichbleibende Erträge sind für eine Produktion qualitativ hochwertiger Weine notwendig und spielen daher eine Schlüsselrolle in der Rebenzüchtung. Der Fokus dieser Studie liegt daher auf Ertragsmerkmalen wie der Beerengröße, Anzahl der Beeren pro Traube und Menge der Trauben pro Weinstock. Die verwandten Merkmale Traubenarchitektur und das Verhältnis von generativem und vegetativem Wachstum wurden zusätzlich bearbeitet. Die Beurteilung von Ertragsmerkmalen auf Einzelstockniveau ist aufgrund der genotypischen Varianz und der Vielfältigkeit des betrachteten Merkmals komplex und zeitintensiv. Als erster Schritt in Richtung Hochdurchsatz (HT) Phänotypisierung von Ertragsmerkmalen wurden zwei voll automatische Bildinterpretationsverfahren für die Anwendung im Labor entwickelt. Das Cluster Analysis Tool (CAT) ermöglicht die bildgestützte Erfassung der Traubenlänge, -breite und -kompaktheit, sowie der Beerengröße. Informationen über Anzahl, Größe (Länge, Breite) und das Volumen der einzelnen Beeren liefert das Berry Analysis Tool (BAT). Beide Programme ermöglichen eine gleichzeitige Erhebung mehrerer, präziser phänotypischer Merkmale und sind dabei schnell, benutzerfreundlich und kostengünstig. Die Möglichkeit, den Vorder- und Hintergrund in einem Freilandbild zu unterscheiden, ist besonders in einem frühen Entwicklungsstadium der Rebe aufgrund der fehlenden Laubwand schwierig. Eine Möglichkeit, die beiden Ebenen in der Bildanalyse zu trennen, ist daher unerlässlich. Es wurde eine berührungsfreie, schnelle sowie objektive Methode zur Bestimmung des Winterschnittholzgewichts, welches das vegetative Wachstum der Rebe beschreibt, entwickelt. In einem innovativen Ansatz wurde unter Kombination von Tiefenkarten und Bildsegmentierung die sichtbare Winterholzfläche im Bild bestimmt. Im Zuge dieser Arbeit wurde die erste HT Phänotypisierungspipeline für die Rebenzüchtung aufgebaut. Sie umfasst die automatisierte Bildaufnahme im Freiland unter Einsatz des PHENObots, das Datenmanagement mit Datenanalyse sowie die Interpretation des erhaltenen phänotypischen Datensatzes. Die Basis des PHENObots ist ein automatisiert gesteuertes Raupenfahrzeug. Des Weiteren umfasst er ein Multi-Kamera- System, ein RTK-GPS-System und einen Computer zur Datenspeicherung. Eine eigens entwickelte Software verbindet die Bilddaten mit der Standortreferenz. Diese Referenz wird anschließend für das Datenmanagement in einer Datenbank verwendet. Um die Funktionalität der Phänotypisierungspipeline zu demonstrieren, wurden die Merkmale Beerengröße und -farbe im Rebsortiment des Geilweilerhofes unter Verwendung des Berries In Vineyard (BIVcolor) Programms erfasst. Im Durschnitt werden 20 Sekunden pro Weinstock für die Bildaufnahme im Feld benötigt, gefolgt von der Extraktion der Merkmale mittels automatischer, objektiver und präziser Bildauswertung. Im Zuge dieses Versuches konnten mit dem PHENObot 2700 Weinstöcke in 12 Stunden erfasst werden, gefolgt von einer automatischen Bestimmung der Merkmale Beerengröße und -farbe aus den Bildern. Damit konnte die grundsätzliche Machbarkeit bewiesen werden. Diese Pilotpipeline bietet nun die Möglichkeit zur Entwicklung weiterer innovativer Programme zur Erhebung neuer Merkmale sowie die Integration zusätzlicher Sensoren auf dem PHENObot.Grapevine is grown on about 1% of the German agricultural area requiring one third of all fungicides sprayed due to pathogens being introduced within the 19th century. In spite of this requirement for viticulture a reduction is necessary to improve sustainability. This objective can be achieved by growing fungus resistant grapevine cultivars. The development of new cultivars, however, is very time-consuming, taking 20 to 25 years. In recent years the breeding process could be increased considerably by using marker assisted selection (MAS). Further improvements of MAS applications in grapevine breeding will come along with developing of faster and more cost efficient high-throughput (HT) genotyping methods.Complementary to genotyping techniques the quality, objectivity and precision of current phenotyping methods is limited and HT phenotyping methods need to be developed to further increase the efficiency of grapevine breeding through sensor assisted selection. Many different types of sensors technologies are available ranging from visible light sensors (Red Green Blue (RGB) cameras), multispectral, hyperspectral, thermal, and fluorescence cameras to three dimensional (3D) camera and laser scan approaches. Phenotyping can either be done under controlled environments (growth chamber, greenhouse) or can take place in the field, with a decreasing level of standardization. Except for young seedlings, grapevine as a perennial plant needs ultimately to be screened in the field. From a methodological point of view a variety of challenges need to be considered like the variable light conditions, the similarity of fore- and background, and in the canopy hidden traits.The assessment of phenotypic data in grapevine breeding is traditionally done directly in the field by visual estimations. In general the BBCH scale is used to acquire and classify the stages of annual plant development or OIV descriptors are applied to assess the phenotypes into classes. Phenotyping is strongly limited by time, costs and the subjectivity of records. Therefore, only a comparably small set of genotypes is evaluated for certain traits within the breeding process. Due to that limitation, automation, precision and objectivity of phenotypic data evaluation is crucial in order to (1) reduce the existing phenotyping bottleneck, (2) increase the efficiency of grapevine breeding, (3) assist further genetic studies and (4) ensure improved vineyard management. In this theses emphasis was put on the following aspects: Balanced and stable yields are important to ensure a high quality wine production playing a key role in grapevine breeding. Therefore, the main focus of this study is on phenotyping different parameters of yield such as berry size, number of berries per cluster, and number of clusters per vine. Additionally, related traits like cluster architecture and vine balance (relation between vegetative and generative growth) were considered. Quantifying yield parameters on a single vine level is challenging. Complex shapes and slight variations between genotypes make it difficult and very time-consuming.As a first step towards HT phenotyping of yield parameters two fully automatic image interpretation tools have been developed for an application under controlled laboratory conditions to assess individual yield parameters. Using the Cluster Analysis Tool (CAT) four important phenotypic traits can be detected in one image: Cluster length, cluster width, berry size and cluster compactness. The utilization of the Berry Analysis Tool (BAT) provides information on number, size (length and width), and volume of grapevine berries. Both tools offer a fast, user-friendly and cheap procedure to provide several precise phenotypic features of berries and clusters at once with dimensional units in a shorter period of time compared to manual measurements.The similarity of fore- and background in an image captured under field conditions is especially difficult and crucial for image analysis at an early grapevine developmental stage due to the missing canopy. To detect the dormant pruning wood weight, partly determining vine balance, a fast and non-invasive tool for objective data acquisition in the field was developed. In an innovative approach it combines depth map calculation and image segmentation to subtract the background of the vine obtaining the pruning area visible in the image. For the implementation of HT field phenotyping in grapevine breeding a phenotyping pipeline has been set up. It ranges from the automated image acquisition directly in the field using the PHENObot, to data management, data analysis and the interpretation of obtained phenotypic data for grapevine breeding aims. The PHENObot consists of an automated guided tracked vehicle system, a calibrated multi camera system, a Real-Time-Kinematic GPS system and a computer for image data handling. Particularly developed software was applied in order to acquire geo referenced images directly in the vineyard. The geo-reference is afterwards used for the post-processing data management in a database. As phenotypic traits to be analysed within the phenotyping pipeline the detection of berries and the determination of the berry size and colour were considered. The highthroughput phenotyping pipeline was tested in the grapevine repository at Geilweilerhof to extract the characteristics of berry size and berry colour using the Berries In Vineyards (BIVcolor) tool. Image data acquisition took about 20 seconds per vine, which afterwards was followed by the automatic image analysis to extract objective and precise phenotypic data. In was possible to capture images of 2700 vines within 12 hours using the PHENObot and subsequently automatic analysis of the images and extracting berry size and berry colour. With this analysis proof of principle was demonstrated. The pilot pipeline providesthe basis for further development of additional evaluation modules as well as the integration of other sensors

Opportunities and limitations of crop phenotyping in southern european countries

ReviewThe Mediterranean climate is characterized by hot dry summers and frequent droughts. Mediterranean crops are frequently subjected to high evapotranspiration demands, soil water deficits, high temperatures, and photo-oxidative stress. These conditions will become more severe due to global warming which poses major challenges to the sustainability of the agricultural sector in Mediterranean countries. Selection of crop varieties adapted to future climatic conditions and more tolerant to extreme climatic events is urgently required. Plant phenotyping is a crucial approach to address these challenges. High-throughput plant phenotyping (HTPP) helps to monitor the performance of improved genotypes and is one of the most effective strategies to improve the sustainability of agricultural production. In spite of the remarkable progress in basic knowledge and technology of plant phenotyping, there are still several practical, financial, and political constraints to implement HTPP approaches in field and controlled conditions across the Mediterranean. The European panorama of phenotyping is heterogeneous and integration of phenotyping data across different scales and translation of “phytotron research” to the field, and from model species to crops, remain major challenges. Moreover, solutions specifically tailored to Mediterranean agriculture (e.g., crops and environmental stresses) are in high demand, as the region is vulnerable to climate change and to desertification processes. The specific phenotyping requirements of Mediterranean crops have not yet been fully identified. The high cost of HTPP infrastructures is a major limiting factor, though the limited availability of skilled personnel may also impair its implementation in Mediterranean countries. We propose that the lack of suitable phenotyping infrastructures is hindering the development of new Mediterranean agricultural varieties and will negatively affect future competitiveness of the agricultural sector. We provide an overview of the heterogeneous panorama of phenotyping within Mediterranean countries, describing the state of the art of agricultural production, breeding initiatives, and phenotyping capabilities in five countries: Italy, Greece, Portugal, Spain, and Turkey. We characterize some of the main impediments for development of plant phenotyping in those countries and identify strategies to overcome barriers and maximize the benefits of phenotyping and modeling approaches to Mediterranean agriculture and related sustainabilityinfo:eu-repo/semantics/publishedVersio

Initial steps for high-throughput phenotyping in vineyards

The evaluation of phenotypic characters of grapevines is required directly in vineyards and is strongly limited by time, costs and the subjectivity of person in charge. Sensor-based techniques are prerequisite in order to allow non-invasive phenotyping of individual plant traits, to increase the quantity of object records and to reduce error variation. Thus, a Prototype-Image-Acquisition-System (PIAS) was developed for semi-automated capture of geo-referenced images in an experimental vineyard. Different strategies were tested for image interpretation using MATLAB®. The interpretation of images from the vineyard with real background is more practice-oriented but requires the calculation of depth maps. Different image analysis tools were verified in order to enable contactless and non-invasive detection of bud burst and quantification of shoots at an early developmental stage (BBCH 10) and enable fast and accurate determination of the grapevine berry size at BBCH 89. Depending on the time of image acquisition at BBCH 10 up to 94 % of green shoots were visible in images. The mean berry size (BBCH 89) was recorded non-invasively with a precision of 1 mm.

Constraint-based automated reconstruction of grape bunches from 3D range data for high-throughput phenotyping

With increasing global population, the resources for agriculture required to feed the growing number of people are becoming scarce. Estimates expect that by 2050, 60 % more food will be necessary. Nowadays, 70 % of fresh water is used by agriculture and experts see no potential for new land to use for crop plants. This means that existing land has to be used efficiently in a sustainable way. To support this, plant breeders aim at the improvement of yield, quality, disease-resistance, and other important characteristics of the crops. Reports show that grapevine cultivation uses more than three times of the amount of fungicides than the cultivation of fruit trees or vegetables. This is caused by grapevine being prone to various fungal diseases and pests that quickly spread over fields. A loose grape bunch architecture is one of the most important physical barriers that make the establishment of a fungal infection less likely. The grape bunch architecture is mostly defined by the inner stem skeleton. The phenotyping of grape bunches refers to the measurement of the phenotypes, i.e., the observable traits of a plant, like the diameter of berries or the lengths of stems. Because of their perishable nature, grape bunches have to be processed in a relatively short time. On the other hand, genetic analyses require data from a large number of them. Manual phenotyping is error-prone and highly labor- and time-intensive, yielding the need for automated, high-throughput methods. The objective of this thesis is to develop a completely automated pipeline that gets as input a 3D pointcloud showing a grape bunch and computes a 3D reconstruction of the complete grape bunch, including the inner stem skeleton. The result is a 3D estimation of the grape bunch that represents not only dimensions (e.g. berry diameters) or statistics (e.g. the number of berries), but the geometry and topology as well. All architectural (i.e., geometrical and topological) traits can be derived from this complete 3D reconstruction. We aim at high-throughput phenotyping by automatizing all steps and removing any requirement for interaction with the user, while still providing an interface for a detailed visualization and possible adjustments of the parameters. There are several challenges to this task: ripe grape bunches are subject to a high amount of self-occlusion, rendering a direct reconstruction of the stem skeleton impossible. The stem skeleton structure is complex, thus, the manual creation of training data is hard. We aim at a cross-cultivation approach and there is high variability between cultivars and even between grape bunches of the same cultivar. Thus, we cannot rely on statistical distributions for single plant organ dimensions. We employ geometrical and topological constraints to meet the challenge of cross-cultivar optimization and foster efficient sampling of infinitely large hypotheses spaces, resulting in Pearson correlation coefficients between 0.7 and 0.9 for established traits traditionally used by breeders. The active working time is reduced by a factor of 12. We evaluate the pipeline for the application on scans taken in a lab environment and in the field

A Next-generation Marker Genotyping Platform (AmpSeq) in Heterozygous Crops: A Case Study for Marker-assisted Selection in Grapevine

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates traitassociated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads ( ⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had o10% missing data, and read depth had a median of 220–244 × . Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, lowcost and easily automated analysis

Potential phenotyping methodologies to assess inter- and intravarietal variability and to select grapevine genotypes tolerant to abiotic stress

ReviewPlant phenotyping is an emerging science that combines multiple methodologies and protocols to measure plant traits (e.g., growth, morphology, architecture, function, and composition) at multiple scales of organization. Manual phenotyping remains as a major bottleneck to the advance of plant and crop breeding. Such constraint fostered the development of high throughput plant phenotyping (HTPP), which is largely based on imaging approaches and automatized data retrieval and processing. Field phenotyping still poses major challenges and the progress of HTPP for field conditions can be relevant to support selection and breeding of grapevine. The aim of this review is to discuss potential and current methods to improve field phenotyping of grapevine to support characterization of inter- and intravarietal diversity. Vitis vinifera has a large genetic diversity that needs characterization, and the availability of methods to support selection of plant material (polyclonal or clonal) able to withstand abiotic stress is paramount. Besides being time consuming, complex and expensive, field experiments are also affected by heterogeneous and uncontrolled climate and soil conditions, mostly due to the large areas of the trials and to the high number of traits to be observed in a number of individuals ranging from hundreds to thousands. Therefore, adequate field experimental design and data gathering methodologies are crucial to obtain reliable data. Some of the major challenges posed to grapevine selection programs for tolerance to water and heat stress are described herein. Useful traits for selection and related field phenotyping methodologies are described and their adequacy for large scale screening is discussedinfo:eu-repo/semantics/publishedVersio

Deep Learning for Plant Stress Phenotyping: Trends and Future Perspectives

Deep learning (DL), a subset of machine learning approaches, has emerged as a versatile tool to assimilate large amounts of heterogeneous data and provide reliable predictions of complex and uncertain phenomena. These tools are increasingly being used by the plant science community to make sense of the large datasets now regularly collected via high-throughput phenotyping and genotyping. We review recent work where DL principles have been utilized for digital image–based plant stress phenotyping. We provide a comparative assessment of DL tools against other existing techniques, with respect to decision accuracy, data size requirement, and applicability in various scenarios. Finally, we outline several avenues of research leveraging current and future DL tools in plant science