Sep 19, 2024
Version 2
FruitRescue! - Apple phenotyping V.2
This protocol is a draft, published without a DOI.
- Mathieu Brisson1,
- Amandine Cornille2
- 1GQE Le Moulon, INRAE: Gif-sur-Yvette, FR;
- 2GQE Le Moulon, CNRS: Gif-sur-Yvette, FR
Protocol Citation: Mathieu Brisson, Amandine Cornille 2024. FruitRescue! - Apple phenotyping. protocols.io https://protocols.io/view/fruitrescue-apple-phenotyping-dk684zhwVersion created by Mathieu Brisson
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: July 19, 2024
Last Modified: September 19, 2024
Protocol Integer ID: 107456
Keywords: fruit tree, fitness, dualex, phenology, fruitrescue, genomic offset, phenotyping, apple
Funders Acknowledgement:
BNP Climate & Biodiversity Initiative
Disclaimer
This draft might change in the future, please notify us for any addition or modification you would like to share.
Abstract
The FruitRescue project aims to create a pipeline to predict fruit trees' genomic offset. To confirm the pipeline predictions, tree fitness parameters are measured in crop and wild tree orchards along a longitudinal gradient. Fitness is assessed through measurements of trunk diameter and the Nitrogen Balance Index (NBI), as well as monitoring phenological parameters such as bud break, flowering time, fruit set, and crop load.
This protocol is meant for Apple tree phenotyping.
Image Attribution
Pictures credited from INRAE come from Frédérique Didelot, UE Horti, INRAE, Beaucouzé France.
Pictures credited from GQE - Le Moulon come from Amandine Hansart and Mathieu Brisson, GQE, INRAE, Gif-sur-Yvette, France.
Guidelines
Orchard Management
In each orchard, standard cultural management practices should be followed as typically implemented on the site. One exception is to be noted : to prevent influencing bitter pit, calcium spraying is not applied.
Each year, a summary of these treatments will be sent to Mathieu Brisson and Amandine Cornille.
During winter a pruning might be required between November and December.
In June, thinning by hand might be required. A maximum of 2 fruit per cluster should remain.
Data management
Throughout the years, you will collect a large amount of data. To ensure good quality and readability of the data when opening spreadsheets on your computer, it is crucial to be vigilant.
Here are some important guidelines concerning the data export step:
- File Type: Always check the file type and favor .tsv or .csv for single sheet documents.
- Date Formatting: Format the sheet's cells as text for dates in ISO 8601 (YYYY-MM-DD) format to facilitate reading across multiple computers and software.
- Raw Data Backup: Always make a copy of the raw data file before opening it.
- Cell and Decimal Separators: Check the cell and decimal separators before opening the file. For example, Dualex ".csv" output files contain data separated by semicolons, and opening the file carelessly could lead to the removal of the decimal separator.
- Data Integrity: It is better to reference the original file in another sheet rather than writing directly inside it.
- Data Structure: It is easier to understand the data when it is represented as a list rather than a map. Fill the given template with the data you have. If you have trouble filling it properly, leave it blank and contact the team who can provide you help.
After the data acquisition and eventual formatting, you will need to send it by mail to Mathieu Brisson, cc Amandine Cornille.
Materials
Marking Spray
Caliper
Equipment
S_Cal EVO Smart Caliper
NAME
Digital Caliper
TYPE
Sylvac
BRAND
810-1506
SKU
LINK
Max scale : 150mm, IP67, Bluetooth
SPECIFICATIONS
Dualex clamps
The optical chlorophyll meters used are the Dualex clamps (Cerovic. et al. 2012 ;
https://metos.at/en/dualex/). For use, see: Youtube - Le Dualex - IFVSudOuest
Safety warnings
There are no anticipated warnings or risks associated with this protocol.
Ethics statement
This research does not involve the use of animals.
Before start
Emails concerning the progress of the project should be sent to Mathieu Brisson cc Amandine Cornille.
Mathieu Brisson
mathieu.brisson@inrae.fr
+33 (0) 6 41 55 24 32
-
Amandine Cornille
amandine.cornille@nyu.edu
Trunk diameter
Trunk diameter
The trunk diameter is an indicator of the tree vigor (Waring 1987). Genetic information as well as environmental cues including abiotic and biotic stresses can modify the source-sink relationships and therefore modify the trunk growth during the year. Tree adaptation to its location can thus be assessed through this measurement.
For grafted trees, a white mark is placed 50cm above the grafting point with marking spray. For ungrafted trees, the mark is placed 50cm from the ground.
After cold hardening, during winter, and 1 month after flowering during summer using a digital caliper, measure the trunk diameter on the uppermost part of the white mark : make a first measurement then a second at 90°. The two measurements will then be averaged.
Measurement of the trunk diameter.
(a) measure of the diameter above the grafting point (grafted trees might have two marks).
(b) We measure the trunk at a given angle (fingers acting as the caliper) then we measure 90° from the first measurement (c).
Note
Using bluetooth and the digital caliper you can use the Sylvac App on your phone or tablet (in the Google Store or in the Apple Store).
You can thus easily use your favorite method to export the results in a .csv sheet from the Sylvac app or to directly write it in a spreadsheet software or in the FieldBook app.
If you choose to export the result from the Sylvac App, be sure to keep an ordered record of the trees you measured. To speed up the data formatting you can then either use the OFFSET functions of your spreadsheet software or use your favorite scripting language to format the data (we can provide you with a R script on demand).
Dualex
Dualex
1 month after the end flowering, in general from the end of May to the end of July. The Dualex measures Chlorophyll and Flavonoid content and enables the calculation of the Nitrogen Balance Index (NBI). These elements are used to assess the tree photosynthetic activity and nitrogen nutrition (Cerovic et al. 2012).
From the base to the top of the tree, the leaves are randomly chosen among those most exposed to light. The dualex measurements are done on the upper side of the leaves. 10 different leaves are measured. When trees don't have enough leaves, take notes of the trees with not enough leaves.
There are some points of vigilence when taking measurements with the Dualex :
- ID your Dualex if you use more than one.
- Write down Line and Number of each tree associated with the group given by the Dualex (and eventually Dualex ID).
- Write down the reason of a missing measurement (not enough leaves, tree death...)
- Always look at the Dualex screen after each measurements to check on the integrity of the data (no star (*) should appear on the screen).
Note
Do not open a Dualex ".csv" file without having a copy : opening it without the right settings can remove the decimal separator of the data. Do not attempt to modify the files : use a proper script or macro to summarize the results and use a secondary file to describe the Date, Group, Dualex ID, tree position and tree ID.
Phenology
Phenology
Bud break / Bud burst
After cold hardening, buds become dormant. Entering in endodormancy, buds are in a stage of physiological inhibition of bursting (Lang, 1987). Only heat accumulation can lift this stage of growth inhibition, even in favorable conditions, switching the buds to their ecodormant stage. During the ecodormancy, buds are physiologically capable of sensing their environment but the bud break is repressed until conditions are favorable (Fadon et al. 2020). The lifting of the ecodormancy encompassing the bud swelling and burst stage is therefore a fitness trait or an indicator of local adaptation (Anderson et al. 2012)
For apple trees, the burst usually occurs in february. Measure trees at stage 07/53 of the BBCH scale corresponding to stage of bud burst (Bagglioni B/C stages).
Head twice a week to the orchard and record the stage of all trees.
Flowering and fruit set
Flowering is a critical step for reproduction success. Its timing and its intensity has long term effect on multiple life history trait (Pallas et al. 2018). Genetic, physiological and environmental factors are thus controlling this complex set of traits that can have a long term effect on the survival and reproduction of trees . Flowering Time intertwined in this complex set of characters is therefore a critical indicator of tree fitness in regard of climate change (Anderson et al. 2012).
Flowering Time
From march to may, apple trees start flowering. Measure trees at stage 59 to 61 of the BBCH scale (Bagglioni E2/F1) when 10% of the flower have bloomed.
Head twice a week to the orchard and record the stage of all trees.
Flowering intensity
Flowering intensity or floribundity will be recorded as the number of bloomed flowers over the maximum number of possible flowers (%). The maximum number of possible flowers is set when all the buds have flowered. The trees are graded when they reach 50% of Flowering and it can be then re-evaluated at 100%. The most important thing is not to look at the flower number but to see if the flower distribution on the tree is according to the number of buds.
1- 0%
3- 25%
5- 50%
7- 75%
9- 100%
Examples:
- A young apple tree sprouts 4 new leaves. Each sprout then produces a flower; the intensity would be 100%.
- Another tree also grows 4 sprouts but only half of the tree's top flowers; the intensity will be 2 or 12,5%.
- If the top of this same tree flowers entirely, then the intensity is 5 or 50%.
- A very small tree with a single sprout that sees its top part entirely flowering is 9 or 100%.
Further information on Flowering Intensity :
A. Small tree intensively blossoming : FI=9
B. This tree looks like it is intensively blossoming but a cluster in the foreground bears no flowers. This position has to be counted. FI=7
C. At first sight this tree doesn't seem to be intensively flowering but almost all clusters are bearing flowers : FI=8
The fruit set rate
When pollination is successful, flowers will develop into swollen, immature and very small green fruits but when unsuccessful, the flowers that did not receive enough compatible pollen will not develop into a fruit and look like small green stars.
The number of fruits are counted 2 to 3 weeks after flowering (=mean fruit number by clusters after abscission). This fruit set is the quantitative measurement of the proportion of flowers developing into fruits.
This will allow to calculate the percentage of fruit set. This is the percentage of flowers that become fruit.
% fruit set per tree = (# fruit / # Flowering buds)* 100
Crop Load
Environmental and Internal cues can also lead to fruit drop after pollination, diminishing the total fruit set in the weeks following pollination and establishing the tree's crop load.
Estimation of the crop load is done after the June fruit drop but before hand thinning. It is expressed in percent of present fruits compared to the maximum possible number of fruits. The tree express its maximum possible number of fruits when bearing fruits on each buds. We use the following scale :
1 = 0% of fruit set meaning 0 fruits on the majority of the tree's cluster
2 = 1 fruit per cluster for most of them
3 = 25% of fruit set meaning most cluster bear 2 fruits
4 = 40% mix of 2-3 fruits per cluster but a majority of 2 fruits per cluster
5 = 50% of fruit set
6 = 60% 3 and 4 fruits per cluster but a majority of 4 fruits per cluster.
7 = 75% of fruit set 3, 4 et 5 fruits per cluster
8 = 4-5 fruits per cluster depending on the tree
9 = 100% of fruit set, all the fruits have developed on all cluster
Fruits
Fruits
Fruits play a crucial role in the maturation, protection and the dispersal of the seeds and thus the effective reproduction of the mother plant. Fruit pigmentation can be a protection against stresses (Espley and Jaakola 2023) or a signal perceived by animals feeding on them in order to disseminate the seeds (Steyn, 2008). Furthermore the fruit is also a major sink thus reflecting resources allocation and overall tree fitness.
Fruit sample weight
When apples are ripen 15 fruits are sampled. The fruits sample weight is measured with a weighting scale. The weight is written in grams.
Fruit sample size variability among the sample.
0- The sample is more homogeneous (more than 3-4 fruits seem to have a similar size)
1- The sample is more heterogeneous
Fruit background color
The fruit background color is estimated by using a scale from 0 to 2 on average by tree.
Scale for the measure of background color.
Relative surface of red color cover on the background
Estimation of the % of surface red color above the background color, given by a scale from 0 to 5, mean by tree.
Scale for the measure of the relative surface of red color cover.Scale for the measure of the relative surface of red color cover.
Heterogeneity of the red color cover
Grade the color diversity intensity among the fruit sample either homogeneous (0) or heterogeneous (1).
Protocol references
The protocol has been written based on the protocol from the RefPop project:
Jung, Michaela, Morgane Roth, Maria José Aranzana, Annemarie Auwerkerken, Marco Bink, Caroline Denancé, Christian Dujak, et al. 2020. “The Apple REFPOP—a Reference Population for Genomics-Assisted Breeding in Apple.” Horticulture Research. Oxford University Press (OUP). doi:10.1038/s41438-020-00408-8.
Anderson, Jill T., David W. Inouye, Amy M. McKinney, Robert I. Colautti, and Tom Mitchell-Olds. 2012. “Phenotypic Plasticity and Adaptive Evolution Contribute to Advancing Flowering Phenology in Response to Climate Change.” Proceedings of the Royal Society B: Biological Sciences. The Royal Society. doi:10.1098/rspb.2012.1051.
Cerovic, Zoran G., Guillaume Masdoumier, NaÏma Ben Ghozlen, and Gwendal Latouche. 2012. “A New Optical Leaf‐clip Meter for Simultaneous Non‐destructive Assessment of Leaf Chlorophyll and Epidermal Flavonoids.” Physiologia Plantarum. Wiley. doi:10.1111/j.1399-3054.2012.01639.x.
Espley, Richard V., and Laura Jaakola. 2023. “The Role of Environmental Stress in Fruit Pigmentation.” Plant, Cell & Environment. Wiley. doi:10.1111/pce.14684.
Fadón, Erica, Eduardo Fernandez, Helen Behn, and Eike Luedeling. 2020. “A Conceptual Framework for Winter Dormancy in Deciduous Trees.” Agronomy. MDPI AG. doi:10.3390/agronomy10020241.
Lang, Gregory A. 1987. “Dormancy: A New Universal Terminology.” HortScience. American Society for Horticultural Science. doi:10.21273/hortsci.22.5.817.
Pallas, Benoît, Sylvie Bluy, Jérôme Ngao, Sébastien Martinez, Anne Clément-Vidal, Jean-Jacques Kelner, and Evelyne Costes. 2018. “Growth and Carbon Balance Are Differently Regulated by Tree and Shoot Fruiting
Contexts: An Integrative Study on Apple Genotypes with Contrasted Bearing Patterns.” Edited by Joào Pereira. Tree Physiology. Oxford University Press (OUP). doi:10.1093/treephys/tpx166.
Steyn, W. J. 2008. “Prevalence and Functions of Anthocyanins in Fruits.” Anthocyanins. Springer New York. doi:10.1007/978-0-387-77335-3_4.
Thomson, James D. 2010. “Flowering Phenology, Fruiting Success and Progressive Deterioration of Pollination in an Early-Flowering Geophyte.” Philosophical Transactions of the Royal Society B: Biological Sciences. The Royal Society. doi:10.1098/rstb.2010.0115.
Waring, R. H. 1987. “Characteristics of Trees Predisposed to Die.” BioScience. Oxford University Press (OUP). doi:10.2307/1310667.