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Molecular Breeding is a Powerful Approach to Accelerate Genetic Gain, the Final Target of Plant BreedingDr. Ivan Schuster PhD PAG XVIII

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• In a free concept, Plant Breeding is the art and the science of creating new genetic combinations and select the best combinations.

• For more than a century, the best combinations were selected based on phenotypic evaluation. Accurate phenotypic selection is challenging due Environmental effects

and GxE interaction. Statistical models can help increase accuracy in phenotypic selection by

isolating environmental effects and managing GxE interactions. But not all presuppositions of statistical models can be attended every time.

• Using DNA Molecular Markers can increase the accuracy of selection in Plant Breeding. Selection of genes or QTLs using molecular markers is not influenced by

the environment.

Molecular Markers in Plant Breeding

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• Regardless of the methods used, all plant breeding goals can be summarized into one main goal: Genetic Gain.

• Phenotypic and Molecular Breeding are complementary and can create lots of synergies in Plant Breeding.

• The key to successful plant breeding in the 21st century lies in the best possible integration between molecular breeding and classical breeding.

• Regardless of technologies used, “Plant Breeding is made in the Field”. • New technologies helps to:

• Have the better genotypes to be evaluated in he field.• Shorten the time to create new varieties/hybrids• Improve the quality of new genotypes• Accelerate Genetic Gain• Others…

• But the final decision continue to be made by field evaluation

Molecular Markers in Plant Breeding

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• Genome Wide Selection concept already have more 20 years• It is also being used in the industry for almost 10 years.

• But just Big Companies, with high level of investments, currently have well stablished Genomic Selection breeding approach in their breeding programs

• Current Target Genotyping by Sequence technologies and cost allow to mid company also use GWS in plant breeding

• In 2020-decade Genomic Selection will be more popular in Plant Breeding.

Genomic Selection

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Creating Variability(Discovery)

Selecting Better Recombinants

Testing

Basic of Plant Breeding

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Example on Corn

Basics of Plant Breeding

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• Intensive use of molecular markers in early generation• Lower heritability

• Less intense use of molecular markers in late generation• Higher heritability

Plant Breeding in XXI Century

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Ion Torrent AgriSeqTM

Genotyping System

Genotyping Chip

Basics:Template Prep with Emulsion PCR – emPCRDetection by ∆pH

Molecular Analysis – Targeted GBS

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Real Time Sequence detection by ∆pH

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Steps for Targeted GBS

1. DNA Targets AmplificationAmplify target regions of DNA samples. This amplification depends of primers number.

2. Prepare Amplicons For Barcode AdditionPartially digestion of amplicons. FUPA Enzyme.

3. Add Barcodes and AdaptersEach fragment generated in the library is ligated to the unique sequence (Barcode).

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Day 2

Sample Multiplexing

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Emulsion PCR - emPCR

Day 3

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Add Nucleotide – Wash – Read pHDay 3

Sequencing

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Genotyping quality parameters

Sequencing Quality Control

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Processing Output File

Day 4

Data Processing

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Workflow

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The Breeding Funel • Molecular Markers can be used to predict the performance of new hybrids before field evaluation.

• The first generation of Hybrid Selection is done in virtual environment, by computers. It makes possible to evaluate

millions of hybrids and select only the best ones to be evaluated in the field.

The Breeding Funnel with Molecular Breeding

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Using the Molecular Data for Breeding Improvement

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Example on Corn

Genomic Prediction

Basic of Plant Breeding

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AccuracyValidating Population

Predictive Model(GGV) –Discovery Population

Genotyping and Phenotyping

Training Population

Field testing of selected hybrids

Selection of Virtual Hybrids

Estimating GGV of Virtual Hybrids using the Prediction Model

Virtual CrossesGenotypingThousands of

Inbreed Lines

Genome Prediction Theory

Using Molecular data for GWS

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Model:y = Xb + Wm + e

y is the vector of phenotypic observation,b is the vector of fixed effect, to be calculated (the average of the phenotypic trait)m is the vector of aleatory effect of markers, to be calculated (effects of each marker in the model)e is the vector of residual effects, X and W are incidence matrix for b and m. Matrix of incidence W contain the values* -1, 0 and 1 based on marker genotypeMatrix of incidence X is a vector of ones, with the dimension of the number od hybrids.

*Other ways to code is use the values 2, 1 and 0 or 0-2p, 1-2p and 2-2p.

Prediction Model: RR-BLUP/GWS

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Marker Data – W matrix Phenotypic Data y matrix

Vector of 1sX matrix

Example of Data

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• The equation of mixed models to predict b and m via RR-BLUP is equivalent to:

y = Xb + Wm + e𝑋𝑋𝑋𝑋𝑋 𝑋𝑋𝑋𝑋𝑋

𝑋𝑋𝑋𝑋𝑋 𝑋𝑋′𝑋𝑋 + 𝐼𝐼1 − ℎ𝑎𝑎2

ℎ𝑎𝑎2/∑𝑖𝑖=1𝑛𝑛 2𝑝𝑝𝑖𝑖𝑞𝑞𝑖𝑖

�𝑏𝑏�𝑚𝑚

= 𝑋𝑋𝑋𝑋𝑋𝑋𝑋𝑋𝑋𝑋

h2 = Heritability, obtained from the datap and q = Allele1 and allele2 frequency in each marker

RR-BLUP/GWS to estimate Marker Value

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The genetic genomic value (GGV) of the individual j is given by:

𝐺𝐺𝐺𝐺𝑉𝑉 = �𝑋𝑋𝑗𝑗 = �𝑖𝑖

𝑤𝑤𝑖𝑖𝑗𝑗 �𝑚𝑚𝑖𝑖

The component wij is the element i of the line j from Matrix W, related to individual j, of the population to be predicted.mi is the effect of the marker i obtained from the specific Model.

Using the Model to Predict the Hybrids Performance

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�𝑚𝑚 =

Example of Estimated Marker Effects

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Marker Effects

X =

Marker Scores Predicted Hybrids

𝐺𝐺𝐺𝐺𝑉𝑉 = �𝑋𝑋𝑗𝑗 = �𝑖𝑖

𝑤𝑤𝑖𝑖𝑗𝑗 �𝑚𝑚𝑖𝑖

Hybrids YieldHybrid1 11.794Hybrid2 16.461Hybrid3 13.848Hybrid4 13.351Hybrid5 5.351Hybrid6 11.423Hybrid7 15.247Hybrid8 16.016Hybrid9 14.540Hybrid10 10.588Hybrid11 16.329Hybrid12 13.716Hybrid13 17.151Hybrid14 11.658Hybrid15 12.118Hybrid16 8.511Hybrid17 15.471Hybrid18 11.610Hybrid19 16.741Hybrid20 15.311Hybrid21 13.600Hybrid22 16.321Hybrid23 14.184Hybrid24 15.627Hybrid25 16.485Hybrid26 13.854Hybrid27 18.413

Predicting New (untested) Hybrids

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Prediction

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∆𝐺𝐺 =𝑖𝑖ℎ𝜎𝜎𝑎𝑎𝑛𝑛

• Using genomic predictions the size of breeding populations can be greatly increased, which can increase selection intensity (i)

• As molecular markers are unaffected by the environment, marker selection has high heritability (ℎ = ℎ2)

• Using molecular markers, it is possible to quantify and manage the variability (and genetic variance) of populations.

• Good hybrid predictions can reduce the number of years of field testing, reducing the time of each breeding generation.

Molecular Breeding and Genetic Gain

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• Plant breeding in XXI Century still have the main target of plant breeding: Genetic Gain• Plant Breeding in XXI Century still have the main basic stages: Create Variability –

Select – Test• The way of breeders is doing plant breeding changed, and continue to change: Variability can still be obtained by germplasm introduction and crossing And can also be obtained by Gene Editing, Transgenic approaches, etc.

Germplasm classification and Heterotic Group identification can be made in days, instead year, with high level of accuracy.

Selections can be accelerated using Genomic Predictions, and skip some field testing. Selection can be made in millions of hybrids, in a virtual environment, and

advance just the most promising hybrids for field testing. But testing still need to be done in the field.

Final Considerations

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• The Tool Box of Plant breeders in XXI Century have more, and more precise tools.

• The new tools can increase every single component of the Breeders Equation, and result in accelerated Genetic Gain.

• Plant Breeding is, since the beginning, a multidisciplinary activity. Plant Breeding in XXI Century included even more disciplines in Pant

Breeding, amplifying the multidisciplinary.• A Key point for the success in Plant Breeding in XXI Century is the interaction

and the synergy between all of the new disciplines with the traditional disciplines used for decades.

Final Considerations