Download Recurrent divergent selection in alfalfa

Improved saccharification efficiency of alfalfa for
bioethanol production
Annick Bertrand, Yves Castonguay, Annie Claessens, Jean Cloutier
Agriculture and Agri-Food Canada, Québec City
Perennial forage crops
Perennial vs annual crops:
Require fewer inputs,
Increase carbon sequestration
Low management
Reduce erosion
Grow on marginal lands
Alfalfa
 Low reliance on N fertilizer (legume)
 High yield (6 t/ha)
 Drought resistant (deep root system)
 Amenable to genetic improvement
Harvest fractionation of alfalfa
Aerial part
Stems
Ethanol production
 High cellulose content
(ethanol)
Leaves
Feed protein co-product
 High protein content (30%)
 Co-products (pinitol,
flavonoids)
Bioethanol production from perennial alfalfa
To increase the competitivity of alfalfa as feedstock for
ethanol production, there is a need for the development
of new genetic resources with:
 High sugar content
 High stem saccharification
• Screening test using commercial enzymes
 Value-added co-products
High throughput screening
Enzymatic cocktail for stem degradation
 Accellerase 1500 (Genencor)
Cellulase and xylanase activity (XC and XY additives)
 Pectinex 3XL (Sigma) added for alfalfa
Pectinase, cellulase et hemicellulase
Near-infrared reflectance spectroscopy



Prediction of physicochemical parameters
Minimal sample preparation
High throughput screening
Stem degradability = Enzyme-released glucose
Enzyme-released glucose (NIRS)
180
160
Fréquence
Frequency
140
120
100
80
60
40
D-
D+
20
0
80
90
100
110
120
130
140
150
Glucose enzymatique prédit (gglucose
kg-1 parois)
Enzyme-released





160
170
Duceppe, Bertrand et al.
(2010)
Efficient high throughput screening
Rapidly characterizes CW degradability of 1200 genotypes
Large genetic diversity for stem degradability
Selection of 20 genotypes with high (D+) and 20 with low (D-) degradability
Crosses of selected genotypes
Intercrossing 20 D+ and 20 D- genotypes
• Three cycles of recurrent divergent selection in two genetic backgrounds
• Conventional breeding (no GMO)
Stem degradability is genetically inherited
Enzyme-released glucose (mg g-1 DW)
positive (D+)
negative (D-)
Heritability =0.26
Heritability =0.39
Number of cycles of recurrent selection
• 25% increase in stem degradability
• No chemical pre-treatment
• No GMO
Field validation at three sites with contrasting pedoclimatic conditions in Québec
b
a
b
Enzyme-released glucose (mg g-1 DW)
Probing the genome of contrasted genetic material
+
-
Number of cycles of recurrent selection
Marker-Assisted Selection (MAS) for higher CW
degradability
• Use of the unique genetic material generated by recurrent
divergent selection
• Identification of genomic regions affecting stem degradability
• Development of molecular markers to accelerate the
identification of highly degradable plants
Random genome amplification

Sequence-related amplified polymorphisms (SRAP)

PCR approach targeting coding sequences


Base on two primer pairs (17-18 nucleotides)
 One rich in GC content (exons)
 One rich in AATT content (introns)
Uncover numerous polymorphic regions over the entire
genome
Polymorphism analyses

Populations



Pools



Biomass type : Orca
Winter-hardy type: 54V54
Genotypes with high (D+) or low (D-) stem degradability
Pool of DNA of 50 for each population
PCR approach

42 SRAP primer pairs
Initial screening
DNA pool (50 genotypes) of initial cultivars(0), and Cycle 2 positive (2+) and negative (2-)
Two genetic backgrounds: 54V54 and Orca
54V54
Me4-R14
M
-2
0
+2
M
-2
0
Orca
F16-R7
F9-R7
+2
M
-2
0
+2
• Polymorphic bands either increase or decrease in intensity
• Response could vary according to genetic background
F16-em4
M
-2
0
+2
Polymorphism Me2-R10 positively associated with increased stem degradability in both backgrounds
Me2-R10
M
54V54
-2
0
+2
Orca
M
-2
0
+2
SRAP primers combination: Me2-R10
54V54 Cycle 2M -2
0
N= 2
+2
54V54 Cycle 2+
M
-2
0
+2
N= 10
Band intensity reflects the number of genotypes with polymorphisms in each population
Selection based on polymorphic markers
• DNA extraction in 250 genotypes of each population.
• Score of genotypes with six different polymorphic markers in
54V54 and Orca (presence or abscence)
• Selection of 25 genotypes with more than two polymorphisms
• Intercrossing selected genotypes
• Proceeded to a second cycle in order to increase the frequency of
the selected markers in new populations
Next steps
• Stem degradability assessment of progenies after 2 cycles of
MAS: to confirm the link between Markers and degradability
Impact of MAS on stem degradability
• Next generation sequencing (GBS): for genome-wide identification
of regions affecting stem degradability
Scientists
Annick Bertrand
Annie Claessens
Yves Castonguay
Patrice Audy
Research team
Plant biochemistry
Plant breeding
Molecular physiology
Molecular biology
Post-doctorate
Solen Rocher
Molecular genetics
Research assistants
Josée Bourassa
Sandra Delaney
Jean Cloutier
Josée Michaud
Marie-Claude Pépin
AAFC © 2012
Plant biochemistry
Plant biochemistry
Molecular genetics
Molecular physiology
Plant breeding
Influence de la lignine
•
•
Large variabilité génétique
Relation négative entre la quantité de lignine
et la quantité de glucose libéré (Jung et al 1997)
Sélection pour une plus grande dégradabilité de la
fibre basée sur les marqueurs moléculaires
NOI 2652
Validation de différences dans la
fréquence génotypique
• Score 45 génotypes des populations 2+ et 2dans chaque background
• 10 marqueurs validés
Screening des deux popualtions
• Extraction de l’ADN de 500 génotypes de
chaque population
• Score de 250 génotypes avec 5 marqueurs
dans 54V54 et 6 marqueurs dans Orca
• 25 génotypes sélectionnés pour les
croisements
Prochaines étapes
• Croisements polycross de 25 plants de Orca et
de 54V54 (en cours)
• Purification et clonage des séquences SRAP
polymorphiques (hiver 2014)
• Second cycle de sélection MAS dans les deux
backgrounds (Été-automne 2014).
• Croisements polycross du cycle 2 à l’hiver
2015.
Stem degradability is genetically inherited
Heritability =0.39 for 54V54
Heritability =0.26 for Orca
Parents
(20 for each group)
Progenies
(110 for each group)
Context
 Over 2 millions ha of agricultural land in
forage production in Québec
 Knowledge, machinery, the infrastructure to cultivate harvest
and store perennial forage crops
 Bioenergy represent new opportunities for farmers, processors,
and rural communities.
Recherche de polymorphismes de l’ADN génomique associés à la dégradabilité
SRAP (Sequence-related amplified polymorphisms)
35°C
50°C
Enzyme-released glucose (mg g-1 DW)
Stem degradability is genetically inherited
Heritability =0.26
Heritability =0.39
Number of cycles of recurrent selection
Recurrent divergent selection in alfalfa
•
•
•
•
Assessment of 1200 genotypes (no pre-treatment, highly lignified stem base)
Large genetic diversity for stem degradability in alfalfa
Selection of 20 genotypes with high (D+) and low (D-) degradability
Crosses of selected genotypes
Enzyme-released glucose (mg g-1 DW)
Stem degradability is genetically inherited
Heritability =0.26
Heritability =0.39
Number of cycles of recurrent selection
• 25% increase in stem degradability
• No chemical pre-treatment
• No GMO