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 sequestrationLow managementReduce erosion Grow on marginal lands

Low reliance on N fertilizer (legume)High yield (6 t/ha)Drought resistant (deep root system)Amenable to genetic improvement

Alfalfa

Harvest fractionation of alfalfa

Aerial part

StemsEthanol production

Leaves

Feed protein co-product

High cellulose content (ethanol)

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 contentHigh stem saccharification

• Screening test using commercial enzymes

Value-added co-products

High throughput screening

Accellerase 1500 (Genencor)Cellulase and xylanase activity (XC and XY additives)

Pectinex 3XL (Sigma) added for alfalfaPectinase, cellulase et hemicellulase

Enzymatic cocktail for stem degradation

Prediction of physicochemical parameters Minimal sample preparation High throughput screening

Near-infrared reflectance spectroscopy

Stem degradability = Enzyme-released glucoseStem degradability = Enzyme-released glucose

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

Enzyme-released glucose (NIRS)

0

20

40

60

80

100

120

140

160

180

80 90 100 110 120 130 140 150 160 170

Glucose enzymatique prédit (g kg-1 parois)

Fréq

uenc

e

D+D-

Duceppe, Bertrand et al. (2010)

Fre

quen

cy

Enzyme-released glucose

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

Heritability =0.26

Heritability =0.39

En

zym

e-r

ele

ase

d g

luco

se (

mg

g-1 D

W)

Number of cycles of recurrent selection

• 25% increase in stem degradability• No chemical pre-treatment• No GMO

positive (D+)

negative (D-)

Field validation at three sites with contrasting pedo-climatic conditions in Québec

b ba

Probing the genome of contrasted genetic materialE

nzy

me

-re

lea

sed

glu

cose

(m

g g

-1 D

W)

Number of cycles of recurrent selection

+

-

Marker-Assisted Selection (MAS) for higher CW Marker-Assisted Selection (MAS) for higher CW degradabilitydegradability

• 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 amplificationRandom genome amplification

Sequence-related amplified polymorphisms (SRAP)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 analysesPolymorphism analyses

PopulationsPopulations Biomass type : Orca Winter-hardy type: 54V54

PoolsPools Genotypes with high (D+) or low (D-) stem degradability Pool of DNA of 50 for each population

PCR approachPCR approach 42 SRAP primer pairs

Initial screening

-2 0 +2Me4-R14

MF9-R7

-2 0 +2M

54V54

DNA pool (50 genotypes) of initial cultivars(0), and Cycle 2 positive (2+) and negative (2-)Two genetic backgrounds: 54V54 and Orca

-2 0 +2F16-R7

MF16-em4

-2 0 +2M

Orca

• Polymorphic bands either increase or decrease in intensity• Response could vary according to genetic background

Me2-R10

-2 0 +2Orca

-2 0 +254V54

M M

Polymorphism Me2-R10 positively associated with increased stem degradability in both backgrounds

54V54 Cycle 2-

54V54 Cycle 2+

N= 2

N= 10

SRAP primers combination: Me2-R10

-2 0 +2M

-2 0 +2M

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

Josée Bourassa Plant biochemistry

Sandra Delaney Plant biochemistry

Jean Cloutier Molecular genetics

Josée Michaud Molecular physiology

Marie-Claude Pépin Plant breeding

Annick Bertrand Plant biochemistry

Annie Claessens Plant breeding

Yves Castonguay Molecular physiology

Patrice Audy Molecular biology

Research teamResearch teamScientistsScientists

Post-doctorate

Solen RocherSolen Rocher Molecular genetics

Research assistants

© 2012AAFC

• Large variabilité génétique• Relation négative entre la quantité de lignine

et la quantité de glucose libéré (Jung et al 1997)

Influence de la lignine

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 2- dans 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.

(20 for each group) (110 for each group)

Parents Progenies

Stem degradability is genetically inherited

Heritability =0.39 for 54V54

Heritability =0.26 for Orca

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.

Context

Recherche de polymorphismes de l’ADN génomique associés à la dégradabilité

SRAP (Sequence-related amplified polymorphisms)

35°C

50°C

Stem degradability is genetically inherited

Heritability =0.26

Heritability =0.39

En

zym

e-r

ele

ase

d g

luco

se (

mg

g-1 D

W)

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

Stem degradability is genetically inherited

Heritability =0.26

Heritability =0.39

En

zym

e-r

ele

ase

d g

luco

se (

mg

g-1 D

W)

Number of cycles of recurrent selection

• 25% increase in stem degradability• No chemical pre-treatment• No GMO