Title

Improved saccharification efficiency of alfalfa for bioethanol production Annick Bertrand, Yves Castonguay, Annie Claessens, Jean Cloutier Agriculture and Agri-Food Canada, Qubec 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 Frquence Frequency 140 120 100 80 60 40 D-

D+ 20 0 80 90 100 110 120

130 140 150 160 Glucose enzymatique prdit (g kg parois) Enzyme-released glucose -1

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 170 Duceppe, Bertrand et al. (2010)

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 Qubec 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 +2 N= 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 Jose Bourassa Sandra Delaney Jean Cloutier Jose Michaud

Marie-Claude Ppin AAFC 2012 Plant biochemistry Plant biochemistry Molecular genetics Molecular physiology Plant breeding Influence de la lignine Large variabilit gntique

Relation ngative entre la quantit de lignine et la quantit de glucose libr (Jung et al 1997) Slection pour une plus grande dgradabilit de la fibre base sur les marqueurs molculaires NOI 2652 Validation de diffrences dans la frquence gnotypique Score 45 gnotypes des populations 2+ et 2dans chaque background 10 marqueurs valids Screening des deux popualtions Extraction de lADN de 500 gnotypes de

chaque population Score de 250 gnotypes avec 5 marqueurs dans 54V54 et 6 marqueurs dans Orca 25 gnotypes slectionns pour les croisements Prochaines tapes Croisements polycross de 25 plants de Orca et de 54V54 (en cours) Purification et clonage des squences SRAP polymorphiques (hiver 2014) Second cycle de slection MAS dans les deux backgrounds (t-automne 2014). Croisements polycross du cycle 2 lhiver 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 Qubec

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 lADN gnomique associs la dgradabilit SRAP (Sequence-related amplified polymorphisms) 35C 50C 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

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