Breeding alfalfa for cold tolerance in

Canada

Annick Bertrand, Annie Claessens, Solen Rocher

Agriculture and Agri-Food Canada, Québec City

© 2018 Agriculture and Agri-Food Canada

Background – Forages in Canada

• Perennial forages are grown on 29.2M ha in Canada

– Approx. 44% of total agricultural acreage; 60% of dairy diets; 80% of beef

diets

– Eastern Canada: 80% of Canadian milk; ~ 2 million hectares (excluding

unseeded pastures)

• Profitability of dairy and beef productions is directly linked to forage yield

(including persistence) and nutritive value

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Perennials overwintering: Ideal scenario

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Exposure to sublethal temperatures

Harsh winter conditions for alfalfa production in Canada

• Establishement of a field nursery

Agriculture and Agri-Food Canada

Classical breeding for winter survival

• Differential winter survival of genotypes

Field selection of plants that survive winter:

- Long, costly and unpredictable process (test winter)

• Hand-made crosses of selected genotypes

Test winter

© 2018

Improvement of freezing tolerance

Freezing-stress selection approach:

-Allows repeated cycles of recurrent selection more rapidly, under

controlled conditions;

-Scheme can be applied to various perennial species

-Development of unique populations;

-Improved genetic material can be used to uncover molecular markers:

identification of variations within the genome that are associated to the

response to selection.

Selection for freezing tolerance (TF)

1500 genotypes grown for four weeks under controlled conditions 16 h photoperiod, 21/17 oC day/night temperature (d/n)

Transfer at 2oC for two weeks

Transfer at -2oC for two weeks

Progressive decrease of temperature to approx. LT50 (Stress 1)

Two additional freezing stress applied Elimination of half of the plants after each stress

50 best performing genotypes intercrosssed to generate TF1 population

Process repeated each year within the new TF population created

4-wk regrowth

Bertrand et al. 2014.Methods in Molecular Biology 1166

Selection by freezing stress

Growth chambers

Differential survival

Walk-in freezers

Hand-made crosses

Agriculture and Agri-Food Canada

Agriculture and Agri-Food Canada

Improvement > 5oC

Red clover

Christie

C -10 -12 -14 -16 -18 -20

C-TF7

© 2018

Apica

Apica-TF7

-26 -24 -22 -16 C -18 -20 -28oC

Alfalfa

A-TF4 A-TF2 A-TF0

© 2018 AAFC

Field validation after a test winter

A-TF4 A-TF2 A-TF0

© 2018 AAFC

Field validation after a test winter

Since freezing tolerance is typically

linked with dormancy, we proceeded

with a simultaneous selection for

reduced dormancy

3000 genotypes grown for four weeks under controlled conditions 16 h photoperiod, 21/17 oC day/night temperature (d/n)

Transfer at 2oC for two weeks

Transfer at -2oC for two weeks

Progressive decrease of temperature to approx. LT50 (Stress 1)

Two additional freezing stress applied Elimination of half of the plants after each stress

50 best performing genotypes intercrosssed to generate TF1 population

Process repeated each year within the new RD-TF population created

Selection for reduced dormancy (RD) and freezing tolerance (TF)

4-wk regrowth under 12 h-photoperiod at 18/15 oC d/n temperature

Selection of the most rapidly growing genotypes

B

• Significant reduction of dormancy winthin two genetic backgrounds after only one cycle of

selection

• Validated under contrasting climatic conditions: Normandin (48.8oN, 72.5333oW; cold humid),

Swift Current (50.2oN, 107.7oW; cold semi-arid)

Field validation under contrasting climatic conditions:

B B

B B B

Eastern Canada: Normandin

Western Canada: Swift Current

Alfalfa

Cryoprotective sugars increase during fall hardening and are significantly higher

in selected populations.

Freezing tolerance and underlying mechanisms are improved by selection

• Freezing tolerance can be improved by recurrent selection.

• Recurrent selection affects marker frequency (SRAP) and candidate gene

expression (SRAP-cDNA).

• Marker Assisted Recurrent Selection could be used to selectively increase

frequency of favorable alleles, and accelerate breeding progress.

• High throughput genotyping is required to identify genes controlling a quantitative

traits like freezing tolerance.

Link between freezing tolerance and molecular changes

The GBS SNP-Calling Reference Optional Pipeline (GBS-SNP-CROP)

• Genotyping-by-Sequencing strategy

• Using the reference genome of a related species (M. truncatula)

Genome wide characterisation of DNA variations

Objectives

- Genome-wide SNP discovery using high-throughput genotyping of recurrently selected

populations of alfalfa

- Assess the effect of recurrent selection on allele frequencies and genetic diversity in two

genetic background (cv. Apica and Evolution)

- Identify shifts in SNP frequency between TF-0 and recurrently selected populations

- Track genome regions affected by recurrent selection in two genetic backgrounds (cultivars) Apica -TF0 Apica –TF5

SNP

discovery

Rocher et al. 2015. PLOS one

Apica

(100 TF0|100 TF5)

Evolution

(95 TF0|89 TF4)

623 152 903

good reads

579 556 416

good reads

12 703 SNP /

200 genotypes

11 201 SNP /

184 genotypes

DNA library

Sequencing

(Illumina HiSeq2500)

GBS-SNP-CROP

(call SNP) M. truncatula genome v4.0 as reference

Statistical analysis (R) Data filtering

Plant material

GBS-SNP-CROP (Parsed, trimmed and aligned reads)

RAD-seq (GBS) of recurrently selected populations workflow

4 libraries

384 genotypes

1 467123 598 reads

(312 to 410 M reads/library)

8577 SNP /

200 genotypes

7446 SNP /

183 genotypes

Effect of recurrent selection on genetic differentiation between

initial and recurrently selected populations

Apica Evolution

-> RS selected populations are genetically differentiated from initial cultivars on PCo2 axis

-> This genetic differentiation is slight (2% of total variation explained by Pco2 axis)

Agriculture and Agri-Food Canada © 2018 Statistical analysis – R packages « Radiator » and « vcfR »

Several SNP under selection pressure distributed along the genome

Number of SNP responding to selection is threshold-dependant

Same regions contain clusters of SNP responding to selection in both backgrounds

=> Statistics must be refined.

Agriculture and Agri-Food Canada © 2018 Statistical analysis – R packages « Radiator » and « vcfR »

Conclusions

• We developped efficient indoor methods of joint selection for

improved freezing tolerance and reduced dormancy.

• Populations recurently selected for superior freezing tolerance are

genetically differentiated from initial cultivar.

• Several SNP, distributed along the genome, are affected by

selection.

• Common regions responded to selection in both genetic

backgrounds.

Agriculture and Agri-Food Canada © 2018

Future steps

• Precisely identify molecular markers associated with freezing tolerance and fall dormancy

• Detect candidate genes linked with these markers, identify favorables alleles of these genes

• Accelerate breeding progress for these two traits using marker assisted selection to:

- increase frequency of favorable alleles

- dissociate both traits

Agriculture and Agri-Food Canada © 2018

Thanks to the team!

We would like to thank the Canadian beef cluster of Agriculture and Agri-Food Canada (AAFC)

and competitive grants of AAFC for financial support.

Agriculture and Agri-Food Canada © 2018

Thank you!