Genomic advancements for canola

Genomic advancements for canola

Elodie Gazave1, Erica E. Tassone2, Megan Wingerson3, James B. Davis3, John M. Dyer2, Matthew A. Jenks5, Jack Brown3 & Michael A. Gore1 Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA 2 Plant Physiology and Genetics Research Unit, U.S. Arid Land Agricultural Research Center, USDAARS, Maricopa, AZ, USA 3 Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID, USA 4 Keygene N.V., Wageningen, Netherlands 5 Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, USA 1

Genetically improve Brassica napus feedstocks to enhance: ďƒź Oil yield and quality stability in western U.S. production conditions and ďƒź Compatibility with HRJ fuel conversion processes (reduce oil processing costs and optimize conversion efficiency.)

Sequenced 882 Brassica napus accessions Identify genetic variants associated with our traits of interest (e.g. yield, oil composition, etc.)

Outline  Building the puzzle.  Develop a powerful data set.  Assess phenotypic variation.  Complete genotypic association studies.

Germplasm Base/Training Populations Source: USDA/Ames; Dutch Collection; UI Collection Spring V Winter

Winter Types 652 Lines G’house Seed & DNA

Spring Types 230 Lines G’house Seed & DNA

882 accessions sent to genotyping by sequencing (GBS): Generated 5 Terabytes of sequencing data

Our goal: Build 882 puzzles & find tiny variations that differentiate each puzzle (genetic variants, single nucleotide polymorphisms [SNPs])

Building the puzzle Brassica oleracea

Whole genome triplication

4.

6 YA M 20 M YA

Whole genome duplication

Arabidopsis thaliana

Brassica rapa

Building the puzzle Brassica oleracea

Whole genome triplication

Brassica napus 4.

6 YA M 20 M YA

Whole genome duplication

Polyploidy 7,500 YA

Arabidopsis thaliana

Brassica rapa

Building the puzzle Challenge 1:

• Ancestral whole genome duplication & triplication. • Each individual puzzle contain 6 partially similar copies of the same piece

5 genes, with 6 copies resembling each other

Building the puzzle Challenge 2:

As a result of polyploidization, each individual puzzle is made of 2 almost identical copies of the same image.

Goal: Build 882 such puzzles and

find differences among them (SNPs)

Accession 1

Accession 2

etc … Accession 3

Population Structure: Principal coordinate (PCO) analysis “Asian” winter cluster (Japan, China, S. Korea, etc.)

Spring “Western Europe” winter cluster (Poland, Germany, Sweden, Latvia, UK, Netherlands, France, etc. )

PCOs based on 260 SNPs from SNP array data set

Genetic Diversity C- subgenome (inherited from B. oleracea)

A- subgenome (inherited from B. rapa)

e op ur

e op ur

Spring

i W

er t n

ia s A

rE te in W

rE te in W

i W

er t n

ia s A

Spring

Germplasm Base/Training Populations Source: USDA/Ames; Dutch Collection; UI Collection Spring V Winter

Winter Types 652 Lines 2012-3 Field Data

652 Lines G’house Seed & DNA

652 Lines 2013-4 Field (ID) Data & Seed

652 Lines 2014-5 Field Data Iowa

Spring Types

652 Lines Field Data Minissota

652 Lines G’house Seed Mx 652 Lines Field (x2) Data Idaho

230 Lines 2012 Field Data & Seed

230 Lines G’house Seed & DNA

230 Lines 2013 Field Data

232 Lines Field Data

230 Lines Field (x2) Data

230 Lines Iowa 2014 Field Data

232 Lines Field Data

230 Lines Field (x2) Data

230 Lines Iowa 2015 Field Data Ames

230 Lines Field Data Akron

230 Lines Field (x2) Data Idaho

Seed Yield – Spring Germplasm Average Year-Site Moscow 2012 980 Genesee 2012 1,172 Moscow 2013 961 Genesee 2013 861 Moscow 2014 916 Genesee 2014 1,209 Moscow 2015 1,812 Genesee 2015 1,630 Average

Maximum Minimum

2,507 2,935 1,735 2,512 1,839 2,213 2,639 2,786

0 0 0 0 91 400 839 561

Heritabilities Gene 2012

0.26

Mosc 2013

0.28

0.29

Gene 2013

0.21

0.21

0.11

Mosc 2014

0.11

0.16

0.03

0.11

Gene 2014

0.09

0.14

0.03

0.18

0.35

Mosc 2015

0.32

0.23

0.24

0.58

0.27

0.14

Gene 2015

0.13

0.25

0.23

0.43

0.22

0.36

0.36

Mosc 2012

Gene 2013

Mosc 2013

Gene 2014

Mosc 2014

Gene 2014

Mosc 2014

Average 2 h = 0.226

Heritabilities Gene 2012

0.26

Mosc 2013

0.28

0.29

Gene 2013

0.21

0.21

0.11

Mosc 2014

0.11

0.16

0.03

0.11

Gene 2014

0.09

0.14

0.03

0.18

0.35

Mosc 2015

0.32

0.23

0.24

0.18

0.27

0.14

Gene 2015

0.13

0.25

0.23

0.43

0.22

0.36

0.36

Mosc 2012

Gene 2013

Mosc 2013

Gene 2014

Mosc 2014

Gene 2014

Mosc 2014

Sites within years 2 h =0.236

Heritabilities Gene 2012

0.26

Mosc 2013

0.28

0.29

Gene 2013

0.21

0.21

0.11

Mosc 2014

0.11

0.16

0.03

0.11

Gene 2014

0.09

0.14

0.03

0.18

0.35

Mosc 2015

0.32

0.23

0.24

0.18

0.27

0.14

Gene 2015

0.13

0.25

0.23

0.43

0.22

0.36

0.36

Mosc 2012

Gene 2013

Mosc 2013

Gene 2014

Mosc 2014

Gene 2014

Mosc 2014

Within sites 2 h = 0.235

Heritabilities Gene 2012

0.26

Mosc 2013

0.28

0.29

Gene 2013

0.21

0.21

0.11

Mosc 2014

0.11

0.16

0.03

0.11

Gene 2014

0.09

0.14

0.03

0.18

0.35

Mosc 2015

0.32

0.23

0.24

0.18

0.27

0.14

Gene 2015

0.13

0.25

0.23

0.43

0.22

0.36

0.36

Mosc 2012

Gene 2013

Mosc 2013

Gene 2014

Mosc 2014

Gene 2014

Mosc 2014

Between sites 2 h = 0.123

Seed Yield – Spring Germplasm

Genotype 03.IL.5.6.1 04.SC.28.4.3 3789.RR Python.CL 05SC11A1.35.2 05SI13A5JB.8.16 05SC1A4.10.1 03.IH.4.12.2 07.SC.38.16 05SC11A1.2.6

Mosc Gene Gene Mosc Avera Ra Mosc Genes Mosc Genes ow- seesee- owge nk ow-13 ee-13 ow-14 ee-15 12 12 14 15 1,949 1,898 1,881 1,871 1,866 1,832 1,761 1,751 1,733 1,733

1 2 3 4 5 6 7 8 9 10

20 49 21 11 22 5 16 18 1 9

6 2 4 3 9 14 40 60 8 10

37 102 33 98 76 13 3 97 47 54

15 45 .

17 10 9 12 .

103 38

7 2 22 24 26 44 82 3 23 90

6 1 21 53 19 163 20 40 68 83

6 42 36 5 1 12 63 32 29 8

6 3 14 13 22 16 11 17 72 27

Seed Yield – Spring Germplasm

Genotype 03.IL.5.6.1 04.SC.28.4.3 3789.RR Python.CL 05SC11A1.35.2 05SI13A5JB.8.16 05SC1A4.10.1 03.IH.4.12.2 07.SC.38.16 05SC11A1.2.6

Mosc Gene Gene Mosc Avera Ra Mosc Genes Mosc Genes ow- seesee- owge nk ow-13 ee-13 ow-14 ee-15 12 12 14 15 1,949 1,898 1,881 1,871 1,866 1,832 1,761 1,751 1,733 1,733

1 2 3 4 5 6 7 8 9 10

20 49 21 11 22 5 16 18 1 9

6 2 4 3 9 14 40 60 8 10

37 102 33 98 76 13 3 97 47 54

15 45 .

17 10 9 12 .

103 38

7 2 22 24 26 44 82 3 23 90

6 1 21 53 19 163 20 40 68 83

6 42 36 5 1 12 63 32 29 8

6 3 14 13 22 16 11 17 72 27

Seed Yield – Spring Germplasm

Genotype Cara Empire 3789.RR Python.CL 05SC11A1.35.2 05SI13A5JB.8.16 05SC1A4.10.1 03.IH.4.12.2 07.SC.38.16 05SC11A1.2.6

Mosc Gene Gene Mosc Avera Ra Mosc Genes Mosc Genes ow- seesee- owge nk ow-13 ee-13 ow-14 ee-15 12 12 14 15 1,949 1,898 1,881 1,871 1,866 1,832 1,761 1,751 1,733 1,733

1 2 3 4 5 6 7 8 9 10

20 49 21 11 22 5 16 18 1 9

6 2 4 3 9 14 40 60 8 10

37 102 33 98 76 13 3 97 47 54

15 45 .

17 10 9 12 .

103 38

7 2 22 24 26 44 82 3 23 90

6 1 21 53 19 163 20 40 68 83

6 42 36 5 1 12 63 32 29 8

6 3 14 13 22 16 11 17 72 27

WINTER GERMPLASM Yield Identifier

2012-13

2014-15

Mean Yield Yield ------------------ kg ha-1 ---------------------

Mean

1,476

1,701

1,251

Maximum

7,065

8,974

6,999

Minimum

0

0

0

WINTER GERMPLASM Yield 2012-13 2014-15 Identifier Mean Rank Yield Rank Yield Rank -------------------- kg ha-1 --------------------PI-384.536 7,065 1 9,532 1 4,598 27 PI-612.846 6,539 2 8,047 4 5,031 15 Visby 6,448 3 8,974 2 3,923 61 PI-458.970 6,007 4 8,279 3 3,735 66 PI-535.862 5,595 5 7,718 5 3,472 75 Ames-061.00 5,545 6 4,305 65 6,784 2 PI-535.852 5,440 7 6,503 9 4,376 36 Ames-156.52 5,437 8 6,811 7 4,063 50 PI-537.302 5,218 9 4,754 47 5,683 7 PI-531.276 5,211 10 6,410 11 4,011 55

Genotype v Phenotype

Coincidence ≠Causation

Observed – Log 10 (p)

o Best Linear Unbiased Estimates (BLUE’s)→ observed yield values corrected for environment. o Incorporated within-spring population structure and kindship as covariate in the GWA’s. o Top 20 low and high yielding accessions from 2014.  stronger year and site effect.

P-value = 0.07

Expected – Log10 (p)

–Log10 (p)

Manhattan plot

These 2 SNP’s are located in a Brassica napus gene annotation: GSBRNA2G00091535001.

Blasting the B. napus peptid against Arabidopsis thaliana:

 Top 2 hits: CONTAINS InterPro

DOMAIN/s: EF-Hand 1, calcium-binding site (InterPro:IPR018247), EF-HAND 2 (InterPro:IPR018249), EF-hand-like domain (InterPro:IPR011992), Calcium-binding EFhand (InterPro:IPR002048)

 Proteins with Calcium-binding EFhand structures have been shown to be key participant in oil bodies formation and stability in many plants.

Genetic & Phenotypic Combined Optimum oil profile for biofuel production: ďƒź High saturated & monounsaturated fatty acids (erucic acid, C22:1, oleic acid, C18:1). ďƒź Low polyunsaturated fatty acids (18:2 & 18:3) & low glucosinolates.

Measured 12 VLCFA compounds: Looked for SNPs associated with these traits (GWAS)

Retrieve known hits Chromosome C03 C22:1

QTL interval for Erucic acid FAE1 gene

… and discover new ones?

Chromosome C02 C18:0

plastid C2:0-ACP C4:0-ACP

Kas1 C16: 0-ACP

K as2 C18:

3-ketoacyl-acyl carrier protein synthase I (KAS I)

Questions

USDA-NIFA/DOE, Title 9008 Biomass Research and Development Initiative (BRDI) Grant