Genetic Variability and association mapping for phenolic acids in a collection of tetraploid wheats

1. Dr. Barbara Laddomada Lecce - January 15th. 2016

2. 1. T. monococcum
2. T. urartu
3. T. aestivum
4. T. turgidum
5. T. timopheevii
6. T. zhukovskyi
Family: Poaceae
Tribe: Triticeae
Genus: Triticum
Wheats
Durum wheat
(Triticum turgidum subsp. durum)
Bread wheat
(Triticum astivum subsp. aestivum)
Wheat 712 Mt
Corn 974 Mt
Rice 475 Mt
Global wheat production

3. Source: Int. Grain Council, 2014
Quantity (Mt)
EU-27 7.3
Italy 3.9
France 1.4
Greece 1.1
Spain 0.7
Canada 4.9
Turkey 2.2
USA 1.6
Algery 1.7
Kazakhstan 2.0
Mexico 2.3
Syria 0.8
Morocco 1.4
Tunisia 1.2
India 1.2
Australia 0.5
Others 6.0
WORLD TOTAL 33.1
Top durum wheat producers
Durum wheat
(Triticum turgidum subsp. durum)

4. Durum wheat
Durum wheat major breeding program aim to increase:
• Grain protein content and composition
• Yellow pigment
• Grain Yield
• Pathogen resistance
• Abiotic stress tolerance
CONTENT OF BIOACTIVE COMPOUNDS
Triticum turgidum subsp. durum

5. T. turgidum subsp. durum
genome AABB
Sterile (genome AB)
T. turgidum subsp. dicoccum
genome AABB
xT. urartu
2n=2x=14
genome AA
Aegilops subsp
2n=2x=14
genome BB
T. turgidum subsp. dicoccoides
genome AABB
Chromosomic doubling
DiploidsTetraploiploids Durum (tetraploid) wheats
T. tauschii
genome DD
x
T. aestivum subsp. aestivum
genome AABBDD
Sterile (genome ABD)
Esaploids
Triticum turgidum L. subsps. (2n=4x=28)

6. Wheat genome size
1 532 4 6 7
A
Genome size: 4900 Mbp
Diploids (2n=2x=14)
Genome AA
Genome size: 13000 Mbp
Tetraploids (2n=4x=28)
Genome AABB B
Genome size: 17000 Mbp
Esaploids (2n=6x=42)
Genome AABBDD
D
Wheat chromosomes

7. Whole wheat grain
Bran
Embryo
Endosperm
Whole wheat products
White products
Endosperm
Endosperm
Carbohydrates
Proteins
Embryo
Vitamins
Minerals
Phenolic acids
Bran
Fibers
Vitamins
Minerals
Phenolic acids

8. Wheat bran tissues FA DHD DHT SA p-CA TOTAL
Bran 5.26 1.01 0.24 0.25 0.09 6.85
Endosperm 0.10 0.03 0.00 0.01 0.00 0.14
Aleurone 8.17 1.07 0.11 0.44 0.21 10 .00
Scutellum 3.48 0.37 0.03 0.01 0.01 3.90
Embryonic axis 0.31 0.09 0.01 0.00 0.00 0.41
Contents of principal phenolic acids (mg g-1 dry matter) in wheat grain districts
Phenolic acids
Cinnamic acids
R1=R4=H. R2=OCH3. R3=OH. vanillic acid
R2=R3=R4=OH. R1=H. gallic acid
R2=R3=R4=H. R1=H. salicilic acid
R2=R4=OCH3. R1=H. R3=OH. syringic acid
Hydroxy benzoic acids
R1=R3=H. R2=OH. coumaric acid
R1=R2=OH. R3=H. caffeic acid
R1=OCH3. R2=OH. R3=H. ferulic acid
R1=R3=OCH3. R2=OH. sinapic acid
FA. ferulic acid; DHD. dehydrodimers of ferulic acid; DHT. dehydrotrimers of ferulic acid; p-CA. p-coumaric acid; SA. sinapic acid

9. Aims of the work
 Variation for phenolic acids in a tetraploid wheat collection;
 Heritability of phenolic acids in tetraploid wheats;
 Detection of genetic loci controlling phenolic acids through
association with SNP markers

10. Plant material
• Valenzano (Bari. Italy)
• 2 year evaluations
• RCB design, 3 replications
I. subsp. durum (65 varieties)
II. subsp. turgidum (12 accessions)
III. subsp. turanicum (8 accessions)
IV. subsp. polonicum (8 accessions)
V. subsp. carthlicum (3 accessions )
VI. subsp. dicoccum (9 accessions)
VII. subsp. dicoccoides (7 accessions)
Triticum turgidum L. (112 genotypes)

11. n
n
n
Phenolic acid analysis
 Hexane
 NaOH hydrolysis
 Acidification
 Ethyl acetate
HPLC-DAD analysis

12. Variability for
total phenolic acidsμgg-1dm
0
200
400
600
800
1000
1200
1400
1600
2012= 730 μg g-1 dm
Min = 340 μg g-1 dm
Max = 1470 μg g-1 dm
T. turgidum accessions
0
200
400
600
800
1000
1200
1400
1600
1800
μgg-1dm
2013= 802 μg g-1 dm
Min = 540 μg g-1 dm
Max = 1700 μg g-1 dm

13. T. turgidum
subspecies
N° of
accessions
Year of
release
Total phenolic acids
Mean Sd Min-Max
subsp. durum 30 1991-2008 807.6a 153.8 587.0 – 1243.0
subsp. durum 27 1971-1990 871.0b 205.3 593.0 – 1585.0
subsp. durum 8 before 1971 794.0abc 94.7 651.0 – 952.2
subsp. turanicum 8 654.1d 91.0 513.8 – 835.6
subsp. polonicum 8 713.8ad 117.6 545.5 – 867.4
subsp. turgidum 12 694.8cd 123.9 527.7 – 959.2
subsp. carthlicum 3 712.8ad 163.0 551.6 – 864.0
subsp. dicoccum 9 635.1cd 117.7 440.1 – 773.8
subsp. dicoccoides 7 670.1d 84.1 562.4 – 798.4
Variability for
total phenolic acids
Different superscripted letters indicate statistically significant differences in total phenolic
acid content content mean (p < 0.05)

14. Most abundant
phenolic acids
0
200
400
600
800
Ferulic acid Sinapic acid
2012
μgg-1dm
a
b ac bc
bd
ac
a
b
cd
bce
bc
bc
b
bc
0
200
400
600
800
1000
Ferulic acid Sinapic acid
μgg-1dm
2013
a
b
ac ac
b
adad
a
b
bc
ad
be
bc
be
Ferulic acid
Sinapic acid

15. Less represented
phenolic acids
p-Coumaric acid >
Vanillic acid >
Syringic acid >
p-Hydroxibenzoic acid
d
0
10
20
30
40
50
durum turanicum polonicum turgidum carthlicum dicoccum dicoccoides
p-Coumaric acid Vanillic acid
Syringic acid p-hydroxybenzoic acid
μgg-1dm
2012
a a b
bc ab
ab
d
a ab ab
ab
ab
c
ab
a b b b bc
ab
ad
a
b
ab
d
ae
bcbc
0
10
20
30
40
50
60
70
durum turanicum polonicum turgidum carthlicum dicoccum dicoccoides
p-Coumaric acid Vanillic acid
Syringic acid p-hydroxybenzoic acid
2013
μgg-1dm
a
a
a a
b
ab
abb
c
b
abb
d
bc
ab
b
ab
ab
ab
bc
c
ab
c ab
c
d
abad

16. 0
200
400
600
800
1000
1200
1400
1600
1800
Pedroso Ambral Mida Primadur Tito
2012 2013
Tetraploids with highest
phenolic acid contentμgg-1dm

17. Phenolic acid profile
Ferulic acid >
Vanillic acid >
p-Coumaric acid
Sinapic acid >
Ferulic acid >
Vanillic acid >
p-Hydroxibenzoic >
Syringic acid >
p-Coumaric acid
Ferulic acid >
p-Coumaric >
Vanillic acid >
Syringic acid >
p-Hydroxibenzoic acid
~25%~1% ~75%
Soluble free Soluble free and conjugated Insoluble bound
0.7 - 1.7 μg g-1 dm 0.8 - 100 μg g-1 dm 450-800 μg g-1 dm
Ethanol : water 80:20 Ethanol : water 80:20 plus NaOH hydrolysis NaOH hydrolysis

18. Source of
variation
d.f. p-Hydroxy
benzoic acid
Vanillic
acid
Syringic
acid
p-Coumaric
acid
Ferulic
acid
Sinapic
acid
Total
phenolic acids
Year (Y) 1 3.702*** 19.445*** 1.098*** 401.927*** 476395.935*** 3391.107*** 604268.532***
Genotype (G) 110 6.541*** 50.426*** 7.382*** 391.356*** 71766.494*** 5112.566*** 94561.467***
Y x G 110 1.776*** 11.102*** 3.964*** 133.104*** 15333.427*** 2513.087*** 27884.704***
Error 222 0.016 0.133 0.039 1.234 155.780 13.374 235.223
h2
B 0.65 0.69 0.48 0.60 0.70 0.50 0.63
Combined analysis of variance and heritability (h2
B) of phenolic acids in a tetraploid wheat collection
evaluaed at Valenzano (Bari. Italy) in 2012 and 2013
*** Significant differences at 0.001 p value. d.f: degree of freedom
Estimation of variance quote attributable to genetic factors
Phenotypic variation
Variation due to genetic
components
Variation due to
environment and error
= +
Heritability (h2
B) of phenolic acids

19. SNPs genotyping
Marker-trait correlation
by TASSEL 3.0
(Mixed Linear Model)
Identification of genetic loci that
contribute to phenolic acid variation
Phenotypic measurements
Tetraploid wheat germplasm collection
Association mapping
for phenolic acids
Measurements
of population characteristics
(structure and relatedness)

20. Genome-specific functional SNP markers were utilized
to discover genetic loci for phenolic acids
90K iSelect array by Illumina iScan
1.0 cm
20 - 50 µm
one
oligonucleotide
sequence per “pixel”49 - 400
chips/wafer
High-throughput genotyping technologies

21. Chromosome localization of SNPs
associated with total phenolic acid content
Chromosomes
Probability
Colored spots represent SNPs distributed along the wheat chromosomes

22. We extracted phenolics from wheat bran by
ultrasound-assisted technologies, and used
the aqueous extract as ingredient to make
rigatoni
Overcoming the adverse effects of
bran on the quality of end-products
ultrasound-assisted
extraction at 20°C for 25
min in a pilot plant
assembled by Weal (Milano,
Italy)
Phenolic acid Content (mg g-1dm.)
p-Hydroxy benzoic acid n.d.
Vanillic acid n.d.
Syringic acid n.d.
Sinapic acid n.d.
Ferulic acid 0.31±0.01
p-Coumaric acid 0.25±0.01
o-Coumaric acid n.d.

23. Semolina was processed with either aqueous
bran extract (supplemented pasta) or water
(control) into ‘mezzi rigatoni’-shaped pasta.
The content of phenolic substances of the supplemented
pasta was in the range reported for whole meal pasta; and
it was significantly higher in both the supplemented types
than in the corresponding controls.
30
40
50
60
70
80
90
100
30
40
50
60
70
80
90
100
0 100 200 300 400 500
RelativeHumidity(%)
Temperature(°C)
Time (min)
relative humidity Temperature
30
40
50
60
70
80
90
100
30
40
50
60
70
80
90
100
0 100 200 300 400 500
RelativeHumidity(%)
Temperature(°C)
Time (min)
Temperature relative humidity
Variations of temperature and relative
humidity in the drier during HT1 (top) and HT2
(bottom) pasta drying programs.
Effect of pasta supplementation
with wheat bran aqueous extracts
Pasta was dried according to two different
drying diagrams (HT1 and HT2).

24. Conclusions
o Significant effect of genotype was observed for all phenolic acids
o Durum cultivars showed the highest amounts of phenolic acids
o Further improvement of phenolic acid content in elite wheat
germplasm could be possible through breeding programs
o 29 SNPs were found associated with individual phenolic acids and
phenolic acid content that could be used for breeding purposes
o The content of phenolic substances was increased in pasta by the
addition of aqueous extract from durum wheat byproducts.

25. Laddomada et al 2015. Wheat bran phenolic acids: bioavailability and stability in
whole wheat-based foods. Molecules. 20. 15666-15685.
Laddomada et al 2015. Phytochemical characterization and anti-inflammatory activity
of extracts from the whole-meal flour of Italian durum wheat cultivars. Int J Mol Sci. 16.
3512-3527.
Publications
Pasqualone et al 2015. Effect of supplementation with wheat bran aqueous extracts
obtained by ultrasound-assisted technologies on the sensory properties and the
antioxidant activity of dry pasta. Nat Prod Commun. 10 (10). 1739-1742.
Laddomada et al 2016. Genetic variation for phenolic acids concentration and
composition in a tetraploid wheat (Triticum turgidum L.) collection. Gen Res Crop
Evol. in press.
Durante et al 2012. Effects of sodium alginate bead encapsulation on the storage
stability of durum wheat (Triticum durum Desf.) bran oil extracted by supercritical
CO2. J Agric Food Chem 60 (42) 10689-95.
Rawat N. Laddomada B. Gill BS. 2013. Genomics of cereal-based functional foods.
Cereal Genomics II. Varshney R.K. and Gupta P.K. (Eds). pp 247-274. Springer
Sciences+Business Media Dordrecht. Netherlands. DOI: 10.1007/978-94-007-6401-
9_10.

26. Acknowledgements
• Dr. Barbara Laddomada
• Dr. Miriana Durante
• Leone D’Amico
• Dr. Giovanni Mita
• Dr. Sofia Caretto
• Anna Maria Pascali
• Salvatore Lisi
Di.S.S.P.A. University of Bari
• Prof. Antonio Blanco
• Dr. Agata Gadaleta
• Prof. Antonella Pasqualone
CNR ISPA. Lecce
• Prof. Marcello S. Lenucci
DiSteBA. Università del Salento
CNR ISPA. Bari
• Dr. Fiorenza Minerivini
• Dr. Angela Cardinali
• Prof. Bikram S. Gill
HPI Project. Enhancing plant health value
and functional food value of wheat

27. Thank you for your attention!