Identification of new allelic variants for freezing

tolerance in winter wheat

WRKY71 and ZCCT2 genes

Radvilė Nagrockaitė-Lelešienė

Dr. Rita Armonienė

Dr. Gintaras Brazauskas

Wheat is a major export crop in Lithuania

• Wheat covers ca. 850 thous. ha (44 % of arable) of which

more than 600 thous. ha is under winter wheat;

• ca. 45 to 55 % of wheat produced is exported

annualy;

• The main export directions are: North Africa,

Middle East and other EU countries;

• Poor overwintering during harsh winters considerably limits wheat

production, e.g. 58% of winter wheat crop had perished over

2013/2014 winter.

Relevance

• Winter hardiness directly affects economic revenue;

• Freezing tolerance is one of the major components of winter

hardiness;

• Freezing tolerance pathways are still not fully understood.

Methods

Targeting Induced Local

Lesions in Genomes (TILLING)

M2 generation

DNA isolation

DNA sequence analysis

M1 generation

Two winter wheat

genotypes M2 generation seeds

M3 generation seeds

High Resolution

Melting analysis

Snow removal – the most “effective” method to

determine “real” freezing tolerance

7

1. Cultivation: 30 seeds of each genotype

for each temperature

2. Acclimation: 2 weeks in 2°C; 200

µmol m-2/s-1 PAR, 12/12 h photoperiod

3. Freezing test: at -4; -6; -8; -10; -12; -14;

-16; -18 temperatures °C

4. Evaluation of survival after 21

days

LT50 value

Results

Three genes were chosen to screen

for induced mutations with relevance

to freezing tolerance:

• Transcription factor gene WRKY71;

• Vernalization gene ZCCT2;

• Sucrose synthase gene SS1.

Transcription factor gene with

WRKY DNA-binding domain (WRKY71)

• A member of a large family of transcription factors;

• Involved in response to abiotic stress;

• Regulates developmental processes, e.g. flowering initiation;

• Operates by binding to the W-box (TTGACC/T) present

in their target promoters.

Four mutations were identified in

WRKY71

*p < 0,05

Missense mutation in WRKY71 gene reduce

freezing tolerance in winter wheat

-12,02 -11,3

-10,51

-14

-12

-10

-8

-6

-4

-2

0

WT M775 M490

Te

mp

era

ture

°C

Genotypes

LT50

*

• One of the VRN2 locus genes;

• Comprised of zinc finger in the first exon and the CCT

domain in the second exon;

• Acts as flowering repressor;

• Associated with spring growth habit in winter wheat.

ZCCT2

Four mutations were identified in

ZCCT2

Missense mutations in ZCCT2 gene reduce

freezing tolerance in winter wheat

-11,18 -10,77 -10,58 -10,23

-14

-12

-10

-8

-6

-4

-2

0

WT M775 M878 M822

Te

mp

era

ture

°C

Genotypes

LT50

Sucrose synthase gene (SS1)

• Activates sucrose accumulation and its activity increases

during cold acclimation in roots and leaves;

• Was found to be a metabolic bottleneck under challenging

environmental conditions;

• Sucrose contributes to the stabilization of membranes and

protects cells from freezing and dehydration.

Two mutations were identified in

SS1 gene

The expression level of SS1 gene in crown and

leaves during cold acclimation

Nonsense mutation in SS1 gene reduces

freezing tolerance in winter wheat

-14,62 -13,82

-16

-14

-12

-10

-8

-6

-4

-2

0

WT M631

Te

mp

era

ture

°C

Genotype

LT50

A total of 9 mutations were identified in three studied genes.

Most (6 out of 9) of the mutations under study had a negative

effect on freezing tolerance in winter wheat as revealed by

artificial freezing testing.

Further work is under way to determine functional properties of

the mutations revealed.

Conclusion

Thank you for your attention