Identification of loci and genes responsible for sodium and chloride - - PowerPoint PPT Presentation

Jake Dunlevy, Deidre Blackmore, Everard Edwards, Rob Walker and Mandy Walker Sam Henderson, Yu Wu, Matthew Gilliham

Identification of loci and genes responsible for sodium and chloride exclusion in rootstocks for use in marker assisted selection

Salinity - a growing problem in Australia

• 90% of Australian vineyards rely on irrigation
• Salts (Na+ and Cl-) can build up over time from repeat application

and evaporation of irrigation water containing these ions

• Particular issue in areas of poor water quality, limited water

supply and high evaporative demand

• Predicted to worsen as climate change modelling predicts

lower rainfall and increased extreme heat events in the future

Na+ Cl-

Impacts of salinity on grapevines

Osmotic stress - roots must work harder to extract

water - stunts growth/reduces yield

Ionic stress - stunts growth/reduces yield

Cl- toxicity Na+ toxicity

Salty berries - reduces wine quality

Na+ Cl-

Some rootstocks can limit the translocation

• f Na+ and Cl-from roots to the shoot

Ion exclusion

Marker Assisted Selection

Root knot nematode resistance Phylloxera Resistance New Rootstocks for Australia

Rootstock breeding strategy

Na+ exclusion Cl- exclusion

Traditional selection

Other desirable traits

• First we need to identify markers

for Cl- exclusion and Na+ exclusion x2 x2

K51-40

A test cross for Cl- exclusion

0.0 0.4 0.8 1.2 1.6

K51-40 140 Ruggeri

Leaf Cl- concentration

(%dry weight)

140 Ruggeri

x

F1 population n=60

• In 1985 a cross was made between a poor Cl- excluder, K51-40

and a strong Cl- excluder 140 Ruggeri

SSR Genotyping In 2014

True hybrids n=40

• V. champinii x V. riparia
• V. berlandieri x V. rupestris

The Plant Accelerator

Salt screen 2 week duration 100mM Cl- 60mM Na+ 3 reps per genotype Automated watering to weight daily - ensures identical salt treatments Multiple Smarthouses - allows multiple temperature treatments Daily imaging - allows detailed growth analysis

0.0 0.5 1.0 1.5 2.0 2.5

140 Ruggeri MC 215-63 MC 215-09 MC 215-26 MC 215-28 MC 215-55 MI 07-46 MC 215-33 MC 215-19 MI 07-47 MC 215-37 MC 215-29 MC 215-25 MI 07-29 MC 215-64 MC 215-54 MC 215-27 MI 07-49 MC 215-04 MC 215-43 MI 07-23 MI 07-27 MI 07-34 MC 215-13 MC 215-23 MC 215-39 MC 215-78 MC 215-69 MI 07-36 MI 07-44 MC 215-45 MC 215-71 MC 215-60 MC 215-10 MI 07-39 MC 215-40 MC 215-01 MC 215-49 MC 215-30 MC 215-76 K51-40 MI 07-33

Leaf Cl- concentration

(% dry weight)

K51-40 X 140 Ruggeri F1 hybrids

Results - Cl- exclusion

• Continuous variation suggests control by multiple genes

140 Ruggeri K51-40

0.00 0.05 0.10 0.15 0.20 0.25

Leaf Na+ concentration (% dry weight) K51-40 x 140 Ruggeri F1 hybrids

Results - Na+ accumulation

140 Ruggeri K51-40

• Skewed variation suggests control by a major locus
• Transgressive variation implies each parent is heterozygous

A major QTL for Na+ exclusion

• Genotype by sequencing

identified 4,000+ SNP markers

• A single major QTL was found which

explains up to 70% of the variation in Na+ exclusion

• Six HKT1 genes in the locus stood out as

likely candidates

Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

Nae locus

• A consensus map was constructed based
• n 514 SNP markers aligned to the

reference genome

HKT – High affinity K+ transporter

• Previously shown to be responsible for Na+

exclusion in other crop species

• Wheat, Rice, Tomato, Maize
• HKT1 transporter proteins remove Na+ ions

from the xylem flow

• This transport activity reduces the amount of

Na+ transported to the leaves

Cotsaftis et al., (2012) PLoS ONE 7(7) e39865

Role of HKT1 in cereals

HKT – High affinity K+ transporter

Are they expressed in roots?

15.4 Mb

HKT1.7 HKT1.1 HKT1.8 HKT1.6 HKT1.2 HKT1.3

15.5 Mb 15.6 Mb 15.7 Mb

Chromosome 11

Do these genes encode functional Na+ transporters?

• All six of the HKTs in the reference genome are located

in the Na+ exclusion locus

• Are one or more responsible for the Na+ exclusion trait?

Functional characterisation

Electrode to inject current Electrode to measure internal potential (voltage)

Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+

HKT1.1 HKT1.3 Xenopus laevis oocytes

(African clawed frog)

HKT1 proteins were expressed in oocytes and then tested for Na+ transport

0.00 0.05 0.10 0.15 0.20 0.25

Leaf Na+ concentration

(% dry weight)

EK ER EK eR eK ER eK eR

E - dominant for good Exclusion e - recessive for poor exclusion

Four unique alleles of HKT1.1

K51-40 140 Ruggeri Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

K51-40 140 Ruggeri

Amino acid differences between the predicted proteins

Characterisation of HKT1.1 allelic variants

K51-40 140 Ruggeri EK eK ER eR

Do the dominant and recessive alleles encode proteins with different rates of Na+ transport?

Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

Yeast toxicity assay

ER

0.5 mM 50 mM Na+ concentration in growth medium Empty vector

eR EK eK

• Expression of Na+ transporters in yeast

cells leads to growth inhibition due to toxic levels of Na+ uptake

Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

• The extent of growth inhibition reflects

the rate of Na+ transport

Which amino acid is responsible?

EK eK eK D537G eK R534S eK D537G/R534S eK eK D537G eK R534S eK D537G/R534S 534 537

• We mutagenized and tested the effect of two amino acid residues

Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

(V. champinii x V. riparia) (V. berlandieri x V. rupestris)

K51-40 140 Ruggeri

Origins of HKT1.1 alleles

E e E E E e E E

SNP position based on HKT1;1 coding sequence (bp) 77 210 229 313 317 386 430 446 487 672 739 795 831 849 868 906 940 948 1039 1172 1173 1502 1535 1536 1600 1610 VisHKT1;1-e K A G G G C G A G G A T G C G T G A G T T C A G G C A K51-40

• V. champinii

A G G G/A C G/A A G G/CA/G T G C G T G A G T T C A G G/A C/A A/G VisHKT1;1-E K G G T A C A A T C G G G T G C A A G T A T A G G A G

• V. riparia

A G G A C A A G C G T G T G C A A G T A T A G G A G VisHKT1;1-E R A A G A G A G G C G T G C G T G T T C A T G G A A G 140

• V. berlandieri

A A G A G A G G C G T G C G T G T T C A T G G A A G Ruggeri VisHKT1;1-e R A G G G C G A G G A T A C A T G A G T T C A A G C A

• V. rupestris

A G G G C G A G G A T G C G T G A G T T C A A/G G C/A A/G

Henderson and Dunlevy et al., (2018) New Phytologist, 217 (3) p1113

• We sequenced HKT1.1 from accessions of the four parent species to

determine the origin of r=each of the four alleles

Acknowledgements

Matthew Gilliham Sam Henderson Yu Wu Mandy Walker Rob Walker Everard Edwards Deidre Blackmore Harley Smith Lauren Hooper + many more

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