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

Identification of loci and genes responsible for sodium and chloride exclusion in rootstocks for use in marker assisted selection Jake Dunlevy, Deidre Blackmore, Everard Edwards, Rob Walker and Mandy Walker Sam Henderson, Yu Wu, Matthew Gilliham

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

Impacts of salinity on grapevines Ionic stress - stunts growth/reduces yield Cl - toxicity Na + toxicity Na + Cl - Salty berries - reduces wine quality Osmotic stress - roots must work harder to extract water - stunts growth/reduces yield

Some rootstocks can limit the translocation of Na + and Cl - from roots to the shoot Na + Cl - Ion exclusion

Rootstock breeding strategy x2 x2 Root knot Phylloxera Cl - Na + Marker Assisted Selection nematode Resistance exclusion exclusion resistance Traditional selection Other desirable traits • First we need to identify markers New Rootstocks for for Cl - exclusion and Na + exclusion Australia

A test cross for Cl - exclusion • In 1985 a cross was made between a poor Cl - excluder, K51-40 and a strong Cl - excluder 140 Ruggeri V. champinii x V. riparia V. berlandieri x V. rupestris x K51-40 140 Ruggeri 1.6 Leaf Cl- concentration 1.2 (%dry weight) 0.8 F 1 population n=60 0.4 SSR Genotyping 0.0 In 2014 K51-40 140 Ruggeri True hybrids n=40

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

Leaf Cl - concentration Results - Cl - exclusion (% dry weight) 0.0 0.5 1.0 1.5 2.0 2.5 140 Ruggeri 140 Ruggeri MC 215-63 MC 215-09 • MC 215-26 MC 215-28 Continuous variation suggests control by multiple genes 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 K51-40 X 140 Ruggeri F 1 hybrids 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 K51-40 MC 215-30 MC 215-76 K51-40 MI 07-33

Results - Na + accumulation • Skewed variation suggests control by a major locus • Transgressive variation implies each parent is heterozygous 0.25 0.20 Leaf Na + concentration (% dry weight) 0.15 0.10 140 Ruggeri K51-40 0.05 0.00 MC 215-10 MI 07-49 MI 07-46 MI 07-34 MI 07-44 MC 215-54 MC 215-27 MC 215-76 MC 215-40 MI 07-23 MC 215-1 MC 215-60 MC 215-9 MC 215-33 MC 215-69 MC 215-19 MI 07-27 MC 215-26 MI 07-29 MI 07-39 R-140 MC 215-39 K51-40 MC 215-71 MC 215-23 MC 215-28 MC 215-78 MC 215-49 MI 07-47 MC 215-13 MI 07-36 MC 215-37 MC 215-64 MC 215-55 MC 215-43 MC 215-63 MC 215-4 MC 215-29 MC 215-45 MC 215-30 MC 215-25 MI 07-33 K51-40 x 140 Ruggeri F 1 hybrids

A major QTL for Na + exclusion • Genotype by sequencing identified 4,000+ SNP markers • A consensus map was constructed based on 514 SNP markers aligned to the Nae locus reference genome • 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

HKT – High affinity K + transporter Role of HKT1 in cereals • 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

HKT – High affinity K + transporter • 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? Chromosome 11 15.4 Mb 15.5 Mb 15.6 Mb 15.7 Mb HKT1.1 HKT1.3 HKT1.2 HKT1.7 HKT1.6 HKT1.8 Are they expressed in roots? Do these genes encode functional Na + transporters?

Functional characterisation Xenopus laevis oocytes (African clawed frog) HKT1.1 HKT1 proteins were expressed in oocytes and then tested for Na + transport Electrode to Electrode to measure internal inject potential (voltage) current HKT1.3 Na + Na + Na + Na + Na + Na + Na + Na + Na + Na + Na + Na +

Four unique alleles of HKT1.1 Amino acid differences between the predicted proteins K51-40 140 Ruggeri 0.25 Leaf Na + concentration E K E R 0.20 E - dominant for good Exclusion E K e R (% dry weight) 0.15 e - recessive for poor exclusion e K E R 0.10 e K e R 0.05 0.00 MC 215-10 MI 07-49 MI 07-46 MI 07-34 MI 07-44 MC 215-54MC 215-27MC 215-76MC 215-40 MI 07-23 MC 215-1 MC 215-60 MC 215-9 MC 215-33MC 215-69MC 215-19 MI 07-27 MC 215-26 MI 07-29 MI 07-39 R-140 MC 215-39 K51-40 MC 215-71MC 215-23MC 215-28MC 215-78MC 215-49 MI 07-47 MC 215-13 MI 07-36 MC 215-37MC 215-64MC 215-55MC 215-43MC 215-63 MC 215-4 MC 215-29MC 215-45MC 215-30MC 215-25 MI 07-33 140 Ruggeri K51-40 Henderson and Dunlevy et al ., (2018) New Phytologist , 217 ( 3 ) p1113

Characterisation of HKT1.1 allelic variants Do the dominant and recessive alleles encode proteins with different rates of Na + transport? 140 Ruggeri K51-40 E R e R E K e K Henderson and Dunlevy et al ., (2018) New Phytologist , 217 ( 3 ) p1113

Yeast toxicity assay Na + concentration in growth medium • Expression of Na + transporters in yeast 0.5 mM 50 mM cells leads to growth inhibition due to toxic levels of Na + uptake Empty vector E R • The extent of growth inhibition reflects the rate of Na + transport e R E K e K Henderson and Dunlevy et al ., (2018) New Phytologist , 217 ( 3 ) p1113

Which amino acid is responsible? • We mutagenized and tested the effect of two amino acid residues 534 537 E K e K e K D537G e K R534S e K D537G/R534S e K e K D537G e K R534S e K D537G/R534S Henderson and Dunlevy et al ., (2018) New Phytologist , 217 ( 3 ) p1113

Origins of HKT1.1 alleles • We sequenced HKT1.1 from accessions of the four parent species to determine the origin of r=each of the four alleles 140 Ruggeri K51-40 ( V. champinii x V. riparia ) ( V. berlandieri x V. rupestris ) E E E e E E E e SNP position based on HKT1;1 coding sequence (bp) 1039 1172 1173 1502 1535 1536 1600 1610 210 229 313 317 386 430 446 487 672 739 795 831 849 868 906 940 948 77 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 VisHKT1;1-e R Ruggeri 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

Acknowledgements Matthew Gilliham Mandy Walker Sam Henderson Rob Walker Yu Wu Everard Edwards Deidre Blackmore Harley Smith Lauren Hooper + many more Thank you for listening!