Anetta Kuczyńska. Institute of Plant Genetics, Polish Academy of Science, Poznań, Poland INNOVATIVE ECONOMY NATIONAL COHESION STRATEGY

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1 Anetta Kuczyńska Institute of Plant Genetics, Polish Academy of Science, Poznań, Poland

2 POLAPGEN-BD: Biotechnological tools for breeding cereals with increased resistance to drought Task 2. Genetic maps and the location of QTLs associated with barley resistance to the water deficiency Project carried out under Innovative Economy Programme , Action 1.3, Subaction within the subject Biological progress in agriculture and environment protection

3 THE AIM OF THE PROJECT 1. The general aim of the project was to enhance competitiveness of breeding companies producing cereal varieties and development of biotechnological services in Poland. This is the action that will lead to a biological progress. 2. Environmental protection. 3. Creating prototype biotechnological services, which could be provided to breeders and agricultural producers.

4 TOPICS & PARTNERS A system approach is achieved by adopting a model of tolerance to drought stress containing ecophysiological, morphological, anatomical, metabolic, proteomic, and molecular levels considered in the context of genetics.

5 THE AIM OF THE STUDY Mapping of quantitative trait loci (QTL) affecting yield and its components in European/Syrian spring barley (Hordeum vulgare L.) cross combinations under optimal and water shortage conditions. genotyping From phenotyping QTL

6 PLANT MATERIAL Three populations consisted of 100 RILs (F 8 ) each, generated via SSD technique in POLAPGEN project: Lubuski x Cam/B1/CI, Maresi x Cam/B1/CI, Georgie x Harmal. Maresi (German, semidwarf malting variety) Cam/B1/CI 08887/CI (Syrian variety)

7 RIL POPULATIONS - OBTAINING Fig. Steps in the procedure of obtaining RIL combined with in vitro culture: a) spikes and immature kernels from which the embryos were dissected; b) embryos placed on B5 medium; c) embryos developed after 4 days of culture; d) plantlets after 8 days of in vitro culture; e) plants transplanted in pots; f) spike plants after flowering stage

8 GREENHOUSE EXPERIMENT the soil was taken from the Grabów Experimental Station (E 21º 39, N 51º 21 ); the soil texture and properties were characterized by the Institute of Agrophisics, Lublin; fertilization according to the Institute of Soil Science and Plant Cultivation, Puławy; water retention curve pf; The pf value is defined as a logarithm of the pressure p (expressed in cm of water head) necessary for water removal from soil capillaries. levels of soil moisture: 1. pf optimal conditions 2. pf water deficit

9 EARLY DROUGHT (TREATMENT I) Water shortage was imposed at the three leaf stage (phase 13 in the BBCH scale) and maintained for 10 days.

10 LATE DROUGHT (TREATMENT II) Water shortage was imposed at the flag leaf stage (phase 37 in the BBCH scale) and maintained for 14 days.

11 MEASURMENTS OF SOIL MOISTURE pf 3,2 3,4 3,6 3,8 4,2 Humidity %w/w 8,0 6,75 5,7 4,75 3,6

12 Traits PHENOTYPING 1. Grain weight per main spike (g) 2. Grain weight per lateral spike (g) 3. Grain weight per plant (g) grain weight (g) 5. Length of main spike (cm) 6. Length of lateral spike (cm) 7. Number of grains per main spike 8. Number of grains per lateral spike 9. Number of spikelets per main spike 10. Number of spikelets per lateral spike 11. Fertility of main spike 12. Fertility of lateral spike 13. Length of main stem (cm) 14. Number of productive tillers per plant 15. Number of tillers per plant 16. Productivity of tillers 17. Straw weight per plant (g) 18. Heading stage (days) Plants secured for biometric observations Average of 10 plants

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14 ConsensusGBS GBM GBM Bmag ABC Bmag ABC ABC Bmag HVM GBM ABC Bmag EBmac EBmac ABC ConsensusGBS ABC ABC GBM1087 ConsensusGBS GBM H Bmag EBmac ConsensusGBS ABC Bmac Bmag GBM EBmac ABC Bmag AF022725A 23.5 ConsensusGBS scssr07970a 26.4 GBM ABC EBmatc HVCMA ABC Bmac ABC ABC Bmac0167 ABC EBmac ABC GBM Bmag HVM Bmag EBmac ConsensusGBS ABC Bmac Bmag H_ GBM EBmac ABC Bmag AF022725A 23.5 ConsensusGBS scssr07970a 26.4 GBM ABC EBmatc HVCMA ABC Bmac ABC ABC Bmac0167 ABC EBmac ABC H_ GBM Bmag HVM H_ Bmag EBmac ConsensusGBS ABC Bmac Bmag H_ H 7H.1 7H.2 7H scssr ABC H_4 GBM1426 scssr15334 GBM GBM Bmag Bmag ABC ABC ABC GBM GBM ConsensusGBS ABC ConsensusGBS GMS ABC04352-pHv ConsensusGBS GMS H_ GBM Bmac ABC ABC ConsensusGBS Bmac0306a Bmac ConsensusGBS ABC H_ scssr ABC H_4 GBM1426 scssr15334 GBM GBM Bmag Bmag ABC ABC ABC GBM GBM ConsensusGBS H_ sgbs a scssr ABC GBM1426 scssr15334 GBM GBM Bmag Bmag ABC ABC ABC GBM GBM ConsensusGBS ABC ConsensusGBS GMS ABC04352-pHv ConsensusGBS GMS EBmac ConsensusGBS Bmag ConsensusGBS GBM ABC ConsensusGBS ABC scssr H.3 5H.1 5H.2

15 1H 2H 3H 4H 5H 6H 7H A consensus linkage map of barley constructed using three RIL mapping populations and used for QTL analysis. Mikołajczak K. et al. Quantitative trait loci for yield and yield-related traits in spring barley populations derived from crosses between European and Syrian cultivars. under review

16 EXPERIMENTS CARRIED OUT IN THE GREENHOUSE Variation of the spike morphology

17 EXPERIMENTS CARRIED OUT IN THE FIELD

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19 Setup experiment 1436PK Phenotyping of 95 RIL barley lines plus 4 control varieties 8x replicates (2xcarrier per line; 4-pot config per carrier) 2x treatment conditions (control normal watering, stress conditions no watering) Pre-cultivation phase: 4 th of August 3 rd of September Sowing of seeds in 96-well tray, soil mixture (Substrate 2 + Sand, 4:1) 24x seeds of each genotype distributed over 24x trays (1xreplicate/tray) Greenhouse climate conditions: 16 C day/14 C night temperature >65% relative humidity Long day conditions ( ) additional light Continuous watering of trays as required (manually done by gardeners) Transplanting of the plants in individual pots Transplanting of single plants in 3.5L pots for LemnaTec system Soil mixture (Substrate 2 + Sand, 4:1) Greenhouse climate conditions: 16 C day/14 C night temperature >65% relative humidity Long day conditions ( ) additional light Continuous watering of pots as required (manually done by gardeners)

20 Phenotyping: 3 rd of September 8 th October Transfer pots from greenhouse to LemnaTec phenotyping facilities 28das (days after sowing) Greenhouse climate conditions: 20 C day/16 C night temperature >65% relative humidity Long day conditions ( ) additional light Automatically watering based on watering regime (target weight) Start watering Start imaging Start stress (35das) No/low watering to 20% field capacity Normal watering 70% FC (control) Start recovery phase Adding 300ml absolute watering to each pot additional target watering to reach 90% FC Fertilization, Combo Hakaphos blau (200ml absolute volume per plant) Fertilization, Combo Hakaphos blau (200ml absolute volume per plant) Rotation of the carriers: each day for one lane, every 3 rd day in between the lane for 11 positions

21 All lines (RILs, varieties) (left) and varieties only (right)

22 All lines (RILs, varieties) (left) and varieties only (right)

23 All lines (RILs, varieties) (left) and varieties only (right)

24 Post-cultivation: from 7 th of October Finish phenotyping, transfer of a selection of plants (around 800 plants) for post-cultivation, harvest Fig. Biometric analysis of spikes: A main spikes collected from a pot; B measurement of spikelets number per spike; C measurement of spike length

25 PC2 (15.28%) EPPN Plant Phenotyping Symposium, Barcelona 2015 PC1 (36.34%) Principal component biplot for traits observed after harvest; green lines non-stressed, red lines stressed

26 Institute of Plant Genetics Polish Academy of Science: Maria Surma Tadeusz Adamski Paweł Krajewski Karolina Krystkowiak Krzysztof Mikołajczak Piotr Ogrodowicz Hanna Ćwiek Leibniz Institute of Plant Genetics and Crop Plant Research, OT Gatersleben, Germany: Astrid Junker, Kathleen Weigelt-Fischer, Thomas Altmann

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