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Jauhar ali. vol 4. screening for abiotic and biotic stress tolerances

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Jauhar ali. vol 4. screening for abiotic and biotic stress tolerances

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Jauhar ali. vol 4. screening for abiotic and biotic stress tolerances

  1. 1. SCREENING FOR ABIOTIC & BIOTIC STRESS TOLERANCES: Key to successful molecular breeding -2 (Salinity) Jauhar Ali Plant Breeder, Senior Scientist IRRI-GSR Project Leader & Regional Coordinator (Asia) PBGB, IRRI (J.Ali@irri.org)
  2. 2. Salt-affected type Electrical conductivity ECe (dSm-1)# Exchangeable Sodium Percentage ESP (%) Sodium Absorption Ratio SAR pHs Saline > 4 < 15 < 13 < 8.8 Sodic < 4 > 15 > 13 8.5-10.5 SALINITY Sodic < 4 > 15 > 13 8.5-10.5 Saline - sodic > 4 > 15 Variable > 8.5 # at 25°C ESP = Exchangable Sodium x 100 Cation Exchange Capacity SAR Na Ca Mg = + + + +2 2 2
  3. 3. •White leaf tip followed by tip burning (salinity) •Stunted plant growth •Low tillering ability •Spikelet sterility and poor seed setting •Poor grain filling •Low harvest index •Lesser florets per panicle Salinity morphological symptoms observed in natural sites/salinity hot spots •Lesser florets per panicle •Lesser 1000 grain weight •Lower grain yield •Change in flowering duration •Leaf rolling •White leaf blotches •Poor root growth •Patchy growth in field RK Singh PBC 2006
  4. 4. First symptom “Leaf tip burning” “Leaf tip burning extends toward base through Lamina” “Ultimate death of leaf – always from oldest to youngest” Salinity symptoms at the vegetative stage RK Singh PBC 2006
  5. 5. Effect of salinity at reproductive stage – papery sterile spikelets RK Singh PBC 2006
  6. 6. Effect of salinity at Reproductive stage – Spikelet Sterility RK Singh PBC 2006
  7. 7. 4. Sequestration of the toxic ions to vacuole or cell wall – cell level compartmentation 3. Excretion of salt through salt glands, salt-hairs or bladders – in most halophytes Predominant salt-tolerance mechanisms operating in plant R.K.Singh Plant Breeding Course, April 06 1. Restricting the entry of toxic ions at root level - Exclusion 2. Transporting the toxic ions to stem, leaf sheath or older leaves – plant level compartmentation Na+ Cl-
  8. 8. Physiology: traits associated with salinity tolerance Regulation of uptake Compartmentation In old tissue Upregualtion of antioxidants Responsive stomata Protective metabolites Polyamines, dehydrins, Earliness R.K.Singh Plant Breeding Course, April 06 antioxidants Vigorous growth [Na+ ] OsmoprotectantsAOSS K+ AtNHX1 H+ Na+ Vacuolar Na+ /H+ SOS1 Na+ H+ Plasma Na+ /H+ AVP1 H+ PPiase Compartmentation within tissue (tissue tolerance) glyoxalates Source : A.M. Ismail
  9. 9. Salinity screeningSalinity screeningSalinity screeningSalinity screening • GSR salinity screening was previously done in greenhouse under salinized SNAP nutrient solution • For 2 seasons now, salinity screening moved saline prone on- farm site (Infanta, Quezon, Philippines).Philippines).
  10. 10. Salinity Screening: salinized nutrient solutionSalinity Screening: salinized nutrient solutionSalinity Screening: salinized nutrient solutionSalinity Screening: salinized nutrient solution Populations (BC1F2) Single plant selections (BC1F3-5) Day 0: Soaking (pre-germinate seeds at 35OC for 2 days & Room Temp. for 1 day) Day 21: 2nd Salinization (to EC 18 dS/m by adding NaCl, pH5.0) (Score for EC 12 using SES) (maintain EC & pH level daily) Day 35: Conditioning Day 3: Sowing (3 seeds/hole in styro-seedling floats) (over the SNAP nutrient solution 1% SNAP A + 1% SNAP B, pH 5.0) Day 7: 1st Salinization (to EC 12 dS/m by adding NaCl, pH5.0) (maintain EC & pH level daily) Day 35: Conditioning (Change SNAP solution with fresh water) (Score for EC 18 using SES) Day 37: Transplanting (of surviving seedlings) (Single seedling per hill) Maturity: Harvesting (Single plant harvesting)
  11. 11. Populations (BC1F2) Single plant selections (BC1F3-4) Seeding (on-farm salinity site) Seedling stage salinity Salinity Screening: OnSalinity Screening: OnSalinity Screening: OnSalinity Screening: On----farm saline prone sitefarm saline prone sitefarm saline prone sitefarm saline prone site Promising populations and lines from seedling stage screening. Transplanting (21 day-old seedlings) (Single seedling per hill) Seedling stage salinity treatment (10-21 DAS) (seedbeds are irrigated with sea water) (EC level is monitored using data logger) Scoring & Selection (21 DAS) (promising populations and lines are EC level monitoring (EC reading varies from 6 to 15 mS/cm) (Sea water is irrigated on to the field when field EC level is low) Selection & harvesting of survivals (Maturity)
  12. 12. Drought & salinity tolerance trait pyramidingtrait pyramiding
  13. 13. Objectives • To understand the salinity tolerance in relation to drought tolerance • To identify & map the salinity tolerance QTLs using DT selected ILs from varied donorusing DT selected ILs from varied donor sources • To find ST mechanistic pathways adopting time related mapping approach.
  14. 14. Materials & methods • DT selected material under lowland drought conditions during DS2000-non target trait mapping • 150 ILs BC2F4 population of 16 crosses, RP(IR64, Teqing & NPT); tolerant(Pokkali), susceptible check (IR29) 2 replications • Controlled growth conditions (29oC/21oC day /night temperature & 75% RH) immediately after 2 days of germination (30oc)in distilled water in petri plates, to initial salinization in plastic tubs with styrofoam floats • 3days at EC of 6 dSm-1increased to EC 12 dSm-1 and maintained upto 23 days with NaCl into Yoshida(1976) nutrient solution with pH 5.0 • 23 DAIS salinised nutrient solution with 150 ILs PA• 23 DAIS salinised nutrient solution with 150 ILs PA score at 10, 16, 23 DAIS and survival % • Tissue extracted in 10ml of 0.1N acetic acid & Na, K measured in atomic absorption spectrophotometer • QTL mapping using145 well distributed SSR markers and phenotypic scores with PROC GLM –ANOVA using SAS • Normalised LD coefficient and GGT was used to find close associations of the introgressions for DT and related to ST
  15. 15. SOURCESSOURCES 10D10D 16D16D 23D23D SURVIVAL%SURVIVAL% IR64(RP)IR64(RP) 55 55 55 57.557.5 TEQING(RP)TEQING(RP) 33 55 77 7.57.5 NPT(RP)NPT(RP) 55 55 55 8585 BINAMBINAM 11 11 33 9090 Phenotypic acceptability scores(1-9 scale) on 10,16, 23 d & survival % of Recurrent Parent(RP), Donor Parent(DP),tolerant & susceptible checks. BR24BR24 11 33 33 7777 FR13AFR13A 33 33 66 87.587.5 OM1723OM1723 33 44 66 57.557.5 STYHSTYH 55 66 55 8888 TYPE3TYPE3 55 66 55 7676 HANNONGHANNONG 11 22 66 7070 ZIHUI 100ZIHUI 100 55 66 77 6565 POKKALIPOKKALI (Tolerant check)(Tolerant check) 11 22 33 100100 IR29IR29 (Susceptible check)(Susceptible check) 77 77 99 00
  16. 16. Results & Discussion • Salinity and drought share certain common regions for tolerance • Identified closely associated QTL bins over different chromosomes related to salinity • Is there any role of such strongly associated bins in the developmental response to salinity stress? • Time related mapping of ST: switching on & off of genes/QTLs against• Time related mapping of ST: switching on & off of genes/QTLs against salinity stress over a given time frame. QTLs for Na-K uptake identified. • ILs identified with enhanced salinity tolerance over check variety Pokkali in elite IR64 & Teqing background and currently being field tested. • Closely related non target traits such as salinity can be mapped using such an approach
  17. 17. Relative salinity tolerance of introgression lines at 10,16 and 23DAIS 60 80 100 Frequency highly tolerant tolerant moderately tolerant 0 20 40 10DAIS 16DAIS 23DAIS Days after initial salinisation Frequency moderately tolerant susceptible highly susceptible
  18. 18. IL 62 Pokkali Highly tolerant IL 62 (Teqing x Type 3) in the foreground of Tolerant check Pokkali & susceptible check IR29 After 23 days in EC12.0 dSm-1 IR29
  19. 19. IR29 roots Pokkali roots IR29 roots ILs showing better new root growth Uniformity to salinity stress
  20. 20. RM2002.8 RM84RM32326.2 RM22028.4 RM27237.3 RM24357.3 RM58358.9 RM2371.6 RM49379.7 RM594.9 RM488101.4 RM237RM246115.2 1 RM21114.4 RM834.7 RM17447.5 RM14549.8 RM27RM30066.0 RM32468.9 RM26275.0 RM47592.5 RM183112.0 2 RM600.0 RM1327.0 RM23115.7 RM54535.3 RM3653.1 RM764.0 RM23276.7 RM25179.1 RM282100.6 3 10 10 16 23 SU23 10 16 SU RM33521.5 RM26135.4 RM18550.8 RM47153.8 RM41756.2 RM14268.5 RM56473.1 RM17780.1 RM25299.0 RM241106.2 4 RN RK RNK SN SN SK SK SK SNK SNK RN RN RK RNK23 RM297131.2 RM128134.8 RM302147.8 RM265155.9 RM431178.3 RM14194.1 RM263127.5 RM525143.7 RM6154.7 RM530158.0 RM112166.0 RM250170.1 RM166183.0 RM213186.4 RM207191.2 RM16131.5 RM135RM426157.3 RM55168.2 RM520191.6 RM293193.4 RM143207.3 RM565215.5 RM570221.1 RM227224.2 10 10 10 16 16 23 23 10 16 23 10 16 23 SU SU 10 16 23 10 16 23 Teqing x Binam SU SUSU SU 2310 16 23SU IR64 x Binam RM303116.9 RM317 RM348118.3 RM131 RM349148.8 RM280152.3 SU23 ST QTL region IR64XBinam-Shoot Na, K, Na/K TQ X Binam-Shoot Na, K, Na/K IR64 X Binam-Root Na, K, Na/K TQ X Binam-Root Na, K, Na/K RN SNSK RN SKSNK RN RN RK RK RN RN
  21. 21. 8 RM1220.0 RM41326.7 RM1328.6 RM19434.7 RM43743.4 RM28956.7 RM16957.9 RM16380.1 5 OSR193.2 RM1907.4 RM51020.8 RM20425.1 RM22526.2 RM31433.6 RM25337.0 RM21738.7 RM53945.1 RM13651.2 RM52761.2 RM374.3 RM54175.5 6 10 10 23 16 23 SU RM511.1 RM4813.2 RM8217.7 RM125RM18024.8 RM1147.0 OSR2250.1 RM33661.0 RM1063.5 OSR467.1 RM35173.3 7 OSR300.0 RM4075.7 RM529.4 RM3828.0 RM2552.2 RM137RM33169.0 RM22380.5 10 16 23 SU 23 23 SU SN SK SNK RN SN SN RN RN RN RN RNK RNK RM161 RM30596.9 RM188100.6 RM421111.2 RM178118.8 RM87129.2 RM334141.8 RM45499.3 RM162108.3 RM528121.6 RM30125.4 RM340133.5 RM412142.4 RM141 RM103145.2 10 16 23 10 16 23 SU SUSU Teqing x Binam 10 16 23 10 16 23 SU SU IR64 x Binam 16 23 RM23488.2 RM1890.4 RM118RM42996.9 RM172115.3 RM248116.6 RM22380.5 RM210RM53190.3 RM80 RM149103.7 RM502121.8 RM264RM281128.6 10 16 10 16 23 SU SU IR64XBinam-Shoot Na, K, Na/K TQ X Binam-Shoot Na, K, Na/K IR64 X Binam-Root Na, K, Na/K TQ X Binam-Root Na, K, Na/K RN SN SNSK SNK RN RN RK
  22. 22. 16 RM22211.3 RM24415.0 RM21617.6 RM23925.2 RM46746.8 RM18458.3 RM27159.4 OSR3368.0 RM26969.6 10 RM286OSR60.0 OSR12.4 RM33227.9 RM16737.5 RM12041.7 RM20254.0 11 RM4150.0 RM45.2 RM1920.9 RM24732.3 RM49134.3 RM10149.5 12 10 10 10 10 16 16 23 23 23 23 SU SU SU 10 16 16 23 23 SU OSR280.0 RM21911.7 RM52413.2 RM342RM4114.3 RM105RM32132.1 RM56647.7 RM25766.1 9 10 16 16 23 SU RM311 SN SKSNK SN SNK SN SKSNK RN RNSNK RN RN RN RK RK SU RM26969.6 RM25870.8 RM29487.1 RM22893.0 RM48497.3 RM333110.4 RM496113.0 RM28768.6 RM20973.9 RM22977.8 RM2185.7 RM206102.9 RM254110.0 RM224120.1 RM144123.2 RM31365.5 RM30974.5 RM46375.5 RM27091.3 RM235102.8 RM17109.1 10 16 16 23 RM24273.3 RM27877.5 RM20181.2 RM21599.4 RM245112.3 RM205114.7 23 SU 10 16 23 10 16 23 SU SU 10 16 23 10 16 23 Teqing x Binam SU SUSU IR64 x Binam IR64XBinam-Shoot Na, K, Na/K TQ X Binam-Shoot Na, K, Na/K IR64 X Binam-Root Na, K, Na/K TQ X Binam-Root Na, K, Na/K SNRN
  23. 23. Salinity tolerance QTLs • 23 QTL segments identified governing salinity tolerance out of which 19 were common for both the crosses • Na-K relationship with salinity tolerance fitted well with PA scores & survival %. • Chromosome 1,2,6,8,10,11 showed major salinity QTL segments with• Chromosome 1,2,6,8,10,11 showed major salinity QTL segments with improved salinity tolerance. • 17 QTL segment introgressions from Binam donor improved tolerance in Teqing background while only 7 in case of IR64 RP. • Na- exclusion mechanism in both root & shoot was introgressed from Binam donor into Teqing background primarily responsible for improved salinity tolerance.
  24. 24. Bin SSR marker 10d 16d 23d SUR 10d 16d 23d SUR 1.1 RM200 -0.83 -1.18 22.5*** 1.6 RM14 0.9** 0.99** 0.57* -5.5* 0.63 3.6 RM16 1.3** 1.37** 1.17* 1.36 6.3 RM539 -0.82* -0.70 6.5 RM3 1.3** 1.365** 0.90 1.4** 7.2 RM11 -0.83 -0.97 9.83 12* 7.4 RM234 -0.83 1.55* IR64 x Binam Teqing x Binam Introgression gene effects for salinity tolerance 7.4 RM234 -0.83 1.55* 8.1 RM407 0.55 1.55* 8.5 RM223 0.46 0.55* 0.63 8.5 RM210 0.45 0.55* 0.78 9.1 RM321 -0.55 -0.88** 1.36 10.2 RM239 -0.83 -0.97 9.83 0.80 0.90 10.4 RM271 1.3** 1.37** 1.17* -1.18 22.5*** 11.1 OSR1 -0.75 0.80 11.2 RM332 -0.96*** -0.88** -0.71* 6.22 0.78 11.5 RM209 0.54 0.90 1.4** 11.6 RM21 0.82* 1.18** 0.90 1.4** 12.1 RM4 -0.83 -0.97 9.83 0.78 Additive effects indicating levels of F value significance at 10%=without astrix; 5%=* ; 1%=** ; 0.1% =***Additive effects indicating levels of F value significance at 10%=without astrix; 5%=* ; 1%=** ; 0.1% =***
  25. 25. IR64/Binam Teqing/Binam chromosome 1 chromosome 1 QTL detected Common QTL detected Additive effects direction 14(4,4,5,82.5% );6(4,3,4,95%);3(3,3,3,95%);8(5,6,6,80%); 7(5,5,4,90%) 2(1,4,5,80% );5(1,3,7,42.5%);7(1,1,3,92.5%);9(3,3,2,95%) Individual No.( PA score on 10,16,23d,sur%)
  26. 26. IR64/Binam Teqing/Binam chromosome 11 chromosome 11 QTL detected Common QTL detectedAdditive effects direction 14(4,4,5,82.5% );6(4,3,4,95%); 2(5,5,4,82.5%); 9(7,7,7,62.5%) 9(3,3,2,95%) 7(1,1,3,92.5%); 5(1,3,7,42.5%) Individual No.( PA score on 10,16,23d,sur%)
  27. 27. Salinity in relation to drought tolerance • A separate group of closely associated Bins for DT was identified and was distinctively different from Salinity tolerance for both the crosses. • Up regulated Bins seems to be quite strongly associated with both DT & ST and were common for both the crosses. • Down regulated Bins showed many putative ST QTLs for TeqingxBinam cross improving ST in comparison to IR64xBinam cross. • 40% of DT Bins coincided with ST in IR64xBinam while 31.3% for TeqingxBinam cross.
  28. 28. CROSSCROSS IL CodeIL Code ShootShoot NaNa ShootShoot KK ShootShoot Na/KNa/K RootRoot NaNa RootRoot KK RootRoot Na/KNa/K SES DAISSES DAIS survivalsurvival %% PhenotypePhenotype avgavg avgavg avgavg avgavg avgavg avgavg 1010 1616 2323 IR64XBINAMIR64XBINAM 1SDL 91SDL 9--11 2.262.26 1.881.88 1.211.21 3.543.54 0.940.94 3.783.78 55 77 33 90.090.0 TolerantTolerant IR64XBINAMIR64XBINAM 2SDL 92SDL 9--22 2.112.11 2.402.40 0.900.90 3.503.50 0.870.87 4.044.04 55 55 44 82.582.5 TolerantTolerant IR64XBINAMIR64XBINAM 3SDL 93SDL 9--33 2.672.67 1.651.65 1.621.62 3.753.75 0.870.87 4.344.34 33 33 33 95.095.0 TolerantTolerant IR64XBINAMIR64XBINAM 6SDL 96SDL 9--66 2.692.69 1.851.85 1.481.48 3.333.33 0.690.69 4.974.97 44 33 44 95.095.0 TolerantTolerant IR64XBINAMIR64XBINAM 7SDL 97SDL 9--77 2.322.32 1.851.85 1.261.26 3.613.61 0.760.76 4.894.89 55 55 44 90.090.0 TolerantTolerant TeqingXBINAMTeqingXBINAM 1SDL 101SDL 10--11 2.782.78 1.371.37 2.032.03 3.373.37 0.680.68 4.924.92 55 55 44 82.582.5 TolerantTolerant TeqingXBINAMTeqingXBINAM 7SDL107SDL10--77 2.332.33 2.112.11 1.101.10 3.203.20 0.650.65 4.974.97 11 11 33 92.592.5 TolerantTolerant Pyramided introgression lines showing improved salinity tolerance TeqingXBINAMTeqingXBINAM 8SDL 108SDL 10--88 2.622.62 1.921.92 1.361.36 3.553.55 0.850.85 4.164.16 33 44 44 82.582.5 TolerantTolerant TeqingXBINAMTeqingXBINAM 9SDL 109SDL 10--99 2.392.39 1.571.57 1.581.58 2.982.98 0.660.66 4.504.50 33 33 22 95.095.0 Highly TolerantHighly Tolerant BinamBinam 3.463.46 1.931.93 1.791.79 3.353.35 0.770.77 4.584.58 11 11 33 90.090.0 TolerantTolerant OM1723OM1723 3.093.09 1.861.86 1.661.66 3.113.11 0.600.60 5.315.31 33 44 66 57.557.5 Moderately tolerantModerately tolerant FR13AFR13A 3.353.35 1.831.83 1.831.83 4.074.07 0.600.60 6.716.71 33 33 66 87.587.5 Moderately tolerantModerately tolerant HaoannongHaoannong 3.113.11 1.901.90 1.691.69 3.453.45 0.530.53 6.526.52 11 22 66 70.070.0 Moderately tolerantModerately tolerant IR64IR64 3.353.35 1.881.88 1.781.78 3.823.82 0.990.99 4.104.10 55 55 55 57.557.5 Moderately tolerantModerately tolerant TEQINGTEQING 2.922.92 1.991.99 1.471.47 3.343.34 0.720.72 4.654.65 33 55 77 7.57.5 SusceptibleSusceptible NPTNPT 2.432.43 2.192.19 1.111.11 3.433.43 0.710.71 4.864.86 55 55 55 85.085.0 Moderately tolerantModerately tolerant PokkaliPokkali 2.102.10 2.522.52 0.840.84 3.433.43 0.830.83 4.324.32 11 22 33 100.0100.0 TolerantTolerant IR29IR29 4.654.65 1.461.46 3.203.20 3.973.97 0.430.43 9.309.30 77 99 99 0.00.0 Highly susceptibleHighly susceptible
  29. 29. What ? • Salinity tolerance is a complex trait and needs to be understood in a wholistic manner. • Molecular QTL pyramiding of ST & DT in select ILs was feasible. • Salinity and drought has relatively similar pathways for tolerance mechanisms and needs to be addressed together to achieve success in breeding superior stress tolerant rice varieties. • Dissection of salinity tolerance QTLs from drought tolerant material will open up new research avenues in the future.

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