Genomics and Developing World Agriculture. John Witcombe CAZS Natural Resources University Wales, Bangor

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1 Genomics and Developing World Agriculture John Witcombe CAZS Natural Resources University Wales, Bangor

2 Molecular markers 1. Pearl millet - downy mildew and drought tolerance 2. Upland rice drought tolerance and yield Marker assisted selection (MAS) Marker evaluated selection (MES) Genetic diversity

3 1. Pearl millet 15 years from start to farmers fields Make a map Find QTLs for target traits Do MAS in target genotype Enter lines in trials Get varietal release 15 years of continuous funding from DFID PSP

4 Demand led research: Downy mildew disease and drought were targeted as the two major constraints. Both traits are also difficult to breed Pyramiding resistance genes is impracticable with conventional methods. Possible with MAS. Drought resistance breeding involves expensive and difficult screening nurseries. MAS was assumed to be more efficient.

5 Downy mildew: single-cross hybrids are the most widely grown pearl millet cultivar type in India Every popular single cross hybrid in India has eventually succumbed to downy mildew Devastating epidemics that cause great economic losses Drought is most common abiotic stress

6 Make a map Started in 1990 (JIC/ICRISAT) Published in 1994 Based on two crosses Had 181 loci over 300 cm Based on RFLPs Added e.g., AFLPs and SSRs ,461, ,513,573, , Nra 756, ,161,49 891, ,386 (1) 223.1, , ,672.2, , , ,477,466, , 848, , (2) , , 443,Xugt , 662, (3) , Xugt ,180,51, , , , , (4) , ,612 84, Xugt , , , 737,579, (5) ,815, , , , Xpsr39(Fbp) 421.3, (6) (7) (A) , ,526,

7 Find QTLs: run in parallel with map development By the time map was published QTLs for downy mildew resistance were already identified (Bangor) Later mapping was done for drought tolerance (IGER)

8 Selecting a target: The extra early hybrid HHB 67 Most popular public-sector hybrid in India Cultivated on about 0.5 M ha Early, high yield, seed easy to produce

9 MAS MAS at ICRISAT for resistance to downy mildew MAS backcrossing in both male and female parents (using RFLPs) Trials of new versions of the hybrid for yield, similarity to HHB 67, and disease resistance

10 QTLs used for 843A the seed parent ICMP P 7-3 ICML 22 (conventional)

11 QTLs used for H 77/833-2 pollinator Bristles Tall Drought tolerance ICMP 451

12 Disease resistance of seed parent 843B was improved by P7-3 QTLs (New Delhi) (Patancheru) (Jodhpur) ICMB % 56% 38% 843B (control) 95% 89% 95% P7-3 (donor) 0.5% 26% 5%

13 Disease resistance of pollinator (H 77/833-2) was improved by ICMP 451 QTLs New Delhi Patancheru Jodhpur ICMR % 50% 50% ICMR % 39% 33% H 77/833-2 (=control) 94% 93% 99% ICMP 451 4% 50% 46%

14 ICMR hybrids yielded more (6 environments, rainy season 2001) Hybrid name Parents Grain yield (t/ha) Time to 50% flower (d) Plant height (cm) HHB A x H 77/ ICMH A x ICMR ICMH A x ICMR Rate of gain for yield as good or better than conventional breeding for yield! Was not a target trait. ICMH released as HHB 67 Improved in 2005 First MAS product released in India

15 Benefits from HHB 67 Improved Stopping a DM epidemic = loss of 28 million (30% of the harvest from 0.5 M ha at 0.7 t/ha at Rs 1850 per tonne) About 10 million pounds additional yield every year From a 10% increase in grain yield per hectare (the minimum expected from the new hybrid)

16 Breeding for yield in droughtstressed environments: Drought tolerance is difficult to assess as requires repeated field evaluation Difficult to repeat across seasons or sites - rain is unpredictable Progress from conventional breeding has been slow

17 Screen genotyped testcrosses to identify QTLs for performance under drought stress conditions Multi-year, replicated, testcross trials over several stress treatments and a non-stressed control, In each E, assess phenology and components of grain and biomass yield Map QTLs for each trait in each environment (performance) Regress out maturity and yield potential and QTL map residuals (tolerance per se)

18 LG 2 M322 M394 PRLT allele accounts for over 20% of the variation Stress performance Drought tolerance per se M214 M321 M592 M443 M356 M cm Grain yield seed mass 3 Harvest index 3 Panicle grain no. 1 Leaf rolling 1 Grain yield seed mass 3 Harvest index 3 Biomass yield 1 QTLs identified in three drought experiments

19 MABC PLRT LG2 allele backcrossed into the pollinator of HHB 67 and hybrids tested for yield under stress and nonstress conditions

20 Grain yield improved in stressed and non-stressed Es, summer 2002 with no maturity increase 1.5 PRLT PRLT+ 843 PRLT PRLT Early drought stress 1 Grain yield (t/ha) (37) (35) (38) (37) (36) 3 Less droughted HHB 67 (36) ICMH (34) ICMH (38) ICMH (37) ICMH (36) Grain Increase (days to flower) Higher grain yield (and biomass yield) from QTL

21 Strategic conclusions Now practical to use MAS for incremental enhancement of selected pearl millet hybrids. Breeding programmes are more efficient since resources concentrate on improving popular cultivars that best meet farmers needs

22 Strategic conclusions The unexpected power of backcrossing. High gains for non-target traits (+yield when DM the target) have been achieved. This result also found in rice. Modified backcross breeding a good idea

23 Funding Has illustrated the success of long-term centrally-funded research Collaboration between ARIs (JIC, IGER, CAZS), IARCs (ICRISAT), and NARS (HAU, TNAU)

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25 Drought resistance in upland rice Mapping population was developed from parents with contrasting drought tolerance mechanisms QTLs for drought-related root traits were identified Price et al TAG 109: 49-56

26 Targets for MAS in rice Root length & mass RG532 RG173 RZ995 e18m43.7 e18m43.6 e12m45.4 C178 e18m43.5 R cm C1370 G393 R cm C86 C949 RZ14 R G338 C39 G89b R1440 G20 C451 R1357 RG650 C507 e12m37.14 e12m36.12 RG351 Aroma RG509 RG83 RG171 G45 e18m43.8 G39/RG139 G57 C601 RG256 Root length, thickness & penetration R902 e12m36.7 G1010 C225 e12m37.10 e18m43.4 G2132 G1073 G187 R2676 R202 RG598 R662 e12m37.4 C643 RG409 RG191 e12m45.1 RG745 e12m36.10 G144 e12m36.16 RZ474 C136 R1618 G164 R1164 e12m36x R79 R1687 G385 e18m43.26 e12m36.13 G1085 e18m43.25 e12m36.4 C506 Root length, thickness & mass e12m35.25 e18m43.17 RG190 C734 RG449 C513 RG163 C1016 MAFF22/23 RG620 C701 G89d RG257 e12m37.2 G1082 C16 C223 RZ390 R3166 R2232 R569 RG13 C624 C43 RZ70 54 cm RG119 RG346 R642 RZ141 e18m43z e12m36.2 G320 e15m35.14 G44 e12m45y e12m36.15 RG2 C189 e12m36.6 G1465 e12m37.12 Root length & penetration C76 RZ516 e12m36.1 RG cm e12m36.18 R2654 e12m37.7 e12m37.6 RG778 RZ682 G24 CD0127 e12m37.13 RM247 G cm R617 RG341 e12m36.14 R1933 C449 RG543 RG181/C901

27 MAS for root traits in rice These 4 QTLs for roots were markerassisted backcrossed into unmapped Indian variety (Kalinga III). 6 years, 3000 marker assays and 323 breeding lines to. Produce a set of near-isogenic lines with different introgressed regions for testing

28 Results: Root length Only 1 of the 4 QTLs (QTL9) significantly increased root length (under both stress and non-stress conditions). Steele et al TAG 112:

29 Results: Grain and straw yield The lines with different combinations of QTLs roots were tested under rainfed conditions in India

30 Results Small proportion of Azucena (the donor) into Kalinga III increased grain yield and straw yield BUT it made no difference whether the QTLs were there or not! i.e. the BC control without QTLs was as good as the BCs with the QTLs

31 Grain yield Straw yield (t/ha) (t/ha) PY 81 No QTL control PY 82 (QTLs 2, 11) PY 83 (QTLs 2, 9, 11) PY 84 (QTLs 2, 11) Kalinga III (recurrent P) LSD 5%

32 Conclusions of MAS for root traits Introgression of Azucena into Kalinga III significantly increased grain and straw yield BUT could be random introductions was not worth effort of QTL analysis and MAS for traits associated with yield The BC lines may be released for medium land environments in eastern India.

33 MES from few cross breeding

34 Many crosses K plants (size of pie) Few crosses K Plants Many crosses (m)( Small populations (n) Few crosses (m)( Large populations (n)( - bulks

35 Breeding Strategy few crosses (smart, clever, carefully chosen) very big populations bulk population breeding selection in target environment immediate testing of varieties with farmers

36 A Failed or Stalled Green Terai Stalled (25-year-old varieties) Revolution Eastern plateau Failed (landraces) High Barind Tract Stalled (25-year-old varieties)

37 After only 8 years: High Yield Gains Nepal 20% (extra 0.6 t/ha) India 54% (extra 0.5 t/ha) Bangladesh 36% (extra 1 t/ha)

38 Barkhe 3004 Masuli

39 Success Four crosses made and three are successful Total of five varieties released or recommended Many more adopted by farmers Probability that 3 out of 4 succeed P <

40 Marker-evaluated selection Kalinga III (rainfed upland) crossed to IR64 (high yielding, lowland) Selected in upland and lowland Evaluated products (varieties) with molecular markers

41 Shifts in allele frequencies were high Allele frequencies (so far 44 marker loci) were tested in 28 selected lines from Kalinga III/IR64 cross. Compared to 50 F2 (unselected control). Kalinga III Upland parent Ashoka 200F Upland variety F2 Control population Kalinga III IR64 Barkhe 3004 Lowland variety IR64 Lowland parent Proportion of Kalinga III alleles (%) Steele et al TAG 109:

42 Shifts in allele frequencies Shifts in allele frequency were highest in India upland where drought was most severe. At 44 SSR markers no IR64 alleles found at high frequency in the best upland varieties (Ashoka 228 and Ashoka 200F) Very low LD. One new marker has IR64 alleles in 25% of lines within Ashoka varieties Finding the introgressed regions is the goal!

43 Markers and parents: Variability in four rice lines from same cross: Kalinga III/Radha 32 Similarity coefficients Judi 572 Judi 575 Judi 579 Judi 582 Judi Judi Judi Judi

44 Summary MAS for secondary traits such as roots is not promising MES is a promising technique (has theoretical advantages over QTL analysis) Evidence that backcross breeding is an effective technique (small, non-targeted introgressions produce significant improvements) Diversity studies of practical use

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