Presentacion de 11th Asian Maize Conference which took place in Beijing, China from November 7 – 11, 2011.

1. INCORPORATION OF
DOWNY MILDEW
RESISTANCE IN TARGETED
MAIZE
BREEDING/PARENTAL
LINES
HF Galvez, CB Pascual, AKB Malijan,
AO Canama,, RB Quilloy, PH Manguiat

2. Corn: Philippines’ most important
cereal crop next to rice
Without fungicide, downy mildew
severely affects corn production (80-
100% loss)
 Metalaxyl fungicide
 Pre-treatment of seed materials
Philippine downy mildew (PDM) in corn
is caused by Peronosclerospora
philippinensis Weston (Shaw)

3. C8 Metalaxyl fungicide and application
rga01_1
36.2  Human health/environment hazard
umc150
13.5
7.6
srga3
umc048
 Additional expense or cost in production
2.9 asg52
1.6 1.6 umc1960 1.6 umc1141
Greatly affects seed viability
1.6 umc1728 1.6 umc1149
5.9

umc089
umc002
9.9
umc012
31.0
phi014
 PDM resistance reported
16.6
umc120
10.0
phi125 Use of resistant varieties remains most
35.5
rga17_2
effective and economical; resistance sources
177.1 cM available
DNA markers mapped in maize genome
Chromosome locations of likely genes (QTL)
for PDM resistance mapped in P345 x Pi23-
maize population

4. Project Objectives
 To fine-map the chromosomal locations of likely genes
(QTL) for DMR
 To validate the QTLs in multi-location disease
screenings under natural infection of downy mildew
 To identify resistance gene orthologs and develop PCR-
based DNA markers specific to the DMR genes
 To establish recombinant inbred line population with
differential disease reaction to downy mildew
 To isolate and characterize the cDNA sequences of the
DMR genes

5. Marker-assisted Purification of Maize Parent Lines
PDM Resistant Lines
Morphological (rouging) then SSR
marker purification
SSR markers from each maize
P345 Nei9008 chromosome
Susceptible/Recipient 2 Cycles: bulk then individual plant
Inbred Lines marker-assisted purification
A line D line
E line FG lines
HJ lines

6. Fine mapping and validation of DMR-QTL
Validation of DMR-QTL
• Plant materials (Pi23 x P345)
- BC1F1 - genotype data (AMBIONET, 2000)
-BC1F3 - phenotype data (Isabela and Bukidnon)
• Method of QTL detection (QTL Cartographer software)
- Single marker analysis
- Composite interval mapping (CIM)

7. Establish genetic structure of RIL mapping population
118 (Pi23 x P345) BC1F2 families
Select 3-5 phenotypic variants
(plants) per family
 Plant height/habit
 Leaf morphology
P345 (R) Parent Pi23 (S) Parent  Tassel and silk color
Bulk and de-bulk SSR analysis for
genotype variants
 SSR markers from each maize
chromosome
(Pi23 x P345) BC1F7 seed production
215 (Pi23 x P345) BC1F3, then ear-to-
row up to BC1F7

8. Multi-location disease evaluation for DMR
- Natural downy mildew (DM) infection with pre-infected
spreader (sweet corn) plants
- % disease incidence (% plants infected/line)
-period of evaluation – 14, 21, 28, 35, 42 & 49 DAE
• Ilagan, Isabela (DA-Isabela Experimental Station)
-152 (Pi23xP345)BC1F3 lines
- parents and sweet corn as susceptible check
• Musuan, Bukidnon (Central Mindanao University)
-197 BC1F3 lines
- parents and sweet corn as susceptible check

9. Multi-location disease evaluation for DMR
- Final multi-locations trial
- 153 BC1F6 lines, parents and sweet corn
• Kabacan, North Cotabato (Univ. of Southern Mindanao)
- use of reported virulent DM isolate (Carmen)
• Banga, South Cotabato (ACM Genetics)
- collaboration with private seed producer

10. RESULTS
Comparative QTL - UPLB, Isabela and Bukidnon
using (Pi23xP345)BC1F3
- Chromosome 8 (major QTL) – UPLB & Bukidnon
- Chromosome 9 – Isabela only
- Chromosome 1 – UPLB and Isabela
- Chromosme 4 - UPLB only

11. Multilocation QTL analysis of corn RIL population (Pi23 x P345)BC1F6
Chrom. Trial site QTL Flanking QTL LRb Genetic Effects R2(%)c
No. detected marker positiona Additive Dominance
1 Isabela rga01_2 –umc67 0.3054 15.93 -6.14 1.15 13.49
2 Bukidnon csu109-bnl8.44 0.2505 11.71 -9.47 0.53 14.90
South Cotabato csu54-umc055 1.1588 20.75 -0.06 - 23.59
3 North Cotabato umc121-phi36 0.0001 11.64 -5.48 0.03 8.92
5 North Cotabato phi91-phi116 2.3954 31.82 -9.87 0.08 29.86
South Cotabato bng1386 - 1.9283 16.21 0.057 61.25 20.4
umc104_1
6 Los Baños umc65-umc59 1.3699 11.79 10.75 4.55 9.88
7 North Cotabato rga16-rga19 0.9461 12.86 -6.19 0.16 11.64
8 Los Baños rga01_1-umc150 0.0001 20.58 20.95 7.67 52.69
9 Isabela umc105-umc113 1.278 24.28 8.32 1.27 24.77
South Cotabato umc81-phi61 0.5471 22.13 -0.081 78.33 25.2
a Position of QTL based on composite interval mapping (CIM)
b Peak value of the maximum-likelihood-ratio(LR) test statistic observed for the QTL
c Proportion of phenotypic variance explained by the QTL

12. Testcrossing and line conversion for DM resistant
maize variety
- DMR-Pi23 x Pi17 and DMR-Pi23
- Pi23 – recurrent parent; Pi17 – other parent for hybrid
• Initial set of candidate DMR-Pi23 lines
- molecular, field DMR (Isabela) & nursery DMR (UPLB)
- 5 BC1F6 lines

13. Marker-assisted introgression of DMR
Marker-assisted line purification
-2 cycles of morphological data
+ SSRs (10 maize chromosomes)
Genuine hybrids (SSR markers)
- Initial SSR analysis, 87% confirmed genuine hybrids
No. of No. of
Total SSR No. of true F1
Entry heterozygous homozygous
loci screened hybrid plants
SSRs SSRs
P345 x Pi17 10 10 0 2/2
P345 x Ca00314 10 9 1 14/19
Nei9008 x Ca00314 10 7 3 31/33

14. Crossing Block: DMR Maize
Hybrids/Converted Lines
20 (Pi23 x P345) BC1F6 PDM resistant lines
Pi23 parent (backcross for line conversion)
Other maize parental line/s: good SCA for
test-Hybrid cross

15. Test-Hybrids: Screenhouse DMR Screening and PYT
Initial cross: 4 test hybrids (Isabela and Bukidnon selections)
1301A and 1304B candidate hybrids:
 Screenhouse DMR and preliminary agro-morpho (yield)
MAS for QTL markers
 Had 0% DM incidence
16 more test hybrids for evaluation

16. Conclusion
• Corn microsatellite (SSR and EST-SSR) and resistance
gene analog (RGA) markers were successfully used to:
purify parent lines and hybridization crosses to
incorporate DMR derived from P345 and Nei9008
Re-establish genetic structure of RIL population for
genetic mapping and in combined MAS breeding
schemes
• Resistance QTL were validated in multi-location DM
screening. The major QTL in Chrom 8 was detected in
UPLB. The mapping of multiple and location specific
QTL suggests variation among DM isolates
• Best performing DM resistant lines (Pi23xP345)BC1F7
and hybrids (1301A and 1304B) were identified and will
be released as a registered new corn variety and/or
genetic stock.

17. MARKER-ASSISTED BREEDING: GENE
PYRAMIDING
Philippine Downy Mildew Resistance (PDMR)
Bacterial Stalk Rot Resistance (BSRR)
Quality Protein Maize (QPM)

18. A BLESSED DAY!