INCORPORATION OF DOWNY MILDEW RESISTANCE IN TARGETED MAIZE BREEDING/PARENTAL LINES HF Galvez, CB Pascual, AKB Malijan, AO Canama,, RB Quilloy, PH Manguiat
INCORPORATION OF DOWNY MILDEW
RESISTANCE IN TARGETED MAIZE
BREEDING/PARENTAL LINES
HF Galvez, CB Pascual, AKB Malijan, AO Canama,, RB Quilloy, PH Manguiat
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)
Metalaxyl fungicide and application
Human health/environment hazard
Additional expense or cost in production
Greatly affects seed viability
PDM resistance reported
Use of resistant varieties remains most
effective and economical; resistance sources
available
DNA markers mapped in maize genome
Chromosome locations of likely genes (QTL)
for PDM resistance mapped in P345 x Pi23-
maize population
36.2
rga01_1
C8
umc150
srga313.5
umc0487.6asg522.9
1.6 1.6 umc1960 1.6 umc11411.6 umc1728 1.6 umc1149
umc0895.9umc002
9.9umc012
31.0
phi014
umc120
16.6
10.0phi125
35.5
rga17_2
177.1 cM
36.2
rga01_1
C8
umc150
srga313.5
umc0487.6asg522.9
1.6 1.6 umc1960 1.6 umc11411.6 umc1728 1.6 umc1149
umc0895.9umc002
9.9umc012
31.0
phi014
umc120
16.6
10.0phi125
35.5
rga17_2
177.1 cM
Project Objectives
To fine-map the chromosomal locations of likely genes(QTL) for DMR
To validate the QTLs in multi-location diseasescreenings 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 withdifferential disease reaction to downy mildew
To isolate and characterize the cDNA sequences of theDMR genes
P345 Nei9008
PDM Resistant Lines
Susceptible/Recipient
Inbred Lines
A line
E line FG lines
HJ lines
D line
Marker-assisted Purification of Maize Parent Lines
Morphological (rouging) then SSR
marker purification
SSR markers from each maize
chromosome
2 Cycles: bulk then individual plant
marker-assisted purification
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)
Establish genetic structure of RIL mapping population
P345 (R) Parent Pi23 (S) Parent
(Pi23 x P345) BC1F7 seed production
118 (Pi23 x P345) BC1F2 families
Select 3-5 phenotypic variants
(plants) per family
Plant height/habit
Leaf morphology
Tassel and silk color
Bulk and de-bulk SSR analysis for
genotype variants
SSR markers from each maize
chromosome
215 (Pi23 x P345) BC1F3, then ear-to-
row up to BC1F7
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
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
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
Multilocation QTL analysis of corn RIL population (Pi23 x P345)BC1F6
Chrom.No.
Trial site QTL detected
Flanking marker
QTL positiona
LRb Genetic Effects R2(%)c
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 -umc104_1
1.9283 16.21 0.057 61.25 20.4
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 QTLc Proportion of phenotypic variance explained by the QTL
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
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
Entry Total SSR
loci screened
No. of heterozygous
SSRs
No. of homozygous
SSRs
No. of true F1 hybrid plants
P345 x Pi17 10 10 0 2/2
P345 x Ca00314 10 9 1 14/19
Nei9008 x Ca00314 10 7 3 31/33
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
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
Conclusion• Corn microsatellite (SSR and EST-SSR) and resistance
gene analog (RGA) markers were successfully used to:
• 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
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
• 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.
MARKER-ASSISTED BREEDING: GENE PYRAMIDING
Philippine Downy Mildew Resistance (PDMR)
Bacterial Stalk Rot Resistance (BSRR)
Quality Protein Maize (QPM)
A BLESSED DAY!