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!