GRM 2013: Marker-assisted breeding for improving phosphorus-use efficiency and tolerance to aluminum toxicity in maize -- S Gudu

Marker-assisted breeding for improving phosphorus-use efficiency and tolerance to

aluminum toxicity in maize.

Presentation by: Samuel Gudu

Scientific Team: S. Gudu, E. Ouma, T. Matonyei, R. Okalebo, C.Othieno, P. Kisinyo, B. Were, A. Onkware, E. Too, J.Agalo, J. Ochuodho (Moi University) Dickson Ligeyo

(KARI) Sidney Parentoni, Claudia Guimaraes, Leon Kochian, Jurandir Magalhaes, Vera Alves, Sylvia Sausa, Lauro Guimaraes (EMBRAPA)

Mathias Wissuwa (JIRCAS), Abdel Ismail, Sigrid Heurer (IRRI) Leon Kochian, Lyza Maron, Miguel Pineros, Jiping Liu and Ed Buckler (USDA-

ARS/CORNELL, USA).

Research Problem: Low maize yield

• Small Scale farmers obtain very low maize yields (<1.0 t/ha) versus 5-8 ton/ha under research conditions and is declining; yet 90% of population depend it as a staple food.

• Overall annual production of 2.8 million metric tons against a consumption of 3.2 million metric tons.

• The 0.4million metric ton annual deficit met through importation and food relief

Constraints to Maize productivity in Kenya

• Low soil fertility/acidity characterized by deficiency of major nutrients (eg P, Ca) and acidity (eg Al, Mn)

• Biotic stresses namely foliar diseases (eg. maize lethal necrotic viris, MSV), Striga and insect pests(eg. Stem borer)

• Frequent drought challenge

• Limited use of recommended inputs (eg fertilizers, low potential cultivars) and husbandry practices.

Distribution of acid soils and maize growing areas in Kenya

4

Distribution of Acid Soils in Kenya (Kanyanjua et al., 2002)

Maize Growing areas of Kenya (Mohammed and Anderwood, 2004)

Some Properties of Acid soils from maize growing areas of Kenya

Sampling

sites

Soil

pH

(H2O)

Olsen P

(mg/kg)

N

(%)

C

(%)

Exch. Cations (Cmol/kg)

ECEC

Cmol/kg

% Al

Sat

Specific

Gravity

Soil Texture (%)

Textural

Class Ca Mg K

Al

Sand Clay Silt

Sega 4.65 2.13 0.14 1.61 2.75 1.10 0.45 2.07 6.37 32.50 2.39 56 30 14 Sand clay loam

Bumala 4.62 2.74 0.16 2.35 3.15 2.05 0.37 2.01 7.58 26.52 2.33 56 28 16 Sand clay loam

Kuinet 4.55 4.48 0.21 2.86 2.69 0.89 0.74 2.24 6.56 34.15 2.29 58 24 18 Sand clay loam

Kavutiri 4.07 6.08 0.36 3.51 1.35 0.10 0.27 4.29 6.01 71.38 1.74 70 8 22 Sand loam

Kangema 4.69 6.00 0.24 2.31 2.30 1.35 0.33 3.32 7.30 45.48 1.82 66 14 20 Sand loam

Kerugoya 4.85 23.18 0.39 3.39 1.95 1.40 0.42 2.71 6.48 41.82 1.90 64 8 28 Sand loam

Source: Kisinyo et al., 2011

Rationale for using breeding for tolerance to soil acidity

• Liming option to minimize Al toxicity is not a sustainable owing to low access and adoption

• P supplementation is not sustainable option owing to high P fixation in Kenyan acid soils and high cost of P which makes farmers not to use at all or use less than recommended rates.

• Use of genotypes capable of utilizing fixed P and withstand high Al saturation

• Conventional breeding for tolerance to Al & P deficiency is slow/less precise hence, MAS tools

Project objectives 1. Screening of Kenyan maize germplasm for Al tolerance in

nutrient solution, for ZmMATE gene expression, and for P efficiency in the field

2. Development of maize topcrosses for assessing yield performance under acid soil conditions in Kenya

3. Evaluation of Kenyan maize topcrosses, synthetics and hybrids for Al tolerance and P efficiency in the field

4. Marker assisted selection for genes/QTLs to improve Al tolerance and P-use efficiency in locally adapted maize germplasm

5. Develop mapping populations using the highly aluminum tolerant Kenyan sources, 203B, K4 and/or CON 5

6. Training and capacity building

Objective 1 a): Aluminium tolerance of various genotypes

Relative net root growth of selected 20 inbred lines after 3 days of growth in nutrient solution culture with Al

Objective 1a: Screening of Kenyan maize germplasm for Al tolerance

05

101520253035404550

1.05 -1.16

0.95 -1.04

0.80 -0.94

0.68 -0.79

0.57 -0.67

0.49 -0.56

0.40 -0.48

0.30 -0.39

0.20 -0.29

0.10 -0.19

Freq

uenc

y

Range of RNRG

Means for RNRG of 235 Kenyan inbred lines screened for Al tolerance in nutrient solution. 26% are highly tolerant ranging from 0.80 -1.16

Regression analysis of ZmMATE1 against RNRG

Only 16.11 % in ZmMATE1 expression can be predicted from observations of RNRG.

Even most Al tolerant lines from Kenya express low level of ZmMate1 gene compared to Brazilian lines

Responsive Non responsive

P-In

effic

ient

P-

effic

ient

+P

-P

+P

-P

Inbreds grown without P

Objective 1c: Screening of Kenyan maize germplasm for P-efficiency in the field

POOLB26 X MUL817

MUL817 X MUL125

With added P

No added P

Added 26kg P No added P

P-efficient line

Grain yield % yield Plant height Ear height Days to 50% (t/ha) Reduction (cm) (cm) Tasselling TopCrosses P Cntrl P Cntrl P Cntrl P Cntrl

MUL 211XS558-27-2-1XR12C9 9.7a 5.3ab 45.88 228.3a 212.3a-d 104.7ab 89.0b-e 74a-d- 75a-c

MUL125XMUL863XS558-2-2-3-7 6.6a 3.5b 46.68 182.0c-e 166.3e 75.7c-g 65.7fg 64d 65cd

H505 6.2ab 3.5b 44.53 185.3c-e 174.0e 70.7e-g 68.0fg 66cd 69a-d

MUL 211XMUL822XR12C12 4.3ab 3.3b 22.01 214.0a-c 210.7a-d 95.7a-c 92.3b-e 74a-d- 76a-c

MUL211XMUL822XR12C11 5.1ab 3.1b 39.45 209.0a-d 202.3b-e 100.0ab 93.0a-d 70a-d 75a-c MUL211XMUL216XR12C7 4.2ab 3.0b 28.27 219.3a-c 216.0a-c 100.0ab 93.3a-d 75a-c 79a MUL817XMUL991XR12C9 9.2a 3.0b 67.39 210.7a-d 208.3a-d 95.0a-d 91.0b-e 67b-d 68b-d MUL863XMUL204XR12C7 3.7ab 2.9b 22.19 214.3a-d 202.0b-e 98.3ab 86.3b-f 72a-d 75a-c H502 3.3b 2.7b 19.34 178.0de 168.7e 59.7g 56.7g 67b-d 69a-d

MUL1007XS558-2-2-3-7XR11C10 5.5ab 2.6b 53.21 191.3b-e 189.3b-e 86.7b-f 80.0b-f 66cd 69a-d

MUL852XGXR12C12 4.5ab 2.5b 43.3 246.3a 232.0a 119.3a 109.0a 78ab 78ab MUL863XMUL996XR11C10 4.2ab 2.4b 42.38 189.0b-e 183.3c-e 77.0c-g 74.3c-g 67b-d 71a-d MUL211XS558-27-2-1XR12C7 4.2ab 2.4b 44.08 229.0a 208.7a-d 108.7ab 91.7b-e 80a 80a

MUL211XS558-27-2-1XR12C12 4.0ab 2.3b 42.78 209.3a-d 199.0b-e 102.3ab 91.0b-e 73a-d 78ab

MUL817XMUL991XR12C7 3.4b 2.1b 37.09 207.3a-d 170.7e 86.3b-f 67.0fg 63d 70a-d MUL125XMUL863XR11C10 4.3ab 1.9b 55.58 183.7c-e 175.0e 73.7d-g 71.3d-g 67b-d 73a-d Treatment mean 5.1 2.9 206.1 194.9 90.9 82.5 70 73 Grand mean 4.02 4.02 200.5 200.5 86.7 86.7 72 72 CV 26 22 8.3 7.3 11.4 11.9 6.2 7.6 SE 1.36 0.57 9.6 8.38 5.83 5.81 2.5 3.2 SED 1.93 0.8 13.6 11.86 8.25 8.22 3.6 4.5

Performance of Topcrosses under acid soil at Sega

Performance of Topcrosses under acid soil at Bumala

Grain yield % yield Plant height Ear height Days to 50%

(t/ha) Reduction (cm) (cm) Tasselling

TopCrosses P Cntrl P Cntrl P Cntrl P Cntrl

MUL211XMUL822XR12C11 5.4a 4.0ab 26.43 253.0ab 251.3a-c 117.0ab 109.3a-c 67a-c 65bc

MUL125XMUL863XR11C10 4.4ab 4.0ab 9.61 222.0a-e 215.7a-e 93.3a-e 91.0a-e 63c 66a-c

MUL 211XS558-27-2-1XR12C9 7.0a 3.9ab 43.31 249.0a-c 237.3a-d 109.0a-c 101.3a-c 67a-c 71a-c

MUL817XMUL991XR12C9 6.1a 3.9ab 35.53 246.3a-c 213.0a-e 115.0ab 85.3b-e 63c 64c

MUL1007XS558-2-2-3-7XR11C10 5.6a 3.2ab 42.53 241.3a-c 199.3b-e 106.0a-c 79.3b-e 64c 68a-c

MUL211XMUL216XR12C7 5.0ab 3.1ab 37.02 290.0a 247.0a-c 132.7a 102.3a-c 67a-c 69a-c

MUL817XMUL991XR12C7 3.7ab 3.0ab 18.63 209.0b-e 202.0b-e 88.0a-e 81.3b-e 63c 65bc

MUL125XMUL863XS558-2-2-3-7 4.6ab 2.8b 38.61 201.0b-e 158.7ab 69.7c-e 54.7e 62c 65bc

MUL 211XMUL822XR12C12 5.2a 2.8b 46.51 239.7a-d 212.3a-e 107.7a-c 88.3a-e 72ab 77a

MUL863XMUL996XR11C10 4.3ab 2.8b 35.60 229.3a-d 203.0b-e 85.7b-e 82.0b-e 65bc 67a-c

H505 5.0ab 2.7b 46.71 214.3a-e 174.7de 81.7b-e 64.0de 64c 66a-c

MUL863XMUL204XR12C7 4.7ab 2.6b 44.61 277.3a 248.7a-c 133.0a 113.7ab 66a-c 74a

H502 3.5ab 2.6b 24.50 167.0e 166.3e 61.0e 59.0e 63c 65a-c

MUL852XGXR12C12 4.9ab 2.2b 55.06 274.0a 234.7a-d 132.3a 116.3ab 67a-c 72ab

MUL211XS558-27-2-1XR12C12 4.4ab 2.1b 51.70 247.0a-c 213.0a-e 124.3a 96.3a-d 70a-c 71a-c

MUL211XS558-27-2-1XR12C7 3.4ab 1.8b 46.51 250.3a-c 216.3a-e 115.3ab 94.0a-e 68a-c 80a Treatment mean 4.8 3.0 238.2 212.1 104.5 88.6 66 69 Grand mean 3.9 3.9 225.1 225.1 96.6 96.6 67 67 CV (%) 17.0 21.0 12 12.5 17.3 18.9 5 8 SE 0.9 0.5 16.2 15.2 10.2 7.4 2 3 SED 1.2 0.8 22.9 21.4 14.4 10.5 3 5

OBJECTIVE 4: Marker-Assisted selection for genes/QTLs to improve Al tolerance and P-efficiency in Locally adapted germplasm

4a) Developed single crosses pyramiding Al and P under field evaluation in acid soils at Chepkoilel site. Just harvested, yield data will be available later

0

2

4

6

8

10

12

14

16

18

Rela

tive

ZmM

ATE1

Exp

ress

ion

ZmMATE1 relative expression levels among 40 accessions after 6 hours of exposure to Al3+ ions at an activity of {39} µM in nutrient solution culture

OBJECTIVE 4b (i): Introgression of ZmMATE/Al tolerance QTLs in Kenyan elite lines using MABC

Entry Genotype Cross RRL (Al) RRL (0) RNRG

1 SYN AL X R12C10-1 Single cross 34.79 109.70 0.32



4 R11C10 X SYN AL X R11C10-4 Back cross 90.43 96.64 0.94





9 R12C10 X SYN AL X R12C10 Back cross 87.53 100.02 0.88



12 SYN AL X AO89 X AO89-2 Back cross 47.40 128.37 0.37


14 SYN Al X AO89 X AO89-5 Back cross 71.12 129.79 0.55


16 AO89 Parent 56.40 179.00 0.32

OBJECTIVE 4b (ii): Aluminium tolerance of introgression material based on solution culture phenotyping

Genetic map constructed with 183 markers SNPs and the Al tolerance QTLs, which are shown as red lines.

SNP Markers Chromosom Posistion (Mbp)

f P R2 individual

PZAO3613-1 1 2.914 14.19 0.0002 5.76

PZA00356-8 1 263.637 13.94 0.0003 5.94

PZA00996-1 5 37.789 3.87 0.0500 0.85

PHM14046-9 8 169.471 7.47 0.0070 2.92

PHM229-15 9 30.003 12.66 0.0005 4.67

PHM5740-9 10 8.773 14.07 0.0002 6.23

R2 TOTAL ADJUSTED 26.85

Markers associated with Al tolerance in maize detected by multiple regression analysis. SNP marker in bold were coincident with Al tolerance QTLs

Objective 5b: Mapping P-efficiency QTLs in Kenyan maize germplasm

HSL3 x 5046-2 X MUL 229

F2 cobs

HSL3 x 5046-2 X MUL 229 (F1)

MUL 229 (P2) HSL3 x 5046-2 (P1)

F1 and parental cobs

KML036 XS396-16-1 (P-efficient, sensitive respectively) 230 F2 genotyped (Kbioscience, UK) using 466 polymorphic SNPs

Distribution of 239 polymorphic SNP markers on the ten maize linkage groups

Chromosome Number of markers Length (cM) Average Length(cM) Chromosome1 20 154.34 7.72Chromosome2 36 425.42 11.82Chromosome3 27 138.89 5.14Chromosome4 20 243.36 12.17Chromosome5 22 118.06 5.37Chromosome6 21 117.81 5.61Chromosome7 9 413.94 45.99Chromosome8 43 331.81 7.72Chromosome9 26 145.45 5.59Chromosome10 15 166.42 11.09WholeGenome 239 2255.5 9.44

Maize Lethal Necrosis Disease interfered with F2:3 maize population

Phenotyping for QTL association mapping for P is ongoing…

Advancing F2 to F2:3 at Migori site in April-Sept. 2013

Objective 6: Training and Capacity Building

Activity • Recruitment of 2 PhD students to undertake

training in molecular breeding Status • Evans Ouma for the Phosphorus studies • Thomas Matonyei for Al work • The students are currently finalizing their studies

(Matonyei writing thesis while Ouma will be conducting his last experiments in March, 2014

SUMMARY OF PRODUCTS

1. Fifty five highly aluminium tolerant inbred lines developed 2. Ten highly phosphorus efficient inbred lines developed 3. Forty top-cross hybrids tolerant to aluminium and low P

developed (1 registered, 3 undergoing registration by Kenya Plant Health Inspectorate Service at NPT, others are being assessed: Phenotyping needed)

4. Twenty eight single cross hybrids (from lines tolerant to both Al toxicity/P deficiency developed (need phenotyping in laboratory and field needed)

5. Three locally adapted Kenyan germplasm introgressed with ZmMate1 gene from Brazil

6. Identified 6 SNP markers associated with Al tolerance QTLs 7. Two Kenyan PhD students are finalizing their studies

Summary of work to be done • Mapping of QTLs associated with P efficiency in

some Kenyan maize • Validation of QTLs for Al tolerance and P

efficiency • Pyramiding QTLs for Al toxicity tolerance and P

efficiency to generate better hybrids and synthetics

• Pyramiding ZmMATE1 (CATETO, Brazil) X 203B (Kenya) for enhanced tolerance to Al toxicity may go beyond 2014

• Phenotyping & Registration of Al/P efficient varieties for farmers remaning

Acknowledgements

• The Generation Challenge Program for funding.

• Our research collaborators for input and support.

• Workshop organizers for invitation to participate in the workshop.

KENYAN PROJECT TEAM MEMBERS

Thank you all

Generation Challenge programme (GCP)

Technology

GRM 2013: Marker-assisted breeding for improving phosphorus-use efficiency and tolerance to aluminum toxicity in maize -- S Gudu