1. 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).

2. 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

3. 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.

4. 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)

5. 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
ClassCa 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

6. 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

7. 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

8. 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

9. Objective 1a: Screening of Kenyan maize germplasm for Al tolerance
0
5
10
15
20
25
30
35
40
45
50
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
Frequency
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

10. 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

11. Responsive Non responsive
P-InefficientP-efficient
+
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 PNo added P
P-efficient line

12. 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

13. 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

14. 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

15. 0
2
4
6
8
10
12
14
16
18
RelativeZmMATE1Expression
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

16. Entry Genotype Cross RRL (Al) RRL (0) RNRG
1
SYN AL X R12C10-1 Single cross 34.79 109.70 0.32
2 SYN AL X R12C10-10 Single cross 75.17 127.03 0.59
3 SYN AL X R12C10-8 Single cross 89.77 106.50 0.84
4 R11C10 X SYN AL X R11C10-4 Back cross 90.43 96.64 0.94
5
R11C10 X SYN AL X R11C10-5 Back cross 97.75 136.50 0.72
6 R11C10 X SYN AL X R11C10-7 Back cross 82.58 104.56 0.79
7 R11C10 X SYN AL X R11C10-8 Back cross 96.33 133.83 0.72
8 R11C10 X SYN AL X R11C10-9 Back cross 94.00 151.87 0.62
9
R12C10 X SYN AL X R12C10 Back cross 87.53 100.02 0.88
10 R12C10 X SYN AL X R12C10-5 Back cross 67.21 119.05 0.56
11 R12C10 X SYN AL X R12C10-4 Back cross 89.07 114.80 0.78
12 SYN AL X AO89 X AO89-2 Back cross 47.40 128.37 0.37
13
SYN AL X AO89 X AO89-3 Back cross 66.80 107.20 0.62
14 SYN Al X AO89 X AO89-5 Back cross 71.12 129.79 0.55
15 SYN AL X AO89 X AO89-40 Back cross 69.67 120.93 0.58
16 AO89 Parent 56.40 179.00 0.32
OBJECTIVE 4b (ii): Aluminium tolerance of introgression material based on
solution culture phenotyping

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

18. 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

19. 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

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

21. Maize Lethal Necrosis Disease
interfered with F2:3 maize population

22. Phenotyping for QTL association mapping for P is ongoing…
Advancing F2 to F2:3 at Migori site in April-Sept. 2013

23. 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

24. 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

25. 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

26. Acknowledgements
• The Generation Challenge Program for
funding.
• Our research collaborators for input and
support.
• Workshop organizers for invitation to
participate in the workshop.

27. KENYAN PROJECT TEAM MEMBERS

28. Thank you all
Generation Challenge
programme (GCP)