3rd Africa Rice Congress Theme 1: Climate resilient rice Mini symposium: towards improved resistance to abiotic stresses Author: Drame et al.

1. Introgression of phosphorus
uptake 1 (Pup1) QTL into rice
varieties locally adapted in
sub-Saharan Africa
Khady Nani Dramé et al.
Africa Rice Center – ESA regional office, Tanzania
k.drame@cgiar.org

2. Outline
1. Introduction - Phosphorus (P) deficiency in sub-Saharan Africa
2. Distribution of Pup1 QTL in African germplasm
3. Status of Pup1 introgression into selected rice varieties
4. Genomic organization of Pup1 locus in O. glaberrima species
5. Conclusion - The way forward

3. P-deficiency in SSA
• Plant-available P deficient in many soils
- low levels of P (inherent or depleted),
- high P-sorption capacity (530 million ha, ~25% of land area)
• Annual fertilizer consumption in Africa = 0.8% (1.29 Mt) of global
fertilizer consumption (IFDC, 2013)
• P-fertilizer price is peaking and it is a finite resource
unaffordable for smallholders
Development of cultivars with enhanced tolerance to P-deficiency to
improve rice yield in a cost effective and sustainable way

4. Genetic approach to mitigate P-deficiency
One of the most successful to date: identification and characterization of Pup1 QTL
Pup1 identified in
Kasalath on chr. 12
Pup1 explained close to 80%
of the variation observed
Pup1 locus includes an INDEL
absent from Nipponbare genome
15
YEARS
LATER
Pup1 major determinant is a
kinase gene located in the INDEL
root growth and development

5. Pup1 gene-based markers
Pup1 locus on Chr. 12 aligned
in Nipponbare and Kasalath
Chin et al. (2010)
•
K1 and K20-1: for both markers, we
could not separate the N and K alleles
which differ by only 3 bp
•
K20-2 digested with Bsp1286I: reliable
marker
•
K46-1, K46-2 and K52 amplified as
expected
Profile of K20-2 amplicons digested with Bsp1286I
K
K
K
K
N
K
K
Profile of K46-1 amplicons (dominant marker)
N
K
N
N
K
N
K
K20-2, K46-1 and K52 were selected
as diagnostic markers of Pup1

6. Distribution of Pup1 in African germplasm
Species
Number
Ecology of adaptation
O. sativa japonica
19
Upland
O. sativa indica
17
Lowland (rainfed and irrigated)
Interspecific O. sativa x O. glaberrima (NERICA)
18
Upland
Interspecific O. sativa x O. glaberrima (NERICA-L)
60
Lowland
O. glaberrima
31
Lowland, Upland and Floating
O. barthii
3
Upland
90
80
Average frequency of Kasalath
(K), Nipponbare (N) and unknown
(other) alleles across loci targeted
by K20-2, K46-1 and K52 markers
70
60
50
Mean K
40
Mean N
30
Mean other
20
10
0
O. sativa
japonica
O. sativa
indica
NERICA
NERICA-L
O.
O. barthii
glaberrima

7. Pup1 transfer into selected upland varieties
Donor
Recipients lacking PSTOL1
(targeted by K46-1)
2011
2012
2013
Kasalath
NERICA 1, 4, 10
Dourado-Precoce
WAB 96-1-1
WAB 189-B-B-B-8-HB
WAB 515-B-16-A2-2
Pup1 survey
Genotype
selection
Genotype
selection
BC1F1 from
7 crosses
BC2F1 from
4 crosses
F1 lines
1. Foreground selection (chr. 12), Pup1-gene based markers

8. … Genotype selection
2. Recombinant selection (chr. 12) flanking Pup1
8 markers tested
8 markers tested
16.05 Mb
15.63 Mb
15.47 Mb
15,31 Mb
15.28 Mb
14.93 Mb
Pup1
Two polymorphic makers
selected at 5’ end and one
marker at 3’ end
3. Background selection (chr. 1 to 12)
281 SSR markers tested and 104 to 112 polymorphic markers identified
384 SNP markers tested and 246 to 277 polymorphic markers identified

9. Current status – Pup1 MABC
Combination for
F1 generation
No. of No. of seeds No. of lines No. lines No. of “true” No of lines
No. of BC1F1
crosses obtained genotyped with Pup1
F1
backcrossed seeds obtained
NERICA 1/Kasalath
4
54
31
27
26
17
1048
NERICA 4/Kasalath
1
19
4
3
3
2
487
NERICA 10/Kasalath
2
41
15
10
10
9
547
DOURADO/Kasalath
1
90
27
14
14
12
930
WAB 96-1-1/Kasalath
1
16
2
1
1
1
586
WAB 189-/Kasalath
2
155
22
10
7
7
1084
WAB 515-/Kasalath
5
111
24
14
13
9
1590
Combination for BC1F1
generation
NERICA 1/Kasalath
NERICA 4/Kasalath
NERICA 10/Kasalath
WAB 515-/Kasalath
No. of lines
sown
No. of lines
genotyped
No. of lines
with Pup1
477
472
477
477
360
321
283
405
128
177
85
196
No. of
No of BC2F1
recombinants seeds obtained
34
24
35
24
1004
1412
829
2178

10. The hidden allele
African germplasm genotyped with K46-1 (PSTOL1 marker)
Pariasca-Tanaka et al. (2013)
L
N
1
N
2
N
3
N
4
N
5
N
6
N
7
N N N N N N N
Amplicons sequenced
8 9 1 1 1 1 1
0 1 2 3 4
N
1
5
N
1
6
N
1
8
N
1
7
W
5
0
C
G
W W
1 1
0 8
4
A new story starts…
N
b
K
a
s

11. Distribution of Pup1 alleles at OsPSTOL1
• New Pup1 allele found in CG14 – different from Kasalath Pup1 by 35 nt and new
primers specific of each allele designed (JIRCAS)
• Allele specific primers used to genotype 145 samples from AfricaRice
Pup 1a –Kasalath allele
K46-1fw
Total
K
C
N
U
O. glaberrima
31
1
29
1
0
O. barthii
3
0
3
0
0
O. sativa indica*
14
5
1
9
0
O. sativa japonica
19
3
14
2
0
Ksp-3rv
Duplexed
CGsp-2fw primer pairs
Upland NERICA
18
3
15
0
0
K46-1rv
Lowland NERICA*
60
13
5
43
0
Ksp-3rv
342bp
Pup 1b – CG14 allele
CGsp-2fw
Single
primers
pairs
K46-1rv
258bp
K46-1fw
C
C
K
C
C
C
C
K
K
K
K
N
K
N
K = Kasalath allele at PSTOL1
C = CG14 allele;
N = Nipponbare allele
U = unknown
* = in these groups, one sample has both K and C allele at OsPSTOL1

12. What about the other Pup1 genes?
First survey in O. glaberrima (32) showed:
K20-2 locus is absent
K46-1 revealed a different allele
K52 locus is largely present
Chin et al. (2010)

13. What about the other Pup1 genes?
First survey in O. glaberrima showed:
K20-2 locus is absent
K46-1 revealed a different allele
K52 locus is largely present
Chin et al. (2010)
???
Different sequences?
Missing genes?

14. Comparison of Pup1 genes between Kasalath and CG14
BLAST search against O. glaberrima genomic sequence for each Pup1 gene
Nipponbare
OsPupK01
OsPupK05
OsPupK20
OsPupK66
OsPupK29
INDEL
Kasalath
OsPupK01
OsPupK05
OsPupK20
OsPupK29
OsPupK43
OsPSTOL1
OsPupK59
OsPupK52
OsPupK45
OsPupK66
INDEL
CG14
OsPupK01
OsPupK05
OsPupK43
OsPSTOL1
OsPupK59
OsPupK52
OsPupK45
OsPupK66
Some of the genes present in Pup1 region (Kasalath) are missing from CG14
genome either partially or completely but the INDEL is present contrary to
Nipponbare where the INDEL is missing

15. The way forward
• Evaluation of Pup1-introgression lines developed (BC2F3) even
though Pup1 is present in the targeted varieties (except WAB515)
• Assessment of the efficiency of CG14-allele at OsPSTOL1 vs
Kasalath allele
• Development of new Pup1-introgression lines (K or C allele) in the
background of lowland rice varieties
• Use of new Pup1 donors more adapted and with better grain quality
than Kasalath in next Pup1 MABC.
upland - IAC165, IR12979, N15, N16, N18
lowland - BW348-1, Saro5, Gambiaka, NL15, NL43 (and 10 other NL)
• Search for new sources of P-deficiency tolerance (mainly PUE)

16. Acknowledgements
“A single finger can not lift a stone”
Acknowledgements to all contributors
 Donor – Japan (Japan Rice Breeding Project, 2010-2014)
 Collaborators – AfricaRice, JIRCAS, IRRI
 Support staff

17. Thank you for
your attention