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
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
• 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
P-deficiency in SSA
Development of cultivars with enhanced tolerance to P-deficiency to improve rice yield in a cost effective and sustainable way
One of the most successful to date: identification and characterization of Pup1 QTL
Genetic approach to mitigate P-deficiency
Pup1 explained close to 80% of the variation observed
Pup1 identified in Kasalath on chr. 12
Pup1 locus includes an INDEL absent from Nipponbare genome
Pup1 major determinant is a kinase gene located in the INDEL root growth and development
15 YEARS LATER
Pup1 gene-based markers
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
KKKKKK N
Profile of K20-2 amplicons digested with Bsp1286I
K20-2, K46-1 and K52 were selected as diagnostic markers of Pup1
Pup1 locus on Chr. 12 aligned in Nipponbare and Kasalath
Profile of K46-1 amplicons (dominant marker)
K N N K NN K
Distribution of Pup1 in African germplasm
O. sativa
japonica
O. sativa
indica
NERICA
NERICA-L
O. glab
errim
a
O. bart
hii0
10
20
30
40
50
60
70
80
90
Mean KMean NMean other
Average frequency of Kasalath (K), Nipponbare (N) and unknown (other) alleles across loci targeted by K20-2, K46-1 and K52 markers
Species Number Ecology of adaptationO. 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
Pup1 transfer into selected upland varieties
Donor Recipients lacking PSTOL1 (targeted by K46-1)
2011 2012 2013
Kasalath NERICA 1, 4, 10 Dourado-PrecoceWAB 96-1-1 WAB 189-B-B-B-8-HB WAB 515-B-16-A2-2
Pup1 survey
F1 lines
Genotype selection
BC1F1 from 7 crosses
Genotype selection
BC2F1 from 4 crosses
1. Foreground selection (chr. 12), Pup1-gene based markers
2. Recombinant selection (chr. 12) flanking Pup1
15
.28
Mb
14
.93
Mb
8 markers tested
16
.05
Mb
15
.63
Mb
8 markers testedPup1
15
.47
Mb
15
,31
Mb
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
Two polymorphic makers selected at 5’ end and one
marker at 3’ end
… Genotype selection
Current status – Pup1 MABC
Combination for BC1F1 generation
No. of lines sown
No. of lines genotyped
No. of lines with Pup1
No. of recombinants
No of BC2F1 seeds obtained
NERICA 1/Kasalath 477 360 128 34 1004
NERICA 4/Kasalath 472 321 177 24 1412
NERICA 10/Kasalath 477 283 85 35 829
WAB 515-/Kasalath 477 405 196 24 2178
Combination for F1 generation
No. of crosses
No. of seeds obtained
No. of lines genotyped
No. lines with Pup1
No. of “true” F1
No of lines backcrossed
No. of BC1F1 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
0% DMSO
5% DMSO
10% DMSO
A new story starts…
African germplasm genotyped with K46-1 (PSTOL1 marker)
The hidden allele
Pariasca-Tanaka et al. (2013)
Amplicons sequenced
• 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
Distribution of Pup1 alleles at OsPSTOL1
Pup 1a –Kasalath allele
Pup 1b – CG14 allele
K46-1fw
Ksp-3rv
CGsp-2fw
K46-1rv
K46-1fw
Ksp-3rv
342bp
CGsp-2fw
K46-1rv
258bp
Single primers pairs
Duplexed primer pairs
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
Upland NERICA 18 3 15 0 0
Lowland NERICA* 60 13 5 43 0
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
C C K C C C C K K K K N K N
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)
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?
Comparison of Pup1 genes between Kasalath and CG14
BLAST search against O. glaberrima genomic sequence for each Pup1 gene
INDEL
OsPupK43 OsPSTOL1OsPupK45
OsPupK59 OsPupK66
Kasalath
OsPupK01 OsPupK05
CG14
OsPupK01 OsPupK05 OsPupK20 OsPupK29 OsPupK66Nipponbare
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
OsPupK52OsPupK01 OsPupK05 OsPupK20 OsPupK29
INDEL
OsPupK43 OsPSTOL1 OsPupK66OsPupK45
OsPupK59OsPupK52
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)
Acknowledgements
Donor – Japan (Japan Rice Breeding Project, 2010-2014)
Collaborators – AfricaRice, JIRCAS, IRRI
Support staff
“A single finger can not lift a stone” Acknowledgements to all contributors
Thank you for
your attention