Introgression of phosphorus
uptake 1 (Pup1) QTL into rice
varieties locally adapted in
sub-Saharan Africa
Khady Nani Dramé...
Outline
1. Introduction - Phosphorus (P) deficiency in sub-Saharan Africa

2. Distribution of Pup1 QTL in African germplas...
P-deficiency in SSA
• Plant-available P deficient in many soils
- low levels of P (inherent or depleted),
- high P-sorptio...
Genetic approach to mitigate P-deficiency
One of the most successful to date: identification and characterization of Pup1 ...
Pup1 gene-based markers
Pup1 locus on Chr. 12 aligned
in Nipponbare and Kasalath

Chin et al. (2010)

•

K1 and K20-1: for...
Distribution of Pup1 in African germplasm
Species

Number

Ecology of adaptation

O. sativa japonica

19

Upland

O. sativ...
Pup1 transfer into selected upland varieties
Donor

Recipients lacking PSTOL1
(targeted by K46-1)

2011

2012

2013

Kasal...
… Genotype selection
2. Recombinant selection (chr. 12) flanking Pup1
8 markers tested

8 markers tested
16.05 Mb

15.63 M...
Current status – Pup1 MABC
Combination for
F1 generation

No. of No. of seeds No. of lines No. lines No. of “true” No of l...
The hidden allele
African germplasm genotyped with K46-1 (PSTOL1 marker)

Pariasca-Tanaka et al. (2013)

L

N
1

N
2

N
3
...
Distribution of Pup1 alleles at OsPSTOL1
• New Pup1 allele found in CG14 – different from Kasalath Pup1 by 35 nt and new
p...
What about the other Pup1 genes?
First survey in O. glaberrima (32) showed:
K20-2 locus is absent
K46-1 revealed a differe...
What about the other Pup1 genes?
First survey in O. glaberrima showed:
K20-2 locus is absent
K46-1 revealed a different al...
Comparison of Pup1 genes between Kasalath and CG14
BLAST search against O. glaberrima genomic sequence for each Pup1 gene
...
The way forward
• Evaluation of Pup1-introgression lines developed (BC2F3) even
though Pup1 is present in the targeted var...
Acknowledgements
“A single finger can not lift a stone”

Acknowledgements to all contributors

 Donor – Japan (Japan Rice...
Thank you for
your attention
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Th1_Introgression of phosphorus uptake 1 (Pup1) QTL into rice varieties locally adapted in sub-Saharan Africa

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

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  • Phosphorus (P) is limiting for crop yield on > 30% of the world’s arable land World resources of inexpensive P may be depleted by 2050annual average P loss in SSA = 2.5 kg /ha (Sachez, 2012) nutrient loss cost 4 bilion USD
  • PSTOL1 was confirmed as a novel serine/threonine kinase gene whose overexpression in transgenic IR64 and Nipponbare significantly enhanced root growth and yield (60%)PSTOL1 regulates early crown root development and root growth, thereby enabling plants to acquire more phosphorus and other nutrients.Successfully transferred in Nipponbare, IR64, IR74 and 3 Indonesian varieties through MABC
  • Upland NERICAs and parents Field trials - Japan (2009, 2010) / JIRCAS - Togo (2009, 2010) / AfricaRice - Cotonou (2011) / AfricaRiceHydroponics - Cotonou (2011) / AfricaRicePopular upland varieties Field trials - Japan (2011) / JIRCAS - Ghana at SARI and Kumasi (2011) / JIRCAS - Cotonou (2011) / AfricaRice
  • Th1_Introgression of phosphorus uptake 1 (Pup1) QTL into rice varieties locally adapted in sub-Saharan Africa

    1. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 17. Thank you for your attention

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