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

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