This project aims to improve rice production on acid soils by identifying rice aluminum (Al) tolerance genes. Researchers found rice does not use the root tip Al exclusion mechanism seen in other cereals. Through linkage and genome-wide association analyses, they identified the ART1 transcription factor and Nrat1 Al transporter genes as contributing to Al tolerance. Near-isogenic lines were generated containing tolerant alleles of four QTL regions to use for breeding. Variation in Nrat1 was shown to result in tolerant and sensitive versions affecting Al uptake. The research provides markers, genes, and lines to improve rice Al tolerance.

1. GCP Project G7009.07:
Cloning, characterization and
validation of AltSB/Al tolerance in
rice
PI: Leon Kochian, USDA-ARS, Cornell University
Co-PI: Susan McCouch, Department of Plant
Breeding, Cornell University

2. Comparative Genomics Challenge Initiative
• Use discovery of sorghum Al tolerance gene, SbMATE, to identify rice Al tolerance genes
for improving rice production on acid soils.
• Found out early in this project that rice, the most Al tolerant cereal, doesn’t use the
root tip Al exclusion mechanism based on Al-activated organic acid release as a major Al
tolerance mechanism as in sorghum, wheat, barley, rye and to a degree, maize.
Al Exclusion Via Organic Acid Release
• Rice truly tolerates Al accumulated in root tip cell wall and
symplasm.
• No correlation between root tip citrate/malate release and
Al tolerance.
• Ma’s lab has cloned several candidate rice Al tolerance
genes involved in Al uptake, cell wall modification, and ART1
which may regulate expression of other tolerance genes

3. Ma (2007) Syndrome of Al toxicity & diversity of Al
resistance in higher plants. Int. Rev. Cyt. 264:225-253
Candidate Rice Al Tolerance Genes
Identified via Forward Genetics Approaches
• Art1: Al regulated transcription
factor that activates the
expression of candidate
tolerance genes.
• STAR1/2: PM ABC transporter
that exports UDP-Glucose to the
cell wall, possibly reducing Al
binding.
• FRL4: MATE citrate transporter
(homolog of SbMATE) that plays
a minor role in tolerance.
• Nrat1 & ALS1: Al transporters
that absorb Al from cell wall
(Nrat1) and sequester it in
vacuole.
• Ma found that none of these
genes mapped to previously
identified Al tolerance QTL

4. Conducted Joint Linkage-Genome Wide
Association Analysis of Rice Al Tolerance
Trait Index Chr QTL Flanking Markers LOD Allele effect R2
TRG 1 Alttrg1.1 RM319/RM315 4.56 Azucena 0.095
TRG 2 Alttrg2.1 RM526/RM318 3.44 Ir64 0.059
LRG 9 Altlrg9.1 RM257/RM160 6.57 Azucena 0.165
TRG 12 Alttrg12.1 RM453/RM512 7.85 Azucena 0.193
QTL Analysis of IR64 x Azucena RIL population
(Azucena is Al tolerant trop japonica and Ir64 is Al sensitive indica)
• Conducted high resolution mapping of largest QTL on chr 12.
• Gene responsible for chr 12 QTL is ART1, the zinc-finger
transcription factor that activates the expression of candidate
rice Al tolerance genes in response to Al.
• Generating NILs with tolerant ART1 allele (Azucena)
introgressed into Al sensitive IR64 and also reciprocal NIL.
• Valuable breeding line for improving indica Al tolerance and to
study effect of different ART1 alleles on expression of other
tolerance genes
• Also studying ART1 allelic diversity in rice association panel to
identify best alleles. Evaluating phenotypic impact via rice
transformation

5. GWAS of Aluminum Tolerance
All /EMMA
indica
aus
trop jap
temp jap
MATE Efflux
protein
MATE Efflux
protein
Root
elongation
STAR2
Isocitrate
lyase
Isocitrate lyase
Nrat candidate
gene
8 SNPs; p=2.8E-07
Ferriportin
domain protein
Iron-sulfur binding
Magnesium-transporter
ART1
C2H2 TF
Outlier Analysis
1 Million SNP Chip

6. Generation of Near Isogenic Lines
for QTL on Chr 1, 2, 9 and 12
• Will have NILs for each of these QTL in both IR64
and Azucena backgrounds by mid 2014.
• Am pyramiding the superior alleles for the 4 QTL
(3 from Azucena and 1 from IR64) in IR64.
• Hope to also pyramid these QTL with superior
OsPSTOL1 (Pup1) allele
• Breeding lines for increasing rice Al tolerance and
acid soil tolerance.
• Experimental materials for identifying novel Al
tolerance mechanisms/genes (chr 1,2, and 9)

7. GWAS Analysis of the PM Al Transporter: Nrat1
GWAS Peak on chr 2
colocalizes with Nrat1
and explains 40% of
the variation in aus Al
tolerance.
Zoom-in view of
GWAS peak co-
localizing with
Nrat1 in aus. Most
significant SNP is
marked by an
asterisk. Nrat1 is
indicated by a red
circle.Dr. Jianyong Li

8. ** ** *
Sensitive Aus
Tolerant Aus
Indica
Japonica
Region Promoter Coding sequence
SNPs M2 M3 M4 M5 M6 M8 M9 M12 M13 M15
Location(MSU V7) 2661026 2661163 2661328 2661476 2661525 1662980 1663056 1663674 1664197 1664242
Sensitive aus G G T C G T A A T C
Tolerant aus, indica and
japonica
A T A T T C G G C T
Tolerant and Sensitive Nrat1 Haplotypes Due
to SNPs in both Promoter and Coding Regions
Al tolerance of aus lines harboring
tolerant and sensitive Nrat1 haplotypes.
SNPs in tolerant
allele associated
both with
increased Nrat1
expression and
higher Al uptake
Al-Induced Nrat1
Expression

9. Functional characterization of Nrat1 in yeast. A: time
course of yeast growth in Al (50 µM). B: Al uptake in yeast
cells exposed to 50 to 200 µM Al. Black symbols/bars: cells
expressing the empty vector (control); white symbols/ bars:
cells expressing tolerant Nrat1-1; gray symbols/bars: cells
expressing sensitive Nrat1-2.
CONTROL
150 µM Al
Col 2a 2b 1a 1b
Root growth of representative plants from
four independent transgenic lines over-
expressing Nrat1, grown in the absence
(top) or presence (bottom) of Al. Col, wild-
type Columbia; 2a, 2b: lines expressing
sensitive Nrat1-2; 1a, 1b, lines expressing
tolerant Nrat1-1.
Functional Analysis of Tolerant and Sensitive Nrat1
Alleles in Yeast and Transgenic Arabidopsis
Hap1: Tol. Allele
Hap2: Sens. Allele
Sensitive
Allele
Tolerant
Allele

10. • Joint linkage-GWA analysis of rice Al tolerance has validated
candidate genes as bona fide Al tolerance genes (ART1 and Nrat1).
• Linkage analysis identified ART1 and GWA analysis identified Nrat1.
• This analysis has also identified a number of novel candidate Al
tolerance regions possibly involved in cell wall tolerance and Al
transport.
• Variation in Nrat1 DNA sequence results in tolerant and sensitive
versions of this PM Al uptake transporter. The tolerant Nrat1
transporter mediates greater Al influx across the root cell PM,
resulting in lower Al levels in the cell wall.
• Al uptake via Nrat1 appears to be a major Al tolerance mechanism.
There may be functional Nrat1 orthologs in maize (work by Claudia
Guimaraes) and sorghum.
Summary

11. PRODUCTS
• Markers flanking Al tolerance QTL on rice chr 1,2 , 9
and 12
• NILs harboring tolerant alleles of each of the 4 QTL
regions in Al both IR64 and Azucena background s as
well as all 4 QTL will be completed in 2014 to use for
breeding for improved Al tolerance.
• NILs harboring QTL on chr 1,2 and 9 a resource for
identifying rice novel Al tolerance genes and
mechanisms.
• Sequence for tolerant and sensitive alleles of two
major Al tolerance genes, ART1 and Nrat1.
• Transgenic wheat and maize expressing the tolerant
Nrat1 allele.