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Genome-Wide Association Study of Symbiotic Nitrogen Fixation Effectiveness
in Medicago tuncatula
Raul Huertas, PhD
Humans
Importance of legumes
symbiotic nitrogen fixation
Livestock Soil conservation
2
Symbiotic nitrogen fixation
Exchangefixed/reduced carbon
forfixed/reduced nitrogen
Difficult to integrate in genomics-assistedbreeding strategies
Many plant genes are known to be required for SNF Few associated with SNF effectiveness
50 MMt N/year=$30 Billions
N2
NH3
CO2
CH2O
ATP + e-
3
Genomics-assisted breedingGenomic Selection
Marker-assisted selection
Inbred lines
Marker associated with desirable trait Genomic Estimated Breeding Values
Genome wide predictionTILLING
Non- Segregating
Genomics approaches for plant improvement
Adapted from Pandey et al., 2016. Front Plant Sci. 7:455
Bi-parental (QTL) and multi-parent association mapping
MAGICNAM
RILs,NILs
Segregating
Training population
Genome-wide association studies (GWAS)
Genotyping + PhenotypingGenome-wide association
studies (GWAS)
Germplasm
4
Mutants
M. truncatula(Stanton-Geddes et al., 2013; Kang et al., 2015; Le Signor et al., 2017; Kang et al., 2018)
Successful use of GWAS in legumes
Alfalfa, common bean, soybean, chickpea, pigeonpea and cowpea(Dhanapal et al., 2015; Kamfwa et al., 2015; Sonah et al., 2015; Upadhyaya et al., 2015; Varshney et al., 2017;Xu et al., 2017; Biazzi et al., 2017; Liu and Yu, 2017; Ravelombola et al., 2017; Yu et al., 2017; Li et al., 2018;Resende et al., 2018; Ravelombola et al., 2018)
Agronomic traits evaluated• Phenology and morphology• Forage quality• Seed development and quality• Tolerance to biotic and abiotic stresses• Performance under nutrient limitations (phosphorus)• SNF - nodulation• SNF effectiveness
5
Identify genes or genomic regions in M. truncatula thataccount for variation in SNF effectiveness in nature.
Establish proof-of-concept for genomic-based plant-breeding approaches to enhance SNF in forage and croplegumes.
Objectives
6
Medicago truncatulaHAPMAP PROJECT
266 accessions sequenced
20X coverage in 28 accessions 5X coverage or higher in 238 accessions
High-density SNP-based genetic map
http://www.medicagohapmap.org/
Population structure and allele frequency
Medicago truncatula Hapmap population
7
Low SNF-E
Control SNFSNFControl
Intermediate SNF-E
SNF Effectiveness: Impact of SNF on plant growth
SNF = low mineral-N + Rhizobia Control = Optimal mineral-N
SNFControl
High SNF-E
Control SNF
High SNF-E
Defining our trait(s) of interest
8
Non-inoculated +S. meliloti 1021 +S. meliloti KH46c +S. medicae WSM419
SNF with individual rhizobia strains
Phenotyping SNF effectiveness witha mixture of Rhizobia strains:KH46c and WSM419
Phenotyping for SNF effectivenessReduced mineral N
0 . 0
0 . 1
0 . 2
0 . 3
0 . 4
S u f f i c i e n t N
n o n - i n o c u l u m
a
R e d u c e d N
+ M i x t u r e
a b
R e d u c e d N R e d u c e d N
+ H K 4 6 c + W S M 4 1 9
b b
Control[+ N]
DW
(g p
lant
-1)
9
SNF
effe
ctiv
enes
s (%
)
Hapmap accession lines H
M183
HM
1 1 7
HM
1 5 6
HM
1 5 0
HM
0 8 4
HM
0 9 1
HM
1 5 4
HM
2 6 8
HM
0 4 0
HM
0 4 8
HM
1 5 9
HM
3 1 0
HM
0 6 7
HM
3 0 6
HM
2 8 9
HM
0 6 6
HM
0 3 5
HM
1 4 8
HM
0 8 5
HM
1 2 4
HM
1 5 3
HM
1 8 9
HM
0 8 0
HM
1 4 9
HM
2 7 1
HM
3 1 1
HM
1 8 1
HM
0 9 3
HM
2 5 3
HM
0 5 4
HM
1 0 1
HM
1 2 7
HM
0 6 9
HM
1 6 6
HM
1 7 8
HM
0 4 3
HM
2 9 4
HM
0 4 5
HM
1 0 8
HM
1 7 0
HM
2 0 7
HM
2 5 6
HM
1 8 2
HM
3 0 2
HM
0 4 1
HM
2 0 6
HM
0 1 1
HM
1 9 7
HM
1 3 4
HM
0 7 1
HM
1 8 7
HM
2 7 0
HM
1 5 7
HM
1 5 5
HM
0 9 8
HM
2 9 3
HM
2 0 8
HM
2 6 9
HM
0 7 5
HM
1 6 3
HM
2 9 0
HM
0 4 9
HM
3 0 0
HM
3 0 1
HM
1 2 9
HM
1 7 7
HM
0 7 0
HM
0 6 8
HM
1 0 9
HM
2 0 2
HM
1 3 1
HM
1 8 4
HM
2 9 5
HM
0 7 6
HM
0 6 4
HM
1 5 1
HM
1 3 8
HM
0 2 8
HM
1 7 9
HM
1 6 7
HM
1 9 1
HM
2 9 9
HM
2 8 7
HM
0 5 1
HM
3 1 4
HM
2 7 7
HM
3 1 3
HM
0 8 9
HM
1 8 8
HM
0 9 9
HM
1 1 4
HM
1 4 7
HM
1 7 5
HM
3 0 8
HM
1 0 7
HM
0 4 7
HM
1 1 9
HM
2 6 2
HM
1 8 5
HM
0 7 4
HM
1 7 2
HM
0 6 0
HM
0 8 2
HM
0 5 6
HM
2 8 8
HM
2 7 6
HM
0 4 2
HM
0 5 9
HM
1 7 6
HM
1 2 0
HM
0 6 1
HM
0 3 4
HM
1 3 0
HM
1 6 9
HM
0 0 7
HM
1 5 2
HM
1 1 8
HM
0 8 8
HM
0 9 2
HM
1 6 0
HM
1 2 6
HM
1 4 5
HM
0 1 0
HM
3 0 9
HM
1 2 8
HM
2 5 9
HM
0 5 5
HM
1 2 1
HM
0 4 6
HM
2 0 9
HM
3 1 2
HM
1 1 1
HM
0 8 3
HM
0 3 1
HM
0 8 6
HM
0 3 6
HM
3 0 5
HM
0 5 2
HM
1 9 2
HM
0 4 4
HM
1 8 6
HM
2 6 0
HM
1 1 2
HM
0 7 8
HM
0 7 2
HM
0 3 8
HM
2 9 7
HM
1 1 5
HM
0 9 5
HM
0 9 7
HM
1 2 2
HM
0 8 1
HM
1 3 9
HM
1 6 5
HM
0 5 7
HM
0 7 3
HM
1 8 0
HM
3 1 5
HM
1 7 3
HM
1 9 8
HM
1 6 1
HM
2 9 62 0
4 0
6 0
8 0
1 0 0High
Intermediate
Low
Control SNF
High SNF-E
Control SNF
Low SNF-E
Freq
uenc
y
SNF-E (%)
High natural variation for SNF effectiveness
10
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .20 .0
0 .2
0 .4
0 .6
0 .8 r = 0.80p value = 0.0001
Bio
mas
s (g
DW
pla
nt-1
)[S
NF]
Biomass (g DW plant-1)[Control]
0 2 0 4 0 6 0 8 0 10 00 .0
0 .2
0 .4
0 .6
0 .8 r = 0.27p value = 0.0001
Bio
mas
s (g
DW
pla
nt-1
)[S
NF]
SNF-E (%)
0 2 0 4 0 6 0 8 0 10 00 .0
0 .2
0 .4
0 .6
0 .8
1 .0
1 .2 r = - 0.31p value = 0.0001
Bio
mas
s (g
DW
pla
nt-1
)[C
ontr
ol]
SNF-E (%)
SNF = low mineral-N + Rhizobia Control = Optimal mineral-N
SNF-E is not determined by plant size
11
-Log
10(P
-Val
ue)
Chromosome
Manhattan plot for SNF effectiveness
12
Tejada-Jimenez et al., 2017. Medicago truncatula MOT1.3 is a plasma membrane molybdenumtransporter required for nitrogenase activity in root nodules.New Phytol, 216(4):1223-1235.
Sulfate transporter-like protein
Induced (x30) during nodulation
Nodule
Root
RNA-sed data
Potentially-interesting gene: MtMOT1.3
13
Preliminary list of potentially-interesting genes
14
Established experimental conditions to evaluate SNF effectivenessin the Medicado truncatula Hapmap population.
High natural variation for SNF effectiveness among accessions.
GWAS utilized 1.7 million high-quality SNPs. Clusters of SNPsassociated with SNF effectiveness on chromosomes 3, 4 and 7.
About 25 potentially-interesting genes have been identified, someof which have been selected for further characterization.
Summary
15
Integrate data from the last biological replicate (ongoing)
Validation of selected candidate genes:- Expression patterns analysis of best and worst accessions.
Ongoing and future directions
Implement in a legume crop- Contact rhuertas@noble.org for potential collaborations
16
GWAS for SNF effectiveness under phosphorus limitationconditions (ongoing).
Michael Udvardi, PhDIvone Torres-Jerez
Functional Genomics LabYun Kang, PhD
Noble Research Institute
Molecular Biological Materials Core FacilityGreenhouse Staff
Ag Services and Research Operations
Acknowledgements
17
Genome-Wide Association Study of Symbiotic Nitrogen Fixation Effectiveness
in Medicago tuncatula
Raul Huertas, PhD
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