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