Download pdf - by Kelvin Kamfwa, Karen Cichy and Jim Kellygl2016conf.iita.org/wp-content/uploads/2016/03/Understanding-the... · by Kelvin Kamfwa, Karen Cichy and Jim Kelly,, Symbiotic N fixation

Understanding the genetic basis of symbiotic nitrogen fixation in

common bean using genomic and transcriptomic analyses

by

Kelvin Kamfwa, Karen Cichy and Jim Kelly

,,

Symbiotic N fixation (SNF) in Beans

• Symbiotic r/ship between

bacteria and legumes

• Occurs in nodules

• SNF is genetically complex

• Adequate genetic variability exists

• But genetic basis of this

variability is poorly understood

Research questions on genetic basis of SNF variability

• Understanding molecular genetic architecture of SNF variability in beans:

– What genomic regions and/ or genes underlie SNF variability? – How much control do these regions/genes have on SNF variability – Which tissues are they expressed in? – What molecular functions/mechanisms they are involved in?

This genetic knowledge can be used to develop breeding strategies & molecular resources

Studies to understand genetic basis of SNF variability in beans

• Three independent, but complementary studies : 1. Genome-wide association analysis of agronomic traits

& SNF in bean 2. Identification of QTL for SNF in beans 3. Transcriptome analysis of two recombinant Inbred

Lines of beans contrasting for SNF

Genome-Wide Association Study for Agronomic traits &

SNF

• Andean Diversity Panel comprised of 270 genotypes

• Landraces, breeding lines and varieties (globally)

• Phenotyped for SNF and associated

traits in the field and Greenhouse

GH & Field Phenotyping Diversity Panel of 270 genotypes GH (Year: 2012 & 2013):

Inoculated: Rhizobia tropici (CIAT 1899) N-free nutrient solution

Nitrogen derived from atmosphere (Ndfa)

quantified at flowering Field (Year: 2012 & 2013)

Montcalm Research farm, MI Low N site Non-Nodulating bean mutant as control

G51396A

Phenotyping for Ndfa: 15N Natural Abundance

• Ndfa measured on shoot biomass flowering and on seed

Chromosome

-Log

10(P

)

GH_12

Field_13

GH_14

Ndfa

Kamfwa et al. 2015 Theor. Appl. Genet.

Consistently identified sig SNPs on Pv03, 07, 09 across envns & tissues

R2: Pv07=0.11 Pv09=0.12

R2: Pv03=0.23 Pv07=0.12 Pv09=0.14

R2: Pv03=0.12 Pv09=0.20

-Log

10(P

)

MLM: 5300 SNPs

Conclusion on GWAS

• Genomic regions (Pv03, 07, 09) for SNF consistently

identified across tissue types & environment

– Suggesting stable contribution to SNF variability

Identification of QTL for SNF in beans

• 188 F4:5 RILs of Solwezi x AO-1012-29-3A • Solwezi: Landrace widely grown in

Zambia

• AO-1012-29-3A from Puerto Rico : Light red kidney, resistant to bruchids

• Solwezi superior to AO-1012-29-3A in SNF

• Evaluated for SNF in 2014 and 2015 in the GH at MSU

Solwezi AO-1012-29-3A

Pv07 Pv09

QTL for Ndfa Identified on Pv07 and Pv09

ss7156491280.0ss7156484141.5ss7156484221.7

ss7156457455.1

ss71564863813.8ss71564797714.0ss71564797814.1ss71564664214.4ss71564700616.7ss71564882717.7ss71564855719.2ss71564571219.6ss71564712921.6ss71564713021.7ss71564861022.8ss71564645324.1ss71564570524.2ss71564644724.5ss71564853926.8ss71564875527.5ss71564603828.6ss71564604329.4ss71564604129.6ss71564769031.1

ss71564816835.1

ss71564636136.7


ss71564717064.1

ND

FA9.1

BM

9.10 1 2 3 4 5 6 7

ss7156463530.0

ss7156486926.3

ss7156486369.4

ss71564715914.3ss71564568317.1ss71564839718.6ss71564838918.7ss71564839619.5ss71564839221.1ss71564568624.4ss71564715825.7ss71564772730.6ss71564521335.6ss71564602138.5ss71564602838.6ss71564603438.8ss71564603639.1ss71564857339.2ss71564857439.3ss71564603040.7ss71564906742.2ss71564804444.0ss71564858047.0ss71564856547.3ss71564857047.4ss71564602947.6ss71564601247.9ss71564523548.3ss71564601148.4ss71564584249.3ss71564584349.9ss71564584050.2ss71564584750.5ss71564583950.6ss71564523653.3ss71563920653.6ss71564523954.3ss71564661155.5ss71564660955.7ss71564661255.8ss71564661356.0ss71564677456.6ss71563934156.8ss71564521059.8ss71564520960.2ss71564522261.1ss71564521861.8ss71564522562.1ss71564523162.6ss71564523062.7

ND

FA7.1

BM

7.2 %N

7.1

RD

W7.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Several co-localizations on Pv07: Pleiotropy or linkage??

R2 for Ndfa: Pv07=0.14 Pv09=0.08

Chromosome

GH_12

Field_13

GH_14

GWAS & QTL Mapping Corroborate on Pv09

ss7156491280.0ss7156484141.5ss7156484221.7

ss7156457455.1


ss71564816835.1

ss71564636136.7


ss71564717064.1

ND

FA9.1

BM

9.10 1 2 3 4 5 6 7

Pv09

Conclusion for QTL mapping

• On Pv09 genomic regions identified in GWAS and

QTL mapping overlapped – Stable QTL across genetic backgrounds

• SP36 and SP118 F4:5 RILs selected from

Solwezi x AO-1012-29-3A • What genes underpin contrasting

differences of SP36 & SP118? • RNA: leaves, nodules and roots of SP36

and SP118 grown under N fixing and non-fixing condition

• Sequenced using Illumina technology • Analyses

– Differentially expressed genes between

SP36 and SP118 (DESeq2) – Overlaying expression over GWAS results

to identify candidate genes at GWAS peaks

Transcriptome Analysis of Two RILs of Bean Contrasting for Symbiotic Nitrogen Fixation

Chromosome

GH_13

0

5

10

15

20

25

30

35

40

Nor

mal

ized

read

cou

nt

Field_13

GH_13

Overlaying RNA-seq on GWAS data: Pv07 & Pv09

Phvul.007G048000 (MADS BOX TF): 4/51 genes within 400 kb of GWAS peak on Pv07

0

50

100

150

200

250

300

Nor

mal

ized

read

cou

nt

Phvul.009G137500 (WRKY TF): In 400 kbp of GWAS peak on Pv09 there are 44 genes but only WRKY was DE

Acknowledgments Dr. Jim Kelly Dr. Karen Cichy University of Zambia Members of Kelly and Cichy Labs