W. Strek*, R. Tomala, B. Cichy, M. Lukaszewicz, A. Olejniczak
by Kelvin Kamfwa, Karen Cichy and Jim...
Transcript of by Kelvin Kamfwa, Karen Cichy and Jim...
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
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
ss71564564241.5ss71564563841.8ss71564563242.0ss71564918542.9ss71564762443.0ss71564762343.1ss71564563145.5ss71564565146.1ss71564939750.1ss71564940150.2ss71564563450.3ss71563922650.4ss71564560750.5ss71564561350.6ss71563959652.3ss71563959752.6ss71564030053.0ss71564051953.6ss71564718355.9ss71564718456.3
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
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
ss71564564241.5ss71564563841.8ss71564563242.0ss71564918542.9ss71564762443.0ss71564762343.1ss71564563145.5ss71564565146.1ss71564939750.1ss71564940150.2ss71564563450.3ss71563922650.4ss71564560750.5ss71564561350.6ss71563959652.3ss71563959752.6ss71564030053.0ss71564051953.6ss71564718355.9ss71564718456.3
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