by Kelvin Kamfwa, Karen Cichy and Jim Kelvin Kamfwa, Karen Cichy and Jim Kelly,, Symbiotic N fixation (SNF) in Beans • Symbiotic r/ship between bacteria and legumes • Occurs

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Text of by Kelvin Kamfwa, Karen Cichy and Jim Kelvin Kamfwa, Karen Cichy and Jim Kelly,, Symbiotic N...

  • 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

    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

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

    Understanding the genetic basis of symbiotic nitrogen fixation in common bean using genomic and transcriptomic analysesbyKelvin Kamfwa, Karen Cichy and Jim Kelly,, Symbiotic N fixation (SNF) in BeansResearch questions on genetic basis of SNF variabilityStudies to understand genetic basis of SNF variability in beansGenome-Wide Association Study for Agronomic traits & SNF GH & Field Phenotyping Phenotyping for Ndfa: 15N Natural Abundance Slide Number 8Conclusion on GWAS Identification of QTL for SNF in beansSlide Number 11Slide Number 12Conclusion for QTL mapping Slide Number 14Slide Number 15Slide Number 16