Investigating the Regulation of Grain Protein Concentration in the IPSRIs: Precision Phenotyping using Fluorescent Promoter Reporter Transgene Expression Christine Lucas 1 , Han Zhao 2 , Mayandi Sivaguru 3 , Steve Moose 1 1 Department of Crop Sciences; University of Illinois; Urbana, IL 61801 2 Jiangsu Academy of Agricultural Sciences, Nanjing City, China 3 Institute of Genomic Biology; University of Illinois; Urbana, IL 61801 Maize protein is deficient in several essential amino acids due to their low proportion within the α-zein storage proteins that constitute the majority (50-60%) of total protein in the kernel. Reducing α-zein protein accumulation is an important goal of Quality Protein Maize (QPM) breeders worldwide. QTL mapping efforts suggest that grain protein is governed by many genes with small phenotypic effects, but genetic regulators still remain largely unknown. NIR has been used, but it is known to be influenced by environmental factors and measures an indirect phenotype. An alternative strategy described here is a precision phenotyping method that involves a red fluorescent protein reporter, mRFP, fused to the regulatory sequences from a single α-zein gene. The mRFP transgene was crossed to the Illinois Protein Strain Recombinant Inbreds (IPSRIs), an advanced intermated RIL mapping population derived from the cross of Illinois High Protein (IHP) and Illinois Low Protein (ILP), and three years of phenotypic data have been collected on the IPSRIs using both NIR and mRFP methods. Candidate genes in the zein pathway and the asparagine cycling pathway, which exhibit allele frequency shifts and expression variation in both the IPSRIs and the IHP and ILP populations, were associated with both phenotypic data sets. The results indicate that the mRFP phenotype allows for the separation of genetic factors that indirectly influence α-zein accumulation, such as those that alter starch synthesis. GBS data on the IPSRIs will allow for a genome-wide association study using both sets of phenotypic data.
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Investigating the Regulation of Grain Protein Concentration in the IPSRIs: Precision Phenotyping using Fluorescent Promoter Reporter Transgene
Expression
Christine Lucas1, Han Zhao2, Mayandi Sivaguru3, Steve Moose1 1Department of Crop Sciences; University of Illinois; Urbana, IL 61801
2Jiangsu Academy of Agricultural Sciences, Nanjing City, China 3Institute of Genomic Biology; University of Illinois; Urbana, IL 61801
Maize protein is deficient in several essential amino acids due to their low proportion within the α-zein storage proteins that constitute the majority (50-60%) of total protein in the kernel. Reducing α-zein protein accumulation is an important goal of Quality Protein Maize (QPM) breeders worldwide. QTL mapping efforts suggest that grain protein is governed by many genes with small phenotypic effects, but genetic regulators still remain largely unknown. NIR has been used, but it is known to be influenced by environmental factors and measures an indirect phenotype. An alternative strategy described here is a precision phenotyping method that involves a red fluorescent protein reporter, mRFP, fused to the regulatory sequences from a single α-zein gene. The mRFP transgene was crossed to the Illinois Protein Strain Recombinant Inbreds (IPSRIs), an advanced intermated RIL mapping population derived from the cross of Illinois High Protein (IHP) and Illinois Low Protein (ILP), and three years of phenotypic data have been collected on the IPSRIs using both NIR and mRFP methods. Candidate genes in the zein pathway and the asparagine cycling pathway, which exhibit allele frequency shifts and expression variation in both the IPSRIs and the IHP and ILP populations, were associated with both phenotypic data sets. The results indicate that the mRFP phenotype allows for the separation of genetic factors that indirectly influence α-zein accumulation, such as those that alter starch synthesis. GBS data on the IPSRIs will allow for a genome-wide association study using both sets of phenotypic data.
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Christine Lucas 49th Illinois Corn Breeders’ School
March 4, 2013
Investigating the regulation of grain protein concentration in the IPSRIs: Precision phenotyping using fluorescent
promoter reporter transgene expression
Outline Summary of previous findings Background
– Illinois Long-term Selection Experiment
Materials and Methods – NIR Phenotype – mRFP Phenotype
Results of Candidate Gene Associations with: – Zein Pathway Genes – Asparagine Cycling Pathway Genes
Comparison of Results using each Method (NIR vs. mRFP) Conclusions Ongoing work
– GWAS
Genetic architecture of kernel composition traits
• Reviewed in Moose et al., 2004 • Goldman et al., 1993 • Dijkhuizen et al., 1998 • Sene et al., 2001 • Dudley et al., 2004 • Dudley et al., 2007 • Cook et al., 2012
– 21-26 QTL for kernel composition traits using NAM
Main Conclusions with respect to genetic architecture: Tens to Hundreds of genes with small phenotypic effects.
ANOVA Table Red/Green R-Square Coef. Var Root MSE Mean
0.95 11.95 0.25 2.1
Source DF Type III SS Mean Sq. F-Value Pr>F Year 2 12.71 6.36 100.51 <.0001 Geno 138 85.18 0.62 9.76 <.0001 Earrep 9 2.42 0.27 4.25 <.0001 Year*Geno 239 34.08 0.14 2.25 <.0001 Year*Earrep 2 0.45 0.23 3.58 0.0301 Geno*Earrep 165 13.81 0.08 1.32 0.0372
mRFP Phenotypic Data 2010
2011
2012
Red Channel/ Green Channel Year Mean Range Variance Std Dev. Coeff. Var. 2010 2.09 2.46 0.15 0.38 18.42 2011 1.93 2.60 0.18 0.42 21.91 2012 2.29 4.68 0.37 0.61 26.56
Association of Asparagine Cycling Pathway Genes with mRFP in the IPSRIs
Why are we detecting different genes using mRFP vs. NIR?
C:N Balance in Seed Starch (biomass/ yield) Protein Zein
mRFP RANK 2010 2011 2012
NIR
RAN
K 2010 -0.07537 0.163234 0.158943 2011 0.470143 0.287994 2012 N/A
Rank correlation between NIR and mRFP NIR mRFP
PBF O2 22-kD zein 19-kD zein Asparaginase
AS3 bZIP Asparaginase
Sum
mar
y
We can use both methods to dissect apart the role of each pathway in regulating not just protein %, but α-zein protein %, as well as starch %.
Conclusions 1.) % zein is regulated by plant N:
– Asparagine cycling genes significant – No variation in color (mRFP) of segregating ear – Why there is a maternal effect. To change protein, select for different aspect of
ASN cycling pathway in the plant. Can make selections earlier in season without making crosses.
2.) % zein is regulated within the kernel as well, but this is the end product of the ASN cycling pathway:
– Zein pathway genes significant 3.) We confirm previous results of many genes, but they may not all have small effects.
– AS3 is an example of one gene with a large effect (1.4%). 4.) The mRFP precision phenotyping method works, but measures different aspects of zein regulation than the NIR method. 5.) The IPSRI mapping population is useful for testing if candidate genes in zein and ASN pathways were targets of selection in the ILTSE, but more markers needed across genome……….
Ongoing Work: GWAS using Genotyping By Sequencing (GBS)
Goals • To provide a significance threshold for candidate
genes • To identify new QTL regulating
A.) floury2/ 22-kD α-zein (mRFP phenotype) B.) protein, starch, oil (NIR phenotype),
• but not just QTL!!!! Genes! • Ultimate goal is to improve protein quality and to
understand regulatory variation underlying complex traits. Sequencing by the Institute of Genomic Diversity (Cornell University)
SNP Calling by Ed Buckler’s lab (Cornell University)
Acknowledgements UIUC PI Stephen Moose Committee members Pat Brown A. Lane Rayburn Sandra Rodriguez-Zas SDS-PAGE & qRT-PCR Han Zhao AS3, ASNase, bZIP marker development Farag Ibraheem Yuhe Liu
Phenotypic Data Collection Michael Zinder Jarai Carter Susann Uphoff Loren Goodrich Michael O’Mara Dominique Thomas mRFP Method Development (Institute of Genomic Biology UIUC) Mayandi (Shiv) Sivaguru Glen Fried
Graduate Student Support Wesley Barber Yuhe Liu …….many more! GBS (Cornell University) Institute of Genomic Diversity Ed Buckler’s lab IPSRI Development John Dudley Monsanto Moose Lab
Funding for this research is from the USDA. Support for Christine is from the William B. and Nancy L. Ambrose Fellowship