FOODCROPS.VN 2017. Sarah M Potts. Identification of QTL and candidate genes for plant density tolerance in maize

1. Identification of QTL and candidate genes for plant density tolerance in maize
Sarah M. Potts, Rita H. Mumm, and Martin O. Bohn, Department of Crop Sciences; University
of Illinois at Urbana-Champaign.
Though demand for grain continues to rise, the amount of available agricultural land is
unlikely to change in the near future. To continue to increase crop yields to meet these growing
demands, it is suggested that improving yield will need to be achieved by increasing yield on a
per-unit basis. This can be achieved by raising plant density while retaining current ‘per plant’
yields. Furthermore, a portion of historical yield increases have been suggested as resulting from
increased plant density tolerance (Duvick 2005).
An initial plant density tolerance survey was conducted to ensure comprehensive
coverage of the many traits that may possibly be involved in plant density tolerance (Mansfield
and Mumm 2014). This survey identified six parent inbreds that were high yielders at high
density and 30 traits that were associated with plant density tolerance. Recombinant inbred line
(RIL) families derived from these inbreds were used to create a connected population of 320
testcross hybrids for the purposes of quantitative trait loci (QTL) mapping and candidate gene
identification. Yield trials of the hybrids were conducted at two locations in 2012 and three
locations in 2013, to evaluate the traits found to be associated with plant density tolerance in the
initial survey. The RIL parents of the 320 hybrids were genotyped using genotype-by-
sequencing (GBS) technology, and hybrid genotypes were inferred. Phenotypic and genotypic
analyses have been conducted and numerous QTL have been identified for traits correlated with
grain yield in the phenotypic analyses.

2. Identification of plant
density tolerance QTL in
maize
Sarah Potts| PhD Candidate
Martin Bohn, Rita Mumm| Advisors University of Illinois at Urbana-Champaign

3. Outline
 Background
 Objectives
 Materials and methods
 Results
 Conclusions
 Future work
 Questions Picture by Carrie Butts

4. Research Introduction
 Increasing demand for grain
 No change in arable land likely
 Previous yield trends
Duvick (2005)

5. Research Introduction
 Previous studies have focused on single traits or
categories of traits for plant density tolerance

6. The five categories hypothesized to
underlie plant density tolerance
• Photosynthetic capacity
• Growth response
• Plant architecture
• Source-sink relationship
• General stress tolerance

7. Initial Density Survey
 12 ex-PVP parents
 North Carolina Design II
 6 densities (19k-54k ppa)
 48 phenotypic traits measured
 3 environments over 2 years

8. Initial Density Survey
 48 phenotypic traits 30 traits
 5 top yielding hybrids
 Yields over 195 bu/acre
Mansfield and Mumm (2014, Crop Sci)
Mean hybrid grain yield across environments and densities

9. Objectives
 Detect QTL for plant density tolerance
 Identify candidate genes for plant density tolerance

10. Materials and Methods
 Connected population
 Alpha design, IBD
 Field evaluation
 30 phenotypic traits measured
 47,000 ppa
 5 environments over 2 years
 Genotyping
 QTL and GWAS methodology

11. Connected Population
 Top yielding inbred
parents of 5 top yielding
survey hybrids
 216 unique parents
 90% DH
 10% SSD
 320 testcross hybrids
Top yielding female inbreds Top yielding male inbreds
XY XZ YZ
AB
AX AY AX AZ AY AZ
BX BY BX BZ BY BZ
AC
AX AY AX AZ AY AZ
CX CY CX CZ CY CZ
BC
BX BY BX BZ BY BZ
CX CY CX CZ CY CZ

12. Field Design and Model
 Experimental design
 Incomplete block design- alpha (0,1)
 20 blocks of 16 genotypes (2x8 plots = 40’x40’)
 Model
𝑦 = 𝑢 + 𝑒𝑖 + 𝑟𝑗(𝑖) + 𝑏 𝑘(𝑖𝑖) + 𝑔𝑙 + 𝑔𝑙 𝑥 𝑒𝑖 + 𝑔𝑙 𝑥 𝑟𝑗(𝑖)
Photos by Tim Mies

13. Field Evaluation
Staygreen (rating)
Days to canopy
closure (Days)
Anthesis silking
interval (Days)
Ear leaf area (cm2)
Leaf angle
(degrees)
Barrenness
(count)

14. Field Evaluation
YieldUp. stem Diam (mm) Ear phenotyping
Zipper effect
Rows/ear Kernels/row
Kernel width
Kernel length
Kernel depth

15. Materials and Methods
Genotype by sequencing data
 DNA extracted from 216 unique RILs
 Quality control
 High concentration
 Low shearing
 Processed by Cornell Institute for Genomic Diversity
 Run included over 32,000 genotypes
 More genotypes in run = more powerful
 ~ 2.2 million SNPs per RIL in original data set
 Hybrids inferred from inbred genotypes

16. Materials and Methods
QTL mapping – collaboration with NRGene
 Average coverage of x 0.01 for each genotype
 Imputed with Hidden Markov Model (HMM)
 Selected only markers which differed between SSS
and NSS parents of each subpopulation
 Between 7,500 and 8,168 markers per subpopulation
 T-tests for marker trait associations, with false
discovery rate (FDR) test to determine reliable QTL

17. Materials and Methods
Genome Wide Association Study
 Filtered for 20% missing marker data in TASSEL
 2,673 SNPs
 Also working on imputation
 GAPIT software for GWAS
 Using both K matrix (GAPIT) and Q matrix (STRUCTURE)

18. Results
 Tassel attributes
Trait H2
Tassel branch number 0.95
Days to anthesis 0.95
Days to silking 0.94
Central spike length 0.93
Leaf angle 0.91
Kernel width 0.90
Ear height 0.89
Total leaf area 0.89
Kernel length 0.86
Tassel weight 0.85
Staygreen 0.85
Number of rows per ear 0.85
Kernel depth 0.84
Ear width 0.83
Plant height 0.83
Ear length 0.80
Broad sense heritability
 Highest heritability
 Kernel dimensions
 Leaf measurements
 Heights
 Flowering time

19. 83 & 171 BpA 92 & 166
BpA
96 & 171 BpA
77 & 185 BpA 91 & 178 BpA 87 & 187 BpA
99 & 176
BpA
98 & 167
BpA
92 & 185 BpA
Combined Yield Family Means

20. Correlations with Grain Yield
Trait 2012 r value 2013 r value Survey r value
Leaf area to prod. 1 g grain -0.83*** -0.82*** -0.62***
Percent barren plants -0.63*** -0.30*** -0.52***
Zipper effect -0.17** NS -0.44***
Percent root lodged 0.20*** NS -0.43***
Anthesis-silking interval -0.58*** NS -0.42***
Kernel width -0.22*** -0.25*** NS
Kernel depth -0.39*** NS NS
Kernel length NS 0.33*** 0.50***
Kernels/row 0.40*** NS 0.42***
Rows/ear 0.23*** 0.27*** 0.54***
Kernels per plant 0.41*** 0.33*** 0.51***
Staygreen -0.25*** 0.22*** 0.45***
Days to canopy closure NS -0.32*** 0.54***
Upper stem diameter NS NS 0.69***
Leaf angle NS 0.30*** 0.71***
** Significant at the 0.01 probability level
***Significant at the 0.001 probability level

21. QTL Results
243
6 5 8
Single env, single pop Multi env QTL
Multi pop QTL Multi env & pop
31
21
24
33
29
13
39
16
23
14
0 10 20 30 40
1
2
3
4
5
6
7
8
9
10Chromosome
Number of QTL
Number of QTL
Preliminary QTL analysis: Collaboration with NRGene

22. QTL Results
Env Family (Subpopulation) Trait Chromosome σ2 var explained LOD
MF1500 B73PHG39 x PHG47PHG84 Rows/ear Chrom 2 0.232 1.78
MF1500 B73PHG39 x LH82PHG84 Rows/ear Chrom 2 0.361 3.21
MF400 B73PHG39 x PHG47PHG84 Rows/ear Chrom 2 0.382 3.24
MF400 B73PHG39 x LH82PHG47 Plant height Chrom 6 0.368 3.29
S600 B73PHG39 x PHG47PHG84 Plant height Chrom 6 0.424 3.84
MF400 B73PHG39 x PHG47PHG84 Plant height Chrom 6 0.428 3.76
S800 B73PHG39 x LH82PHG47 Staygreen Chrom 4 0.394 3.15
MonA6 B73PHG39 x PHG47PHG84 Staygreen Chrom 4 0.559 3.73
MF400 B73PHG39 x PHG47PHG84 ASI Chrom 9 0.488 3.05
S800 B73PHJ40 x LH82PHG47 ASI Chrom 9 0.62 3.78
S600 B73PHG39 x LH82PHG47 Ear height Chrom 3 0.409 3.43
MonA6 B73PHJ40 x PHG47PHG84 Ear height Chrom 3 0.66 3.51
MF400 B73PHJ40 x LH82PHG47 Staygreen Chrom 9 0.526 3.08
MonA6 B73PHG39 x PHG47PHG84 Staygreen Chrom 9 0.705 5.04

23. QTL Results
Chr1
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
Chr8
Chr9 Chr10

24. GWAS Results

25. GWAS Results

26. Concluding Remarks
 Verified findings of Mansfield and Mumm (2014)
 Similar correlations between Yield and Leaf Area to
Produce 1 g Grain, Percent Barren Plants, Anthesis
Silking Interval, Kernel Length, Kernels/Row,
Rows/Ear, Kernels/Plant, Leaf Angle
 Discrepancies between this study and initial survey
 Upper stem diameter NS
 Percent root lodged was 0.20*** and NS correlated
with yield in 2012 and 2013, but -0.43*** in survey
 Staygreen was -0.25*** in 2012, but 0.22*** in 2013
 Days to canopy closure was NS and -0.32*** in 2012
and 2013, but 0.54*** in survey

27. Concluding Remarks
 Most important traits for plant density tolerance:
 Low leaf area to produce 1 g grain (LATP)
 Long, narrow kernels
 In dry years, short ASI is beneficial
 In wetter years, upright leaf angle is beneficial
 QTL identified in QTL analysis
 Over 240 QTL identified
 6 high confidence QTL selected for further study
 1 NRGene QTL confirmed in GWAS
 2 GWAS QTL identified for candidate gene analysis

28. Future Work
 Ongoing collaboration with NRGene
 Refining marker filtering and GWAS study
 Examine alignment, especially near telomeres
 Quantitative genetics analysis
 Candidate gene analysis
 Fine mapping
 Candidate gene validation

29. Acknowledgements
Committee
 Dr. Martin Bohn
 Dr. Rita Mumm
 Dr. Fred Below
 Dr. Pat Brown
Staff
 Nicole Yana
 Graduate students
 Undergraduate workers
 UIUC farm crew
Others
 NRGene
 Cornell IGD
 Below lab
 Bradley lab
 Brian Mansfield
Funding
This project is funded by the USDA National
Institute of Food and Agriculture. The
authors would also like to gratefully
acknowledge the Illinois Plant Breeding
Center, and student funding from the
Illinois Corn Marketing Board Fellowship in
Plant Breeding, the Pioneer Hi-Bred Plant
Breeding Fellowship, and the Illinois
Chapter of ARCS® (Achievement Rewards
for College Scientists) Foundation, Inc.

30. Questions?

31. Initial density survey
Inbred Group Background
B73 SSS Iowa Stiff Stalk Synthetic
LH1 SSS Iowa Stiff Stalk Synthetic; B37 type
PHG39 SSS Maize Amargo/Iowa Stiff Stalk Synthetic; B37/B14 type
PHJ40 SSS Iowa Stiff Stalk Synthetic
LH123HT NSS Pioneer Hybrid 3535
LH82 NSS Minn13/Krug derived/W153 derived
Mo17 NSS Lancaster
PH207 NSS Iodent/Long Ear OPV/Minn13
PHG35 NSS Oh07-Midland/Iodent/Linstrom Long Ear/Minn13
PHG47 NSS Oh43/Iodent*WF9/MKSDTA C10 Synthetic
PHG84 NSS Oh07-Midland/Minn13/Iodent/ReidYD/OsterlandYD/
Lancaster/Pioneer Female Composite OPV
PHZ51 NSS Minn13/Iodent/ReidYD/OsterlandYD/Lancaster/South
US Land Race Synthetic/FunksG4949/Midland
Modified from Johnson (2008)