GRM 2013: Cloning, characterization and validation of PUP1/P efficiency in maize -- L Kochian

GCP Project Number: G7010.03.01: Cloning, characterization and validation

of PUP1/P efficiency in maize

PI: Leon Kochian, USDA-ARS/Cornell University, USA Co-Pi’s: Claudia Guimaraes, Sidney Parentoni, Jurandir Magalhães, Vera Alves, Maria José Vasconcelos, Sylvia Sousa, Roberto Noda, Embrapa Maize and Sorghum, Sete Lagoas, Brazil Lyza Maron, Miguel Pineros, Jiping Liu, Randy Clark, Ed Buckler, Jon Shaff, USDA-ARS/Cornell, USA Sam Gudu, Moi University/KARI, Eldoret, Kenya Mathias Wissuwa, JIRCAS, Tsukuba, Ibaraki, Japan

• Identification of OsPSTOL1 (Pup-1) orthologs in maize • QTL/gene mapping for P use efficiency in maize in the field and in hydroponics • Inheritance studies on maize root architecture under high and low P • Validation of maize PSTOL1 candidate genes and if necessary, novel P efficiency QTL (if maize PSTOL1 homologues are not functional in P efficiency)

Project Objectives

Gene Identification Physical position Identity (%) Coverage (%) E-value

GRMZM2G451147 ZmPSTOL1 Chr8: 152.043.859 70 97 3.4e-131

GRMZM2G164612 ZmPSTOL2 Chr8: 152.100.275 70 97 2.3e-127

AC193632.2_FGP002 ZmPSTOL3 Chr4: 39.792.602 69 95 2.0e-105

GRMZM2G448672 ZmPSTOL4 Chr3: 206.918.421 66 97 4.7e-186

GRMZM2G412760 ZmPSTOL5 Chr3: 20.172.140 55 99 5.1e-104

GRMZM2G172396 ZmPSTOL6 Chr8: 13.267.001 55 99 9.6e-123

OsPSTOL1 Ortholog Identification in Maize • Using OsPSTOL1 as a query, six predicted genes were found in the maize genome sharing more than 55% of amino acid sequence identity with OsPSTOL1. • These genes were located on chromosomes 3, 4 and 8, at physical positions described in Table below. • Genetic markers for the six ZmPSTOL genes were generated and mapped on a linkage map for a L3 (P efficient) x L22 (P inefficient) RIL population.

Phylogeny of Maize PSTOL1 Orthologs

• Phylogenetic tree of OsPSTOL1 related sequences, including predicted protein sequences from maize, rice and Arabidopsis. • The predicted maize proteins share more than 55% sequence identity with OsPSTOL1. • These predicted maize proteins cluster together with OsPSTOL1 and Arabidopsis SNC4 and PR5, suggesting like OsPSTOL1 they are serine/threonine receptor-like kinases of the LRK10L-2 subfamily.

Maize Test Cross Hybrids Derived from Embrapa Elite Diversity Panel Phenotyped for P efficiency •321 testcross hybrids were evaluated in the field over two years under low and high P. • High variability in yield under low & high P was observed as well as a significant differences in grain yield under high vs. low P • Phenotypic data will be used for association analysis and genomic selection

Low P High P

QTL Mapping of Maize P Efficiency in the Field • RIL population derived from L3 (P efficient) x L53 (P inefficient) was backcrossed to the parental lines and then phenotyped for P efficiency traits in the field on low P and sufficient P field sites. • P efficiency traits determined and mapped were P acquisition efficiency (PAE; grain produced/amount soil available P), P use efficiency (PUE; amount plant P/amount soil available P), and P utilization efficiency (PUTIL; grain produced/amount plant P). • Six QTLs were identified for PUE, six for PAE and five for PUTIL . • Most of the QTLs mapped for PUE were coincident with the genomic regions mapped for PAE. This agrees with the high correlation (0.89) between these traits, which were also highly correlated with grain yield under low P, 0.96 and 0.85, respectively. • This result indicates that P use efficiency is mainly due to P acquisition efficiency, as was also found by Parentoni et al. (Maydica 55:1; 2010). • None of the QTLs for P utilization efficiency were coincident with the other P efficiency indexes, suggesting that different genes are involved in P utilization.

QTL Mapping of Maize P Efficiency in the Field (con’t)

QTL Mapping of Maize P Efficiency and 2D Root Traits in Hydroponics

• The L3 x L22 maize RIL population was grown in paper pouches moistened with low P and sufficient P nutrient solution and roots were digitally imaged and root traits quantified using our RootReader 2D platform (Clark et al, Plant Cell Envir 36: 454; 2013).

• Root and shoot dry weight and P accumulation were also quantified and QTL mapping was conducted on P efficiency traits (PUE, PAE, PUTIL) and root traits.

• Out of 32 root traits, four were selected for mapping analysis based on De Sousa et al. (Functional Plant Biol; 2012): length (cm), volume (cm3), volume of fine roots (1.0<d≤2.0 mm) (cm3) and root surface area (cm2).

Root Phenotyping Tools

Growth: Hydroponics (Al tolerance)

Agar Plates (Zn nutrition)

Pouches (P nutrition, Salinity stress)

Sand Pots (RSA validation, P nutrition)

Capture and Analysis: Digital Photography (Single images)

RootReader2D Software calculates range of root growth traits on both whole root system and specific traits

Visit: www.plantmineralnutrition.net

Overall Efficiency: 1000’s of plants per day

2D Phenotyping Platform 3D Phenotyping Platform

Clark et al., Plant Physiol2012

Growth: Gel Cylinders (RSA - root system architecture)

*New: Hydroponics (RSA, Stress/functional studies)

Capture and Analysis: Digital Photography (Image sequences)

RootReader3D Software reconstructs series of 2D images into a 3D RSA and computes specific root traits

Overall Efficiency: ~100 plants per day

Clark et al., Plant, Cell & Envir2011

Root Imaging Pipeline – Imaging RSA in 2-D Root Growth & Imaging

RootReader 2D Software

Clark et al. 2012. High-throughput 2D root system phenotyping platform facilitates genetic analysis of root growth and development. Plant Cell Environ.

QTL Mapping of Maize P Efficiency and 2D Root Traits in Hydroponics

• The L3 x L22 maize RIL population was grown in paper pouches moistened with low P and sufficient P nutrient solution and roots were digitally imaged and root traits quantified using our RootReader 2D platform (Clark et al, Plant Cell Envir 36: 454; 2013).

• Root and shoot dry weight and P accumulation were also quantified and QTL mapping was conducted on P efficiency traits (PUE, PAE, PUTIL) and root traits.

• Out of 32 root traits, four were selected for mapping analysis based on De Sousa et al. (Functional Plant Biol; 2012): length (cm), volume (cm3), volume of fine roots (1.0<d≤2.0 mm) (cm3) and root surface area (cm2).

Co-localization of P Efficiency QTL from Field Studies with P Efficiency and Root Trait QTL from Nutrient Solution Phenotyping

Hydroponics Hydroponics

Field Field

A B

Co-localization of P Efficiency QTL from Field Studies with P Efficiency and Root Trait QTL from Nutrient Solution Phenotyping

Chr 7

Chr 7

Hydroponics Hydroponics

Chr 8

Field Field

• A region from 209 to 272 cM on chromosome 1: Co-localization of QTLs controlling PUE, PAE and P utilization efficiency (PUTIL) in the field with a multiple-trait QTL for root morphology and PAE in nutrient solution • A region spanning 82 - 95 cM on chromosome 3: Co-localization of QTL controlling PUE and PAE in the field with a multiple-trait QTL for root morphology and PAE in nutrient solution. • A region from 77 to 83 cM on chromosome 7: Co-localization of QTL controlling PUE and PAE in the field with a QTL for root diameter. • A region spanning 100 – 127 cM on chromosome 8: Co-localization of QTL controlling PUE and PAE in the field with QTLs for PAE, root length and root surface area in nutrient solution.

The Combined Analysis of QTL Mapping for Root Traits and P Efficiency Indices in the Field Based On Grain Yield Has Led Us To Focus on Four Genomic Regions

Colocalization of ZmPSTOL1 Orthologs with Maize P Efficiency and/or Root Trait QTL

ZmPSTOL Expression in Roots & Shoots of P Efficient (L3) and P Inefficient (L22) Maize

• ZmPSTOL1, 4 and 6 preferentially expressed in roots. • ZmPSTOL1 and 4 expression increases in response to P deficiency. • ZmPSTOL4 preferentially expressed in roots of P efficient L3 - colocalizes with root traits and not P efficiency traits. • ZmPSTOL1 only rice Pup1 homolog that colocalizes with PAE and PUE. It’s expression is specific to roots and is induced by low P plant status. • ZmPSTOL1 expression is exclusively in roots of P inefficient L22, but the superior allele for this chr 8 PAE and PUE QTL donated by L22. • ZmPSTOL1 is most similar in sequence of the maize orthologs to OsPSTOL1.

Shallow Intermediate Deep

What Is the Ideal Root Architecture P Efficiency in Low P Soils?

[P]

[H+]

P Efficient Soybean Line Dr. Hong Liao’s group, Root Biology Center, SCAU, Guangzhou

3D RSA Imaging System

• Stationary camera with fixed capture settings that is synchronized to a turntable via a LabVIEW interface and digital controller

• 100 images captured per root system, 3.6° of rotation between images

• Capture time of approximately 10 minutes per root system

3D Reconstruction Process via RootReader 3D

Thresholded rotational image sequence consisting of 40-100 2D images Perspective back projection of 2D root points from

each 2D image into a temporary 3D voxel volume

Transformation of each temporary voxel into a final voxel volume

Adaptive thresholding of each horizontal cross section through final voxel volume to generate 3D root model

Germplasm and Screening Mapping Results

Genetic Mapping of RSA

Peak SNP -66kb -33kb 0 +33kb +66kb

“A” “B” SNP Allele SNP Allele

n=39 n=118 QTL QTL

Germplasm and Screening Mapping Results

Genetic Mapping of RSA

Peak SNP -66kb -33kb 0 +33kb +66kb

“A” “B” SNP Allele SNP Allele

n=44 n=107 QTL QTL

Germplasm and Screening Mapping Results

Genetic Mapping of RSA

• Subpopulation SNPs selected from within 3kb of the peak Indica SNP

Aus Indica

Temperate Japonica Tropical Japonica

All Subpops

n=211 n=360 n=9 n=80 n=44 n=107

n=10 n=169 n=7 n=162

QTL QTL

The Gel-Based Root Growth System Has Its Limitations

• Roots of some plant species such as maize & sorghum as well as fine rooted species such as canola don’t grow well in the gel cylinders

• Labor and cost intensive • Can’t easily impose different nutrient regimes • Limited to work with fairly small root systems

(young plants)

Transition From Gel to Hydroponics

M8 Rice line Nipponbare parent

12 day old rice grown in low P nutrient solution

3D Imaging/Analysis of RSA in Hydroponics

•We can use the hydroponic systems for 3-D imaging because our software subtracts out the mesh and reconstructs the images •Are using this hydroponic system with 3D black plastic mesh to screen sorghum populations (270 lines) and to correlate RSA traits with physiological data. •Can use much larger vessels than with the gel and still maintain rapid throughput. • Important design for longer growth periods, as crown roots may be important for water acquisition and they don’t appear until around 12 days.

Dr. Alexandre Falcão computer wizard

3 D Printers

The growth system is created from ABS plastic mesh circles made with a 3-D printer.

The mesh system serves to constrain the roots, but not to impede their growth.

Three-D RSA Reconstruction of 100 Two-D Sorghum Root Images (15 Day Old Plant)

• Barbara Hufnagel from Jurandir Magalhaes’s lab is currently in our lab working with our staff to phenotype and quantify RSA 3D traits for the sorghum association panel. • We will be set up to phenotype maize RSA for this project in early 2014.

Products • Due to the more upstream nature of this project, the products are still in the pipeline and will be forthcoming starting later in 2014. • Claudia is generating NILs for specific P efficiency QTL for verification of QTL effects and as a breeding resource. Pyramiding of multiple QTL in NILs will have greater potential for impact. • Work ongoing to validate via association mapping analysis and more in depth molecular physiological investigations of candidate ZmPSTOL1 genes to identify OsPSTOL1 orthologs involved in maize P efficiency. • Ultimately will have breeding lines for improved P efficiency. • Catalog of bi-parental and GWA QTL & markers for root system architecture traits that may play a role in maize P efficiency.