Source: BOYCE THOMPSON INSTITUTE submitted to
INVESTIGATIONS INTO THE GENETIC AND PHYSIOLOGICAL MECHANISMS OF ALUMINUM TOLERANCE IN RICE (ORYZA SPP.)
 
PROJECT DIRECTOR: Kochian, L. V. McCouch, S. R.
 
PERFORMING ORGANIZATION
Robert Holley Center for Agriculture and Health
BOYCE THOMPSON INSTITUTE
ITHACA,NY 14853
 
NON TECHNICAL SUMMARY: Aluminum (Al) is the most abundant metal in the earth's crust and in highly acidic soils (pH<5) is solubilized as the trivalent cation, which is very phytotoxic to plant roots. Al toxicity is the primary limitation to crop production on approximately 50% of the world's potentially arable lands, including ~20% of North America as well as in many developing countries, where food security is most tenuous. Rice is a staple crop for half the world's population and is a major U.S. agricultural export. Rice is significantly more Al tolerant than other grasses, including maize, wheat and sorghum, but our understanding of the genetic, molecular and physiological mechanisms of rice Al tolerance is still limited. With higher levels of Al tolerance and abundant genetic and genomic resources, rice is a good model to investigate cereal Al tolerance. The goal of this research is to identify the major genes involved in Al resistance in rice and provide a better understanding of the physiological mechanisms of Al resistance. The activities outlined in this proposal will allow us to: 1) Identify, clone, and characterize the gene(s) underlying a major and novel rice Al tolerance QTL we have recently identified. 2) Develop near isogenic lines (NILs) containing the major Al tolerance QTL for use in breeding and physiological studies. 3) Conduct full-genome association mapping using 500 O. sativa and O. rufipogon genotypes to identify novel chromosomal regions (50-500kb) conferring Al tolerance in rice. 4) Determine and characterize the physiological mechanism(s) underlying rice Al tolerance. The findings from the proposed research on the elucidation of the molecular and physiological basis of the very high Al tolerance expressed in rice, will allow for more efficient breeding of acid soil-tolerant rice varieties and also provide important new molecular and genetic resources for improving acid soil tolerance of cereal crops in general.
 
OBJECTIVES: The long-term goal of this research is the development of crops with superior performance on acidic soils based on a comprehensive understanding of the genetic and physiological mechanisms underlying plant response to aluminum (Al) stress. The high levels of Al tolerance exhibited by rice, the extensive genetic resources and the public availability of a genome sequence, make rice an excellent model to meet the goals of this research. The goals and objectives of this proposal are: 1) Identify, clone, and characterize the gene underlying a major rice Al tolerance QTL: The major rice Al tolerance QTL on chromosome 12 we recently identified will be fine-mapped and cloned using an advanced backcross population. 2) Develop near isogenic lines (NILs) containing major Al tolerance QTL for use in breeding and physiological studies, and field evaluation on Al toxic acid soils. 3) Identify additional Al tolerance loci through full-genome association mapping. 4) Investigate the physiological mechanism(s) underlying Al tolerance in rice and begin to understand how Al is toxic to root growth. Our preliminary data provides strong evidence that rice utilizes novel Al tolerance mechanisms much different than the Al-activated root organic acid exudation employed by sorghum, maize and wheat. The expected outputs will focus on dissemination of the research findings via publication in high profile, peer-reviewed journals, and presentations at international and national scientific meetings. Also, the propsoed research will be a mechanism by which young scientists are trained in crop genomics research and the translation of the findings from this research to facilitate crop improvement.
 
APPROACH: The methods employed for each of the 4 research objectives are as follows. 1) Identify, clone, and characterize the gene underlying a major rice Al tolerance QTL: The major rice Al tolerance QTL on chromosome 12 we recently identified will be fine-mapped and cloned using an advanced backcross population. The identified gene(s) will be sequenced to identify functional polymorphism(s) and expression analysis will be conducted to determine Al-induction and tissue location. Sequence information will determine the evolutionary history, subpopulation origin and distribution of the allele. 2) Develop near isogenic lines (NILs) containing major Al QTL for use in breeding and physiological studies, and field evaluation on Al toxic acid soils: NILs will be produced to introgress the tolerant allele(s) into a sensitive background for use in applied breeding programs and in physiological experiments. An NIL that introgresses the sensitive allele into the tolerant background will also be produced for use in physiological experiments aimed at understanding the physiological mechanism of the tolerance gene. Field evaluation of these lines will be conducted on Al toxic acid soils. 3) Identify additional Al tolerance loci through full-genome association mapping: A full-genome association mapping platform (450 accessions genotyped with 44,000 SNPs) recently developed in the McCouch lab will be utilized to identify Al tolerance loci conferring Al tolerance across diverse germplasm. This panel represents the genetic diversity of O. sativa and O. rufipogon and will allow detection of Al tolerance loci with an expected resolution of 50kb (estimated LD in O. rufipogon), 100kb (LD in Indica), or up to 1Mb (LD in Japonica). This analysis will enable us to identify candidate regions for future fine-mapping and for immediate use in applied breeding programs. 4) Investigate the physiological mechanism(s) underlying Al tolerance in rice and begin to understand how Al is toxic to root growth: Our preliminary data provides strong evidence that rice utilizes a novel Al tolerance mechanism. We will confirm whether the sorghum Al tolerance MATE gene (AltSB) is involved in Al tolerance in rice by evaluating knockout mutants of the rice MATE homolog. We will screen mutants that map within rice Al tolerance QTL to determine if there is a significant difference in tolerance. Second, under conditions of Al toxicity, we will investigate whether inhibition of root growth is due to the toxicity of proteins involved in cell expansion and whether rice proteins are capable of functioning at higher Al concentrations compared to maize, wheat, and sorghum. Lastly, we will also investigate whether root growth inhibition by Al is due to alterations in cell wall composition/structure resulting from Al toxicity and/or if cell wall modifications are responsible for differences in Al tolerance within rice or between rice and other cereals.
 
CRIS NUMBER: 0219809 SUBFILE: CRIS
PROJECT NUMBER: NYR-2009-02273 SPONSOR AGENCY: NIFA
PROJECT TYPE: AFRI COMPETITIVE GRANT PROJECT STATUS: NEW MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jan 15, 2010 TERMINATION DATE: Jan 14, 2013

GRANT PROGRAM: PLANTS & ENVIRONMENT
GRANT PROGRAM AREA: Natural Resources

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
201153010202.216%
201153010402.217%
201153010802.217%
203153010202.216%
203153010402.217%
203153010802.217%

CLASSIFICATION HEADINGS
KA203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
S1530 - Rice
F1020 - Physiology
F1080 - Genetics
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 80%
APPLIED 20%
DEVELOPMENTAL (N/A)%

KEYWORDS: aluminum toxicity~aluminum tolerance~cereals~acid soils~rice~root growth~root organic acid exudation~gene cloning~map based cloning~assocation mapping

PROGRESS: Jan 15, 2010 TO Jan 14, 2011
OUTPUTS: 1) To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Also, a novel hydroponic solution was developed and found to be far superior to the Yoshida's rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize, sorghum, and rice. 2) Multiple rice Al tolerance QTL studies have identified a region on chr 1 that is in close proximity to the rice MATE that is a homolog of the sorghum Al tolerance gene (SbMATE) we previously identified, leading to the hypothesis that this gene may be underlying these QTL. SbMATE functions in sorghum Al tolerance as an Al-activated root citrate efflux transporter that excludes Al from the root tip, with differences in Al tolerance across sorghum genotypes directly related to gene expression. Quantitative RT-PCR was conducted to determine if differences in rice MATE gene expression correlated with differences in rice Al tolerance. 3) We used the rice Al tolerance phenotyping system described above to phenotype 374 diverse rice accessions that are members of the NSF-TV rice association panel in Dr. McCouch's lab. This involved digitally imaging and quantifying root growth in more than 10,000 seedlings grown hydroponically under +/-Al conditions. The rice association panel had already been genotyped with 44,000 SNPs as the basis for GWA studies; once the rice association panel was scored for Al tolerance, we then conducted genome-wide association (GWA) analysis. We also phenotyped two bi-parental populations for Al tolerance and QTL mapping of Al tolerance was conducted. Our findings have been shared with the research community via the following presentations: Leon Kochian: "Imaging and Quantifying Whole Root Systems for Genome-Wide Analysis of Root System Architecture", Root Genomics Symposium, Plant and Animal Genome (PAG) Meeting, San Diego, January, 2011 Leon Kochian: "The Comparative Genomics Challenge Initiative - Translational Research for Improving Aluminum Tolerance and Phosphorous efficiency in Cereals", Generation Challenge Program Symposium, , Plant and Animal Genome (PAG) Meeting, San Diego, January, 2011. Leon Kochian: "Molecular and Genetic Regulation of Cereal Aluminum Tolerance", Department of Soil Science and Agricultural Chemistry, University of Agricultural Sciences, Bangalore, India, November 29, 2010. Leon Kochian: "Fighting fire with fire: Plants tolerate acid soils by releasing organic acids" at the Roots of Agriculture Symposium at the 2010 AAAS meeting, San Diego, CA, 2010. Leon Kochian: Opening keynote speaker at the 2010 Plant Biotech Denmark Symposium with the talk entitled "Adaptive strategies for plant responses to toxic metals in the soil", Copenhagen, Denmark, March 4, 2010. PARTICIPANTS: Adam Famoso completed his PhD in the Department of Plant Breeding working on this project between the McCouch and Kochian labs. He was the lead scientist both on the physiological, molecular and genetic analyses of rice Al tolerance. He was first author on both publications generated in the first year of this project. Dr. Famoso is currently a plant breeder with Pioneer. Randy Clark is a PhD student in the Department of Biological and Environmental Engineering in the Kochian lab. He developed the high throughput rice imaging system and the software and other computational tools to phenotype rice whole root systems for Al tolerance. To date, more than 20,000 rice seedlings have been phenotyped for Al tolerance. Juan David Arbelez is a first year PhD student in Plant Breeding in the McCouch and Kochian labs. He is working on the fine scale mapping and cloning of the large Al tolerance QTL on chr 12. He also is generating NILs harboring a number of the Al tolerance QTL for further analysis and also to generate breeding lines for improving rice Al tolerance. Jianyong Li is a new postdoctoral researcher who just joined the project (replacing Adam Famoso who left for Pioneer after completing his PhD). He will be working on the molecular aspects of the project while Juan David Arbelez will focus on the genetics/breeding aspects of the project James Jones-Rounds is a technician who worked between the McCouch and Kochian labs who made a major contribution to the phenotyping of rice seedlings from both the rice diversity panel and two bi-parental mapping populations. He currently is applying to graduate school for his PhD in biology. Joseph Gage is a Cornell University undergraduate biology student who assisted Adam Famoso on the root phenotyping work. Shelina Gautamais a Cornell University undergraduate biology student who assisted in isolation of DNA from roots of the 346 line diversity panel and also the bi-parental mapping populations. Kengo Onishi is a high school student from Ithaca High who assisted on the phenotyping of rice Al tolerance. TARGET AUDIENCES: In addition to the training of PhD students, postdocs, and technicians, through this prject we have mentored both undergraduate students, and a high school student. Joseph Gage is a Cornell University undergraduate biology student who assisted Adam Famoso on the root phenotyping work. Shelina Gautama is a female Cornell University undergraduate biology student who assisted in isolation of DNA from roots of the 346 line diversity panel and also the bi-parental mapping populations. Kengo Onishi is a high school student from Ithaca High who assisted on the phenotyping of rice Al tolerance. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2010-01-15 TO 2011-01-14 1) Rice was found to be significantly more tolerant than maize, wheat, and sorghum at all Al concentrations; the mean rice was 2-6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of >20 rice genotypes and compared to two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. We found that there is no correlation between Al exclusion from the rice root apex and root growth in Al. Furthermore, there was no correlation between root citrate or malate exudation, Al tolerance, and root tip Al levels. This indicates that the roots of tolerant rice varieties can continue to grow even with significant Al accumulation into the root tip. Thus, rice must employ unique mechanisms of Al tolerance not found in other cereal species. 2) With regards to qRT-PCR analysis of the rice ortholog of the sorghum Al tolerance gene, SbMATE, we found a lack of correlation between rice MATE gene expression and Al tolerance. Thus we conclude that the rice homolog of the sorghum Al tolerance gene is not involved in mediating rice Al tolerance, which agrees with the above described lack of correlation between tolerance and root tip exclusion of Al. 3) For the GWA analysis of rice Al tolerance, subpopulation structure explained 57% of the phenotypic variation and the mean Al tolerance in Japonica was twice that of Indica. Forty-seven regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Nine of these regions colocalized with a priori candidate genes and ten co-localized with previously identified QTLs. Three regions corresponding to Al sensitive rice mutants (ART1, STAR2, Nrat1) were identified through biparental QTL mapping or GWA to be involved in natural variation of rice Al tolerance. Haplotype analysis around the Nrat1 gene identified susceptible and tolerant haplotypes explaining 40% of the Al tolerance variation within the aus subpopulation and sequence analysis of Nrat1 identified two nonsynonymous mutations specific to Al sensitive aus accessions. 4) GWA analysis discovered more phenotype-genotype associations and provided higher resolution, but QTL mapping of bi-parental populations identified critical rare and/or subpopulation-specific alleles not detected by GWA analysis. Mapping using Indica/Japonica populations identified QTLs associated with transgressive variation where susceptible parent alleles enhanced Al tolerance in the tolerant Japonica background. This work supports the hypothesis that selectively introgressing alleles across subpopulations is an efficient approach for trait enhancement in plant breeding programs and demonstrates the fundamental importance of subpopulation in interpreting and manipulating the genetics of complex traits in rice

PUBLICATION INFORMATION: 2010-01-15 TO 2011-01-14
Famoso AN, Clark RT, Shaff JE, Craft E, McCouch SR, Kochian LV. 2010. Development of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanisms. Plant Physiol.153: 1678 - 1691.

PROJECT CONTACT INFORMATION
NAME: Kochian, L. V.
PHONE: 607-255-2454
FAX: 607-255-2459