Progress 01/15/10 to 01/14/14
Outputs
Target Audience: Our findings have been shared with the research community via the following presentations: 1) 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. 2) 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; 3) 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; 4) 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; 5) 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; 6) Leon Kochian: Invited symposium speaker on "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; 7) Leon Kochian: Invited Symposium Speaker on "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; 8) Leon Kochian: Invited speaker on "Update on research on rice aluminum tolerance and maize phosphorous efficiency projects ", Generation Challenge Program Comparative Genomics Challenge Initiative Annual Meeting, Sete Lagoas, Brazil, March 21, 2011; 9) Leon Kochian: Invited speaker on "Molecular mechanisms of crop adaptation to acid soils", 2cd International Symposium on Integrated Plant Biology, Lanzhou, China, August 26, 2011; 9) Susan McCouch: Plenary speaker on "Natural Variation In Rice: Mixing, Matching, Keeping, Sharing", Plant and Animal Genome (PAG) Meeting, San Diego, January, 2012; 10) Leon Kochian: Invited key note speaker on "Using Molecular Approaches to Improve Cereal Crops for Adaptation to Marginal Soils" at the 1st Biotechnology World Congress, Dubai, United Arab Emirates, February 14, 15, 2012; 11) Leon Kochian: Invited seminar speaker on "Using Molecular, Genetic & Physiological Approaches to Improve Cereal Crops on Marginal Soils", Department of Aridland Agriculture, United Arab Emirates University, Al Ain, UAE, February 16, 2012; 12) Leon Kochian: Invited plenary speaker on "Root Genomics Research Aimed at Improving Crop Adaptation to Resource Limited Environments" at the 2012 Water for Food Conference, Lincoln, Nebraska, May 30-June 1, 2012; 13) Leon Kochian: Invited speaker on "How Plant Roots Respond to and Neutralize Toxic Metals in the Soil" at the Plant Abiotic Stress Symposium at the 2012 American Society of Plant Biologists Annual Meeting, Austin, TX, on July 21, 2012; 14) Leon Kochian: Invited seminar speaker on "Molecular, Genetic & Physiological Investigations of Cereal Crop Adaptations to Soil Abiotic Stresses" at the Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, September 3, 2012; 15) Leon Kochian: Invited symposium speaker on "Variation in cereal aluminum tolerance is due to transcriptional and post-transcriptional regulation of cereal aluminum tolerance genes", Abiotic Stress Workshop, Plant and Animal Genome XXI Meeting, Jan 13, 2013, San Diego, CA Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 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 in PloS Genetics and Plant Physiology. Dr. Famoso is currently a plant breeder with Pioneer Hybrid. Randy Clark was 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 which enabled the team to phenotype more than 40,000 rice seedlings for Al tolerance. He was first author on the publication in Plant, Cell and Environment in the second year of the project on the development of the RootReader 2D platform used to phenotype rice seedlings. Dr. Clark is currently a plant physiologist with Pioneer Hybrid. Juan David Arbelaez has just completed his PhD in Plant Breeding in Dr. McCouch's lab working on this project. He conducted the fine scale mapping and cloning of the large Al tolerance QTL on chr 12. He also has generated the 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 postdoctoral researcher who joined the project in the second year. He has been the lead on the research characterizing and identifying functional genetic variants in the root Al uptake transporter, Nrat1, and showed that this variation pays a significant role in overall genetic variation in rice Al tolerance. His work was published in the last year of this project in PNAS. James Jones-Rounds was 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 has moved to the Department of Neurobiology at Cornell, as neurobiology is his direct field of interest and he hopes to attend graduate school in neurobiology. Anjali Merchant replaced Mr. Jones-Rounds as the technician on the project and worked with Juan Daivid Arbaleaz and others on the genotyping of rice lines. She is currently in a PhD program in biology at Yale University. We have had a number of undergraduate students on the project. Joseph Gage was a Cornell University undergraduate biology student who assisted Adam Famoso on the root phenotyping work. Shelina Gautamais was 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. Huang Chun was a Cornell undergraduate student who worked Juan David Arbalaez on the phenotyping and genotyping of the rice fine-scale mapping populations. Taylor Apolostico was a Cornell undergraduate student who worked with Dr. Li on the cloning and molecular characterization of rice Nrat1. We also had 2 Ithaca High School students who worked on the project. Kengo Onishi was a high school student from Ithaca High who worked full time for the summer of 2012 and assisted on the phenotyping of rice Al tolerance. Aksash Garg is a high school student from Ithaca High who worked full time for the summer of 2014 assisting several people on the project on growing plants and on the phenotyping of rice Al tolerance. How have the results been disseminated to communities of interest? Our findings have been shared with the research community via the following presentations: 1) 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. 2) 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; 3) 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; 4) 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; 5) 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; 6) Leon Kochian: Invited symposium speaker on "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; 7) Leon Kochian: Invited Symposium Speaker on "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; 8) Leon Kochian: Invited speaker on "Update on research on rice aluminum tolerance and maize phosphorous efficiency projects ", Generation Challenge Program Comparative Genomics Challenge Initiative Annual Meeting, Sete Lagoas, Brazil, March 21, 2011; 9) Leon Kochian: Invited speaker on "Molecular mechanisms of crop adaptation to acid soils", 2cd International Symposium on Integrated Plant Biology, Lanzhou, China, August 26, 2011; 9) Susan McCouch: Plenary speaker on "Natural Variation In Rice: Mixing, Matching, Keeping, Sharing", Plant and Animal Genome (PAG) Meeting, San Diego, January, 2012; 10) Leon Kochian: Invited key note speaker on "Using Molecular Approaches to Improve Cereal Crops for Adaptation to Marginal Soils" at the 1st Biotechnology World Congress, Dubai, United Arab Emirates, February 14, 15, 2012; 11) Leon Kochian: Invited seminar speaker on "Using Molecular, Genetic & Physiological Approaches to Improve Cereal Crops on Marginal Soils", Department of Aridland Agriculture, United Arab Emirates University, Al Ain, UAE, February 16, 2012; 12) Leon Kochian: Invited plenary speaker on "Root Genomics Research Aimed at Improving Crop Adaptation to Resource Limited Environments" at the 2012 Water for Food Conference, Lincoln, Nebraska, May 30-June 1, 2012; 13) Leon Kochian: Invited speaker on "How Plant Roots Respond to and Neutralize Toxic Metals in the Soil" at the Plant Abiotic Stress Symposium at the 2012 American Society of Plant Biologists Annual Meeting, Austin, TX, on July 21, 2012; 14) Leon Kochian: Invited seminar speaker on "Molecular, Genetic & Physiological Investigations of Cereal Crop Adaptations to Soil Abiotic Stresses" at the Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, September 3, 2012; 15) Leon Kochian: Invited symposium speaker on "Variation in cereal aluminum tolerance is due to transcriptional and post-transcriptional regulation of cereal aluminum tolerance genes", Abiotic Stress Workshop, Plant and Animal Genome XXI Meeting, Jan 13, 2013, San Diego, CA, Also, some of the findings from this project were used in Dr. Kochian's lectures on plant aluminum tolerance in his Cornell graduate plant mineral nutrition course entitled "Crop and Soil Science/Plant Biology (CSS/BIOP) 6420 MINERAL NUTRITION: FROM PLANTS TO HUMANS. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Nearly half of the world's potentially arable lands are highly acidic, and on these acid soils aluminum (Al) toxicity severely damages roots, resulting in major yield losses. Approximately 20% of the US soils and ~50% of the lands in the tropics and subtropics are highly acidic. Thus acid soils are prevalent in developing countries where food security is most tenuous. Rice is the most Al tolerant cereal yet until this project, not that much was known about genetic variation in Al tolerance genes. The major discoveries made in this project was the demonstration that several candidate rice Al tolerance genes exhibit significant and functional genetic variation, and that this genetic variation can be exploited to improve yields of cereal crops on acid soils. This should have a significant impact in improving agriculture in developing countries. Objective 1. Fine mapping of a major Al tolerance QTL: We have identified a large effect Al tolerance QTL (20% of variation) on chromosome 12 (Famoso et al (2011) which we found colocalizes with the candidate tolerance gene, ART1, 2011), a transcription factor that is known to regulate expression of a suite of other candidate Al tolerance genes in response to Al (Huang et al., 2011, Xia et al., 2010, Yamaji et al., 2009 and Yokosho et al., 2011). Using ~450 recombinants selected from the 4,000 plants generated to fine-map this region, we reduced the candidate region from 3 Mbp to 300 Kbp. We subsequently generated a ~4,000-plant population to further narrow down the QTL region to a 30 Kbp interval. This region contains six putative gene models but only ART1 is related to Al tolerance. We strongly believe that ART1 is the gene conferring the natural Al tolerance between IR64 and Azucena at the Alt12.1 QTL. We are now testing this will proceed to test this hypothesis using a transgenic approaches. Objective 2. Development of NILs harboring Al tolerance QTL: We are close to completion on the development of reciprocal near isogenic lines (NILs) targeting four major Al tolerance QTLs we identified from QTL mapping for Al tolerance reported in Famoso et al (2011) on chr 1, 2, 9 and 12. We used RILs from the IR64 x Azucena population to generate 8 NILs, each containing one of the 4 QTL in both parental backgrounds. 200 BC2 plants from the IR64 background and 200 BC1 from the Azucena background were grown. This 400-plant population was subjected to marker assisted 'positive' selection for each allele of interest. Using this information, BC2 plants in the IR64 and Azucena background were selected for backcrossing. These plants contained one, two, three or recombinant fragments of the QTLs of interest. BC3 crosses in the IR64 and Azucena background were selected to generate a population of plants. Again positive selection was performed and plants were selected to genotype their genetic backgrounds. This confirmed that our selected lines carried the target fragment of interest, and also allowed us to estimate the proportion of residual genome from the donor parent that remained in the genetic background of the recurrent parent. Using these data, we were able to select the best lines for further backcrossing. We determined that 96.2% of the recurrent genome had been recovered from the IR64 background and 91.1% had been recovered in the plants with the Azucena background. Objective 3: GWA analysis of rice Al tolerance: GWA analysis was conducted for Al tolerance for 383 diverse rice accessions from McCouch's rice association panel genotyped with her 44K rice SNP chip (Zhao et al., 2011). For the GWA analysis based on the 44K SNP chip, forty-eight regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Four of these regions co-localized with a priori candidate genes and two highly significant regions co-localized with previously identified QTLs. Three regions corresponding to induced Al sensitive rice mutants (ART1, STAR2, Nrat1) were identified through bi-parental QTL mapping or GWA to be involved in natural variation for Al tolerance. The same rice panel was re-genotyped with McCouch's subsequent 1 million rice SNP chip and the GWA was re-run. This resulted in identification of a number of additional significant associations, including some of those obtained as QTL from our bi-parental mapping, including ART1. We subsequently focused on a second large GWA peak on chr 2 that accounts for 40% of the variation in Al tolerance in the aus sub-pop and co-locates with a putative Al uptake transporter, Nrat1(Xia et al, 2010).Our subsequent detailed analysis of this peak is our best example of the value of linking functional genomics and natural variation in rice Al tolerance. We found that an aus-specific Al sensitive haplotype in this region is functionally related to the Nrat1 gene.It was previously that Nrat1 (for Nramp aluminum transporter), is an Al transporter localized to the plasma membrane of root cells, which when knocked out, enhances Al sensitivity (Xia et al, 2010). This is consistent with this transporter serving to mediate Al uptake by moving it directly into root cells, presumably into the vacuole, and away from the root cell wall (Xia et al, 2010). Our haplotype analysis of the GWA region on chromosome 2 and sequence analysis of the Nrat1 gene identified putative sensitive and tolerant haplotypes that implicate the Nrat1 gene, and further identified two putative functional polymorphisms specific to the Al sensitive aus accessions. We have verified these tolerant and sensitive haplotypes; the sensitive Nrat1 haplotype is only found in the 9 very Al sensitive aus lines. All of the more tolerant aus lines, as well as all of the indica and japonica lines (which are more tolerant than aus) carry the tolerant Nrat1 haplotype. We cloned the tolerant and sensitive Nrat1 versions, expressed them in yeast, and then measured Al tolerance and Al uptake in Nrat-expressing yeast in comparison to yeast expressing the empty pYES vector. Yeast expressing both versions of the Nrat gene were more Al sensitive than yeast expressing the empty vector, which is consistent with the Nrat transporter mediating Al uptake into yeast, which do not have an appropriate internal tolerance mechanism to deal with the Al being transported into the cytoplasm (storage in the vacuole in rice root cells). Furthermore, yeast expressing the more sensitive Nrat1 allele were significantly less Al sensitive than yeast expressing the tolerant Nrat1 variants. This is consistent with the Nrat1's from the more Al tolerant rice lines being able to transport more Al from the cell wall into the rice root symplasm, where a tonoplast transporter would then sequester the Al in the vacuole (see, Huang et al, 2011). This was verified from yeast Al uptake studies where we found that the Nrat1 from the more Al tolerant rice lines transports significantly more Al across the yeast plasma membrane than does the Nrat1from the Al sensitive aus lines, while yeast expressing either Nrat1 haplotype take up more Al than yeast expressing the empty vector. This is the first direct evidence that genetic variation in the coding sequence of a rice Al tolerance gene underlies the variation in Al tolerance. We have shown via site-directed mutagenesis followed by functional expression in yeast that any of the 4 amino acid alterations in the Nrat1 Al uptake transporter are sufficient to decrease Al uptake and Al tolerance. We hypothesize that because rice does not exclude Al from the root tip, and 90% of the root tip Al is in the cell wall, there must be rice Al tolerance mechanisms functioning to protect the cell wall. The integration of genome-wide diversity analysis with functional genomics approaches allowed us to verify this is a novel plant Al tolerance mechanism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Famoso AN; Zhao Z; Clark RT; Tung C-W; Wright M; Bustamante C; Kochian LV & McCouch SR. 2011. Genetic architecture of aluminum tolerance in rice (O. sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genetics 7(8): e1002221
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
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 Physiol153: 16781691.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Clark RT, Famoso AN, Zhao K, Shaff JE, Craft JE, Bustamante CD, McCouch SR, Aneshansley DJ, Kochian LV. 2013. High-throughput 2D root system phenotyping platform facilitates genetic analysis of root growth and development. Plant Cell Envir 36: 454-466.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Li JY, Liu J, Dong D, Jia X, McCouch SR, Kochian LV. 2014. Natural variation in Nramp (NRAT1) expression and function that play a key role in rice aluminum tolerance. Proc Natl Acad Sci USA 111: 6503-6508.
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Progress 01/15/12 to 01/14/13
Outputs
Target Audience: In addition to the training of 1 postdoc, Dr. Jianyong Li, 1 PhD student, Juan David Arbalaez, and 1 technician, Anjali Merchant, through this project we have mentored both Cornell undergraduate students, and summer undergraduate interns, and an Ithaca High School undergraduate. Taylor Apostilico is a Cornell University undergraduate biology student who assisted Jianyong Li on the research on rice Nrat1. A second Cornell udnergrad, and Haun Chung, was mentored by Juan David Arbalaez and worked on phenotyping and genotyping the fine-scale mapping populations for the cloning of the Al tolerance QTL on chr 12. Juan David also mentored our technician, Anjali Marchant, on genotyping rice lines for the map-based cloning and the NIL generation. Akshath Garg is an Ithaca High School student who worked during the summer of 2012 and assisted on the phenotyping of rice Al tolerance, working with Dr. Kochian. With regards to presentations of the research from this grant at scientific meetings: 1) Leon Kochian: Invited key note speaker on "Using Molecular Approaches to Improve Cereal Crops for Adaptation to Marginal Soils" at the 1st Biotechnology World Congress, Dubai, United Arab Emirates, February 14, 15, 2012; 2) Leon Kochian, Invited seminar speaker on "Using Molecular, Genetic & Physiological Approaches to Improve Cereal Crops on Marginal Soils", Department of Aridland Agriculture, United Arab Emirates University, Al Ain, UAE, February 16, 2012; 3) Leon Kochian, Invited plenary speaker on "Root Genomics Research Aimed at Improving Crop Adaptation to Resource Limited Environments" at the 2012 Water for Food Conference, Lincoln, Nebraska, May 30-June 1, 2012; 4) Leon Kochian, Invited speaker on "How Plant Roots Respond to and Neutralize Toxic Metals in the Soil" at the Plant Abiotic Stress Symposium at the 2012 American Society of Plant Biologists Annual Meeting, Austin, TX, on July 21, 2012; 5) Leon Kochian, Invited seminar speaker on "Molecular, Genetic & Physiological Investigations of Cereal Crop Adaptations to Soil Abiotic Stresses" at the Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, September 3, 2012; 6) Leon Kochian, Invited symposium speaker on "Variation in cereal aluminum tolerance is due to transcriptional and post-transcriptional regulation of cereal aluminum tolerance genes", Abiotic Stress Workshop, Plant and Animal Genome XXI Meeting, Jan 13, 2013, San Diego, CA Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Juan David Arbelez is a 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. He mentored Anjali marchant, on SNP-based genotyping tools used for both the map-based cloning and NIL generation projects. Juan David also mentored Cornell undergrad student, Haun Chung, who assisted him with phenotyping and genotyping of the rice fine-scale mapping populations. Dr. Jianyong Li is a postdoctoral researcher working in the Kochian and McCouch labs on the role of Nrat1 in rice Al tolerance, and the role of rice cell wall proteins in Al tolerance. He mentored Cornell undergraduate student Taylor Apolostico, who worked with him on the cloning and molecular characterization of rice Nrat1, the novel Al uptake transporter innvolved in rice root cell wall Al tolerance. How have the results been disseminated to communities of interest? Some of the findings from this project were used in Dr. Kochian's lectures on plant aluminum tolerance in his graduate plant mineral nutrition course entitled Crop and Soil Science/Plant Biology (CSS/BIOP) 6420 MINERAL NUTRITION: FROM PLANTS TO HUMANS. Also, a number of talks on this research were presented by Dr. Kochian at US and international scientific meetings. These included: 1) Invited key note speaker on "Using Molecular Approaches to Improve Cereal Crops for Adaptation to Marginal Soils" at the 1st Biotechnology World Congress, Dubai, United Arab Emirates, February 14, 15, 2012; 2) Invited seminar speaker on "Using Molecular, Genetic & Physiological Approaches to Improve Cereal Crops on Marginal Soils", Department of Aridland Agriculture, United Arab Emirates University, Al Ain, UAE, February 16, 2012; 3) Invited plenary speaker on "Root Genomics Research Aimed at Improving Crop Adaptation to Resource Limited Environments" at the 2012 Water for Food Conference, Lincoln, Nebraska, May 30-June 1, 2012; 4) Invited speaker on "How Plant Roots Respond to and Neutralize Toxic Metals in the Soil" at the Plant Abiotic Stress Symposium at the 2012 American Society of Plant Biologists Annual Meeting, Austin, TX, on July 21, 2012; 5) Invited seminar speaker on "Molecular, Genetic & Physiological Investigations of Cereal Crop Adaptations to Soil Abiotic Stresses" at the Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China, September 3, 2012; 6) Invited symposium speaker on "Variation in cereal aluminum tolerance is due to transcriptional and post-transcriptional regulation of cereal aluminum tolerance genes", Abiotic Stress Workshop, Plant and Animal Genome XXI Meeting, Jan 13, 2013, San Diego, CA What do you plan to do during the next reporting period to accomplish the goals? This project is ending but our renewal proposal entitled "Dissecting the Genetic, Molecular and Physiological Basis of Aluminum Tolerance in Rice: Implications for Cereal Improvement" has been funded. This will enable us to continue this research and build upon the progress made in this project.
Impacts
What was accomplished under these goals?
Rice is the most aluminum (Al) tolerant of all cereals and thus is a good source of novel genes underlying physiological mechanisms of Al tolerance that can be used to improve the Al tolerance of rice and other crops to improve crop yields on acid soils that are a significant fraction of the developing countries in S America, Sub-Saharan Africa and Asia. In this project, we have map-based cloned a major and novel Al tolerance QTL and found that ART1, a transcription factor that regulates Al-inducible expression of other rice Al toelrance genes, was responsible for this QTL. This is a significant finding, as it deomnstrates that significant genetic variation for this major Al tolerance gene exisits that cna be exploited via molecular breeding and biotechnology to enhance rice and ther crop sepcies Al tolerance. In this year, we nearly completed all the work for objectives 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) and Objective 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). Objective 1. Fine mapping of a major Al tolerance QTL: One of the project goals is to fine map and clone the gene responsible for the largest effect Al tolerance QTL on chromosome 12 identified in Famoso et al (2011). This QTL, Alt12.1, explains around 20% of the Al tolerance in the biparental RIL population study between IR64 and Azucena and we found that it colocalizes with the candidate gene ART1 (Tutsui et al., 2011), a C2H2 zinc finger transcription factor that is known to regulate ~31 other genes, several of which have been characterized from cloning of Al sensitive mutants and confirmed to confer Al tolerance or sensitivity (Huang et al., 2011, Xia et al., 2010, Yamaji et al., 2009 and Yokosho et al., 2011). Using ~450 recombinants selected from the 4,000 plants generated to fine-map this region, we reduced the candidate region from 3 Mbp to 300 Kbp during the first year of this project. We subsequently generated a ~4,000-plant population to further narrow down the QTL region. Using markers K_3.28, ART1_3.58 and Indel_3.62 we were able to identify 60 recombinant lines across the 300 Kbp candidate region. Three more markers were genotyped across the region in the 60 recombinants to generate genotypic groups for mapping purposes. The progeny of these 60 lines were phenotyped for Al tolerance and their scores were used for fine mapping (F2-F3 strategy). Using this information we were able to reduce the candidate region to a 30 Kbp interval between marker K_3.57 and marker Indel_3.62. This region contains six putative gene models including ART1. Of the 6 putative genes, only ART1 has been reported to be related to Al tolerance (Huang et al., 2009, Huang et al., 2011, Xia et al., 2010 and Yokosho et al., 2011). We strongly believe that ART1 is the gene conferring the natural Al tolerance between IR64 and Azucena at the Alt12.1 QTL. We are now testing this will proceed to test this hypothesis using a transgenic approach over the next 12 months. This will ionvolve complementation studies and expression analysis will be carried out using the IR64 and Azucena alleles to confirm our hypothesis and to explore the relationship among these naturally occurring ART1 alleles and other genes reported to be involved in the Al tolerance mechanism in rice. All previous work has utilized induced mutations rather than natural alleles to study this Al tolerance pathway, so our work will contribute a new perspective to our understanding of Al tolerance in rice. Objective 2. Development of NILs harboring Al tolerance QTL: We first continued development of a set of reciprocal near isogenic lines (NILs) targeting four major Al tolerance QTLs we identified from the previous bi-parental QTL mapping for Al tolerance reported in Famoso et al (2011) on chr 1, 2, 9 and 12. We used five RILs: RIL48, RIL56, RIL186, RIL241 AND RIL252) extracted from a biparental mapping population IR64 (Oryza sativa subpopulation indica) x Azucena (Oryza sativa subpopulation tropical japonica)) to generate 8 near isogenic lines (NILs), each containing one of 4 QTL for Al tolerance. Each QTL was backcrossed into bothIR64 and Azucena backgrounds. In the previous year, we completed two generations of backcrossing for the NILs in the IR64 background, and one generation of backcrossing for the NILs in the Azucena background. Then, 200 BC2 plants from the IR64 background and 200 BC1 from the Azucena background were grown. This 400-plant population was subjected to marker assisted 'positive' selection for each allele of interest using a set of 12 SNP KASPar markers (three markers per QTL) that flank each of the QTL regions in chr 1, 2, 9 and 12. Using this information, 21 BC2 plants in the IR64 background and 29 in the Azucena background were selected for backcrossing. These plants contained one, two, three or recombinant fragments of the QTLs of interest. Between 20 and 30 seeds were obtained on average for each cross.Subsequently, 13 of the BC3 crosses in the IR64 background and 15 of the BC2 crosses in the Azucena background were selected to generate a population of 336 plants, 12 plants per cross. Again positive selection was performed using the set of KASpar markers and 94 plants were selected for background checking, 47 in the IR64 background and 47 in the Azucena background. In collaboration with Dr. Michael Thomson at the International Rice Research Institute (IRRI) in the Philippines, we utilized the 384-SNP BeadArray Golden Gate™ assay (OPA3.1) optimized for indica x japonica crosses to screen DNA samples extracted from the 94 selected plants and two parent controls. The 384-SNP data allowed us to confirm that our selected lines carried the target fragment of interest, and also allowed us to estimate the proportion of residual genome from the donor parent that remained in the genetic background of the recurrent parent for each set of NILs. Using this data, we were able to select the best lines for further backcrossing. Out of 384 SNPs on the assay, 258 SNPs were polymorphic between IR64 and Azucena and were informative for our work. These SNPs were evenly distributed across the genome and allowed us to estimate that 96.21% of the recurrent genome had been recovered in the 47 plants selected from the IR64 background and 91.13% had been recovered in the 48 plants from the Azucena background. These results conformed to theoretical expectations, based on the original percentage of recurrent parent in each of the five RILs selected for developing the NILs, and the number of backcross generations. Currently these 94 plants are being backcrossed at Cornell University (see Figure 6). We expect this will be the last generation of backcrossing. BC3 and BC4 seed will be harvested and planted again at Cornell . This generation will again be genotyped using KASPar markers to determine which plants carry the introgressions of interest, and we will harvest selfed seed from selected plants. Then, the selfed generation will again be planted and KASPar markers will be used to identify plants that are homozygous for the target introgressions. Seed from reciprocal NILs carrying homozygous introgressions at the 4 target QTLs will be harvested in bulk in late summer 2013.
Publications
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Progress 01/15/11 to 01/14/12
Outputs
OUTPUTS: Obj 1) To clone the major rice aluminum (Al) tolerance QTL on chr 12 identified from a Azucena (tol) x IR64 (sens) RIL population, BC1F3 plants from a cross between a RIL harboring the QTL and IR64 were genotyped with 6 SNPs that cover the ~3 Mbp QTL region. 430 recombinants plants were identified and their progeny phenotyped to narrow down the candidate region to 300 Kbp. 4000 BC1F4 plants were generated from the previous BC1F3 recombinants and genotyped with 6 SNPs evenly distributed across the 300 Kbp region. Then, 56 recombinants plants for the region were identified and their progeny phenotyped to map and narrow down the region to a 30 Kbp interval where a very likely candidate Al tolerance gene was identified. Obj 2) NILs that contain four different Al tol QTL from our earlier mapping study are being developed. Three of these QTL on chr 1, 9 and 12 are alleles conferred by the tolerant parent, Azucena, and 1 QTL on chr 2 is conferred by sensitive IR64, indicative of transgressive variation. Three backcross cycles have been carried out and 48 plants have been selected using markers that contain either 1 Azucena allele or a combination of them. 4 other NILs are being developed each containing 1 of the IR64 alleles by backcrossing them with Azucena. 2 backcross cycles have been completed and 48 plants have been positively selected using markers that contain either 1 IR64 allele or a combination of the IR64 alleles. The NILs will be ready for studies by summer 2013. Obj 3) In the 2cd year, the NSF-TV rice diversity panel that was used for Al tolerance GWA analysis in year 1 (genotyped with 44K SNP chip), was re-genotyped with a 1 million SNP chip and GWA analysis re-run. This analysis generated considerable novel rice Al tolerance information as described below in Outcomes. Obj 4) We have shown for a GWA region on chr 2 that the aus-specific Al sensitive haplotype is related to an Nramp transporter that was recently identified as Nrat1, a root plasma membrane Al transporter, which when knocked out increases Al sensitivity. This is consistent with Nrat1 mediating Al uptake away from the root cell wall into the root cell cytoplasm, where it presumably is sequestered in the vacuole. Sequence analysis of Nrat1 identified putative sensitive and tolerant haplotypes as well as possible functional polymorphisms specific to the Al sensitive aus accessions. These data provide valuable information for identifying Nrat alleles that can be used to test the hypothesis that rice Al tolerance is conferred in part by reducing Al concentrations in the cell wall. To test the hypothesis, we selected 12 Al tolerant and sensitive aus, japonica and indica lines from the NSF-TV diversity panel. Aus lines are generally much more Al sensitive than japonica rice. The Nrat1 gene was cloned and sequenced from these lines and then we expressed the Al tolerant and sensitive Nrat1 alleles in yeast and quantified yeast Al uptake and tolerance. Finally, we measured Al levels in the root tip cell wall and symplasm in the 12 rice lines to see if the tolerant versus sensitive Nrat1 alleles are associated with altered Al content in the rice root cell wall or symplasm. PARTICIPANTS: Randy Clark is a 5th year PhD student in the Department of Biological and Environmental Engineering in the Kochian and McCouch labs. 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 second 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. Dr. Jianyong Li is a postdoctoral researcher working in the Kochian and McCouch labs on the role of Nrat1 in rice Al tolerance, and the role of rice cell wall proteins in Al tolerance. he replaced Dr. Adam Famoso, who was the lead researcher on the project, who after completing his PhD with us left in the fall of 2010 to take a position with Pioneer. James Jones-Rounds is a technician who worked between the Kochian and McCouch labs and made a major contribution to the phenotyping of rice seedlings from both the rice diversity panel and two bi-parental mapping populations. He has moved to the Department of Neurobiology at Cornell, as neurobiology is his direct field of interest and he hopes to attend graduate school in neurobiology. TARGET AUDIENCES: In addition to the training of PhD students, postdocs, and technicians, through this project we have mentored both undergraduate students, and a high school student. Melissa major is a Cornell University undergraduate biology student who assisted Jianyong Li on the research on rice Nrat1. Kengo Onishi is a high school student from Ithaca High who worked full time during the summer of 2011 and assisted on the phenotyping of rice Al tolerance. Leon Kochian: Invited symposium speaker on "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: Invited Symposium Speaker on "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: Invited speaker on "Update on research on rice aluminum tolerance and maize phosphorous efficiency projects ", Generation Challenge Program Comparative Genomics Challenge Initiative Annual Meeting, Sete Lagoas, Brazil, March 21, 2011. Leon Kochian: Invited speaker on "Molecular mechanisms of crop adaptation to acid soils", 2cd International Symposium on Integrated Plant Biology, Lanzhou, China, August 26, 2011. Susan McCouch: Plenary speaker on "Natural Variation In Rice: Mixing, Matching, Keeping, Sharing", Plant and Animal Genome (PAG) Meeting, San Diego, January, 2012. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Obj 1) For the map-based cloning of the major Al tolerance QTL on chr 12, fine scale mapping enabled us to narrow down the candidate region to a 30 Kbp interval that co-localizes with ART1 (and other 5 genes). ART1 is a transcription factor previously identified by Ma's lab that controls the expression of a number of rice Al tolerance genes and is quite likely the candidate gene for the Alt12.1 QTL. This same region was identified from our GWAS study on rice Al tolerance. We now are working via complementation analysis to verify that ART1 is responsible for the Alt12.1 QTL. This is the 1st demonstration that variation in ART1 is associated with rice Al tolerance and sets the stage for improving rice Al tolerance via molecular breeding based on superior ART1 alleles. Obj 3) The new GWA analysis of rice Al tolerance using the 1 M SNP chip yielded exciting new information, including identification of a number of novel Al tolerance QTL that co-localize with candidate genes suggested from previous studies, and novel QTL associated with new candidate genes. Furthermore, the new GWA analysis yielded QTL with much better spatial resolution compared to the Al tolerance QTL identified from the 44K SNP genotyping. Finally, the new GWA analysis yielded very interesting information regarding outliers -lines especially within the Japonica and Indica subpopulations that are considerably more Al tolerant or sensitive than the mean for the specific subpop. The very interesting observation here is that many of these outlier lines harbor introgressions from other subpopulations that co-localize with specific Al tolerance QTL. Obj 4) An excellent example of the value of linking functional genomics and natural variation comes from the GWA analysis of rice PM localized Al transporter Nrat1, and we have shown this year that the Nrat1 alleles from Al sensitive aus lines all share the same 4 amino acid mutations, while the Al tolerant aus and all of the indica and japonica lines (which are more Al tolerant) all express the same wild type Al tolerant Nrat1 haplotype. The wild type Nrat1 mediated higher levels of yeast Al uptake than did the mutant version from Al sensitive aus, which was associated with greater Al sensitivity. This increased sensitivity must be due to the lack of an additional tonoplast Al transporter that in rice functions to sequester the absorbed Al into the root cell vacuole. The use of site directed mutagenesis to change each of the four "mutant" amino acids back to the wild type AA showed that all 4 mutations are required to reduce Nrat1-mediated Al uptake across the plasma membrane associated with the reduced rice Al tolerance in the selected aus lines. Finally, in planta experiments showed that the more Al sensitive aus lines maintained higher cell wall Al levels and lower symplastic Al levels. This indicates that the amino acid alterations in the Al sensitive aus haplotypes alter Nrat1 transport function, such that the transporter is less effective at moving Al from the cell wall to the cell symplasm, and the reduction in cell wall Al mediated by Nrat1 in more tolerant rice lines is a bona fide mechanism of rice Al tolerance.
Publications
- Famoso AN; Zhao Z; Clark RT; Tung C-W; Wright M; Bustamante C; Kochian LV & McCouch SR. 2011. Genetic architecture of aluminum tolerance in rice (O. sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genetics 7(8): e1002221
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Progress 01/15/10 to 01/14/11
Outputs
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.
Impacts
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
Publications
- 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.
- Famoso AN; Zhao Z; Clark RT; Tung C-W; Wright M; Bustamante C; Kochian LV & McCouch SR. 2011. Genetic architecture of aluminum tolerance in rice (O. sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genetics (In Press).
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