Progress 08/15/06 to 08/14/10
Outputs
OUTPUTS: Outputs from this grant included the performance and analysis of experiments that provide insight into the hormonal controls of root branching in Arabidopsis and tomato. Results from these studies were presented at a number of conference and symposia during this grant period. These included oral presentations at the Auxin 2008 Meeting, the 8th International Conference on the Plant Hormone Ethylene, the American Society of Plant Biology (ASPB) national meeting and the Northeastern regional meeting, the North Carolina Plant Molecular Biology Retreat, and departmental seminars at the University of Tennessee, University of North Carolina at Asheville, University of Florida, Duke University, Penn State University, Clemson University, and the Danforth Plant Science Center. The work was also reported in poster presentations at several meetings of the American Society of Plant Biology meeting, the 8th International Conference on the Plant Hormone Ethylene, the Gordon Research Conference on Plant Metabolic Engineering, and the FASEB Mechanisms of Plant Development Conference. The PI also participated in a work shop for high school teachers with a presentation on Genetically Modified Crops and in tutoring students at Paisley IB Magnet School for the Science Olympiad Program. Finally, this project supported the establishment of a Wake Forest University Campus Garden that is used for growth of tomatoes for research as well as education outreach and for food for the WFU Campus Kitchen, a group that provides meals for organizations that serve the disadvantaged. This work was cited in articles directed to the general public which were published in the Winston Salem Journal, the Raleigh Observer, and Wake Forest University publications. PARTICIPANTS: This project has trained undergraduates, graduate students, and post-doctoral researchers. Three graduate students have been trained by this project and have now completed their degrees. Poornima Sukumar's PhD project focused on the examination of root gravitropism and adventitious root formation and the roles of flavonoids and ethylene in controlling these processes. Mary Beth Lovin's PhD examined the regulation of lateral root branching by environmental changes that may modulate auxin synthesis and/or transport to enhance root formation. Cassie Mattox's MS research explored the mechanisms by which auxin transport protein localization controls root branching in Arabidopsis. Two Post-doctoral research associates have worked on this project. Sangeeta Negi's focused on the role of ethylene in regulation of lateral root branching in tomato and Arabidopsis and isolation and characterization of tomato mutants with altered root branching patterns. Daniel Lewis focused on the mechanisms that control flavonoid accumulation and thereby regulate auxin transport during root formation and gravitropism and development of methods for examination of auxin transport and quantitative analyses of gene expression. Two undergraduates have also worked on this project, Kevin Cooper and Jonathan Isley, with both students now enrolled in graduate programs in plant biology. A number of collaborations were formed during this project. With Dr. Maria Ivanchenko, Oregon State University, we have published two articles on the regulation of Arabidopsis root architecture by ethylene. Dr. Harry Klee, University of Florida and Dr. Jim Giovannoni, Cornell University, have provided genetic resources and insight which aided our characterization of root architecture in these tomato mutants. We collaborated with Dr. Alan Jones, University of North Carolina, and Dr. Ralph Panstruga, Max Planck Institute for Plant Breeding, to characterize auxin transport in mlo thigotropic mutants. Dr. Jerry Cohen, University of Minnesota, has invited us to visit his laboratory to perform free IAA measurements and along with a graduate student in his laboratory, Xing Liu, who are coauthors on our recent publication on ethylene regulation of root architecture in tomato. We have collaborated with Dr. Joe Kieber, University of North Carolina, to quantify ethylene levels in mutants, Dr. Alison DeLong, Brown University, and Dr. Abidur Rahman, Iwate University, Japan, in genetic analysis of gravitropism. TARGET AUDIENCES: The target audience for this research is the scientific community and communication occurs through publications and presentations. Additionally, throughout the period of this grant, Dr. Muday has worked with undergraduates, middle school and high school students, and secondary school teachers. She developed an undergraduate course in Plant Physiology and has taught lectures on Plant GMOs to teachers. She also participated in teaching secondary school students a variety of scientific principles through interaction with the Science Olympiad Team at a local IB Magnet school. This school has 66% minority enrollment with student participation on Science Olympiad mirroring the diversity of the school. Finally, she participated, with the assistance of USDA funds, in establishing a WFU Campus Garden that is used for growth of mutant tomatoes to generate seeds for ongoing experiments and for growing more than 50 varieties of heirloom tomatoes for educational outreach and for use by Campus Kitchen to provide produce used in meals distributed through organizations that serve the disadvantaged in our community. These tomatoes were used development of exercises to teach plant biology and genetics to 3rd, 7th, 9th, and 11th grade students using heirloom tomatoes, bringing these lessons to near 500 students. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The outcomes and impacts from this work are primary focused on scientific knowledge resulting from experimental results. During this grant period, we have completed the first step in understanding the effects of the plant hormone ethylene on root formation and gravitropism in Arabidopsis and tomato and have published or submitted six papers in this area. These papers show that ethylene inhibits lateral root initiation and elongation and that this effect is mediated by well know signaling proteins. We have found that lateral roots are regulated in very similar ways in Arabidopsis and tomato, but that there are fundamental differences in ethylene regulated adventitious root formation in these two species. We have also provided insight into the ethylene controls of root and hypocotyl gravitropic response in Arabidopsis. We have also characterized a new tomato mutant with altered root branching to provide insight into genetic controls of root architecture in this crop plant. Finally, we have identified hormonal controls of expression of the genes encoding flavonoid biosynthesis enzymes that leads to enhanced synthesis of flavonols, which in turn regulate root growth and development. Training of four graduate students and two post-doctoral researchers has been enhanced by this project. We have also greatly expanded our agricultural capabilities through establishment of the Wake Forest University Campus Garden, which we have utilized for tomato seed propagation. We have also optimized new techniques in live imaging of roots using both laser scanning confocal microscopy and video imaging. We have also established reproducible techniques for Quantitative Real Time Polymerase Chain Reaction for use with RNA samples derived from both Arabidopsis and tomato. We have also developed methods for examination of auxin transport and this has resulted in a technique oriented publication. We have also established collaborations with a number of investigators in which we have applied the techniques developed with USDA support resulting in collaborative publications in areas including light and mechanical controls of root growth orientation.
Publications
- Lewis, DR, Ramirez, MV, Miller, ND, Keith, R, Helm, R, Winkel, BSJ, Muday, GK (2010) Auxin and ethylene induce distinct flavonol accumulation patterns through independent transcriptional networks: Plant Cell: In review
- Negi, S, Sukumar, P, Liu, X, Cohen, JD, and Muday, GK (2010) Genetic dissection of the role of ethylene in regulating auxin dependent lateral and adventitious root formation in tomato. Plant Journal 61: 3-15
- Chen, Z, Noir, S. Kwaiitaal, M, Hartmann, HA, Wu, M-J, Mudgel, Y, Sukumar, P, Muday, GK, Panstruga, R, and Jones, AM (2009) Two seven-transmembrane domain MLO proteins co-function in root Thigmomorphogenesis. Plant Cell: 21:1972-1991
- Lewis, DR and Muday, GK (2009) Measurement of auxin transport in Arabidopsis thaliana Nature Protocols: 4: 437-451
- Sukumar, P, Edwards, KS, Rahman, A, and DeLong, A, and Muday, GK (2009) PINOID kinase regulates root gravitropism through modulation of PIN2-dependent basipetal auxin transport in Arabidopsis thaliana. Plant Physiol: 150: 722-735
- Negi, S, Ivanchenko, MG, and Muday, GK (2008) Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana. Plant J. 55: 175-187
- Ivanchenko, MG, Muday, GK, and Dubrovsky, JG. (2008) Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana. Plant J. 55: 335-347
- Buer, CS, Muday, GK, Djordjevic, MA (2008) Implications of long-distance flavonoid movement in Arabidopsis thaliana. Plant Signaling and Behavior: 3: 415-417
- Buer, CS, Muday, GK, Djordjevic, MA (2007) Flavonoids are transported long distances in Arabidopsis. Plant Physiol. 145: 478-490
- Muday, GK and Rahman, A (2007) Auxin transport and the integration of gravitropic growth. In Plant Tropisms: eds Gilroy, S and Masson, P, Blackwell Publishing pp 47-78.
- Muday, GK, Brady, SR, Argueso, C, Deruere, J, Kieber, JJ, and DeLong, A. (2006) RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling. Plant Physiology: 141: 1617-1629
- Sibout, R, Sukumar, P, Hettiarachchi, C, Holm, M, Muday, GK and Hardtke, CS (2006) Opposite root growth phenotypes of hy5 vs. hy5 hyh correlate with increased constitutive auxin signaling. PLOS Genetics: 2:1898-1911
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Outputs from this grant included the performance and analysis of experiments that provide insight into the hormonal controls of root branching in Arabidopsis and tomato. Results from these studies were presented at a number of conference and symposia during this year. These included oral presentations at the Auxin 2008 Meeting, the 8th International Conference on the Plant Hormone Ethylene, Wake Forest University Perspectives in Biology Symposia, the North Carolina Plant Molecular Biology Retreat, and departmental seminars at the University of Tennessee, University of North Carolina at Asheville, and in poster presentations at the American Society of Plant Biology meeting and the 8th International Conference on the Plant Hormone Ethylene, and the Gordon Research Conference on Plant Metabolic Engineering. The PI also participated in a work shop for high school teachers with a presentation on Genetically Modified Crops and in tutoring students at Paisley IB Magnet School for the Science Olympiad Program. Finally, this project supported the establishment of a Wake Forest University Campus Garden that is used for growth of tomatoes for research as well as education outreach and for food for the WFU Campus Kitchen, a group that provides meals for organizations that serve the disadvantaged. PARTICIPANTS: This project has trained undergraduates, graduate students, and post-doctoral researchers. Kevin Cooper is undergraduate working on the regulation of root architecture by flavonoids and Jonathan Isley worked on examination of the localization of auxin transport proteins under conditions and in mutants that exhibit altered auxin transport. Three graduate students have been trained by this project. Poornima Sukumar's projects include the examination of root gravitropism and adventitious root formation and the roles of flavonoids and ethylene in controlling these processes. Mary Beth Lovin, who recently completed her PhD, has examined the regulation of lateral root branching by environmental changes that may modulate auxin synthesis and/or transport to enhance root formation. Cassie Mattox, who recently completed her MS, explored the mechanisms by which auxin transport protein localization controls root branching in Arabidopsis. Two Post-doctoral research associates have worked on this project. Sangeeta Negi's projects focus on the role of ethylene in regulation of lateral root branching in tomato and Arabidopsis and isolation and characterization of tomato mutants with root branching patterns. Daniel Lewis focused on the mechanisms that control flavonoid accumulation and thereby regulate auxin transport during root formation and gravitropism. A number of collaborations were formed during this project. With Dr. Maria Ivanchenko, Oregon State University, we have published two articles on the regulation of Arabidopsis root architecture by ethylene. Dr. Harry Klee, University of Florida and Dr. Jim Giovannoni, Cornell University, have provided generic resources and greatly encouraged the characterization of root architecture in these tomato mutants. We collaborated with Dr. Alan Jones, University of North Carolina, and Dr. Ralph Panstruga, Max Planck Institute for Plant Breeding, to characterize auxin transport in mlo thigotropic mutants. Dr. Jerry Cohen, University of Minnesota, has invited us to visit his laboratory to perform free IAA measurements and along with a graduate student in his laboratory, Xing Liu, is a coauthor on our recent publication on ethylene regulation of root architecture in tomato. We have collaborated with Dr. Joe Kieber, University of North Carolina, to quantify ethylene levels in mutants. TARGET AUDIENCES: The target audience for this research is the scientific community and communication occurs through publications and presentations. Additionally, throughout the period of this grant, Dr. Muday has worked with undergraduates, middle school and high school students, and secondary school teachers. She developed an undergraduate course in Plant Physiology and has taught lectures on Plant GMOs to teachers. She also participated in teaching secondary school students a variety of scientific principles through interaction with the Science Olympiad Team at a local IB Magnet school. This school has 66% minority enrollment with student participation on Science Olympiad mirroring the diversity of the school. Finally, this year she participated, with the assistance of USDA funds, in establishing a WFU Campus Garden that is used for growth of mutant tomatoes to generate seeds for ongoing experiments and grew over 50 varieties of heirloom tomatoes for educational outreach and for use by Campus Kitchen to provide produce used in meals distributed through organizations that serve the disadvantaged in our community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The outcomes and impacts from this work are primary focused on scientific knowledge resulting from experimental results. During this project period, we have completed the first step in understanding the effects of the plant hormone ethylene on root formation in Arabidopsis and tomato and have published three papers in this area. These papers show that ethylene inhibits lateral root initiation and elongation and that this effect is mediated by well know signaling proteins. We have found that lateral roots are regulated in very similar ways in Arabidopsis and tomato, but that there are fundamental differences in ethylene regulated adventitious root formation in these two species. We have also characterized a new tomato mutant with altered root branching to provide insight into genetic controls of root architecture in this crop plant. Finally, we have identified hormonal controls of expression of the genes encoding flavonoid biosynthesis enzymes that leads to enhanced synthesis of flavonols, which in turn regulate root growth and development. Training of four graduate students and two post-doctoral researchers has been enhanced by this project. We have also greatly expanded our agricultural capabilities through a collaboration with the Children's Home and Farm and establishment of the Wake Forest University Campus Garden, as we have utilized these sites for a third season of tomato seed propagation. We have also optimized new techniques in live imaging of roots using both laser scanning confocal microscopy and video imaging and established reproducible techniques for qRT-PCR for use with RNA samples derived from both Arabidopsis and tomato.
Publications
- Negi, S, Sukumar, P, Liu, X, Cohen, JD, and Muday, GK (2009) Genetic dissection of the role of ethylene in regulating auxin dependent lateral and adventitious root formation in tomato. Plant Journal. In press
- Chen, Z, Noir, S. Kwaiitaal, M, Hartmann, HA, Wu, M-J, Mudgel, Y, Sukumar, P, Muday, GK, Panstrug, R, and Jones, AM (2009) Two seven-transmembrane domain MLO proteins co-function in root Thigmomorphogenesis. Plant Cell: 21:1972-1991.
- Lewis, DR and Muday, GK (2009) Measurement of auxin transport in Arabidopsis thaliana Nature Protocols: 4: 437-451.
- Sukumar, P, Edwards, KS, Rahman, A, and DeLong, A, and Muday, GK (2009) PINOID kinase regulates root gravitropism through modulation of PIN2-dependent basipetal auxin transport in Arabidopsis thaliana. Plant Physiol: 150: 722-735.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Outputs from this grant included the performance and analysis of experiments that provide insight into the hormonal controls of root branching in Arabidopsis and tomato. Results from these studies were presented at a number of conference and symposia during this year. These included oral presentations at the USDA Project Director's meeting, the Auxin 2008 Meeting, 5th International Symposium on Adventitious Root Formation, the North Carolina Plant Molecular Biology Retreat, and departmental seminars at Pennsylvania State University, University of Minnesota, and the University of Florida and in poster presentations at the American Society of Plant Biology meeting and the 19th International Conference on Arabidopsis Research. The PI also participated in a work shop for high school teachers with a presentation on Genetically Modified Crops and in tutoring students at Paisley IB Magnet School for the Science Olympiad Program. Finally, this work was disseminated through the above activities, but was also the subject of a newspaper article that appeared in the Winston-Salem Journal in September, 2008. PARTICIPANTS: This project has trained undergraduates, graduate students, and post-doctoral researchers. Melissa Lyle is a undergraduate lab member who is working on the process of adventitious root development and mechanisms that regulate auxin transport in the hypocotyl to enhance root formation. Kevin Cooper is another undergraduate working on the regulation of root architecture by flavonoids. These undergraduates have been introduced to the process of scientific experimentation and have worked with both the PI to learn the theory of their projects and with other laboratory members to learn the methods employed to ask scientific questions. Three graduate students have been trained by this project. Poornima Sukumar's projects include the examination of root gravitropism and adventitious root formation and the roles of flavonoids and ethylene in controlling these processes. Mary Beth Lovin is examining the regulation of lateral root branching by environmental changes that may modulate auxin synthesis and/or transport to enhance root formation. Cassie Mattox is exploring the mechanisms by which auxin transport protein localization controls root branching in Arabidopsis. These students have been trained in the scientific process, beginning with synthesis of the literature, definition of research questions, and written overviews of planned experiments. All three students are learning to write scientific papers summarizing their findings. These students have all presented their work at scientific meetings through both poster and oral presentations. Two Post-doctoral research associates have worked on this project. Sangeeta Negi's projects focus on the role of ethylene in regulation of lateral root branching in tomato and Arabidopsis and isolation and characterization of tomato mutants with root branching patterns. Daniel Lewis focuses on the mechanisms that control flavonoid accumulation and thereby regulate auxin transport during root formation and gravitropism. Post-doctoral researchers have had opportunities to plan an experimental outline, write papers and grants, and present their findings in both poster and oral presentations, publish their work, and train more junior laboratory members. A number of collaborations were formed during this project. With Dr. Maria Ivanchenko, Oregon State University, we have published two articles on the regulation of root architecture by ethylene. Dr. Harry Klee, University of Florida and Dr. Jim Giovannoni, Cornell University, have provided generic resources and greatly encouraged the characterization of root architecture in these tomato mutants. Dr. Jerry Cohen, University of Minnesota, has allowed us to visit his laboratory to perform free IAA measurements. We have collaborated with Dr. Joe Kieber, University of North Carolina, to quantify ethylene levels in mutants. Finally, we have developed a collaborative effort with the Methodist Children's Home and Farm, in which they have shared land for propagation of tomato plants to obtain sufficient seeds in exchange for us to grow and contribute additional produce for consumption by their students and for their weekly farmer's market. TARGET AUDIENCES: Throughout the period of this grant, Dr. Muday has worked with undergraduates, middle school and high school students, and secondary school teachers. She developed an undergraduate course in Plant Physiology and has taught lectures on Plant GMOs to teachers. She also participated in teaching secondary school students a variety of scientific principles through interaction with the Science Olympiad Team at a local IB Magnet school. This school has 66% minority enrollment with student participation on Science Olympiad mirroring the diversity of the school. Finally, the genetic variants of tomato utilized in this study have been grown for seed at the Methodist Children's Home and Farm. A number of members of the community have learned about plant hormones and their role in fruit ripening and plant architecture as a result of this study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The outcomes and impacts from this work are primary focused on scientific knowledge resulting from experimental results. During this project period, we have completed the first step in understanding the effects of the plant hormone ethylene on root formation in Arabidopsis and have published two papers in this area. These papers show that ethylene inhibits lateral root initiation and elongation and that this effect is mediated by well know signaling proteins. We are working on our two additional papers examining the regulation of root development in tomato. We have found that lateral roots are regulated in very similar ways in Arabidopsis and tomato, but that there are fundamental differences in ethylene regulated adventitious root formation in these two species. We have also characterized a new tomato mutant with altered root branching to provide insight into genetic controls of root architecture in this crop plant. Training of three graduate students and two post-doctoral researchers has been enhanced by this project. We have also greatly expanded our agricultural capabilities through a collaboration with the Children's Home and Farm, as we have utilized one of their farm sites for a second and larger scale season of tomato seed propagation and contributed to their training program with production of produce for consumption and sale by the students living there. We have also optimized new techniques in live imaging of roots using both laser scanning confocal microscopy and video imaging. We have also established reproducible techniques for Quantitative Real Time Polymerase Chain Reaction for use with RNA samples derived from both Arabidopsis and tomato.
Publications
- Negi, S, Ivanchenko, MG, and Muday, GK (2008) Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana. Plant J. 55: 175-187
- Ivanchenko, MG, Muday, GK, and Dubrovsky, JG. (2008) Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana. Plant J. 55: 335-347
- Buer, CS, Muday, GK, Djordjevic, MA (2008) Implications of long-distance flavonoid movement in Arabidopsis thaliana. Plant Signaling and Behavior: 3: 415-417
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Progress 08/15/06 to 08/14/07
Outputs
During year one of this grant, we have made significant progress on the Arabidopsis experiments and have almost completed our field season to increase the available seeds from tomato mutants to pursue the tomato experiments in depth. This proposal focused on cross talk between auxin and ethylene on three processes that define root architecture: Root gravitropism, lateral root branching, and adventitious root formation. We have now published three papers that address the proposed experiments in this grant. The first paper addresses one of the primary objectives of this proposal and shows that Arabidopsis root gravitropism is inhibited by ethylene and that this inhibition requires flavonoid synthesis. The second paper illustrates that flavonoid precursors can be transported long distances in plants and this understanding is critical for building models on the function of flavonoids. Third, we have collaborated with Dr. Christian Hardtke to test the possibility that the
phenotypic alterations in the hy5 mutant are linked to altered flavonoid synthesis. The graduate student supported by this project, Poornima Sukumar, has developed a system to examine adventitious root formation in Arabidopsis, which leads to adventitious root formation on the hypocotyl with predictable numbers and position. She has examined the role of auxin and ethylene in regulation of formation of adventitious roots, and demonstrated an absolute requirement of auxin transport in this process. Ethylene negatively regulates adventitious root formation and reduces the effect of exogenous IAA in stimulation of adventitious root formation. The tt4 mutant, which makes no flavonoids, has enhanced adventitious root formation, consistent with enhanced IAA transport in the mutant and a positive role of auxin in regulation of adventitious root formation. Poornima presented these results at the ASPB meeting and we have begun to write a manuscript describing these results. The post-doc
supported by this project, Sangeeta Negi, is examining the role of ethylene in regulation of lateral root formation in Arabidopsis and tomato. She has clearly demonstrated that the ethylene precursor, ACC, reduces root branching in Arabidopsis and tomato and that mutants of both species isolated for ethylene insensitive growth, also show ethylene insensitive lateral root formation. Furthermore, mutants with enhanced ethylene synthesis and constitutive signaling show reduced lateral root formation. Sangeeta has shown that treatments that alter ethylene abundance or response also alter acropetal IAA transport. The ethylene mutants still respond to exogenous auxin, which stimulates lateral root formation, suggesting that the effect of ethylene may be directly tied to regulation of IAA transport, rather than the ability of roots to respond to auxin. Consistent with this finding, mutants that are defective in flavonoid synthesis, which negatively regulate IAA transport, are much less
sensitive to the effect of ACC. Sangeeta presented these results at the ASPB meeting and we are currently in the initial stages of writing a paper describing these results.
Impacts
Root architecture, which reflects growth orientation and branching patterns, has a profound impact on plants' productivity and stress-resistance. Genetic variants of crop plants that enhance root branching are more drought- and stress-tolerant. Similarly, a strong root gravitropic response allows plants to penetrate the soil more deeply, thereby minimizing dehydration and maximizing nutrient recovery. Plant propagation relies heavily on the ability of shoot cuttings to effectively generate adventitious roots, yet the mechanisms behind the dramatic variation between species in the propensity to form adventitious roots are unknown. Despite their obvious agricultural importance, very little is understood about the molecular mechanisms that control root orientation and branching. This research is focused on examining the hormonal controls of root architecture to understand the mechanisms that control branch formation and root orientation, with a focus that is primarily
genetic. We are using the model plant species, Arabidopsis thaliana, and the crop species, Lycopersicon esculentum (tomato), for these studies. We have plants of both species with defects in genes that control the physiological responses to the plant hormones auxin and ethylene and are characterizing the root architecture in these mutants, as well as using these mutants to identify the mechanisms by which these hormones control root architecture. Identification of genes that control these processes can lead to improvement of crops to enhance root formation and thereby enhance growth and crop yields.
Publications
- Buer, CS, Sukumar, P, and Muday, GK (2006) Ethylene induced flavonoid synthesis modulates root gravitropism. Plant Physiology: 140: 1384-1396
- Buer, CS, Muday, GK, Djordjevic, MA (2007) Flavonoids are transported long distances in Arabidopsis. Plant Phys. Published on line. August 24, 2007; 10.1104/pp.107.101824
- Sibout, R, Sukumar, P, Hettiarachchi, C, Holm, M, Muday, GK and Hardtke, CS (2006) Opposite root growth phenotypes of hy5 vs. hy5 hyh correlate with increased constitutive auxin signaling. PLOS Genetics: 2:1898-1911
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