GROWTH AND DEVELOPMENT OF RHIZOMES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0220680
• Grant No.: 2010-65116-20458
• Proposal No.: 2009-03477
• Project Start Date: Jan 1, 2010
• Project End Date: Dec 31, 2013
• Grant Year: 2010
• Program Code: [91414]- Plant Biology: Growth and Development

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
Crop & Soil Sciences

Non Technical Summary
This proposal addresses the growth and development of rhizomes, combined vegetative/reproductive structures that are a valuable asset in establishment and persistence of productive stands of biomass, forage, and turfgrasses already cultivated on >60 million acres and likely to increase. Rhizomes also enhance "weediness" of many invasive plants that infest millions of acres of crops and native grasslands, causing economic losses and hindering utilization of potentially valuable genes from exotic relatives in crop improvement. The basic gene set associated with rhizome development appears widespread and information about rhizomatousness from botanical models extrapolates well. Guided by new studies to clarify rhizome developmental biology and ontogeny, laser microdissection coupled with massively parallel sequencing will yield a very precise and essentially complete profile of genes and genetic networks functioning during rhizome development. Genetic analysis of a novel plant population that segregates for rhizomatousness improving those explained in prior studies. Our findings may enable development of novel methods to enhance productivity of desirable plants and control undesirable ones, also training young scientists in integrative contemporary approaches to addressing agricultural challenges.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2300	1050	67%
206	2300	1080	33%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2300 - Weeds;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;

Keywords

qtl

perennial

weed

invasive

Goals / Objectives
Our long-term goal is to implement new approaches to regulating growth and development of rhizomes. Identification of genes, regulatory elements, and biochemical functions that are important to rhizome development but dispensable to other plant processes would provide the foundation to search for plant growth regulators that specifically target rhizomes. Such targeting of growth regulation to rhizomes might provide for control of rhizomatous weeds even in closely related crops, for example Johnsongrass (S. halepense) in sorghum. Moreover, better understanding of rhizome development may also benefit improvement of turf and forage grasses, and development of plant genotypes optimal for biomass/biofeedstock production on marginal lands. In the proposed funding period, we will build on our prior results by testing appropriate null hypotheses to further explore the following questions: (1) What are the cellular processes that are crucial for development of rhizomes, a hybrid reproductive/storage organ (2) What is the spectrum of genetic polymorphisms (QTLs) that perturb developmental pathways leading to the formation and growth of rhizomes (3) What are the intersections between cellular processes/developmental signals and genomic locations implicated in growth and development of rhizomes

Project Methods
Methods to be applied toward the respective goals are as follows: (1) What are the cellular processes that are crucial for development of rhizomes, a hybrid reproductive/storage organ Transcript profiling based on Solexa (or other) sequencing is expected to yield ~5 million tags (reads) for each of six samples. These voluminous expression tags, collectively sufficient to sample each sorghum gene an average of 1000 times if genes were equally expressed (which of course they will not be), will permit us to dramatically improve knowledge of relative expression patterns of specific gene functional groups in the rhizotranscriptome, also revealing how these patterns relate to changes in expression profiles over the course of rhizome development. Of especially high value is expected to be the identification of an appreciable set of developmental markers (differentially expressed genes) necessary to future studies to precisely pin down the point at which vegetative buds are committed to tiller versus rhizome development. (2) What is the spectrum of genetic polymorphisms (QTLs) that perturb developmental pathways leading to the formation and growth of rhizomes We will map and phenotype a new recombinant inbred line (RIL) population derived from the S. bicolor x S. propinquum cross, substantially improving on early-generation characterization of the number, locations, and phenotypic effects of QTLs contributing to the formation and growth of rhizomes in an F2 population. To our knowledge, this is the first recombinant inbred (RIL) population in any taxon that segregates for rhizomatousness. Because RIL genotypes are reproducible, this population will provide the means to conduct studies that require destructive sampling, for example investigating both endogenous biochemical-physiological parameters and exogenous experimental treatments that could not be studied on single F2 plants. (3) What are the intersections between cellular processes/developmental signals and genomic locations implicated in growth and development of rhizomes How will we bridge the gap from linked markers and expression patterns to determinant genes First, the interspecific RILs (above) provide a foundation for fine mapping and positional cloning of specific QTLs by conventional approaches. In support of this goal, the ability to identify intersections between expression data and genetic polymorphisms (QTLs; aim 2) from our unique RIL population provides one means of narrowing the field of candidates for genes that may be directly responsible for the unique patterns of growth, development, and metabolism that differentiate rhizomes from stems or other plant organs. Voluminous expression tags (aim 1) will permit us to investigate how expression of particular genes and/or gene functional groups (for example, groups of genes sharing common Pfam domains) are related to rhizome QTLs (both the genome-wide set and individual QTLs), how these patterns change over the course of rhizome development, and to what degree these patterns are supported by data from additional taxa (such as ginger and turmeric, see below).

Progress 01/01/10 to 12/31/13

Outputs
Target Audience: Outcomes are salient to both fundamental and applied agriscience researchers, as well as private sector research and development teams. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? MS student W. Kong is the primary person supported by this project at UGA, and has been a major participant in phenotyping and genetic mapping. Ms Kong completed her MS thesis, gave two posters at PAG (2012-3), and one workshop talk at PAG (2014) based on the project. She has now undertaken PhD studies in the PI's lab. PhD student Chengbo Zhou has been a major player in S. halepense LD analysis PhD student H. Jin has been a major participant in genetic mapping Several undergraduates have participated in phenotyping and genotyping (C. Pillay, E. Giattina, D. Rassler, J. Perry, A. Kerr) Cornell:. Robyn Johnston, postdoctoral researcher analyzed rhizome development and performing laser-microdissection of rhizome for RNA-seq analyses. Endia LaNia Blunt is conducting in situ hybridization to validate expression patterns of selected genes. This commitment helped to engage the US Department of Energy Joint Genome Institute, to provide whole-genome Illumina sequence for S. propinquum and S. halepense. The PI has developed collaboration with S. Cox, The Land Institute, to explore the transmission genetics of a cross between tetraploid S. bicolor (via an unreduced gamete) and S. halepense. This cross may provide a supplementary and complementary means to compare rhizomatousness at the diploid (S. propinquum) and tetraploid (S. halepense) levels. How have the results been disseminated to communities of interest? Both the PI and co-PI have presented the work at relevant professional meetings, Ms Kong has presented several posters, and refereed publications are published (1) and in progress (at least 2 more). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Outputs To provide necessary knowledge of the development and ontogeny of rhizomes, we have analyzed rhizome development in Sorghum halepense and S. propinquum. In both species, primary rhizomes develop from buds in the axils of photosynthetic leaves, and grow in a horizontal or downward direction. Rhizome apices may subsequently reorient to a vertical growth habit and initiate leafy shoots, indicating that they are not committed to one specific identity. This developmental plasticity makes it difficult to determine the identity of any individual rhizome apex. However, we have discovered that axillary buds on rhizome segments can be released from apical dominance by excision of the primary rhizome apex. In S. halepense these buds will develop as green vertical shoots if the rhizome segment is excised from the parent plant, or as secondary rhizomes if the segment remains attached to the parent plant. We have utilized this system to select apices of known identity for laser capture and RNA sequencing. This study has revealed differences between S. halepense and S. propinquum that may contribute to the increased rhizomatousness of S. halepense. S. propinquum initiates many leafy axillary shoots (tillers), but is slower to begin initiating rhizomes than S. halepense. The rhizomes initiated by S. propinquum plants reorient to vertical (leafy shoot) growth more rapidly than in S. halepense. In S. propinquum, unlike in S. halepense, axillary buds on primary rhizomes may develop as leafy shoots, even when the rhizome remains attached to the parent plant. Given that the goal of this project is to identify targets for the control of S. halepense, and that rhizome development seems to be controlled differently in S. propinquum than in S. halepense, we have conducted transcript profiling in S. halepense. We grew S. halepense plants from the Gypsum-9 line, and completed tissue collections and laser captures. We collected meristems from buds induced to develop either as rhizomes or as leafy shoots and compared transcripts in these two meristem types. We collected two replicates of each meristem type, with 10 meristems per replicate. This should provide sufficient RNA for subsequent RNA amplification and generation of libraries for RNA sequencing. We have published the S. bicolor x S. propinquum RIL population, also obtaining three years of phenotypic data on this population, substantially improving on early-generation (F2) characterization of the number, locations, and phenotypic effects of QTLs contributing to the formation and growth of rhizomes. To our knowledge, this is the first recombinant inbred (RIL) population in any taxon that segregates for rhizomatousness. A 30x whole-genome Illumina sequence has been produced for S. propinquum, and another for S. halepense. We are beginning to explore fine-scale levels of LD in additional regions of Johnsongrass populations that we have previously analyzed with RFLPs (Morrell et al., 2005). Outcomes These studies shed new light on the basic developmental biology and ontogeny of rhizomes. The RIL set is likely to be of value for genetic dissection of a wide range of traits.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wenqian Kong, Valorie H. Goff, Changsoo Kim, Andrew H. Paterson 2014. Genetic Analysis of Vegetative Branching and Rhizomatousness in Sorghum. Talk in Forage and Turf grasses workshop, PAG.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: GENETIC ANALYSIS OF PLANT ARCHITECTURE IN SORGHUM, by Wenqian Kong
• Type: Journal Articles Status: Other Year Published: 2014 Citation: Wenqian Kong, Huizhe Jin, Changsoo Kim, Valorie H. Goff, Tae-Ho Lee and Andrew H. Paterson. 201#. Quantitative Trait Analysis of Branching of Recombinant Inbred Lines for Sorghum bicolor � S. propinquum.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We have analyzed rhizome development in Sorghum halepense and S. propinquum. Primary rhizomes develop from buds in the axils of photosynthetic leaves, and grow in a horizontal or downward direction. Rhizome apices may subsequently reorient to a vertical growth habit and initiate leafy shoots, indicating that they are not committed to one specific identity. This developmental plasticity makes it difficult to determine the identity of any individual rhizome apex. However, we have discovered that axillary buds on rhizome segments can be released from apical dominance by excision of the primary rhizome apex. In S. halepense these buds will develop as green vertical shoots if the rhizome segment is excised from the parent plant, or as secondary rhizomes if the segment remains attached to the parent plant. We intend to utilize this system to select apices of known identity for laser capture and RNA sequencing. This study has revealed differences between S. halepense and S. propinquum that may contribute to the increased rhizomatousness of S. halepense. S. propinquum initiates many leafy axillary shoots (tillers), but is slower to begin initiating rhizomes than S. halepense. The rhizomes initiated by S. propinquum plants reorient to vertical (leafy shoot) growth more rapidly than in S. halepense. In S. propinquum, unlike in S. halepense, axillary buds on primary rhizomes may develop as leafy shoots, even when the rhizome remains attached to the parent plant. To identify targets for the control of S. halepense, we have recently completed the labwork for transcript profiling in S. halepense genotype Gypsum-9 line. Using laser capture microdissection, we collected meristems from buds induced to develop either as rhizomes or as leafy shoots and are now comparing transcripts in these two meristem types. We collected two replicates of each meristem type, with 10 meristems per replicate. Counts of sequencing reads that passed QC have been completed, and data are being analyzed in a variety of ways. We have put ca. 200 SSR markers on a new recombinant inbred line (RIL) population derived from the S. bicolor x S. propinquum cross, also obtaining three years of phenotypic data on progeny of this cross, substantially improving on early-generation characterization of the number, locations, and phenotypic effects of QTLs contributing to the formation and growth of rhizomes in an F2 population in which phenotyping was complicated not only by heterozygosity and inability to replicate, but also by complicating factors such as segregation for photoperiodic flowering. To our knowledge, this is the first recombinant inbred (RIL) population in any taxon that segregates for rhizomatousness. The map has now been published (Kong et al, 2013) and QTL analysis of rhizome-related traits is advanced, one chapter in Ms Kong's thesis that is expected to be defended in May of 2013. A 30x whole-genome Illumina sequence has been produced for S. propinquum, and another is being produced for S. halepense. We are beginning to explore fine-scale levels of LD in additional regions of Johnsongrass populations that we have previously analyzed with RFLPs (Morrell et al., 2005). PARTICIPANTS: MS student W. Kong is the primary person supported by this project at UGA, and has been a major participant in phenotyping and genetic mapping. She is near completion of her M.S., and plans to continue toward a Ph.D. in the PI's lab. PhD student Chengbo Zhou has been a significant player in S. halepense LD analysis. PhD student H. Jin has been a major participant in genetic mapping. Several undergraduates have participated in phenotyping and genotyping (C. Pillay, E. Giattina, D. Rassler, J. Perry, A. Kerr) Cornell:. Robyn Johnston, postdoctoral researcher is analyzing rhizome development and performing laser-microdissection of rhizome for RNA-seq analyses This commitment helped to engage the US Department of Energy Joint Genome Institute, to provide whole-genome Illumina sequence for S. propinquum and S. halepense. The PI has developed collaboration with S. Cox, The Land Institute, to explore the transmission genetics of a cross between tetraploid S. bicolor (via an unreduced gamete) and S. halepense. This cross may provide a supplementary and complementary means to compare rhizomatousness at the diploid (S. propinquum) and tetraploid (S. halepense) levels. TARGET AUDIENCES: Outcomes are expected to be salient to both fundamental and applied agriscience researchers, as well as private sector research and development teams. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We anticipate that these studies, when complete, will shed new light on the basic developmental biology and ontogeny of rhizomes. In addition, the RIL set that is being characterized is likely to be of value for genetic dissection of a wide range of traits. We have provided one example in our publication (flowering time) and have begun to work on additional traits.

Publications

• Genetic Analysis of Vegetative Branching and Rhizomatousness in Sorghum, Wenqian Kong and Andrew H. Paterson, Poster at PAG 2013 (based on 2012 results).
• Wenqian Kong and Huizhe Jin, Cleve D. Franks, Changsoo Kim, Rajib Bandopadhyay, Mukesh K. Rana, Susan A. Auckland, Valorie H. Goff, Lisa K. Rainville, Gloria B. Burow, Charles Woodfin, John J. Burke, Andrew H. Paterson 2013. Genetic analysis of recombinant inbred lines for Sorghum bicolor x S. propinquum. G3 3:101-108. doi:10.1534/g3.112.004499 (written and accepted for publication in 2012).

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: To provide necessary knowledge of the development and ontogeny of rhizomes, we have analyzed rhizome development in Sorghum halepense and S. propinquum. In both species, primary rhizomes develop from buds in the axils of photosynthetic leaves, and grow in a horizontal or downward direction. Rhizome apices may subsequently reorient to a vertical growth habit and initiate leafy shoots, indicating that they are not committed to one specific identity. This developmental plasticity makes it difficult to determine the identity of any individual rhizome apex. However, we have discovered that axillary buds on rhizome segments can be released from apical dominance by excision of the primary rhizome apex. In S. halepense these buds will develop as green vertical shoots if the rhizome segment is excised from the parent plant, or as secondary rhizomes if the segment remains attached to the parent plant. We intend to utilize this system to select apices of known identity for laser capture and RNA sequencing. This study has revealed differences between S. halepense and S. propinquum that may contribute to the increased rhizomatousness of S. halepense. S. propinquum initiates many leafy axillary shoots (tillers), but is slower to begin initiating rhizomes than S. halepense. The rhizomes initiated by S. propinquum plants reorient to vertical (leafy shoot) growth more rapidly than in S. halepense. In S. propinquum, unlike in S. halepense, axillary buds on primary rhizomes may develop as leafy shoots, even when the rhizome remains attached to the parent plant. Given that the goal of this project is to identify targets for the control of S. halepense, and that rhizome development seems to be controlled differently in S. propinquum than in S. halepense, we have decided to conduct transcript profiling in S. halepense. We are growing S. halepense plants from the Gypsum-9 line, and we aim to have tissue collections and laser captures completed by late 2011. We will collect meristems from buds induced to develop either as rhizomes or as leafy shoots and compare transcripts in these two meristem types. We will collect two replicates of each meristem type, with 10 meristems per replicate. This should provide sufficient RNA for subsequent RNA amplification and generation of libraries for RNA sequencing. We have put ca. 200 SSR markers on the S. bicolor x S. propinquum RIL population, also obtaining two years of phenotypic data on this population, substantially improving on early-generation (F2) characterization of the number, locations, and phenotypic effects of QTLs contributing to the formation and growth of rhizomes. To our knowledge, this is the first recombinant inbred (RIL) population in any taxon that segregates for rhizomatousness. We estimate that an additional 50-100 markers are needed to complete the map (these are in progress). A 30x whole-genome Illumina sequence has been produced for S. propinquum, and another is being produced for S. halepense. We are beginning to explore fine-scale levels of LD in additional regions of Johnsongrass populations that we have previously analyzed with RFLPs (Morrell et al., 2005). PARTICIPANTS: MS student W. Kong is the primary person supported by this project at UGA, and has been a major participant in phenotyping and genetic mapping. PhD student Chengbo Zhou has been a major player in S. halepense LD analysis PhD student H. Jin has been a major participant in genetic mapping Several undergraduates have participated in phenotyping and genotyping (C. Pillay, E. Giattina, D. Rassler, J. Perry, A. Kerr) Cornell:. Robyn Johnston, postdoctoral researcher is analyzing rhizome development and performing laser-microdissection of rhizome for RNA-seq analyses This commitment helped to engage the US Department of Energy Joint Genome Institute, to provide whole-genome Illumina sequence for S. propinquum and S. halepense. The PI has developed collaboration with S. Cox, The Land Institute, to explore the transmission genetics of a cross between tetraploid S. bicolor (via an unreduced gamete) and S. halepense. This cross may provide a supplementary and complementary means to compare rhizomatousness at the diploid (S. propinquum) and tetraploid (S. halepense) levels. TARGET AUDIENCES: Outcomes are expected to be salient to both fundamental and applied agriscience researchers, as well as private sector research and development teams. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We anticipate that these studies, when complete, will shed new light on the basic developmental biology and ontogeny of rhizomes. In addition, the RIL set that is being characterized is likely to be of value for genetic dissection of a wide range of traits.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: To provide necessary knowledge of the development and ontogeny of rhizomes, we have analyzed rhizome development in Sorghum halepense and S. propinquum. In both species, primary rhizomes develop from buds in the axils of photosynthetic leaves, and grow in a horizontal or downward direction. Rhizome apices may subsequently reorient to a vertical growth habit and initiate leafy shoots, indicating that they are not committed to one specific identity. This developmental plasticity makes it difficult to determine the identity of any individual rhizome apex. However, we have discovered that axillary buds on rhizome segments can be released from apical dominance by excision of the primary rhizome apex. In S. halepense these buds will develop as green vertical shoots if the rhizome segment is excised from the parent plant, or as secondary rhizomes if the segment remains attached to the parent plant. We intend to utilize this system to select apices of known identity for laser capture and RNA sequencing. This study has revealed differences between S. halepense and S. propinquum that may contribute to the increased rhizomatousness of S. halepense. S. propinquum initiates many leafy axillary shoots (tillers), but is slower to begin initiating rhizomes than S. halepense. The rhizomes initiated by S. propinquum plants reorient to vertical (leafy shoot) growth more rapidly than in S. halepense. In S. propinquum, unlike in S. halepense, axillary buds on primary rhizomes may develop as leafy shoots, even when the rhizome remains attached to the parent plant. Given that the goal of this project is to identify targets for the control of S. halepense, and that rhizome development seems to be controlled differently in S. propinquum than in S. halepense, we have decided to conduct transcript profiling in S. halepense. We are growing S. halepense plants from the Gypsum-9 line, and we aim to have tissue collections and laser captures completed by late 2011. We will collect meristems from buds induced to develop either as rhizomes or as leafy shoots and compare transcripts in these two meristem types. We will collect two replicates of each meristem type, with 10 meristems per replicate. This should provide sufficient RNA for subsequent RNA amplification and generation of libraries for RNA sequencing. We have put ca. 200 SSR markers on the S. bicolor x S. propinquum RIL population, also obtaining two years of phenotypic data on this population, substantially improving on early-generation (F2) characterization of the number, locations, and phenotypic effects of QTLs contributing to the formation and growth of rhizomes. To our knowledge, this is the first recombinant inbred (RIL) population in any taxon that segregates for rhizomatousness. We estimate that an additional 50-100 markers are needed to complete the map (these are in progress). A 30x whole-genome Illumina sequence has been produced for S. propinquum, and another is being produced for S. halepense. We are beginning to explore fine-scale levels of LD in additional regions of Johnsongrass populations that we have previously analyzed with RFLPs (Morrell et al., 2005). PARTICIPANTS: MS student W. Kong is the primary person supported by this project at UGA, and has been a major participant in phenotyping and genetic mapping. PhD student Chengbo Zhou has been a major player in S. halepense LD analysis PhD student H. Jin has been a major participant in genetic mapping Several undergraduates have participated in phenotyping and genotyping (C. Pillay, E. Giattina, D. Rassler, J. Perry, A. Kerr) Cornell:. Robyn Johnston, postdoctoral researcher is analyzing rhizome development and performing laser-microdissection of rhizome for RNA-seq analyses This commitment helped to engage the US Department of Energy Joint Genome Institute, to provide whole-genome Illumina sequence for S. propinquum and S. halepense. The PI has developed collaboration with S. Cox, The Land Institute, to explore the transmission genetics of a cross between tetraploid S. bicolor (via an unreduced gamete) and S. halepense. This cross may provide a supplementary and complementary means to compare rhizomatousness at the diploid (S. propinquum) and tetraploid (S. halepense) levels. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Given that the goal of this project is to identify targets for the control of S. halepense, and that rhizome development seems to be controlled differently in S. propinquum than in S. halepense, we have decided to conduct transcript profiling in S. halepense rather than S. propinquum.

Impacts
We anticipate that these studies, when complete, will shed new light on the basic developmental biology and ontogeny of rhizomes. In addition, the RIL set that is being characterized is likely to be of value for genetic dissection of a wide range of traits.

Publications

• No publications reported this period