Progress 10/01/15 to 02/17/16

Outputs
Target Audience:Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided Sandra Truong, a Genetics graduate student, training in the area of modeling sorghum drought tolerance traits. The project also provided Dr. Brock Weers continued professional development in the area of root traits and QTL analysis. How have the results been disseminated to communities of interest?A paper describing the development of a crop model for energy sorghum and use of the model for examining the potential utility of VPD-limited transpiration traits. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Identify traits that improve plant performance in water-limited environments. Identify QTL/genes and biochemical mechanisms that improve crop yield in drought prone environments.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: McCormick, RF, Truong, SK, Mullet, JE. 3D Sorghum Reconstructions from Depth Images Identify QTL Regulating Shoot Architecture. Plant Physiol. 2016;172 (2):823-834. doi: 10.1104/pp.16.00948. PubMed PMID:27528244. PubMed Central PMC5047103.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kebrom, TH, Mullet, JE. Transcriptome Profiling of Tiller Buds Provides New Insights into PhyB Regulation of Tillering and Indeterminate Growth in Sorghum. Plant Physiol. 2016;170 (4):2232-50. doi: 10.1104/pp.16.00014. PubMed PMID:26893475. PubMed Central PMC4824614.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Truong, S.K., McCormick, R.F., Mullet, J.E. (2017) Bioenergy Sorghum Crop Model Predicts VPD-Limited Transpiration Traits Enhance Biomass Yield in Water-Limited Environments. Frontiers in Plant Science (In press)

Progress 02/18/11 to 02/17/16

Outputs
Target Audience:Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided Sandra Truong, a Genetics graduate student, training in the area of modeling sorghum drought tolerance traits. The project also provided Dr. Brock Weers continued professional development in the area of root traits and QTL analysis. How have the results been disseminated to communities of interest?A paper describing the development of a crop model for energy sorghum and use of the model for examining the potential utility of VPD-limited transpiration traits. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Identify traits that improve plant performance in water-limited environments. 2. Identify QTL/genes and biochemical mechanisms that improve crop yield in drought prone environments.

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?A paper describing the new stay-green and dhurrin QTL was published. What do you plan to do during the next reporting period to accomplish the goals?An RNAseq approach will be used to characterize the molecular basis of stay-green 2.

Impacts
What was accomplished under these goals? A new QTL for stay-green drought tolerance was identified that aligned with a QTL for variation in leaf content of dhurrin. Genes for dhurrin biosynthesis were identified in the QTL and alleles that could explain the molecular basis of the QTL were identified

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Hayes, C.M., Weers, B.D., Thakran, M., Burow, G., Xin, Z., Emendack, Y., Burke, J.J., Rooney, W.L., Mullet, J.E. (2015) Discovery of a Dhurrin QTL in Sorghum: Co-localization of Dhurrin Biosynthesis and a Novel Stay-green QTL. Crop Science 56: 1-9.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A post-doc was trained. How have the results been disseminated to communities of interest? A publication describing the physiological basis of stay-green drought tolerance was published. What do you plan to do during the next reporting period to accomplish the goals? Continue anlaysis of sorghum populations for drought tolerance traits and QTL.

Impacts
What was accomplished under these goals? A root pipe lysimeter system was used to analyze and characterize the physiological basis of Stg2, an important locus conferring drought tolerance. QTL for root traits were identified.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Borrell, A.K., Van Oosterom, E.J., Mullet, J.E., George-Jaeggli, B., Jordan, D.R., Klein, P.E., and Hammer, G.L. (2014) Stay-green alleles individually enhance grain yield in sorghum under drought by modifying canopy development and water use patterns. New Phytologist (doi: 10.1111/nph.12869).

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. Changes/Problems: None What opportunities for training and professional development has the project provided? One undergraduate student, Kevin Bredemyer, was trained in various aspects of drought tolerance research and research training of a post-doc Dr. Brook Weers continued. How have the results been disseminated to communities of interest? A paper on the physiological basis of the stay-green drought tolerance trait was submitted. What do you plan to do during the next reporting period to accomplish the goals? QTL for root angle will be identified and the genetic basis of this trait investigated.

Impacts
What was accomplished under these goals? A root pipe lysimeter system was further automated and expanded to enable population screening for traits that contribute to water use efficiency and drought resilience. QTL for root architecture, root growth in response to water deficit, and stay-green were identified and analyzed. Progress towards fine mapping the genes underlying Stay-Green locus 2 was made.

Publications

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

Outputs
OUTPUTS: A root pipe system developed in 2011 was used to analysis of water use and root system architecture in sorghum genotypes that vary in drought tolerance. QTL for flowering time, root architecture, root growth in response to water deficit are being identified using several different populations. Map-based cloning of key genes that regulate these traits is underway. PARTICIPANTS: Dr. John Mullet, Professor Dr. Brock Weers, Post-doctoral scientist TARGET AUDIENCES: Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Producers of corn and other crops that may benefit from improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Near Isogenic Lines (NILs) that contain QTL for stay-green drought tolerance in sorghum have been constructed and are being used to characterize the physiology and molecular basis of improved drought tolerance and water use efficiency. The genetic basis of root system architecture and its impact on drought avoidance investigated in this project could further increase yield in water-limited environments for both grain sorghum and energy crops.

Publications

• No publications reported this period

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

Outputs
OUTPUTS: A root pipe system was developed for the analysis of water use and root systems in sorghum genotypes that vary in drought tolerance. QTL for flowering time, root architecture, root growth in response to water deficit, and stay-green were identified and analyzed. Progress was made towards the identification of genes underlying stay-green drought tolerance. PARTICIPANTS: Dr. John Mullet, Professor Dr. Brock Weers, Post-doctoral scientist Ms. Shanshan Yang, Graduate student TARGET AUDIENCES: Sorghum breeders and producers will benefit from sorghum hybrids with improved drought tolerance. Subsistence farmers in Africa could also benefit from the development of sorghum inbreds with higher productivity in water limited environments. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
QTL that control stay-green drought tolerance in sorghum have been identified and this information is being used to create sorghum genotypes with improved drought tolerance. The genetic basis of root system architecture and its impact on drought avoidance investigated in this project could further increase yield in water-limited environments.

Publications

• No publications reported this period

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

Outputs
OUTPUTS: QTL that control stay-green drought tolerance in sorghum have been identified. This information is being used to create sorghum genotypes with improved drought tolerance. The genetic basis of root system architecture and its impact on drought avoidance now being analyzed could further increase yield in water limited environments. PARTICIPANTS: John Mullet TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Digital genotyping technology was improved and used to create new genetic maps for analysis of QTL for drought traits. QTL for flowering time, root architecture, root growth in response to water deficit, and stay-green were identified and analyzed. Improved methods for root analysis were developed. Progress was made towards the identification of genes underlying stay-green.

Publications

• Borrell, A., Jordan, D., George-Jaeggli. B., Mace, E., Hamlet, S., Hammer, G., Mclean, G., Van Oosterom, E., Hunt, C., Klein, P., Mullet. J.E. (2010) Fine Mapping Candidate Genes for Stay-Green in Sorghum; Simplicity Beyond Complexity Summer Crops Research Conference.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: A sorghum RIL population segregating for variation in grain yield, flowering time, leaf area, height, root traits, and drought tolerance was characterized in several locations. A high-resolution genetic map based on the RIL population was created using a new sequence-based genotyping technology termed Digital Genotyping. The map contained ~600 DGA markers spanning all linkage groups. The DGA map was used locate QTL for a wide range of traits including those listed above. This analysis will allow the relationship between canopy and root traits to be related to variation in pre- and post-flowering drought tolerance in sorghum. PARTICIPANTS: John Mullet, Professor Patricia Klein, Associate Professor Brock Weers, Ph.D. student TARGET AUDIENCES: The information obtained through this project was disseminated to the public and industry through public presentations. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research is contributing to fundamental knowledge of gene regulatory networks that control growth, development and drought tolerance in sorghum. The traits and genetic loci identified will be used to increase yield and yield stability through marker-assisted breeding for improved drought tolerance.

Publications

• No publications reported this period

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Progress was achieved towards understanding the genetic and physiological basis of pre-flowering and post-flowering drought tolerance in sorghum. A number of key traits were identified (leaf area, root length, tillering, leaf chlorophyll content) that distinguish BTx642, a source of post-flowering stay-green drought tolerance, and Tx7000, a source of pre-flowering drought tolerance. QTL controlling these traits were mapped in a RIL population derived from a cross of these parental lines. QTL analysis will allow variation in the traits analyzed to be related to variation in pre- and post-flowering drought responses that segregate in this population. PARTICIPANTS: John Mullet, Professor Brock Weers, Ph.D. student TARGET AUDIENCES: The information obtained through this project was disseminated to the public and industry through publications and public presentations. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This research is identifying sorghum genes and trait loci involved in drought tolerance that will increase yield and yield stability through marker-assisted breeding for improved drought tolerance.

Publications

• Mullet, J.E. (2008) Traits and Genes for Plant Drought Tolerance. IN: Biotechnology in Agriculture and Forestry, Molecular Genetic Approaches to Maize Improvement, Vol. 63, pp. 55-64. Springer Verlag (Alan L. Kriz, Brian A. Larkins (eds.).

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Progress was achieved towards map-based cloning several genes for sorghum drought tolerance. Genes that modulate sorghum's stay-green trait were analyzed further in order to understand their mode of action and origin. Several different genetic sources of the sorghum stay-green trait were analyzed using molecular marker technology. This analysis indicated that the different sources of stay-green are likely of independent origin and may control different traits. Further progress was also made on the characterization of sorghum genes that are regulated in response to water deficit and the plant hormone ABA. PARTICIPANTS: John Mullet, Professor Karen Harris, Ph.D. student Brock Weers, Ph. D. student TARGET AUDIENCES: The information obtained through this project were disseminated to the public and industry through publications and public presentations. PROJECT MODIFICATIONS: None

Impacts
This research is building the infrastructure for sorghum genome research, a key enabling technology needed to understand the genetic basis of sorghum drought tolerance. Identification of sorghum genes and trait loci involved in drought tolerance will increase yield and yield stability through marker-assisted breeding for improved drought tolerance.

Publications

• Rooney, W.L., Blumenthal, J., Bean, B., Muller, J.E. (2007) Designing sorghum as a dedicated bioenergy feedstock. Biofuels Bioproducts and Biorefining 1:147-157.

Progress 01/01/06 to 12/31/06

Outputs
Progress report from 01/01/06 to 12/31/06: A new genome-wide sequence-based technology was developed for assessing genetic diversity among sorghum genotypes. Progress was also achieved towards map-based cloning several genes for sorghum drought tolerance. Differences in gene expression between sorghum genotypes differing in drought tolerance traits were identified. This discovery was followed up using eQTL analysis in order to determine if the differences in expression were caused by mutations acting cis or trans- to the gene targets.

Impacts
This research is building the infrastructure for sorghum genome research, a key enabling technology needed to understand the genetic basis of sorghum drought tolerance. Identification of sorghum genes involved in drought tolerance will increase yield and yield stability.

Publications

• Harris, K., Subudhi, P.K., Borrell, A., Jordan, D., Rosenow, D., Nguyen, H., Klein, P., Klein R., Mullet, J.E. (2006) Sorghum stay-green QTL individually reduce post-flowering drought-induced leaf senescence. J. Exp. Biol. (In press).
• Borrell, A., Jordan, D., Mullet, J.E., Henzell, B., Hammer, G. (2006) Drought Adaptation in Sorghum, Chapter 10, pp. 335-401. Drought Adaptation in Cereals, Jean-Marcel Ribaut (editor), Food Products Press, the Haworth Press, Inc., New York.

Progress 01/01/05 to 12/31/05

Outputs
Progress report from 01/01/05 to 12/31/05: The architecture of sorghum chromosomes was analyzed using fluorescence in situ hybridization with large insert clones from the TAMU-ARS genetic map. This allowed the development of a universal nomenclature for sorghum chromosomes based on size. In addition, euchromatic chromosomal regions of high gene density and pericentromeric heterochromatic regions in each sorghum chromosome were identified. The expression of 12,000 sorghum genes was characterized in plants treated with hormones involved in plant response to pests as well as osmotic stress. This analysis identified a large number of genes, biochemical pathways, and regulons that mediate sorghum's response to biotic and abiotic stress.

Impacts
This research is building the infrastructure for sorghum genome research, a key enabling technology needed to understand the genetic basis of sorghum drought tolerance. Identification of sorghum genes involved in drought tolerance will increase yield and yield stability.

Publications

• Buchanan, C. D., Lim, S., Salzman, R., Giannis, Klein, R., Cordonnier-Pratt, M.M., Pratt, L.H., Klein, P.E., Mullet, J.E. (2005) Sorghum bicolor's transcriptome response to dehydration, ABA, and high salt. Plant Mol. Biol 58: 699-720.

Progress 01/01/04 to 12/31/04

Outputs
The architecture of sorghum chromosomes was characterized using Fluorescence In Situ Hybridization of sorghum BACs that had been mapped on the sorghum genetic map. This showed that most sorghum chromosomes have euchromatic arms and a large pericentromeric region of heterochromatin. A universal nomenclature for sorghum chromosomes was developed based on chromosome size. In addition, sorghum gene expression changes associated with osmotic stress and plant hormones was characterized using microarray technology.

Impacts
The construction of an integrated genetic and physical map of the sorghum genome provides a powerful tool for trait mapping and gene discovery. This research is revealing fundamental features of the sorghum genome, accelerating the discovery of agriculturally important genes, and aiding the analysis of diversity in the sorghum germplasm collection.

Publications

• Salzman, R.A., Brady, J., Buchanan, C., Klein, P., Klein, R., Pratt, L, Cordonnier-Pratt, M.M., Mullet, J.E. (2005) Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiol. (In press).
• Klein, R.R., Klein, P.E., Mullet, J.E., Minx, P., Rooney, W.L., Schertz, K.R. (2005) Fertility restorer locus RF1 of sorghum (Sorghum bicolor L.) encodes a pentacotripeptide repeat protein not present in the syntenic region of rice chromosome 12. Theoretical and Applied Genetics (In press).
• Kim, J.S., Klein, P.E., Klein, R.R., Price, H.J., Mullet, J.E., Stelly, D.M. (2005) Chromosome identification and nomenclature of Sorghum bicolor. Genetics (In press).
• Kim, J-S., Klein, P.E., Klein, R.R., Price, J.H., Mullet, J.E., Stelly, D.M. (2005) Molecular cytogenetic maps of sorghum linkage groups B and H. Genetics (In press).
• Buchanan, C.D., Klein, P.E., Mullet, J.E. (2004) Phylogenetic analysis of 5-non-coding regions from the ABA responsive RAB gene family of sorghum, maize and rice provides insight into the composition, organization and function of cis-regulatory modules. Genetics 10: 1639-1654.

Progress 01/01/03 to 12/31/03

Outputs
The TAMU-ARS sorghum genome map was refined during 2003. The architecture of several additional sorghum chromosomes were analyzed using BAC-based florescence in situ hybridization. Genes encoded by 850 mapped BACs spanning the entire genome were identified and aligned to the rice genome sequence by a directed type of sequence-scanning. The sorghum genome map is being used to map and isolate a number of agriculturally important genes including genes that regulate flowering time, resistance to aluminum, resistance to disease and insects, and genes that contribute to sorghum's drought tolerance. The sorghum genome map was also used to aid the analysis of the apomixis locus in Pennisetum and diversity in the sorghum germplasm collection.

Impacts
The construction of an integrated genetic and physical map of the sorghum genome provides a powerful tool for trait mapping and gene discovery. This research is revealing fundamental features of the sorghum genome, accelerating the discovery of agriculturally important genes, and aiding the analysis of diversity in the sorghum germplasm collection.

Publications

• Akiyama, Y., Conner, J.A., Goel, S., Morishige, D., Mullet, J.E., Hanna, W.W., Ozias-Akins, P. (2004) High resolution physical mapping by FISH reveals extensive chromosomal heteromorphism and demarcation by repetitive sequences of the genomic region associated with apomixis in Pennisetum squamulatum. PNAS (In press).
• Klein, P.E., Klein, R.R., Vrebalov, J., Mullet, J.E. (2003) Sequence-based alignment of sorghum chromosome 3 and rice chromosome 1 reveals extensive conservation of gene order and one major chromosomal rearrangement. Plant J. 34:605-621.
• Menz, M.A., Klein, R.R., Unruh, N.C., Rooney, W.L., Klein, P.E., Mullet, J.E. (2004) Genetic diversity of public inbreds of sorghum using mapped AFLP and SSR markers. Crop Science (In press).

Progress 01/01/02 to 12/31/02

Outputs
The TAMU-ARS sorghum genome map was developed further during 2002. A new BAC library from IS3620C was constructed and pooled in six dimensions. The BAC DNA pools were used to link additional BAC contigs to the genome map. The architecture of four additional sorghum chromosomes was analyzed using BAC-based florescence in situ hybridization. Genes encoded by mapped BACs were identified by a directed type of sequence-scanning. The BAC derived sequences were processed using Phred/Phrap and subjected to BLASTX analysis. Protein coding genes identified through this process were aligned with the rice genome sequence using BLASTN. Analysis of sequences from 118 BACs mapped on sorghum chromosome 3 aligned this chromosome with rice chromosome 1 and identified one major rearrangement. The sorghum genome map is being used to map and isolate a number of agriculturally important genes including genes that regulate flowering time,resistance to aluminum, resistance to disease and insects, and genes that contribute to sorghum's drought tolerance.

Impacts
The construction of an integrated genetic and physical map of the sorghum genome provides a powerful tool for trait mapping and gene discovery. This research is revealing fundamental features of the sorghum genome and will accelerate the discovery of agriculturally important genes from sorghum.

Publications

• Morishige, D.T., Childs, K.L., Moore, L.D., Mullet, J.E. (2002) Targeted analysis of orthologous phytochrome A regions of the sorghum, maize and rice genomes using comparative gene island sequencing. Plant Phys. 130: 1614-1625.
• Islam-Faridi, M.N., Childs, K.L., Klein, P.E., Hodnett, G., Menz, M.A., Klein, R.R., Rooney, W.L., Mullet, J.E., Stelly, D.M., Price, H.J. (2002)Cytogenetics of sorghum chromosome 1: FISH with mapped BACs. Genetics 161:345-353.
• Kim, J-S., Childs, K.L., Faridi, N-I., Menz, M.A., Klein, R.R., Klein, P.E., Price, H.J., Mullet, J.E., Stelly, D.M. (2002) Integrated karyotyping of sorghum by in situ hybridization of landed BACs. Genome 45: 402-412.
• Menz, M.A., Klein, R.R., Mullet, J.E., Obert, J.A., Unruh, N.C., Klein, P.E. (2002) A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP, RFLP and SSR markers. Plant Mol. Biol. 48: 483-499.
• Morgan, P.W., Finlayson, S.A., Childs, K.L., Mullet, J.E., Rooney, W.L. (2002) Opportunities to improve adaptability and yield in grasses: Lessons from sorghum. Crop Sci. 42: 1791-1799.

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

Outputs
A high resolution genetic map of the sorghum genome was created primarily using AFLP technology. The map contained 2454 AFLP loci, 136 SSR loci, and 203 loci based on cDNAs and genomic clones. A population of 136 recombinant inbred lines derived from BTx623 and IS3620C was used for map construction. A Web site http://SorghumGenome.tamu.edu, has been created to provide all the necessary information contained in the map. The sorghum genetic map was used to aid the mapping of the Rf1 gene for fertility restoration in sorghum. The map, and BACs linked to the map, have also been used to characterize the relationship betweeen physical and genetic distance along linkage group A, and to determine the relationship between linkage groups and chromosomes in sorghum. A new technology was developed that allows the selective acquisition of gene sequences from BAC clones. Direct selection technology was developed to assist the identification of genes encoded by BAC clones from any region of the sorghum genome.

Impacts
The construction of a high density genetic map provides a powerful tool for gene discovery in sorghum. This research is revealing fundamental features of the sorghum genome and providing insights that will aid in the identification, isolation and utilization of sorghum genes involved in plant response to water deficit.

Publications

• Childs, K.L., Klein, R.R., Klein, P.E., Morishige, D.T., Mullet, J.E. (2001) Mapping genes on an integrated sorghum genetic and physical map using cDNA selection technology. The Plant Journal. 27: 243-255.
• Klein, R.R., Klein, P.E., Chhabra, A.K., Dong, J., Pammi, S., Childs, K.L.,Mullet, J.E., Rooney, W.R., Schertz, K.F. (2001) Molecular mapping of the rf1 gene for pollen fertility restoration in sorghum (Sorghum bicolor L.). Theor. Appl. Genet. 102 (2001) 8, 1206-1212.

Progress 01/01/00 to 12/31/00

Outputs
Sorghum EST sequences and clones were obtained from the University of Georgia. A set of 16 plates containing 1088 different ESTs were acquired and the cDNAs were rearrayed using a Qbot. The clone inserts were PCR amplified and will be used to create microarrays for analysis of genes induced by abiotic stress. RNA has been isolated from plants exposed to water deficit. This RNA will be labeled and used to probe the microarrays. A high throughput method was developed for building an integrated genetic and physical map of the sorghum genome. A 5x library of BAC clones was fingerprinted and pooled in six different ways for parallel contig mapping. Analysis of 200 primer combinations identified over 3,000 contigs and mapped 1,000 onto the sorghum genetic map. This map will be used to accelerate the localization and isolation of genes involved in sorghum's adaptation to adverse environments.

Impacts
(N/A)

Publications

• Klein, P.E., Klein, R.R., Cartinhour, S.W., Ulanch, P.E., Dong, J., Obert,J.A., Morishige, D.T., Schlueter, S.D., Childs, K.L., Ale, M., and Mullet,J.E. 2000 A High-throughput AFLP-bsed Method for Constructing Integrated Genetic and Physical Maps: Progress Toward a Sorghum Genome Map. Genome Research 10:789-807.

Progress 01/01/99 to 12/31/99

Outputs
During the past year, our research has been focused on mapping two genes for maturity in sorghum. In order to facilitate future research on these genes, we have begun construction of a physical map of the sorghum genome. An existing BAC library was established and expanded and methods for constructing a physical map was analyzed. Over the past several years, we have mapped several genes on the sorghum genome that control flowering time, height, disease resistance, and drought tolerance. Several thousand BAC DNAs were isolated and fingerprinted. In addition, pools of DNAs from BACs in the library were constructed so that PCR based genome markers could be added to the physical map.

Impacts
(N/A)

Publications

• Klein, P.E., Klein, R.R., Cartinhour, S.W., Ulanch, P.E., Dong, J.M., Obert, J.A., Morishige, D.T., Schlueter, S.D., Childs, K.L., Ale, M., Mullet, J.E. (2000) A High Throughput AFLP-based Method for Constructing Integrated Genetic and Physical Maps: Progress Towards a Sorghum Genome Map. Genome Research (Submitted).
• Finlayson, S.A., Lee, I.-J., Mullet, J.E., Morgan, P.W. (1999) The Mechanism of Rhythmic Ethylene Production in Sorghum. The Role of Phytochrome B and Simulated Shading. Plant Physiol. 119: 1-7.
• Crasta, O., Xu, W., Rosenow, D.T., Mullet, J.E., Nguyen, H.T. (1999) Mapping of Post-Flowering Drought Resistance Traits in Grain Sorghum: Association of QTLs Influencing Premature Senescence and Maturity. Mol. Gen. Genet. 262:579-588.

Progress 01/01/98 to 12/31/98

Outputs
The overall goal of this research is to identify genes that contribute to drought tolerance in sorghum and other crops. Many loci that contribute to drought tolerance are being mapped including genes for the stay green trait in sorghum and gene for maturity. In order to accelerate trait locus mapping, and to isolate the corresponding genes, we have begun development of a sorghum physical map. Additional sorghum BAC libraries were prepared, 37,000 now available, and methods for high through put DNA extraction were developed. DNAs from 25,000 BACs were fingerprinted and images scanned into a computer based analysis system. Overlapping fingerprints predict BACs that form contigs. A second technology for physical map construction was also developed termed SAS-DNA Technology. This method identifies overlapping BACs using unique amplifiable DNAs.

Impacts
(N/A)

Publications

• Klein, R.R., Morishige, D.L, Klein, P.E., Dong, J., Mullet, J.E. , 1998. High Throughput BAC DNA isolation for Physical Map Construction of Sorghum. Plant Mol. Biol. Reporter.

Progress 01/01/97 to 12/31/97

Outputs
Over the past several years, we have mapped several genes on the sorghum genome that control flowering time, height, disease resistance, and drought tolerance. During the past year, this research has been focused on mapping two genes for maturity in sorghum. To facilitate future research on these genes, we have begun construction of a physical map of the sorghum genome. An existing BAC library was expanded and methods for constructing a physical map were analyzed. Several thousand BAC DNAs were isolated and fingerprinted. In addition, pools of DNAs from BACs in the library were constructed so that PCR based genome markers could be added to the physical map.

Impacts
(N/A)

Publications

• WHITSITT, M.S., COLLINS, R.G., MULLET, J.E. 1997. Modulation of Dehydration Tolerance in Soybean Seedlings;Dehydrin Mat1 is Induced by Dehydration But Not Abscisic Acid. Plant Phys. 114:917-925.

Progress 01/01/96 to 12/30/96

Outputs
Three genes that are induced by water deficit in pea were identified in sorghum.Partial sequences of the corresponding cDNAs were obtained and used a probes and for genetic mapping. All three genes, SbDha1 (encodes an aldehyde dehydrogenase), SbMip1 ( encodes a membrane intrinsic protein) and SbDhn2 (encodes a dehydrin), were up regulated in response to water deficit. SbDhn2 and SbMipl were also activated by the hormone ABA. SbDhn2 and SbDha1 were located on the sorghum linkage map because these genes may be responsible for differences in drought tolerance in sorghum and gene mapping may eventually be related to trait locus identification. Studies on sybean dehydrins and dehydration tolerance were completed and a publication submitted during the past year.

Impacts
(N/A)

Publications

• WHITSITT, M. S., MULLET, J.E. 1997. The Expression and Mapping of Drought Responsive Genes in Sorghum bicolor. (Molecular Breeding, submitted).
• WHITSITT, M.S., COLLINS, R. G., MULLET, J.E. 1997. Modulation of Dehydration Tolerance in Soybean Seedlings. Dehydrin Matl is Induced by Dehydration but not ABA. (Plant Phys., submitted).

Progress 01/01/95 to 12/30/95

Outputs
1. Cloned a dehydrin cDNA from sorghum. This gene was sequenced and was found tobe induced in response to water deficit and ABA. The gene is being located on the sorghum genetic map. 2. Dehyrin or maturation genes (MAT1) were acquired for use in the analysis of soybean plants to dehydration. The soybean dehydrin gene was not induced by ABA or mild water deficit. In contrast, dehydration of plants to a water loss of 25% caused gene induction. 3. Soybean plant response to dehydration was examined. Control plants could survive (50% survival) approximately 40% dehydration. In contrast, plants pretreated with ABA, mild or severe water deficit could survive more severe dehydration. This assay reveals adaptive mechanisms that allow soybean plants to survive dehydration. 4. Soybean vegetative storage protein genes (Vsp) are induced in response to water deficit. Homologous genes were isolated from Arabidopsis and their expression patterns studied. Interestingly, Arabidopsis has at least two Vsp genes and the expression of these genes is similar to that observed in soybean. Vsp cis-elements were analyzed in a separate study.

Impacts
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Publications

Progress 01/01/94 to 12/30/94

Outputs
1. Cloned a dehydrin cDNA from sorghum. This gene was sequenced and was found tobe induced in response to water deficit and ABA. The gene is being located on the sorghum genetic map. 2. Dehydrin or maturation genes (MAT1) were acquired for use in the analysis of soybean plants to dehydration. The soybean dehydrin gene was not induced by ABA or mild water deficit. In contrast, dehydration of plants to a water loss of 25% caused gene induction. 3. Soybean plant response to dehydration was examined. Control plants could survive (50% survival) approximately 40% dehydration. In contrast, plants pretreated with ABA, mild or severe water deficit could survive more severe dehydration. This assay reveals adaptive mechanisms that allow soybean plants to survive dehydration. 4. Soybean vegetative storage protein genes (Vsp) are induced in response to water deficit. Homologous genes were isolated from Arabidopsis and their expression patterns studied. interestingly, Arabidopsis has at least two Vsp genes and the expression of these genes is similar to that observed in soybean. Vsp cis-elements were analyzed in a separate study.

Impacts
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Publications

Progress 01/01/93 to 12/30/93

Outputs
One long term objective of this research is to map and identify genes which contribute to drought tolerance in sorghum. As a first step toward this objective, a sorghum RFLP genetic map has been constructed and populations which segregate for traits relating to drought tolerance set up. We have recently optimized a new method for genetic mapping and gene tagging for sorghum. This method, involves PCR amplification of DNA using 10 bp DNAs. The polymorphic DNAS revealed by these assays are called RAPDs (random amplified polymorphic DNA) (Williams et al, 1980). A protocol for RAPD detection was devised for sorghum and the optimized procedure was used to map several RAPDs on the sorghum genetic map. The utility of this procedure was also found to extend to examination of different genotypes and the tagging of genes using bulked segregant analysis. Other projects advanced during this research period include mapping the gene(s) controlling the `stay green' trait in sorghum, cloning a gene encoding phytochrome from sorghum and cloning a gene from the dehydrin family of genes. Research on `stay green' in sorghum was initiated because plants which `stay green' post-flowering in sorghum show enhanced drought tolerance during this phase of development. 90 plants from an F6 population were examined and 118 RFLP probes identified which segregate in this population. This research will continue into 1994 in order to combine phenotypic data and refine the mapping of genes controlling this trait.

Impacts
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Publications

Progress 01/01/92 to 12/30/92

Outputs
Over the past several years we have isolated and characterized several genes which show altered expression in response to water deficit. These genes include 7a, a putative water channel, 15a which encodes a thiol protease, 26g which encodes an aldehyde reductase, several dehydrins and several genes which encode cell wall proteins or unknown proteins. In the last year we have begun a major new initiative to relate these water deficit inducible genes to drought tolerance observed in sorghum under field conditions. Several crop breeders in Texas have identified sorghum lines which are drought tolerant either during the seedling state, at heading or post-flowering. The genes which contribute to field drought tolerance can be localized in the sorghum genome using RFLP or RAPD analysis. If genes which are induced in response to water deficit are also mapped on the sorghum genome, then the relationship between these two types of information can be obtained. Therefore, over the past year we have worked on methods to optimize RAPD mapping of the sorghum genome. Optimal conditions for DNA extraction, PCR analysis and gel separations have been worked out and the first 10 RAPD markers mapped on the sorghum RFLP map. We have also made progress in isolating several cDNAs corresponding to genes induced in response to water deficit from sorghum. These genes will be mapped on the sorghum genetic map in the near future.

Impacts
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Publications

Progress 01/01/91 to 12/30/91

Outputs
(i) Growth of stems is differentially inhibited relative to roots in plants exposed to water deficit. We investigated changes in mRNA levels in stems which were inhibited by water deficit. Several cDNAs were isolated and sequenced. Genes encoding tubulin and actin were down-regulated by water deficit whereas sbPRP-1 mRNA accumulated. Changes in the expression of genes encoding cell wall proteins may be important in growth control. (ii) ABA biosynthesis in plants in response to water deficit involves a dioxygenase which converts violaxanthin to xanthoxin. One candidate for the dioxygenase is lipoxygenase. Therefore we cloned several vegetative lipoxygenases and characterized their response to water deficit. The two classes of lox examined were induced by water deficit. The genes were also induced by wounding and jasmonic acid. (iii) Jasmonic acid is a key regulator of vsp gene expression. We examined if JA/MeJA levels increase in wounded tissue using a quantitative assay. The levels of JA and MeJA increased within 2 hrs post wounding and this correlated with induction of vsp, chs and genes encoding the PRPs.

Impacts
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Publications

Progress 01/01/90 to 12/30/90

Outputs
The expression of vspA and vspB genes encoding soybean vegetative storage proteins was studied during seedling development and in response to water deficit, tissue wounding, and jasmonic acid treatment. vspA and vspB encode VSP-alpha and VSP-Beta, 28-kilodalton and 31-kilodalton vacuole-localized polypeptides that are 80% homologous. vspA and vspB mRNAs could be distinguished on RNA blots using 3'-end probes. vspA mRNA was threefold to sevenfold more abundant than vspB mRNA in leaves, about equal expression was observed in stems, and vspB mRNA exceeded vspA in roots. Transcripts were not detected in dry seeds but appeared in intact or excised seedling axes between 12 hr and 24 hr after initiation of imbibition. Both transcripts were highly abundant in the meristematic region of seedling stems and in developing leaves but were rare in mature stems, leaves, and roots. In situ localization showed that vsp transcripts were found throughout the hypocotyl hook but were concentrated in cells associated with the epidermis and vascular bundles. Water deficit caused increased vsp mRNA levels in leaves and stems, which suggests that inhibition of growth necessitates temporary storage of amino acids. Wounding induced primarily vspB mRNA in etiolated seedlings, whereas both vspA and vspB mRNA levels increased in wounded leaves. Jasmonic acid and methyl jasmonate were potent inducers of vsp gene expression in cell cultures, developing axes, leaves, and roots.

Impacts
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Publications

Progress 01/01/89 to 12/30/89

Outputs
We have investigated the expression of two genes (vspA, vspB) which encode soybean vegetative storage proteins in soybean seedlings during development and response to water deficit, wounding and jasmonic acid treatment. The proteins enoded by vspA and vspB are 80% homologous, 28 kDa and 31 kDa respectively, and are localized in vacuoles and to a lessor extent, the cell wall. Gene specific RNA probes do not detect vsp mRNA in dry seeds but both vspA and vspB mRNA appear in the seedling axis between 12 and 24 hrs after imbibition. In 3 day-old dark-grown seedlings, both genes are highly expressed in the apical portion of the stem, cotyledons and plumule with only low expression in non-growing stem regions and roots. In situ localization of vsp mRNA revealed accumulation in the epidermal layer of the stem and in cells near the vascular bundles. Protein encoded by the vsp genes was also localized in these regions perhaps reflecting a storage protein function (near vascular bundles) and a role in growth (epidermal layer). Water deficit and ABA treatment increased vspA and decreased vspB mRNA levels to a small extent but did not perturb the distribution of vsp mRNA in stems and roots. In contrast, methyl jasmonate significantly increased levels of vspA mRNA in non-growing regions of the stem and root. This treatment increased vspB mRNA levels in mature stems, mature roots and root tips to even a greater extent.

Impacts
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Publications

Progress 01/01/88 to 12/30/88

Outputs
Two proteins which are induced in soybean seedlings exposed to water deficit were analyzed. Antibodies were prepared and cDNA libraries screened. cDNAs to 28 kDa and 31 kDa proteins were isolated. The 28 kDa protein corresponds to a cell wall localized protein whereas preliminary data shows both the 28 and 31 are vacuolar localized. An analysis of the role of ABA in root/shoot ratio changes observed in water deficient plants was initiated. ABA could mimic the drought-induced changes. The mode of action is yet unclear. Initial characteriztion of 3 turgor-responsive genes was uundertaken. Sequence analysis showed that these genes have not been previously identified. Regulation of chloroplast translation was examined in 8 day-old dark-grown barley. Evidence indicates regulation of initation.

Impacts
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Publications

Progress 01/01/87 to 12/30/87

Outputs
Work was completed on glutathione reductase, a key enzyme in oxygen detoxification in chloroplasts. It was found that the enzyme could be inactivated by a light-dependent mechanism but the nature of the modification was not clarified. Recent work has focused on growth inhibition in water-deficient soybean seedlings. Poly A RNA populations and polysomes were monitored in stressed plants (Mason et al., 1987). cDNA libraries have been prepared and screened to provide cDNA specific for stem development. These genes will be used to characterize the growth response in water-limited seedlings.

Impacts
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Publications

Progress 01/01/86 to 12/30/86

Outputs
Glutathione reductase, a key enzyme in the oxygen detoxification pathway in chloroplasts, was isolated and characterized. The native holoenzyme was found to consist of two 60,000 dalton subunits. A shift in mobility on SDS gels to 72,000 daltons was observed in the absence of reducing agents. The purified enzyme was inactivated when incubated in the presence of washed thylakoid membranes and light. Inactivation correlated with the loss of enzyme binding to ADP-sepharose. Antibodies to chloroplast glutathione reductase were prepared and used to show that enzyme inactivation was not due to proteolysis nor charge modification. Light-induced inactivation of glutathione reductase was also dependent on photosynthetic electron transport. Studies on the effect of abscisic acid on chloroplast biogenesis in barley leaves were initiated.

Impacts
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Publications

Progress 01/01/85 to 12/30/85

Outputs
Glutathione reductase, a key enzyme in the oxygen detoxification pathway in chloroplasts, was isolated and characterized. The native holoenzyme was found to consist of two 60,000 dalton subunits. A shift in mobility on SDS gels to 72,000 daltons was observed in the absence of reducing agents. The purified enzyme was inactivated when incubated in the presence of washed thylakoid membranes and light. Inactivation correlated with the loss of enzyme binding to ADP-sepharose. Field grown cotton accumulated proteins which comigrate with radiolabeled heat shock proteins when subjected to high temperatures. High temperature effects were induced in droughted cotton due to stomatal closure and increased leaf temperature. This is the first report of significant accumulation of heat shock proteins in higher plants.

Impacts
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Publications

Progress 01/01/84 to 12/30/84

Outputs
Plants respond to stress of various types by inducing and/or modifying biochemical systems which allow the plant to survive and produce seed. We have analyzed cotton plant response to high temperatures both in field grown plants and in seedlings grown in controlled environments. We have found that field grown cotton which is exposed to daily temperatures in excess of 37C for 2-4 weeks accumulate 8 polypeptides which are not found in irrigated controls which do not experience canopy temperatures greater than 32C. The polypeptides observed are also radiolabeled if seedlings are exposed to short periods of high temperature. The isolation and characterization of these "heat shock" polypetides is underway. Our lab is also studying plant responses to drought. Abscisic acid is known to be increased 10-40 fold in plants which have been dehydrated to the point of zero-turger. We are initiating studies on the biochemistry of abscisic acid synthesis and the regulation of its induction by drought. To study this problem we have developed analytical and elisa assays for abscisic acid and have developed a controlled system in which induction will be studied. Our next experimental phase will deal with the biosynthetic pathway to abscisic acid and whether transcription and/or translation is involved in induction of this stress-hormone.

Impacts
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Publications

Progress 01/01/83 to 12/30/83

Outputs
This Hatch project which concerns plant chloroplast responses to water stress, was initiated in September 1983. Therefore the stage of work is reported here. The initial phase of this research inolves a survey to determine if new polypeptides are induced in chloroplasts during exposure of plants to heat, water or salt stress. Preliminary results on total translation products revealed that several new products are induced by each stress. Further experiments are being done to characterize the specific changes occurring in plastids and whether regulation occurs at the transcriptional or translational level.

Impacts
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Publications