Progress 03/01/05 to 02/28/11

Outputs
OUTPUTS: OUTPUTS: (1) Structural organization of the wheat genome. Sequencing and annotation of randomly selected BAC clones and of BAC contigs of ~1Mb in size have provided insight into the structure and the evolution of the wheat genome. Ca. 37% of genes in the wheat genome are present in multi-gene islands. Multi-gene islands occur in proximal as well as distal chromosome regions but there is a trend for the number of genes per gene island to decrease and the inter-island distances to increase from the distal to proximal regions. Comparative analyses with Brachypodium, rice and sorghum have shown that the rate of gene duplications and insertions correlates with genome size. Larger genomes thus not only contain higher levels of repetitive DNA than smaller genomes, but also have undergone an increase in the size of the gene space. (2) Isolating the pearl millet d2 dwarfing gene. A d2 gene candidate was identified using a combination of three approaches. Genetic mapping in ~1500 F2 progeny derived from a cross between a tall (D2D2) and a dwarf (d2d2) inbred line, haplotyping of several dwarf and tall lines with markers closely linked to the d2 phenotype and comparative analysis of the pearl millet d2 region with the orthologous region in sorghum identified a 410 kb region in the sorghum genome carrying 40 genes. Once of the 40 genes was ABCB1, a P-glycoprotein involved in auxin transport. Mutations in ABCB1 had previously been shown to cause dwarfisms in both sorghum (dw3 phenotype) and maize (br1 mutant). RT-PCR with primers developed against sorghum ABCB1 showed that the pearl millet ortholog was expressed in the stem of the tall pearl millet inbred ICMP 451 but not in the dwarf inbred Tift 23DB. (3) Mapping pearl millet fertility restoration gene(s). Because of fertility problems in this mapping population, this project has been abandoned. Instead, we have started work on mapping a flowering mutant in pearl millet which was identified in an F2 population generated from a cross between the African line 02GH884 and a US inbred, Tift 454. The cross was originally developed to map the stay-green trait provided by 02GH884. The mutant phenotype is characterized by a cone-shaped panicle, sterility and profuse tillering from the nodes and segregates 3:1 (wild type:mutant) in the population, indicating control by a single recessive gene. Mapping of this trait is in progress. (4) Genetic analyses in finger millet. A biodiversity study was conducted using SSR markers and comprising 79 Eleusine coracana subsp. coracana accessions, 14 E. coracana subsp. africana accessions, 2 E. indica and 1 E. kigeziensis accession. The data showed a clear population structure differentiating (1) the wild accessions, (2) the cultivated accessions from Africa and (3) the cultivated accessions from Asia. A finger millet genetic map was constructed and a comparative analysis of the finger millet genome with other grass genomes was conducted. PARTICIPANTS: Katrien M. Devos - Principal Investigator; Alicia Massa - Post-doctoral Research Associate; Xiangyang Xu - Post-doctoral Research Associate; Trudi Thomas - Research Technician; Rajiv Parvathaneni - Graduate Student; Vinod Jakkula - Graduate Student; Chris Papadopoulos - Graduate Student (left the graduate program); Srinivasachary - Visiting Graduate Student (6 months); Collaborators: Jeff Wilson, USDA-ARS, Tifton; Jeff Bennetzen, UGA; Jan Dvorak, UC Davis; Agnes Chan, JCVI; Pablo Rabinowicz, University of Maryland; Olin Anderson, USDA-ARS, Albany; Phillip San Miguel, Purdue University; Mathews Dida, a finger millet breeder from Kenya was trained in molecular techniques (18 months); Ignatius Angarawai, a pearl millet breeder from Lake Chad Institute, Nigeria, was trained in molecular techniques (1 year); Nethra N, a researcher from University of Agricultural Sciences, Bangalore, was trained in molecular techniques (6 months). TARGET AUDIENCES: The wheat and millet scientific communities (academics and breeders). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The mapping, isolation and functional characterization of genes controlling traits of agronomic importance will enhance our understanding of plant developmental processes and ultimately benefit the farmers and consumers through the adoption of new varieties and new management strategies. Studies on the phenotypic and genotypic biodiversity of germplasm will inform breeders as to the best line combinations to use to improve cultivars. The basic wheat research will provide essential data on the organization of the wheat genome and its relationship to B. distachyon and other grasses that are needed to develop strategies for sequencing the large bread wheat genome. Having the sequence of the wheat genome available will facilitate genomics research, including the isolation and functional characterization of genes, which, in turn, will help in varietal improvement. Our projects also aim to enhance understanding of the evolution of grass genomes and to provide information on the mechanisms that plants may use to adapt to diverse habitats.

Publications

• Parvathaneni R, Jakkula V, Padi FK, Faure S, Nethra N, Pontaroli AC, Wu X, Bennetzen JL, Devos KM (2013) Fine-mapping and identification of a candidate gene underlying the d2 dwarfing phenotype in pearl millet, Cenchrus americanus (L.) Morrone. G3 3:563-572 (2013)
• Gottlieb A*, Muller H-G*, Massa A, Wanjugi H, Deal K, You F, Xu X, Guo YQ, Luo M, Anderson O, Chan A, Rabinowicz P, Devos KM*, Dvorak J* (2012) The insular organization of gene space in grass genomes and its evolution. PLoSOne 8:e54101 Authors with * contributed equally to the paper
• Massa AN, Wanjugi H, Deal KR, O'Brien K, You FM, Maiti R, Chan AP, Gu YQ, Luo MC, Anderson OA, Rabinowicz PD, Dvorak J, Devos KM (2011) Gene space dynamics during the evolution of Aegilops tauschii, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor genomes. Mol Biol Evol 28:2537-2547
• Devos KM (2010) Grass genome organization and evolution. Current Opinion in Plant Biol 12:139-145
• Devos KM, Dolezel J, Feuillet C (2009) Genome organization and comparative genomics. In: Wheat, Science and Trade (Carver B, ed) pp.327-367; Devos KM (2009) Grass genome organization and evolution. Current Opinion in Plant Biol (in press)
• Devos KM, Costa de Oliveira A, Xu X, Estill JC, Estep M, Jogi A, Morales M, Pinheiro J, San Miguel P, Bennetzen JL (2008) Structure and organization of the wheat genome - the number of genes in the hexaploid wheat genome. In: Appels R, Esatwood R, Lagudah E, Langridge P, Mackay M, McIntyre L, Sharp P (eds) Proc 11th Int Wheat Genet Symp. Sydney, Sydney University Press, eO24, pp1-5
• Srinivasachary, Dida MM, Gale MD, Devos KM (2007) Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes. Theor Appl Genet 115:489-499
• McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Somers DJ, Anderson OA (2007) Catalogue of gene symbols for wheat: 2007 Supplement. Annu Wheat Newslet 53:159-180
• Dida MM, Srinivasachary, Ramakrishnan S, Bennetzen JL, Devos KM (2006) The genetic map of finger millet, Eleusine coracana. Theor Appl Genetics 114:321-332
• Devos KM, Hanna WW, Ozias-Akins P (2006) Pearl Millet. In: Genome Mapping and Molecular Breeding in Plants Vol.1: Cereals and Millets (Kole C, ed), pp. 303-323
• Dida MM, Devos KM (2006) Finger Millet. In: Genome Mapping and Molecular Breeding in Plants Vol.1: Cereals and Millets (Kole C, ed), pp. 333-343
• McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Anderson OA (2006) Catalogue of gene symbols for wheat: 2006 Supplement. Annu Wheat Newslet 52:208-230
• McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Somers DJ, Anderson OA (2008) Catalogue of gene symbols for wheat: 2008 Supplement. Annu Wheat Newslet 54:209-225
• Nagy ED, Lee T-C, Ramakrishna W, Xu Z, Klein PE, SanMiguel P, Cheng C-P, Li J, Devos KM, Schertz K, Dunkle L, Bennetzen JL. (2007) Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin. Theor Appl Genet 114:961-970
• Devos KM (2005) Updating the crop circles. Current Opinion in Plant Biol 8:155-162
• Devos KM, Ma J, Pontaroli AC, Pratt LH, Bennetzen JL (2005) Analysis and mapping of randomly chosen BAC clones from hexaploid bread wheat. Proc. Natl Acad Sci 102:19243-19248

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

Outputs
OUTPUTS: (1) Structural organization of the wheat genome. A total of 188 hexaploid wheat BAC clones have been annotated and mapped, and a further 28 BAC clones have been annotated but not mapped. Our results demonstrate that ca. 50% of the genes are present in multi-gene islands, and that multi-gene islands occur in proximal as well as distal chromosome regions. The results provide new insights into the organization of genes in the bread wheat genome. We are also assessing the extent of colinearity of Ae. tauschii, the D-genome donor to bread wheat with Brachypodium distachyon, a potential model for wheat, and with rice and sorghum. 13 Ae. tauschii contigs with a size of several hundred kb have been sequenced. BAC ordering has been completed for all sequenced contigs, and gene annotation has been completed for 9 contigs. Our data show that the Ae. tauschii genome had undergone multiple rearrangements and is the least stable genome of the 4 genomes analyzed. (2) Isolating the pearl millet d2 gene. Fine mapping of the d2 gene in two mapping populations showed that the d2 gene was located in a linkage block of several markers that did not display recombination events in the mapping populations analyzed (~1,500 F2 plants). Comparative analysis of the d2 region with other grasses showed that the orthologous regions in maize and sorghum carried the Br1 and dw3 genes, respectively. In a preliminary experiment, RT-PCR with primers for these candidate genes showed that the pearl millet ortholog was expressed in internodes of two tall pearl millet inbred lines during flowering but not in the internodes of 3 dwarf inbreds. A pearl millet BAC library has been screened for the presence of a BAC clone containing a Br1/dw3 ortholog. A single BAC clone has been identified for sequencing. (3) Mapping pearl millet fertility restoration gene(s). Because of fertility problems in this mapping population, this project has been abandoned. Instead, we have started work on mapping the stay-green trait, which provides longer photosynthetic activity which will hopefully translate into higher yields under drought conditions. A mapping population was generated from a cross between inbreds 02GH884 and Tift 454 by Jeff Wilson, USDA-ARS, Tifton. Mapping of this population is in progress. The population was also phenotyped in 2009 by Jeff Wilson for a range of traits, including stay-green, flowering and seed size. PARTICIPANTS: Katrien M. Devos - Principal Investigator; Trudi Thomas - Research Technician; Rajiv Parvathaneni - Graduate Student; Collaborators: Jeff Wilson, USDA-ARS, Tifton; Ignatius Angarawai, a pearl millet breeder from Lake Chad Institute, Nigeria, was trained in molecular techniques (1 year); Nethra N, a researcher from University of Agricultural Sciences, Bangalore, was trained in molecular techniques (6 months) TARGET AUDIENCES: The wheat and millet scientific communities (academics and breeders). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The mapping, isolation and functional characterization of genes controlling traits of agronomic importance will ultimately benefit the farmers and consumers through the adoption of new varieties and new management strategies. The basic wheat research will provide essential data on the organization of the wheat genome and its relationship to B. distachyon and other grasses that are needed to develop strategies for sequencing the large bread wheat genome. Having the sequence of the wheat genome available will facilitate genomics research, including the isolation and functional characterization of genes, which, in turn, will help in varietal improvement.

Publications

• Devos KM, Dolezel J, Feuillet C (2009) Genome organization and comparative genomics. In: Wheat, Science and Trade (Carver B, ed) pp.327-367; Devos KM (2009) Grass genome organization and evolution. Current Opinion in Plant Biol (in press)

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

Outputs
OUTPUTS: 1.Structural organization of the wheat genome. My lab has annotated 69 and mapped 180 of 200 sequenced BAC clones. Our results suggest that gene densities in the distal regions are considerable lower (1 gene/54kb) than previously published values (1 gene/5-20 kb). We are also assessing the extent of colinearity between wheat and Brachypodium distachyon, a potential model for wheat. We have shotgun sequenced and annotated a contig of 11 BAC clones and conducted a comparative analysis with the sequenced B. distachyon and rice genomes. Some disruption in colinearity was identified between Ae. tauschii and both B. distachyon and rice, indicating that the rearrangement had taken place in Ae. tauschii. The results are important to help design strategies on how to sequence the 17,000 Mb wheat genome. 2.Isolating the pearl millet d2 gene. Using the rice and sorghum genomic sequences, markers were developed for the d2 region. Nine markers were mapped to the d2 interval. Phenotyping of F3 plants derived by selfing 24 informative F2 plants that carried a recombination event in the d2 region provided the genotype at the D2 locus for these plants. Combining the scores at the D2 locus with those of other markers showed that eight of the nine markers cosegregated with d2. The markers that cosegregate with D2 span a region of at least 389 kb in sorghum. The closest proximal marker to D2 remains 224C4, the closest distal marker is PSM344. We are currently in the process of constructing a physical map for the D2 region. We are also assessing a second mapping population of some 550 F2 plants for recombination events in the 224C4 - PSM344 region. 3.Mapping pearl millet fertility restoration gene(s). The cross Tift454-7 x 99-72-4 was selected for mapping. The project is in collaboration with Jeff Wilson, USDA-ARS, Tifton, who is conducting the phenotypic analysis. Phenotyping of the F2 lines is done by testcrossing each F2 line onto both an A1 and A4 CMS line. The amount of selfed seed set on the testcross lines provides the genotype at the restorer loci. For unknown reasons, a large proportion of the F2 lines were sterile. Marker analyses are being conducted on the 48 lines that provided sufficient pollen for testcrossing. Another 150 F2 lines are being grown for testcrossing. Ten markers have been mapped. 4.Finger millet biodiversity analysis. The analysis of the genotypic and phenotypic data generated for a collection of 79 Eleusine coracana subsp. coracana accessions, 14 E. coracana subsp. africana accessions, 2 E. indica and 1 E. kigeziensis accessions has been completed. The data show that a clear population structure exists within the E. coracana germplasm with distinct populations being formed by (1) the wild accessions, (2) the cultivated accessions from Africa and (3) the cultivated accessions from Asia. Introgression between African and Indian material was observed, mainly as a result of targeted breeding to expand the germplasm basis on the Asian and African continents, and also between the African cultivated and wild accessions. 5.Finger millet-Comparative mapping. A manuscript on the finger millet-rice comparative map was published in 2007. PARTICIPANTS: Katrien M. Devos - Principal Investigator; Trudi Thomas - Research Technician; Chris Papadopoulos - Graduate Student; Collaborators: Jeff Wilson, USDA-ARS, Tifton

Impacts
The mapping, isolation and functional characterization of genes controlling traits of agronomic importance will ultimately benefit the farmers and consumers through the adoption of new varieties and new management strategies. The basic wheat research will provide essential data on the organization of the wheat genome and its relationship to B. distachyon that are needed to develop strategies for sequencing the large bread wheat genome. Having the sequence of the wheat genome available will facilitate genomics research, including the isolation and functional characterization of genes, which, in turn, will help in varietal improvement.

Publications

• Nagy ED, Lee T-C, Ramakrishna W, Xu Z, Klein PE, SanMiguel P, Cheng C-P, Li J, Devos KM, Schertz K, Dunkle L, Bennetzen JL. (2007) Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin. Theor Appl Genet 114:961-970
• Srinivasachary, Dida MM, Gale MD, Devos KM (2007) Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes. Theor Appl Genet 115:489-499
• McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris CF, Appels R, Somers DJ, Anderson OA (2007) Catalogue of gene symbols for wheat: 2007 Supplement. Annu Wheat Newslet 53:159-180

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

Outputs
Wheat. I.1. Chromosomal translocations. Two markers that flank the 4L/5L breakpoints on 4AL and 5AL most closely have been cloned and sequenced from 4 genomes with and 3 without the translocation. Aligned coding regions were used to generate phylogenetic trees. The lack of clustering between the genomes with and without the 4L/5L translocation demonstrates that the translocation is of polyphyletic origin. We initiated a collaboration with J. Dvorak, UC Davis, to develop BAC contigs across the breakpoint regions. Two separate contigs were identified for the breakpoint region on 5D. To establish the physical size of this region, we are collaborating with J. Dolezel, Czech Republic, to conduct in situ hybridization on flow-sorted and stretched wheat chromosomes with BAC clones from the two contigs. I.2.Structural organization of the wheat genome. My lab has annotated 69 of the 111 sequenced BAC clones (total number to be sequenced is 220). 45 BAC clones have been mapped to chromosome bins using deletion lines. Our results suggest that gene densities in the distal regions are considerable lower (1 gene/54kb) than previously published values (1 gene/5-20 kb). The results are important to help design strategies on how to sequence the 17,000 Mb wheat genome. I.3.Isolate of Rht12. No progress. Pearl millet. II.1. Isolation of d2 and d4. d2: 930 plants have been screened for recombination events in the 2.5 cM interval spanning d2. Twenty eight recombinants were identified and have been genotyped with 4 markers, previously shown to be located within the d2 region. F2:3 plants are growing in the glasshouse to provide information on the allelic composition of the d2 height genes in the corresponding F2 plants. This will allow us to precisely map d2, a first step to isolating the d2 gene. d4 and Br1: Attempts to clone the Br1 gene by PCR have failed. A pearl millet fosmid library is currently under construction of a D4D4Br1Br1 genotype and will be screened for the presence of d4 and Br1. II.2. Mapping fertility restoration gene(s). Four crosses, generated between Tift 454 (a restorer line for A1 cytoplasm) and either 99-70 or 99-72 lines (restorer lines for A4 cytoplasm) by Jeff Wilson, were analyzed for polymorphism levels. DNA has been extracted of the cross with the highest levels of variation. II.3. Mapping striga resistance genes. No progress. II.4.Recombination study. No progress. Finger millet. III.1. Biodiversity analysis. The genotypic data generated in the finger millet germplasm collection have been analyzed. I am currently working with J. Leebens-Mack, UGA, to analyze the phenotypic data, produced by our collaborators in Kenya and Uganda, in a phylogenetic context. We have also developed an additional set of some 100 microsatellites. The set of newly developed SSRs have been screened for amplification and variation in our finger millet mapping population. III.2. Comparative mapping. A manuscript on the finger millet genetic map was published in January 2007. A second manuscript on the finger millet-rice comparative map is under review for Theor and Appl Genet. Addition of SSRs and tef markers on the finger millet map is continuing.

Impacts
The mapping, isolation and functional characterization of genes controlling traits of agronomic importance will ultimately benefit the farmers and consumers through the adoption of new varieties and new management strategies. The basic wheat research will provide essential data on the organization of the wheat genome that is needed to develop strategies for sequencing the large bread wheat genome. Having the sequence of the wheat genome available will facilitate genomics research, including the isolation and functional characterization of genes, which, in turn, will help in varietal improvement.

Publications

• Dida MM, Srinivasachary, Ramakrishnan S, Bennetzen JL, Devos KM (2006) The genetic map of finger millet, Eleusine coracana. Theor Appl Genetics 114:321-332
• Devos KM, Hanna WW, Ozias-Akins P (2006) Pearl Millet. In: Genome Mapping and Molecular Breeding in Plants Vol.1: Cereals and Millets (Kole C, ed), pp. 303-323
• Dida MM, Devos KM (2006) Finger Millet. In: Genome Mapping and Molecular Breeding in Plants Vol.1: Cereals and Millets (Kole C, ed), pp. 333-343

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

Outputs
I.1. Work was initiated to isolate the breakpoint of the 4L/5L translocation that characterizes several Triticeae species. Using the rice genomic information, we narrowed the breakpoints on chromosomes 4L and 5L down to less than 20 kb in rice. The two markers that flank the breakpoints most closely are being cloned from six species, three with and three without the translocation. Following sequence analysis, phylogenetic trees will be constructed with each of the sequences to demonstrate that the translocation is of polyphyletic origin. I.2. Grant funds have been obtained from the National Science Foundation to sequence, annotate and map 220 randomly selected BAC clones from hexaploid wheat to get insight into the organization of the wheat genome. Because a proportion of the BAC clones are expected to consist of only repetitive DNA, we developed a technique whereby repeat boundaries are used as unique markers. This technique, together with the analysis and mapping of the first four sequenced BAC clones has been published in 2005 in Proceedings of the National Academy of Sciences. II.1. d2: The first 200 F2 plants of a population of 2000 have been screened for recombination events in the 2.5 cM interval spanning d2. Three recombinant plants were identified. d4 and Br1: Primers were generated against the candidate genes for d4 and Br1, identified from rice based on their comparative map position and putative function. Complete cosegregation was obtained in a F2 population of 60 plants between the OSD4 marker and d4, and between the OSBR1 marker and Br1. The pearl millet ortholog of OSD4 has been cloned and sequenced. Work is underway to also clone the pearl millet ortholog of OSBR1. II.2. Jeff Wilson has developed two mapping populations that segregate for the A1 and A4 fertility restoration genes. II.4. The mapping population segregating for the linked phenotypic markers d2 (semi-dwarfism) and Rp (purple plant color) has been generated and F2 seed is available. III.1. A set of 60 finger millet accessions have been evaluated in the field in Kenya and Uganda for morphological characteristics, and have been genotyped with some 50 SSR markers at UGA. Data analysis is in progress. III.2. The rice-finger millet genetic maps have been completed and two publications are in progress. In addition, markers mapped in the close relative tef have been obtained from Mark Sorrells, Cornell University. All tef markers obtained (some 150) have been screened for variation between MD-20 and Okhale-1, the parents of our finger millet populations. So far, 30 tef markers have been mapped in finger millet.

Impacts
The mapping, isolation and functional characterization of genes controlling traits of agronomic importance will ultimately benefit the farmers and consumers through the adoption of new varieties and new management strategies.

Publications

• Devos KM, Ma J, Pontaroli AC, Pratt LH, Bennetzen JL (2005) Analysis and mapping of randomly chosen BAC clones from hexaploid bread wheat. Proc. Natl Acad Sci 102:19243-19248