Ca. Liberibacter asiaticus is the putative fastidious bacterial causal agent of citrus greening disease, also known as Huanglongbing (HLB), translated from Chinese as yellow dragon disease. The HLB bacterial pathogen is indigenous to Asia but has been introduced and dispersed to citrus throughout the Americas. A related bacterium that is indigenous to the Americas causes damage to potato (zebra chip) and tomato (vein-greening) and other solanaceous hosts. The causal agents are propagative and circulative in the psyllid vector, Diaphorina citri (Kuwayama) and Bactericera cockerelli (Sulc.), the Asian citrus and potato (or tomato) psyllid, respectively. The specific psyllid proteins that are indirectly or directly involved in the circulative, propagative transmission pathway are not known. However, if proteins were known that function at key points in the pathway e.g. post-ingestion, infection, biofilm formation, nutrition, circulation, and/or acquisition were known, such knowledge could be exploited to knock out their expression and abate pathogen transmission. To this end a combined approach involving functional genomics and anatomical localization of the bacterium is being implemented. Results indicate that Ca. Liberibacter establishes biofilms on the outer surfaces of the alimentary canal and salivary glands of the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama and the potato psyllid (PP) Bactericera cockerelli Sulc. In silico transcript profiling of infected and uninfected ACP and PP identified a number of mis-expressed, unique transcripts (unitrans). Functional predictions (gene ontology associations) implicate certain of these unitrans in Ca. Liberibacter infection of the psyllid host and/or in psyllid-mediated Ca. Liberibacter transmission processes.

Citrus greening is the most destructive disease of citrus crops worldwide. The introduced Asian citrus psyllid (ACP) Diaphorina citri Kuwayama transmits the (putative) causal bacterium, Candidatus Liberibacter asiaticus. A close relative, Ca. L. solanacearum, is the pathogen associated with Zebra chip disease of potato and vein-greening disease of tomato. It is both transmitted by and propagative in the endemic (western U.S) potato psyllid (PoP) Bactericerca cockerelli Sulc. The PoP occurs widely in the western U.S. and so has been used as a parallel study system for the quarantined ACP-greening complex. To identify proteins involved in global psyllid-Ca. Liberibacter interactions, the ACP and PoP transcriptomes were sequenced, yielding a total of 45,976 and 82,224 Illumina unique ACP and PoP transcripts, respectively. Cluster analysis revealed a high degree of sequence and transcript conservation suggestive of roles in core growth and developmental processes, providing the first molecular snapshot of the specific psyllid genes responsive to parasite invasion and circulation in the host. Evidence of inter-psyllid molecular conservation substantiates the suitability of PoP as a study system for ACP-Ca. L. asiatcus. Comparative in silico expression analysis within and between psyllid species revealed predicted functions involved in Ca. Liberibacter parasitism that were both unique and shared in common among adult and nymphal instars. In addition, functional characterization based on Gene Ontology analysis has revealed a number of genes associated with host-parasite interactions that could mediate Ca. Liberibacter infection, propagation, and circulation in the psyllid, as well as transmission processes.

Rice blast disease, caused by the filamentous fungus Magnaporthe grisea, is a serious and recurrent problem in all rice-growing regions of the world (Talbot, 2003; Valent & Chumley, 1991). It is estimated that each year enough rice is destroyed by rice blast disease to feed 60 million people. Control of this disease is difficult; new host-specific forms develop quickly to overcome host resistance and chemical control is typically not cost effective (Ou, 1987). Infections occur when fungal spores land and attach themselves to leaves using a special adhesive released from the tip of each spore (Hamer et al., 1988). The germinating spore develops an appressorium, a specialized infection cell, which generates enormous turgor pressure – up to 8 MPa – that ruptures the leaf cuticle allowing invasion of the underlying leaf tissue (de Jong et al., 1997; Dean, 1997). Subsequent colonization of the leaf produces disease lesions from which the fungus sporulates and spreads to new plants. When rice blast infects young rice seedlings, whole plants often die, while spread of the disease to the stems, nodes or panicle of older plants results in nearly total loss of the rice grain. Recent reports have further shown that the fungus has the capacity to infect plant roots (Sesma & Osbourn, 2004). Different host-limited forms of Magnaporthe also infect a broad range of grass species including wheat, barley and millet.

Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages.

Approximately 185,000 Gossypium EST sequences comprising > 94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety Of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A(T) and D-T genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics.

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

To better understand the molecular basis of the defense response against the rice blast fungus ( Magnaporthe grisea), a large- scale expressed sequence tag ( EST) sequencing approach was used to identify genes involved in the early infection stages in rice ( Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen- challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full- length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum ( Sorghum bicolor) ESTs than to barley ( Hordeum vulgare), wheat ( Triticum aestivum), and maize ( Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.

Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied.

Many clone-based physical maps have been built with the FingerPrinted Contig (FPC) software, which is written in C and runs locally for fast and flexible analysis. If the maps were viewable only from FPC, they would not be as useful to the whole community since FPC must be installed on the user machine and the database downloaded. Hence, we have created a set of Web tools so users can easily view the FPC data and perform salient queries with standard browsers. This set includes the following four programs: WebFPC, a view of the contigs; WebChrom, the location of the contigs and genetic markers along the chromosome; WebBSS, locating user-supplied sequence on the map; and WebFCmp, comparing fingerprints. For additional FPC support, we have developed an FPC module for BioPerl and an FPC browser using the Generic Model Organism Project (GMOD) genome browser (GBrowse), where the FPC BioPerl module generates the data files for input into GBrowse. This provides an alternative to the WebChrom/WebFPC view. These tools are available to download along with documentation. The tools have been implemented for both the rice (Oryza sativa) and maize (Zea mays) FPC maps, which both contain the locations of clones, markers, genetic markers, and sequenced clone (along with links to sites that contain additional information).

Single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) are becoming important genetic markers for major crop species. In this study we demonstrate their utility for locating fingerprint contigs (FPCs) to the genetic map. To derive SNP and InDel markers, we amplified genomic regions corresponding to 3000 unigenes across 12 maize (Zea mays L.) lines, of which 194 unigenes (6.4%) showed size polymorphism InDels between B73 and Mo17 on agarose gels. The analysis of these InDels in 83 diverse inbred lines showed that InDels are often multiallelic markers in maize. Single nucleotide polymorphism discovery conducted on 592 unigenes revealed that 44% of the unigenes contained B73/Mo17 SNPs, while 8% showed no sequence variation among the 12 inbred lines. On average, SNPs and InDels occurred every 73 and 309 bp, respectively. Multiple SNPs within unigenes led to a SNP-haplotype genetic diversity of 0.61 among inbreds. The unigenes were previously assigned to maize FPCs by overgo hybridization. From this set of unigenes, 311 (133 SNP and 178 InDel) loci were mapped on the intermated B73 X Mo17 (IBM) high-resolution mapping population. These markers provided unambiguous anchoring of 129 FPCs and orientation for 30 contigs. The FPC anchored map of maize will be useful for map-based cloning, for genome sequencing efforts in maize, and for comparative genomics in grasses. The amplification primers for all mapped InDel and SNP loci, the diversity information for SNPs and InDels, and the corresponding overgoes to anchor bacterial artificial chromosome (BAC) contigs are provided as genetic resources.

Rice (Oryza sativa L.) chromosome 3 is evolutionarily conserved across the cultivated cereals and shares large blocks of synteny with maize and sorghum, which diverged from rice more than 50 million years ago. To begin to completely understand this chromosome, we sequenced, finished, and annotated 36.1 Mb ( approximately 97%) from O. sativa subsp. japonica cv Nipponbare. Annotation features of the chromosome include 5915 genes, of which 913 are related to transposable elements. A putative function could be assigned to 3064 genes, with another 757 genes annotated as expressed, leaving 2094 that encode hypothetical proteins. Similarity searches against the proteome of Arabidopsis thaliana revealed putative homologs for 67% of the chromosome 3 proteins. Further searches of a nonredundant amino acid database, the Pfam domain database, plant Expressed Sequence Tags, and genomic assemblies from sorghum and maize revealed only 853 nontransposable element related proteins from chromosome 3 that lacked similarity to other known sequences. Interestingly, 426 of these have a paralog within the rice genome. A comparative physical map of the wild progenitor species, Oryza nivara, with japonica chromosome 3 revealed a high degree of sequence identity and synteny between these two species, which diverged approximately 10,000 years ago. Although no major rearrangements were detected, the deduced size of the O. nivara chromosome 3 was 21% smaller than that of japonica. Synteny between rice and other cereals using an integrated maize physical map and wheat genetic map was strikingly high, further supporting the use of rice and, in particular, chromosome 3, as a model for comparative studies among the cereals.

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

Rice is the world's most important food crop and a model for cereal research. At 430 megabases in size, its genome is the most compact of the cereals. We report the sequence of chromosome 10, the smallest of the 12 rice chromosomes (22.4 megabases), which contains 3471 genes. Chromosome 10 contains considerable heterochromatin with an enrichment of repetitive elements on 10S and an enrichment of expressed genes on 10L. Multiple insertions from organellar genomes were detected. Collinearity was apparent between rice chromosome 10 and sorghum and maize. Comparison between the draft and finished sequence demonstrates the importance of finished sequence.

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint-based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome ( approximately 90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.

Crop plant research is poised to make revolutionary strides including the following: cloning target genes based on their function and/or their position in the genome; documenting all genes and their interplay; defining and exploring all the existing genetic diversity in a species; and using