Transcriptome reprogramming due to the introduction of a barley telosome into bread wheat affects more barley genes than wheat.

Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat-barley 7HL addition line allowed the transcriptional impact both on 7HL genes of a non-native genetic background and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35 301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up-regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down-regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials.

Characterization of a hypoallergenic wheat line lacking ω-5 gliadin.

BACKGROUND: There is no curative treatment for wheat-dependent exercise-induced anaphylaxis (WDEIA). ω-5 Gliadin is one of the dominant allergens affecting WDEIA patients. The use of ω-5 gliadin-free wheat flour in the regular diet is considered one of the prophylactic approaches against the elicitation of allergic symptoms and sensitization to ω-5 gliadin. We sought to find hypoallergenic bread wheat (or common wheat) that lacked the genes encoding ω-5 gliadin and to evaluate its in vitro allergenicity. We also aimed to evaluate the sensitization ability of one of the selected hypoallergenic wheat lines by using a possible animal model of wheat allergy. METHODS: We screened the deletion lines of bread wheat by western blotting to ascertain common wheat lines lacking the ω-5 gliadin locus. The deletion lines we used have partial deficiency of chromosome 1B (Endo and Gill, 1996). To assess sensitization ability of gluten from the selected deletion line, guinea pigs were fed with either the gluten from the selected deletion line or commercially available gluten, and allergic score was evaluated after challenging the same gluten preparations. RESULTS: We found that a deletion line 1BS-18 had the least deficiency of chromosome 1B among the deletion stocks lacking the ω-5 gliadin locus. The challenge test using the guinea pigs revealed that the symptoms induced by application of the 1BS-18 gluten were much less than that of commercially available gluten. CONCLUSIONS: The deletion line 1BS-18, which lacked the ω-5 gliadin locus, is likely to have a low sensitization capacity in the guinea pig. The use of the wheat products of the 1BS-18 line in daily life may provide a feasible solution for the onset of wheat allergy.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Homoeologous relationship of rye chromosome arms as detected with wheat PLUG markers.

Based on the similarity in gene structure between rice and wheat, the polymerase chain reaction (PCR)-based landmark unique gene (PLUG) system enabled us to design primer sets that amplify wheat genic sequences including introns. From the previously reported wheat PLUG markers, we chose 144 markers that are distributed on different chromosomes and in known chromosomal regions (bins) to obtain rye-specific PCR-based markers. We conducted PCR with the 144 primer sets and the template of the Imperial rye genomic DNA and found that 131 (91.0%) primer sets successfully amplified PCR products. Of the 131 PLUG markers, 110 (76.4%) markers showed rye-specific PCR amplification with or without restriction enzyme digestion. We assigned 79 of the 110 markers to seven rye chromosomes (1R to 7R) using seven wheat-rye (cv. Imperial) chromosome addition and substitution lines: 12 to 1R, 8 to 2R, 11 to 3R, 8 to 4R, 16 to 5R, 12 to 6R, and 12 to 7R. Furthermore, we located their positions on the short or long (L) chromosome arm, using 13 Imperial rye telosomic lines of common wheat (except for 3RL). Referring to the chromosome bin locations of the 79 PLUG markers in wheat, we deduced the syntenic relationships between rye and wheat chromosomes. We also discussed chromosomal rearrangements in the rye genome with reference to the cytologically visible chromosomal gaps.

Gametocidal genes: from a discovery to the application in wheat breeding.

Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.

PCR and sequence analysis of barley chromosome 2H subjected to the gametocidal action of chromosome 2C.

Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.

Dissection of barley chromosome 4H in common wheat by the gametocidal system and cytological mapping of chromosome 4H with EST markers.

We used two gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley chromosome 4H added to common wheat. The Gc chromosome induced chromosomal structural rearrangements in the progeny of the 4H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying rearranged 4H chromosomes and characterized the rearranged chromosomes by sequential C-banding and in situ hybridization. We established 60 dissection lines of common wheat carrying single rearranged 4H chromosomes. The rearranged 4H chromosomes had either a deletion or a translocation or a complicated structural change. The breakpoints were distributed in the short arm, centromere and the long arm at a rough ratio of 2:2:1. We conducted PCR analysis using the dissection lines and 93 EST markers specific to chromosome 4H. Based on the PCR result, we constructed a cytological map of chromosome 4H with 18 regions separated by the breakpoints of the rearranged chromosomes. Thirty-seven markers were present in the short arm and 56 in the long arm, and about 70% of the markers were present in no more than the distal 25.6% and 43.1% regions of the short and long arms, respectively. It is noteworthy that nine of the short-arm markers and 13 of the long-arm markers existed in the small subtelomeric regions at both ends characterized by the HvT01 sequences. We reconstructed a genetic map using 38 of the 93 markers that was used to construct the cytological map of chromosome 4H. The order of the markers on the genetic map was almost the same as that on the cytological map. On the genetic map, no markers were available in the pericentromeric region, but on the cytological map, 14 markers were present in the proximal region, and one of the markers was in the centromeric region of the short arm.

Molecular mapping of the suppressor gene Igc1 to the gametocidal gene Gc3-C1 in common wheat.

Several species of the genus Aegilops, wild relatives of wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) carry gametocidal (Gc) genes. Gc genes kill the gametes without themselves by causing chromosomal breakage during post-meiotic cell divisions, and therefore are strong segregation distorters. The Gc gene Gc3-C1 derived from chromosome 3C of Ae. triuncialis (2n = 4x = 28, CCUU) induces chromosomal breakage in wheat cultivar 'Chinese Spring' (CS) but not in cultivar 'Norin 26' (N26). This cultivar-specific inhibition of Gc function is caused by a suppressor gene Igc1 located on chromosome 3B of N26. Igc1 is presumed to be a modified Gc gene without breakage function because of its homoeology to Gc3-C1. Here we report the results of linkage and physical mapping of Igc1 to help elucidate the molecular mechanisms underlying Gc action. Segregation analysis of the phenotypic data in BC(1)F(1) mapping population of the cross between (CSxN26)F(1) and CS + 3C" showed a 1:1 segregation ratio indicating that Igc1 is a dominant gene. In the linkage analysis, three molecular marker loci Xgwm285, Xgwm376, and Xcfp1886 cosegregated with the Igc1 locus. Bin mapping assigned the loci Xgwm285 and Xcfp1886 to bin C-3BS1-0.33 and Xgwm376 to bin C-3BL2-0.22. Physical mapping using Gc-induced chromosomal deletion lines of chromosome 3B of N26 revealed that the Igc1 locus resides in 52.0% or 2.1% of bins C-3BS1-0.33 and C-3BL2-0.22, respectively. Pericentromeric localization of Igc1 in chromosome 3B of N26 may have a positive effect to keep the two-component system of the Gc action. Map-based cloning approach to isolate the Igc1 may be difficult because recombination is depleted in the pericentromeric region. As is shown in this study, the combination of genetic and physical mapping offers high efficiency to identify the regions where genes are located especially in regions with low levels of recombination.

Development of self-fertile deletion homozygous and ditelosomic lines for the long arm of chromosome 2A in common wheat.

Most deletions for the short arm of chromosome 2A (2AS), and the telocentric chromosome for the long arm of chromosome 2A (2AL), are available only in the heterozygous condition in 'Chinese Spring' hexaploid wheat. This is due to the female sterility, and therefore self-sterility, of their homozygotes, caused by the partial or entire loss of the 2AS chromosome arm on which genes for normal synapsis and female fertility are located. On the other hand, a D-genome disomic substitution line 2D(2A) of 'Langdon' tetraploid wheat, in which chromosome 2D is disomically substituted for chromosome 2A, is available (i.e., self-fertile) despite chromosome 2A being missing in this line. This fact indicates that another gene for female fertility must be present in Langdon 2D(2A). We attempted to develop self-fertile 2AS homozygous deletion and ditelosomic 2AL lines by transferring this female fertility gene, through a series of crosses and cytological screening, from Langdon 2D(2A) to the two aneuploid lines. We finally obtained self-fertile 2AS homozygous deletion and ditelosomic 2AL lines. These lines displayed normal meiotic chromosome pairing and lacked all 12 of the 2AS markers used for PCR analysis.

Cytological dissection and molecular characterization of chromosome 1R derived from 'Burgas 2' common wheat.

Rye chromosome 1R harbors many agronomically important genes such as resistance genes for rusts. Using the gametocidal system, we dissected the 1R chromosome substituted for wheat chromosome 1B in a common wheat cultivar 'Burgas 2'. The gametocidal system induces chromosomal breakage in the 1R chromosome, as well as in wheat chromosomes. We cytologically examined a pool of prescreened common wheat plants that had been shown to have single or multiple rearranged 1R chromosomes and established 95 common wheat lines carrying single 1R segments. We conducted PCR analysis of these lines, termed '1R dissection lines', using 10 PCR-based 1R-specific markers. We mapped the 10 PCR-based markers along the 1R chromosome with the breakpoints of the 1R dissection lines. Based on the PCR result and the positions of the primary and secondary constrictions, we could separate the breakpoints of the rearranged 1R chromosomes into 12 regions along the 1R chromosome. On the other hand, using the breakpoints, we could separate the PCR-based markers from each other except for two markers. These dissection lines are useful in mapping DNA markers and may facilitate the construction of contig maps.

Dissection of barley chromosome 3H in common wheat and a comparison of 3H physical and genetic maps.

We used the gametocidal system to dissect a barley chromosome 3H added to common wheat. The gametocidal system induced chromosomal structural changes in the 3H addition line of common wheat, and we cytologically screened for rearranged chromosomes involving the 3H chromosome by in situ hybridization (FISH/GISH). We established 50 common wheat lines carrying single rearranged (or dissected) 3H chromosomes of independent origin. The dissected 3H chromosomes were either deletions or translocations with wheat chromosomes, and their breakpoints were in the centromere/the long arm/the short arm in a rough ratio of 1:2:2. We used these so-called 3H dissection lines to map 36 EST markers that were polymorphic between euploid common wheat and the 3H addition line and that had been used for the construction of a 3H genetic map. We conducted PCR analysis to detect the EST markers in the dissection lines. The results of the PCR analysis, which mostly corresponded to the retained or lost segments of the dissected 3H chromosomes, allowed us to place the 36 EST markers into 20 chromosomal regions flanked by the breakpoints of the dissected chromosomes. We compared this physical map constructed in this study with a 3H genetic map constructed using the same EST markers. The order of all EST markers was consistent between the two maps. We briefly discuss on the advantage of the physical mapping using dissection lines over genetic mapping.

Chromosome Arm Locations of Barley Sucrose Transporter Gene in Transgenic Winter Wheat Lines.

Three transgenic HOSUT lines of winter wheat, HOSUT12, HOSUT20, and HOSUT24, each harbor a single copy of the cDNA for the barley sucrose transporter gene HvSUT1 (SUT), which was fused to the barley endosperm-specific Hordein B1 promoter (HO; the HOSUT transgene). Previously, flow cytometry combined with PCR analysis demonstrated that the HOSUT transgene had been integrated into different wheat chromosomes: 7A, 5D, and 4A in HOSUT12, HOSUT20, and HOSUT24, respectively. In order to confirm the chromosomal location of the HOSUT transgene by a cytological approach using wheat aneuploid stocks, we crossed corresponding nullisomic-tetrasomic lines with the three HOSUT lines, namely nullisomic 7A-tetrasomic 7B with HOSUT12, nullisomic 5D-tetrasomic 5B with HOSUT20, and nullisomic 4A-tetrasomic 4B with HOSUT24. We examined the resulting chromosomal constitutions and the presence of the HOSUT transgene in the F2 progeny by means of chromosome banding and PCR. The chromosome banding patterns of the critical chromosomes in the original HOSUT lines showed no difference from those of the corresponding wild type chromosomes. The presence or absence of the critical chromosomes completely corresponded to the presence or absence of the HOSUT transgene in the F2 plants. Investigating telocentric chromosomes occurred in the F2 progeny, which were derived from the respective critical HOSUT chromosomes, we found that the HOSUT transgene was individually integrated on the long arms of chromosomes 4A, 7A, and 5D in the three HOSUT lines. Thus, in this study we verified the chromosomal locations of the transgene, which had previously been determined by flow cytometry, and moreover revealed the chromosome-arm locations of the HOSUT transgene in the HOSUT lines.

Development of Deletion Lines for Chromosome 3D of Bread Wheat.

The identification of genes of agronomic interest in bread wheat (Triticum aestivum L.) is hampered by its allopolyploid nature (2n = 6x = 42; AABBDD) and its very large genome, which is largely covered by transposable elements. However, owing to this complex structure, aneuploid stocks can be developed in which fragments or entire chromosomes are missing, sometimes resulting in visible phenotypes that help in the cloning of affected genes. In this study, the 2C gametocidal chromosome from Aegilops cylindrica was used to develop a set of 113 deletion lines for chromosome 3D in the reference cultivar Chinese Spring. Eighty-four markers were used to show that the deletions evenly covered chromosome 3D and ranged from 6.5 to 357 Mb. Cytogenetic analyses confirmed that the physical size of the deletions correlated well with the known molecular size deduced from the reference sequence. This new genetic stock will be useful for positional cloning of genes on chromosome 3D, especially for Ph2 affecting homoeologous pairing in bread wheat.

Dissection of rye chromosome 1R in common wheat.

Rye chromosome 1R contains many agronomically useful genes. Physical dissection of chromosome 1R into segments would be useful in mapping 1R-specific DNA markers and in assembling DNA clones into contig maps. We applied the gametocidal system to produce rearranged 1R chromosomes of Imperial rye (1R(i)) added to common wheat. We identified rearranged 1R(i) chromosomes and established 55 1R(i) dissection lines of common wheat carrying a single rearranged 1R(i) chromosome. Fifty-two of the rearranged 1R(i) chromosomes had single breakpoints and three had double breakpoints. The 58 breakpoints were distributed in the short arm excluding the satellite (12 breakpoints), in the satellite (4), in the long arm (28), and in the centromere (14). Out of the 55 lines, nine were homozygous for the rearranged 1R(i) chromosomes, and the remaining lines were hemizygous. We developed 26 PCR-based EST markers that were specific to the 1R(i) chromosome, and nine of them amplified 1R(i) arm-specific PCR products without restriction-enzyme digestion. Using the nine EST markers and two previously reported 1R-specific markers, we characterized the 55 1R(i) dissection lines, and also proved that we can select critical progeny plants carrying specific rearranged 1R(i) chromosomes by PCR, without cytological screening, in 48 out of the 55 hemizygous dissection lines.

Molecular characterization of benzoxazinone-deficient mutation in diploid wheat.

Benzoxazinones (Bxs) are representative defensive compounds in gramineous plants, including wheat (genus Triticum) and its wild relative species (genus Aegilops). Bx production was found to be variable among three diploid wheat species with the same A genome as hexaploid wheat (2n=6x=42, genomes AABBDD). All accessions of Triticum monococcum (2n=2x=14, AA) and Triticum urartu (2n=2x=14, AA) accumulated Bxs, but 18 out of 28 accessions of Triticum boeoticum (2n=2x=14, AA) were Bx-deficient. Bx-deficient accessions were grouped into two types by genomic PCR analysis of the five Bx biosynthetic loci (TbBx1-TbBx5): those retaining all five loci (type I) and those lacking TbBx3 and TbBx4 loci (type II). Despite the Bx-deficient phenotype, all five TbBx genes were transcribed in the type-I accessions. The Bx deficiency in one accession of type I was due to the disintegration of the TbBx1, TbBx4 and TbBx5 genes due to insertions or deletions in their coding sequences. The TbBx2 and TbBx3 genes of those accessions had the complete sequences of the functional enzymes. In the type-II accessions, the remaining three genes, TbBx1, TbBx2 and TbBx5, were all transcribed, with the exception of two accessions in which either TbBx1 or TbBx5 was not transcribed. The TbBx1 coding sequence of the type-II accessions was also disintegrated, like that of the type-I accessions. These findings suggest that the Bx deficiency in T. boeoticum first resulted from disintegration of the TbBx1 coding sequence, followed by transcription failure, disintegration of the coding sequences and elimination of the TbBx1-TbBx5 genes.

Dissection of barley chromosome 5H in common wheat.

We dissected barley chromosome 5H added to common wheat by a genetic method or the gametocidal system. Firstly, we induced chromosomal breaks in the offspring of a 5H addition line of common wheat carrying a gametocidal chromosome and cytologically screened for plants with structural chromosomal changes involving 5H, such as deletions and translocations. Secondly, we screened the progeny of such plants to establish common wheat lines carrying structurally changed chromosomes containing single segments of the dissected 5H. Using 23 representative 5H dissection lines, we physically mapped 97 barley EST markers assigned to 5H. The ESTs fell into 20 regions of 5H between the breakpoints of the 23 dissected segments, distributing rather evenly along the chromosome, with significantly higher frequency in the distal region of the long arm. The ESTs, in turn, allowed us to distinguish the breakpoints of dissected 5H segments. We demonstrated by PCR (polymerase chain reaction), as well as by in situ hybridization, that these dissected 5H segments were stably transmitted in the dissection lines. We discuss the usefulness of the 5H dissection lines for physical mapping of DNA markers. These 5H dissection lines are available from National BioResource Projects-Wheat, Japan.

Chromosome arm location of the genes for the biosynthesis of hordatines in barley.

Hordatines A and B, the strong antifungal compounds in barley (Hordeum vulgare), are biosynthesized from p-coumaroyl- and feruloyl-CoA and agmatine by two successive reactions catalyzed by agmatine coumaroyltransferase (ACT) and peroxidase. ACT catalyzes the formation of agmatine conjugates (p-coumaroylagmatine and feruloylagmatine) from precursor CoAs and agmatine, and peroxidase catalyzes the oxidative coupling of agmatine conjugates to form hordatines. Our previous study demonstrated that the short arm of barley chromosome 2H (2HS) is responsible for the biosynthesis of hordatines. In the present study, however, barley genes encoding the ACT (HvACT) and a peroxidase (HvPrx7) were found to be located on the long arm of 2H (2HL). The amounts of hordatines and precursor agmatine conjugates were analyzed in wheat (Triticum aestivum) and wheat lines carrying a whole 2H chromosome, 2HS or 2HL. The addition of 2H and 2HL elevated the levels of agmatine conjugates in wheat. This could be attributed to the HvACT on 2HL. However, the content of agmatine conjugates increased also in the 2HS addition line, suggesting the presence of another unidentified ACT gene on 2HS. Hordatines were detected in wheat, but their content was by far lower than those in barley. The 2H and 2HS addition lines accumulated substantial amounts of hordatines, while the 2HL addition line accumulated them as little as wheat did in spite of the substantial transcription of the HvPrx7 gene on 2HL and of the increased accumulation of the precursor agmatine conjugates. These facts suggest that the HvPrx7 gene on 2HL is not involved in the hordatine biosynthesis and that unidentified peroxidase gene responsible for the hordatine biosynthesis is located on 2HS in barley.

KEGG bioinformatics resource for plant genomics research.

Kyoto Encyclopedia of Genes and Genomes (KEGG) is a bioinformatics resource for understanding biological function from a genomic perspective. It is a multispecies, integrated resource consisting of genomic, chemical, and network information, with cross-references to numerous outside databases and containing a complete set of building blocks (genes and molecules) and wiring diagrams (biological pathways) to represent cellular functions. KEGG consists of a suite of databases: PATHWAY, GENES/Sequence Similarity Database (SSDB), Biomolecular Relations in Information Transmission and Expression (BRITE), and LIGAND, which is a composite database of COMPOUND, DRUG, GLYCAN, REACTION, REPAIR, and ENZYME. Two new databases have been recently added to KEGG: DGENES (for draft genomes) and EGENES (for expressed-sequence tag [EST] data). EGENES is a knowledge base system for efficient analysis of organism-specific ESTs, including publicly available plant ESTs. EGENES links the genomic information with higher order functional information in a single database. The genomic information stored in EGENES is a collection of EST contigs, produced by assembling the public ESTs. In this chapter, we will introduce KEGG and discuss its importance for the plant research community by focusing on EGENES. Because all the resources in KEGG follow the same architecture and design, an appraisal of EGENES should give readers an idea of the available information stored in KEGG and how to use them efficiently.

Chromosomal Allocation of DNA Sequences in Wheat Using Flow-Sorted Chromosomes.

Flow cytometry enables chromosomes to be sorted into different groups based on their characteristics, such as relative DNA content and the presence of repetitive DNA sequences. Despite the recent progress in the analysis of plant genome organization and chromosome structure, there is a need for easy methods to assign DNA sequences to individual chromosomes. Here, we describe an easy way to allocate genes or DNA sequences to chromosomes in wheat using flow-sorted chromosomes combined with fluorescence in situ hybridization and PCR analyses.

The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat.

BACKGROUND: Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches. RESULTS: We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template. CONCLUSIONS: Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.