Large homogeneous genome regions (isochores) in soybean [glycine max (L.) merr].

The landscape of plant genomes, while slowly being characterized and defined, is still composed primarily of regions of undefined function. Many eukaryotic genomes contain isochore regions, mosaics of homogeneous GC content that can abruptly change from one neighboring isochore to the next. Isochores are broken into families that are characterized by their GC levels. We identified 4,339 compositionally distinct domains and 331 of these were identified as long homogeneous genome regions (LHGRs). We assigned these to four families based on finite mixture models of GC content. We then characterized each family with respect to exon length, gene content, and transposable elements. The LHGR pattern of soybeans is unique in that while the majority of the genes within LHGRs are found within a single LHGR family with a narrow GC range (Family B), that family is not the highest in GC content as seen in vertebrates and invertebrates. Instead Family B has a mean GC content of 35%. The range of GC content for all LHGRs is 16-59% GC which is a larger range than what is typical of vertebrates. This is the first study in which LHGRs have been identified in soybeans and the functions of the genes within the LHGRs have been analyzed.

Placing paleopolyploidy in relation to taxon divergence: a phylogenetic analysis in legumes using 39 gene families.

Young polyploid events are easily diagnosed by various methods, but older polyploid events become increasingly difficult to identify as chromosomal rearrangements, tandem gene or partial chromosome duplications, changes in substitution rates among duplicated genes, pseudogenization or locus loss, and interlocus interactions complicate the means of inferring past genetic events. Genomic data have provided valuable information about the polyploid history of numerous species, but on their own fail to show whether related species, each with a polyploid past, share a particular polyploid event. A phylogenetic approach provides a powerful method to determine this but many processes may mislead investigators. These processes can affect individual gene trees, but most likely will not affect all genes, and almost certainly will not affect all genes in the same way. Thus, a multigene approach, which combines the large-scale aspect of genomics with the resolution of phylogenetics, has the power to overcome these difficulties and allow us to infer genomic events further into the past than would otherwise be possible. Previous work using synonymous distances among gene pairs within species has shown evidence for large-scale duplications in the legumes Glycine max and Medicago truncatula. We present a case study using 39 gene families, each with three or four members in G. max and the putative orthologues in M. truncatula, rooted using Arabidopsis thaliana. We tested whether the gene duplications in these legumes occurred separately in each lineage after their divergence (Hypothesis 1), or whether they share a round of gene duplications (Hypothesis 2). Many more gene family topologies supported Hypothesis 2 over Hypothesis 1 (11 and 2, respectively), even after synonymous distance analysis revealed that some topologies were providing misleading results. Only ca. 33% of genes examined support either hypothesis, which strongly suggests that single gene family approaches may be insufficient when studying ancient events with nuclear DNA. Our results suggest that G. max and M. truncatula, along with approximately 7000 other legume species from the same clade, share an ancient round of gene duplications, either due to polyploidy or to some other process.

Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.

A new integrated genetic linkage map of the soybean.

A total of 391 simple sequence repeat (SSR) markers designed from genomic DNA libraries, 24 derived from existing GenBank genes or ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences were developed. In contrast to SSRs derived from EST sequences, those derived from genomic libraries were a superior source of polymorphic markers, given that the mean number of tandem repeats in the former was significantly less than that of the latter ( P<0.01). The 420 newly developed SSRs were mapped in one or more of five soybean mapping populations: "Minsoy" x "Noir 1", "Minsoy" x "Archer", "Archer" x "Noir 1", "Clark" x "Harosoy", and A81-356022 x PI468916. The JoinMap software package was used to combine the five maps into an integrated genetic map spanning 2,523.6 cM of Kosambi map distance across 20 linkage groups that contained 1,849 markers, including 1,015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, six AFLPs, ten isozymes, and 12 others. The number of new SSR markers added to each linkage group ranged from 12 to 29. In the integrated map, the ratio of SSR marker number to linkage group map distance did not differ among 18 of the 20 linkage groups; however, the SSRs were not uniformly spaced over a linkage group, clusters of SSRs with very limited recombination were frequently present. These clusters of SSRs may be indicative of gene-rich regions of soybean, as has been suggested by a number of recent studies, indicating the significant association of genes and SSRs. Development of SSR markers from map-referenced BAC clones was a very effective means of targeting markers to marker-scarce positions in the genome.

Estimates of conserved microsynteny among the genomes of Glycine max, Medicago truncatula and Arabidopsis thaliana.

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.

Residential and recreational acquisition of possible estuary-associated syndrome: a new approach to successful diagnosis and treatment.

Evidence suggests that the estuarine dinoflagellates, Pfiesteria piscicida Steidinger & Burkholder and P. shumwayae Glasgow & Burkholder, members of the toxic Pfiesteria complex (TPC), may release one or more toxins that kill fish and adversely affect human health. In the current study we investigated the potential for undiagnosed cases of possible estuary-associated syndrome (PEAS), as termed by the Centers for Disease Control and Prevention (CDC), in a population that had residential and/or recreational exposure to TPC-affected estuaries, but that did not have direct contact with fish kills or lesioned fish. Age-adjusted visual contrast sensitivity (VCS) was significantly lower and the presence of PEAS-associated symptoms was much higher in the estuary cohort (n = 77) than in combined-control cohorts (n = 87), one without exposure to bodies of water (n = 53) and one with exposure to marine waters (n = 34). In the estuary cohort, 37 individuals met the CDC case definition for PEAS and had significantly lower VCS than non-PEAS cases. The VCS improved and symptoms abated after 2 weeks of treatment with cholestyramine. Cholestyramine, the original drug approved for treatment of hypercholesterolemia, has previously been reported to enhance the elimination rates of a variety of toxins, presumably by interruption of enterohepatic recirculation through toxin entrapment in its polymeric structure and/or anion-exchange process. Control studies showed that repeated VCS testing alone did not improve VCS scores and that cholestyramine treatment did not affect VCS in patients with elevated cholesterol levels. These results suggested that a) susceptible individuals may acquire PEAS through residential and/or recreational contact with TPC-affected estuaries in the absence of an active fish kill; b) VCS is a useful indicator in PEAS diagnosis and treatment monitoring; and c) PEAS can be effectively treated with cholestyramine. Because the study did not use population sampling techniques, the results do not indicate PEAS prevalence. Furthermore, definitive diagnosis of PEAS and association with TPC toxin(s) must await identification of, and a serologic test for, the putative TPC toxin(s).

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Possible estuary-associated syndrome: symptoms, vision, and treatment.

The human illness designated as possible estuarine-associated syndrome (PEAS) by the Centers for Disease Control and Prevention (CDC) has been associated with exposure to estuaries inhabited by toxin-forming dinoflagellates, including members of the fish-killing toxic Pfiesteria complex (TPC), Pfiesteria piscicida and Pfiesteria shumwayae. Humans may be exposed through direct contact with estuarine water or by inhalation of aerosolized or volatilized toxin(s). The five cases reported here demonstrate the full spectrum of symptoms experienced during acute and chronic stages of this suspected neurotoxin-mediated illness. The nonspecific symptoms most commonly reported are cough, secretory diarrhea, headache, fatigue, memory impairment, rash, difficulty in concentrating, light sensitivity, burning skin upon water contact, muscle ache, and abdominal pain. Less frequently encountered symptoms are upper airway obstruction, shortness of breath, confusion, red or tearing eyes, weakness, and vertigo. Some patients experience as few as four of these symptoms. The discovery that an indicator of visual pattern-detection ability, visual contrast sensitivity (VCS), is sharply reduced in affected individuals has provided an objective indicator that is useful in diagnosing and monitoring PEAS. VCS deficits are present in both acute and chronic PEAS, and VCS recovers during cholestyramine treatment coincident with symptom abatement. Although PEAS cannot yet be definitively associated with TPC exposure, resolution with cholestyramine treatment suggests a neurotoxin-mediated illness.

Genetic analyses of two independent chlorophyll-deficient mutants identified among the progeny of a single chimeric foliage soybean plant.

Chimeric (variegated) foliage plants are frequently observed in many species. In soybean [Glycine max(L.) Merr.], progeny of chimeric plants are a source of nuclear and cytoplasmically inherited mutants. Self-pollinated progeny of a single chimeric plant derived from tissue culture of PI 427099 (Jilin 3) included plants with green foliage, chimeric foliage, yellow foliage (viable), and yellow foliage (lethal). Our objectives were to determine (1) inheritance, linkage, and allelism of the lethal-yellow mutant with known chlorophyll-deficient mutants; (2) inheritance, linkage, and allelism of the viable-yellow mutant with known chlorophyll-deficient mutants; (3) allelism of the lethal-yellow mutant with the viable-yellow mutant; and (4) male and female gamete transmission of the viable-yellow mutant trait. The viable-yellow mutant was allelic to T323, y20 y20 (Ames 2) Mdh1-n Mdh1-n (Ames 2) and was assigned genetic type collection number T361 and gene symbol y20 y20 (Ames 24) Mdh1-n Mdh1-n (Ames 22). The lethal-yellow mutant was allelic to T225H (Y18 y18) and was assigned genetic type collection number T362H and gene symbol Y18 y18 (Ames 2). T225H became Y18 y18 (Ames 1). The two chlorophyll-deficient mutants were not linked to each other. There was no significant difference in F(1) male or female gamete transmission of the viable-yellow mutant. However, many cross-combinations gave significant deviations from the expected 3 green plants:1 viable-yellow plant in the F(2) generation. The allelism of these two chlorophyll-deficient mutants with mutants T225H and T323, derived from putative transposable element systems, is intriguing. An explanation of this phenomenon awaits molecular experimentation.

Genome organization in dicots: genome duplication in Arabidopsis and synteny between soybean and Arabidopsis.

Synteny between soybean and Arabidopsis was studied by using conceptual translations of DNA sequences from loci that map to soybean linkage groups A2, J, and L. Synteny was found between these linkage groups and all four of the Arabidopsis chromosomes, where GenBank contained enough sequence for synteny to be identified confidently. Soybean linkage group A2 (soyA2) and Arabidopsis chromosome I showed significant synteny over almost their entire lengths, with only 2-3 chromosomal rearrangements required to bring the maps into substantial agreement. Smaller blocks of synteny were identified between soyA2 and Arabidopsis chromosomes IV and V (near the RPP5 and RPP8 genes) and between soyA2 and Arabidopsis chromosomes I and V (near the PhyA and PhyC genes). These subchromosomal syntenic regions were themselves homeologous, suggesting that Arabidopsis has undergone a number of segmental duplications or possibly a complete genome duplication during its evolution. Homologies between the homeologous soybean linkage groups J and L and Arabidopsis chromosomes II and IV also revealed evidence of segmental duplication in Arabidopsis. Further support for this hypothesis was provided by the observation of very close linkage in Arabidopsis of homologs of soybean Vsp27 and Bng181 (three locations) and purple acid phosphatase-like sequences and homologs of soybean A256 (five locations). Simulations show that the synteny and duplications we report are unlikely to have arisen by chance during our analysis of the homology reports.

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.

Analysis and mapping of gene families encoding beta-1,3-glucanases of soybean.

Oligonucleotide primers designed for conserved sequences from coding regions of beta-1,3-glucanase genes from different species were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing and cross-hybridization of amplification products indicated that at least 12 classes of beta-1,3-glucanase genes exist in the soybean. Members of classes mapped to 34 loci on five different linkage groups using an F(2) population of 56 individuals. beta-1,3-Glucanase genes are clustered onto regions of five linkage groups. Data suggest that more closely related genes are clustered together on one linkage group or on duplicated regions of linkage groups. Northern blot analyses performed on total RNA from root, stem, leaf, pod, flower bud, and hypocotyl using DNA probes for the different classes of beta-1,3-glucanase genes revealed that the mRNA levels of all classes were low in young leaves. SGlu2, SGlu4, SGlu7, and SGlu12 mRNA were highly accumulated in young roots and hypocotyls. SGlu7 mRNA also accumulated in pods and flower buds.

Learning and memory difficulties after environmental exposure to waterways containing toxin-producing Pfiesteria or Pfiesteria-like dinoflagellates.

BACKGROUND: At the beginning of autumn, 1996, fish with "punched-out" skin lesions and erratic behaviour associated with exposure to toxins produced by Pfiesteria piscicida or Pfiesteria-like dinoflagellate species were seen in the Pocomoke River and adjacent waterways on the eastern shore of the Chesapeake Bay in Maryland, USA. In August, 1997, fish kills associated with Pfiesteria occurred in these same areas. People who had had contact with affected waterways reported symptoms, including memory difficulties, which raises questions about the human-health impact of environmental exposure to Pfiesteria toxins. METHODS: We assessed 24 people who had been exposed. We collected data on exposure history and symptoms, did a complete medical and laboratory assessment (13 people), and carried out a neuropsychological screening battery. Performance on neuropsychological measures was compared with a matched control group. RESULTS: People with high exposure were significantly more likely than occupationally matched controls to complain of neuropsychological symptoms (including new or increased forgetfulness); headache; and skin lesions or a burning sensation of skin on contact with water. No consistent physical or laboratory abnormalities were found. However, exposed people had significantly reduced scores on the Rey Auditory Verbal Learning and Stroop Color-Word tests (indicative of difficulties with learning and higher cognitive function), and the Grooved Pegboard task. There was a dose-response effect with the lowest scores among people with the highest exposure. By 3-6 months after cessation of exposure, all those assessed had test scores that had returned to within normal ranges. INTERPRETATION: People with environmental exposure to waterways in which Pfiesteria toxins are present are at risk of developing a reversible clinical syndrome characterised by difficulties with learning and higher cognitive functions. Risk of illness is directly related to degree of exposure, with the most prominent symptoms and signs occurring among people with chronic daily exposure to affected waterways.

Diagnosis of Pfiesteria-human illness syndrome.

The first case reports of human illness caused by exposure to Pfiesteria piscicida toxin(s) acquired outside of a laboratory are reported. Though Pfiesteria, a toxin-forming dinoflagellate, is responsible for killing billions of fish in estuaries in North Carolina, its role in human illness has remained controversial, in part due to lack of identification of the toxin. A recent fish kill in the rivers of the lower Eastern Shore has permitted careful investigation and identification of a distinct clinical syndrome resulting from exposure to the Pfiesteria toxin--Pfiesteria human illness syndrome (PHIS). Patients have memory losses, cognitive impairments, headaches, skin rashes, abdominal pain, secretory diarrhea, conjunctival irritation, and bronchospasm. Not all patients have all elements of the syndrome.

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.

Phylogenetic utility of histone H3 intron sequences in the perennial relatives of soybean (Glycine: Leguminosae).

Histone H3 loci form a large multigene family in most plant species. In Glycine, some of these loci possess introns, whose sequences can provide characters for assessing phylogenetic relationships among species of the genus. Phylogenetic analyses of two closely related H3-B loci revealed a complex evolutionary pattern, producing trees from which species relationships could not be inferred readily. The single H3-D locus, in contrast, provided data suitable for the construction of gene trees whose topologies were sufficiently similar to other hypotheses of relationships within the subgenus Glycine to give confidence that evolution at this locus is tracking species phylogenies. H3-D topologies identified several of the same groupings found in previous phylogenetic studies using the chloroplast genome. However, histone H3-D and chloroplast genome data sets were in other respects incongruent, as revealed by both topological differences and numerical measures of congruence. The principal difference involved Glycine falcata, whose chloroplast genome belongs to one of the three strongly supported clades in the subgenus, but whose histone H3-D allele was sister to those of the remaining members of the subgenus. The H3-D topology is more in keeping with the morphologically, ecologically, and genetically divergent nature of this species. The H3-D locus appears to be a useful source of phylogenetic characters for interspecific studies in Glycine, providing resolution among taxa whose relationships were unresolved in previous studies.

Resistance gene analogs are conserved and clustered in soybean.

Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.

Genome duplication in soybean (Glycine subgenus soja).

Restriction fragment length polymorphism mapping data from nine populations (Glycine max x G. soja and G. max x G. max) of the Glycine subgenus soja genome led to the identification of many duplicated segments of the genome. Linkage groups contained up to 33 markers that were duplicated on other linkage groups. The size of homoeologous regions ranged from 1.5 to 106.4 cM, with an average size of 45.3 cM. We observed segments in the soybean genome that were present in as many as six copies with an average of 2.55 duplications per segment. The presence of nested duplications suggests that at least one of the original genomes may have undergone an additional round of tetraploidization. Tetraploidization, along with large internal duplications, accounts for the highly duplicated nature of the genome of the subgenus. Quantitative trait loci for seed protein and oil showed correspondence across homoeologous regions, suggesting that the genes or gene families contributing to seed composition have retained similar functions throughout the evolution of the chromosomes.

Organization of the histone H3 genes in soybean, barley and wheat.

Several variants of the replacement histone H3 genes from soybean, barley and wheat have been cloned and sequenced. Analysis of segregating populations in barley and soybean, as well as analysis of clones isolated from a soybean genomic library, suggested that these genes are dispersed throughout the genome. Several genes contains introns located in similar positions, but of different lengths and sequence. Comparison of mRNA levels in different tissues revealed that the intron-containing and intronless genes have different expression patterns. The distribution of the introns in the histone H3 genes across several plant species suggests that some of the introns might have been lost during the evolution of the gene family. Sequence divergence among introns and gene-flanking sequences in cloned gene variants allowed us to use them as specific probes for localizing individual gene copies and analyzing the genomic distribution of these variants across a range of genotypes.