Nuclear genomes of higher plants vary enormously in size. A substantial proportion of this variation is due to polyploidy, and it is assumed that 50% or more of angiosperms are polyploids. However, although all plants, at the diploid level, require the same number of genes and regulatory DNA sequences for physiological processes like germination, growth, flowering and reproduction, the total amount of the chromosomal DNA still ranges from some 130 Mbp in the model species Arabidopsis thaliana L. up to more than 25,000 Mbp in the timber species Pinus elliottii Engelm. var. elliottii. The differences in complexity of diploid nuclear genomes over several orders of magnitude (Bennett and Leitch, 1995) are caused by repetitive DNA which consists of repeated sequences varying in size from dinucleotides to motifs longer than 10,000 bp and which is present in copy numbers from many hundreds to hundred thousands.

This paper reports results obtained from microsatellite DNA analysis of genetic structure for populations of the native fungus Cronartium quercuum f. sp fusiforme infecting loblolly pine (Pinus taeda L.) over much of this host's natural range. Mostly all fusiform rust galls formed under field conditions are produced as a result of infection and colonization by haploid mycelium originating from a single basidiospore of C. quercuum fusiforme. If multiple infections do occur, then only a single haplotype must ultimately dominate and be responsible for gall formation. High levels of microsatellite variability exist in C. quercuum fusiforme and most of this variation occurs within local populations (average 88.4%). A statistically significant proportion, however, is found among populations, and the magnitude of this differentiation is closely associated with geographic distance between populations. Unweighted pair-group mean analysis and principal components analysis both indicate that at least four genetically distinct regional groups of C. quercuum fusiforme exist in the south Atlantic and Gulf coastal plains. In summary, the distribution of genetic variability in C. quercuum fusiforme is consistent with a hypothesis of at least four metapopulations with gene flow occurring less among regions than among populations within regions, and where overall levels of gene migration are related to geographic distance between populations.

Photochemical quenching, nonphotochemical quenching, and yield of photosystem II were measured on seedlings of full-sibling, open-, and self-pollinated slash pine (Pinus elliottii Engelm. var. elliottii) families. Our results reveal that genetic variation in photochemical quenching and yield of photosystem II exists within this species. The pattern of variation found in these traits is consistent with the variance profile expected to occur as a result of segregation among nuclear genes. Variation among families accounted for 17% of the total variation observed in photochemical quenching, whereas the component for trees within families made up slightly more than 25% of the total. Less variation, both among families as well as among trees within families, was found for yield of photosystem II. A strikingly different pattern was observed for nonphotochemical quenching. Other than the error term, only pretreatment effects contributed significantly to the variation observed. This suggests that nonphotochemical quenching is largely influenced by environmental factors. With regard to associations between fluorescence and growth traits, both height and diameter growth were found to be positively correlated with photochemical quenching (0.36 and 0.33, respectively) when selfed and open-pollinated families were analyzed along with control-pollinated families.

The abundance and genomic organization of six simple sequence repeats, consisting of di-, tri-, and tetranucleotide sequence motifs, and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC) 5 and (GACA) 4 are present in only low copy numbers. The variation in abundance of simple sequence motifs reflects a similar situation to that found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus . The hybridization pattern of Picea species, bald cypress and gingko were different from the patterns detected in the Pinu s species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG) n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three- needle pine genomes, such as slash pine ( Pinus elliottii Engelm. var. elliottii ), compared to P. strobus, Picea species, bald cypress and gingko. P. elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome.Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity, such to (CA) 8 , (GA) 8 , (GGAT) 4 and (GATA) 4 , and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated.

Single-urediniospore cultures of the fusiform rust fungus were used to inoculate seedlings from 10 full-sib families of a five-parent slash pine diallel at two different times in 1994. The presence or absence of fusiform rust galls was recorded for each inoculated seedling at 9 months postinoculation, and percent infection levels for each family-inoculum-time combination were used for detecting differences among host families and fungal cultures and for identifying differential interactions. The existence of differential interactions between two or more fungal cultures and two or more host families verifies that complementary gene action does exist in this pathosystem. Some host families may be excluded from more detailed interaction studies on the basis of their redundancy and lack of participation in differential interactions.

DNA was extracted from dried specimens of nine taxa of the Cantharellaceae. Approximately 325 bases near the 5′ end of the nuclear 28S ribosomal gene were sequenced, and the sequences were compared. Sequence analyses demonstrated that Cantharellus and Craterellus should be treated as distinct genera. The phylogeny generated using parsimony suggests that Cantharellus tubaeformis and Pseudocraterellus sinuosus should be considered species of Craterellus . As a result of this study, we recognize the first placement of these two species in Craterellus by Fries and Quélet. A reassessment of the morphological characters used to separate Cantharellus and Craterellus is indicated.

-Seedlings from 20, full-sib families of a five-parent slash pine diallel were inoculated using two, single urediniospore-derived cultures of the fusiform rust fungus on two different dates during the 1994 growing season. Presence or absence of fusiform rust galls was recorded for each inoculated seedling at nine months postinoculation and percent infection levels for each family:inoculum:date combination were calculated. The complementary genetics model normally requires clonal material of either the host or pathogen in order to assign genotypes to identified complementary gene pairs. Diallel data, however, allows these assignments to be made even when both the host and pathogen populations are segregating. For the July data, three pairs of complementary genes are thought to explain the observed percent infection levels among the host families and fungus cultures. The putative genotypes of the fungus cultures WLP-10 and CCA-2 are A 1 A 1 :A2a2 :a3 a3 and A l a i : A2a2:A3A3, respectively. The respective host genotypes for parents 8-7, 9-2, 18-26, 18-62, and 18-27 are R I R] :r3r3 , R i r 1 :r3 r3 , r i r i :R2r2/r2r2 :r3 r3 , r 1 r 1 :r2r2/R2r2 :r3 r3 , and R 1 r 1 :R3r3 . The homozygous dominant reaction gene of parent 8-7 at locus 1, complemented by the homozygous dominant avirulence gene in culture WLP-10, is thought to be the reason that families with 8-7 as one of the parents appear to be so resistant when challenged with WLP-10. Two additional complementary gene pairs can be hypothesized if the May data is included for families 9-2 x 18-26 and 18-27 x 18-26. The identification of these gene pairs using both the May and July inoculation data suggest the existence of temperature-sensitive genes in this pathosystem. A Chi-square analysis of the July inoculation data using a complementary model with four gene pairs indicate a good fit between expected and observed percentage infection levels. The implications of these findings on rust screening and deployment are discussed.

ABSTRACT A three-generation American chestnut x Chinese chestnut pedigree was used to construct a genetic linkage map for chestnut and to investigate the control of resistance to Endothia parasitica (chestnut blight fungus). DNA genotypes for 241 polymorphic markers (eight isozymes, 17 restriction fragment length polymorphisms [RFLPs], and 216 random amplified polymorphic DNAs [RAPDs]) were assayed on an F(2) family consisting of 102 individuals. Of these markers, 196 were segregating as expected and, subsequently, used for primary linkage mapping. Two isozymes, 12 RFLPs, and 170 RAPDs were mapped to 12 linkage groups spanning a total genetic distance of 530.1 Kosambi centimorgans. F(2) plants were evaluated for a response to E. parasitica infection by directly inoculating them with two unique fungal isolates and measuring canker expansion over a period of 3.5 months. Results were compared with the marker genotype data, thereby identifying genomic regions significantly associated with a resistance response. Single-marker or nonsimultaneous analyses of variance identified seven genomic regions that appear to have an effect on host response. Multiple-marker or simultaneous models suggest that three of these regions have a significant effect on host response, together explaining as much as 42.2% of the total variation for canker size. At each of the three putative resistance loci, alleles derived from the Chinese chestnut grandparent were associated with smaller canker size, or higher levels of resistance.

We have investigated the physical distribution of the reverse transcriptase genes of Ty1-copia-like retrotransposable elements from 12 plant species belonging to different subdivisions by hybridization in situ on chromosome preparations. Ty1-copia-like elements showed different and non-random hybridization patterns. A dispersed distribution throughout most of the chromosomes with reduced hybridization at some regions or with some weak clustering at other regions was found in Allium cepa, Beta vulgaris, Brassica campestris, Brassica oleracea, Pennisetum glaucum, Pinus elliottii, Selaginella apoda, Vicia faba and Vicia narbonensis. Reduced hybridization occured mainly at centromeric regions, nucleolus-organizing regions and regions known to be mainly composed of tandemly repeated sequences. In the fern Pteris cretica the retroelements showed a dispersed genomic organization with clustering at some chromosomal regions and whole chromosomes showing little signal. In Arabidopsis thaliana and Cicer arietinum, Ty1-copia-like elements were found in clusters at the paracentromeric heterochromatin, a novel organization for a repetitive element in A. thaliana. New retroelement families were isolated from A. thaliana and from Beta vulgaris. Alignment of the deduced peptide sequences with Ty1-copia-like elements from other plants showed considerable divergence which was used to calculate their relationships, indicating the value of reverse transcriptase gene analysis in phylogenetic and biodiversity studies.

Seedlings from 8 sources of slash pine (Pinus elliottii var. elliottii and var. densa) and from 2 sources each of Caribbean pine (P. caribaea var. caribaea) and West Indian pine (P. occidentalis) were inoculated using inocula obtained from 2 sources of Cronartium quercuum f. sp. fusiforme, the causal agent of fusiform rust disease. The percentage of infection, evident primarily as galls and sporulation of pycnia of C. q. fusiforme, differed significantly within sources of slash pine and among species. The percentage of infection of all inoculated shoots was highest on slash pine (P. e. densa) seedlings from south Florida (92%) and lowest on P. occidentalis (30%). Among slash pine seedlings, the general trend was more pycnia sporulation on seedlings from sources nearest the origins of inocula, with the only exception being on those of P. e. densa from the most southern source, which showed abundant sporulation of pycnia similar to seedlings P. c. caribaea from a nearby source. No sporulation occurred on seedlings of P. occidentalis. Differences among families within sources of slash pine were significant for percentages of infection and sporulation of pycnia on all shoots inoculated using C. q. fusiforme. Because sporulation of pycnia is a prerequisite for fecundity in C. q. fusiforme, the results suggest strong selection on natural inoculum for infection and fertility among and within sources of slash pine. Breeding strategies currently recognize families of slash pine that minimize damage due to fusiform rust disease, but new strategies might consider limiting pathogen reproduction.

Seedlings of wind-pollinated families from 6 sources of slash pine (Pinus elliottii var. elliottii and var. densa) were tested for development of fusiform rust disease using 2 sources of Cronartium quercuum f. sp. fusiforme inocula. The seed source origins ranged from 24.5°N latitude at the southern tip of Florida (P. e. densa), to 30.25 °N latitude, in north central Florida (P. e. elliottii). All seedlings received basidiospores of both inocula; inoculum of each source was applied to a single, separate shoot. Differences among varieties of slash pine were significant, with less pycnial sporulation present on seedlings from southern seed sources. Differences between inocula were significant within P. e. elliottii only, suggesting increased specificity for P. e. elliottii hosts and these inocula, although inocula x family-within-seed-source interactions were not significant. Heritability estimates for infection or sporulation on an individual seedling basis ranged from 0 to 0.45 within P. e. densa and from 0.20 to 0.59 within P. e. elliottii. On a family mean basis, heritability estimates were higher, ranging up to 0.58 within P. e. densa and 0.71 within P. e. elliottii. Diverse sources of reaction to C. q. fusiforme appear to be present in P. e. densa, suggesting a backcross breeding approach in which genes for reaction are introgressed into fast-growing populations of P. e. elliottii. The relatively large individual tree-based heritability estimates in P. e. elliottii should help to expedite the introgression process.

Seedlings of wind-pollinated families from 6 sources of slash pine (Pinus elliottii var. elliottii and var. densa) were tested for development of fusiform rust disease using 2 sources of Cronartium quercuum f. sp. fusiforme inocula. The seed source origins ranged from 24.5 degrees N latitude at the southern tip of Florida (P. e. densa), to 30.25 degrees N latitude, in north central Florida (P. e. elliottii). All seedlings received basidiospores of both inocula; inoculum of each source was applied to a single, separate shoot. Differences among varieties of slash pine were significant, with less pycnial sporulation present on seedlings from southern seed sources. Differences between inocula were significant within P. e. elliottii only, suggesting increased specificity for P. e. elliottii hosts and these inocula, although inocula x family-within-seed-source interactions were not significant. Heritability estimates for infection or sporulation on an individual seedling basis ranged from 0 to 0.45 within P. e. densa and from 0.20 to 0.59 within P. e. elliottii. On a family mean basis, heritability estimates were higher, ranging up to 0.58 within P. e. densa and 0.71 within P. e. elliottii. Diverse sources of reaction to C. q. fusiforme appear to be present in P. e. densa suggesting a backcross breeding approach in which genes for reaction are introgressed into fast-growing populations of P. e. elliottii. The relatively large individual tree-based heritability estimates in P. e. elliottii should help to expedite the introgression process.

Slash pine is native to the southeastern USA, but is commercially valuable world-wide as a timber-, fiber- and resin-producing species. Breeding objectives emphasize selection for fusiform rust disease resistance. Identification of markers linked to genetic factors conditioning specificity should expand our knowledge of disease development. Towards this end, random amplified polymorphic DNA (RAPD) markers were identified and mapped in a tree hypothesized to be homozygous dominant for resistance at one locus and homozygous recessive at another. Because the DNA prepared for analysis was from haploid maternally-inherited, megagametophyte tissue of seeds, RAPD markers were observed as either present or absent. The analysis revealed 13 linkage groups of three or more loci, ranging in size from 28 to 68 cM, and nine linked pairs. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, range from 2,880 to 3,360 cM, and equal 6.0-6.9 x 10(6) bp/cM (physical size > 20,000 Mbp). Using a 30 cM map scale and including unlinked markers, ends of linkage groups, and linked pairs, the RAPD markers account for approximately 2,160 cM or 64-75% of the genome. Mapping 80 additional RAPD markers placed 131 loci total in 20 linkage groups of three or more loci, nearly doubling the coverage in the groups to a genetic map distance of approximately 1,347 cM. Two other slash pine trees also have been RAPD mapped. DNA-DNA in situ hybridization and cytochemical staining are being used to integrate the genetic recombinational maps. A karyotype and ideogram have been prepared for slash pine (2n = 2x = 24); metaphase chromosome preparations show 11 pairs of long metacentric chromosomes and one shorter pair of submetacentric chromosomes. Patterns of fluorescence in situ hybridization to genes for the large and small rRNA subunits and fluorochrome banding patterns using the GC-base-specific chromomycin A3 (CMA) and AT-base-specific 4',6-diamidino-2-phenylindole (DAPI) allowed all twelve pairs of chromosomes to be identified and a standard karyotype established. A family of sequences associated with (TTTAGGG)n related repeats has been identified in slash pine using a labeled synthetic oligonucleotide probe. Fluorescence in situ hybridization shows a weak signal at telomeres and significantly stronger intensity at non-telomeric sites. The most common non-telomeric location was in the pericentric regions of chromosomes; interstitial sites of hybridization were relatively common. Microsatellite DNAs, an abundant retrotransposon-like element, and total genomic in situ hybridization and species and chromosome specific DNAs are being evaluated for analyses of interspecific hybrids and chromosome evolution between related species. Interest in low and single copy sequences is increasing.

Spermogonia of Cronartium quercuum f.sp. fusiforme developed just beneath the bark on galled regions of infected pine seedlings. Spermogonia consist of flattened, spreading, island-like masses of fungal tissue covered with a thin layer of liquid containing large numbers of spermatia. Spermatia arose in an annellophoric fashion from the tips of long, slender sporogenous cells produced in a distinct layer. Each sporogenous cell contained a large prominent nucleus that underwent mitosis as each spermatium initial developed. One of the resulting nuclei moved into the initial while the other remained in the sporogenous cell. Once a spermatium was delimited, it was pushed away from the tip of the sporogenous cell as another spermatium initial developed below it. Once delimited, a spermatium underwent specific morphological changes as it matured. A mature spermatium was subpyriform in shape and surrounded by a thin wall. In addition to a single large nucleus each spermatium contained ribosomes, mitochondria, lipid bodies, strands of endoplasmic reticulum, vacuole-like inclusions, and many small vesicles that packed its base. Keywords: transmission electron microscopy, pycnidia, pycnidiospores, spermogonia.

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

A karyotype and idiogram were prepared for slash pine (Pines elliottii Engelm, var, elliottii; 2n = 2x = 24) using 26 mitotic metaphase cells from root-tips of seedlings; each metaphase had 11 pairs of long metacentric chromosomes and one shorter pair of submetacentric chromosomes. Fluorescent in situ hybridization showed seven pairs of chromosomes with intercalary sites for genes of 18S-5.8S-25S rRNA (18S-25S rDNA) and one pair with a paracentromeric site. Probes of 5S rDNA localized a major site of hybridization on another pair of chromosomes, whereas two minor sites occurred on other chromosomes, one with and one without a site of 18S-25S rDNA. After in situ hybridization, chromosomes were stained with the GC-base-specific fluorochrome chromomycin A(3) (CMA). Positive bands at intercalary sites were detected only on two pairs of chromosomes, paracentromeric sites only on three pairs of chromosomes, and bands at both regions on four pairs, Many bands of CMA showed at sites of hybridization of 18S-25S rDNA but one major band occurred at a centromere without an rDNA site. After staining with 4',6-diamidino-2-phenylindole (DAPI), bands appeared at AT-rich intercalary and/or centromeric regions of nearly all chromosomes. A characteristic double band of DAPI occurred near the centromere of one pair of chromosomes. Patterns of fluorescence in situ hybridization and fluorochrome banding allowed us to identify all 12 pairs of chromosomes and to establish a standard karyotype. We propose that the numbers assigned to the chromosomes be used for homologous group designations in slash pine and as the framework for homoeologous group designations of chromosomes in other species of pine and perhaps other genera of conifers.

Genetic linkages were identified between the two mating-type factors and 115 RAPD marker loci in a population of 96 homokaryons from a single basidiome of the ectomycorrhizal Laccaria bicolor . A genetic linkage map with 15 linkage groups and three linked pairs is presented. On this basis, the size of the nuclear genome of L. bicolor is estimated to be approximately 3667 recombination units. The RAPD markers provided approximately 57% coverage of the genome and may enable fingerprint identification of laboratory cultures and field isolates for ecological studies. The map will aid studies on the ectomycorrhizal interaction and population variation in L. bicolor .

We examined the population genetic structure of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton using single spore homokaryotic cultures from 33 basidiomes collected in northern Minnesota in association with red pine (Pinus resinosa Ait.), jack pine (Pinus banksiana Lamb.), and trembling aspen (Populus tremuloides Michx.) of three age-classes (0–20 years, 21–40 years, and > 41 years). Mating competence between cultures of isolates, as determined by the presence of clamp connections, revealed the presence of two subpopulations that were not freely interbreeding, one composed of 29 dikaryons, the other of 3 dikaryons. Phenetic cluster analysis using random amplified polymorphic DNA markers did not reveal differentiation between these subpopulations. Clustering failed to reveal genetically distinct groups based on incompatibility group, tree host species, or geographic origin of isolates. Key words: heterogenic incompatibility, RAPD, population genetics.

A set of 420 random, 10-base, oligonucleotide primers was screened for random amplified polymorphic DNA (RAPD) fragments within a sample of eight megagametophyte DNAs of a single slash pine (Pinus elliottii Engelm. var. elliottii) tree. The apparently repeatable RAPD fragments were further characterized within a sample of 68 megagametophytes from the same tree. Fragments segregating in a 1∶1, present-to-absent, ratio were classified and mapped using multi-point linkage analysis. The analysis revealed 13 linkage groups of at least three loci, ranging in size from 28 to 68 cM, and nine linked pairs of loci. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, ranged from 2880 to 3360 cM. Using a 30-cM map scale and including the 24 unlinked markers and the ends of the 13 linkage groups and nine linked pairs, the set of RAPD markers accounts for approximately 2160 cM or 64–75% of the genome. This extent of genomic coverage should allow for the efficient mapping of genes responsible for a reaction to the causal agent of fusiform rust disease, Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme.

Random amplified polymorphic DNA markers were developed for Cronartium quercuum f. sp. fusiforme, causal agent of fusiform rust disease on pines in the southern United States. Genetic linkages were identified between 103 marker loci in a population of 48 gametothalli and drops of spermatia produced from a single urediniospore culture. A preliminary genetic linkage map with eight linkage groups and 12 linked pairs is presented. Based on the extent of linkage detected among loci, the size of the nuclear genome of C. q. f. sp. fusiforme is estimated to be approximately 5100 recombination units. The 103 marker loci characterized are estimated to provide approximately 40% coverage of the genome. The random amplified polymorphic DNA markers may be of immediate utility in fingerprint identification of laboratory cultures and field isolates for epidemiological studies. Even this preliminary genetic linkage map has great potential for aiding in studies on the host-pathogen interaction in fusiform rust disease and monitoring variation in pathogen populations.