The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18s-5.8s-25s rRNA genes, 5s rRNA genes,telomere-like sequences,and a family of centromeric repetitive DNA sequences

A knowledge of genome organization is important for understanding how genomes function and evolve, and provide information likely to be useful in plant breeding programmes involving hybridization and genetic manipulation. Molecular techniques, including in situ hybridization, molecular cloning and DNA sequencing, are proving valuable tools to investigate the structure, organization, and diversity of chromosomes in agricultural crops. Heterologous labelled 18 s-5.8 s-25 s (pTa71) and 5 s rDNAs (pTa794) were used for in situ hybridization on Vigna unguiculata (L.) Walp. chromosomes. Hybridization with 18 s-5.8 s-25 s rRNA gene probes occurred at the same chromosomal sites which were positive to the CMA fluorochrome. Silver staining of nucleolar-organizing regions indicated that all the rDNA sites detected using the 18 s-5.8 s-25 s rRNA gene probe possessed active genes. Degenerate telomeric repeats gave hybridization signals at the telomeres of most chromosomes and no intercalary sites were detected at metaphase; the sequences appear to have no preferential distribution in interphase nuclei. A repetitive DraI family from V. unguiculata was cloned (pVuKB1) and characterized. The DraI repeat is 488 nucleotides long, AT rich (74%), and hybridized on all chromosomes in the centromeric areas. The presence of this sequence family was investigated by Southern hybridization in different Vigna species and other Leguminoseae. It was only detected in V. unguiculata, and hence represents a species-specific DNA sequence.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Quantitative karyotyping of three diploid Brassica species by imaging methods and localization of 45s rDNA loci on the identified chromosomes

 Chromosomes of the three diploid Brassica species, B. rapa (AA), B. nigra (BB) and B. oleracea (CC), were identified based on their morphological characteristics, especially on the condensation pattern appearing at the somatic pro-metaphase stage. The morphological features of the pro-metaphase chromosomes of the three Brassica spp. were quantified by imaging methods using chromosome image analyzing system II (CHIAS 2). As a result, quantitative chromosome maps or idiograms of the three diploid Brassica spp. were developed. The fluorescence in situ hybridization (FISH) method revealed the location of 45s rDNA (the 26s-5.8s-18s ribosomal RNA gene cluster) on the chromosomes involved. The number of 45s rDNA loci in the B. rapa, B. nigra and B. oleracea are five, three and two, respectively. The loci detected were systematically mapped on the idiograms of the three Brassica spp. Received: 5 September 1997 / Accepted: 6 October 1997     

Cytological studies of African cultivated rice,Oryza glaberrima

African cultivated rice, Oryza glaberrima Steud., was cytologically characterized by using both karyotype analysis and molecular cytology. The somatic chromosomes resemble those of Asian cultivated rice, Oryza sauva L., in general morphology, although some minor differences were noted. Multicolor fluorescence in situ hybridization (McFISH) with chromosomes detected one 45s (17s-5.8s-25s) ribosomal RNA gene locus (45s rDNA) and one 5s ribosomal RNA gene locus (5s rDNA) in the chromosome complement. The 45s rDNA and 5s rDNA loci were physically mapped to the distal end of the short arm of chromosome 9 and to the proximal region of the short arm of chromosome 11 respectively, as in O. sativa. Based on the cytological observations and the physical map of the rDNA loci, the chromosomal organization of O.glaberrima and O. sativa seems to be very similar.     

Detection and physical mapping of the 18S-5.8S-26S rDNA and the pKFJ660 probe with microsatellite sequences derived from the rice blast fungus (Magnaporthe grisea) in conifer species

Sequences homologous to the pKFJ660 probe, a fragment of DNA derived from the rice blast fungus (Magnaporthe grisea) carrying TC/AG repeat microsatellite sequences and 30 bp direct repeats were identified in the genome of Picea (spruce) and Pinus (pine) species by fluorescence in situ hybridization (FISH) and slot blot analyses. Slot blot analysis using the pKFJ660 probe revealed hybridization signals with genomic DNAs from various pine and spruce species. Further analyses indicated that the copy number of the (AG)30 motif was higher than 5 x 10(4) per plant genome for all plant samples tested, but the copy number of the sequences homologous to the whole pKFJ660 probe varies considerably among the 25 plant species tested. In situ hybridization of metaphase chromosomes from Pinus resinosa, P. banksiana and P. strobus showed the presence of sequences homologous to this probe on several chromosomes in a dispersed pattern. Major signals were observed on a few chromosomes indicating that some of these sequences are clustered in specific genomic locations. The locations of these repeats were compared to those of 18S-5.8S-26S rDNA in pine species. Chromosomal distribution of 18S-5.8S-26S rDNA varied among the three pine species (P. resinosa, P. banksiana and P. strobus) studied. Ribosomal DNA (rDNA) sites were identified on 14 to 20 chromosomes in these pine species.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the Section Linum (Syn. Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18S–5.8S–26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA were colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum, L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

Classifying seedlots of Picea sitchensis and P. glauca in zones of introgression using restriction analysis of chloroplast DNA

Summary Chloroplast DNA (cpDNA) restriction analysis was used to classify five reforestation seedlots as to species. The material included two Sitka spruce (Picea sitchensis (Bong.) Carr.), one white spruce (P. glauca (Moench) Voss) from interior British Columbia, and two putative hybrid seedlots from the coast-interior introgression zone in British Columbia. The cpDNA patterns generated by Bam-HI and Bc1-I from individual trees of Sitka spruce, white spruce, western white spruce (P. glauca var. albertiana (S. Brown)), and Engelmann spruce (P. engelmanni (Parry)) were species-specific. They were used as reference patterns for comparisons. In addition, two controlled crosses between white and Sitka spruce were analyzed to demonstrate the paternal inheritance of cpDNA in spruces. The cpDNA restriction patterns for the five seedlots were obtained from composite samples of seedlings from each lot and compared to the typical cpDNA patterns of each species. Restriction patterns for the two Sitka spruce seedlots agreed with those from the Sitka spruce tree, while patterns for the white spruce seedlots from British Columbia agreed with those from the white spruce tree, lacking evidence of any Engelmann spruce component in the sample. On the other hand, one putative hybrid seedlot showed cpDNA patterns similar to white spruce while the other showed fragments unique to both Sitka and white spruce, indicating that this was a hybrid seedlot. The analysis of cpDNA restriction polymorphism has proven to be an effective tool for classifying seedlots in regions of introgression. To our knowledge, these results provide the first demonstration of the use of cpDNA analysis for solving practical forestry problems.     

Heterochromatin and rDNA patterns in Solanum species of the Morelloid and Dulcamaroid clades (Solanaceae)

The heterochromatin distribution and the position of 18-5.8-26S, and 5S rDNA loci were determined in 13 species of Solanum of the Morelloid and Dulcamaroid clades. The CMA/DAPI staining and FISH were employed. Two types of constitutive heterochromatin were determined: CMA⁺/DAPI^? associated to NOR and CMA⁺/DAPI^? distributed as terminal bands. In the Morelloid clade, CMA⁺/DAPI^? bands were found in five species while in the Dulcamaroid clade, only S. angustifidum presented this feature. In the Morelloid clade, two to four 18-5.8-26S rDNA loci occupied terminal positions and two rDNA 5S loci were found with variable positions (terminal, intercalary, and centromeric). In the Dulcamaroid clade, two terminal 18-5.8-26S rDNA loci were detected with the exception of S. salicifolium which possessed four such loci and two to four 5S rDNA loci. Solanum crispum is the only species possessing the 5S in synteny with 18-5.8-26S rDNA loci. Karyotype features chromosome banding pattern as well as the location of ribosomal genes which varied among the species, reflecting the chromosome differentiation and evolutionary divergence. The findings obtained contributed to the development of tools that can be used for establishing chromosomic homeologies among species and hence to clarify their taxonomic relationships.     

Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.     

Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database

The online resource http://www.plantrdnadatabase.com/ stores information on the number, chromosomal locations and structure of the 5S and 18S‐5.8S‐26S (35S) ribosomal DNAs (rDNA) in plants. This resource was exploited to study relationships between rDNA locus number, distribution, the occurrence of linked (L‐type) and separated (S‐type) 5S and 35S rDNA units, chromosome number, genome size and ploidy level. The analyses presented summarise current knowledge on rDNA locus numbers and distribution in plants. We analysed 2949 karyotypes, from 1791 species and 86 plant families, and performed ancestral character state reconstructions. The ancestral karyotype (2n = 16) has two terminal 35S sites and two interstitial 5S sites, while the median (2n = 24) presents four terminal 35S sites and three interstitial 5S sites. Whilst 86.57% of karyotypes show S‐type organisation (ancestral condition), the L‐type arrangement has arisen independently several times during plant evolution. A non‐terminal position of 35S rDNA was found in about 25% of single‐locus karyotypes, suggesting that terminal locations are not essential for functionality and expression. Single‐locus karyotypes are very common, even in polyploids. In this regard, polyploidy is followed by subsequent locus loss. This results in a decrease in locus number per monoploid genome, forming part of the diploidisation process returning polyploids to a diploid‐like state over time.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Physical mapping of ribosomal RNA genes in peonies (Paeonia,Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S–5.8S–26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.     

Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines

Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.This revised version was published online in April 2005 with corrections to Fig. 2.     

Chromosomal localization of 18S and 5S rDNA using FISH in the genus <Emphasis Type="Italic">Tor</Emphasis> (Pisces,Cyprinidae)

Dual color fluorescence in situ hybridization (FISH) was performed to study the simultaneous chromosomal localization of 18S and 5S ribosomal genes in the genus Tor for the first time. The 18S and 5S rDNAs in four Tor species were amplified, sequenced and mapped on the metaphase chromosomes. The number and distribution of 18S and 5S rDNA clusters were examined on metaphase chromosome spreads using FISH. The specimens of T. chelynoides, T. putitora and T. progeneius showed six bright fluorescent signals of 18S rDNA and T. tor exhibited ten such signals. The 5S rDNA signals were present only on one pair of chromosomes in all the four Tor species. Ag-NORs were observed on two pairs of chromosomes in T. chelynoides, T. putitora, T. progeneius and four pairs in T. tor. Comparison of the observed 18S rDNA FISH signals and Ag-NORs strongly suggested a possible inactivation of NORs localized at the telomeres of a subtelocentric and telocentric chromosome pairs in all four species. The 5S rDNA contained an identical 120 bp long coding region and 81 bp long highly divergent non-transcribed spacers in all species examined. 18S and 5S rDNA sequencing and chromosomal localization can be a useful genetic marker in species identification as well as phylogenetic and evolutionary studies.     

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

Physical mapping of 18S-5.8S-26S and 5S ribosomal RNA gene families in three important vetches (Vicia species) and their allied taxa constituting three species complexes

The most-important vetch species, Vicia narbonensis (narbon vetch, section Faba), Vicia villosa (hairy vetch, section Cracca) and Vicia sativa (common vetch, section Vicia) and their close relatives (often difficult to circumscribe into distinct taxa) constitute respectively, Narbonensis, Villosa and Sativa species complexes in the genus Vicia. The distribution of the 18S-5.8S-26S (18S-26S) and 5S ribosomal RNA (rRNA) gene families on the chromosomes of 19 (2n=2x=10,12,14) of the 24 species and subspecies belonging to the three species complexes, and Vicia bithynica (2n=12, section Faba) and Vicia hybrida (2n=12, section Hypechusa) was studied by fluorescence in situ hybridization (FISH) with pTa 71 (18S-26S rDNA) and pTa 794 (5S rDNA) DNA clones. Computer – aided chromosome analysis was performed on the basis of chromosome length, the arm-length ratio and the position of the hybridization signals. The positions of the four (2+2) signals of the two rRNA gene families were similar between each of the three, as well as two subspecies of V. narbonensis and Vicia johannis, respectively. Two major 18S-26S rDNA loci were found in the nucleolus organiser regions (NORs) of each of the species except V. hybrida, where it was present in two out of four SAT chromosomes. In addition to major NORs, two minor loci have been physically mapped at the centromeric regions of chromosomes of group 1 in Vicia amphicarpa, Vicia macrocarpa and V. sativa, and two NORs of group 5 in V. hybrida, and on the long arms of group 4 in V. bithynica. Two or four 5S rDNA loci, observed in the short arms of groups 2–4 and 5, and 18S-26S rDNA loci were located in different chromosomes of all the species within the Narbonensis and Villosa species complexes, and Vicia angustifolia of the Sativa species complex. In the remaining six species of the Sativa species complex, and V. bithynica and V. hybrida, the two or four 5S rDNA sites were present in chromosomes which harbor 18S-26S rRNA genes. The tandemly repeated 5S rDNA sites, located at the proximal part of the long arm of groups 3–5, were diagnostic for V. angustifolia, Vicia cordata, Vicia incisa, V. macrocarpa, Vicia nigra and V. sativa of the Sativa species complex. In V. amphicarpa of the same complex, the tandem repeats were located at the distal part of the long arms of group 3. Variability in the number, size and location of two ribosomal DNA probes could generally distinguish species within the Narbonensis and Sativa species complex, V. bithynica and V. hybrida. With respect to the four species of the Villosa species complex the karyotypes could not be identified individually on the basis of the distribution of two ribosomal gene families in three out of seven pairs of chromosomes. Received: 18 October 2000 / Accepted: 20 March 2001     

Inheritance of chloroplast and mitochondrial DNA in Picea and composition of hybrids from introgression zones

Summary The cloning of white spruce (Picea glauca) mitochondrial DNA homologous to the cytochrome oxidase II and ATPase genes of maize is described. These probes were used to define restriction fragment length polymorphisms which distinguish the white, Engelmann (P. engelmannii) and Sitka spruce (P. sitchensis) populations that occur in British Columbia. Analysis of progeny from crosses between the species revealed that mitochondrial DNA was maternally inherited in all cases (32 progeny from five independent crosses). The inheritance of chloroplast DNA was determined using a probe described previously; in this case, all progeny exhibited paternal inheritance (27 progeny from four crosses). Mitochondrial and chloroplast probes were used to test trees from zones of introgression between coastal (Sitka) and interior spruces (white and Engelmann). In most cases mitochondria and chloroplasts within individuals were contributed by different species. The data shows that there is a significant Sitka spruce component in trees east of the coastal watershed in British Columbia.