Localization of BAC clones on mitotic chromosomes of <Emphasis Type="Italic">Musa acuminata</Emphasis> using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their ‘low-copy’ subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.     

<Emphasis Type="Italic">Agropyron elongatum</Emphasis> chromatin localization on the wheat chromosomes in an introgression line

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F₅ line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1–3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid II-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-03₁₂₉₆ from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Identification of parental genomes and genomic organization in <Emphasis Type="Italic">Aster microcephalus</Emphasis> var. <Emphasis Type="Italic">ovatus</Emphasis>

The karyotype of diploid Aster iinumae is morphologically similar to that of diploid Aster ageratoides var. ageratoides, however, its chromosome size is apparently smaller (S-type chromosomes versus L-type chromosomes, respectively). The hybrid origin of tetraploid Aster microcephalus var. ovatus (LS-type chromosomes) has previously been suggested by cytogenetics and chloroplast DNA (cp DNA) data. The cp DNA phylogeny also implies that the S-type chromosome is apomorphic, which means that genome size reduction occurred on the evolutionary way to A. iinumae. In this study, we have demonstrated that the chromosome size difference does not depend on the intensity of chromosome condensation but on the DNA content. The simultaneous genomic in situ hybridization (GISH) results show the similarity between S-type chromosomes of A. iinumae and A. microcephalus var. ovatus, and between L-type chromosomes of A. ageratoides and A. microcephalus var. ovatus, which provide additional evidence for A. microcephalus var. ovatus being a tetraploid amphidiploid produced by hybridization between S-type chromosomes and L-type chromosomes. The distribution patterns of Ty1-copia-like retrotransposons were similar in L- and S-type chromosomes. The copies of this retrotransposon dispersed uniformly on all chromosomes, and it is not yet apparent how the Ty1-copia-like retrotransposon affects the size difference between them.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

FISH-aimed karyotype analysis in <Emphasis Type="Italic">Aconitum</Emphasis> subgen. <Emphasis Type="Italic">Aconitum</Emphasis> reveals excessive rDNA sites in tetraploid taxa

The location of 5S and 35S rDNA sequences in chromosomes of four Aconitum subsp. Aconitum species was analyzed after fluorescence in situ hybridization (FISH). Both in diploids (2n?=?2x?=?16; Aconitum variegatum, A. degenii) and tetraploids (2n?=?4×?=?32; A. firmum, A. plicatum), rDNA repeats were localized exclusively on the shorter arms of chromosomes, in subterminal or pericentromeric sites. All analyzed species showed similar basal genome size (Cx?=?5.31–5.71 pg). The most striking features of tetraploid karyotypes were the conservation of diploid rDNA loci and emergence of many additional 5S rDNA clusters. Chromosomal distribution of excessive ribosomal sites suggests their role in the secondary diploidization of tetraploid karyotypes.     

Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, <Emphasis Type="Italic">Pomphorhynchus laevis</Emphasis> and <Emphasis Type="Italic">Pomphorhynchus tereticollis</Emphasis> (Acanthocephala)

We studied distribution of ribosomal DNA (rDNA) sequences along with chromosomal location of the nucleolar organizer regions (NORs) in males of two fish parasites, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Fluorescence in situ hybridization with 18S rDNA probe identified two clusters of rDNA in each species, but revealed a remarkable difference in their location on chromosomes. In P. laevis, the rDNA-FISH signals were found in long arms of the first chromosome pair and in short arms of the second pair. Whereas in P. tereticollis, rDNA clusters were located in long arms of both the first and second chromosome pairs. The divergent location of rDNA clusters in the chromosome No. 2 supports current classification of P. tereticollis, previously considered a synonym of P. laevis, as a separate species. A possible scenario of the second chromosome rearrangement during karyotype evolution of the two species involves two successive pericentric inversions. In both species, one or two prominent nucleoli were apparent within interphase nuclei stained with either silver nitrate or a fluorescent dye YOYO-1. However, a single large nucleolus was observed in early stages of mitosis and meiosis I regardless the number of rDNA clusters. Nevertheless, two bivalents with silver-stained NORs in diakinesis and two silver-stained sites in early prophase II nuclei indicated that all NORs are active. This means that each Pomphorhynchus NOR generates a nucleolus, but the resulting nucleoli have a strong tendency to associate in a large body.     

Analysis of remote asymmetric somatic hybrids between common wheat and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Callus-derived protoplasts of common wheat (Triticum aestivum L. cv. Hesheng 3) irradiated with ultraviolet light were fused by using the PEG method with cell suspension-derived protoplasts of Arabidopsis thaliana. Regenerated calli and green plants resembling that of wheat were obtained. The hybrid nature of putative calli and plants were confirmed by isozyme, random amplified polymorphic DNA and genomic in situ hybridization (GISH) analyses. GISH results indicated that 1∼3 small chromosome fragments of A. thaliana were found introgression into the terminals of wheat chromosomes, forming highly asymmetric hybrids. Cytoplasmic genome tests did not show any cytoplasmic genetic materials from A. thaliana. However, variations from the normal wheat cytoplasmic genome were found, indicating recombination or rearrangement occurred during the process of somatic hybridization. The chromosome elimination in the asymmetric somatic hybridization of remote phylogenetic relationship was discussed. A miniature inverted-repeat transposable element related sequence was found by chance in the hybrids which might accompany and impact the process of somatic hybridization. Jingyao Deng and Haifeng Cui provided same contribution to this work.     

New PCR based markers allowed to identify <Emphasis Type="Italic">Secale</Emphasis> chromatin in wheat-<Emphasis Type="Italic">Secale africanum</Emphasis> introgression lines

The genus of Secale has many agronomically important characters. In order to use the best of this species, markers tracking the rye chromatin incorporated into wheat must be developed. In this study, one rye genome-specific random amplified polymorphic DNA (RAPD) marker was isolated from Secale africanum (R^a genome). Two cloned markers, named OPP13₁₁₆₅ and OPP13₆₆₂, were 1165 bp and 662 bp, respectively. Sequence analysis revealed that OPP13₁₁₆₅ was highly homologous to a part of a new class of transposon-like gene called the Revolver family, and OPP13₆₆₂ was partially similar to LTR gypsy-like retrotransposon. Fluorescence in situ hybridization (FISH) showed only OPP13₁₁₆₅ localized within the whole arms of rye except their terminal regions and no signal was detected on wheat chromosomes, while OPP13₆₆₂ had no hybridization signal detected on wheat and rye genomes. Based on these sequences, two pairs of sequence-characterized amplified region (SCAR) primers were designed, and the resulted SCAR markers were able to target both cultivated and wild Secale species. The FISH patterns and the two SCAR markers should be able to identify and track all wheat-rye translocation lines, especially the S. africanum chromatin.     

Telomere and 45S rDNA sequences are structurally linked on the chromosomes in <Emphasis Type="Italic">Chrysanthemum segetum</Emphasis> L.

Some reports have shown that nucleolar organizer regions are located at the telomeric region and have a structural connection with telomeres at the cellular level in many organisms. In this study, we found that all 45S ribosomal DNA (rDNA) signals were located at telomeric regions on the chromosomes in Chrysanthemum segetum L., and the 45S rDNA showed distinct signal patterns on different metaphase chromosome spreads. The bicolor fluorescence in situ hybridization experiment on the extended fibers revealed that telomere repeats were structurally connected with or interspersed into rDNA sequences. The close cytological structure relation between rDNA and telomere sequences led us to use PCR with combinations of the telomere primer and the rDNA primer to obtain some fragments, which were flanked by different rDNA and telomere primer sequences. One representative clone CHS2 contains closely connected rDNA and telomere sequences, suggesting that the telomere sequence invaded into the conserved rDNA sequence. In addition, the sequences of some PCR clones were flanked by the single telomeric primer sequence or the rDNA primer sequence. These results suggested that homologous recombination occurred between tandem repeat units of rDNA sequences or telomere repeats at the chromosome terminus.     

The origin of the C-genome and cytoplasm of <Emphasis Type="Italic">Avena</Emphasis> polyploids

The contribution of C-genome diploid species to the evolution of polyploid oats was studied using C-genome ITS-specific primers. SCAR analysis among Avena accessions confirmed the presence of C-genome ITS1-5.8S-ITS2 sequences in the genome of AACC and AACCDD polyploids. In situ hybridization and screening of more than a thousand rRNA clones in Avena polyploid species containing the C-genome revealed substantial C-genome rRNA sequence elimination. C-genome clones sequenced and Maximum Likelihood Parsimony analysis revealed close proximity to Avena ventricosa ITS1-5.8S-ITS2 sequences, providing strong evidence of the latter's active role in the evolution of tetraploid and hexaploid oats. In addition, cloning and sequencing of the chloroplastic trnL intron among the most representative Avena species verified the maternal origin of A-genome for the AACC interspecific hybrid formation, which was the genetic bridge for the establishment of cultivated hexaploid oats.     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

Localization of repetitive DNA in the chromosomes of <Emphasis Type="Italic">Microtus agrestis</Emphasis> by means of <Emphasis Type="Italic">in situ</Emphasis> hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.     

A comparative cytogenetic study of the tetraploid oat species with the A and C genomes: <Emphasis Type="Italic">Avena insularis,A. magna</Emphasis>, and <Emphasis Type="Italic">A. murphyi</Emphasis>

C-banding of chromosomes and in situ hybridization with the probes pTa71 and pTa794 were used for a comparative cytogenetic study of the three tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi. These species were similar in the structure and C-banding patterns of several chromosomes as well as in the location of the loci 5S rRNA genes and major NOR sites; however, they differed in the number and localization of minor 45S rDNA loci as well as in the morphology and distribution of heterochromatin in some chromosomes. According to the data obtained, A. insularis is closer to A. magna, whereas A. murphyi is somewhat separated from these two species. Presumably, all the three studied species originated from the same tetraploid ancestor, and their divergence is connected with various species-specific chromosome rearrangements. The evolution of A. murphyi is likely to have occurred independently of the other two species.     

Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia,Muridae, <Emphasis Type="Italic">Sylvaemus</Emphasis>)

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.     

Characterization of an Alien Chromosome of <Emphasis Type="Italic">Oryza officinalis</Emphasis> Transferred the Genomic and Cytological Environment of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Distant hybridization between rice species and their genome interactions have become the hot points of genetic research. The new formed hybrid was unstable and many of the alien chromosomes would be excluded out in the meiosis procedure. In this study, we investigated the phenomenon that the monosomic alien addition lines (MAALs) of Oryza officinalis (CC)–Oryza sativa (AA) inherited in the genomic and cytoplasmic environments of cultivated rice. In a series of backcross programs, every time the MAALs were backcrossed with the recurrent parent Hejiang19, we get a portion of the backcross progenies that were similar to the female parent MAALs in morphological characters (with rolled leaves) and chromosome constitutions (2n + 1). The monosomic alien chromosome exhibited stable inheritance in the genomic and cytoplasmic environments of cultivated rice. Molecular biology and fluorescence in situ hybridization (FISH) analyses on mitotic cells showed that the alien chromosomes in MAALs were inclined to be transferred integrated into the backcross progenies with rolled leaves and no big fragments of alien chromosomes introgressing into the chromosomes of cultivated rice. By FISH analysis on the meiotic cells, we proved that the single alien chromosome performing nonstandard meiosis and behaving like a dispensable genetic factor, which would be the cytological reasons for the stable inheritance of alien chromosomes.     

Molecular mapping of stripe rust resistance gene <Emphasis Type="Italic">YrHu</Emphasis> derived from <Emphasis Type="Italic">Psathyrostachys huashanica</Emphasis>

Wheat stripe rust is a destructive disease that affects most wheat-growing areas worldwide. Resistance genes from related species and genera add to the genetic diversity available to wheat breeding programs. The stripe rust-resistant introgression line H9020-17-25-6-4 was developed from a cross of resistant Psathyrostachys huashanica with the susceptible wheat cultivar 7182. H9020-17-25-6-4 is resistant to all existing Chinese stripe rust races, including the three most widely virulent races, CYR32, CYR33, and V26. We attempted to characterize this new line by genomic in situ hybridization (GISH) and genetic analysis. GISH using P. huashanica genomic DNA as a probe indicated that the translocated segment was too small to be detected. Genetic analysis involving F₁, F₂, and F_2:3 materials derived from a cross of Mingxian 169 and H9020-17-25-6-4 indicated that a single dominant gene from H9020-17-25-6-4, temporarily designated YrHu, conferred resistance to CYR29 and CYR33. A genetic map consisting of four simple sequence repeat, two sequence-tagged site (STS), and two sequence-related amplified polymorphism markers was constructed. YrHu was located on the short arm of chromosome 3A and was about 0.7 and 1.5 cM proximal to EST-STS markers BG604577 and BE489244, respectively. Both the gene and the closely linked markers could be used in marker-assisted selection.     

Comparative analysis of diploid species of <Emphasis Type="Italic">Avena</Emphasis> L. using cytogenetic and biochemical markers: <Emphasis Type="Italic">Avena canariensis</Emphasis> Baum et Fedak and <Emphasis Type="Italic">A. longiglumis</Emphasis> Dur.

The diploid oat species containing the A genome of two types (Al and Ac) were studied by electrophoresis of grain storage proteins (avenins), chromosome C-banding, and in situ hybridization with probes pTa71 and pTa794. The karyotypes of the studied species displayed similar C-banding patterns but differed in size and morphology of several chromosomes, presumably, resulting from structural rearrangements that took place during the divergence of A genomes from a common ancestor. In situ hybridization demonstrated an identical location of the 45S and 5S rRNA gene loci in Avena canariensis and A. longiglumis similar to that in the A. strigosa genome. However, the 5S rDNA locus in A. longiglumis (5S rDNA1) was considerably decreased in the chromosome 3Al long arm. The analysis demonstrated that these oat species were similar in the avenin component composition, although individual accessions differed in the electrophoretic mobilities of certain components. A considerable similarity of A. canariensis and A. longiglumis to the Avena diploid species carrying the As genome variant was demonstrated.     

Localization of <Emphasis Type="Italic">Dras1</Emphasis> gene in polytene chromosomes of sibling species of <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

The Dras1 gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and their hybrids. A 1037-bp fragment of Dras1 gene from the D. virilis genome was used as the probe. The gene sequence was localized in the region of a 25 A-B disk in chromosome 2 (in accordance with the D. virilis polytene chromosome map (Gubenko and Evgen’ev, 1984).     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.