Rapid divergence of repetitive DNAs in Brassica relatives.

Centromeric, subtelomeric, and telomeric repetitive DNAs were characterized in Brassica species and the related Raphanus sativus and Arabidopsis thaliana. In general, rapid divergence of the repeats was found. The centromeric tandem satellite repeats were differentially distributed in the species studied, suggesting that centromeric repeats have diverged during the evolution of the A/C and B genome lineages. Sequence analysis of centromeric repeats suggested rapid evolution. Pericentromere-associated retrotransposons were identified and showed divergence during the evolution of the lineages as centromeric repeats. A novel subtelomeric tandem repeat from B. nigra was found to be conserved across the diploid Brassica genomes; however, this sequence was not identified in the related species. In contrast to previous studies, interstitial telomere-like repeats were identified in the pericentromeres of Brassica chromosomes, and these repeats may be associated with genomic stability. These results provide insight into genome evolution during polyploidization in Brassica and divergence within the Brassicaceae.

Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli.

Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.

Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences.

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

Integration of cytogenetic and genetic linkage maps unveils the physical architecture of tomato chromosome 2.

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 microm, with 1 microm equivalent to 540 kb. BAC-FISH resolved a pair of loci that were 3.7-3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of approximately 200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Extensive chromosomal polymorphism revealed by ribosomal DNA and satellite DNA loci in 13 Citrus species.

Little is known about the chromosomal variability and polymorphism existing in mitotic chromosomes of Citrus, mainly due to lack of reliable chromosomal markers and small chromosome size. To test the hypothesis of chromosomal polymorphism and provide the foundation of the genome organization in the Citrus cultivars, we have developed molecular cytogenetic markers for 13 Citrus species collected from Jeju island, Korea. In this study, we demonstrated that the chromosomal locations of cytogenetic markers are quite variable and extremely polymorphic, in contrast to the previous studies. The data obtained in this study will be of utmost importance in cytological systematics and karyotyping of the Citrus species.

Molecular cloning of peroxidase cDNAs from dehydration-treated fibrous roots of sweetpotato and their differential expression in response to stress.

Three peroxidase (POD) cDNAs were isolated from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas) plant via the screening of a cDNA library, and their expressions were assessed to characterize functions of each POD in relation to environmental stress. Three PODs were divided into two groups, designated the basic PODs (swpb4, swpb5) and the anionic PODs (swpa7), on the basis of the pI values of mature proteins. Fluorescence microscope analysis indicated that three PODs are secreted into the extracellular space. RTPCR analysis revealed that POD genes have diverse expression patterns in a variety of plant tissues. Swpb4 was abundantly expressed in stem tissues, whereas the expression levels of swpb5 and swpa7 transcripts were high in fibrous and thick pigmented roots. Swpb4 and swpa7 showed abundant expression levels in suspension cultured cells. Three POD genes responded differently in the leaf and fibrous roots in response to a variety of stresses including dehydration, temperature stress, stress-associated chemicals, and pathogenic bacteria.

Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco.

Plant peroxidases (POD) reduce hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. Extracellular POD can also induce H(2)O(2) production and may perform a significant function in responses to environmental stresses via the regulation of H(2)O(2) in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542-552 2003; Jang et al. in Plant Physiol Biochem 42:451-455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H(2)O(2 )was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H(2)O(2) production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H(2)O(2)-regulated stress response signaling pathway.

Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of sweetpotato.

A new dehydration responsive element-binding (DREB) protein gene encoding for an AP2/EREBP-type transcription factor was isolated by screening of the cDNA library for dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Its cDNA (referred to as swDREB1) fragment of 1206bp was sequenced from, which a 257 amino acid residue protein was deduced with a predicted molecular weight of 28.17kDa. A search of the protein BLAST database revealed that this protein can be classified as a typical member of a DREB subfamily. RT-PCR and northern analyses revealed diverse expression patterns of the swDREB1 gene in various tissues of intact sweetpotato plant, and in leaves and fibrous roots exposed to different stresses. The swDREB1 gene was highly expressed in stems and tuberous roots. In fibrous roots, its mRNA accumulation profiles clearly showed strong expression under various abiotic stress conditions such as dehydration, chilling, salt, methyl viologen (MV), and cadmium (Cd) treatment, whereas it did not respond to abscisic acid (ABA) or copper (Cu) treatment. The above results indicate that swDREB1 may be involved in the process of the plant response to diverse abiotic stresses through an ABA-independent pathway.

Differential expression of 10 sweetpotato peroxidases in response to sulfur dioxide, ozone, and ultraviolet radiation.

Secretory class III plant peroxidase (POD, EC 1.11.1.7) is believed to function in diverse physiological processes, including responses to various environmental stresses. To understand the function of each POD in terms of air pollutants and UV radiation, changes in POD activity and expression of 10 POD genes isolated from cell cultures of sweetpotato (Ipomoea batatas) were investigated in the leaves of sweetpotato after treatment with sulfur dioxide (SO(2) 500ppb, 8h/day for 5 days), ozone (O(3) 200ppb, 8h/day for 6 days), and ultraviolet radiation (UV-B 0.6mWm(-2) for 24h, UV-C 0.16mWm(-2) for 24h). All treatments significantly reduced the PSII photosynthetic efficiency (F(v)/F(m)). POD-specific activities (units/mg protein) were increased in leaves treated with SO(2) and O(3) by 5.2- and 7.1-fold, respectively, compared to control leaves. UV-B and UV-C also increased POD activities by 3.0- and 2.4-fold, respectively. As determined by RT-PCR analysis, 10 POD genes showed differential expression patterns upon treatment with air pollutants and UV radiation. Among the POD genes, swpa1, swpa2, and swpa4 were strongly induced following each of the treatments. Interestingly, basic POD genes (swpb1, swpb2, and swpb3) were highly expressed following SO(2) treatment only, whereas neutral swpn1 was highly induced following O(3) treatment only. These results indicated that some specific POD isoenzymes might be specifically involved in the defense mechanism against oxidative stress induced by air pollutants and UV radiation in sweetpotato plants.

The reference genetic linkage map for the multinational Brassica rapa genome sequencing project.

We describe the construction of a reference genetic linkage map for the Brassica A genome, which will form the backbone for anchoring sequence contigs for the Multinational Brassica rapa Genome Sequencing Project. Seventy-eight doubled haploid lines derived from anther culture of the F(1) of a cross between two diverse Chinese cabbage (B. rapa ssp. pekinensis) inbred lines, 'Chiifu-401-42' (C) and 'Kenshin-402-43' (K) were used to construct the map. The map comprises a total of 556 markers, including 278 AFLP, 235 SSR, 25 RAPD and 18 ESTP, STS and CAPS markers. Ten linkage groups were identified and designated as R1-R10 through alignment and orientation using SSR markers in common with existing B. napus reference linkage maps. The total length of the linkage map was 1,182 cM with an average interval of 2.83 cM between adjacent loci. The length of linkage groups ranged from 81 to 161 cM for R04 and R06, respectively. The use of 235 SSR markers allowed us to align the A-genome chromosomes of B. napus with those of B. rapa ssp. pekinensis. The development of this map is vital to the integration of genome sequence and genetic information and will enable the international research community to share resources and data for the improvement of B. rapa and other cultivated Brassica species.

Suppression of RICE TELOMERE BINDING PROTEIN 1 results in severe and gradual developmental defects accompanied by genome instability in rice.

Although several potential telomere binding proteins have been identified in higher plants, their in vivo functions are still unknown at the plant level. Both knockout and antisense mutants of RICE TELOMERE BINDING PROTEIN1 (RTBP1) exhibited markedly longer telomeres relative to those of the wild type, indicating that the amount of functional RTBP1 is inversely correlated with telomere length. rtbp1 plants displayed progressive and severe developmental abnormalities in both germination and postgermination growth of vegetative organs over four generations (G1 to G4). Reproductive organ formation, including panicles, stamens, and spikelets, was also gradually and severely impaired in G1 to G4 mutants. Up to 11.4, 17.2, and 26.7% of anaphases in G2, G3, and G4 mutant pollen mother cells, respectively, exhibited one or more chromosomal fusions, and this progressively increasing aberrant morphology was correlated with an increased frequency of anaphase bridges containing telomeric repeat DNA. Furthermore, 35S:anti-RTBP1 plants expressing lower levels of RTBP1 mRNA exhibited developmental phenotypes intermediate between the wild type and mutants in all aspects examined, including telomere length, vegetative and reproductive growth, and degree of genomic anomaly. These results suggest that RTBP1 plays dual roles in rice (Oryza sativa), as both a negative regulator of telomere length and one of positive and functional components for proper architecture of telomeres.

Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts.

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6-2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.

Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants.

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.

A Survey of the Brassica rapa genome by BAC-end sequence analysis and comparison with Arabidopsis thaliana.

Brassica rapa ssp. pekinensis (Chinese cabbage) is an economically important crop and a model plant for studies on polyploidization and phenotypic evolution. To gain an insight into the structure of the B. rapa genome we analyzed 12,017 BAC-end sequences for the presence of transposable elements (TEs), SSRs, centromeric satellite repeats and genes, and similarity to the closely related genome of Arabidopsis thaliana. TEs were estimated to occupy 14% of the genome, with 12.3% of the genome represented by retrotransposons. It was estimated that the B. rapa genome contains 43,000 genes, 1.6 times greater than the genome of A. thaliana. A number of centromeric satellite sequences, representing variations of a 176-bp consensus sequence, were identified. This sequence has undergone rapid evolution within the B. rapa genome and has diverged among the related species of Brassicaceae. A study of SSRs demonstrated a non-random distribution with a greater abundance within predicted intergenic regions. Our results provide an initial characterization of the genome of B. rapa and provide the basis for detailed analysis through whole-genome sequencing.

A high-resolution karyotype of cucumber (Cucumis sativus L. 'Winter Long') revealed by C-banding, pachytene analysis, and RAPD-aided fluorescence in situ hybridization.

Using molecular cytogenetic DNA markers, C-banding, pachytene analysis, and fluorescence in situ hybridization (FISH), a high-resolution karyotype was established in the cucumber. C-banding showed distinct hetero chromatic bands on the pericentromeric, telomeric, and intercalary regions of the chromosomes. The C-banding patterns were also consistent with the morphology of 4'-6-diamino-2-phenylindole dihydrochloride (DAPI)-stained pachytene chromosomes. Two repetitive DNA fragments, CsRP1 and CsRP2, were obtained by PCR and localized on the mitotic metaphase and meiotic pachytene chromosomes. CsRP1 was detected on the pericentromeric heterochromatic regions of all chromosomes, except chromosome 1. CsRP2 was detected on 5 (chromosomes 1, 2, 3, 4, and 7) of 7 chromosomes. All homologous chromosome pairs could be distinguished by FISH using 2 RAPD markers. This is the first report on molecular karyotyping of mitotic and meiotic spreads of cucumber.

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa.

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.

Functional characterization of NtCEF1, an AP2/EREBP-type transcriptional activator highly expressed in tobacco callus.

Using PCR-select cDNA subtraction, we identified the genes that are predominantly expressed in the shooty callus induced by suppression of the CHRK1 receptor-like kinase gene. One of the identified genes encoded a novel AP2/EREBP-type transcription factor, and it was highly expressed in various types of tobacco callus including the CHRK1 transgenic callus, hence designated as Nicotiana tabacum Callus-Expressing Factor 1 NtCEF1. The NtCEF1-GFP fusion protein was localized in the nucleus. The full length and the C-terminal acidic region of NtCEF1 could function as a transactivator in yeast, when fused to the LexA DNA binding domain. Expression of the NtCEF1 gene was induced by ethylene and by various abiotic stresses. Gel retardation assay revealed that NtCEF1 could bind specifically to the GCC box as well as to the C/DRE motif, albeit less strongly. Interestingly, NtCEF1 overexpression in Arabidopsis resulted in constitutive expression of various ethylene-responsive and defense genes that contain the GCC box in the promoter-but none of the genes containing the upstream C/DRE elements-indicating that NtCEF1 preferentially recognizes the GCC box in vivo. Furthermore, the NtCEF1-overexpressing Arabidopsis plants exhibited enhanced resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Taken together, these results suggest that NtCEF1 is a transcription factor preferentially activating the GCC box-containing defense genes, and that it modulates increased resistance against the biotic stress by activation of the downstream gene expression.

A high-resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolor fluorescence in situ hybridization.

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2 n=20) was constructed based on the 4'-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 microm to 3.30 microm. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 microm to 51.3 microm, with a total of 385.3 microm, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.

Characterization of the plastid-encoded carboxyltransferase subunit (accD) gene of potato.

The plastid accD gene encoding the carboxyltransferase b subunit of acetyl-coenzyme A carboxylase (ACCase) was cloned from potato. Potato accD (saccD) is 2487 bp in length with a 614 bp 5 cent upstream promoter region and an ORF of 1524 bp, corresponding to a polypeptide of 507 amino acids. The N-terminal region lacks recognizable motifs, while the C-terminal regions contains five motifs. Among these is motif II, PLIIVCASGGARMQE, the sole motif present in all available accD sequences of plants and animals, and of E. coli, suggesting that this motif may correspond to the catalytic site. saccD has the typical prokaryotic promoter signatures, TTGACA and TATCAA, which are -35 and -10-like sequences for plastid-encoded RNA polymerase (PEP), at positions -184 and -160, respectively. However, it seems to be transcribed by the nucleus-encoded RNA polymerase because it is expressed in tuber and root, and in the dark (under crippled PEP conditions) and its transcription initiation sites do not correspond to those of PEP. saccD is expressed in all potato tissues, i.e., leaf, stem, root, and tuber, and its transcript is produced at a similar rate in the light and dark, at different developmental stages, and during growth in the presence of different sugars and carbon sources. Taken together, our results suggest that potato accD is a housekeeping gene constitutively expressed in both chloroplast and amyloplast.

Construction of a garlic BAC library and chromosomal assignment of BAC clones using the FISH technique.

For molecular and cytogenetic studies, two partial bacterial artificial chromosome (BAC) libraries of the garlic cultivar Allium sativum L. 'Danyang' were constructed using high molecular weight (HMW) garlic DNA, the pBAC1-SACB1 vector, and the pIndigoBAC536 vector. The average insert size of the BAC library was about 90 kb. The sequence compositions of the BAC clones were characterized by Southern hybridization with garlic genomic DNA and a repetitive sequence clone of garlic. Two BAC clones with weak signals (thus implying mostly unique sequences), GBC2-5e and GBC2-4d, were selected for FISH analysis. FISH analysis localized the GBC2-5e (approximately 100 kb) BAC clone on the long arm of garlic chromosome 7. The other BAC clone, GBC2-4d (approximately 110 kb), gave rise to discrete FISH signals on a mid-size early metaphase chromosome. The FISH screening with BAC clones proved to be a useful resource for molecular cytogenetic studies of garlic, and will be useful for further mapping and sequencing studies of important genes of this plant.