A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

稻属不同染色体组着丝粒BAC克隆的分离和鉴定

着丝粒在真核生物有丝分裂和减数分裂染色体正常的分离和传递中起着重要的作用。通过构建5个稻属二倍体野生种的基因组BAC文库, 采用菌落杂交和FISH技术, 筛选和鉴定了各染色体组着丝粒克隆, 并且分析了这些克隆在不同基因组间的共杂交情况, 结果表明: (1) C染色体组的野生种O. officinalis 和F染色体组的野生种O. brachyantha具有各自着丝粒特异的卫星DNA序列, 并且O. brachyantha着丝粒还具有特异的逆转座子序列; (2) A、B和E染色体组的野生稻O. glaberrima、O. punctata和O. australiensis着丝粒区域都含有与栽培稻着丝粒重复序列CentO和CRR同源的序列; (3) C染色体组野生稻O. officinalis的2条体细胞染色体着丝粒具有CentO的同源序列, 同时也发现其所有着丝粒区域都包含栽培稻CRR的同源序列。这些结果对克隆稻属不同染色体组的着丝粒序列、研究不同染色体组间着丝粒的进化关系和稻属不同着丝粒DNA序列与功能之间的关系均具有重要意义。     

The compact Brachypodium genome conserves centromeric regions of a common ancestor with wheat and rice

The evolution of five chromosomes of Brachypodium distachyon from a 12-chromosome ancestor of all grasses by dysploidy raises an interesting question about the fate of redundant centromeres. Three independent but complementary approaches were pursued to study centromeric region homologies among the chromosomes of Brachypodium, wheat, and rice. The genes present in pericentromeres of the basic set of seven chromosomes of wheat and the Triticeae, and the 80 rice centromeric genes spanning the CENH3 binding domain of centromeres 3, 4, 5, 7, and 8 were used as “anchor” markers to identify centromere locations in the B. distachyon chromosomes. A total of 53 B. distachyon bacterial artificial chromosome (BAC) clones anchored by wheat pericentromeric expressed sequence tags (ESTs) were used as probes for BAC-fluorescence in situ hybridization (FISH) analysis of B. distachyon mitotic chromosomes. Integrated sequence alignment and BAC-FISH data were used to determine the approximate positions of active and inactive centromeres in the five B. distachyon chromosomes. The following syntenic relationships of the centromeres for Brachypodium (Bd), rice (R), and wheat (W) were evident: Bd1-R6, Bd2-R5-W1, Bd3-R10, Bd4-R11-W4, and Bd5-R4. Six rice centromeres syntenic to five wheat centromeres were inactive in Brachypodium chromosomes. The conservation of centromere gene synteny among several sets of homologous centromeres of three species indicates that active genes can persist in ancient centromeres with more than 40 million years of shared evolutionary history. Annotation of a BAC contig spanning an inactive centromere in chromosome Bd3 which is syntenic to rice Cen8 and W7 pericentromeres, along with BAC FISH data from inactive centromeres revealed that the centromere inactivation was accompanied by the loss of centromeric retrotransposons and turnover of centromere-specific satellites during Bd chromosome evolution.     

A Ty3/gypsy retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes

A 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library by Jiang et al. (1996, Proc. Natl Acad. Sci. USA, 93, 14210-14213). We have amplified a partially homologous 809 bp sequence from barely genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescent in situ hybridization (FISH). Sequence analysis showed this barley homolog of pSau3A9 to have high similarity to the integrase region of the polyprotein gene of Ty3/gypsy group retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described by Aragón-Alcaide et al. (1996, Chromosoma, 105, 261-268) and may represent all or part of the long-terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.     

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.     

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.     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Physical arrangement of retrotransposon-related repeats in centromeric regions of wheat

Cereal centromeres commonly contain many repetitive sequences that are derived from Ty3/gypsy retrotransposon. FISH analysis using a large DNA insert library of wheat identified a 67-kb clone (R11H) that showed strong hybridization signals on the centromeres. The R11H clone contains Ty3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified. Subsequently, we isolated additional 23 large-insert clones which also contained the integrase and CCS1 sequences. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca. 55-kb interval on average. This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.     

A fine physical map of the rice chromosome 5

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1–3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for ~150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Chia-Hsiung Cheng and Mei-Chu Chung have equal contributions.     

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.Communicated by P.B. Moens     

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).     

A repetitive DNA sequence associated with the centromeres of Chironomus pallidivittatus.

A clone containing centromere-associated DNA from Chironomus pallidivittatus was obtained by microdissection-microcloning. It hybridizes to the centromeric end of one chromosome and exclusively to regions in the three remaining, metacentric chromosomes to which centromeres have previously been localized on cytological grounds. In the metacentric positions the hybridization can be assigned to thin bands. The clone contains 155bp tandem repeats and short flanking regions represented in all of the centromeres. Titration experiments show that the four centromeres together contain 200kb of 155bp repeat per genome. In a line of tissue culture cells the amounts are increased by a factor 1.5-2, resulting in proportionately extended arrays of tandem repeats. Each repeat contains two invertrepeats surrounding a region containing only AT base pairs, a feature with some similarity to functionally essential elements in the Saccharomyces cerevisiae centromere.     

Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.).

A diminutive rye chromosome (midget) in wheat was used as a model system to isolate a highly reiterated centromeric sequence from a rye chromosome. Fluorescence in situ hybridization (FISH) shows this sequence localized within all rye centromeres and no signal was detected on wheat chromosomes. DNA sequencing of the repetitive element has revealed the presence of some catalytic domains and signature motifs typical of retrotransposon genes and has been called the Bilby family, representing a diverged family of retrotransposon-like elements. Extensive DNA database searching revealed some sequence similarity to centromeric retrotransposons from wheat, barley, and centromeric repetitive sequences from rice. Very low levels of signal were observed when Bilby was used as a probe against barley, and no signal was detected with rice DNA during Southern hybridization. The abundance of Bilby in rye indicates that this family may have diverged from other distantly related centromeric retrotransposons or incorporated in the centromere but rapidly evolved in rye during speciation. The isolation of a rye retrotransposon also allowed the analysis of centromeric breakpoints in wheat-rye translocation lines. A quantitative analysis shows that the breakpoint in IDS.1RL and 1DL.1RS and recombinant lines containing proximal rye chromatin have a portion of the rye centromere that may contribute to the normal function of the centromeric region.     

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

The centromere composition of multiple repetitive sequences on rice chromosome 5

The large-scale primary structure of the centromeric region of rice chromosome 5 was analyzed, the first example in a cereal species. The yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs aligned on the centromere of rice chromosome 5 (CEN5) covered a distance of more than 670 kb. Strong suppression of genetic recombination, one of the features of a functional centromere, occurred along the contig region. The most remarkable feature of CEN5 is the composition of the multiple repetitive elements. Oryza-specific RCS2 short tandem repeats were clustered along less than 100 kb at one end of the contig. At least 15 copies of the conserved domain of the 1.9 kb RCE1 centromeric repeats, which are similar to the long terminal repeats (LTRs) of gypsy-type retrotransposon RIRE7, were dispersed mainly in 320 kb stretches next to RCS2 tandem clusters. Many copies of the LTR-like sequences of RIRE3 and RIRE8, another gypsy-type retrotransposon, were also found throughout the contig. On the other hand, the gagpol region was less conserved in the contig. These results indicate that the rice centromere is composed of multiple repetitive sequences with the RCS2 tandem cluster probably being situated as the core of a functional centromere of some hundreds of kilobases to megabases in length.     

Organization of the 1.9-kb repeat unit RCE1 in the centromeric region of rice chromosomes

This paper presents the first report on the structure of a 14-kb centromere sequence in a cereal genome that includes 1.9-kb direct repeats. The cereal centromeric sequence (CCS1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which serves as the binding site for the centromere-specific protein CENP-B in human. To isolate centromeric units from rice (Oryza sativa L.), we performed PCR using the CENP-B box-like sequences (CBLS) as primers. A 264-bp clone was amplified by this method, and called RCS1516. It appeared to be a novel member of the CCS1 family, sharing about 60% identity with the CCS1 sequences of other cereals. Then, a 14-kb genomic clone, λRCB11, carrying the RCS1516 sequence was isolated and sequenced. It was found to contain three copies of a 1.9-kb direct repeat, RCE1, separated by 5.1- and 1.7-kb. A 300-bp sequence at the 3′ end of RCE1 is highly conserved in all three copies (>90%) and is almost identical to the RCS1516 sequence including the CBLS motif. The copy number of RCE1 was estimated to range from 10² to 10³ in the haploid genome of rice. Cloned RCE1 units were used for fluorescent in situ hybridization (FISH) analysis, and signals were observed on almost every primary constriction of rice chromosomes. Thus it was concluded that RCE1 is a significant component of the rice centromere. The λRCB11 clone contained at least four A/T-rich regions, which are candidate for matrix attachment regions (MARs), in the sequences between the RCE1 repeats. Other elements that are homologous to the short centromeric repetitive sequences pSau3A9 and pRG5, detected in both sorghum and rice, were also found in the clone. Received: 9 June 1998 / Accepted: 16 September 1998     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research.

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23,808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes.