Construction of a BAC library of Korean ginseng and initial analysis of BAC-end sequences

We estimated the genome size of Korean ginseng ( Panax ginseng C.A. Meyer), a medicinal herb, constructed a Hin dIII BAC library, and analyzed BAC-end sequences to provide an initial characterization of the library. The 1C nuclear DNA content of Korean ginseng was estimated to be 3.33 pg (3.12×10³ Mb). The BAC library consists of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents. Sequencing of 2167 BAC clones generated 2492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2%, 20.9% and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. A comparison of the functional categories represented by the protein-coding regions found in BAC-end sequences with those of Arabidopsis revealed that proteins pertaining to energy metabolism, subcellular localization, cofactor requirement and transport facilitation were more highly represented in the P. ginseng sample. In addition, a sequence encoding a glucosyltransferase-like protein implicated in the ginsenoside biosynthesis pathway was also found. The majority of the transposable element sequences found belonged to the gypsy type (67.6%), followed by copia (11.7%) and LINE (8.0%) retrotransposons, whereas DNA transposons accounted for only 2.1% of the total in our sequence sample. Higher levels of transposable elements than protein-coding regions suggest that mobile elements have played an important role in the evolution of the genome of Korean ginseng, and contributed significantly to its complexity. We also identified 103 microsatellites with 3–38 repeats in their motifs. The BAC library and BAC-end sequences will serve as a useful resource for physical mapping, positional cloning and genome sequencing of P. ginseng.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean (Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.     

Genome-wide BAC-end sequencing of <Emphasis Type="Italic">Cucumis melo</Emphasis> using two BAC libraries

Background

Although melon (Cucumis melo L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library.

Results

We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes.

Conclusion

The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes.

     

Identification of Repetitive Elements in the Genome of Oreochromis niloticus: Tilapia Repeat Masker

The large-scale bacterial artificial chromosome-end sequencing project of Nile tilapia (Oreochromis niloticus) has generated extensive sequence data that allowed the examination of the repeat content in this fish genome and building of a repeat library specific for this species. This library was established based on Tilapiini repeat sequences from GenBank, sequences orthologous to the repeat library of zebrafish in Repbase, and novel repeats detected by genome analysis using MIRA assembler. We estimate that repeats constitute about 14% of the tilapia genome and also give estimates for the occurrence of the different repeats based on the Basic Local Alignment Search Tool searches within the database of known tilapia sequences. The frequent occurrence of novel repeats in the tilapia genome indicates the importance of using the species-specific repeat masker prior to sequence analyses. A web tool based on the RepeatMasker software was designed to assist tilapia genomics.     

Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.     

A major invasion of transposable elements accounts for the large size of the <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">tritici</Emphasis> genome

Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115 kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6 Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174 Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequence.     

Fosmid Library Construction and Initial Analysis of End Sequences in Zhikong Scallop (<Emphasis Type="Italic">Chlamys farreri</Emphasis>)

Zhikong scallop (Chlamys farreri Jones et Preston, 1904) is one of the most commercially important bivalves in China, but research on its genome is underdeveloped. In this study, we constructed the first Zhikong scallop fosmid library, and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between two and eight positive clones, and none of those tested was absent from the library. End-sequencing of 480 individual clones generated 828 sequences after trimming, with an average sequence length of 624 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 213 (25.72%) and 44 (5.31%) significant hits (E < e⁻⁵), respectively. Repetitive sequences analysis resulted in 375 repeats, accounting for 15.84% of total length, which were composed of interspersed repetitive sequences, tandem repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of the Zhikong scallop genome.     

Functional features of a single chromosome arm in wheat (1AL) determined from its structure

Bread wheat (Triticum aestivum L.) is one of the most important crops globally and a high priority for genetic improvement, but its large and complex genome has been seen as intractable to whole genome sequencing. Isolation of individual wheat chromosome arms has facilitated large-scale sequence analyses. However, so far there is no such survey of sequences from the A genome of wheat. Greater understanding of an A chromosome could facilitate wheat improvement and future sequencing of the entire genome. We have constructed BAC library from the long arm of T. aestivum chromosome 1A (1AL) and obtained BAC end sequences from 7,470 clones encompassing the arm. We obtained 13,445 (89.99%) useful sequences with a cumulative length of 7.57 Mb, representing 1.43% of 1AL and about 0.14% of the entire A genome. The GC content of the sequences was 44.7%, and 90% of the chromosome was estimated to comprise repeat sequences, while just over 1% encoded expressed genes. From the sequence data, we identified a large number of sites suitable for development of molecular markers (362 SSR and 6,948 ISBP) which will have utility for mapping this chromosome and for marker assisted breeding. From 44 putative ISBP markers tested 23 (52.3%) were found to be useful. The BAC end sequence data also enabled the identification of genes and syntenic blocks specific to chromosome 1AL, suggesting regions of particular functional interest and targets for future research.     

Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5′ and 3′ ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e ⁻⁵), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.     

Begin at the beginning: A BAC-end view of the passion fruit (Passiflora) genome

Background

The passion fruit (Passiflora edulis) is a tropical crop of economic importance both for juice production and consumption as fresh fruit. The juice is also used in concentrate blends that are consumed worldwide. However, very little is known about the genome of the species. Therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. In this study, we have constructed a large-insert BAC library and provided the first view on the structure and content of the passion fruit genome, using BAC-end sequence (BES) data as a major resource.

Results

The library consisted of 82,944 clones and its levels of organellar DNA were very low. The library represents six haploid genome equivalents, and the average insert size was 108 kb. To check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight Passiflora gene sequences available in public databases. BACs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four BACs in three chromosomes (n = 9). Then, we explored 10,000 BES and we identified reads likely to contain repetitive mobile elements (19.6% of all BES), simple sequence repeats and putative proteins, and to estimate the GC content (~42%) of the reads. Around 9.6% of all BES were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed (940). The vast majority of the top-hits made by our sequences were to Populus trichocarpa (24.8% of the total occurrences), Theobroma cacao (21.6%), Ricinus communis (14.3%), Vitis vinifera (6.5%) and Prunus persica (3.8%).

Conclusions

We generated the first large-insert library for a member of Passifloraceae. This BAC library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. Remarkably, a number of BAC-end pair sequences could be mapped to intervals of the sequenced Arabidopsis thaliana, V. vinifera and P. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-816) contains supplementary material, which is available to authorized users.     

Exploiting BAC-end sequences for the mining,characterization and utility of new short sequences repeat (SSR) markers in Citrus

The aim of this study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs), and to evaluate their transferability, discriminating capacity of genotypes and mapping ability in Citrus. A set of 1,281 simple sequence repeat (SSR) markers were developed from the 46,339 Citrus clementina BAC-end sequences (BES), of them 20.67% contained SSR longer than 20 bp, corresponding to roughly one perfect SSR per 2.04 kb. The most abundant motifs were di-nucleotide (16.82%) repeats. Among all repeat motifs (TA/AT)n is the most abundant (8.38%), followed by (AG/CT)n (4.51%). Most of the BES-SSR are located in the non-coding region, but 1.3% of BES-SSRs were found to be associated with transposable element (TE). A total of 400 novel SSR primer pairs were synthesized and their transferability and polymorphism tested on a set of 16 Citrus and Citrus relative’s species. Among these 333 (83.25%) were successfully amplified and 260 (65.00%) showed cross-species transferability with Poncirus trifoliata and Fortunella sp. These cross-species transferable markers could be useful for cultivar identification, for genomic study of Citrus, Poncirus and Fortunella sp. Utility of the developed SSR marker was demonstrated by identifying a set of 118 markers each for construction of linkage map of Citrus reticulata and Poncirus trifoliata. Genetic diversity and phylogenetic relationship among 40 Citrus and its related species were conducted with the aid of 25 randomly selected SSR primer pairs and results revealed that citrus genomic SSRs are superior to genic SSR for genetic diversity and germplasm characterization of Citrus spp.     

The first insight into the <Emphasis Type="Italic">Taxus</Emphasis> genome via fosmid library construction and end sequencing

Taxus mairei is a critically endangered and commercially important cultured medicinal gymnosperm in China and forms an important medicinal resource, but the research of its genome is absent. In this study, we constructed a T. mairei fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of one million clones with an average insert size of about 39 kb, amounting to 3.9 genome equivalents. Fosmid stability assays indicate that T. mairei DNA was stable during propagation in the fosmid system. End sequencing of both 5′ and 3′ ends of 968 individual clones generated 1,923 sequences after trimming, with an average sequence length of 839 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 560 (29.1%) significant hits (E < e⁻⁵). Repetitive sequences analysis revealed that 20.8% of end sequences are repetitive elements, which were composed of retroelements, DNA transposons, satellites, simple repeats, and low complexity sequences. The distribution pattern of various repeat types was found to be more similar to the gymnosperm Pinus and Picea than to the monocot and dicot. The satellites of T. mairei were significantly longer than those of P. taeda and P. glauca. The tetra-nucleotide repeats of T. mairei were much longer than those of P. glauca and P. taeda. The fosmid library and the fosmid end sequences, for the first time, will serve as a useful resource for large-scale genome sequencing, physical mapping, SSR marker development and positional cloning, and provide a better understanding of the Taxus genome.     

Development and analysis of a germline BAC resource for the sea lamprey, a vertebrate that undergoes substantial chromatin diminution

Over the last several years, the sea lamprey (Petromyzon marinus) has grown substantially as a model for understanding the evolutionary fundaments and capacity of vertebrate developmental and genome biology. Recent work on the lamprey genome has resulted in a preliminary assembly of the lamprey genome and led to the realization that nearly all somatic cell lineages undergo extensive programmed rearrangements. Here we describe the development of a bacterial artificial chromosome (BAC) resource for lamprey germline DNA and use sequence information from this resource to probe the subchromosomal structure of the lamprey genome. The arrayed germline BAC library represents ∼10× coverage of the lamprey genome. Analyses of BAC-end sequences reveal that the lamprey genome possesses a high content of repetitive sequences (relative to human), which show strong clustering at the subchromosomal level. This pattern is not unexpected given that the sea lamprey genome is dispersed across a large number of chromosomes (n ∼ 99) and suggests a low-copy DNA targeting strategy for efficiently generating informative paired-BAC-end linkages from highly repetitive genomes. This library therefore represents a new and biologically informed resource for understanding the structure of the lamprey genome and the biology of programmed genome rearrangement.     

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.     

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene‐enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous–nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome‐wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri‐nucleotide repeats were most common among the SSRs and were overrepresented in the protein‐coding sequences, we found that selection against indels of tri‐nucleotide repeats was relatively weak in both African and Asian rice. Our genome‐wide sequencing of O. glaberrima and in‐depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.     

BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution

Coffee is one of the world’s most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.     

BAC libraries of Triticum urartu, Aegilops speltoides and Ae. tauschii, the diploid ancestors of polyploid wheat

Triticum urartu, Aegilops speltoides and Ae. tauschii are respectively the immediate diploid sources, or their closest relatives, of the A, B and D genomes of polyploid wheats. Here we report the construction and characterization of arrayed large-insert libraries in a bacterial artificial chromosome (BAC) vector, one for each of these diploid species. The libraries are equivalent to 3.7, 5.4 and 4.1 of the T. urartu, Ae. speltoides, Ae. tauschii genomes, respectively. The predicted levels of genome coverage were confirmed by library hybridization with single-copy genes. The libraries were used to estimate the proportion of known repeated nucleotide sequences and gene content in each genome by BAC-end sequencing. Repeated sequence families previously detected in Triticeae accounted for 57, 61 and 57% of the T. urartu, Ae. speltoides and Ae. tauschii genomes, and coding regions accounted for 5.8, 4.5 and 4.8%, respectively.     

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.     

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low‐ and high‐copy sequences

Recent advances have highlighted the ubiquity of whole‐genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid‐like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph‐based clustering of next‐generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low‐copy repeats. The loss of high‐copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus‐like Ty3/Gypsy retroelements and some low‐complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low‐ and high‐copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low‐copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high‐copy‐number sequences.