A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm² filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe. A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomic applications in barley. Received: 20 September 1999 / Accepted: 25 March 2000     

Assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley

Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third‐generation sequencer (PacBio), we de novo assembled a 4.84‐Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59‐Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high‐confidence’ genes, 31 564 were validated by RNA‐sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low‐diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.     

Genotyping Trichomonas vaginalis

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.     

A whole-genome snapshot of 454 sequences exposes the composition of the barley genome and provides evidence for parallel evolution of genome size in wheat and barley

The genomes of barley and wheat, two of the world's most important crops, are very large and complex due to their high content of repetitive DNA. In order to obtain a whole-genome sequence sample, we performed two runs of 454 (GS20) sequencing on genomic DNA of barley cv. Morex, which yielded approximately 1% of a haploid genome equivalent. Almost 60% of the sequences comprised known transposable element (TE) families, and another 9% represented novel repetitive sequences. We also discovered high amounts of low-complexity DNA and non-genic low-copy DNA. We identified almost 2300 protein coding gene sequences and more than 660 putative conserved non-coding sequences. Comparison of the 454 reads with previously published genomic sequences suggested that TE families are distributed unequally along chromosomes. This was confirmed by in situ hybridizations of selected TEs. A comparison of these data for the barley genome with a large sample of publicly available wheat sequences showed that several TE families that are highly abundant in wheat are absent from the barley genome. This finding implies that the TE composition of their genomes differs dramatically, despite their very similar genome size and their close phylogenetic relationship.     

Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers

We have extended our earlier work to show that individual 14–20mer peptide nucleic acid probes directed against interspersed α-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant α-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program ``whole genome' approach based on ∼400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, ∼240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome ``bar coding'. Potential application of PNA probes for investigating repeat structure and function is also discussed. Received: 2 September 1999 / Accepted: 16 December 1999     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Using high-density exon arrays to profile gene expression in closely related species

Global comparisons of gene expression profiles between species provide significant insight into gene regulation, evolutionary processes and disease mechanisms. In this work, we describe a flexible and intuitive approach for global expression profiling of closely related species, using high-density exon arrays designed for a single reference genome. The high-density probe coverage of exon arrays allows us to select identical sets of perfect-match probes to measure expression levels of orthologous genes. This eliminates a serious confounding factor in probe affinity effects of species-specific microarray probes, and enables direct comparisons of estimated expression indexes across species. Using a newly designed Affymetrix exon array, with eight probes per exon for approximately 315 000 exons in the human genome, we conducted expression profiling in corresponding tissues from humans, chimpanzees and rhesus macaques. Quantitative real-time PCR analysis of differentially expressed candidate genes is highly concordant with microarray data, yielding a validation rate of 21/22 for human versus chimpanzee differences, and 11/11 for human versus rhesus differences. This method has the potential to greatly facilitate biomedical and evolutionary studies of gene expression in nonhuman primates and can be easily extended to expression array design and comparative analysis of other animals and plants.     

Variation in the ratio of physical to genetic distance in intervals adjacent to theMla locus on barley chromosome 1H

Variants of the pulsed-field gel electrophoresis technique were used in conjunction with two-dimensional DNA gel electrophoresis (2-DDGE) to determine the ratio of physical to genetic distance in two genetically defined intervals on barley chromosome 1H.2-DDGE analysis demonstrated that two loci that define a 0.3 cM interval, as determined by hybridization with BCD249, reside on a single 450-kbMluI fragment. This result indicates a maximum ratio of physical to genetic distance in this interval of 1500 kb/cM as compared to 3.7–4.2 Mb/cM for the barley genome as a whole. High molecular weight (HMW) DNA restricted withNotI and probed sequentially with MWG068 and BCD249 yield diffuse bands at approximately 2.8 Mb and 3.0 Mb in the C.I. 16151 and C.I. 16155 parental lines, respectively. These results suggest the maximum ratio of physical to genetic distance in the interval defined by these probes is 7.8 Mb/cM. unique HMW DNA restriction fragment length polymorphisms (RFLP) were attributed to the presence of recombination breakpoints. Data from the recombination breakpoint analysis were used to estimate a ratio of physical to genetic distance of 2.5 Mb/cM in theXbcd249.2-Xmwg068 interval and 0.465 Mb/cM in theXbcd249.1-Xbcd249.2 interval. Both physical linkage and recombination breakpoint analysis indicate theXbcd249.1-Xbcd249.2 interval is approximately five-fold smaller, physically, than theXbcd249.2-Xmwg068 interval.     

Physical mapping of the barley stem rust resistance gene rpg4

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

A bacterial artificial chromosome library for sugarcane

Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm² filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library’s potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene. Received: 12 September 1998 / Accepted: 12 March 1999     

Localization of DNA probes for human ribosomal genes on barley chromosomes]

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.     

Towards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle

The barley stem rust resistance genes Rpg1 and rpg4 were mapped in barley on chromosomes 1P and 7M, respectively and the syntenous rice chromosomes identified as 6P and 3P by mapping common probes in barley and rice. Rice yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones were used to isolate probes mapping to the barley Rpg1 region. The rice BAC isolated with the pM13 probe was a particularly excellent source of probes. A high-resolution map of the Rpg1 region was established with 1400 gametes yielding a map density of 3.6 markers per 0.1 cM. A detailed physical map was established for the rice BAC fragment containing the Rpg1-flanking markers pM13 and B24. This fragment covers a barley genetic distance of 0.6 cM and a rice DNA physical distance of ca. 70 kb. The distribution of barley cross-overs in relation to the rice DNA physical distances was extremely uneven. The barley genetic distance between the pM13 marker and Rpg1 was 0.1 cM per ca. 55 kb, while on the proximal side it was 0.5 cm per ca. 15 kb. Three probes from the distal end of the pM13 BAC mapped 3.0 cm proximal of Rpg1 and out of synteny with rice. These experiments confirm the validity of using large insert rice clones as probe sources to saturate small barley (and other large genome cereals) genome regions with markers. They also establish a note of caution that even in regions of high microsynteny, there may be small DNA fragments that have transposed and are no longer in syntenous positions.     

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).