Production of a complete set of wheat-barley group-7 chromosome recombinants with increased grain ß-glucan content.

KEY MESSAGE: Wheat-barley group-7 recombinant chromosomes were selected using molecular cytogenetics and SNP markers; increased grain ß-glucan content was observed in wheat plants with two and four copies of HvCslF6. The soluble dietary fiber (1-3)(1-4) mixed linked ß-D-glucan from cereal grains is a valuable component of a healthy diet, which reduces risks of coronary disease and diabetes. Although wheat is an important cereal crop providing a substantial portion of daily calories and protein intake in the human diet, it has a low level of ß-glucan. Owing to the plasticity of the polyploid wheat genome, agronomically important traits absent in the wheat primary gene pool can be introgressed from distant relatives. Barley (Hordeum vulgare L.) has a high grain ß-glucan content. Earlier, we introgressed this trait into wheat in the form of whole arm compensating Robertsonian translocations (RobT) involving group-7 chromosomes of barley and all three sub-genomes of hexaploid wheat (Triticum aestivum L). In the presented research, we shortened the barley 7HL arms in these RobTs to small pericentromeric segments, using induced wheat-barley homoeologous recombination. The recombinants were selected using SNP markers and molecular cytogenetics. Plants, comprising barley cellulose synthase-like F6 gene (HvCslF6), responsible for ß-glucan synthesis, had a higher grain ß-glucan content than the wheat control. Three wheat-barley group-7 recombinant chromosomes involving the A, B and D sub-genomes laid the basis for a multiple-copy gene introgression to hexaploid wheat. It is hypothesized that further increases in the ß-glucan content in wheat grain can be obtained by increasing the number of HvCslF6 copies through combining several recombinant chromosomes in one line. The wheat lines with four copies of HvCslF6 exceeded the ß-glucan content of the lines with two copies.

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships.

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

The Agropyron cristatum karyotype, chromosome structure and cross-genome homoeology as revealed by fluorescence in situ hybridization with tandem repeats and wheat single-gene probes.

KEY MESSAGE: Fluorescence in situ hybridization with probes for 45 cDNAs and five tandem repeats revealed homoeologous relationships of Agropyron cristatum with wheat. The results will contribute to alien gene introgression in wheat improvement. Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat and a promising source of novel genes for wheat improvement. To date, identification of A. cristatum chromosomes has not been possible, and its molecular karyotype has not been available. Furthermore, homoeologous relationship between the genomes of A. cristatum and wheat has not been determined. To develop chromosome-specific landmarks, A. cristatum genomic DNA was sequenced, and new tandem repeats were discovered. Their distribution on mitotic chromosomes was studied by fluorescence in situ hybridization (FISH), which revealed specific patterns for five repeats in addition to 5S and 45S ribosomal DNA and rye subtelomeric repeats pSc119.2 and pSc200. FISH with one tandem repeat together with 45S rDNA enabled identification of all A. cristatum chromosomes. To analyze the structure and cross-species homoeology of A. cristatum chromosomes with wheat, probes for 45 mapped wheat cDNAs covering all seven chromosome groups were localized by FISH. Thirty-four cDNAs hybridized to homoeologous chromosomes of A. cristatum, nine hybridized to homoeologous and non-homoeologous chromosomes, and two hybridized to unique positions on non-homoeologous chromosomes. FISH using single-gene probes revealed that the wheat-A. cristatum collinearity was distorted, and important structural rearrangements were observed for chromosomes 2P, 4P, 5P, 6P and 7P. Chromosomal inversions were found for pericentric region of 4P and whole chromosome arm 6PL. Furthermore, reciprocal translocations between 2PS and 4PL were detected. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of crested wheatgrass in alien gene introgression into wheat.

Chromosome Rearrangements Caused by Double Monosomy in Wheat-Barley Group-7 Substitution Lines.

Interspecific or introgressive hybridization is one of the driving forces in plant speciation, producing allopolyploids or diploids with rearranged genomes. The process of karyotype reshaping following homoploid interspecific hybridization has not been studied experimentally. Interspecific hybridization is widely used in plant breeding to increase genetic diversity and introgress new traits. Numerous introgression stocks were developed for hexaploid wheat Triticum aestivum L. (2n = 6x = 42, genome AABBDD). Double monosomic lines, containing one alien chromosome from the tertiary gene pool of wheat and one homoeologous wheat chromosome, represent a simplified model for studying chromosome rearrangements caused by interspecific hybridization. The pairing of a chromosome from the tertiary gene pool with a wheat homoeologue is restricted by the activity of the wheat Ph1 gene, thus, rearrangements caused by chromosome breakage followed by the fusion of the broken arms can be expected. We analyzed chromosome aberrations in 4 sets of lines that originated from double monosomics of barley (Hordeum vulgare L.) chromosome 7H and wheat group-7 chromosomes with dicentric or ring chromosomes. The dynamics of wheat-barley dicentric chromosomes during plant development was followed and an increased diversity of rearrangements was observed. Besides the targeted group-7 chromosomes, other wheat chromosomes were involved in rearrangements, as chromosomes broken in the centromeric region fused with other broken chromosomes. In some cells, multi-centric chromosomes were observed. The structure and dosage of the introgressed barley chromatin was changed. The transmission of the rearrangements to the progenies was analyzed. The observed aberrations emphasize the importance of cytogenetic screening in gene introgression projects.

Development of a complete set of wheat-barley group-7 Robertsonian translocation chromosomes conferring an increased content of ß-glucan.

KEY MESSAGE: A complete set of six compensating Robertsonian translocation chromosomes involving barley chromosome 7H and three chromosomes of hexaploid wheat was produced. Grain ß-glucan content increased in lines containing 7HL. Many valuable genes for agronomic performance, disease resistance and increased yield have been transferred from relative species to wheat (Triticum aestivum L.) through whole-arm Robertsonian translocations (RobT). Although of a great value, the sets of available translocations from barley (Hordeum vulgare L.) are limited. Here, we present the production of a complete set of six compensating RobT chromosomes involving barley chromosome 7H and three group-7 chromosomes of wheat. The barley group-7 long-arm RobTs had a higher grain ß-glucan content compared to the wheat control. The ß-glucan levels varied depending on the temperature and were higher under hot conditions. Implicated in this increase, the barley cellulose synthase-like F6 gene (CslF6) responsible for ß-glucan synthesis was physically mapped near the centromere in the long arm of barley chromosome 7H. Likewise, wheat CslF6 homoeologs were mapped near the centromere in the long arms of all group-7 wheat chromosomes. With the set of novel wheat-barley translocations, we demonstrate a valuable increase of ß-glucan, along with a resource of genetic stocks that are likely to carry many other important genes from barley into wheat.

Major structural genomic alterations can be associated with hybrid speciation in Aegilops markgrafii (Triticeae).

During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross-species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next-generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross-genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter- and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.

Homoeologous recombination-based transfer and molecular cytogenetic mapping of a wheat streak mosaic virus and Triticum mosaic virus resistance gene Wsm3 from Thinopyrum intermedium to wheat.

KEY MESSAGE: Here, we report the production of a wheat- Thinopyrum intermedium recombinant stock conferring resistance to wheat streak mosaic virus and Triticum mosaic virus. Wheat streak mosaic caused by the wheat streak mosaic virus (WSMV) is an important disease of bread wheat (Triticum aestivum) worldwide. To date, only three genes conferring resistance to WSMV have been named and two, Wsm1 and Wsm3, were derived from the distantly related wild relative Thinopyrum intermedium. Wsm3 is only available in the form of a compensating wheat-Th. intermedium whole-arm Robertsonian translocation T7BS·7S#3L. Whole-arm alien transfers usually suffer from linkage drag, which prevents their use in cultivar improvement. Here, we report ph1b-induced homoeologous recombination to shorten the Th. intermedium segment and recover a recombinant chromosome consisting of the short arm of wheat chromosome 7B, part of the long arm of 7B, and the distal 43% of the long arm derived from the Th. intermedium chromosome arm 7S#3L. The recombinant chromosome T7BS·7BL-7S#3L confers resistance to WSMV at 18 and 24 °C and also confers resistance to Triticum mosaic virus, but only at 18 °C. Wsm3 is the only gene conferring resistance to WSMV at a high temperature level of 24 °C. We also developed a user-friendly molecular marker that will allow to monitor the transfer of Wsm3 in breeding programs. Wsm3 is presently being transferred to adapted hard red winter wheat cultivars and can be used directly in wheat improvement.

Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus.

Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A tuberculatus In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A tuberculatus.

Chromosome engineering, mapping, and transferring of resistance to Fusarium head blight disease from Elymus tsukushiensis into wheat.

KEY MESSAGE: This manuscript describes the transfer and molecular cytogenetic characterization of a novel source of Fusarium head blight resistance in wheat. Fusarium head blight (FHB) caused by the fungus Fusarium graminearum Schwabe [telomorph = Gibberella zeae (Schwein. Fr.) Petch] is an important disease of bread wheat, Triticum aestivum L. (2n = 6x = 42, AABBDD) worldwide. Wheat has limited resistance to FHB controlled by many loci and new sources of resistance are urgently needed. The perennial grass Elymus tsukushiensis thrives in the warm and humid regions of China and Japan and is immune to FHB. Here, we report the transfer and mapping of a major gene Fhb6 from E. tsukushiensis to wheat. Fhb6 was mapped to the subterminal region in the short arm of chromosome 1E(ts)#1S of E. tsukushiensis. Chromosome engineering was used to replace corresponding homoeologous region of chromosome 1AS of wheat with the Fhb6 associated chromatin derived from 1E(ts)#1S of E. tsukushiensis. Fhb6 appears to be new locus for wheat as previous studies have not detected any FHB resistance QTL in this chromosome region. Plant progenies homozygous for Fhb6 had a disease severity rating of 7 % compared to 35 % for the null progenies. Fhb6 has been tagged with molecular markers for marker-assisted breeding and pyramiding of resistance loci for effective control of FHB.

Resistance to the Ug99 Race Group of Puccinia graminis f. sp. tritici in Wheat-Intra/intergeneric Hybrid Derivatives.

New races of Puccinia graminis f. sp. tritici, the causal agent of stem rust, threaten global wheat production. In particular, races belonging to the Ug99 race group significantly contribute to yield loss in several African nations. Genetic resistance remains the most effective means of controlling this disease. A collection of 546 wheat-intra- and intergeneric hybrids developed by W. J. Sando (United States Department of Agriculture, Beltsville, MD) was screened with eight races of P. graminis f. sp. tritici, including races TTKSK, TTKST, TTTSK, TRTTF, TTTTF, TPMKC, RKQQC, and QTHJC. There were 152 accessions resistant to one or more races and 29 accessions resistant to TTKSK, TTKST, and TTTSK. Of these 29 accessions, 9 were resistant to all races, 14 had infection type patterns that were indistinguishable from cultivars possessing Sr9h and Sr42, 2 were indistinguishable from accessions with SrTmp, and 4 did not display resistant patterns of accessions with any known Sr gene. Three accessions (604981, 605286, and 611932) characterized cytogenetically were disomic substitution lines, each with a single Thinopyrum ponticum chromosome pair. One accession (606057) was a disomic substitution or addition line with two pairs of T. ponticum chromosomes. In total, seven accessions are postulated to contain novel stem rust resistance genes. This research indicates the value of extant collections of wheat-intergeneric hybrids as sources of disease resistance genes.

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae.

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement. To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6 x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1 U-6 U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3 L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.

Labeling meiotic chromosomes in maize with fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) can be used to visualize chromosomal features using repetitive or single gene probes above a minimum target size. When applied to meiosis, each chromosome of the karyotypic complement can be identified, which can facilitate an understanding of the interrelationship of different chromosomes during this process. On the other hand, the pachytene stage of early meiosis is characterized by slightly but not strongly condensed chromosomes that permit more detailed analyses of adjacent features than can be achieved with somatic metaphase chromosomes.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number.

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world's potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99).

The emergence of the highly virulent Ug99 race complex of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Eriks. and Henn.) threatens wheat (Triticum aestivum L.) production worldwide. One of the effective genes against the Ug99 race complex is Sr44, which was derived from Thinopyrum intermedium (Host) Barkworth and D.R. Dewey and mapped to the short arm of 7J (designated 7J#1S) present in the noncompensating T7DS-7J#1L∙7J#1S translocation. Noncompensating wheat-alien translocations are known to cause genomic duplications and deficiencies leading to poor agronomic performance, precluding their direct use in wheat improvement. The present study was initiated to produce compensating wheat-Th. intermedium Robertsonian translocations with Sr44 resistance. One compensating RobT was identified consisting of the wheat 7DL arm translocated to the Th. intermedium 7J#1S arm resulting in T7DL∙7J#1S. The T7DL∙7J#1S stock was designated as TA5657. The 7DL∙7J#1S stock carries Sr44 and has resistance to the Ug99 race complex. This compensating RobT with Sr44 resistance may be useful in wheat improvement. In addition, we identified an unnamed stem rust resistance gene located on the 7J#1L arm that confers resistance not only to Ug99, but also to race TRTTF, which is virulent to Sr44. However, the action of the second gene can be modified by the presence of suppressors in the recipient wheat cultivars.

Suspension column for recovery and separation of substances using ultrasound-assisted retention of bead sorbents.

A novel approach to sorption recovery and separation of different substances is proposed which is based on the use of suspended bead sorbents instead of conventional packed beds of such sorbents. This makes it possible to employ small-sized beads which are trapped in a low-pressure column due to ultrasound-assisted retention, without any frits to hold the sorption material. A flow system including a separation mini-column, named herein a suspension column, has been developed and tested by the studies of solid phase extraction (SPE) of trace metals from bi-distilled water and sea water using a 150-µL column with a silica-based sorbent containing iminodiacetic groups (DIAPAK IDA) and having a grain size of 6 µm. The adsorption properties of DIAPAK IDA suspension (9.5mg) were evaluated through adsorption/desorption experiments, where the effect of solution pH and eluent on the SPE of trace metals were examined by ICP-MS or ICP-AES measurements. When sample solution was adjusted to pH 8.0 and 1 mol L(-1) nitric acid was used as eluent, very good recoveries of more than 90% were obtained for a number of elements in a single-step extraction. To demonstrate the versatility of the approach proposed and to show another advantage of ultrasonic field (acceleration of sorbate/sorbent interaction), a similar system was used for heterogeneous immunoassays of some antigens in ultrasonic field using agarose sorbents modified by corresponding antibodies. It has been shown that immunoglobulins, chlamidia, and brucellos bacteria can be quantitatively adsorbed on 15-µm sorbent (15 particles in 50 µL) and directly determined in a 50-µL mini-chamber using fluorescence detection.

Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat.

Fluorescence in situ hybridization (FISH) is a useful tool for physical mapping of chromosomes and studying evolutionary chromosome rearrangements. Here we report a robust method for single-copy gene FISH for wheat. FISH probes were developed from cDNA of cytosolic acetyl-CoA carboxylase (ACCase) gene (Acc-2) and mapped on chromosomes of bread wheat, Triticum aestivum L. (2n = 6x = 42, AABBDD), and related diploid and tetraploid species. Another nine full-length (FL) cDNA FISH probes were mapped and used to identify chromosomes of wheat species. The Acc-2 probe was detected on the long arms of each of the homoeologous group 3 chromosomes (3A, 3B, and 3D), on 5DL and 4AL of bread wheat, and on homoeologous and nonhomoeologous chromosomes of other species. In the species tested, FISH detected more Acc-2 gene or pseudogene sites than previously found by PCR and Southern hybridization analyses and showed presence/absence polymorphism of Acc-2 sequences. FISH with the Acc-2 probe revealed the 4A-5A translocation, shared by several related diploid and polyploid species and inherited from an ancestral A-genome species, and the T. timopheevii-specific 4A(t)-3A(t) translocation.

Recovery of a telomere-truncated chromosome via a compensating translocation in maize.

Maize-engineered minichromosomes are easily recovered from telomere-truncated B chromosomes but are rarely recovered from A chromosomes. B chromosomes lack known genes, and their truncation products are tolerated and transmitted during meiosis. In contrast, deficiency gametes resulting from truncated A chromosomes prevent their transmission. We report here a de novo compensating translocation that permitted recovery of a large truncation of chromosome 1 in maize. The truncation (trunc-1) and translocation with chromosome 6 (super-6) occurred during telomere-mediated truncation experiments and were characterized using single-gene fluorescent in situ hybridization (FISH) probes. The truncation contained a transgene signal near the end of the broken chromosome and transmitted together with the compensating translocation as a heterozygote to approximately 41%-55% of progeny. Transmission as an addition chromosome occurred in ~15% of progeny. Neither chromosome transmitted through pollen. Transgene expression (Bar) cosegregated with trunc-1 transcriptionally and phenotypically. Meiosis in T1 plants revealed eight bivalents and one tetravalent chain composed of chromosome 1, trunc-1, chromosome 6, and super-6 in diplotene and diakinesis. Our data suggest that de novo compensating translocations allow recovery of truncated A chromosomes by compensating deficiency in female gametes and by affecting chromosome pairing and segregation. The truncated chromosome can be maintained as an extra chromosome or together with the super-6 as a heterozygote.

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution, sensitivity, and banding paint development.

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.