Chromosome landing at the barley Rar1 locus

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene. Received: 9 June 1998 / Accepted: 15 July 1998     

Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.)

Key message

The Ror1 gene was fine-mapped to the pericentric region of barley chromosome 1HL.

Abstract

Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18 cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.     

High-resolution genetic and physical mapping of the Rar1 locus in barley

 The Mla-12-mediated resistance in barley against Erysiphe graminis f. sp. hordei requires for its function the Rar1 gene. High-resolution genetic mapping was accomplished by inspecting more than 4000 plants segregating for Rar1 within an 0.7-cM interval containing the target gene. Marker enrichment in the target region was carried out by an amplified fragment length polymorphism (AFLP)-based search for polymorphic loci using bulked DNA templates from resistant and susceptible recombinants adjacent to Rar1. RFLP markers closely linked to Rar1 were used to investigate the relationship between physical and genetical distances by PFGE Southern analysis, indicating the physical linkage of two genetically separated RFLP loci. Comparative mapping of Rar1-linked RFLP probes in barley and rice identified a break of collinearity in the orthologous chromosome segments. Received: 11 February 1998 / Accepted: 3 March 1998     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400?kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100?kb, spanned by nine overlapping cosmid clones.     

Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region.

In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley. All, except one, were in synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.     

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.     

Construction of a YAC library from barley cultivar Franka and identification of YAC-derived markers linked to the Rh2 gene conferring resistance to scald (Rhynchosporium secalis).

The Rh2 resistance gene of barley (Hordeum vulgare) confers resistance against the scald pathogen (Rhynchosporium secalis). A high-resolution genetic map of the Rh2 region on chromosome I (7H) was established by the use of molecular markers. Tightly linked markers from this region were used to screen existing and a newly constructed yeast artificial chromosome (YAC) library of barley cv. Franka composed of 45,000 clones representing approximately two genome equivalents. Corresponding YAC clones were identified for most markers, indicating that the combined YAC library has good representation of the barley genome. The contiguous sets of YAC clones with the most tightly linked molecular markers represent entry points for map-based cloning of this resistance gene.     

Construction of a porcine YAC library and mapping of the cardiac muscle ryanodine receptor gene to Chromosome 14q22–q23

Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available. Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes. The library was cloned in the amplifiable vector pCGS966. A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates. An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a onefold coverage of the porcine genome. To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes. One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23. The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel.     

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the<Emphasis Type="Italic"> Rps6</Emphasis> region

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36,864 clones, of which 19,956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.     

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.     

Construction of a potato YAC library and identification of clones linked to the disease resistance loci R1 and Gro1

 A yeast artificial chromosome (YAC) library was constructed from high-molecular-weight DNA of potato (Solanum tuberosum). Potato DNA fragments obtained after complete digestion with four different rare-cutter restriction enzymes were cloned using the pYAC-RC vector. The library consists of 21 408 YAC clones with an average insertion size of 140 kb. The frequency of YAC clones having insertions of chloroplast or mitochondrial DNA was estimated to be 0.5% and 0.3%, respectively. The YAC library was screened by PCR with 11 DNA markers detecting single genes or small gene families in the potato genome. YACs for 8 of the 11 markers were detected in the library. Using 2 markers that are linked to the resistance genes R1 and Gro1 of potato, we isolated two individual YAC clones. One of these YAC clones was found to harbour one member of a small family of candidate genes for the nematode resistance gene Gro1. Received : 5 May 1997 / Accepted : 20 May 1997     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones. Received: 13 March 1997 / Accepted: 11 may 1997     

A novel repetitive sequence associated with the centromeric regions of Arabidopsis thaliana chromosomes

The middle repetitive fraction of the Arabidopsis genome has been relatively poorly characterized. We describe here a novel repetitive sequence cloned in the plasmid mi167, and present in ～90 copies in the genome of Arabidopsis thaliana ecotype Columbia. Hybridization analysis to physically mapped YAC clones representing Arabidopsis chromosome 4 revealed four mi167-hybridizing loci, all clustered near the centromere. No other loci were detected on YAC clones covering chromosome 4. In situ hybridisation experiments to Arabidopsis chromosome spreads showed that mi167-hybridizing sequences are clustered at the centromeric heterochromatin of all five chromosomes; on two chromosomes the hybridization appeared to be localised on one arm. Additional mi167-hybridizing loci were detected, one of which was adjacent to a non-centromeric, heterochromatic region. This work supports the idea that the majority of middle repetitive DNA in the Arabidopsis genome is clustered. It also adds to our understanding of the organization of the centromere of Arabidopsis chromosome 4.     

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions.

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10-30% of the colonies were recombinant, as visualized by red-white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE-1 copia-like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector-insert junctions.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 ₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA

YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library. Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries. The coordinates of YAC clones hybridizing to these sequences are given. A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome. None of the EW YAC clones analysed were chimaeric in this way. YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability. These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments.     

Walking, Cloning, and Mapping with YACs in 3q27: Localization of Five ESTs Including Three Members of the Cystatin Gene Family and Identification of CpG Islands

Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescencein situhybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there.