Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.     

A 2-Megabase Physical Contig Incorporating 43 DNA Markers on the Human X Chromosome at p11.23–p11.22 from ZNF21 to DXS255

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23–p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internalAlu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter–ZNF21–DXS7465E (MG66)–DXS7927E (MG81)–WASP, DXS1011E, DXS7467E (MG21)–DXS- 7466E (MG44)–GATA1–DXS7469E (Xp664)–TFE3–SYP (DXS1007E)–Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.     

Large-scale analysis of sequence tags in Xp11.4-11.3 and evaluation of candidate genes for X-linked ocular diseases

The gene-rich region of Xp11.4-Xp11.3 was characterized by increasing the physical marker density. Sequence tags (STSs) were generated by IRS- and DOP-PCR techniques, subsequent cloning, sequencing, and creation of primer pairs for single-copy sites. A total of 224 novel STSs were collected, providing an average marker density of 18 kb in the Xp11.4-Xp11.3 region which is assumed to be approximately 4 Mb in size. Sequence analysis of generated and established STSs via data base searches identified a novel gene highly homologous with the protein phosphatase 1 inhibitor 2 (IPP-2) and two pseudogenes; all of which map to the approximately 1.5 Mb proximal region of the critical region for X-linked congenital stationary night blindness type I (CSNB1) between markers DXS993 and DXS228. Using well-defined DNA panels, 69 STSs were fine-mapped to this approximately 1.5 Mb region, providing a marker coverage of one marker per 22 kb. No allelic loss was observed when the total STS content was applied to patient DNAs by PCR-mediated amplification. However, given the association of this region with a number of inherited ocular diseases, the data presented here provide valuable tools for genetic linkage and large-scale association studies.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

人Xp21．1－p21．3上3．5MbYAC重叠群构建及物理图谱分析

用Ａｌｕ－ＰＣＲ指纹图谱法分析了人Ｘｐ２１．１－ｐ２１．３上一系列的酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ,ＹＡＣ）克隆,发现其中的两个ＹＡＣ克隆构成包含ＤＸＳ１６６位点的重叠群,而且这一重叠群与以前构建的包含ＤＭＤ基因全序列的ＹＡＣ重叠群相连接,ＹＡＣ克隆末端探针交叉杂交证实了这一重叠,使这一ＹＡＣ重叠群至少延伸至ＤＸＳ１６６位点,形成一个跨度为３．５Ｍｂ的ＹＡＣ重叠群。基于这些重叠的ＹＡＣ克隆绘制了这一区域的大尺度限制酶切图谱,并在这一图谱上定位了ＤＸＳ１６６位点,从而确定了ＤＸＳ１６６位点与ＤＭＤ基因的物理关系。这一工作为ＤＭＤ基因的５＇远端调控作用研究及该区域未知基因的克隆奠定了基础。     

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.     

Genetic and physical mapping of Avr1a in Phytophthora sojae

The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F(2) populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F(2) populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F(2) population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojae BAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F(2) progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a.     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation

An integrated large-insert clone map of the region Xq11-q12 is presented. A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb. The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers. Overall marker density across this region is approximately 1 STS/60 kb. Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2). Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints. In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation.     

An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula.

The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a approximately 400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM-1. The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM.microm(-1). These estimates are in good agreement with the empirically determined value of approximately 300 kb.microm(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene.     

A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.     

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.     

Refined Linkage Disequilibrium and Physical Mapping of the Gene Locus for X-Linked Dystonia–Parkinsonism (DYT3)

X-linked dystonia-parkinsonism (XDP) is a recessive disorder characterized by generalized dystonia with some patients exhibiting parkinsonism. The disease gene, DYT3, is located between DXS453 (DXS993) and DXS559, and strongest linkage disequilibrium is found distal to DXS7117 and proximal to DXS559. We have isolated and analyzed four novel polymorphic markers between DXS7117 and DXS559 and, by haplotype analysis, have narrowed the candidate interval to <350 kb. A sequence-ready contig of 700 kb has been constructed spanning DXS7117 to DXS559 and is composed of 35 PACs, BACs, and cosmids. Nine genes and novel ESTs have been mapped into this contig, and mutations in the coding regions and intron-exon borders of two genes have been excluded as the cause of XDP. Several of the other genes and ESTs located within the contig code for proteins implicated in normal brain development and function and are candidates for DYT3.     

A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region.

We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels. Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors. As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones. Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors.     

A bacterial artificial chromosome (BAC) library of sugar beet and a physical map of the region encompassing the bolting gene <Emphasis Type="Italic"> B</Emphasis>

In sugar beet (Beta vulgaris L.), early bolting is caused by a single dominant gene, designated B. Twenty AFLP markers selected from a 7.8-cM segment of the B region on chromosome 2 were used to screen a YAC library, and a first-generation physical map including the B gene, made up of 11 YACs, was established. Because the genome coverage of the YAC library was low, a BAC library was constructed in the vector pBeloBAC11. This library consists of 57,600 clones with an average insert size of 116 kb, corresponding to 8.8 genome equivalents. Screening of the BAC library with chloroplast and mitochondrial DNA probes indicated that less than 0.1% of the clones contained organelle-derived DNA. To fill the gaps in the physical map around the B gene, the BAC library was screened with four AFLP markers and 10 YAC-derived probes. In total, 54 different BACs were identified. Overlaps between BACs were detected by using BAC termini amplified by PCR as probes, and by RFLP fingerprinting. In this way, a minimal tiling path of the central 4.6-cM region was constructed, which consists of 14 BACs. The B locus was localized to a 360-kb contig, a size which makes positional cloning of the gene feasible.     

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.)

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

A sequence-ready BAC/PAC contig and partial transcript map of approximately 1.5 Mb in human chromosome 17q25 comprising multiple disease genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.