A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb

ABSTRACT: BACKGROUND: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of the ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution. RESULTS: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by the DNA fingerprinting, BAC-end sequencing, and the sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826. CONCLUSIONS: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Molecular organization of the canine major histocompatibility complex

The major histocompatibility complex (MHC) is composed of a tightly linked cluster of genes; in dogs, this is referred to as the dog leukocyte antigen (DLA) region. The canine MHC is located on chromosome 12, and several genes within the DLA region have been identified that have significant sequence similarity to their human counterparts. However, in order to characterize other loci in the DLA region, DNA sequencing has begun using a canine bacterial artificial chromosome (BAC) library. Initially 135 BAC clones were isolated from a BAC library using a mixture of human and canine probes. These BAC clones were screened with locus-specific primers in polymerase chain reactions (PCRs). Fifty-six BAC clones were subjected to FingerPrinted Contig (FPC) analysis and several overlapping clones were identified. One BAC clone RP81-231-G24 has been sequenced. Preliminary sequence analysis of this 150 kb clone indicates that it contains the region where the class I and class III regions are joined and encompasses DLA-12a, DLA-53, DLA-12, DLA-64, TNF-alpha, and a canine gene that appears to resemble the HLA class III gene HSPA1A (HSP70-1).     

A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip

Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co‐localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3‐CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α‐ketoglutarate‐dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p‐coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.     

A high‐throughput BAC end analysis protocol (BAC‐anchor) for profiling genome assembly and physical mapping

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.     

Characterization of the turkey MHC chromosome through genetic and physical mapping

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

Fine physical mapping of the rice stripe resistance gene locus, Stvb-i

The Stvb-i gene confers stripe disease resistance to rice. For positional cloning, we constructed a physical map spanning 1.8-cM distance between flanking markers, consisting of 18 bacterial artificial chromosome (BAC) clones, around the Stvb-i locus on rice chromosome 11. The 18 clones were isolated by screening a BAC library derived from a japonica cultivar, Shimokita, with three Stvb-i-linked RFLP markers and DraI-digested DNAs of a yeast artificial chromosome (YAC) clone. The results of Southern hybridization and restriction enzyme analyses indicated that these BAC clones are contiguous and cover about a 700-kb region containing the Stvb-i allele. Utilizing end and internal fragments of the BAC insert DNAs, 33 molecular markers were generated within a small chromosomal region including the Stvb-i locus. Genotyping analysis with these markers for a resistant cultivar and four nearby recombinants selected from 120 F₂ individuals indicated that Stvb-i is contained within an approximately 286-kb region covered with two overlapping BAC clones. Received: 25 August 1999 / Accepted: 16 November 1999     

Extended sequence of the turkey MHC <Emphasis Type="Italic">B</Emphasis>-locus and sequence variation in the highly polymorphic B-G loci

Genetic variation in the major histocompatibility complex (MHC) is directly correlated to differences in disease resistance. Immunity is greatly dependent on highly polymorphic genes in the MHC, such as class I, class II, and class III complement genes. Preliminary studies of wild turkey populations show extreme polymorphisms in a family of genes exclusive to the avian MHC, the class IV or B-G genes. Significance of this variation is unclear as there are few and conflicting studies of the expression of these genes. Confounding understanding of B-G variation is the lack of a complete delineation of the number of loci in the turkey genome. Direct 454 sequencing of a clone from the CHORI-260 BAC library was used to extend the turkey MHC B-locus sequence, identifying five additional complete B-locus genes including two B-G loci. Sequences of the new B-G genes were compared with those of other turkey gene (BG1–3) and sequences available for other galliformes. Phylogenetic analysis shows species-specific gene evolution supporting a birth–death model of evolution for the B-G gene family. Analysis of variation within the signal peptide sequence (exon 1) found two clusters of polymorphism among the turkey B-G genes. Resequencing of exon 1 in a diverse sample including wild, heritage, and commercial turkeys confirmed multiple alleles at each B-G gene. Future studies aim to correlate B-G variation with group and individual immunological differences.     

Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

 To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes. Received: 27 July 1998 / Accepted: 5 August 1998     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome

The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones.     

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Grass Carp

Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.     

Development of SNP markers linked to the <Emphasis Type="Italic">L</Emphasis> locus in <Emphasis Type="Italic">Capsicum</Emphasis> spp. by a comparative genetic analysis

In pepper, the TMV resistance locus L is syntenic to the tomato I2 and the potato R3 loci on chromosome 11. In this report, we identified pepper bacterial artificial chromosome (BAC) clones corresponding to the I2 and R3 loci and developed L-linked markers using the BAC sequence information. A BAC library was screened using the tomato I2C-1 gene as a probe. The resulting clones were sorted further by PCR screening, sequencing, and genetic mapping. A linkage analysis revealed that BAC clone 082F03 could be anchored to the target region near TG36 on chromosome 11. Using the 082F03 sequence, more BAC clones were identified and a BAC contig spanning 224 kb was constructed. Gene prediction analysis showed that there were at least three I2/R3 R gene analogs (RGAs) in the BAC contig. Three DNA markers closely linked (about 1.2 cM) to the L ⁴ gene were developed by using the BAC contig sequence. The single nucleotide polymorphism marker 087H3T7 developed in this study was subjected to linkage analysis in L ⁴ - and L ³ -segregating populations together with previously developed markers. The 189D23M marker, which is known to co-segregate with L ³ , was located on the opposite side of 087H3T7, about 0.7 cM away from L ⁴ . This supports the idea that L ³ and L ⁴ may be different genes closely linked within the region instead of different alleles at the same locus. Finally, use of flanking markers in molecular breeding program for introgression of L ⁴ to elite germplasm against most aggressive tobamoviruses pathotype P_1,2,3 is discussed.