Mapping of the Mod-1 locus on mouse Chromosome 9

A new method for typing the Mod-1 locus on mouse Chromosome (Chr) 9 was developed, based on restriction fragment length polymorphism (RFLP) within a polymerase chain reaction (PCR)-amplified fragment. The new method led us to revise the strain distribution pattern (SDP) of Mod-1 in the BXD (C57BL/6JxDBA/2J) and AKXD (AKR/J x DBA/2J) recombinant inbred (RI) strains. The new SDP eliminates several previously reported examples of double recombination events between Mod-1 and the closest flanking loci in the BXD and AKXD strains. In the BXD strains, the revised SDP of Mod-1 was identical to that of the Mod-1-related D9Rtil locus. Thus, the identity of D9Rtil as a Mod-1-related locus rather than Mod-1 itself is in question. The method was also applied to an interspecific backcross panel between an inbred strain of Mus musculus molossinus (MSM/Ms) and C57BL/6J to map Mod-1 with respect to surrounding microsatellite loci, defining the proximal localization of Mod-1 with respect to D9Mit10 with a genetic distance of 0.6±0.6 cM.     

Genetic mapping and assignment of a long-range repeat cluster to band D of chromosome 1 in Mus musculus and M. spretus.

A cluster (D1Lub1) of a long-range repeat family was mapped to the proximal part of the Giemsa-negative band D in Chromosome 1 of Mus musculus and M. spretus by in situ hybridization with cloned probes of the long-range repeat family. By making use of restriction fragment length polymorphisms in DNAs from interspecific backcross mice, the cluster could be mapped to a position 5.3 +/- 2.1 cM distal to the Inha locus and the same distance proximal to the Bcl-2 locus. D1Lub1 was inseparable in 114 meiotic events from Acrg, Sag, and Akp-3. Taken together, the data may serve as a reference for coordinating the genetic and cytogenetic maps of Mus Chromosome 1. High-copy-number variants of the cluster, which appear cytogenetically as homogeneously staining regions at the same chromosome location, presumably arose by amplification of the long-range repeat family in situ.     

Assignment of the structural gene for argininosuccinate synthetase to proximal mouse chromosome 2

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-1), we have searched for restriction fragment length polymorphisms in the mouse genome using cloned sequences from the mouse arginosuccinate synthetase structural gene. Five restriction fragment length polymorphisms were found among the recombinant inbred progenitor strains AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J. Of these, four polymorphisms were found to distinguish the SWR/J strain from the other six strains, which all had the same fragment. The fifth polymorphism revealed differences among the progenitor strains for recombinant inbred strain sets AKXL, BXD, and SWXL. The strain distribution pattern for this polymorphism indicated close linkage of Ass-1 to Hc (the fifth component of complement) on proximal mouse chromosome 2 with a recombination fraction of 0.016 and a 95% confidence interval of 0.003 to 0.054. These data place Ass-1 in a syntenic group with the genes Hc, Abl, Fpgs, and Ak-1 whose linkage has been conserved between human chromosome 9q and mouse chromosome 2.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Presymptomatic testing for Huntington's disease

Summary Presymptomatic testing for Huntington's disease (HD) is possible through the use of restriction fragment length polymorphisms (RFLPs) at the closely linked D4S10 locus. Recombination between the HD and D4S10 loci will occur in 4%–5% of meioses, and is a well-recognised complication of predictive testing. Recombination between RFLPs within the D4S10 locus is a rare event and can usually be ignored. We report a case where such an intra-locus recombination frustrated attempts to predict the chance of a high-risk individual inheriting the HD gene.     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Identification and mapping of a leaf rust resistance gene in barley line Q21861.

Barley line Q21861 possesses an incompletely dominant gene (RphQ) for resistance to leaf rust caused by Puccinia hordei. To investigate the allelic and linkage relations between RphQ and other known Rph genes, F2 populations from crosses between Q21861 and donors of Rph1 to Rph14 (except for Rph8) were evaluated for leaf rust reaction at the seedling stage. Results indicate that RphQ is either allelic with or closely linked to the Rph2 locus. A doubled haploid population derived from a cross between Q21861 and SM89010 (a leaf rust susceptible line) was used for molecular mapping of the resistance locus. Bulked segregant analysis was used to identify markers linked to RphQ, using random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and sequence tagged sites (STSs). Of 600 decamer primers screened, amplified fragments generated by 9 primers were found to be linked to the RphQ locus; however, only 4 of them were within 10 cM of the target. The RphQ locus was mapped to the centromeric region of chromosome 7, with a linkage distance of 3.5 cM from the RFLP marker CDO749. Rrn2, an RFLP clone from the ribosomal RNA intergenic spacer region, was found to be very closely linked with RphQ, based on bulked segregant analysis. An STS marker, ITS1, derived from Rrn2, was also closely linked (1.6 cM) to RphQ.     

The 5-HT2 serotonin receptor gene Htr-2 is tightly linked to Es-10 on mouse chromosome 14.

Clones coding for the 5-HT2 serotonin receptor were isolated from rat brain cDNA libraries. Using one of the cDNA clones as the probe, mouse genomic DNAs from intersubspecific backcrosses were analyzed by Southern blot hybridization for a restriction fragment length polymorphism. The 5-HT2 serotonin receptor gene, Htr-2, was mapped to mouse Chromosome 14 and is closely linked with the marker Es-10.     

The pericentromeric 21 DNA marker pGSM21 (D21S13) contains an expressed HTF island.

The DNA marker locus D21S13, localized in the 21q11.1-q21 region, has been closely linked to familial Alzheimer's disease. We constructed a physical map of 1.7 Mb around D21S13 using probes pGSM21 and pGSE9. The results indicated that pGSM21 contains recognition sites for at least three rare-cutting restriction enzymes. The clustering of rare-cutting restriction sites is indicative of the presence of an HTF (HpaII tiny fragment) island. Restriction site mapping and methylation analysis proved that pGSM21 contains a methylation-free HTF island. Furthermore, a cDNA correlate has been isolated confirming that pGSM21 is part of an expressed sequence. Today, the gene associated with pGSM21 is the gene closest to the centromere on the 21q arm.     

Marker haplotype association with growth in German cystic fibrosis patients

Summary In 84 families with 101 children with cystic fibrosis (CF) and 103 unaffected siblings, the haplotype of CF chromosomes was determined with six restriction fragment length polymorphism (RFLP) markers that span the CF gene locus. Patient groups with different genotypes in the more distant flanking marker loci MET D, MET H, and D7S8 differed significantly from each other with respect to percentile height and weight, and percentage of weight for height. Patients homozygous 1-1 in met D (TaqI) and met H (TaqI) were thin and tall when homozygous 1-1 in J3.11 (MspI), and small when homozygous 2-2 in J3.11. Heterozygosity in 3.11 and met H and homozygosity 1-1 in met D segregated with the most severe growth retardation. In contrast, growth was normal in patients who were heterozygous in met D and/or had an uncommon KM.19/XV-2c haplotype. Most patients with pancreatic sufficiency and/or borderline sweat test values were carrying rare haplotypes on their CF chromosomes. Adult patients clustered in genotype groups with normal height percentile distributions. This association between haplotype and clinical severity of CF in the German population provides evidence for genetic microheterogeneity of the CF locus, either because of the existence of multiple alleles of the CF gene itself and/or because of the existence of closely linked polymorphic genes that control growth and development and hence modulate the clinical course and prognosis of CF.     

Evidence for close linkage of a mouse light chain marker with the Ly-2,3 locus.

Light chains associated with normal serum immunoglobulin can be resolved into a finite number of discrete focusing bands by isoelectric focusing. Four distinct light chain patterns can be distinguished among the inbred mouse strains. In the present studies inheritance of the characteristic light chain patterns has been studied in the AKXL recombinant inbred lines (derived from C57L/J and AKR/J parental lines) and in the inbred Ly-2a,3a congenic line B6.PL-Ly-2aLy-3a/Cy as well as in individual backcross animals of an incipient Ly-2a,3a congenic strain. Virtually complete concordance was observed for the expression of light chains characteristic of phenotype B (AKR-J-like) and the expression of the Ly-2a,3a allele. This observation indicates that a locus controlling light chain structure and/or expression is closely linked (less than 2.6 map units) to the Ly-2,3 locus on mouse Chromosome 6. The locus controlling normal light chain IF-patterns has been designated Ef1.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

Genetic polymorphism of a bovine t-complex gene (TCP1) linkage to major histocompatibility genes

Southern blot analysis of genomic cattle DNA was carried out using murine cDNA probes representing the Tcp-1 gene of the t complex. Excellent cross-hybridization was obtained, and the probes apparently hybridized to at least two bovine TCP1 genes. Two independent restriction fragment length polymorphisms, each composed of two allelic variants, were detected; the inheritance of the restriction fragment length polymorphisms was confirmed by family data. One of the restriction fragment length polymorphisms, designated TCP1B, was evidently due to a gene duplication and was revealed with any restriction enzyme used. The duplication was found in three different cattle breeds investigated. Family segregation data indicated that TCP1B is linked to major histocompatibility complex genes. The result was consistent with close linkage to the major histocompatibility complex class II DO beta gene, whereas a fairly high recombination frequency was indicated between TCP1B/DO beta and other major histocompatibility complex genes. The result assigns TCP1B to a bovine linkage group previously comprising major histocompatibility complex class I and class II genes and blood group locus M. The similarity between this linkage group and parts of mouse chromosome 17 (t-H-2) and human chromosome 6 (TCP1-HLA) is discussed.     

Mapping of the mouse 86-kDa heat-shock protein expressed gene (Hsp86-1) on chromosome 12 and related genes on chromosomes 3, 4, 9, and 11

The HSP86 gene family in BALB/c, AKR/J, C58/J, and NFS/N inbred mice comprises an intron-containing expressed gene and, depending on the strain, two to four other HSP86-related members that are apparently processed pseudogenes. The expressed gene locus, Hsp86-1, was identified by its sequence identity with the mouse HSP86 cDNA coding region together with the presence of an intron at the same position as in the homologous human gene. Hsp86-1 was mapped 11.6 cM from the immunoglobulin heavy chain gene IgH on Chromosome 12 using an intersubspecies backcross. Two of the other loci that were common to all inbred strains tested, designated Hsp86-ps1 and Hsp86-ps2, were mapped to positions on Chromosomes 11 and 3, respectively. An HSP86-related locus specific to NFS/N and C58/J mice, designated Hsp86-ps3, was mapped on Chromosome 9. Also, an HSP86-related locus that was unique to NFS/N mice, designated Hsp86-ps4, was mapped to Chromosome 4.     

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes

Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.     

Genes for serum amyloid A proteins map to Chromosome 7 in the mouse

Summary Several restriction fragment length variants have been detected among inbred strains using a mouse serum amyloid A cDNA clone. Five variants were shown to segregate as a single genetic unit and were mapped to Chromosome 7 between the glucose phosphate isomerase locus (Gpi-1) and the pink eye dilution locus (p) using recombinant inbred and congenic strains. The finding that no major MspI or BclI restriction fragments were shared between digests of DNAs from a Chromosome 7 congenic strain and its inbred partner, indicate that most, and probably all, sequences detected with the probe are clustered on Chromosome 7. Aneuploid mapping was used to show that the serum amyloid A gene complex (Saa) is proximal to the Chromosome 7 breakpoint in T(7;X)1Ct, a translocation in which the middle third of Chromosome 7 is inserted into the X-chromosome. A survey of inbred strains revealed a single common Saa haplotype and eight rare haplotypes. The complex distribution of 14 different variants suggests that recombination may have played a role in haplotype evolution.This work was supported by grants GM18684 and CA33093 from the National Institute of General Medical Sciences and the National Cancer Institute, respectively.     

Bacteriophage association of streptococcal pyrogenic exotoxin type C.

A gene encoding streptococcal pyrogenic exotoxin type C (SPE C) was isolated from bacteriophage DNA derived from Streptococcus pyogenes CS112. The gene, designated speC2, was shown to reside near the phage attachment site of phage CS112. A restriction endonuclease map of the CS112 phage was generated, and the location and orientation of the speC2 gene were determined. Hybridization analyses of eight SPE C-producing strains revealed restriction fragment length polymorphism of the speC gene-containing DNA fragments and further showed that each speC was linked to a common CS112 phage-derived DNA fragment.     

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.)

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.