Construction of a sequence-tagged high-density genetic map of papaya for comparative structural and evolutionary genomics in brassicales

A high-density genetic map of papaya (Carica papaya L.) was constructed using microsatellite markers derived from BAC end sequences and whole-genome shot gun sequences. Fifty-four F(2) plants derived from varieties AU9 and SunUp were used for linkage mapping. A total of 707 markers, including 706 microsatellite loci and the morphological marker fruit flesh color, were mapped into nine major and three minor linkage groups. The resulting map spanned 1069.9 cM with an average distance of 1.5 cM between adjacent markers. This sequence-based microsatellite map resolved the very large linkage group 2 (LG 2) of the previous high-density map using amplified fragment length polymorphism markers. The nine major LGs of our map represent papaya's haploid nine chromosomes with LG 1 of the sex chromosome being the largest. This map validates the suppression of recombination at the male-specific region of the Y chromosome (MSY) mapped on LG 1 and at potential centromeric regions of other LGs. Segregation distortion was detected in a large region on LG 1 surrounding the MSY region due to the abortion of the YY genotype and in a region of LG6 due to an unknown cause. This high-density sequence-tagged genetic map is being used to integrate genetic and physical maps and to assign genome sequence scaffolds to papaya chromosomes. It provides a framework for comparative structural and evolutional genomic research in the order Brassicales.     

A long-range physical map of human Chromosome 21q22.1 band from the YAC continuum

The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis). A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs). A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb. Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band. The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs. By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1. This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers. More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed. This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1. Received: 1 September 1995 / Accepted: 21 November 1995     

Fine cytogenetical analysis of the band 10a1-2 and the adjoining regions in the Drosophila melanogaster X chromosome

The region 9E1-2 - 10B1-2 of the Drosophila melanogaster X chromosome was analysed under the light (LM) and the electron (EM) microscope using different fixatives and an EM map of the region was constructed. EM analysis revealed 21 bands in the region 9E1-2 - 10B1-2 instead of 36 bands in Bridges' map. This discrepancy mainly results from the fact that 14 bands indicated as "doublets" by Bridges appear as a single bands. No doublets were found in the whole 9B1-2 - 10C1-2 region after fixation of salivary glands in 3% glutaraldehyde, 3% formaldehyde and 3 : 1 ethanol-acetic acid mixture. 45% acetic acid is the only fixative which results in strongly vacuolated appearance of the bands. - The break points of 30 chromosome rearrangements in the region 9E1-2 - 10B1-2 were located under EM or LM within the limits of the EM map of this region.     

A restriction map of IncFIV plasmid R124

A physical and genetic map of the 125.7-kb IncFIV plasmid R124 was constructed using the restriction enzymes Sal1 and EcoR1. Two discrete regions involved in plasmid replication were identified on the plasmid genome. One region was located on a 4.66-kb segment of an EcoR1 fragment at map coordinates 73.87 to 78.53 kb. Another was located within an 8.05-kb segment of an EcoR1 fragment at map coordinates 113.40 to 121.45. This region was very unstable but, when ligated to the 3.21-kb EcoR1 fragment E13 located at map coordinates 18.83 to 22.06 kb, replication was stable. Thus, at least three regions of R124 widely separated around the genome are associated with plasmid replication and stable maintenance. Each of these three regions expressed incompatibility with R124. The Tc resistance gene of R124 was located on the contiguous EcoR1 fragments E8 and E12 located at map coordinates 100.49 to 113.40.     

The Unc-22(iv) Region of Caenorhabditis Elegans: Genetic Analysis of Lethal Mutations

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.     

Unique species of mRNA from adenovirus type 7 early region 1 in cells transformed by adenovirus type 7 DNA fragment.

Adenovirus type 7 (Ad7) early region 1 mRNA species transcribed in rat cell lines transformed by the HindIII-I . J fragment (the left 7.8% of the viral genome) and in human KB cells infected with Ad7 were mapped on the viral genome, using S1 nuclease gel and diazobenzyloxymethyl paper hybridization techniques. At the early stage of productive infection, two mRNA's (950 and 840 nucleotides long) with the common 5' and 3' ends but different internal splicings were mapped from region 1A (map units 1.4 to 4.3), and one mRNA (2,310 nucleotides long, with the internal splicing between map units 9.9 to 10.1) was mapped from region 1B (map units 4.6 to 11.4). At the late stage, these early spliced mRNA's were also found and at least three additional Ad7 mRNA's were identified: 700-nucleotide-long mRNA in region 1A; and 1,100- and nucleotide-long mRNA's in region 1B. In transformed rat cell lines, two early region 1A mRNA's (950 and 840 nucleotides long) were also transcribed. Surprisingly, in addition, several unique Ad7 mRNA's, not found in productivity infected cells, were identified in all of the transformed cell lines. Their molecular sizes and coding sequences varied in individual cell lines. However, these mRNA's had the 5' end-proximal portion in region 1B and the 3' end-proximal portion in region 1A, these portions being transcribed by extending from region 1B to 1A on viral DNA fragments joined in a tandem array in transformed cells.     

Molecular genetic linkage maps of mouse chromosomes 4 and 6.

We have generated a moderate resolution genetic map of mouse chromosomes 4 and 6 utilizing a (C57BL/6J x Mus spretus) F1 x Mus spretus backcross with RFLPs for 31 probes. The map for chromosome 4 covers 77 cM and details a large region of homology to human chromosome 1p. The map establishes the breakpoints in the mouse 4-human 1p region of homology to a 2-cM interval between Ifa and Jun in mouse and to the interval between JUN and ACADM in human. The map for mouse chromosome 6 spans a 65-cM region and contains a large region of homology to human 7q. These maps also provide chromosomal assignment and order for a number of previously unmapped probes. The maps should allow the rapid regional assignment of new markers to mouse chromosomes 4 and 6. In addition, knowledge of the gene order in mouse may prove useful in determining the gene order of the homologous regions in human.     

Polyoma virus defective DNAs. I. Physical maps of a related set of defective molecules (D76, D91, D92).

Three related polyoma virus species, designated D92 (92% the size of full-length polyoma virus DNA), D91 (91%) and D76 (76%) have been analysed and their structures compared with that of polyoma virus A2 DNA. Three independent methods (restriction endonuclease cleavage, depurination fingerprinting and DNA-DNA hybridization) were used in the analysis.The defective DNAs appear to be: (1) entirely composed of viral sequences (no host DNA sequences were detected): (2) made up in part of long continuous sequences of DNA which appear identical to sequences of A2 DNA (D92 contains continuous sequences from 1 to 72 map units on the physical map of A2 DNA; that is, it contains the entire late region and part of the early region of the viral DNA. D91 and D76 contain those same sequences except for a 1% deletion around 18 map units): (3) made up in part of rearranged viral sequences.Several interesting features were noted about the rearranged sequences present in the defective DNAs. Sequences from the region around 67 map units were found linked to other (non-contiguous) regions of the DNA. Sequences from about 72 map units were linked to sequences from about 1 map unit. Multiple copies of sequences from 67 to 72 map units (from around the origin of DNA replication) were found (4 copies in D91 and D92, and 2 copies in D76).     

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.     

Adenine + thymine content of different genes located on the broad host range plasmid RP4

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.     

A radiation hybrid map of 48 loci including the clouston hidrotic ectodermal dysplasia locus in the pericentromeric region of chromosome 13q

To facilitate the identification of the gene responsible for Clouston hidrotic ectodermal dysplasia (HED), we used a chromosome 13-specific radiation hybrid panel to map 54 loci in the HED candidate region. The marker retention data were analyzed using RHMAP version 3. The 54 markers have an average retention frequency of 31.6% with decreasing retention as a function of distance from the centromere. Two-point analysis identified three linkage groups with a threshold lod score of 4.00; one linkage group consisted of 49 loci including the centromeric marker D13Z1 and the telomeric flanking marker for the HED candidate region D13S143. Assuming a centromeric retention model, multipoint maximum likelihood analysis of these 49 loci except D13Z1 provided a 1000:1 framework map ordering 29 loci with 21 unique map positions and approximately 2000 times more likely than the next order. Loci that could not be ordered with this level of support were positioned within a range of adjacent intervals. This map spans 347 cR9000, has an average resolution of 17.3 cR9000, and includes 3 genes (TUBA2, GJbeta2, and FGF-9), 18 ESTs, 19 polymorphic loci, and 8 single-copy DNA segments. Comparison of our RH map to a YAC contig showed an inconsistency in order involving a reversed interval of 6 loci. Fiber-FISH and FISH on interphase nuclei analyses with PACs isolated from this region supported our order. We also describe the isolation of 8 new chromosome 13q polymorphic (CA)n markers that have an average PIC value of 0.67. These data and mapping reagents will facilitate the isolation of disease genes from this region.     

Molecular and physical mapping of a barley gene on chromosome arm 1 HL that causes sterility in hybrids with wheat.

Addition of the long arm of barley chromosome 1H (1HL) to wheat causes severe meiotic abnormalities and complete sterility of the plants. To map the barley gene responsible for the 1H-induced sterility of wheat, a series of addition lines of translocated 1H chromosomes were developed from the crosses between the wheat 'Shinchunaga' and five reciprocal translocation lines derived from the barley line St.13559. Examination of the seed fertility of the addition lines revealed that the sterility gene is located in the interstitial 25% region of the 1HL arm. The genetic location of the sterility gene was also estimated by physically mapping sequence-tagged site (STS) markers and simple-sequence repeat (SSR) markers with known map locations. The sterility gene is designated Shw (sterility in hybrids with wheat). Comparison of the present physical map of 1HL with two previously published genetic maps revealed a paucity of markers in the proximal 30% region and non-random distribution of SSR markers. Two inconsistencies in marker order were found between the present physical map and the consensus genetic map of group 1 chromosomes of Triticeae. On the basis of the effects on meiosis and chromosomal location, the relationship of the present sterility gene with other fertility-related genes of Triticeae is discussed.     

A High-Resolution PAC and BAC Map of the SCA2 Region

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.     

Bovine papilloma virus transcription: polyadenylated RNA species and assessment of the direction of transcription

The bovine papilloma virus type 1 (BPV-1)-specific RNA species were identified in virus-induced bovine warts, hamster tumors, and transformed hamster and mouse cells. In each case two major species were present (1.1 and 1.3 kilobases [kb]). Also two species of 1.6 and 1.8 kb appearing in variable amounts were found. Only in the keratinized periphery of the warts, where virus replication takes place, was it possible to reveal an additional 2-kb RNA species. In this tissue, however, the 1.6-kb species was not detected. The basal part of a bovine wart contained an additional minor, 2.9-kb, BPV-1-specific RNA sequence. By hybridization with purified defined BPV-1 DNA fragments it was shown that most of the coding sequences of the 2-kb species were transcribed from a region between 0.02 and 0.19 map units. The majority of the coding sequences of the smaller species in transformed cells were located in the region between 0.31 and 0.61 map units. The putative 5' ends mapped between 0.72 and 0.96 map units. Oligodeoxythymidylic acid-primed [(32)P]cDNA was synthesized from various RNA preparations to generate probes for the detection of 3' termini of the polyadenylated BPV-1 RNAs. By hybridization across the BPV-1 genome only one signal between the map positions 0.30 and 0.40 was obtained when RNA from transformed cells and from a tumor was used as a template. In contrast, RNA from the periphery of a wart led to the detection of an additional signal which was confined to the region between 0.96 and 1.00 map units. From the arrangement of both the 3' termini and the coding areas along the viral genome it appears that several RNA species are transcribed from one DNA strand.     

水稻耐亚铁毒QTLs的定位

亚铁毒是潜育性水稻土中限制水稻产量的主要因子。利用龙杂8503／IR64的F2和等价的F3群体,在营养液中培养来定位耐亚铁毒的QTLs。通过构建101SSR标记的遗传连锁图谱来确定耐亚铁毒QTLs的位置和特性。借助叶片棕色斑点指数、株高和最大根长3个性状,利用营养液在水稻苗期来评价F2单株、F3群体和亲本龙杂8503、IR64,共检测到叶片棕色斑点指数、株高和最大根长的QTLs20个,分布在水稻的10条染色体上,表明这些性状受多基因控制。控制叶片棕色斑点指数的QTLs分别定位在第1染色体的RM315-RM212、第2染色体的RM6-RM240和第4染色体的RM252-RM451之间。与前人的研究结果比较发现：1）位于第4染色体RM252-RM451之间的控制叶片棕色斑点指数的QTL与水稻功能图谱上控制叶绿素含量减少的QTL的位置一致。另一个位于第1染色体的RM315-RM212之间的控制叶片棕色斑点指数的QTL与水稻功能图谱上位于C178-R2635之间控制叶绿素含量的QTL连锁。2）位于第2染色体RM6-RM240之间的第3个控制叶片棕色斑点指数的QTL与位于RZ58-CD0686的控制钾吸收的QTL连锁。     

A radiation hybrid map of the RN region in pigs demonstrates conserved gene order compared with the human and mouse genomes

We recently constructed a 7000-rad porcine whole-genome radiation hybrid (RH) panel with the primary objective of integrating linkage maps of microsatellites with evolutionary conserved genes into one ordered map. In order to evaluate the resolution of this RH panel, we have now constructed a radiation hybrid map of the Chromosome (Chr) 15q2.3-q2.6 region containing the RN gene. This gene has large effects on glycogen content in muscle and meat quality. Ten microsatellites covering a region of 55 centiMorgans and eight genes (AE3, FN1, IGFBP5, INHA, IRS1, PAX3, TNP1, and VIL1) were placed on the Sscr15 RH map. All the genes, except IRS1, were mapped on the RH map between microsatellites located in 15q2.5. The relative order of AE3 and INHA was inverted on the porcine physical map in comparison with the mouse linkage map. The order of other genes already mapped in the mouse (FN1, IGFBP5, TNP1, VIL1, INHA/AE3, and PAX3) was identical in pigs. We found no clear difference between the gene order on pig Chr 15 and human Chr 2q. Received: 4 November 1998 / Accepted: 8 February 1999     

Identification of three new microsatellite markers in the spinocerebellar ataxia type 2 (SCA2) region and 1.2 Mb physical map

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease recently mapped to chromosome 12q close to the locus D12S84 by genetic linkage analysis. To generate additional genetic markers in the SCA2 region, we constructed a physical map of the region using yeast artificial chomosome (YAC), P1 artificial chromosome (PAC) and cosmid clones. The physical map was found to agree well with the genetic map. Three novel microsatellite markers were isolated and physically mapped. A novel approach to isolate CAG repeats directly from YAC DNAs is described. Received: 25 January 1995 / Revised: 26 September 1995     

Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp)

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.