Continuous linkage map of human chromosome 14 short tandem repeat polymorphisms.

Nine moderately to highly informative short tandem repeat polymorphisms were assigned to chromosome 14 using somatic cell hybrids and were mapped using linkage analysis. The nine markers formed a continuous linkage map covering almost the entire long arm from 14q11.2 to q32. The markers filled a large gap within previously reported linkage maps for this chromosome. Best order of the new loci from q11.2 to q32 was D14S50, D14S54, D14S49, D14S47, D14S52, D14S53, D14S55, D14S48, and D14S51. The order shown for all adjacent pairs of loci was very strongly favored with the exception of loci pair D14S55 and D14S48, for which the order was moderately favored. Map lengths for the nine loci were 142 cM in females and 72 cM in males. Female recombination frequencies exceeded male recombination frequencies in the middle and distal portions of the map.     

A genetic linkage map for Arctic char (Salvelinus alpinus): evidence for higher recombination rates and segregation distortion in hybrid versus pure strain mapping parents.

We constructed a genetic linkage map for Arctic char (Salvelinus alpinus) using two backcrosses between genetically divergent strains. Forty-six linkage groups (expected = 39-41) and 19 homeologous affinities (expected = 25) were identified using 184 microsatellites, 129 amplified fragment length polymorphisms (AFLPs), 13 type I gene markers, and one phenotypic marker, SEX. Twenty-six markers remain unlinked. Female map distance (9.92 Morgans) was substantially higher than male map distance (3.90 Morgans) based on the most complete parental information (i.e., the F1 hybrids). Female recombination rates were often significantly higher than those of males across all pairwise comparisons within homologous chromosomal segments (average female to male ratios within families was 1.69:1). The female hybrid parent had significantly higher recombination rates than the pure strain female parent. Segregation distortion was detected in four linkage groups (4, 8, 13, 20) for both families. In family 3, only the largest fish were sampled for genotyping, suggesting that segregation distortion may represent regions possessing influences on growth. In family 2, almost all cases showing segregation distortion involved markers in the female hybrid parent.     

A study of genetic association with electrophysiological measures related to alcoholism: GAW14 data

Recently, alcohol-related traits have been shown to have a genetic component. Here, we study the association of specific genetic measures in one of the three sets of electrophysiological measures in families with alcoholism distributed as part of the Genetic Analysis Workshop 14 data, the NTTH (non-target case of Visual Oddball experiment for 4 electrode placements) phenotypes: ntth1, ntth2, ntth3, and ntth4. We focused on the analysis of the 786 Affymetrix markers on chromosome 4. Our desire was to find at least a partial answer to the question of whether ntth1, ntth2, ntth3, and ntth4 are separately or jointly genetically controlled, so we studied the principal components that explain most of the covariation of the four quantitative traits. The first principal component, which explains 70% of the covariation, showed association but not genetic linkage to two markers: tsc0272102 and tsc0560854. On the other hand, ntth1 appeared to be the trait driving the variation in the second principal component, which showed association and genetic linkage at markers in four regions: tsc0045058, tsc1213381, tsc0055068, and tsc0051777 at map distances 53.26, 85.42, 89.31, and 172.86, respectively. These results show that the partial answer to our starting question for this brief analysis is that the NTTH phenotypes are not jointly genetically controlled. The component ntth1 displays marked genetic linkage.     

Herpes simplex virus ICP27 activation of stress kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Genes, age, and alcoholism: analysis of GAW14 data

A genetic analysis of age of onset of alcoholism was performed on the Collaborative Study on the Genetics of Alcoholism data released for Genetic Analysis Workshop 14. Our study illustrates an application of the log-normal age of onset model in our software Genetic Epidemiology Models (GEMs). The phenotype ALDX1 of alcoholism was studied. The analysis strategy was to first find the markers of the Affymetrix SNP dataset with significant association with age of onset, and then to perform linkage analysis on them. ALDX1 revealed strong evidence of linkage for marker tsc0041591 on chromosome 2 and suggestive linkage for marker tsc0894042 on chromosome 3. The largest separation in mean ages of onset of ALDX1 was 19.76 and 24.41 between male smokers who are carriers of the risk allele of tsc0041591 and the non-carriers, respectively. Hence, male smokers who are carriers of marker tsc0041591 on chromosome 2 have an average onset of ALDX1 almost 5 years earlier than non-carriers.     

Deletion and linkage mapping of eight markers from the proximal short arm of chromosome 6

Eight chromosome 6p markers (MUT, D6S4, D6S5, D6S19, D6S29, PIM, HLA, and F13A) were regionally mapped using somatic cell hybrid deletion cell lines that retained different regions of chromosome 6p. New restriction fragment length polymorphisms were identified at the D6S5 and PIM loci using newly isolated genomic clones at these loci. Genetic linkage among the eight loci was determined using the 40 CEPH reference families. Linkage analyses showed that these loci are in one linkage group spanning 48 cM in males and 128 cM in females. Using both the deletion mapping data and multipoint linkage analyses, chromosomal order for these loci was determined as centromere-(MUT, D6S4)-(D6S5, D6S19)-(D6S29, PIM)-HLA-F13-A-telomere. Analyses of sex-specific recombination frequencies revealed a higher rate of recombination in females in the region between D6S4 and D6S29, while the recombination rate in males was higher for the interval between D6S29 and the HLA loci.     

Comparative analyses of linkage maps and segregation distortion of two F₂ populations derived from japonica crossed with indica rice

To facilitate genetic research, we constructed two linkage maps by employing two F? populations derived from rice inter-subspecific crosses, japonica Tainung 67 (TNG67)/indica Taichung Sen 10 (TCS10) and japonica TNG67/indica Taichung Sen 17 (TCS17). We established linkage map lengths of 1481.6 cM and 1267.4 cM with average intervals of 13.8 cM and 14.4 cM by using 107 and 88 PCR markers for coverage of 88% of the rice genome in TNG67/TCS10 and TNG67/TCS17, respectively. The discrepancy in genetic maps in the two populations could be due to different cross combinations, crossing-over events, progeny numbers and/or markers. The most plausible explanation was segregation distortion; 18 markers (16.8%) distributed at nine regions of seven chromosomes and 10 markers (11.4%) at four regions of four chromosomes displayed severe segregation distortion (p < 0.01)in TNG67/TCS10 and TNG67/TCS17, respectively. All segregation-distorted markers in these two populations corresponded to reported reproductive barriers, either gametophytic or zygotic genes but not to hybrid breakdown genes. The observed recombination frequency, which was higher or lower than the intrinsic frequency, revealed the association of segregation distortion skewed to the same or different genotypes at the consecutive markers. The segregation distortion, possibly caused by reproductive barriers, affects the evaluation recombination frequencies and consequently the linkage analysis of QTLs and positional cloning.     

Genetic linkage map of Coffea canephora: effect of segregation distortion and analysis of recombination rate in male and female meioses.

Two complementary segregating plant populations of Coffea canephora were produced from the same clone. One population (DH) comprised 92 doubled haploids derived from female gametes, while the other population (TC) was a test cross consisting of 44 individuals derived from male gametes. Based on the DH population, a genetic linkage map comprising 160 loci was constructed. Eleven linkage groups that putatively correspond to the 11 gametic chromosomes of C. canephora were identified. The mapped loci included more than 40 specific sequence-tagged site markers, either single-copy RFLP probes or microsatellites, that could serve as standard landmarks in coffee-genome analyses. Furthermore, comparisons for segregation distortion and recombination frequency between the two populations were performed. Although segregation distortions were observed in both populations, the frequency of loci exhibiting a very pronounced degree of distortion was especially high in the DH population. This observation is consistent with the hypothesis of strong zygotic selection among the DH population. The recombination frequencies in both populations were found to be almost indistinguishable. These results offer evidence in favour of the lack of significant sex differences in recombination in C. canephora.     

A single mutation responsible for temperature-sensitive entry and assembly defects in the VP1-2 protein of herpes simplex virus

Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.     

Multipoint linkage analysis of loci in the proximal long arm of the human X chromosome: application to mapping the choroideremia locus.

Choroideremia (McK30310), an X-linked retinal dystrophy, causes progressive night blindness, visual field constriction, and eventual central blindness in affected males by the third to fourth decade of life. The biochemical basis of the disease is unknown, and prenatal diagnosis is not available. Subregional localization of the choroideremia locus to Xq13-22 was accomplished initially by linkage to two restriction-fragment-length polymorphisms (RFLPs), DXYS1 (Xq13-q21.1) and DXS3 (Xq21.3-22). We have now extended our linkage analysis to 12 families using nine RFLP markers between Xp11.3 and Xq26. Recombination frequencies of 0%-4% were found between choroideremia and five markers (PGK, DXS3, DXYS12, DXS72, and DXYS1) located in Xq13-22. The families were also used to measure recombination frequencies between RFLP loci to provide parameters for the program LINKMAP. Multipoint analysis with LINKMAP provided overwhelming evidence for placing the choroideremia locus within the region bounded by DXS1 (Xq11-13) and DXS17 (Xq21.3-q22). At a finer level of resolution, multipoint analysis suggested that the choroideremia locus was proximal to DXS3 (384:1 odds) rather than distal to it. Data were insufficient, however, to distinguish between a gene order that puts choroideremia between DXS3 and DXYS1 and one that places choroideremia proximal to both RFLP loci. These results provide linkage mapping of choroideremia and RFLP loci in this region that will be of use for further genetic studies as well as for clinical applications in this and other human diseases.     

A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans)

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.     

Exploring the genetic characteristics of 93-11 and Nipponbare recombination inbred lines based on the GoldenGate SNP assay

Understanding genetic characteristics in rice populations will facilitate exploring evolutionary mechanisms and gene cloning. Numerous molecular markers have been utilized in linkage map construction and quantitative trait locus (QTL) mappings. However, segregation-distorted markers were rarely considered, which prevented understanding genetic characteristics in many populations. In this study, we designed a 384-marker GoldenGate SNP array to genotype 283 recombination inbred lines (RILs) derived from 93-11 and Nipponbare Oryza sativa crosses. Using 294 markers that were highly polymorphic between parents, a linkage map with a total genetic distance of 1,583.2 cM was constructed, including 231 segregation-distorted markers. This linkage map was consistent with maps generated by other methods in previous studies. In total, 85 significant quantitative trait loci (QTLs) with phenotypic variation explained (PVE) values≥5% were identified. Among them, 34 QTLs were overlapped with reported genes/QTLs relevant to corresponding traits, and 17 QTLs were overlapped with reported sterility-related genes/QTLs. Our study provides evidence that segregation-distorted markers can be used in linkage map construction and QTL mapping. Moreover, genetic information resulting from this study will help us to understand recombination events and segregation distortion. Furthermore, this study will facilitate gene cloning and understanding mechanism of inter-subspecies hybrid sterility and correlations with important agronomic traits in rice.     

Simian virus 40 chromatin interaction with the capsid proteins

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

High-resolution physical mapping of four microsatellite repeat markers near the RYR1 locus on chromosome 19q13.1 and apparent exclusion of the MHS locus from this region in two malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is a potentially lethal, hereditary disorder of skeletal muscle that may be triggered by inhalation anesthetics and depolarizing muscle relaxants. Defects in the gene encoding the ryanodine receptor (RYR1) localized on human chromosome 19q13.1 have been proposed to be responsible for MHS. Using a chromosome 19-specific human/hamster somatic cell hybrid mapping panel, we were able to determine that four closely linked microsatellite repeat markers bracket RYR1 with the order 19cen-D19S75-D19S191-RYR1-(D19S47, D19S190)-19ter. Application of the four markers to genetic studies of MHS showed recombination between the markers and MHS in two families, with linkage analysis apparently excluding the MHS locus from the RYR1 region of 19q13.1. These results therefore support the recent observations of genetic heterogeneity in MHS.     

An amplified fragment length polymorphism map of the silkworm

The silkworm (Bombyx mori L.) is a lepidopteran insect with a long history of significant agricultural value. We have constructed the first amplified fragment length polymorphism (AFLP) genetic linkage map of the silkworm B. mori at a LOD score of 2.5. The mapping AFLP markers were genotyped in 47 progeny from a backcross population of the cross no. 782 x od100. A total of 1248 (60.7%) polymorphic AFLP markers were detected with 35 PstI/TaqI primer combinations. Each of the primer combinations generated an average of 35.7 polymorphic AFLP markers. A total of 545 (44%) polymorphic markers are consistent with the expected segregation ratio of 1:1 at the significance level of P = 0.05. Of the 545 polymorphic markers, 356 were assigned to 30 linkage groups. The number of markers on linkage groups ranged from 4 to 36. There were 21 major linkage groups with 7-36 markers and 9 relatively small linkage groups with 4-6 markers. The 30 linkage groups varied in length from 37.4 to 691.0 cM. The total length of this AFLP linkage map was 6512 cM. Genetic distances between two neighboring markers on the same linkage group ranged from 0.2 to 47 cM with an average of 18.2 cM. The sex-linked gene od was located between the markers P1T3B40 and P3T3B27 at the end of group 3, indicating that AFLP linkage group 3 was the Z (sex) chromosome. This work provides an essential basic map for constructing a denser linkage map and for mapping genes underlying agronomically important traits in the silkworm B. mori L.     

结合SADF与RAPD标记构建家蚕连锁图

用40个选择性扩增多态性引物和137个随机性扩增多态性引物对家蚕Bombyx mori F₂群体进行分子标记的筛选。获得544个符合遗传分离比的多态性位点,并用Mapmaker软件进行连锁分析。在最小LOD值为4,最大重组值为0.2条件下拆分连锁群,并对处于同一连锁群的位点进行合理排序,计算出位点间的重组值后转换为图谱距离,构建了一个家蚕的分子连锁图。     

Mapping of plumage colour and blood protein loci on the microsatellite linkage map of the Japanese quail

The objective of this work was to map classical markers (plumage colours and blood proteins) on the microsatellite linkage map of the Japanese quail (Coturnix japonica). The segregation data on two plumage colours and three blood proteins were obtained from 25 three-generation families (193 F2 birds). Linkage analysis was carried out for these five classical markers and 80 microsatellite markers. A total of 15 linkage groups that included the five classical loci and 69 of the 80 microsatellite markers were constructed. Using the BLAST homology search against the chicken genome sequence, three quail linkage groups, QL8, QL10 and QL13, were suggested to be homologous to chicken chromosomes GGA9, GGA20 and GGA24, respectively. Two plumage colour loci, black at hatch (Bh) and yellow (Y), and the three blood protein loci, transferrin (Tf), haemoglobin (Hb-1) and prealbumin-1 (Pa-1), were assigned to CJA01, QL10, QL8, CJA14 and QL13, respectively.     

Identification of CpG islands in a physical map encompassing the Friedreich's ataxia locus

The Friedreich's ataxia locus has been previously assigned to chromosome 9q 13-21.1 by the demonstration of tight linkage to two anonymous DNA markers. MCT112 (Z greater than 80, theta = 0) and DR47 (Z greater than 50, theta = 0). The absence of recombination between these three loci has prevented the resolution of gene/probe order in this region, impeding strategies for gene isolation. We report physical mapping over a 4-Mb genomic interval, linking the markers MCT112 and DR47 on a common 460-kb NotI fragment and identifying 11 CpG islands in the 1.7-Mb interval most likely to contain the Friedreich's ataxia locus. Four of these islands were detected only by analysis of three YAC clones spanning a 700-kb interval including the MCT112/DR47 cluster. Without clear evidence of the precise location of the disease locus from recombination events, each of these regions must be considered as specifying a potential "candidate" sequence for the mutated gene.     

Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.