Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the disease haemophilia A has been mapped to the distal portion of Xq, the BclI RFLP makes possible higher-resolution genetic linkage mapping with respect to other polymorphic markers on this portion of the X chromosome. We have established close linkage of the factor VIII gene to several useful RFLP markers, including the highly informative marker St14. These markers should also be useful for prenatal diagnosis of haemophilia A and for detection of its carriers.     

Strategy for the mapping of interactive genes using bulked segregant analysis method and Mapmaker/Exp software

A qualitative trait is usually controlled by a single gene, but it may be sometimes controlled by two or even more genes. This phenomenon is called gene interaction. Rapidly searching for linked mo- lecular markers via bulked segregant analysis (BSA) and then constructing regional linkage map with Mapmaker/Exp has become a common approach to mapping single major genes. However, methods and computer programs developed for mapping single major genes cannot be simply applied to interactive genes because the genetic patterns of gene interac- tions are quite different from that of single-gene in- heritance. Up to now, experimental methods for quickly screening molecular markers linked to inter- active genes and statistical methods and corre- sponding computer softwares for simultaneously analyzing the linkage relationships of multiple mo- lecular markers to an interactive gene have not been available. To solve this problem, in this paper, we propose a strategy for mapping interactive genes using BSA and Mapmaker/Exp. We demonstrate that all interactive genes can be mapped by the 'BSA Mapmaker/Exp' strategy using F2 generation (in a few cases, F3 generation is also needed). As BSA and Mapmaker/Exp have been broadly used in gene mapping studies and are well known by many re- searchers, the strategies proposed in this paper will be useful for practical researches.     

Model for antenatal diagnosis of beta-thalassaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms

Polymorphisms of DNA restriction sites within the human fetal globin genes have been used to identify chromosomes that carry beta-thalassaemia genes in individuals heterozygous for this disease. This has allowed an antenatal diagnosis for beta-thalassaemia to be carried out by observation of the pattern of the inherited polymorphism of a linked DNA sequence not involved in the genetic pathogenesis of the disease. In the populations we have investigated there is no constant pattern of polymorphism that segregates with the beta-thalassaemia gene. The use of linked polymorphisms should, therefore, be applicable to antenatal diagnosis both of beta-thalassaemia and of any other single-gene defect for which there is a DNA probe specific for a sequence linked to the affected locus.     

Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker

An F₂ population developed from theXa-4 near isogenic lines, IR24 and IRBB4, was used for fine mapping of the rice bacterial blight resistance gene,Xa-4. Some restriction fragment length polymorphism (RFLP) markers on the high-density map constructed by Harushima et al. and the amplified DNA fragments homologous to the conserved domains of plant disease resistance (R) genes were used to construct the genetic linkage map around the geneXa-4 by scoring susceptible individuals in the population.Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction (PCR) marker M55. The R gene homologous fragment marker RS13 was found co-segregating withXa-4 by analyzing all the plants in the population. This result opened an approach to map-based cloning of this gene, and marker RS13 can be applied to molecular marker-assisted selection ofXa-4 in rice breeding programs.     

Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which affects approximately 1 in 3,300 males, making it the most common of the neuromuscular dystrophies. The biochemical basis of the disease is unknown and as yet no effective treatment is available. A small number of females are also affected with the disease, and these have been found to carry X; autosome translocations involving variable autosomal sites but always with a breakpoint within band Xp21 of the X chromosome (implicated by other kinds of genetic evidence as the site of the DMD lesion). In these female patients the normal X chromosome is preferentially inactivated, which it is assumed silences their one normal DMD gene, leading to expression of the disease. In one such affected female the autosomal breakpoint lies in the middle of the short arm of chromosome 21, within a cluster of ribosomal RNA genes. Here we have used rRNA sequences as probes to clone the region spanning the translocation breakpoint. A sequence derived from the X-chromosomal portion of the clone detects a restriction fragment length polymorphism (RFLP) which is closely linked to the DMD gene and uncovers chromosomal deletions in some male DMD patients.     

PCR—RFLP analysis of mitochondrial DNA ND5／6 region among3 subspecies of common carp（Cyprinus carpio L.）and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio,Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp(Cyprinus carpio L.)A total of 2.4kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently.The results indicated that each subspecies owned one hyplotype and four restriction enzymes(Dde Ⅰ，HaeⅢ,Taq Ⅰ and MboⅠ)produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.     

DNA typing from single hairs

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.     

Molecular genetic evidence for heterogeneity in manic depression

Manic depression is a severe cyclic mental illness that can be unipolar or bipolar and has a lifetime risk of approximately 7 per 1,000 in most populations. Families with multiple cases of manic depression have been described that are compatible with both autosomal dominant and X-linked modes of genetic transmission. Psychoactive antidepressant and stimulant drugs that help to ameliorate depression and mania are thought to act by affecting catecholamine neurotransmitter systems such as adrenaline, noradrenaline and dopamine, amongst others. Mutations affecting the tyrosine hydroxylase (TH) gene, which encodes the rate-limiting enzyme for the synthesis of these three neurotransmitters, might therefore be responsible for causing the manic depressive phenotype. We have studied three Icelandic kindreds amongst whom it appears that a single autosomal dominant disease allele is segregating. In these families there were 44 cases amongst 73 individuals at risk. Genetic linkage studies were carried out using clones encoding tyrosine hydroxylase the variable portion of the Harvey-ras-1 (HRAS1) locus and the variable region of the insulin gene (INS). All three markers are closely linked on chromosome 11 and were used to observe the segregation of restriction fragment length polymorphisms (RFLPs) in the three affected kindreds. We found no evidence for linkage to these markers in any of the three families. In contrast, Gerhard et al. found linkage between manic depression and HRAS1 in a single large Amish kindred. We conclude that there is genetic heterogeneity of linkage in manic depression. Therefore mutations at different loci are responsible for the manic depressive phenotype in the Amish and in Iceland.     

Mapping of two new brown planthopper resistance genes from wild rice

A brown planthopper (BPH) resistance line, B5, derived its resistance genes from the wild riceOryza officinalis Wall exwatt, was hybridized with Taichung Native 1, a cultivar highly susceptible to BPH. A mapping population composed of randomly selected 167 F₂ individuals was used for determining the BPH resistance genes by the restriction fragment length polymorphism analysis (RFLP). Bulked segregant analysis was conducted to identify RFLP makers linked to the BPH resistance genes in B5. The results indicated that the markers linked to BPH resistance are located at two genomic regions on the long arm of chromosome 3 and the short arm of chromosome 4, respectively. The existence of the two loci was further assessed by the quantitative trait locus (QTL) analysis. We located the two loci at a 3.2 cM interval between G1318 and R1925 on chromosome 3 and a 1.2 cM interval between C820 and S11182 on chromosome 4. Comparison with the BPH genes that have been reported indicated that the BPH resistance genes in B5 are novel. These two genes may be useful BPH resistance resource for rice breeding. Furthermore, the mapping of the two genes is useful for cloning the BPH resistance genes.     

Genetic and physical mapping of AvrPi7, a novel avirulence gene of Magnaporthe oryzae using physical position-ready markers

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating crop diseases worldwide. The avirulence gene corresponding to rice blast resistance gene Pi7 in field isolate CHL346 was inherited as a single gene, designated AvrPi7, in a segregating population consisting of 189 ascospore progenies derived from a cross between field isolates CHL346 and CHL42. In order to determine the chromosomal location of the AvrPi7 locus, a total of 121 simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of reference isolate 70-15 of M. oryzae. Linkage analysis of the locus with these SSR markers showed that eight SSR markers on chromosome 1 were linked to the locus, among which the closest flanking markers MS1-9 and MS1-15 were 3.2 and 16.4 cM from the locus, respectively. For fine mapping, additional PCR-based makers including eight SSR markers and three candidate avirulence gene (CAG) markers were developed in the region flanking both markers. The AvrPi7 locus was genetically delimited within a 1.6-cM region flanked by markers MS1-21 and MS1-22, and co-segregated with the marker CAG2. To construct a physical map of the AvrPi7 locus, molecular markers linked to the Avr gene were mapped on the supercontigs of the ref-erence isolate 70-15 through bioinformation analysis (BIA). Consequently, the AvrPi7 locus was delim-ited to a 75-kb interval flanked by markers MS1-21 and MS1-22 based on the reference sequence. Merodiploids observed in this study are also discussed.     

Close linkage of c-Harvey-ras-1 and the insulin gene to affective disorder is ruled out in three North American pedigrees

Affective disorder (AD) is one of the major forms of functional psychoses. Although the mode of transmission is uncertain, family, twin and adoption studies strongly suggest a genetic involvement. Because a basic biochemical abnormality is not known, direct analysis of the disease using a probe for the defective gene is not possible. However, a specific locus can be tested for its relevance to the aetiology of AD by genetic linkage, using restriction fragment length polymorphisms (RFLPs). Using probes for the c-Ha-ras-1 oncogene and the insulin gene, Gerhard et al. and Egeland et al. found convincing evidence for close linkage between these markers and a locus for AD in a large Old Order Amish pedigree. In an attempt to confirm this finding, we examined three bipolar pedigrees outside the Amish population. Our results indicate the absence of linkage from 0 to 15% recombination frequency between AD and the insulin gene-HRAS1 region in these pedigrees.     

Worldwide migration of amplified insecticide resistance genes in mosquitoes

In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B according to substrate preference, and overproduction of all esterases B is due to gene amplification. Distribution of electrophoretically distinct variants of overproduced esterases A and B is geographically restricted, with the exception of esterases A2 and B2, always found together throughout at least three continents. To determine whether this situation is due to migration or to a high mutation rate, esterase B structural genes and their flanking regions were compared by sequence and/or restriction fragment length polymorphism analysis. Whereas structural genes were similar, flanking regions of electrophoretically dissimilar esterases B varied considerably. In contrast, flanking sequences of esterases B2 from different geographical locations (Africa, Asia, North America) were identical. These results suggest that amplified esterase B2 genes originated from an initial event that has subsequently spread organophosphate insecticide resistance by migration.     

利用线粒体 DNA Cytb 基因 PCR-RFLP 分析方法鉴别青鱼和草鱼

建立了一种以线粒体细胞色素 b（Cyt b）基因为标靶,应用 PCR -RFLP 技术进行草鱼和青鱼种质鉴定的分析方法。设计一对引物 QYCYT -S 和 QYCTY -A 分别对青鱼和草鱼的 Cyt b 基因进行了 PCR 扩增,并选用 BglⅠ和 EcoRⅡ两种限制性内切酶对扩增产物进行酶切分析。结果表明,草鱼和青鱼都可以扩增出1111 bp 的条带,两种酶切检验发现,草鱼扩增产物能被 BglⅠ切成247 bp 和864 bp 两个片段,而青鱼的 PCR 产物能被 EcoRⅡ切成140 bp 和971 bp 两个片段,这表明,mtDNA Cyt b 基因 BglⅠ和EcoRⅡ的酶切位点都可作为鉴定青鱼和草鱼的有效分子标记。利用 PCR -RFLP 分析 mtDNA Cyt b 基因的方法操作简单,是一种快速鉴别草鱼和青鱼的可靠方法。     

The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene

Amyloid beta-protein (AP) is a peptide of relative molecular mass (Mr) 42,000 found in the senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles of patients with Alzheimer's disease and Down's syndrome (trisomy 21). Recent molecular genetic evidence has indicated that AP is encoded as part of a larger protein by a gene on chromosome 21 (refs 5-7). The defect in the inherited autosomal dominant form of Alzheimer's disease, familial Alzheimer's disease (FAD), has been mapped to the same approximate region of chromosome 21 by genetic linkage to anonymous DNA markers, raising the possibility that this gene product, which could be important in the pathogenesis of Alzheimer's disease, is also the site of the inherited defect in FAD (ref. 5). We have determined the pattern of segregation of the AP gene in FAD pedigrees using restriction fragment length polymorphisms. The detection of several recombination events with FAD suggests that the AP gene is not the site of the inherited defect underlying this disorder.     

Fine mapping of the <Emphasis Type="Italic">Ht2 (Helminthosporium turcicum</Emphasis> resistance 2) gene in maize

Fine mapping of Helminthosporium turcicum resistance gene Ht2 is extremely valuable for map-based cloning of the Ht2 gene,gaining a better knowledge of the distribution of resistance genes in maize genome and marker-assisted selection in maize breeding.An F2 mapping population was developed from a cross between a resistant inbred line 77Ht2 and a susceptible inbred line Huobai.With the aid of RFLP marker analyses,the Ht2 gene was mapped between the RFLP markers UMC89 and BNL2.369on chromosome 8,with a genetic distance of 0.9cM to BNL2.369.There was a linkage between SSR markers UMC1202,BNLG1152,UMC1149 and the Ht2 gene by SSR assay,Among the SSR markers,the genetic distance between UMC1149 and the Ht2 gene was 7.2cM,By bulked segregant analysis 7 RAPD-amplified products which were probably linked to the Ht2 gene were selected after screening 450 RAPD primers and converted the single-copy ones into SCAR markers.Linkage analysis showed that the genetic distance between the SCAR marker SD-06633 and the Ht2 gene was 0.4cM.From these results,a part of linkage map around the Ht2 gene was constructed.     

Inversion in the H-2 complex of t-haplotypes in mice

Mouse t-haplotypes demonstrate strong linkage disequilibrium between t-lethal genes and specific H-2 types, presumably a result of recombination suppression between t and normal chromosomes. The observation of free recombination occurring between two complementary t-haplotypes suggested a chromosomal mismatch between t and normal chromosomes. Recent data showing the H-2 complex to be misplaced relative to two other markers, T and tf, in t-haplotypes suggested that chromosomal rearrangement in t-haplotypes might be the basis for their 'mismatch' with the normal chromosome. Here, to analyse the molecular nature of the rearrangement, we have cloned a polymorphic H-2 class I restriction fragment, which had previously been shown to map centromeric to the serologically defined H-2 complex in t-haplotypes. Genetic mapping studies show that this cloned t-DNA is homologous to the H-2 D region of wild-type chromosomes, and that the E alpha Ia gene maps telomeric to this DNA fragment in t-haplotypes, in contrast to its orientation in wild-type chromosomes. These results give molecular evidence for an inversion of H-2 in t-haplotypes, which may be at least partially responsible for recombination suppression and thus for linkage disequilibrium.     

Direct detection of more than 50% of the Duchenne muscular dystrophy mutations by field inversion gels

Duchenne muscular dystrophy (DMD) is an X-linked disorder affecting about 1 in 3,500 males. It is allelic with the milder Becker muscular dystrophy. The biochemical basis for both diseases is unknown and no effective treatment is available. Long-range physical mapping has shown that the DMD gene, localized in Xp21, is extremely large, exceeding 2 million base pairs. Until now, carrier detection and prenatal diagnosis has involved the use of linked restriction fragment length polymorphism markers which detect muscular dystrophy-associated deletions in about 10% of the cases. Field inversion gel electrophoresis (FIGE) allows the detection of structural rearrangements in 21 out of 39 of the DMD patients studied (54%), of which 14 (65%) were not detected by conventional methods. Large deletions seem to make up a much higher fraction of the DMD mutations than so far indicated by other methods. A region prone to deletion was located in the distal half of the gene. FIGE analysis could provide a valuable extension of information for carrier detection and prenatal diagnosis. The technique should be generally applicable to the study of diseases involving structural chromosomal rearrangements.