广西毛南族DXS7133、DXS8378、DXS6789和DXS7423基因座遗传多态性

采用PCR-STR及基因分型技术,对广西毛南族167名(女57,男110)健康无关个体4个X-STR基因座(DXS7133、DXS8378、DXS6789和DXS7423)的遗传多态性进行研究。结果显示4个X-STR基因座分别检出4、5、9、3个等位基因和5、9、18、5种基因型,4个X-STR基因座女性的基因型频率分布均符合Hardy-Weinberg平衡定律(P>0.05)。群体遗传多态性指标为:多态信息含量(PIC)0.9611、男性个体识别率(DPmale)0.9771、女性个体识别率(DPfemale)0.9980、父-母-女三联体非父排除率(MECtrio)0.9611、父-女二联体非父排除率(MECduo)0.8821,显示上述4个X-STR基因座均具有较高多态性,在法医学个人识别、亲权鉴定及群体遗传学研究中有重要应用价值,同时也为人类群体遗传学、法医学等研究提供了广西毛南族群体X-STR基因座的基础数据,丰富了中华民族基因数据库。     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

Linkage analysis of two cloned DNA sequences, DXS197 and DXS207, in hypophosphatemic rickets families

The human X-linked hypophosphatemic rickets gene locus (HYP, formerly HPDR) has been previously localized by linkage analysis to Xp22.31-Xp21.3 and the locus order Xpter-DXS43-HYP-DXS41-Xcen established. Recombination between HYP and these flanking markers is frequently observed and additional markers have been sought. The polymorphic loci DXS197 and DXS207 have been localized to Xpter-Xp11 and Xp22-Xp21, respectively. We have further localized DXS197 to Xpter-Xp21.3 by using a panel of rodent-human hybrid cells and have established the map positions of DXS197 and DXS207 in relation to HYP by linkage studies of hypophosphatemic rickets families. Linkage between DXS197 and the loci DXS43, DXS85, and DXS207 was established with peak lod score values of 6.19, 0 = 0.032; 4.14, 0 = 0.000; and 3.01, 0 = 0.000, respectively. Multilocus linkage analysis mapped the DXS197 and DXS207 loci distal to HYP and demonstrated the locus order Xpter-DXS85-(DXS207, DXS43, DXS197)-HYP-DXS41-Xcen. These additional genetic markers DXS197 and DXS207 will be useful as alternative markers in the genetic counseling of some families.     

中国朝鲜族DXS7132、DXS6854和GATA31E08基因座遗传多态性

利用复合PCR扩增、变性聚丙烯酰胺凝胶电泳和银染方法对261名中国朝鲜族人群3个X-STR基因座研究发现, 在DXS7132、DX6854和GATA31E08中分别检出8个、6个和10个等位基因; 基因频率分别分布在0.006~0.344、0.015~0.450和0.005~0.317之间; 分别检出19、17和21种基因型, 女性个体基因型频率分布均符合Hardy-Weinberg平衡(P>0.05); 观测杂合度分别为0.706, 0.743, 0.772; 多态信息含量分别为0.710, 0.730, 0.670;女性个人识别力分别为0.898, 0.911, 0.857; 男性个人识别力分别为0.766, 0.802, 0.734; 联体单倍型多样性大于0.988。结果表明, 3个基因座均具有较高的杂合度、多态信息量和个人识别力, 是较理想的遗传标记系统, 在研究中国朝鲜族起源和与其他民族之间的亲缘关系等方面具有较高的应用价值, 并且对中国朝鲜族群体的亲子鉴定和个人识别提供宝贵的基础资料。     

Genetic mapping of two loci,DXS454 and DXS458, with respect to the X-linked agammaglobulinemia gene locus

The dinucleotide repeat sequences at the DXS454 and DXS458 loci have been mapped genetically to Xq22, to the interval between DXS3 and DXS17. We have now mapped them with respect to XLA and five other loci, to within the DXS3 to XLA interval. The more precise localisation of these polymorphic loci will be useful for the fine-mapping of disease loci on the long arm of the X chromosome and enable these probes to be used for prenatal diagnosis and carrier status determination in families with XLA.     

Physical and genetic mapping of polymorphic loci in Xq28 (DXS15, DXS52, and DXS134): analysis of a cosmid clone and a yeast artificial chromosome.

Sequences corresponding to the Xq28 loci DXS15, DXS52, DXS134, and DXS130 were shown to be present in a 140-kb yeast artificial chromosome (YAC XY58, isolated by Little et al.). This YAC clone appears to contain a faithful copy of this genomic region, as shown by comparison with human DNA and with a cosmid clone that contains probes St14c (part of the DXS52 sequences) and cpX67 (DXS134). cpX67 and St14c are contained in 11 kb and detect the same MspI RFLP polymorphism. A comparison of the YAC restriction map and pulsed-field gel electrophoresis data leads us to propose the following order of loci: DXS52(VNTR)-DXS33-DXF22S3-DXS130-DXS134 -DXS52-DXS15-DXS52, this whole cluster being comprised within 575 kb. The physical proximity of the DXS15, DXS52, and DXS134 loci led us to reinvestigate recombination events that had been reported between these loci in families from the Centre d'Etude du Polymorphisme Humain. Our results do not support the assumption that this region shows increased recombination.     

A 2-Mb YAC Contig Encompassing Three Loci (DXF34, DXS14, and DXS390) That Lie between Xp11.2 Translocation Breakpoints Associated with Incontinentia Pigmenti Type 1

We demonstrate that all the repeat elements representing the conserved loci DXF34 and DXS390 lie between the X;9 and the X;17 translocation breakpoints associated with incontinentia pigmenti type 1 (IP1). Sequence-tagged sites (STSs) at DXF34S1, DXS14, and DXS390 have been used to isolate YAC clones containing these loci, and a contig of approximately 2 Mb has been constructed. Patterns of hybridization observed in the YAC clones indicate that DXS390 comprises two distinct regions (A and B). The STS at DXS390 detects the A region and includes a polymorphic CA repeat (PIC = 0.25). This expansion of the cloned region around DXF34 and DXS390 will enable the isolation of additional conserved sequences that will help in understanding both the lesions underlying the pathogenesis of IP1 and the size and extent of the man-mouse homologous block defined by DXF34.     

用复合PCR检测DXS7132和DXS6804的单倍型

建立X染色体短串联重复基因座DXS7132和DXS6804的复合扩增系统,并用聚丙烯酰胺凝胶电泳和银染技术进行分型,调查广东汉族人群该二基因座的多态性。结果发现DXS7132有7个等位基因,扩增产物的生长在276-300bp;DXS6804有6个等位基因,扩增产物的长度在177-201bp。序列分析揭示这两个基因座的重复单位均为四核苷酸重复。由于基因组作图信息显示DXS7132和DXS6804之间存在紧密连锁,故用家系分析法确定女性的单体型。在827条染色体（男性211,女性616）中,共发现了33种单体型,女性的二倍体数据符合Hardy-Weinberg平衡。基因多样性、多态性信息量和女孩非父排除率分别达0.9999、0.9440和0.9411。对308个三联体的家系调查表明这两个基因座符合X染色体共显性遗传,未发现突变。结果表明,DXS7231和DXS6804在女孩的亲权认定,特别是缺乏双亲检验的案件中有重要的应用价值。     

5个X-STR基因座荧光复合扩增体系的建立及法医学应用

为了发掘更多多态性高的X染色体短串联重复(X-STR)基因座, 以解决法医学实践中的特殊案例, 建立了一组荧光复合扩增体系, 同时检测DXS6803、DXS981、DXS6809、DXS6789和DXS7132 5个X-STR基因座, 并用ABI PRISM 3100作毛细管电泳和GeneMapper ID 3.1软件进行基因分型, 结果清晰, 灵敏度高, 重复性好。最低检出限为0.25 ng, 10~20 ng模板DNA能得到最佳结果, 在实际检案中能得到满意结果。实验表明, 本体系能为用X-STR基因座解决特殊的亲权鉴定案提供快速鉴定技术, 是常染色体STR、Y-STR等鉴定方法的良好补充, 在法医学实践中有较好的实用价值。     

Genetic mapping of four DNA markers (DXS16, DXS43, DXS85, and DXS143) from the p22 region of the human X chromosome

Summary Linkage analysis of four polymorphic anonymous DNA markers from the Xp22 region was performed using families from the Centre d'Etude du Polymorphisme Humain. The loci DXS43 (pD2) and DXS16 (pXUT23) were found to be tightly linked ( = 0.02 at = 14.96) and proximal to both DXS85 (782) and DXS143 (dic56). Multipoint linkage analysis suggests the order:     

A highly polymorphic dinucleotide repeat on the proximal short arm of the human X chromosome: linkage mapping of the synapsin I/A-raf-1 genes.

A compound (AC)n repeat located 1,000 bp downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA data-base comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. PCR amplification of this synapsin I/A-raf-1 associated repeat by using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a PIC value of .84 and a minimum of eight alleles. Because the synapsin I gene has been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is DXS7SYN/ARAF1TIMPDXS255DXS146, with a relative probability of 5 x 10(8) as compared with the next most likely order. This highly informative repeat should serve as a valuable marker for disease loci mapped to the Xp11 region.     

Genetic analysis of the 10 ChrX STRs loci in Chinese Han nationality from Guangdong province

This study is to explore the polymorphic nature of X-Chromosome short tandem repeats (ChrX STRs) loci, and to determine its application in kinship tests for forensic cases. A new fluorescent multiplex PCR that simultaneously amplifies the 10 ChX STRs loci in the same PCR reaction had been set up. DXS7132, DXS981, DXS6801, DXS6809, DXS6789, DXS7424, DXS101, DXS7133, GATA165B12 and GATA31E08 were analyzed in a sample of 511 (399 males and 112 females) unrelated individuals from Guangdong Han nationality in China. One hundred and one alleles were observed in all the loci. Here, we investigated the allele frequencies and mutation rates of the ten loci, and then made the comparison of allele frequencies distribution among different populations. The results show the ten loci in the multiplex systems may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Anderson Fabry disease,a close linkage with highly polymorphic DNA markers DXS17, DXS87 and DXS88

Summary Anderson Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A deficiency. Hemizygous males and some heterozygous females develop renal failure and cardiovacular complications in early adult life. We have investigated six large UK families to assess the possible linkage of five polymorphic DNA probes to the Anderson Fabry locus, previously localised to Xq21-24. No recombination was found between Anderson Fabry disease and DXS87, DXS88 and DXS17, which gave lod_max=6.4,6.4 and 5.8 respectively at θ=0.00, (upper confidence limit 0.10). DXS3 gave lod_max 2.9 at θ=0.10 (upper confidence limit 0.25). DXYS1 was excluded from linkage. The best fit map (DXYS1/DXS3) θ=0.192 (DXS17/DXS87/DXS88/Anderson Fabry locus) provided no information about the order of loci in parentheses due to the absence of recombinants. The close linkage of DXS17, DXS87 and DXS88, together with α-galactosidade A estimation, can be used for antenatal diagnosis and carrier detection until the application of a gene specific probe has been evaluated.     

PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower

Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.     

Human glandular Kallikrein genes: genetic and physical mapping of the KLK1 locus using a highly polymorphic microsatellite PCR marker.

We describe a highly polymorphic microsatellite repeat sequence, KLK1 AC, which is located 3' to the human glandular kallikrein gene (KLK1) at 19q13.3-13.4. A multiplex PCR was developed to simultaneously genotype the KLK1 AC repeat length polymorphism and a similar repeat at the adjacent APOC2 locus at 19q13.2. Genotypes from these two loci in the 40 large kindred pedigrees from the Centre d'Etude du Polymorphisme Humain were used in conjunction with the background genetic map to establish a multipoint linkage map. The KLK1 locus was also localized physically using somatic cell hybrid DNA templates for polymerase chain reaction analysis. Both genetic and physical mapping studies are consistent with the assignment cen-APOC2-KLK1-D19522-qter. The linkage map places KLK1 approximately 10 cM distal to APOC2. These markers therefore flank the myotonic dystrophy gene and may be useful for diagnosis.     

Localization of the highly polymorphic microsatellite DXS456 on the genetic linkage map of the human X chromosome

The CA repeat microsatellite DXS456, with a heterozygosity of 77%, has been localized by multipoint linkage analysis in relation to 20 other genetic markers. DXS456 mapped to a 4.2-cM interval defined by the flanking markers DXS178 and DXS287. The maximum likelihood order of markers, cen-(DXYS1X/DXYS13X/DXYS2X/DXYS12X)-DXS366 -DXS178-DXS456-DXS287-DXS358-DXS267- qter, is favored by odds greater than 1000:1 over the subset of most likely alternative orders. Linkage of DXS456 can be inferred for at least six disease genes that are known to be linked to markers in the region Xq21.31-Xq25 and the marker will serve as an important index point for orienting these and other disease and marker loci in the region.     

Identification of a U7snRNA homologue mapping to the human Xq27.1 region,between the DXS1232 and DXS119 loci

To contribute to the identification and analysis of novel genes, we undertook the study of a cosmid clone in the Xq27 region of human DNA. The cloned fragment was previously observed to have a high number of evolutionarily conserved sequences. In this genomic stretch of DNA we have identified sequence homologous to the U7 RNA gene including its potential regulatory elements. This paper describes the genomic organisation of this gene and its mapping to the Xq27.1 genomic sub-interval between the DXS1232 and DXS119 loci.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Rat Gene Mapping Using Pcr-Analyzed Microsatellites

One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat x mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F2 crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes.     

Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.