The use of DNA markers to estimate the extent and nature of genetic variability in Solatium tuberosum cultivars

High levels of DNA polymorphism were detected in 27 Solanum tuberosum cultivars examined. Combinations of at least two c-DNA clones have been identified which in conjunction with EcoRI allow important UK potato cultivars to be characterised by their molecular profiles. The widely grown North American cultivar Russett Burbank was also successfully fingerprinted. Estimates of genetic diversity based on restriction fragment length polymorphism (RFLP) data indicate the important role that wild potato species and exotic germplasm have played in the development of the cultivars studied. A graphical method for simultaneously highlighting similarities and differences between genotypes for individual hybridising fragments is presented. This approach is particularly useful in identifying and recording fragments which are unique to certain genotypes. Two potato cultivars: Fiona and Morag produce unique RFLP profiles when digested with EcoRI and EcoRV and probed with a flax ribosomal DNA sequence. Both Fiona and Morag possess incomplete or partial (quantitative) type resistance to G. pallida which was transferred from S. vernei. The preferential transmission of the r-DN A fragments from S. vernei may indicate that this locus is associated with genetic factors controlling resistance to G. pallida.     

aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat

Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.     

An Ala/Thr variation in the coding region of the human cystatin C gene (CST3) detected as a SstII polymorphism

A DNA variation in the coding region of the human cystatin C gene has been detected by direct sequencing. The polymorphism, a G/A transition, leads to an Ala/Thr variation in the penultimate amino acid of the signal peptide. The base substitution results in the loss of a SstII restriction site, thus allowing the design of a rapid polymerase chain reaction assay for analysis of this polymorphism in the population.     

Association analysis of the E-selectin 98G > T polymorphism and the risk of childhood ischemic stroke

Genes related to platelet and arterial endothelial function have been recently considered as independent risk factors for stroke. We aimed to analyze a relationship between the E‐selectin 98G > T polymorphism and stroke in children and to observe the transmission of E‐selectin alleles from heterozygous parents to their affected children. We studied 59 children after stroke, 112 parents, and 87 healthy children. The E‐selectin 98G > T polymorphism was analyzed with the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. The frequency of the 98T allele in patients was almost twofold lower than in controls (5.1% vs. 9.8%, p = 0.145, odds ratios (OR) = 0.49) as well as carriers of the 98T allele (19.5% in controls vs. 8.5% in cases, p = 0.067, OR = 0.38). The G allele of the E‐selectin 98G > T polymorphism was more frequently transmitted to the children after stroke compared to the T allele (68% vs. 32%). In conclusion, we did not confirm the relationship between the 98G > T polymorphism of the E‐selectin gene and childhood ischemic stroke. There is still a need for further studies. Copyright © 2010 John Wiley & Sons, Ltd.     

Human pepsinogen A (PGA): an informative gene complex located at 11q13

Summary Human pepsinogen (PGA) exhibits extensive polymorphism that can be detected both at the protein and the DNA level. We describe here two restriction fragment length polymorphisms, EcoRI and BglII, which provide for the detection of three of the most common PGA haplotypes (A, B, and C) in the United States population. The relationship of these polymorphisms to each PGA haplotype was determined by analysis of DNA from individuals exhibiting the corresponding protein phenotypes and by analysis of a series of human × mouse somatic cell hybrids containing the individual chromosome 11 homologous from heterozygous individuals exhibiting the AB and AC protein phenotypes. The use of the BglII polymorphism in combination with previously described EcoRI polymorphism provides a very informative marker of 11q13.     

No association between the G196A and C270T polymorphism of the brain-derived neurotrophic factor gene and male infertility

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal development and reproductive action. Abnormal expression of BDNF gene has been detected in human sperm and seminal serum. In the present study, we investigated the possible association of G196A and C270T polymorphism of BDNF gene with male infertility. The genotypes of the G196A and C270T polymorphisms were in Hardy-Weinberg equilibrium both in fertile and infertile group. The genotype distribution frequencies were similar between infertile and fertile group. The results showed that the G196A and C270T polymorphism of the BDNF gene is unrelated to the male infertility, at least in a Chinese population.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I)

In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1–2, and 2. Received: 20 March 1996 / Revised: 14 May 1996     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

Prognostic role of the CDNK1B V109G polymorphism in multiple endocrine neoplasia type 1

CDKN1B encodes the cyclin‐dependent kinase inhibitor p27/Kip1. CDKN1B mutations and polymorphisms are involved in tumorigenesis; specifically, the V109G single nucleotide polymorphism has been linked to different tumours with controversial results. Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant syndrome, characterized by the development of different types of neuroendocrine tumours and increased incidence of other malignancies. A clear genotype–phenotype correlation in MEN1 has not been established yet. In this study, we assessed whether the CDKN1B V109G polymorphism was associated with the development of aggressive tumours in 55 consecutive patients affected by MEN1. The polymorphism was investigated by PCR amplification of germline DNA followed by direct sequencing. Baseline and follow‐up data of tumour types and their severity were collected and associated with the genetic data. MEN1‐related aggressive and other malignant tumours of any origin were detected in 16.1% of wild‐type and 33.3% of polymorphism allele‐bearing patients (P = NS). The time interval between birth and the first aggressive tumour was significantly shorter in patients with the CDKN1B V109G polymorphism (median 46 years) than in those without (median not reached; P = 0.03). Similarly, shorter was the time interval between MEN1 diagnosis and age of the first aggressive tumour (P = 0.02). Overall survival could not be estimated as 96% patients were still alive at the time of the study. In conclusion, CDKN1B V109G polymorphism seems to play a role in the development of aggressive tumours in MEN1.     

Entry into S phase is inhibited in two immortal cell lines fused to senescent human diploid cells.

Senescent human diploid cells (HDC) were fused to T98G human glioblastoma cells and to RK13 rabbit kidney cells, and DNA synthesis was analyzed in the heterodikaryons. T98G and RK13 cells are “partially transformed” cell lines that have some characteristics of normal cells, yet are transformed to immortality, i.e., they do not senesce. Previous experiments have shown that “fully transformed” HeLa and SV80 cells induce DNA synthesis in senescent HDC nuclei, whereas normal young HDC do not. Our experiments show that T98G and RK13 cells do not induce DNA synthesis in senescent HDC nuclei. These results demonstrate that the ability to induce DNA synthesis in senescent HDC is not correlated with immortality per se. Our results show further that a T98G cell in S phase at the time of fusion to a senescent HDC will continue to make DNA. However, a T98G cell in G1 phase at the time of fusion is prevented from initiating DNA synthesis. RK13 cells behave similarly to T98G. These results are consistent with the hypothesis that the molecular basis for the senescent phenotype involves a block that prevents cells in G1 phase from entering S phase. Thus, we conclude that the senescent phenotype can be dominant in heterokaryons composed of senescent HDC fused with certain immortal cell lines. To explain the different results obtained with various immortal cell lines, we present a model that suggests that T98G and RK13 cells are immortal because they have lost a normal regulatory factor, whereas HeLa and SV80 are immortal because they have gained a dominant transformation factor.     

天麻基因组微卫星特征分析与分子标记开发

该研究基于第二代测序技术建立了天麻的基因文库,筛选微卫星序列,并对微卫星位点的类型、丰度、长度、偏好性等进行了分析与比较;并为60条重复次数高的微卫星序列设计了引物,运用4个种群80个样本进行了PCR扩增和聚丙烯酰胺凝胶电泳检测。结果表明:(1)天麻基因组测序得到61 048条基因序列,检测出微卫星位点12 107个,其中二核苷酸重复最多、长度变异大。(2)设计的60对微卫星引物中的20对能扩增出清晰条带且有多态性,每个位点的复等位基因数(N_a)在4~14之间,平均为8.40;多态性信息含量(PIC)平均为0.77。该研究开发的天麻微卫星分子标记为开展天麻遗传学研究及种质资源鉴定等工作奠定了基础。     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Polymorphism of transferrin in carp (Cyprinus carpio L.): Genetic determination,isolation, and partial characterization

Seven transferrin variants (A, B, C, D, E, F, and G) have been found in carp sera (Cyprinus carpio L.). Genetic analysis involves five variants and agrees with the hypothesis of simple codominant autosomal inheritance at one transferrin (Tf) locus in spite of the fact that the carp is a tetraploid in relation to other species of the same family. Carp populations from three regions were studied which differed in gene frequencies. Individual populations were in Hardy—Weinberg equilibrium. The polymorphism of carp transferrins can be used for the identification of offspring of single parent pairs, stocked in one pond. Transferrins have been isolated and characterized. Homozygous phenotypes comprised four iron-binding components differing in electrophoretic mobility. This heterogeneity is not caused by sialic acid, which is absent. Amino acid composition, content of hexoses (1 mole/mole of protein) and hexosamines (1 mole/mole of protein), molecular weight (70,000), and the isoelectric point (5.0) have been determined. No N-terminal amino acid could be detected.     

Cross-species transferability of G. arboreum-derived EST-SSRs in the diploid species of Gossypium

Diploid species with a common Gossypium origin are highly diverse in morphology and have been classified into eight genomic groups designated A–G and K. In this study, the transferability of 207 Gossypium arboreum-derived expressed sequence tag-simple sequence repeat (EST-SSR) primer pairs was examined among 25 different diploid accessions representing 7 genomes and 23 Gossypium species. We found that 124 of the 207 (60%) primer pairs produced amplification products in all 25 accessions. The remaining 83 (40%) primer pairs produced amplification in only a subset of species, ranging from 13 to 22 species, which is consistent with some genome- and species-specific amplification. The cross-species amplification of these EST-SSRs in 22 diploid species was 96.5% in 4,554 combinations (207 SSRs×22 species), indicative of a high transferability among the Gossypium species. Furthermore, a high level of polymorphism with an average number of 6.53 alleles per SSR marker was detected. No correlation was found between the repeat motif type and cross-species amplification. DNA sequencing showed that the high-level polymorphism findings was mainly due to changes in the number of repeat motifs and that the high transferability can be attributed to a higher-level conservation in the flanking regions among these diploid Gossypium species. The transferability among these different diploid species presented here can increase the efficiency of transferring genetic information across species and further enhance their introgression into cultivated cotton species by the molecular tagging of important genes existing in these diploid species using the EST-SSR markers.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/ absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.