Modulation of polymorphic loci detection with synthetic tandem repeat variants

The modifications of hybridization patterns were studied when Southern blots, carrying stallions DNA samples, were probed with eight synthetic tandem repeats (STRs), related by sequence variations in the basic unit. Because STRs preferentially crosshybridize with genomic VNTRs, they usually give patterns looking more like DNA fingerprints, but we found that even small modifications in the STR monomer could cause major changes in the hybridization profiles and could induce a shift of fingerprint pattern towards the detection of only one or two loci. This enables the use of STRs as direct genetic markers for linkage analysis, without cloning of the corresponding DNA fragment. Moreover, the set of STR variants can suggest consensus sequences allowing some prediction of the banding pattern.     

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.     

Isolation and characterization of polymorphic simple sequence repeat loci in Armillaria ostoyae

A modified hybridization strategy was used to construct a microsatellite enriched library from DNA of Armillaria ostoyae, a serious root pathogen on pine. Sequence characterization of 19 random clones revealed 12 distinct loci harbouring a repetitive motif. Primer design from the flanking regions allowed for their development as polymerase chain reaction based markers. Polymorphic assessment at both the population and global levels revealed levels of variation useful for genetic studies. The level of cross‐species amplification observed with closely related Armillaria species was high, raising the possible exploitation of these primers across the genus.     

A polymorphic complex dinucleotide repeat at the telomeric D8S7 locus.

Two cosmids isolated from a flow-sorted chromosome 8 library by hybridization with pSW50 (D8S7) were screened for GT microsatellite sequences. Both contained a positive 900-bp Hind III-Xba I fragment. Sequencing revealed a complex dinucleotide repeat. Flanking oligonucleotide primers were synthesized, and the polymerase chain reaction products produced by these primers were typed within the CEPH panel of families. A two-allele polymorphism was identified that is in linkage disequilibrium with a previously described insertion-deletion polymorphism at this locus.     

A new statistic for the analysis of association between trait and polymorphic marker loci

Inference for detecting the existence of an association between a diallelic marker and a trait locus is based on the chi-squared statistic with one degree of freedom. For polymorphic markers with m alleles (2), three approaches are mainly used in practice. First, one may use Pearson's chi-squared statistic with m-1 degrees of freedom (d.f.) but this leads to a loss in test power. Second, one can select an allele to be the most associated and then collapse the other allele categories into a single class. This reduces in a biased way, the locus to a diallelic system. Third, one may use the Terwilliger [J.D. Terwilliger, Am. J. Hum. Genet. 56 (1995) 777] likelihood ratio statistic which has a non-standard unknown limiting probability distribution. In this paper, we propose a new statistic, L(D), based on the second testing approach. We derive the asymptotic probability distribution of L(D) in an easy way. Simulation studies show that L(D) is more powerful than Pearson's chi-squared statistic with m-1 d.f.     

A continuous restriction map from HLA-E to HLA-F. Structural comparison between different HLA-A haplotypes

The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.     

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.     

New polymorphic DNA marker close to the fragile site FRAXA

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.     

Ten new highly polymorphic microsatellite loci in the blood clam Scapharca broughtonii

Ten novel microsatellite loci were isolated from blood clam Scapharca broughtonii, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. The number of alleles ranged from 11 to 23, and the observed and expected heterozygosities ranged from 0.63 to 0.93 and 0.66 to 0.95, respectively. Four loci showed significant Hardy–Weinberg disequilibrium at P < 0.05 level. The high variabilities revealed in this study suggest that microsatellites should prove useful for various genetic investigations.     

Molecular analysis of HLA-A2.4 functional variant KLO: close structural and evolutionary relatedness to the HLA-A2.2 subtype

The structure of an HLA-A2.4 functional variant (A2.4c) expressed on donor KLO has been examined by comparative peptide mapping with other HLA-A2 antigens of known structure and radiochemical sequencing. All the peptide differences between A2.4c and A2.1 could be accounted for by five amino acid changes at positions 9, 43, 66, 95, and 156. The nature of residues 9, 43, and 95 in A2.4c was determined by sequencing to be identical to those in A2.2Y. The nature of residue 156 in A2.4c was also assigned as identical to that in A2.2Y on the basis of the identity of the corresponding peptide in its chromatographic comparison with A2.2Y. Position 66 was unique to A2.4c. It was determined to be an Asn residue instead of the Lys present in all other HLA-A2 antigens of known structure. This was the only detected amino acid difference between A2.4c and A2.2Y. The results indicate that, from a structural point of view, A2.4c is most closely related to the A2.2 subtype antigens and not to other A2.4 antigens. The data are compatible with the assumption that A2.4c was derived from A2.2Y by a single point mutation event.     

A polymorphic dinucleotide repeat in the human IL-10 promoter

The polymorphism data reported in this paper have been submitted to the Human Genome databse and have been assigned the accession number G00-603-930     

Detection of polymorphic loci in complex genomes with synthetic tandem repeats.

Sixty synthetic probes mimicking minisatellite structures have been used on Southern blots bearing a set of DNA samples from a panel of complex genomes. They enable the detection of polymorphic loci in all the species tested and sometimes provide directly usable genetic markers. The general approach reported here should facilitate the study of genetic variability and the efficient development of genetic markers necessary for the mapping of complex genomes.     

Twelve new polymorphic microsatellite loci and PCR multiplexing in the whitefly, Bemisia tabaci

Twelve new dinucleotide microsatellite loci of the whitefly, Bemisia tabaci, were obtained from enriched genomic libraries. Three polymerase chain reaction multiplex sets comprising three, five and four loci were optimized and characterized across 133 B. tabaci females from Israeli rearings and natural populations collected in four Mediterranean countries (Tunisia, France, Spain and Morocco). There were three to 24 alleles per locus and the observed heterozygosity was from 0.084 to 0.420. Deviation from Hardy-Weinberg equilibrium was detected at four loci associated with significant heterozygote deficiencies due to null alleles and presence of subpopulations that were mostly in the Tunisian sample. The 12 loci carried independent information.     

Isolation and characterization of tetranucleotide repeat polymorphic microsatellite loci in Larus saundersi (Aves, Laridae)

Nine polymorphic microsatellite loci were isolated, using tetranucleotide repeat oligonucleotide probes from an enriched DNA library of the globally "vulnerable" Saunders's gull (Larus saundersi), collected from the Yancheng coastal wetland, one of the three remaining breeding sites in China. Six breast muscle tissues and 16 blood samples from 22 gulls and eight eggshell membrane tissues were collected for this analysis. The number of alleles per locus ranged from 4 to 15, with a mean of 8.9. The observed and expected heterozygosities ranged from 0.58 to 0.89 and 0.58 to 0.9, with means of 0.77 and 0.81, respectively. No significant linkage disequilibrium and no divergence from Hardy-Weinberg equilibrium were detected among these loci. Based on Micro-Checker tests, no null alleles are present at any of the loci. The microsatellite loci described here will be valuable for exploring population genetic structure and for other relevant genetic studies of Saunders's gull.