Sequence-tagged Site Analysis Research Articles

Construction of a Genetic Linkage Map Based on Amplified Fragment Length Polymorphism Markers and Development of Sequence-Tagged Site Markers for Marker-Assisted Selection of the Sporeless Trait in the Oyster Mushroom (Pleurotus eryngii)

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.

IDENTIFICATION OF POTATO VARIETIES BY DNA PROFILING

Two DNA profiling methods have been used for variety discrimination in potatoes. Sequence tagged site analysis of microsatellites was shown to be very efficient and was more reliable than inter-SSR PCR analysis. Using only 3 microsatellite primer pairs, it was possible to distinguish between 50 potato varieties.

Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage.

We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.

Characterization and Genomic Mapping of Genes and Pseudogenes of a New Human Protein Tyrosine Phosphatase

Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTP4A bears the signature active site of all PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNAs encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3′-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescencein situhybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11.

DNA profiling techniques for plant variety identification

The unambiguous identification of plant varieties is important in many areas of agriculture and plant biology research. New techniques based on DNA profiling provide novel approaches to varietal identification which offer advantages over traditional morphological comparisons. These advantages include great potential resolving power, data that can be analysed objectively, testing at all stages of development, and cost effectiveness. In this review, the advantages of recently introduced polymerase chain reaction (PCR) based methods are compared with the well-established restriction fragment length polymorphism technique. Two approaches to PCR-based DNA profiling (random amplification of polymorphic DNA, sequence-tagged sites analysis) are compared and contrasted, and statistical methods for the analysis of data are described. The potential use of these techniques for Plant Breeders Rights registration in Australia is outlined.

Construction of rare restriction site (NotI, SacII and ClaI) linking libraries and sequence-tagged site analysis of single-copy clones from a human chromosome-3-specific library

We have constructed rare restriction-site (NotI, SacII and ClaI) chromosome 3 (Chr 3) -specific linking libraries in a plasmid-based vector by mass transfer of a λ phage human Chr-3-specific library (LA03NS01-ATCC57717) into pUC18. Total plasmid DNA isolated from the plasmid-based Chr-3-specific library was digested with either ClaI, NotI or SacII. Linear molecules were separated from undigested circles by pulsed-field polyacrylamide-gel electrophoresis. Purified linear molecules were circularized with T4 DNA ligase and transformed into bacteria. The resulting clones were greatly enriched for sequences recognized by the original restriction endonuclease used for digestion (83 to 95%). These sublibraries are composed of 600 (NotI) 1000 (SacII) or 30000 (ClaI) clones. Thus, this procedure allows for easy isolation of Chr-3-specific DNA clones containing a variety of rare restriction sites. Sequence-tagged site (STS) data are also presented for five site-specifically mapped Chr-3-specific DNA clones. These studies may facilitate the construction of region-specific linking libraries for mapping of various disease-specific loci on Chr 3.