Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.     

IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques

 The BARE-1 retrotransposon is an active, dispersed, and highly abundant component of the genome of barley (Hordeum vulgare) and other species in its genus. Like all retrotransposons of its kind, BARE-1 is bounded by long terminal repeats (LTRs). We have developed two amplification-based marker methods based on the position of given LTRs within the genome. The IRAP (Inter-Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two LTRs using outward-facing primers annealing to LTR target sequences. In REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), amplification between LTRs proximal to simple sequence repeats such as constitute microsatellites produces markers. The methods can distinguish between barley varieties and produce fingerprint patterns for species across the genus. The patterns indicate that although the BARE-1 family of retrotransposons is disperse, these elements are locally clustered or nested and often found near tandem arrays of a simple sequence repeat. Received: 30 June 1998 / Accepted: 21 August 1998     

Genetic variability in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) and in the <Emphasis Type="Italic">Helianthus</Emphasis> genus as assessed by retrotransposon-based molecular markers

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon’s and Jaccard’s indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae)

Spartina x townsendii arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced Spartina alterniflora, native to the eastern seaboard of North America. Duplication of the hybrid genome gave rise to Spartina anglica, a vigorous allopolyploid involved in natural and artificial invasions on several continents. This system allows investigation of the early evolutionary changes that accompany stabilization of new allopolyploid species. Because allopolyploidy may be a genomic shock, eliciting retroelement insertional activity, we examined whether retrotransposons present in the parental species have been activated in the genome of S. anglica. For this purpose we used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposons-microsatellite amplified polymorphism (REMAP) markers, which are multilocus PCR-based methods detecting retrotransposon integration events in the genome. IRAP and REMAP allowed the screening of insertional polymorphisms in populations of S. anglica. The populations are composed mainly of one major multilocus genotype, identical to the first-generation hybrid S. x townsendii. Few new integration sites were encountered in the young allopolyploid genome. We also found strict additivity of the parental subgenomes in the allopolyploid. Both these findings indicate that the genome of S. anglica has not undergone extensive changes since its formation. This contrasts with previous results from the literature, which report rapid structural changes in experimentally resynthesized allopolyploids.     

Efficiency of different PCR-based marker systems for assessment of Iris pumila genetic diversity

We investigated informativeness and effectiveness of different marker types (ISSR, IRAP, REMAP, RGAP and LP-PCR that employ primers based on the conservative sequences of abiotic stress response genes) to study genetic diversity of Iris pumila L. By the number of amplicons per primer, number of polymorphic amplicons per primer and resolving power index (Rp), ISSR-markers were the most efficient followed by LP-PCR-markers. In order of decreasing value of indicators of genetic diversity “the percentage of polymorphic bands”, and “the average Jaccard? genetic distance between plants”, marker systems may be arranged as follows: ISSR > RAPD > LP-PC > RGAP ≈ IRAP. For ISSR-markers, the percentage of polymorphic bands was 1.3–1.7 times higher than for the others, and the average genetic distance was 1.2–1.3 times higher. Different marker systems were ranked by the value of Nei? gene diversity and the Shannon? index as follows: ISSR > RAPD ≈ LP-PCR > RGAP ≈ IRAP, with the highest and the lowest values differing 1.4 times. Genetic population structure was investigated with program Structure 2.3. The data of all marker systems suggest that all genomes under study belonged to one population. The PCoA and cluster analyses based on genetic distances showed distinctions in clustering generated from different markers data and summarized data, as well as the lack of strong clusters. Mantel test revealed significant positive correlation between the matrices of genetic distances generated by the data of almost all marker systems. The strongest correlation was found between RGAP- and IRAP-markers (r = 0.452, p = 0.01) and between RGAP and ISSR (r = 0.430, p = 0.01). ISSR, RAPD and LP-PCR proved to be more effective for the study of I. pumila genetic diversity, nevertheless, joint use of different marker systems will provide a more comprehensive assessment of variation in different genomic regions.