DOI: 10.5281/zenodo.13364281
title: Java tool for PCR, in silico PCR and genotyping
title: in silico PCR genotyping applications

description: We performed in silico PCR analysis of several complete plant genomes using a list of primers corresponding to an inverted repeat sequence of Hordeum-Triticum Athos miniature inverted-repeat transposable element (MITE) sequences. MITE nonautonomous members of Class II element families are derived by internal deletion of autonomous elements, and they are short (70-300 bp in length) and have conserved terminal repeats.

For example, Athos, one of the MITE families described in grasses. Athos element sequences were collected from the genome of Hordeum vulgare, of which there are about 205 per complete genome. The Athos element sequences are highly truncated, including partial loss of terminal inverted repeats in the barley genome. Sequences of terminal inverted repeats contain multiple point mutations, insertions or deletions, which creates a difficulty for the selection of universal primers that would cover all whole copies of this element. Therefore, we selected all unique sequence variants for terminal inverted repeats and used them as primers to identify and obtain complete MITE elements for genomes of other cereals and as a negative control, we used the genome of human and long-horned nomad bee (Nomada hirtipes). Since the sequences of terminal inverted repeats for Athos element were different and quite degenerate, we used all 46 unique variants simultaneously as Forward primer in the analysis. The same primer will act as Forward and also Reverse. The length of the primers was 15 nucleotides, which localise to the furthest region of the terminal inverted repeat at the Athos element. The size for the amplicon in this case could be 30 to 200 nucleotides, including truncated elements with a central part. We used search conditions with control options: type=primer number3errors=0; minlen=30; maxlen=200. The results of this analysis are represented in Table 2. In the genome of Hordeum vulgare we identified 768 Athos and related elements, which is much more than was detected by blast analysis (205 copies for GCF_904849725.1, Blast: RefSeq Genome Database). This is because we detected not only Athos elements but also related MITE elements with overlapping end repeats. 

In our analysis, we could only detect whole Athos and related elements that contained both repeats, whereas the central part could vary. For the Hordeum bulbosum genome, we detected a 1620 record number of complete Athos and related elements compared to other species of the Hordeum family. This corresponds to the doubled genome size of this species compared to other species of the Hordeum family. For wheat genomes (Aegilops tauschii, Triticum dicoccoides), being the most similar to species of the Hordeum family, numerous copies of the related Athos and related elements were detected, with this MITE occurring much more frequently in the wheat genome than in the genome of Hordeum vulgare. It is well observed that the copy number of Athos and related elements directly depends on the genome size; the larger the genome, the greater the copy number of this element detected.