Java tool for PCR, in silico PCR and genotyping

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.本研究针对多个完整植物基因组开展计算机模拟PCR（in silico PCR）分析，所用引物组对应大麦-小麦属Athos微型反向重复转座元件（miniature inverted-repeat transposable element, MITE）序列的反向重复区域。MITE作为II类转座元件家族中的非自主成员，由自主转座元件内部缺失演化而来，其长度较短（70~300 bp）且拥有保守的末端重复序列。 以禾本科中已报道的MITE家族之一——Athos为例：其元件序列从栽培大麦（Hordeum vulgare）基因组中获取，该基因组中该元件的完整拷贝数约为205个。栽培大麦基因组中的Athos元件序列存在严重截短现象，包括末端反向重复序列的部分缺失。末端反向重复序列区域存在多个点突变、插入或缺失事件，这为能够覆盖该元件所有完整拷贝的通用引物设计带来了困难。因此，本研究选取末端反向重复序列的所有独特序列变体作为引物，用于从其他谷类作物基因组中鉴定并获取完整的MITE元件；同时选用人类与长角隧蜂（Nomada hirtipes）基因组作为阴性对照。由于Athos元件的末端反向重复序列存在显著差异且简并性较强，本研究同时将全部46种独特序列变体作为正向引物用于本次分析，且每条引物可同时兼任正向与反向引物。引物长度为15个核苷酸，结合位点位于Athos元件末端反向重复序列的最远端区域。此条件下扩增产物的长度范围为30~200 nt，可覆盖包含截短中央区域的元件。本次分析采用的搜索参数为：type=primer number，errors=0；minlen=30；maxlen=200。本次分析结果详见表2。在栽培大麦基因组中，本研究共鉴定出768个Athos及相关元件，远多于BLAST分析的检测结果（RefSeq基因组数据库GCF_904849725.1版本中仅检测到205个拷贝）。这是因为本研究不仅检测到了Athos元件，还同时鉴定出了末端重复序列存在重叠的相关MITE元件。 本次分析仅能检测到同时包含两个末端重复序列的完整Athos及相关元件，其中央区域序列可存在差异。与大麦属其他物种相比，球茎大麦（Hordeum bulbosum）基因组中被检测到的完整Athos及相关元件数量达1620个，这与该物种的基因组大小为大麦属其他物种两倍的特征相符。对于与大麦属物种亲缘关系最近的小麦族基因组（包括节节麦Aegilops tauschii、野生二粒小麦Triticum dicoccoides），本研究同样检测到了大量Athos相关元件，且该MITE在小麦基因组中的出现频率远高于栽培大麦基因组。研究结果清晰表明，Athos及相关元件的拷贝数与基因组大小直接相关：基因组越大，检测到的该元件拷贝数越多。