Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds

<div><p>The paternally inherited Y chromosome displays the population genetic history of males. While modern domestic horses (<i>Equus caballus</i>) exhibit abundant diversity within maternally inherited mitochondrial DNA, no significant Y-chromosomal sequence diversity has been detected. We used high throughput sequencing technology to identify the first polymorphic Y-chromosomal markers useful for tracing paternal lines. The nucleotide variability of the modern horse Y chromosome is extremely low, resulting in six haplotypes (HT), all clearly distinct from the Przewalski horse (<i>E. przewalskii</i>). The most widespread HT1 is ancestral and the other five haplotypes apparently arose on the background of HT1 by mutation or gene conversion after domestication. Two haplotypes (HT2 and HT3) are widely distributed at high frequencies among modern European horse breeds. Using pedigree information, we trace the distribution of Y-haplotype diversity to particular founders. The mutation leading to HT3 occurred in the germline of the famous English Thoroughbred stallion “Eclipse” or his son or grandson and its prevalence demonstrates the influence of this popular paternal line on modern sport horse breeds. The pervasive introgression of Thoroughbred stallions during the last 200 years to refine autochthonous breeds has strongly affected the distribution of Y-chromosomal variation in modern horse breeds and has led to the replacement of autochthonous Y chromosomes. Only a few northern European breeds bear unique variants at high frequencies or fixed within but not shared among breeds. Our Y-chromosomal data complement the well established mtDNA lineages and document the male side of the genetic history of modern horse breeds and breeding practices.</p> </div

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients

Founders contributing to modern horses.

<p>Stallions with descendants in our dataset are listed, giving their origin, HT and their distribution in extant horse breeds (as estimated from our dataset).</p

Haplotype network of the six modern and two Przewalski horse HTs.

<p>Circles represent the haplotypes with the area proportional to the observed frequency in 20 male horses in the initial Y-chromosomal sequence analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060015#pone.0060015.s014" target="_blank">Table S4</a>). HT1, n = 7 (three Lipizzan, two Arabian, one Shetland pony, one Shire horse); HT2, n = 5 (five Lipizzan); HT3, n = 3 (one Thoroughbred, one Trakehner, one Quarter horse); HT 4 (one Icelandic horse), HT5 (one Norwegian Fjord horse), HT6 (one Shetland pony), HTPrz1 (one Przewalski horse), HTPrz2 (one Przewalski horse). A dashed line between the haplotypes indicates, that the polymorphism is located on the highly variable contig YE3, which was omitted when estimating divergence time and nucleotide diversity.</p

Geographic distribution and history of Y-haplotypes in modern horse breeds.

<p>(a) Geographic distribution of Y-chromosomal haplotypes in a set of modern horse breeds. Only a few important breeds are specified, the full list with information on breeds and HT frequencies is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060015#pone.0060015.s017" target="_blank">Table S7</a>. (b) Origin of modern domestic horse founders deduced from pedigree data. Each founder is represented by a drum with its size proportionally to the number of offspring in the dataset. The number in the drums serve as founder identifiers. Detailed information on founders (name, year of birth, breed, origin, information on import) is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060015#pone-0060015-t001" target="_blank">Table 1</a>. (c) Male introgression routes deduced from the pedigree and the distribution of HT2 and HT3 in our dataset. HT2 (yellow arrows) arrived from South-East at early times and has been spread during the Neapolitan and Oriental introgression waves, but did not reach Northern Europe and the Iberian peninsula. The English wave in red is well documented through pedigree data and the spread of HT3 (red arrows). Due to the ubiquitous occurrence of HT1, this haplotype is not considered. The black solid lines reflect the limits of the observation of HT2 and HT3.</p

Pedigree of Darley Arabians progeny depicting the origin of HT3 from HT2.

<p>Breeds of analysed males are listed on the bottom and the haplotypes of their ancestors are reconstructed (HT2-yellow, HT3-red, unknown-grey). Selected famous stallions are shown by name; dotted lines connect relatives where at least one ancestor is omitted. No descendants from “Pot8os” and “Waxy” were available apart from “Whalebone, 1807”. The mutation leading to HT3 must have occurred either in the germline of stallion “Eclipse” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060015#pone.0060015-Eclipse1" target="_blank">[54]</a> or in his son “Pot8os” or in his grandson “Waxy” and rose to very high frequency in the English Thoroughbred and many sport horse breeds through the progeny of the stallion “Whalebone”.</p

Primer and hydrolysis probe sequences.

<p>¹ GenBank (NCBI) accession number in brackets.</p><p>² 3′-prime allele-specific bases of ARMS primers are indicated by bold and underlined letters, intentional, additional mismatches to increase specificity are underlined.</p><p>Tm: melting temperature, °C; bp: base pair</p><p>Primer and hydrolysis probe sequences.</p

Workflow of SNPase-ARMS qPCR.

<p>Fig 1 shows the workflow of SNPase-ARMS qPCR. 1. SNPase preamplification: with the SNPase polymerase, allele-specific primers amplify the target DNA based on the respective single nucleotide variant (SNV) with extreme sensitivity. In 15 PCR cycles the ratio between target (blue circles) and non-target (orange triangles) DNA is changed towards the target DNA. An exemplary temperature protocol for the <i>BRAF</i> V600E assay is shown. The last PCR cycle ends in a 4°C step to inhibit unspecific elongation. The PCR plate is put on ice immediately afterwards, and kept on ice during the next step. 2. Probe and Polymerase: the reaction tube (PCR plate) is opened (preferentially in a separate room to avoid contamination), and 5′ to 3′ exonuclease active polymerase and hydrolysis probe are added. 3. qPCR: in this step, the already preamplified target gene is amplified by the 5′ to 3′ exonuclease active polymerase. The initial step, 95°C for 15 minutes, inhibits the residual SNPase polymerase, and activates the newly added hot-start polymerase. During the following standard qPCR, the sequence-specific hydrolysis probe is cleaved and a fluorescence signal corresponding to the number of cleaved probes is created (symbolized by blue circles with a yellow corona). An exemplary temperature protocol for the <i>BRAF</i> V600E assay is shown. 4. Analysis: the qPCR is evaluated via the amplification plot. Quantification of positive samples is performed with the standard curve method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.ref037" target="_blank">37</a>] using the ViiA^™ Software, v1.2.4.</p

Poisson distribution at low copy numbers of the <i>BRAF</i> V600E target mutation.

<p>At very low copy numbers, only part of the reaction wells can contain the target gene due to Poisson distribution. Therefore, even under ideal conditions in less than 100% of the reaction wells target DNA can be detected (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.g002" target="_blank">Fig 2</a>). Fig 6 shows the relation between spiked copies (x-axis) and the percentage of positive reactions (y-axis). White bars represent the percentage of reactions that are expected to yield positive signals following ideal Poisson distribution. Light and dark grey columns represent the percentage of reactions that yielded positive signals for <i>BRAF</i> V600E detection in a background of 10⁵ and 2 × 10⁵ wild type copies respectively. Reaction numbers: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.g005" target="_blank">Fig 5</a>. For details on qPCR plate layout see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.g002" target="_blank">Fig 2</a>. While at 10 copies per reaction the number of positive wells nearly represents ideal conditions, at 1 and 3 copies the assay detects less than expected positive samples. Reduction of positive calls below 10 starting copies is common in PCR based methods, even without the demanding conditions of mutation detection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.ref049" target="_blank">49</a>]. Under the extreme sensitivity and specificity constraints tested, the performance of this assay, i.e. correct calling of on average one single mutation per reaction in 2 × 10⁵ wild type DNAs, is unprecedented in qPCR. The reduction of positive calls at very low copy number is the trade-off for extreme specificity. As shown, it can be compensated by the possibility to apply multiple wells per run. The <i>BRAF</i> V600E assay correctly detects and differentiates between 0, 1, 3 and 10 spiked copies both against a background of 10⁵ and 2 × 10⁵ wild type copies in our setting (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142273#pone.0142273.g005" target="_blank">Fig 5</a>).</p