Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

<div><p>Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on <i>Ape</i>KI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (<i>Medicago sativa</i> spp. <i>sativa</i>): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the <i>Medicago truncatula</i> syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.</p></div

3D representation of PCA of 72 plant samples (35 ATF0 and 37 ATF5) genotyped with two SNP loci datasets

<p>A) 72 plant samples genotyped with 11,694 SNP loci and B) 72 plant samples genotyped with 2,732 SNP loci retained after genotype-level filtration for minimum read counts C) Cumulative proportion of variance explained by the first three components in the two SNP loci datasets.</p

PCR primers with respective Tm (°C) for the amplification of genome regions of <i>M</i>. <i>sativa</i> covering SNP loci identified with GBS.

<p>Predicted and observed size of amplified fragments and their location on the <i>Medicago truncatula</i> genome (<i>v4</i>.<i>0</i>) are indicated.</p><p>PCR primers with respective Tm (°C) for the amplification of genome regions of <i>M</i>. <i>sativa</i> covering SNP loci identified with GBS.</p

Example of comparison of GBS and 454 sequencing of TP61949 in eight plant samples.

<p>A) GBS and 454 read counts of each allele (A1|A2); B) predicted tetraploid allelic ratios (convergent ratios in green and discordant ratios in red); C) bi-allelic predicted genotype (A1, A2 and H) before genotype-level filtration and D) after genotype-level filtration of GBS data for minimum read counts (11 reads for homozygous genotypes, 2 reads of each allele for heterozygous genotypes, 0.1 as minimum minor allele frequency). Genotype calls showing concordance (green), discordance (red for GBS homozygotes and orange for GBS heterozygotes) with both sequencing methods or that are missing (white) before and after genotype-level filtration for minimum read counts. A complete representation of validation results for 14 SNP loci in eight plant samples is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131918#pone.0131918.s003" target="_blank">S3 Fig</a>.</p

Haplotypes identified with 454 sequences covering TP91313 in eight plant samples.

<p>Haplotypes defined with 454 sequences with perfect and imperfect match with GBS 64bp sequence are listed separately. Position of SNPs is based on location on <i>M</i>. <i>truncatula</i> reference sequence. SNPs included in UNEAK TP are highlighted in bold. RC of GBS alleles (A1 and A2) and 454 sequences covering each haplotype in the eight genotyped plant samples are indicated. Cumulative number of A1 like and A2 like reads are also presented. SNPs with RC ≥ 5% in individual plant samples were used to define haplotypes. Haplotypes with frequency < 5% in all individual plant samples are not indicated but total read counts supporting those other haplotypes are reported. Haplotypes corresponding to each of the 14 GBS loci are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131918#pone.0131918.s006" target="_blank">S3 Table</a>.</p

Observed frequency distribution of total read counts (A1+A2) supporting each genotype call in ATF0 and ATF5 populations.

<p>Genotype calls (5%) supported by >200 reads are not shown.</p

Development of hospice-palliative care with focus on Germany and the Czech Republic

Vývoj hospicové paliativní péče se zaměřením na Německo a Českou Republiku Development of hospice-palliative care with focus on Germany and the Czech Republic Ruth Kubíčková The aim of the thesis was to map the development, crucial moments and current .shape of hospice-palliative care and simultaneously confirm the thesis that the care of spiritual feature of dying persons has been an integral part of organized development of hospice care since its beginning and nowadays, it is still one of its principal themes. The first chapter deals with branch terminology since its clear definition is essential, not only for dialogue inside the system of palliative care providers and experts, but for interdisciplinary communication and cooperation which is - according to the definition of palliative care - the foundation of its holistic approach to patients. The second and third chapters are concerned with the development of nursing care since the ancient times to the diversification of modern hospice-palliative care at the end of the twentieth century. They deduce that the Christianity, with its central commandment to love thy neighbour as thyself, has changed the attitude to care of suffering persons, both in the nursing care and in shapes and ways of providing care. In the response to revolutionary changes in..

Additional file 3: Table S4a. of A proteome analysis of freezing tolerance in red clover (Trifolium pratense L.)

List of DIGE-spots with homology with sequences in databases that are up-regulated in response to cold acclimation (ANOVA, P < 0.01). (XLSX 27 kb

Additional file 4: Table S4b. of A proteome analysis of freezing tolerance in red clover (Trifolium pratense L.)

List of DIGE-spots with homology with sequences in databases that are down-regulated in response to cold acclimation (ANOVA, P < 0.01) (XLSX 18 kb