A New Genetic Linkage Map of the Zygomycete Fungus <em>Phycomyces blakesleeanus</em>

<div><p><i>Phycomyces blakesleeanus</i> is a member of the subphylum Mucoromycotina. A genetic map was constructed from 121 progeny of a cross between two wild type isolates of <i>P. blakesleeanus</i> with 134 markers. The markers were mostly PCR-RFLPs. Markers were located on 46 scaffolds of the genome sequence, covering more than 97% of the genome. Analysis of the alleles in the progeny revealed nine or 12 linkage groups, depending on the log of the odds (LOD) score, across 1583.4 cM at LOD 5. The linkage groups were overlaid on previous mapping data from crosses between mutants, aided by new identification of the mutations in primary metabolism mutant strains. The molecular marker map, the phenotype map and the genome sequence are overall congruent, with some exceptions. The new genetic map provides a genome-wide estimate for recombination, with the average of 33.2 kb per cM. This frequency is one piece of evidence for meiosis during zygospore development in Mucoromycotina species. At the same time as meiosis, transmission of non-recombinant chromosomes is also evident in the mating process in <i>Phycomyces</i>. The new map provides scaffold ordering for the genome sequence and a platform upon which to identify the genes in mutants that are affected in traits of interest, such as carotene biosynthesis, phototropism or gravitropism, using positional cloning.</p> </div

Comparison between genetic maps of <i>Phycomyces</i>.

<p>The previous mapping data is dark blue, redrawn from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058931#pone.0058931-Eslava4" target="_blank">[29]</a>. The positions of the genes in the new RFLP map (pale blue) are based on the locations of the closest markers to the gene. Five additional linkage groups were established in previous studies and remain to be anchored to the RFLP-based map.</p

Strains used in this study. NG: isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine.

<p>Strains used in this study. NG: isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine.</p

Progeny generated for genetic analysis. The parent named “progeny 82A” is derived from the cross between UBC21 x NRRL1555.

<p>Progeny generated for genetic analysis. The parent named “progeny 82A” is derived from the cross between UBC21 x NRRL1555.</p

Three steps in generating the <i>Phycomyces</i> genetic map.

<p><b>A.</b> Zygospores forming from a cross between UBC21 (+) and NRRL1555 (–) on V8 juice agar medium. The asexual sporangiophores have been removed from the plate for clarity. <b>B.</b> An example of primer design for PCR-RFLP markers. A restriction enzyme site polymorphism (HindIII *; scaffold 38 nucleotide position 361,161) between UBC21 and NRRL1555 was selected, and 550 bp on either side of the site examined by BLAST to ensure it was single copy in the genome and that it had no other restriction enzyme sites that would interfere with allele interpretation. <b>C.</b> HindIII digested PCR products resolved on a 1.2% agarose gel, from 17 progeny (a subset out of 121 total) and the parents NRRL1555 (N) and UBC21 (U) amplified with primers ALID1007-ALID1008. The markers (M) are the Invitrogen 1 kb+ ladder. The NRRL1555 allele generates 509 bp and 98 bp fragments. The UBC21 allele is uncut at 607 bp in size.</p

Mutations identified in strains of <i>Phycomyces</i>.

<p>A. Phenotypes on potato dextrose agar (PDA), PDA +250 mg/L 5-fluorouracil, YNB minimal medium, and YNB supplemented with uracil (20 mg/L) or lysine (30 mg/L) after three days growth at room temperature. The PDA was supplemented with uracil to support growth of the <i>pyrF</i> and <i>pyrG</i> mutants. <b>B.</b> The positions (red lines and strain names) and nature (codons and amino acid residue substitutions) of the mutations in the corresponding genes in these strains. <b>C.</b> Segregation data for <i>lysA</i>. Parents were A914 (A) and for UBC21 (U). Alleles are: for auxotrophy are either <i>A</i> for <i>lysA</i> or WT for wild type, the C or T nucleotide in the <i>lysA</i> gene, for PCR-RFLPs A for NRRL1555 and B UBC21, the <i>sex</i> gene is either <i>sexM</i> (<i>M</i>) or <i>sexP</i> (<i>P</i>), and the phototropism phenotype <i>I</i> for <i>madI</i> or WT for wild type. Progeny with both alleles are designated U and shaded blue and nt indicates a genotypic (rather than phenotypic) marker. <b>D.</b> Segregation data for <i>furA</i>. Parents were A34 (A) and NRRL1555 (N). Markers are: resistant (R) or sensitive (S) to 5-fluorouracil, the A or G nucleotide in the <i>furA</i> gene, <i>madD</i> (<i>D</i>) or wild type (WT) for phototropism, the sex phenotype is either (+) or (–) and equivalent PCR analysis for the <i>sexM</i> (<i>M</i>) or <i>sexP</i> (<i>P</i>) gene.</p

Additional file 2: of Identification of new biomarkers for Acute Respiratory Distress Syndrome by expression-based genome-wide association study

Table S2. Chi-square values of cross-referenced genes. Given that different microarray platforms have multiple probes for a single gene, the cross-referencing of such platforms generates numerous combinations of expression values for a given gene (32160 in this study). Chi-square value was calculated for each of these combinations and combination with the best chi-square value for a given gene was retained, which resulted in 3152 unique gene entries. These genes were linked to human genome and plotted against their location (Fig. 1). (XLS 185 kb

Additional file 1: of Identification of new biomarkers for Acute Respiratory Distress Syndrome by expression-based genome-wide association study

Table S1. Detailed representation of data obtained from GEO. ARDS gene expression submissions were retrieved from GEO using two terms “Acute lung injury” and “Lung injury”, which resulted in 23 and 25 data sets, respectively. These 48 entries were filtered down to 31 entries according to conditions described in Methods. The reason for filtering out an experiment is provided. (XLSX 16 kb

Pathway analysis.

<p>Top canonical pathways as predicted from the 65genes containing the76 SNPs that were identified using χ²tests. Pathway predictions were done using the Core Analysis function of Ingenuity Pathway Analysis. *, P-Value of <0.05 indicates a non-random association between the genes and pathway; **, Ratio of the number of genes in the dataset involved in the pathway to the total number of genes in the pathway.</p><p>Pathway analysis.</p

Participant demographics and comorbidities for the ARDS cases.

<p>*8 samples, 4 exome sequenced and 4 TaqMan genotyped ARDS patients did not have these phenotypes available. An additional 2 exome sequenced ARDS patients did not have ventilator-free day’s data. In addition to the 8 patients missing severity and mortality phenotype data, 2 patients were excluded from the regression because their phenotypes were thought to be missing until after the regressions were completed.</p><p>Participant demographics and comorbidities for the ARDS cases.</p