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Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

ABSTRACT: BACKGROUND: The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo. RESULTS: Illumina sequencing technology and reference based assembly on Red Jungle Fowl genome sequences were used. Results of genome resequencing of pooled DNA of each 10 BL and SL chickens reached 5.1x and 7.0x coverage, respectively. The total number of SNPs was 4.8 and 5.5 million in BL and SL genome, respectively. Through a series of filtering processes, a total of ~1 million unique SNPs were found in the SL alone. Eventually of the 156 reliable marker SNPs, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, 139 genes were chosen for further analysis. Of these, 14 randomly chosen SNPs were examined for SNP verification by PCR and Sanger sequencing to detect SNP positions in 20 BL and 70 SL chickens. The results of the analysis of the 14 SNPs clearly showed differential frequencies of nucleotide bases in the SNP positions between BL and SL chickens. Bioinformatic analysis showed that the 156 most reliable marker SNPs included genes involved in dermatological diseases/conditions such as ADAMTS13, ASPM, ATP6V0A2, BRCA2, COL12A1, GRM5, LRP2, OBSCN, PLAU, RNF168, STAB2, and XIRP1. Intermolecular gene network analysis revealed that candidate genes identified in SLV play a role in networks centered on protein kinases (MAPK, ERK1/2, PKC, PRKDC), phosphatase (PPP1CA), ubiquitinylation (UBC) and amyloid production (APP). CONCLUSIONS: Various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the SLV chicken through genome resequencing. The genetic markers and bioinformatic interpretations of amino acid mutations found in SLV chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility.

SUBMITTER: Jang HM

PROVIDER: S-EPMC4152579 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

Jang Hyeon-Min HM Erf Gisela F GF Rowland Kaylee C KC Kong Byung-Whi BW

BMC genomics 20140823

<h4>Background</h4>The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.<h4>Results</h4>Illumina sequ ...[more]

PMID: 25151476

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Project description:BackgroundNext-generation sequencing technologies are widely used for detection of millions of Single Nucleotide Polymorphisms (SNPs) and also provide a means of assessing their variation. This information is useful for composing subsets of highly informative SNPs for region-specific or genome-wide analysis and to identify mutations regulating phenotypic differences within or between populations. In this study, we investigated the sensitivity of SNP detection and introduced the flanking SNPs value (FSV) as a novel measure for predicting SNP-variability using ~5X genome resequencing with ABI SOLID and DNA pools from two chicken lines divergently selected for juvenile bodyweight.ResultsGenotyping with a 60 K SNP chip revealed polymorphisms within or between two divergently selected chicken lines for 31 363 SNPs, 48% of which were also detected using resequencing of DNA pools. SNP detection using resequencing was more powerful for positions with larger differences in allele frequency between the lines. About 50% of the SNPs with non-reference allele frequencies in the range 0.5-0.6 and 67% of those with frequencies > 0.9 could be detected. On average, ~3.7 SNPs/kb were detected by resequencing, with about 5% lower density on microchromosomes than on macrochromosomes. There was a positive correlation between the observed between-line SNP variation from the 60 K chip analysis and our proposed FSV score computed from the genome resequencing data. The strongest correlations on macrochromosomes and microchromosomes were observed when the FSV was calculated with total flanking regions of 62 kb (correlation 0.55) and 38 kb (correlation 0.45), respectively.ConclusionsGenome resequencing with limited coverage (~5X) using pooled DNA samples and three non-reference reads as a threshold for SNP detection, identified 50 - 67% of the 60 K SNPs with a non-reference allele frequency larger than 0.5. The SNP density was around 5% lower on the microchromosomes, most likely because of their higher gene content. Our proposed method to estimate the SNP variation (FSV) uses additional sequence information to better predict SNP informativity. The FSV scores showed higher correlations for SNPs with a larger difference in allele frequency between the populations. The correlation was strongest on macrochromosomes, probably due to a lower recombination rate.

Project description:Cucumber is an economically important vegetable crop worldwide. Powdery mildew (PM) is one of the most severe diseases that can affect cucumber crops. There have been several research efforts to isolate PM resistance genes for breeding PM-resistant cucumber. In the present study, we used a chromosome segment substitution line, SSL508-28, which carried PM resistance genes from the donor parent, JIN5-508, through twelve generations of backcrossing with a PM-susceptible inbred line, D8. We performed whole-genome resequencing of SSL508-28 and D8 to identify single nucleotide polymorphisms (SNPs), and insertions and deletions (indels). When compared against the reference genome of the inbred cucumber line 9930, a total of 468,616 SNPs and 67,259 indels were identified in SSL508-28, and 537,352 SNPs and 91,698 indels were identified in D8. Of these, 3,014 non-synonymous SNPs and 226 frameshift indels in SSL508-28, and 3,104 non-synonymous SNPs and 251 frameshift indels in D8, were identified. Bioinformatics analysis of these variations revealed a total of 15,682 SNPs and 6,262 indels between SSL508-28 and D8, among which 120 non-synonymous SNPs and 30 frameshift indels in 94 genes were detected between SSL508-28 and D8. Finally, out of these 94 genes, five resistance genes with nucleotide-binding sites and leucine-rich repeat domains were selected for qRT-PCR analysis. This revealed an upregulation of two transcripts, Csa2M435460.1 and Csa5M579560.1, in SSL508-28. Furthermore, the results of qRT-PCR analysis of these two genes in ten PM resistant and ten PM susceptible cucumber lines showed that when exposed to PM, Csa2M435460.1 and Csa5M579560.1 exhibited a higher expression level of resistant lines than susceptible lines. This indicates that Csa2M435460.1 and Csa5M579560.1 are candidate genes for PM resistance in cucumber. In addition, the non-synonymous SNPs in Csa2M435460.1 and Csa5M579560.1, identified in SSL508-28 and D8, might be the key to high PM-resistance in SSL508-28.

Dataset Information

Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

Publications

Genome resequencing and bioinformatic analysis of SNP containing candidate genes in the autoimmune vitiligo Smyth line chicken model.

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