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Genome-wide microsatellite identification in the fungus Anisogramma anomala using Illumina sequencing and genome assembly.

Cai G, Leadbetter CW, Muehlbauer MF, Molnar TJ, Hillman BI - PLoS ONE (2013)

Bottom Line: Primers were successfully designed for 42,923 loci (97%).Twenty-three (9.7%) were found to be perfect polymorphic loci.Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers The State University of New Jersey, New Brunswick, New Jersey, United States of America.

ABSTRACT
High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the "Seq-to-SSR" approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel "Seq-Assembly-SSR" approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.

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Frequency distribution of microsatellite loci in Anisogramma anomala by motif length.
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pone-0082408-g002: Frequency distribution of microsatellite loci in Anisogramma anomala by motif length.

Mentions: The identified microsatellite motifs in A. anomala consist of 37,824 mono- (81.0%), 4,575 di- (9.8%), 3,264 tri- (7.0%), 677 tetra- (1.5%), 111 penta- (0.24%), and 226 hexa- (0.48%) nucleotide motifs (Figure 2). The number of tri-nucleotide motifs is 71% of that of the di-nucleotide motifs, and the number of hexa-nucleotide motifs is more than twice that of the penta-nucleotide motifs. The over-representation of tri- and hexa- motifs may be explained by contribution of coding sequences—these two types of motifs are usually over-represented in coding regions likely because they do not interrupt the open reading frames [33].


Genome-wide microsatellite identification in the fungus Anisogramma anomala using Illumina sequencing and genome assembly.

Cai G, Leadbetter CW, Muehlbauer MF, Molnar TJ, Hillman BI - PLoS ONE (2013)

Frequency distribution of microsatellite loci in Anisogramma anomala by motif length.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842260&req=5

pone-0082408-g002: Frequency distribution of microsatellite loci in Anisogramma anomala by motif length.
Mentions: The identified microsatellite motifs in A. anomala consist of 37,824 mono- (81.0%), 4,575 di- (9.8%), 3,264 tri- (7.0%), 677 tetra- (1.5%), 111 penta- (0.24%), and 226 hexa- (0.48%) nucleotide motifs (Figure 2). The number of tri-nucleotide motifs is 71% of that of the di-nucleotide motifs, and the number of hexa-nucleotide motifs is more than twice that of the penta-nucleotide motifs. The over-representation of tri- and hexa- motifs may be explained by contribution of coding sequences—these two types of motifs are usually over-represented in coding regions likely because they do not interrupt the open reading frames [33].

Bottom Line: Primers were successfully designed for 42,923 loci (97%).Twenty-three (9.7%) were found to be perfect polymorphic loci.Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers The State University of New Jersey, New Brunswick, New Jersey, United States of America.

ABSTRACT
High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the "Seq-to-SSR" approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel "Seq-Assembly-SSR" approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.

Show MeSH
Related in: MedlinePlus