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

Rocher S, Jean M, Castonguay Y, Belzile F - PLoS ONE (2015)

Bottom Line: 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.About 60% had a significant match on the Medicago truncatula syntenic genome.Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche et de Développement sur les Sols et les Grandes Cultures, Agriculture et agroalimentaire Canada, Quebec City (QC), Canada.

ABSTRACT
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 ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): 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 Medicago truncatula 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.

No MeSH data available.


Related in: MedlinePlus

3D representation of PCA of 72 plant samples (35 ATF0 and 37 ATF5) genotyped with two SNP loci datasetsA) 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.
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pone.0131918.g005: 3D representation of PCA of 72 plant samples (35 ATF0 and 37 ATF5) genotyped with two SNP loci datasetsA) 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.

Mentions: Principal component analysis of plant sample distribution in the multivariate space using 11,694 SNP loci showed a clear separation between ATF0 and ATF5 along the first axis, and a slightly higher dispersion of members of the ATF5 population when compared to ATF0 (Fig 5A). PCA performed with the 2,732 SNP loci retained after genotype-level filtration showed a similar discrimination between the two populations (Fig 5B). It is noteworthy that in both datasets, only 4% of the variability within the SNP loci is explained by the first axis, and 10% of the variance is explained by the first three components. A large proportion of the genetic variability remains unstructured (Fig 5C).


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

Rocher S, Jean M, Castonguay Y, Belzile F - PLoS ONE (2015)

3D representation of PCA of 72 plant samples (35 ATF0 and 37 ATF5) genotyped with two SNP loci datasetsA) 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482585&req=5

pone.0131918.g005: 3D representation of PCA of 72 plant samples (35 ATF0 and 37 ATF5) genotyped with two SNP loci datasetsA) 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.
Mentions: Principal component analysis of plant sample distribution in the multivariate space using 11,694 SNP loci showed a clear separation between ATF0 and ATF5 along the first axis, and a slightly higher dispersion of members of the ATF5 population when compared to ATF0 (Fig 5A). PCA performed with the 2,732 SNP loci retained after genotype-level filtration showed a similar discrimination between the two populations (Fig 5B). It is noteworthy that in both datasets, only 4% of the variability within the SNP loci is explained by the first axis, and 10% of the variance is explained by the first three components. A large proportion of the genetic variability remains unstructured (Fig 5C).

Bottom Line: 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.About 60% had a significant match on the Medicago truncatula syntenic genome.Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche et de Développement sur les Sols et les Grandes Cultures, Agriculture et agroalimentaire Canada, Quebec City (QC), Canada.

ABSTRACT
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 ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): 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 Medicago truncatula 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.

No MeSH data available.


Related in: MedlinePlus