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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

Haplotypes identified with 454 sequences covering TP91313 in eight plant samples.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 M. truncatula 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 S3 Table.
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pone.0131918.g003: Haplotypes identified with 454 sequences covering TP91313 in eight plant samples.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 M. truncatula 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 S3 Table.

Mentions: A variable number of SNPs (4 to 43) were detected in the first 400 bp of the retained sequences and were used to define haplotypes as illustrated in Fig 3, S2 Fig and S3 Table. Among those SNPs, ten are affecting ApeKI restriction sites, including five affecting sites that generates GBS reads (TP7278, TP80194, TP91313 and TP31029; S2 Fig). Up to eleven different haplotypes per amplicon were detected among the eight plant samples. However, in most cases no more than four haplotypes containing GBS alleles were predicted in any given plant sample, except for TF5-5 in TP80194 and TP79240, TF0-36 in TP79240 and TF5-20 in TP31029.


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)

Haplotypes identified with 454 sequences covering TP91313 in eight plant samples.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 M. truncatula 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 S3 Table.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131918.g003: Haplotypes identified with 454 sequences covering TP91313 in eight plant samples.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 M. truncatula 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 S3 Table.
Mentions: A variable number of SNPs (4 to 43) were detected in the first 400 bp of the retained sequences and were used to define haplotypes as illustrated in Fig 3, S2 Fig and S3 Table. Among those SNPs, ten are affecting ApeKI restriction sites, including five affecting sites that generates GBS reads (TP7278, TP80194, TP91313 and TP31029; S2 Fig). Up to eleven different haplotypes per amplicon were detected among the eight plant samples. However, in most cases no more than four haplotypes containing GBS alleles were predicted in any given plant sample, except for TF5-5 in TP80194 and TP79240, TF0-36 in TP79240 and TF5-20 in TP31029.

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