Detection and genotyping of restriction fragment associated polymorphisms in polyploid crops with a pseudo-reference sequence: a case study in allotetraploid Brassica napus
Open Access
- 24 May 2013
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Genomics
- Vol. 14 (1), 1-12
- https://doi.org/10.1186/1471-2164-14-346
Abstract
Background The presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops. Results To address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds. Conclusions These results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence.Keywords
This publication has 42 references indexed in Scilit:
- Multiplexed shotgun genotyping for rapid and efficient genetic mappingGenome Research, 2011
- Parent-independent genotyping for constructing an ultrahigh-density linkage map based on population sequencingProceedings of the National Academy of Sciences, 2010
- Comparative Analysis between Homoeologous Genome Segments ofBrassica napusand Its Progenitor Species Reveals Extensive Sequence-Level DivergencePlant Cell, 2009
- High-throughput genotyping by whole-genome resequencingGenome Research, 2009
- Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencingPlant Biotechnology Journal, 2009
- Identification of genetic variants using bar-coded multiplexed sequencingNature Methods, 2008
- SOAP: short oligonucleotide alignment programBioinformatics, 2008
- Sequence-Level Analysis of the Diploidization Process in the TriplicatedFLOWERING LOCUS CRegion ofBrassica rapaPlant Cell, 2006
- Structural variation in the human genomeNature Reviews Genetics, 2006
- Nuclear DNA content of some important plant speciesPlant Molecular Biology Reporter, 1991