Abstract
BackgroundThe 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.ResultsTo 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.ConclusionsThese 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.
Highlights
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
We developed a bioinformatics pipeline termed RFAPtools to unambiguously discriminate between SNPs versus differences of homoeologous sequences in polyploid crops such as oilseed rape without a reference genome sequence
To estimate the number of SacI-MseI fragments generated with ddRADseq in the B. napus genome, we did in silico double digestion of the B. rapa chromosome A03 (31.72 Mb in length) as a representative with the same restriction enzymes and obtained 3,904 SacI-MseI fragments within the size range of 141–420 bp
Summary
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. To overcome the complexity of genomes and improve genotyping through-put, approaches such as complexity reduction of polymorphic sequences (CRoPS), restrictionsite-associated DNA sequencing (RADseq) and genotyping by sequencing (GBS) that coupled reduced representation libraries (RRLs) with NGS technologies have been developed [12,13,14,15,16,17] and extensively used to sequence a similar subset of restriction fragments from multiple individuals for accurate SNP discovery and genotyping at low cost [18]. No computational tool has the ability to unambiguously and efficiently discriminate SNPs from homoeologous sequence differences when both copies of the two homoeologous genomes are being sampled
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
