title: Detection and genotyping of restriction fragment associated polymorphisms in polyploid crops with a pseudo-reference sequence: a case study in allotetraploid Brassica napus.
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.
authors.forename: Xun
authors.initials: X
authors.lastname: Chen
authors.forename: Xuemin
authors.initials: X
authors.lastname: Li
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.forename: Jinsong
authors.initials: J
authors.lastname: Xu
authors.forename: Zhikun
authors.initials: Z
authors.lastname: Wu
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Muhammad
authors.initials: M
authors.lastname: Younas
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Luo
authors.forename: Jiangsheng
authors.initials: J
authors.lastname: Wu
authors.forename: Songnian
authors.initials: S
authors.lastname: Hu
authors.forename: Kede
authors.initials: K
authors.lastname: Liu
mesh_terms: D029688:Brassica napus / Q000235:genetics*; D002874:Chromosome Mapping; D018556:Crops, Agricultural / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D008040:Genetic Linkage; D015698:Genomic Library; D005838:Genotype; D060005:Genotyping Techniques; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011123:Polyploidy; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D018744:DNA, Plant