Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions

Abstract

Background: Homoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.

Results: Sequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina's GoldenGate® Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.

Conclusions: This proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.

Figure 1

Brassica rapa region on chromosome A1 used for design of intergenomic SNPs. (A) Positions of BAC sequences relative to WGS sequence contigs. Green lines mark BAC sequences that were used for assay design, whereas red lines represent sequence contigs from working draft sequences of BAC clones that linked the WGS sequence contigs shown as black lines. Designations 37 – 51 refer to accession numbers AENI01000037.1 – AENI01000051.1. (B) Position of SNP assay sequences used in this study relative to the region of interest.

Figure 2

Analysis of Brassica accessions with oligonucleotide pool assays. Normalised fluorescence intensities and normalised Theta values are indicated on the x- and y-axis, respectively. Values for Brassica napus var. Express, Brassica rapa and Brassica oleracea accessions are displayed in grey, red and blue, respectively. For Brassica rapa and Brassica oleracea two accessions were analysed with four replicates each. Two independent plants with six replicates each were examined for Brassica napus var. Express. Panels A, B and C show the results for assays #637-11, #578-5 and #479-12b, the data for #386-14d, #11396 and #11303 are presented in panels D, E and F, respectively.

Figure 3

Subgenome assignment of BACs. All 57 putative BACs that were identified with the six SNP assays surrounded by the box are shown as rectangles. SNP assays were ordered according to their sequence in the Brassica rapa genome. Two slashes indicate that one or more of the SNP assays used in this study map in the Brassica rapa genome between the ones shown. PCR experiments revealed which of the putative BACs truly map to the regions of interest. Black font marks confirmed BACs, whereas BAC coordinates that were not verified to map to the homoeologous regions of interest are shown in red font. Red, dark blue and black boxes indicate SNP scores that were confirmed by PCR analyses, whereas dotted outlines mark false positive SNP scores. Pink or pale blue boxes correspond to scores that were only revealed by PCR but not by Illumina’s GoldenGate® Genotyping Assay. Red or pink colour represent the A subgenome, C subgenome sequences are displayed in dark or pale blue. Scores of assays for which only one of the assay nucleotides revealed putative BACs are shown as black boxes.

Figure 4

Analysis of BACs with amplicons spanning SNP assays. Sequences of PCR amplicons established for representative BACs are shown below six different SNP assay sequences. Horizontal arrows represent the extent of the assay nucleotides. Vertical arrows mark the positions of the SNP assay nucleotides. Differences between A and C subgenome BACs are highlighted in red and blue, respectively. BAC sequences spanning #11396, #11392a and #11372 are monomorphic at the site of the assay nucleotides, nonetheless A and C subgenome BACs can be distinguished based on sequence differences. For amplicon 11372 the region corresponding to the assay is shown and a region mapping approximately 300 bp apart. For the latter the BAC sequences are shown underneath the amplicon sequence of Brassica napus var. Express.

Figure 5

BAC contigs of homoeologous regions of interest. The order of 52 SNP assays as found in the Brassica rapa genome is indicated at the top. Assays labelled in grey font indicate that a particular assay and/or assay nucleotide did not reveal any BAC that was confirmed to carry sequences corresponding to this assay. BACs that were confirmed to carry sequences corresponding to the SNP assays are displayed as rectangles. Red and dark blue boxes stand for SNP scores that were validated by analyses with PCR amplicons. Pink or pale blue boxes represent false negative SNP scores. Scores corresponding to the A subgenome are shown in red or pink, dark or pale blue boxes are indicative of the C subgenome. Coordinates that belong to the 738 BACs that had been selected initially for the validation studies are labelled in plain black font. BAC contigs are shown as filled rectangles below the names of the assays. Arrows indicate that assay order cannot be determined based on BAC contig data. Coordinates labelled in bold and italics highlight additional BACs that had been integrated at the end of the BAC contigs for the refinements of the contigs. Clear rectangles indicate that BAC contigs were bridged by such additional BACs.