In this study, we developed a procedure for comparative mapping between B. napus and Arabidopsis with SSR markers with the aid of B. rapa and B. oleracea genome sequences. To the best of our knowledge, this is the first report to construct a comparative map among Arabidopsis and three Brassica species with a SSR-based genetic map (Additional files 3, 4, 5 and 6: Figure S1-S4; Additional file 1: Table S1). The SSR markers have been widely used as a preferable type of molecular marker in genetic mapping in Brassica species. However, it was difficult to use a SSR map for comparative mapping with Arabidopsis directly. First, individual SSR primer pairs only have limited sequence information, which renders a direct alignment with Arabidopsis genome ineffective. For example, in a study to construct a mainly SSR-based integrated map in B. napus, Wang et al. found that <2% of the primer pairs could identify homologous regions to Arabidopsis, of which only 50% agreed with those identified using the corresponding SSR clone sequences. Second, high homology between the A and C genomes often results in multiple polymorphic loci in B. napus for a single Arabidopsis gene, which further complicates the comparative analysis between B. napus and Arabidopsis. In this study, we circumvented the two difficulties by making use of recently released genomic sequences of B. rapa and B. oleracea. Through anchoring the SSR loci on B. napus LGs to the B. rapa/B. oleracea genome by e-PCR, we were able to match the B. napus SSR loci with their Arabidopsis homologues, thus making such a comparative mapping feasible. By overcoming the difficulties in comparative mapping using a SSR-based genetic map of B. napus and Arabidopsis genomic sequences, this procedure thus proved a novel idea for a comprehensive comparison among Arabidopsis, B. napus and its two progenitor species, B. rapa and B. oleracea.
To make use of the information derived the SSR loci as much as possible, a less stringent E-value was initially used in this study to identify more putative homologous loci. As indicated by Lukens et al., a less stringent cutoff could result in more non-specific region of homology. However, since our major purpose in this study was to establish colinear relationships between B. napus and Arabidopsis through the conserved blocks, such non-specific homology regions in the initial screening will be re-examined. With the criterion for identification of conserved blocks, such non-specific loci will not affect the determination of the conserved blocks. This is evident through the data listed in Additional file 1: Table S1, in which about 66% of the loci under the less stringent (E-value >1E-05) cutoff eventually were linked to a perspective block, indicating that some weak but biologically relevant sequence relationships could be revealed with such a procedure, which reduces the loss of valuable information from the SSR loci on the B. napus map.
The establishment of such a comparative map offers an effective way to transfer the gene information from model plant Arabidopsis to B. napus, an amphidiploid crop species, as demonstrated by mapping the seed size/weight genes on the B. napus genetic map (Figures 1 and 2). Furthermore, we identified candidate genes for eight TSW QTLs through the mapping (Figures 1 and 2; Additional files 3, 4, 5 and 6: Figure S1-S4). Together, the seed distribution map and the identified candidate genes for mapped TSW QTLs provide valuable information about the genetic control of seed weight in B. napus. Although such a list of seed size/weight genes could be further expanded by including other genes related to the process of seed development, our results do exemplify the universal usefulness of such an approach. A flow diagram for the process is presented in Additional file 11: Figure S6.
Mapping of the seed weight related genes and the candidate genes for TSW QTLs could accelerate the molecular cloning and functional characterization of the QTLs. As shown in Figure 3, the prediction of the candidate genes for several mapped QTLs is accurate. Such a process will allow us to isolate the potential candidate genes for a particular QTL by homologous cloning strategy rather than tedious and time-consuming traditional map-based cloning procedure. On the other hand, by cloning some of predicted potential candidate genes that were even not located in the genetic map, for example AP2 in this study, it is possible to uncover the polymorphic alleles in two parental lines without QTL mapping information (Figure 4). By doing so, we were able to develop an allele-specific marker for one of locus of the AP2 gene in B. napus and place the marker on the corresponding LG (Figure 4). There are three and two copies of the AtAP2 homologues identified in B. rapa (including one copy located on a scaffold) and B. oleracea, respectively (Table 3; Additional file 9: Table S4). Consistently, there are four copies mapped on LGs A1, A3, C1 and C7 of the B. napus genetic map, respectively (Figures 1 and 2). Although the exact molecular significance of the insertion in the cloned BnAP2 allele of SW Hickory is yet to be established, identification of the polymorphic locus between the two parental lines lays foundation for further functional characterization of all the AP2 alleles in the B. napus genome.
The seed weight genetic map revealed the complexity of the genetic control of seed weight in amphidiploid rapeseed. For example, a single TSW QTL may have one or multiple candidate gene(s), such as TSWA2 (with only one gene, GW2, located) and TSWA1 (with 6 genes located) (Figure 1 and Additional file 9: Table S4). Mapping of these candidate genes could apparently narrow down the range of the potential target genes. Of course, even though potential candidate genes are mapped to a locus, this does not imply that they control the trait. The QTL may result from variation in other novel genes which have not been studied in model systems.
It is interesting to notice that some genes showing major effects on seed size/weight in rice and Arabidopsis, such as GS3, GS5, GW2 and MINI3, TTG2, ARF2, IKU2, were located on the minor QTLs regions, or even not in the confidence intervals of previously mapped TSW QTLs (Figures 1 and 2; Additional file 9: Table S4). In addition, no homologue of qSW5/GW5, an important rice seed size gene [56, 57] could be identified in both the A- and C- genome (Table 3). A more comprehensive evaluation of B. napus germplasm is needed to understand whether these genes may exhibit different effects on the studied trait in various species. On the other hand, no candidate genes for two previously mapped major QTLs, TSWA7a and TSWA7b were identified, suggesting that the two QTLs may represent novel determinants for seed weight in amphidiploid B. napus.