Development of DNA Markers for Anchoring BAC Contigs to the Specific Genomic Regions on Chromosome 6B
DNA markers are essential to anchor BAC contigs onto their specific genomic positions. Information of publically available markers was collected from the databases such as “GrainGenes” and “National BioResource Project-Wheat Japan”. There are only about 200 markers such as SSR and RFLP available currently for chromosome 6B within these databases. Considering the chromosome size and number of BAC contigs, it is obvious that this number of markers is far enough to anchor BAC contigs for a physical map with high chromosome coverage. We thereafter used other public marker resources, like PLUG (PCR-based Landmark Unique Gene) marker, that were EST-PCR markers developed by taking advantage of the syntenic gene conservation between rice and wheat (Ishikawa et al. 2007, 2009). The barley GenomeZipper (chromosome 6H) (Mayer et al. 2011) and syntenic relationship among rice (chromosome 2) and Brachypodium (chromosome 3) were also used as a good resource with more than 2,200 markers. Insertion Site-Based Polymorphism (ISBP) marker, which is developed using the junction sequences between transposable elements and their flanking sequences, has been reported as a powerful tool for wheat studies (Paux et al. 2006). Using our survey sequences (wheat- urgi.versailles.inra.fr/Seq-Repository) obtained from 6BS and 6BL, we identified more than 40,000 ISBP marker candidates from both arm survey sequences, and then a higher success rate of markers assigned to chromosome 6B was exhibited than those of other sequence resources (Kaneko et al. in preparation). All markers assigned to chromosome 6B were used for PCR screening of BAC libraries, leading to anchor BAC contigs on the chromosome 6B, which cover about 86 % of the entire chromosome. In general, the ISBP markers were scattered while genic markers tended to be clustered within the gene-baring BAC contigs. These results might reflect the features of wheat genome organization that it is predominantly composed of repetitive elements around small genic region, indicating the efficiency of ISBP markers as anchors because of their randomly distributed pattern on the wheat genome.
The BAC contigs have been assigned to their specific genomic regions using the anchoring markers, which is extremely important because the order of the contigs provides essential information to create a pseudomolecule sequence to reach the final goal of the IWGSC. A genetic map using a recombinant inbred line derived from a cross between CS and winter cultivar 'Mironovskaya 808' (M808) was already developed (Kobayashi et al. 2010), which provided the good frame map with SSRs (Fig. 11.1). We have added the markers mentioned above showing polymorphism between CS and M808 to the 6B genetic map. DNA makers on the genetic map has led to assign BAC contigs covering about 33 % of chromosome 6B in length. Because the pericentromeric region is devoid of recombination event, the marker density of genetic map around the centromere is very low. Aiming to overcome this problem, we employed a method using the chromosome deletion lines
caused by the gametocidal (Gc) system or γ-ray irradiation to map more DNA markers on chromosome 6B. The Gc system-induced chromosomal breakage of 6B
was derived from a cross between CS with monosomic addition of a chromosome 2C from Aegilops cylindrica Host and a nullisomic 6B-tetrasomic 6A (N6BT6A) line of CS. Chromosome mapping using both the new developed deletion lines and previously produced lines by Endo and Gill (1996) led to localize DNA markers more than on 70 loci (Sakaguchi et al. in preparation). Radiation hybrid (RH) mapping is known as a powerful tool for a high-resolution mapping in wheat (Tiwari
et al. 2012). The RH panel was produced through a cross between the N6BT6A and CS with the pollen freshly irradiated by γ-ray (Watanabe et al. in preparation). Using the above RH panel, we have successfully assigned the BAC contigs that represented more than 80 % of the entire physical length of chromosome 6B. From the genic marker information, the contig order along the RH map could provide a
virtual gene order on the chromosome 6B. The results thus allow us to study the relationship between the position of genic markers present on the chromosome 6B BAC contigs and the rice gene order on chromosome 2, which will reveal the microscale rearrangements with a higher degree of resolution than previously identified by comparative mapping using EST (La Rota and Sorrells 2004).
Fig. 11.1 A linkage map of M808 and CS constructed using RILs. Linkage analysis was performed using MAPMAKER/EXP version 3.0b. The threshold for log-likelihood score was set at 3.0, and the genetic distances were calculated with the Kosambi function. The linkage map provides 410 loci of SSR markers, and the total map length is 2814.5 cM with average spacing of 6.9 cM between markers
We have successfully established a physical map of chromosome 6B integrating the BAC contigs and genetic or RH maps using a large number of DNA markers. Our physical map has a high quality and a high resolution, and provides important information on the robust BAC contigs necessary for its genomic sequencing to conduct comparative analysis and prediction of the genomic structure. Currently BAC sequencing is underway using the next-generation sequencer. We will provide a high quality reference sequence to offer an unlimited source on marker development and genome organization of wheat chromosome 6B.