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BAC library construction, marker development and chromosome mapping for genome sequencing of chromosome 6B in common wheat

Research objective

We are in charge of the following three parts of the 6B sequencing project.

  • (1)Construction of the 6B specific BAC libraries
    The goal is to obtain chromosome arm (in our case short and long arms of 6B) specific genomic libraries and genomic DNA. For full coverage of the 6B chromosome we considered that the BAC libraries with 10x coverage should be sound.
  • (2)Development of a large number of DNA markers that will be used for anchoring BAC contigs to chromosome maps
    BAC contigs is being made by the other project team (KGS1003). Our objective here is to develop enough number of DNA markers to anchor BAC contigs. Because the number of BAC contigs was not known at the beginning of the project, we set our goal to develop at least 1500 DNA markers specific to chromosome 6B. We are also trying to develop the DNA markers that distribute evenly along the length of chromosome 6B.
  • (3)Development of chromosome maps of 6B
    We aimed to develop chromosome maps of 6B that are reliable and with high resolution. The map will be used to anchor BAC contigs to chromosome 6B. Thus, we intended to use common marker between BAC screening and map construction. Because the pericentromeric region of wheat chromosome 6B is devoid of recombination events, the resolution of genetic map around the centromere is very low. We are aimed to overcome this problem by employing chromosome deletions caused by the gametocidal system or irradiation.

What we have done

In the two year term of the project what we achieved are

  • (1)Construction of the 6B specific BAC libraries
    We cytologically screened the double ditelosomic 6B line (a wheat aneuploid with sets of telosomes 6BS and 6BL) stored in the National BioResource Project, Japan and propagated them enough to be used for chromosome flow-sorting and BAC library construction by Dr. Jareslav Dolezel, Institute of Experimental Botany, Czech. The BAC libraries were successfully constructed and used for fingerprinting and contig assembly in the other project team (KGS1003). Chromosome arm specific DNA samples were also prepared that are being used in DNA marker assignment to chromosome arms 6BS and 6BL.
  • (2)Development of a large number of DNA markers that will be used for anchoring BAC contigs to chromosome maps
    First we gathered information of publically available DNA markers mapped to chromosome 6B, whose number was as little as 300. We utilized PLUG (PCR-based landmark unique gene) markers, kindly provided by Dr. Toshiki Nakamura (Tohoku Agricultural Research Center). Information from barley genomics provided by Dr. Kazuhiro Sato was used to develop DNA markers that are homologous to genes on barley 6H chromosome. Syntenic relationship among Gramineae species including rice, Brachypodium, and barley was a good source of genic markers. However, the DNA markers successfully assigned to chromosome 6B was 1759 (55.3%). Another potential problem with those markers resides in the fact that the most markers are genic markers that are known to be unevenly distributed on the wheat chromosomes. We decided to use ISBP (insertion site based polymorphic) markers to cover non-genic region of the chromosome because the ISBPs are based on repetitive sequences. We could identify more than 40000 ISBP markers from the chromosome specific survey sequences of 6BS and 6BL. A test set of ISBP markers (1000 markers per chromosome arm) exhibited higher success rate (more than 80%) than other markers.
  • (3)Development of chromosome maps of 6B
    We constructed a genetic map of chromosome 6B by using a RIL (recombinant inbred line) population derived from a cross between cultivars Chinese Spring and Mironovskaya 808. A population consisted of 210 lines provided good frame map with SSRs (Kobayashi et al. 2010). We added ISBP and DArT markers to the map. Besides the genetics map, we constructed a radiation hybrid map and a gametocidal map that are aimed to resolve markers in the pericentromeric region.

We are taking parts of the 6B-sequencing project(circled in red)in collaboration with KGS1003

Members

  • Shuhei NASUDA, Project Leader.
  • Satoko KANEKO
  • Sakaguchi TOYOTAKA
  • Shota WATANABE
  • Takashi R. ENDO

画像:サンプル

  • Shigeo TAKUMI
  • Ryoko OHNO
  • Julio C.M. IEHISA

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  • Katsuyuki HAYAKAWA
  • Chikako ABE

Nisshin Flout Milling Inc.

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