Ozdemir Nehir, Cloutier Sylvie
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Canada.
Genome. 2005 Jun;48(3):401-10. doi: 10.1139/g05-005.
Gliadins and glutenins are storage proteins important in determining the bread-, noodle-, and pasta-making quality of wheat. Glutenins consist of HMW and LMW subunits. The Glu-A3, Glu-B3, and Glu-D3 loci on the short arms of chromosomes 1A, 1B, and 1D, respectively, are the major loci for LMW glutenins. To construct physical maps of the Glu-3 loci, a set of 24 high-density filters representing a 3.1x genome coverage hexaploid wheat BAC library was screened by hybridization using a probe made of 3 LMW glutenin sequences. After 2 rounds of hybridization, a subset of 536 BAC clones were selected and fingerprinted. Three developing seed cDNA libraries were also constructed. A total of 5000-6000 ESTs were generated from each library, assembled into contigs and searched by homology for LMW glutenin sequences. In total, 90 full-length LMW glutenin sequences were found to cluster into 8 distinct groups representing at least 21 different LMW glutenin subunits. A set of 24 pairs of PCR primers was designed from these groups and used as markers on the BAC clones. The combined fingerprinting and marker data were used to build the physical maps using FPC software. A total of 91 contigs comprising 254 clones were obtained and 282 clones remained singletons.
醇溶蛋白和谷蛋白是决定小麦制作面包、面条和意大利面品质的重要贮藏蛋白。谷蛋白由高分子量（HMW）和低分子量（LMW）亚基组成。分别位于1A、1B和1D染色体短臂上的Glu - A3、Glu - B3和Glu - D3位点是LMW谷蛋白的主要位点。为构建Glu - 3位点的物理图谱，使用由3个LMW谷蛋白序列制成的探针，通过杂交筛选了一组代表3.1倍基因组覆盖的六倍体小麦BAC文库的24个高密度滤膜。经过两轮杂交后，选择了536个BAC克隆的子集并进行指纹分析。还构建了3个发育种子cDNA文库。每个文库共产生了5000 - 6000个ESTs，组装成重叠群并通过同源性搜索LMW谷蛋白序列。总共发现90个全长LMW谷蛋白序列聚集成8个不同的组，代表至少21种不同的LMW谷蛋白亚基。从这些组中设计了24对PCR引物，并用作BAC克隆上的标记。结合指纹分析和标记数据，使用FPC软件构建物理图谱。共获得91个包含254个克隆的重叠群，282个克隆仍为单克隆。
