期 刊 ：植物遗传资源学报 2014年 15卷 3期
关键词：簇毛麦；抗病基因类似物(RGAs)；染色体定位；分子标记；
Keywords:Dasypyrum villosum, Resistance gene analogy, Chromosome assign, Molecular marker,
摘 要 ：小麦近缘种簇毛麦携带许多尚未克隆的抗病（R）基因。NBS-LRR类型的R基因占已克隆植物R基因的绝大多数，因此，本研究根据NBS-LRR类型R基因的保守序列设计引物，从簇毛麦基因组DNA和cDNA中扩增获得23条相关序列。基于其中5条抗病基因同源序列（RGAs）H-56/d6，H-66/b2和CDS40设计引物，对小麦、簇毛麦、硬簇双二倍体及其杂种，以及已知携带个别簇毛麦染色体或染色体臂的小麦材料进一步进行PCR扩增，结果表明：3对引物均可对簇毛麦、硬-簇双二倍体进行特异扩增；同时，源于序列H-66/b2的引物可对1VL和6VL染色体臂进行特异扩增；来自序列CDS40的引物可在同时携带1VL和2VS、或同时携带2VS和4V的小麦材料，以及具有6VL的小麦材料中特异扩增，而H-56/d6的引物在携带1VL、2VS、4V和6V染色体臂或染色体的小麦背景中都不能获得目的片段的扩增。这些结果不仅为外源染色体臂在小麦背景中的追踪与鉴定提供了新的分子标记，而且这些标记还与外源染色体或染色体臂上的抗病基因或抗病基因同源物紧密连锁或共分离。
Abstract:Dasypyrum villosum, a wheat wild relative, carries many disease resistance genes which have not been cloned yet. Considering majority of the plant disease resistance (R) proteins belong to the nucleotide-binding-leucine rich repeat (NBS-LRR) -containing R gene family, the conserved sequences of NBS-LRR resistance genes were used to design a primer pair MLA6-1-F/MLA6-1-R in present study. Genomic DNA and cDNA from Dasypyrum villosum were amplified with the primer pair, and in total twenty-three sequences were achieved. They were roughly divided into two groups based on their similarity in sequences with each other aligned by DNAMAN soft and with the resistance genes searched by NCBI BLAST. In Mla/RGAs-like group, twelve sequences had similarities of 91–93% and 87–90% to Aegilops tauschii resistance gene analogy (RGAs) and Triticum monococcum DV92 Mla1 gene, respectively. Another sequence H6 had 85% identity with Hordeum vulgare genes Mla1, Mla6, Mla12. According to the five sequences of the resistance gene analogues (RGAs) H-56/d6, H-66/b2 and CDS40, in which the sequences from both gDNA and cDNA were all obtained, primer pairs were redesigned to amplify common wheat, Dasypyrum villosum, Triticum durum- Dasypyrum villosum amphidiploid and its hybrids, and the wheat lines simultaneously carrying certain Dasypyrum villosum chromosome arms or entire chromosome such as 1VL and 2VS, 2VS and 4V, or 6V, separately. The result showed that the primer pair of H-56/d6 could not amplify an expected fragment from the lines with 1VL, 2VS, 4V and 6V, and primer pair derived from sequence of H-66/b2 could get amplification from Dasypyrum villosum, Triticum durum- Dasypyrum villosum amphidiploid, its hybrids, and the wheat lines carrying Dasypyrum villosum chromosome 1VL and 6VL, but not from the lines with 2VS, 4V and 6VS. While, primer pair designed from the sequence of CDS40 could not amplify an expected fragment specifically from the 6VS translocation line 10SR3109 and Pm97033. The markers not only confer novel molecular markers for tracing the alien chromosome in wheat background, but can also be tightly linked to R gene and R genes clusters, which will lay a foundation for further isolation and identification of the resistance genes from Dasypyrum villosum.