Phytopathology. 2007 Jun;97(6):728-36. doi: 10.1094/PHYTO-97-6-0728.
ABSTRACT To investigate disease resistance gene analogs (RGAs) encoding coiled-coil-nucelotide-binding site-leucine-rich repeats (CC-NBS-LRR) proteins in western white pine, degenerate primers targeting the conserved motifs in the NBS domain were designed to amplify RGAs from genomic DNA and cDNA. Sixty-one distinct RGAs were identified with identities to well-known R proteins of the CC-NBS-LRR subfamily. These RGAs exhibited variation of putative amino acid sequences from 13% to 98%, representing a complex CC-NBS-LRR subfamily. A phylogenetic tree constructed from the amino acid sequence alignment revealed that these 61 RGAs were grouped with other CC-NBS-LRR members from angiosperms, and could be further divided into six classes with an identity threshold of 68%. To map RGAs, RGA polymorphisms and a modified amplified fragment length polymorphism (AFLP) method with incorporated sequences from the NBS domain were used to reveal NBS or NBS-AFLP markers. RGA polymorphism study revealed that three off the identified RGAs were not linked to the Cr2 gene imparting resistance to white pine blister rust. However, the AFLP strategy, using bulk segregant analysis (BSA) and haploid segregation analysis, identified 11 NBS-AFLP markers localized in the Cr2 linkage, the closest two to the gene being 0.41 cM and 1.22 cM away at either side. Eight of these markers showed significant amino acid sequence homologies with RGAs.
摘要 为了研究西方白松中编码卷曲螺旋-核苷酸结合位点-亮氨酸丰富重复（CC-NBS-LRR）蛋白的抗病基因类似物（RGAs），针对 NBS 结构域中的保守基序设计了简并引物，从基因组 DNA 和 cDNA 中扩增 RGAs。从 cDNA 中扩增了 61 个独特的 RGA，与 CC-NBS-LRR 亚家族的已知 R 蛋白具有身份同一性。这些 RGA 表现出假定氨基酸序列的变化从 13%到 98%，代表了一个复杂的 CC-NBS-LRR 亚家族。从氨基酸序列比对构建的系统发育树表明，这 61 个 RGA 与被子植物的其他 CC-NBS-LRR 成员分组在一起，并且可以进一步分为六个类，同一性阈值为 68%。为了对 RGAs 进行作图，使用 RGA 多态性和改良的扩增片段长度多态性（AFLP）方法，结合 NBS 结构域的序列，揭示 NBS 或 NBS-AFLP 标记。RGA 多态性研究表明，鉴定出的 3 个 RGA 与赋予对白松疱锈病抗性的 Cr2 基因无关。然而，使用基于群体的分离分析（BSA）和单倍体分离分析的 AFLP 策略，确定了 11 个 NBS-AFLP 标记定位于 Cr2 连锁，最接近基因的两个标记分别位于两侧 0.41 cM 和 1.22 cM。其中 8 个标记与 RGAs 显示出显著的氨基酸序列同源性。
