De Jong W S, De Jong D M, De Jong H, Kalazich J, Bodis M
Department of Plant Breeding, Cornell University, Ithaca, NY 14853-1901, USA.
Theor Appl Genet. 2003 Nov;107(8):1375-83. doi: 10.1007/s00122-003-1395-9. Epub 2003 Sep 3.
The potato R locus is necessary for the production of red pelargonidin-based anthocyanin pigments in any tissue of the plant, including tuber skin and flower petals. The production of pelargonidins in plants requires the activity of dihydroflavonol 4-reductase (DFR) to catalyze the reduction of dihydrokaempferol into leucopelargonidin. To test the hypothesis that potato R encodes DFR, portions of both dfr alleles were sequenced from a diploid potato clone known to be heterozygous Rr. Sequence comparison revealed a polymorphic BamHI restriction site. The presence or absence of this site was monitored in three diploid populations that segregated for R, as well as in a wide range of tetraploid breeding clones and cultivars, by amplifying a fragment of dfr and digesting the products with BamHI. An identically sized dfr restriction fragment lacking the BamHI site was present in all potato clones that produced red anthocyanin pigments, while the same fragment was absent in many potato clones with white tuber skin and flowers. An independent RFLP test using DraI to detect sequence polymorphism was performed on a subset of the potato clones. This test also revealed dfr-derived bands that were present in all red-colored potatoes and absent in several white clones. The presence of shared restriction fragments in all red-colored potatoes provides strong evidence that R does indeed code for DFR. The data are also consistent with a 48 year-old hypothesis by Dodds and Long, that R was selected just once during the domestication of potato. A cDNA clone corresponding to the red allele of dfr was sequenced and compared to two other alleles. The red allele is predicted to encode a 382 amino acid protein that differs at ten amino acid positions from the gene products of the two alternative alleles. Several of these differences map in a region known to influence DFR substrate specificity in Gerbera.
马铃薯的R基因座对于在植物的任何组织中产生基于红色天竺葵素的花青素色素是必需的，包括块茎表皮和花瓣。植物中天竺葵素的产生需要二氢黄酮醇4-还原酶（DFR）的活性来催化二氢山奈酚还原为无色天竺葵素。为了验证马铃薯R基因编码DFR的假设，从已知为Rr杂合的二倍体马铃薯克隆中对两个dfr等位基因的部分进行了测序。序列比较揭示了一个多态性的BamHI限制性酶切位点。通过扩增dfr片段并用BamHI消化产物，在三个R基因分离的二倍体群体以及广泛的四倍体育种克隆和品种中监测该位点的存在与否。在所有产生红色花青素色素的马铃薯克隆中都存在一个大小相同但缺少BamHI位点的dfr限制性片段，而在许多具有白色块茎表皮和花朵的马铃薯克隆中则不存在该片段。对一部分马铃薯克隆进行了使用DraI检测序列多态性的独立RFLP测试。该测试也揭示了在所有红色马铃薯中存在而在几个白色克隆中不存在的源自dfr的条带。所有红色马铃薯中共享限制性片段的存在提供了强有力的证据，表明R基因确实编码DFR。这些数据也与多兹和朗48年前提出的假设一致，即R基因在马铃薯驯化过程中只被选择了一次。对与dfr红色等位基因对应的cDNA克隆进行了测序，并与另外两个等位基因进行了比较。预测红色等位基因编码一个382个氨基酸的蛋白质，该蛋白质在十个氨基酸位置上与另外两个等位基因的基因产物不同。其中几个差异位于已知影响非洲菊DFR底物特异性的区域。
