United States Department of Agriculture-Agricultural Research Service, Crop Improvement and Protection Research Unit, 1636 East Alisal Street, Salinas, CA 93905, USA.
BMC Plant Biol. 2009 Nov 23;9:135. doi: 10.1186/1471-2229-9-135.
Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection.
A combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the Tvr1 resistance gene on chromosomal linkage group 2. Nine molecular markers, based on expressed sequence tags (EST), were closely linked to Tvr1 in the mapping population, developed from crosses between resistant (Salinas and Salinas 88) and susceptible (Valmaine) cultivars. Sequencing of these markers from a set of 68 cultivars revealed a relatively high level of nucleotide polymorphism (theta = 6.7 x 10-3) and extensive linkage disequilibrium (r(2) = 0.124 at 8 cM) in this region. However, the extent of linkage disequilibrium was affected by population structure and the values were substantially larger when the analysis was performed only for romaine (r(2) = 0.247) and crisphead (r(2) = 0.345) accessions. The association mapping approach revealed that one of the nine markers (Cntg10192) in the Tvr1 region matched exactly with resistant and susceptible phenotypes when tested on a set of 200 L. sativa accessions from all horticultural types of lettuce. The marker-trait association was also confirmed on two accessions of Lactuca serriola - a wild relative of cultivated lettuce. The combination of three single-nucleotide polymorphisms (SNPs) at the Cntg10192 marker identified four haplotypes. Three of the haplotypes were associated with resistance and one of them was always associated with susceptibility to the disease.
We have successfully applied high-resolution DNA melting (HRM) analysis to distinguish all four haplotypes of the Cntg10192 marker in a single analysis. Marker-assisted selection for dieback resistance with HRM is now an integral part of our breeding program that is focused on the development of improved lettuce cultivars.
生菜（Lactuca saliva L.）易患凋萎病，这是一种由呼肠孤病毒科的两种病毒引起的土传病害。凋萎病在罗马生菜和叶用生菜中普遍存在，而现代冰山品种对此病具有抗性。冰山品种的抗性由 Tvr1 基因赋予，该基因是一个单一的显性基因，可提供持久的抗性。本研究描述了该抗性基因的精细定位、Tvr1 区域核苷酸多态性和连锁不平衡分析，以及用于标记辅助选择的分子标记的开发。
经典连锁作图和关联作图的组合使我们能够确定 Tvr1 抗性基因在染色体连锁群 2 上的位置。从抗性品种（Salinas 和 Salinas 88）和感病品种（Valmaine）杂交组合中开发的 9 个基于表达序列标签（EST）的分子标记与 Tvr1 紧密连锁，位于图谱群体中。从一组 68 个品种中对这些标记进行测序，揭示了该区域相对较高水平的核苷酸多态性（theta = 6.7 x 10-3）和广泛的连锁不平衡（r(2) = 0.124，距离 8 cM）。然而，连锁不平衡的程度受到群体结构的影响，当仅对罗马生菜和脆生菜品种进行分析时，其值会大大增加（r(2) = 0.247 和 r(2) = 0.345）。关联作图方法表明，在对来自所有生菜园艺类型的 200 个 L. sativa 品种进行测试时，Tvr1 区域的 9 个标记之一（Cntg10192）与抗性和感病表型完全匹配。该标记与性状的关联也在两种野生生菜（Lactuca serriola）品种上得到了证实。Cntg10192 标记的三个单核苷酸多态性（SNP）的组合确定了四个单倍型。三个单倍型与抗性相关，其中一个单倍型总是与疾病的易感性相关。
我们已成功应用高分辨率 DNA 熔解（HRM）分析在单次分析中区分 Cntg10192 标记的所有四个单倍型。HRM 辅助选择凋萎病抗性现已成为我们专注于开发改良生菜品种的育种计划的一个组成部分。
