Location: Crop Improvement and Protection ResearchTitle: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1.) Author
Submitted to: Biomed Central (BMC) Plant Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/23/2009
Publication Date: 11/23/2009
Citation: Simko, I., Pechenick, D.A., Mchale, L.K., Truco, M.J., Ochoa, O.E., Michelmore, R.W., Scheffler, B.E. 2009. Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1.. Biomed Central (BMC) Plant Biology, Year 2009, volume 9, article number 135. Interpretive Summary: Lettuce dieback is a soil-borne viral disease that is one of the limiting factors for romaine and leaf-type lettuce production in California. Currently, there is no method that effectively reduces, removes, or destroys the virus in infested soil. Thus the best control of lettuce dieback is accomplished by using resistant cultivars. However, development of resistant cultivars up to now has required extensive field-based testing. Our identification of a molecular marker that is tightly linked to the Tvr1 gene conferring durable resistance will reduce the need for field-based screening and accelerate development of resistant cultivars. A combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the resistance gene on chromosomal linkage group 2. Examination of the Tvr1 region revealed a relatively high level of nucleotide polymorphism (for a selfing species) and extensive linkage disequilibrium. One of the markers (Cntg10192) flanking the Tvr1 gene showed 100% accuracy in detecting resistant and susceptible phenotypes in a set of 200 L. sativa accessions from all horticultural types of lettuce and two accessions from L. serriola. A combination of three SNPs in this EST-based marker identified four haplotypes. Three of the haplotypes are related to dieback resistance, while a single haplotype is always associated with susceptibility to the disease. Application of high-resolution DNA melting analysis allowed us to distinguish all four haplotypes of the Cntg10192 marker in a single assay. Since heterozygous state is also easily distinguishable by the HRM analysis, we can identify and select homozygous individuals whose offspring do not segregate for resistance in the following generation. Screening for dieback resistance with this molecular marker is now part of our breeding program. Marker-assisted selection with Cntg10192 is being used to develop improved romaine and leaf-type cultivars resistant to the disease. In addition, we are employing the molecular markers to prevent inadvertent introgression of the susceptible haplotype into the iceberg lettuce gene pool.
Technical Abstract: 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 (' = 6.7 × 10-3) and extensive linkage disequilibrium (r2 = 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 (r2 = 0.247) and crisphead (r2 = 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.