2013 Annual Report
1a.Objectives (from AD-416):
Specific objective is to develop tomato translational genomic tools through transcriptome analysis to identify genes that are associated with virus resistance.
1b.Approach (from AD-416):
The University of Florida tomato breeding program has vast experience in developing improved germplasm with multiple disease resistance as well as other traits such as improved flavor and heat-tolerant fruit setting ability. Examples of resistance genes that were discovered in wild species, introgressed into tomato, genetically characterized, and incorporated into tomato varieties include the I-3 gene conferring resistance to fusarium wilt race 3 (Scott and Jones, 1995), and the Ty-3 and Ty-4 genes conferring resistance to tomato yellow leaf curl virus (TYLCV) and other begomoviruses. Recently they discovered the begomovirus resistance gene from ‘Tyking’ was recessive and located on chromosome 4. Other resistance genes not yet in varieties are the Sw-7 Tomato Spotted Wilt Virus (TSWV) resistance gene and numerous genes conferring resistance to various races of bacterial spot caused by Xanthomonas species. The linkage of high levels of bacterial wilt (Ralstonia solanacearum) with small fruit size was broken in developing the breeding line Fla. 8109. In addition, the Ty-3 and Ty-1 genes have been fine mapped and breeding lines without linkage drag have been developed that should dramatically improve future resistant varieties. Current research involves using SNP genotyping from SolCAP (http://solcap.msu.edu/) and other sources to identify genes of interest and conduct fine mapping. These projects include: fine mapping of TYLCV resistance genes Ty-2 and Ty-4 and locating genes for resistance to Bemisia tabaci, the sweet potato whitefly that vectors begomoviruses, and locating bacterial spot resistance genes from PI 114490 where we have just found six genes. Specifically in the current project, Dr. Scott will generate near isogenic lines (NILs) with resistance to TYLCV and TSWV. For TYLCV screening and phenotyping on the ty-5 gene from Tyking, viruliferous whitefly (B. tabaci) will be used for inoculation and the resistant rating system will be based on symptom expression. For NIL for TSWV, the process will be the same using the Sw-7 gene. TSWV screening and phenotyping will be done following inoculation by thrips on test plants, as well as resistant and susceptible controls. These plant materials or their RNAs will be provided to Ling’s lab for RNA-Seq analysis. Genes involved in tomato responding to virus (TYLCV and TSWV) infection will be identified through differential expression profiling.
Research in this subordinate project is related to inhouse project Objective 3: Characterize and develop host resistance to effectively manage viral and Fusarium wilt diseases on watermelon. In backcrossing the ty-5 gene that provides resistance to tomato yellow leaf curl virus (TYLCV), our approach has recently changed because new information has emerged since this grant started. Israeli colleagues working with our lab on another project have found that the ty-5 resistance allele resulted from a loss of function mutation within a single gene; this mutation likely occurred in a cultivated tomato background, as opposed to it being contained within an introgression from a wild species. We now have molecular markers flanking the gene and these are being used to backcross ty-5 into Fla. 8059. For the backcrossing, we used an F1 with a somewhat complex pedigree as the ty-5 donor; this F1 had one parent that was developed by crossing a line (8879) twice to 8059; the parent on the other side was developed by crossing a ty-5 line (8638B) twice to 8059. The 8638B line also came out of a cross with 8059 in its pedigree. Thus, we started the direct backcrossing with a hybrid that had a considerable amount of 8059 in its background, theoretically about 81%. This hybrid was backcrossed one time before the grant started, the second backcross (BC2) was made in the fall of 2012, and BC3 was made in the spring of 2013. With 3 BCs at present, we would expect to have 87.5% of the 8059 genome if there were no 8059 in the initial donor pedigree. Since there was 8059 in the initial donor, there should be well over 90% of the 8059 genome present at BC3. This fall the BC4 will be made, but we will also self-pollinate the BC3 to attain BC3S1 seed that can be screened for TYLCV in the spring to verify that we have carried the resistance gene.
For backcrossing the Sw-7 gene that provides resistance to tomato spotted wilt virus (TSWV), our donor parent was Fla. 8516. Backcrossing started before the grant began. We made BC4 to 8059 in fall 2012 and BC5 to 8059 in spring 2013. In this case, we should now be at 96.875% of the Fla. 8059 genome. This fall we will make BC6 and we will self-pollinate the BC5 to attain BC5S1 seed. In the spring of 2014, we will screen the BC5S1 population with markers throughout the introgression to identify resistant plants with the smallest introgression that can be used for backcrossing and/or transcriptome marker development. Note that the TSWV screening will necessitate maintaining a colony of viruliferous thrips for inoculation. It is likely that screening of BC5S2 lines will be necessary to verify the BC5S1 results.