Location: Sunflower and Plant Biology Research2011 Annual Report
1a. Objectives (from AD-416)
The goals of this project are to i) identify and validate white mold (WM) resistance QTL from P. coccineus and transfer them into common bean, ii) examine phenotypic interaction among major QTL conferring partial resistance to WM in common bean, and iii) use Phaseolus-Glycine synteny and gene expression studies to leverage the soybean whole genome sequence (and the soon to be available Phaseolus whole genome sequence) for fine-mapping WM resistance QTL and candidate gene discovery.
1b. Approach (from AD-416)
For QTL discovery (Objective 1), three recombinant inbred backcross line populations from P. vulgaris /P. vulgaris x P. coccineus crosses will be used to identify QTL associated with WM resistance as measured in the field and by the straw test. Among the mapped markers will be a set of candidate genes to determine if any co-segregate with QTL. The indentified QTL will be validated in separate populations and environments by comparing among populations to determine if QTL map to the same location, and comparing to existing maps of common bean and soybean. Lines with the QTL will be tested in different field environments and with the straw test to determine if expression is environment-specific. Lastly, the QTL from P. coccineus will be transferred into advanced common bean breeding lines (both snap and dry beans) with stable fertility traits and adequate agronomic performance. Existing and novel QTL (from this study) will be pyramided into various genetic combinations to examine QTL interactions (Objective 2). This will be accomplished by developing inbred line populations from bi-parental crosses designed to combine two or more QTL. The lines will be tested for WM reaction in replicated field and greenhouse environments and assayed for presence absence of QTL-linked markers. Phenotypic effects and interactions among the QTL will be examined by statistical regression of disease reaction on presence/absence of linked QTL markers. Fine-structure mapping and candidate gene discovery (Objective 3) will focus on major WM resistance QTL on chromosomes Pv2, Pv7, and Pv8. The mapping approach will leverage genomic synteny demonstrated between common bean and soybean. Common bean markers linked with the QTL will be located on the soybean whole genome sequence scaffold. Common bean EST contig and singletons (which represent expressed genes) located in the same region of the QTL-linked markers in soybean will be converted to CAPs markers. The development of a minimum set of 25 new EST based CAPS markers will be targeted for each QTL region for high density fine mapping of the QTL intervals in common bean RIL populations segregating for the QTL. Next generation sequencing (Roche 454 or Solexa), will be used to identify genes differentially expressed between resistant and susceptible near-isogenic lines (NILs) for the major Pv 7 and Pv 8 QTL. Inbred lines will be used to examine gene expression for the Pv2 QTL for now because NILs do not exist yet. mRNA will be isolated from stem tissue from infected (24 and 48 h after inoculation) and mock-inoculated resistant and susceptible NILs. mRNA will be sequenced, and the sequence data aligned to determine which genes are differentially expressed. These genes will be mapped in the common bean genome and associations with the target QTL and other WM resistance QTL determined. Genes not located near QTL may be responsible for regulating resistance response. Differential gene expression will be confirmed by quantitative rt PCR.
3. Progress Report
This project was initiated on June 1, 2010, research is ongoing, and the overall objective is the identification of genetic factors (QTL) controlling partial resistance to white mold in common bean. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. This project is focused on the identification, interactions, and fine mapping of white mold resistance-QTL in common bean, specifically performing gene expression profiling of the resistance and susceptible responses in common bean to Sclerotinia infection. We evaluated stem tissue that was infected using the straw test method. ARS collaborators at Prosser, WA conducted all of the plant pathology work. Specifically, several RIL lines carrying either the tolerant or susceptible WM8.3 QTL (chromosome 8 QTL) were evaluated by infecting with the pathogen and collecting stem samples at 24 and 48 hr post-infection. As controls, non-infected, wounded (leaf cut and detached per the methodology), and mock-inoculation (agar plug placed on wound) stem samples were collected. The experiment was performed twice. Total RNA was isolated by ARS collaborators in Prosser. We sent total RNA samples to the National Center for Genomic Research (NCGR) in Santa Fe, NM. NCGR created seven sequencing libraries by pooling RNA within each of the seven treatments. (Mock inoculation RNA samples were pooled for the 24 and 48 hr time points). Each library was sequenced using the IlluminaGAIIx sequencing-by-synthesis technique. Reads of 36-nt were collected. A total of 130,414,421 reads generated a total of 4.8 Gbases of data. All of the reads were contiged using the Velvet software package using kmer values of 23, 25, 27, 29, and 31. This generated a total of 110,278 assembled non-redundant transcripts that represented our reference genome for our additional analyses were performed. The reads within each treatment were then aligned to this reference transcript set.After further analyses, we discovered that 294 genes were significantly up regulated in the 24 hr susceptible response, and 237 were up regulated in the resistant response. Similarly, at 48 hr, 199 genes were up regulated in the susceptible genotypes and 264 in the resistant genotypes. All of these genes have been annotated using the nomenclature inherited from the annotated soybean genome. GO annotation clustering will be performed next, and we are currently beginning RT-PCR analyses to confirm putative responses.