Location: Sunflower and Plant Biology Research2012 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. 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 developed an indel-based map using Population 029C in which previously identified WM8.3 QTL is segregating. The ordering was based on the recently available (to the PI) assembly of the common bean genome sequence. We next performed composite interval QTL mapping with the cofactors selected by the QGene software. Included in the analysis is the experiment-wide LOD score based on 10,000 permutations and results to date suggest a major QTL found on chromosome 8 of common bean. Markers s02137 and s10259 define the boundary of the QTL. These markers physically map at positions 48,811,431 and 49,944,221, respectively on the 1.0 build of the Pv08. This is where we were predicting that WM8.3 is located. On this map, the genetic distance is 9.5 cM. Importantly, we observed that the field and straw test phenotypic scores all uncovered a single QTL at the same position.We have also completed the development of the new set of indel markers for region WM7.1 and those will be mapped to define the QTL region shortly. Regarding the physical mapping of the WM8.3 QTL, we evaluated next generation sequencing data to uncover the introgression region on chromosome 8. Given that the lines used for the RNA-seq analysis differed in the WM8.3 region, and given that this was due to backcross, introgression breeding, we predicted that significant SNP differences on chromosome 8. Results to date, number SNPs in 1.0 Mbp windows across chromosome 8, suggest that there are two introgression regions, on either end of the chromosome. Importantly, the QTL falls within the introgression region on the right end of the chromosome. We have also performed RNA-seq analysis on the WM8.3 locus. The data have been collected and a preliminary analysis identified two genes known to be associated with disease resistance response in Arabidopsis thaliana. One gene is involved in the innate immunity signaling pathway, and the other is a traditional NBS-LRR gene. Both of these map to chromosome 8. In total, ~400 genes were found to be expressed differentially at 24 and 48 hr post infection. We are currently awaiting the final annotation of the common bean genome to perform more definitive analysis. Additionally, the tissue samples for the WM7.1 QTL have been collected, the RNA extracted, and the RNA is currently in the queue at NCGR for sequencing.