Location: Sunflower and Plant Biology Research2013 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 July 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 have completed an indel-based map using Population 029C. In addition, the common bean genome sequence released in August 2013. This enabled us to develop a consensus genetic and physical map of the region where this QTL resides. A cluster of 13 markers were discovered that co-segregate together. These markers define the WM8.3 QTL peak. When these were physically mapped, they were discovered to span almost 40 megabases of the Pv08 chromosome. The reason for this is that the majority of these markers are located in the pericentromeric region of the chromosome. This region has significantly reduced recombination, and given the size of the mapping population, it is expected in retrospect that these would indeed co-segregate. Roughly 1000 genes are located in this region of the chromosome. So the challenge is to determine which of these genes is associated with the QTL. We turned to RNA-seq for further limit the number of potential candidates. One potential candidate, Phvul.008G099500, beginning at position 10,737,731 Mb, is a homolog of AtPUB13, and Arabidopsis gene recently discovered to be involved in defense signaling. A second candidate, Phvul.008G079700, beginning at 7,647,554 Mb, contains a leucinerich repeat at the N terminal. This structure is used to recognize for protein-protein recognition, such as the Cf-2 in tomato. This past year we performed DNA-seq analysis to determine the physical location of the WM7.1 QTL. Two pooled DNA samples consisting of seven tolerant and 6 susceptible lines from the Z0725-11 population, and similarly two pooled DNA samples (six tolerant/ten susceptible lines) from the Z-725-15 were sequenced. SNPs distributions were analyzed to determine regions with skewed distributions, and these regions were considered to be introgressed regions from the WM7.1 QTL. Using joint distributions, the region from 3.4 to 7.8 Mb. This result is consistent with previous research that showed that the phaseolin marker (Phs) located at 4.959 Mb on Pv07. Further work on this population will investigate those genes that are cycling in the resistant and susceptible response following infection by the white mold pathogen.