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 July 1, 2010, and research is ongoing. The overall objective is to develop white mold resistant dry and snap beans using highly resistant accessions of Phaseoluscoccineus as a source. Our primary objective has been to identify and validate white mold (WM) resistance QTL from P. coccineus and transfer them into common bean. For QTL discovery, three recombinant inbred backcross line populations from P. vulgaris /P. vulgaris x P. coccineus crosses have been used to identify QTL associated with WM resistance as measured in the field and by the straw test. For the two populations using the PI433251 P. coccineus donor parent, we have also selected lines on the basis of a large number of significant markers as determined by single factor analysis, as well as significant markers for putative resistance factors on Pv10 . The selected lines are for the 91G/PI255956 backcross inbred (BCIB) population: WMG762, WMG 836, WMG853, WMG861, WMG897, WMG903, and WMG904; for the MO162/PI433251 BCIB population: WMM 619 and WMM688; and for the 91G/PI433251 BCIB population: WMG 308, WMG327, WMG377, and WMG388. During the winter of 2011, a greenhouse straw test was performed on twenty of the most resistant lines from the three interspecific populations to verify resistance. WMM 745 possessed higher levels of resistance than the check G122, though not the moderately resistant recurrent parent M0162. Only two lines (WMG 836, WMG 36) were less resistant than any of the three resistant check varieties (G122, M0162, NY 6020-5). Eighteen of the lines were more resistant than 91G and none showed less resistance than either susceptible check (91G and OSU 5613). These results and those from the field in 2011 indicate that the interspecific lines (with the exception of WMG 36) are fixed for resistance and have been shown to be so over three generations of advance. Crosses were made during the spring of 2011 with 13 interspecific lines possessing markers for identified interspecific QTL to produce 55 cross combinations. The populations were grown in the field and three single plants with at least 96 seeds each were 2 2 harvested from each cross. These were planted in the field in 2012 for generation advance to the F3 by single seed descent. One BCIB line (WMG904) has already been crossed to P. vulgaris G122 and NY6020, and was advanced to the F4 by single seed descent (SSD) in 2011. These two crosses have been planted in 2012 field for one more generation of SSD prior to phenotypic and genotypic analysis.