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, 2008, 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. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Our focus has been the transfer of the high levels of white mold resistence found in P. coccineus (runner bean) into common bean. We have used for this purpose three backcross-inbred populations: OR 91G/PI255956 (115 BC2F4-8 lines), OR 91G/PI433251B (256 BC2F4-7 lines), and M0162/PI433251B (119 BC2F4-7 lines). Analysis of the three populations has been completed. In 2010, we compared our mapping results for the OR 91G/PI433251B (Gx43), and M0162/PI433251B (M0x43) with the previously analyzed 91G/PI 255956 (Gx25) population. The Gx25 population map had QTL on Pv02, Pv06 and Pv09 that collectively explained 34.7% of the phenotypic variation. In the Gx43 population, 112 markers formed nine linkage groups corresponding to 8 of 11 LGs. Single factor analysis of variance (SFA) identified 26 loci significantly associated with white mold resistance. A QTL associated with field severity was identified by Multiple QTL Mapping (MQM) on Pv05 and accounted for 6.4% of the phenotypic variation. In the Mx43 population, 70 of 79 SSRs produced three linkage groups corresponding to Pv02, Pv03 and Pv04. SFA identified 30 loci significantly associated with white mold resistance. A QTL associated with field severity located on Pv02 accounted for 9.8% of the phenotypic variation. Even though polymorphic markers were identified on these linkage groups, Pv01, Pv05, and Pv08 in Gx25, Pv08, Pv09 and Pv11 in Gx43 and Pv01, Pv05-Pv11 for Mx43 were not represented. Severe segregation distortion was observed, with more heterozygotes and fewer homozygous donor alleles than expected. The most logical explanation is that homozygous P. coccineus alleles in a P. vulgaris background reduce fitness compared to heterozygous alleles, the latter out-reproducing the former. Reduced recombination was also associated with segregation distortion leading to the slow elimination of genetic load. In Mx43, several instances of heterosis for resistance were observed.We are now entering the next phase of the research where we validate and characterize QTL and transfer them into economically important common bean backgrounds. To do this, we identified a subset of lines from each population that combine relatively high levels of resistance (as measured in the straw and/or field tests) and the markers associated with the significant QTL in each population.