Identification and Verification of QTL Associated with Frost Tolerance Using Linkage Mapping and GWAS in Winter Faba Bean

Abstract

Frost stress is one of the abiotic stresses that causes a significant reduction in winter faba bean yield in Europe. The main objective of this work is to genetically improve frost tolerance in winter faba bean by identifying and validating QTL associated with frost tolerance to be used in marker-assisted selection (MAS). Two different genetic backgrounds were used: a biparental population (BPP) consisting of 101 inbred lines, and 189 genotypes from single seed descent (SSD) from the Gottingen Winter bean Population (GWBP). All experiments were conducted in a frost growth chamber under controlled conditions. Both populations were genotyped using the same set of 189 SNP markers. Visual scoring for frost stress symptoms was used to define frost tolerance in both populations. In addition, leaf fatty acid composition (FAC) and proline content were analyzed in BPP as physiological traits. QTL mapping (for BPP) and genome wide association studies (for GWBP) were performed to detect QTL associated with frost tolerance. High genetic variation between genotypes, and repeatability estimates, were found for all traits. QTL mapping and GWAS identified new putative QTL associated with promising frost tolerance and related traits. A set of 54 SNP markers common in both genetic backgrounds showed a high genetic diversity with polymorphic information content (PIC) ranging from 0.31 to 0.37 and gene diversity ranging from 0.39 to 0.50. This indicates that these markers may be polymorphic for many faba bean populations. Five SNP markers showed a significant marker-trait association with frost tolerance and related traits in both populations. Moreover, synteny analysis between Medicago truncatula (a model legume) and faba bean genomes was performed to identify candidate genes for these markers. Collinearity was evaluated between the faba bean genetic map constructed in this study and the faba bean consensus map, resulting in identifying possible genomic regions in faba bean which may control frost tolerance genes. The two genetic backgrounds were useful in detecting new variation for improving frost tolerance in winter faba bean. Of the five validated SNP markers, one (VF_Mt3g086600) was found to be associated with frost tolerance and FAC in both populations. This marker was also associated with winter hardiness and high yield in earlier studies. This marker is located in a gene of unknown function.

Keywords: GWAS; QTL mapping; QTL validation; Synteny; faba bean; frost tolerance.

Figure 1

Winter faba bean genetic linkage map based on 117 SNP markers showing location of QTL for frost tolerance and FAC in 101 RILs. Red markers refer to validated QTL for the same trait. Blue marker refer to validated QTL for other frost tolerance or/and FAC.

Figure 2

The Q–Q plot shows the expected −log (P) vs. the −log (P) for AUSPC, LCAF, and LTAF. (A) GLM (B) MLM + kinship.

Figure 3

Graphic representation of QTLs showing interaction effects for proline content in BPP (101 RILs). Black circles refer to QTLs with additive effects. The red line refers to significant interaction between the main effects of two QTLs.

Figure 4

Highest expression of candidate genes in different tissues. The gene expression was considered in tissue which was not exposed to any treatment (under control conditions).

Figure 5

(A) Linkage groups associated with putative QTL for frost tolerance (Arbaoui et al., 2008b), (B) The collinearity between SNP&RAPD-LG18 constructed by Sallam et al. (unpublished data, doi: 10.6084/m9.figshare.3471665, https://figshare.com/s/3aee3d0182a75c7375a9), SNP-LG10 of the genetic map was constructed in the present study, and both RAPD-LG18 and RAPD-LG10 were constructed by Arbaoui et al. (2008b). Markers on SNP-LG10 are located on chromosome 7 of M. truncatula and collinear with FBCM_05.