Carmen Palomino

Assessment of candidate reference genes for expression studies in Vicia faba L. by real-time quantitative PCR

Faba bean (Vicia faba L.) cultivation has declined in recent years due to several factors, including diseases and anti-nutritional compounds in the seeds. The introduction of disease resistance and the elimination of anti-nutritional factors in new varieties are important objectives in any breeding program for the species. Because of the faba bean’s huge genome, it is necessary to rely on synteny with related species in order to identify candidate genes responsible for the character under study. Quantification of expression level of candidate genes could help to validate them. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression level. Real-time quantitative PCR was used for evaluate the expression stability of 11 candidate reference genes. A wide set of samples, including different tissues, genotypes and several inoculations for the most important pathogens were employed. The expression stability of the candidate genes was analyzed using two different algorithms, geNorm and NormFinder, and results obtained from both algorithms were highly correlated for each experimental set. In all cases, either ACT1, CYP2 or ELF1A genes performed as the most stable genes in our experimental sets. They also represent part of the best combination of genes according to the geNorm and NormFinder algorithms. Our data showed the wide expression range of the selected genes, confirming that no single reference gene had a stable expression under these conditions in the faba bean. We recommend the use of ACT1, CYP2 and ELF1A as the most suitable reference genes to normalize gene expression for future studies in V. faba.

Large-Scale Transcriptome Analysis in Faba Bean (Vicia faba L.) under Ascochyta fabae Infection.

Faba bean is an important food crop worldwide. However, progress in faba bean genomics lags far behind that of model systems due to limited availability of genetic and genomic information. Using the Illumina platform the faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to Ascochyta fabae infection have been characterized. De novo transcriptome assembly provided a total of 39,185 different transcripts that were functionally annotated, and among these, 13,266 were assigned to gene ontology against Arabidopsis. Quality of the assembly was validated by RT-qPCR amplification of selected transcripts differentially expressed. Comparison of faba bean transcripts with those of better-characterized plant genomes such as Arabidopsis thaliana, Medicago truncatula and Cicer arietinum revealed a sequence similarity of 68.3%, 72.8% and 81.27%, respectively. Moreover, 39,060 single nucleotide polymorphism (SNP) and 3,669 InDels were identified for genotyping applications. Mapping of the sequence reads generated onto the assembled transcripts showed that 393 and 457 transcripts were overexpressed in the resistant (29H) and susceptible genotype (Vf136), respectively. Transcripts involved in plant-pathogen interactions such as leucine rich proteins (LRR) or plant growth regulators involved in plant adaptation to abiotic and biotic stresses were found to be differently expressed in the resistant line. The results reported here represent the most comprehensive transcript database developed so far in faba bean, providing valuable information that could be used to gain insight into the pathways involved in the resistance mechanism against A. fabae and to identify potential resistance genes to be further used in marker assisted selection.

QTLs for ascochyta blight resistance in faba bean (Vicia faba L.): validation in field and controlled conditions

Abstract. Ascochyta blight is an important disease of faba bean (Vicia faba L.). Yield losses can be as high as 90% and losses of 35–40% are common. The line 29H is one of the most resistant accessions to the pathogen (Ascochyta fabae Speg.) ever described. In this work, we aimed to validate across generations the main quantitative trait loci (QTLs) for ascochyta blight resistance identified in the cross 29H × Vf136 and to test their stability under field conditions. QTLs located on chromosomes II and III have been consistently identified in the recombinant inbred line (RIL) population of this cross, in both controlled (growth chamber) and field conditions and, thus they are good targets for breeding. In addition, a new QTL for disease severity on pods has been located on chromosome VI, but in this case, further validation is still required. A synteny-based approach was used to compare our results with previous QTL works dealing with this pathogen. Our results suggest that the QTL located on chromosome II, named Af2, is the same one reported by other researchers, although it is likely that the donors of resistance differ in the allele conferring the resistance. By contrast, the location of Af3 on chromosome III does not overlap with the position of Af1 reported by other authors, suggesting that Af3 may be an additional source of resistance to ascochyta blight.