Development and validation of a robust KASP marker for zt2 locus in faba bean (Vicia faba)

Faba bean has the potential to become a key food and feed protein crop in many areas of the world. The presence of tannins in its seed coat has limited the deployment of this crop as feed and food. The expression of either of the two recessive genes, zt1 and zt2, causes a great reduction of tannins from the seed coat and results in a white flower phenotype. Molecular markers linked to these loci are fundamental tools for speeding up the breeding of low-tannin varieties. The main aim of this study was to develop and validate a robust molecular marker linked to the zt2 locus. We used 176 recombinant inbred lines of the Disco/2 × ILB 938/2 cross at F₆ and genotyped those using 257 SNP (single nucleotide polymorphism) markers. An SNP marker associated with zt2 locus was found on faba bean chromosome 3 and was used to develop a high-throughput low-cost KASP (kompetitive allele-specific PCR) marker. The KASP marker can successfully discriminate low-tannin faba beans carrying zt2 from those carrying zt1 and wild-type alleles.     

Inheritance and linkage of a gene for testa-imposed seed dormancy in faba bean (Vicia faba L.)

Seed dormancy in the grain legumes may interfere with germination in some situations, yet is not well described. Dormancy in faba bean (Vicia faba L.) was investigated to determine the mode of inheritance and whether the gene or genes controlling this trait are linked to other genes. Seeds produced by 235 recombinant inbred lines at the F₆ generation were used for this study. The parents were the cultivar ‘Optica’ and 172, an Afghanistan landrace which possesses extended seed dormancy. Germination was scored on a seed sample from each recombinant inbred line. Segregation patterns indicated that the trait is monogenic. This gene, named doz, is linked to a gene controlling anthocyanin and proanthocyanidin synthesis, sp-b, with the genetic distance estimated to be about 25 cM. No associations were detected between seed dormancy and pod dehiscence, seed lustre or mean seed weight. The implications of the persistence of a gene for seed dormancy in the gene pool of V. faba for the introgression of traits from landraces and cultivars possessing dormancy and for the origins of domesticated V. faba are discussed.     

Two host genes in Vicia faba for nodulation deficiency with strain specificity for Rhizobium leguminosarum

In greenhouse tests, two inbred lines of faba bean, I 40 and I 25, proved to be nodulation resistant and F₂ progenies were used to determine the genetic control of this nodulation deficiency. The defect is strain-specific for the Rhizobium strains St 48 and St 53. In inbred line I 40, the deficient nodulation character is controlled by a dominant gene which is called Sym-2, while another recessive gene sym-3 is assumed for line I 25; Sym-2 is epistatic over sym-3.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

Shortening the generation cycle in faba bean (Vicia faba) by application of cytokinin and cold stress to assist speed breeding

The aim of this study was to reduce the length of the breeding cycle for faba bean by accelerating seed setting. We examined the mode and time of exogenous 6-benzylaminopurine (BAP) (cytokinin) application, and cold treatments and their combinations in two faba bean genotypes. Acropetal node number of pod and seed set and pollen viability were recorded during the experiments. Application of BAP improved pollen germination. The application of 10^–5 M BAP 4 days after flowering increased seed set at the lower nodes. Cold treatment (8/4°C day/night for 2 days) after the onset of flowering induced the formation of more pods and faster pod set compared to the non-cold treatment. The time to first seed was significantly reduced, and pollen viability was increased in plants exposed to cold treatment. Increased pollen viability also showed a significant positive correlation with seed setting. The combinations of 10^–5 BAP and cold treatment together had similar and independent effects. These results will accelerate plant breeding in faba bean by providing additional tools for reducing generation time.     

Hybrid performance and AFLP- based genetic similarity in faba bean

Successful prediction of heterosis and performance of F₁-hybrids from the genetic similarity of their parents based on molecular markers has been reported in several crops and can be very helpful in hybrid breeding. The relationship between genetic similarities based on amplified fragment length polymorphism (AFLP) of 18 European faba bean lines and their hybrid performance and heterosis was investigated. Parental lines, 62 F₁-hybrids and their F₂-progenies were evaluated in field trials in four environments in Germany for seed yield, 1,000-seed weight and plant height. Results clearly demonstrated a stable superiority of the hybrids over their inbred parents and elite check cultivars, and showed a marked and varying amount of heterosis. Parental seed yield and F₂-hybrid yield were promising as predictors for F₁-hybrids. AFLP analysis of the 18 inbred lines using 26 EcoRI/MseI primer combinations resulted in 1202 polymorphic fragments. Cluster analysis based on genetic similarity estimates unambiguously identified pedigree-related inbred lines. No clear separation of the 18 inbred lines into subgroups was detected. Correlation coefficients between genetic similarity estimates and either heterosis or F₁-hybrid performance were small and not useful. Also correlations between specific genetic similarity and specific combining ability were too small for all traits to be of predictive value. Results showed that AFLP-based genetic similarities are not useful to predict the performance of hybrids or heterosis within the elite European faba bean gene pool.     

Genetic analysis of photosynthesis‐related traits in faba bean (Vicia faba) for crop improvement

Increasing productivity through improvement of photosynthesis in faba bean breeding programmes requires understanding of the genetic control of photosynthesis‐related traits. Hence, we investigated the gene action of leaf area, gas exchange traits, canopy temperature, chlorophyll content, chlorophyll fluorescence parameters and biomass. We chose inbred lines derived from cultivars 'Aurora' (Sweden) and 'Mélodie' (France) along with an Andean accession, ILB 938, crossed them (Aurora/2 × Mélodie/2, ILB 938/2 × Aurora/2 and Mélodie/2 × ILB 938/2), and prepared the six standard generations for quantitative analysis (P₁, P₂, F₁, F₂, B₁, and B₂). Gene action was complex for each trait, involving additive and dominance gene actions and interactions. Additive gene action was important for SPAD, photosynthetic rate, stomatal conductance and F_v/F_m. Dominance effect was important for biomass production. It is suggested that breeders selecting for productivity can maximize genetic gain by selecting early generations for canopy temperature, SPAD and F_v/F_m, then later generations for biomass. The information on genetics of various contributing traits of photosynthesis will assist plant breeders in choosing an appropriate breeding strategy for enhancing productivity in faba bean.     

Effect of density,cultivar and irrigation on spring sown monocrops and intercrops of wheat (Triticum aestivum L.) and faba beans (Vicia faba L.)

Spring-sown intercrops of wheat (Triticum aestivum L.) and faba beans (Vicia faba L.) were grown in three experiments at the University of Reading, UK. One wheat cultivar, Axona, and one (experiment 1) or two bean cultivars (experiments 2 and 3) Scirocco and Maris Bead, were grown as sole crops and intercrops at 50%, 75% and 100% recommended density. Experiments were rainfed but irrigation was an additional treatment in experiment 3. Biomass and seed yields of both wheat and faba beans were greater when monocropped than when intercropped. There was no evidence that radiation use efficiency (RUE) of intercrops was significantly different from sole crops. In all intercrops there was no significant effect of density on biomass, RUE or seed yield, though there were compensating changes in yield components. Seed yields of Maris Bead were significantly greater than Scirocco in experiment 3 but not experiment 2. There was no significant effect of irrigation on RUE or on wheat biomass and seed yield, but there was a trend for irrigation to increase faba bean biomass (P = 0.07) and seed yield (P = 0.06). With later sowing in experiments 2 and 3, time to harvest was shorter and wheat and bean biomass, seed yield and RUE were reduced. All land equivalent ratio (LER) values for both biomass (maximum 1.23) and seed yield (maximum 1.44) were greater than 1, with one exception in experiment 3, indicating that intercrops of wheat and faba beans make more effective use of land than equivalent sole crops. Partial LERs for faba beans were always lower than those of wheat. The tendency was for highest LERs to occur at 75% recommended density.     

Genotypic variation for drought tolerance in Vicia faba

Instability of yield in faba bean is partly caused by drought susceptibility. Four sets with 10-19 faba bean genotypes each were evaluated in multilocal field trials between 1992 and 1996. Stress occurred as natural drought in one experiment and as artificial terminal drought in three experiments. Artificial drought was induced by rain shelters; the control treatment was irrigated. Tolerance was assessed as the ratio of yield under drought (Yd) to well-watered yield (Yw). Highly significant variances between genotypes occurred; heritability of tolerance was 0.51 < h² <0.88. Exotic (North African. Latin American) genotypes were more tolerant than adapted material. Correlations between Yw and Yd were 0.77** < r <0.97**, and variance of Yd was less than one-third of the variance of Yw. Drought tolerance was negatively correlated with Yd (-0.41 < r < -0.22). Relative reduction of plant height due to drought was a promising trait to improve drought tolerance indirectly in two sets. The prospects of improving Yd are good: heritability was 0.68 < h² < 0.86. Genetic improvement of drought tolerance also seems feasible. A specific cross was proposed to create improved material.     

Resistance against broomrapes (Orobanche and Phelipanche spp.) in faba bean (Vicia faba) based in low induction of broomrape seed germination

Broomrapes are weedy root parasitic plants that severely constraint faba bean production. After long and extensive breeding efforts made in several countries only moderately resistant cultivars are available to farmers, being their resistance based on a combination of avoidance and resistance mechanisms. In this work we characterize the resistance mechanism of two faba bean breeding lines selected for resistance to Orobanche crenata. Mini-rhizotron experiments showed that low induction of seed germination is a major component of resistance in these lines against O. crenata, O. foetida and P. aegyptiaca. This is confirmed by in vitro experiments with root exudates. The fact that low induction of germination is similarly operative against O. crenata, O. foetida and P. aegyptiaca reinforce the value of this resistance.     

Genetic Effects on Host × Strain Interaction in the Symbiosis of Vicia faba and Rhizobium leguminosarum

50 Vicia faba lines were inoculated with 4 strains of Rhizobium leguminosarum in all possible combinations under greenhouse conditions in 5 replications. A root-sterile method was used for the cultures. Characters such as plant height, leaf colour, number and colour of nodules, the dry weights of shoot, root and nodules, and the nitrogen content were recorded, after 10 weeks. It was demonstrated by analysis of variance that, for example, for the dry matter of the shoot only 1.9 % of the total phenotypic variance was caused by interaction between Vicia and Rhizobium genotypes, but 24.7 % Was conditioned by the faba bean genotypes and 73.4 % by the Rhizobium strains. Differences of Vicia faba genotypes in their reaction to different Rhizobium strains were detected by joint regression analysis.     

The Nutritive Value of Ten Inbred Lines of Faba Beans (Vicia faba L.) in Relation to their Content of Antinutritional Constituents and Protein Quality

Ten inbred lines of fabe beans (Vicia faba L.) selected according to their quality characters have been investigated for carbohydrates, proteins, and antinutritional compounds. Digestible energy, N-balance trials with growing rats comprising determination of the protein digestibility and biological value were used as criteria in connection with comprehensive chemical-biochemical analysis. The chemical composition of the ten lines showed a considerable diversity as cad the results from the rat crisis. However, the content of vicine and convicine in all or the investigated lines were below the level previously found to have effects on the nutritive value, Starch, protein and fibre were the quantitatively dominating seed constituents, and all showed great variation among the lines. The starch content was not correlated to the quality or nutritive value of the seed, whereas the protein content was negatively correlated to the biological value and net protein utilization. These correlations followed the content of essential amino acids, lysine, threonine and methionine in the faba bean proteins. The content of the sulfur-containing ammo acids cysteine and methionine are especially dominant factors for protein quality. A simple method for total sulphur determination was found nut 10 be a sufficiently reliable technique for evaluating the content of methionine and cysteine in the faba beans. Tannin, insoluble- and total dietary fibre are phenolic aromatic compounds which were negatively correlated with the faba bean quality as expressed by digestible energy, the protein digestibility and the biological value of the faba beans. The results obtained have also revealed, that it is not sufficient to consider tannin as a group in relation to the faba bean quality. We need to separate and evaluate the different types, of phenolics in relation to the variations in quality of faba beans. Some of the low molecular weight (LMW) phenolics in the faba beans seem 10 be involved in inhibitory effects on hydrolase enzymes, chymotrypsin and trypsin. Trypsin and chymotrypsin from different animals were different in their sensitivity to faba bean inhibitors and additional experiments are required to reveal details about these effects. It has also been revealed that fructosans Lind LMW carbohydrates (oligosaccharides) are important in relation to the quality of faba beans.     

Studies on Sowing Depth for Chickpea (Cicer arietinum L.), Faba Bean(Vicia faba L.) and Lentil (Lens culinaris Medik) in a Mediterranean-type Environment of South-western Australia

Pulses such as chickpea, faba bean and lentil have hypogeal emergence and their cotyledons remain where the seed is sown, while only the shoot emerges from the soil surface. The effect of three sowing depths (2.5, 5 and 10 cm) on the growth and yield of these pulses was studied at three locations across three seasons in the cropping regions of south-western Australia, with a Mediterranean-type environment. There was no effect of sowing depth on crop phenology, nodulation or dry matter production for any species. Mean seed yields across sites ranged from 810 to 2073 kg ha⁻¹ for chickpea, 817–3381 kg ha⁻¹ for faba bean, and 1173–2024 kg ha⁻¹ for lentil. In general, deep sowing did not reduce seed yields, and in some instances, seed yield was greater at the deeper sowings for chickpea and faba bean. We conclude that the optimum sowing depth for chickpea and faba bean is 5–8 cm, and for lentil 4–6 cm. Sowing at depth may also improve crop establishment where moisture from summer and autumn rainfall is stored in the subsoil below 5 cm, by reducing damage from herbicides applied immediately before or after sowing, and by improving the survival of Rhizobium inoculated on the seed due to more favourable soil conditions at depth.     

Electrophoresis of the Seed Proteins of Vicia faba L. and Its Immediate Progenitors — A Reappraisal

Electrophoretic patterns of total seed protein analyzed by polyacrylamide gel electrophoresis under non-dissociating and dissociating conditions were found to have diagnostic value for the Vicia species. The close similarity in the seed protein profiles of the cultivated species and Vicia narbonensis narbonensis indicated that this last species can be considered as the immediate progenitor of V. faba. The study also indicated that V. serratifolia should be included as a subspecies in V. narbonensis. The broad bean, Vicia faba, is one of the most important leguminous pulses cultivated over a wide range (from Central Asia to Western Europe and from Ethiopia and Sudan to Scandinavia). Schultz -Motel (1972) was the first to identify remains of V. faba seeds from archeological sites of the Bronze Age, mainly from Central and Southern Europe. Recently, Kislev (1985) found charred seeds of horsebean (V. faba L.) from the 7th millennium B.C. Thus, horsebean should be included among the legumes grown by some early Neolithic people. Zohary and Hopf (1973) have suggested that the wild ancestors of V. faba are species belonging to section Faba. In particular, the French vetch V. narbonensis L., was the promosing candidate; most probably, because the area of distribution of V. narbonensis coincides with that of V. faba. On the other hand, Ieibe (1934), Ladizinsky (1975), and Abdall and Gunzel (1979) refuted the reports that V. narbonensis (and wild species from the section Faba) may be considered as immediate ancestors of the cultivated species of V. faba. These authors reported on the albumin profile of seed protein in Vicia. This study aims to reassess the relationships between V. faba and its taxonomically related species in terms of their storage protein profiles and present some information on the evolution of V. faba.     

Susceptibility of Faba Bean (Vicia faba L.) to Heat Stress During Floral Development and Anthesis

Experiments were conducted over 2 years to quantify the response of faba bean (Vicia faba L.) to heat stress. Potted winter faba bean plants (cv. Wizard) were exposed to temperature treatments (18/10; 22/14; 26/18; 30/22; 34/26 °C day/night) for 5 days during floral development and anthesis. Developmental stages of all flowers were scored prior to stress, plants were grown in exclusion from insect pollinators to prevent pollen movement between flowers, and yield was harvested at an individual pod scale, enabling effects of heat stress to be investigated at a high resolution. Susceptibility to stress differed between floral stages; flowers were most affected during initial green‐bud stages. Yield and pollen germination of flowers present before stress showed threshold relationships to stress, with lethal temperatures (t₅₀) ?28 °C and ~32 °C, while whole plant yield showed a linear negative relationship to stress with high plasticity in yield allocation, such that yield lost at lower nodes was partially compensated at higher nodal positions. Faba bean has many beneficial attributes for sustainable modern cropping systems but these results suggest that yield will be limited by projected climate change, necessitating the development of heat tolerant cultivars, or improved resilience by other mechanisms such as earlier flowering times.     

Significance of Seed Source on Grain Yield in Faba Beans (Vida faba L.) and Dry Peas (Pisum sativum L.)

In order to investigate the significance of seed source on yield and related characters 47 seed lots of seven faba bean varieties and 24 seed lots of five dry pea varieties were tested in three series of field trials. The grain yield of the faba bean varieties tested was considerably modified by seed source, whereas only small and nonsignificant effects of seed source were found in dry peas. In general, seed sources with a high thousand grain weight, a high protein content, high portions of undamaged and healthy seeds, and in faba beans a high degree of cross-fertilization exhibited superior grain yields. Production of high-performance seed proved to be the most difficult for the large-seeded and white-flowered faba bean varieties. Based on a tabular summary on factors affecting effects of seed source problems of seed production in both grain legumes and their impact on yield are discussed.     

Genetic variability of faba bean genotypes for chocolate spot (<Emphasis Type="Italic">Botrytis fabae</Emphasis>) resistance and yield

Faba bean (Vicia faba L.) has high utility as a food and soil fertility improving crop. One of the major fungal pathogens of faba bean is Botrytis fabae, the causative agent of chocolate spot. The disease affects significantly the leaf, stem, pod and seed of faba bean compromise its productivity in the smallholder farming sector. Nonetheless, there are limited resistant/tolerant faba bean varieties available and disease control technology options. Therefore, it was prudent to evaluate faba bean landraces for chocolate spot resistance. Fifty landraces together with ten improved varieties were evaluated both in the field and in the greenhouse under natural and artificial inoculation with previously selected aggressive Botrytis fabae isolate (Iso-016) from West Gojjam, in Ethiopia. There were highly significant differences (p?<?0.001) among the landraces for reaction to the disease and agronomic traits. Significant positive correlation was recorded between reaction of genotypes in the field and greenhouse disease data. The overall mean disease epidemics varied from 92.5 to 697.5 for the area under disease progress curve (AUDPC). The highest level of resistance was found in the ICARDA lines, ILB-4726, ILB-938 and BPL-710. Of all 18 landrace collections displayed significantly lower disease reaction than the susceptible check. However the resistance was moderate. The selected eighteen landraces will be recommended for use in breeding for chocolate resistance. Overall, resistance was highly heritable, suggesting that phenotypic selection can be exploited to improve chocolate spot resistance in faba bean varieties.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.     

Density enhancement of a faba bean genetic linkage map (Vicia faba) based on simple sequence repeats markers

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.     

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F₂ mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F₂ plants were divided into three subpopulations according to the original F₁ plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.