Parental genetic divergence
The comparison between the two parental varieties shows that the difference in terms of length, width and thickness is highly significant (p<0.05). Likewise, the t-test confirms that the sphericity of ‘Zina’ seeds is significantly higher than that of ‘Reina Mora’ (Table 1). Thus, the quantitative marker adopted is polymorphic and makes it possible to discriminate between the two varieties chosen.
Outcrossing rate estimation
Of the 108 plants sown in Sidi Allal Tazi (36 plants/replicate), eight plants of ‘Reina Mora’ (Plant 18 of the first replicate, plants 7, 8, 9 and 10 of the second replicate and plants 4, 12 and 14 of the third replicate) as well as three plants of ‘Zina’ (Plant 8 of the first replicate and plants 1 and 15 of the second replicate) did not survive, during pod formation period. In fact, ‘Reina Mora’ is more adapted to irrigated areas, which must have constrained its development, especially after the occurrence of several heat waves during 2021 growing season. Thus, the first replicate consists of 34 plants of both varieties, instead of 36, while the second and third plots are composed of 30 and 33 plants, respectively.
At the level of replicates, outcrossing rate ranged from 0 to 8.82% over the two locations. The highest average rate was observed in Sidi Allal Tazi (7.22%). In this environment and based on the ‘seed coat color’ marker, two plants of ‘Zina’ have been pollinated by ‘Reina Mora’, namely plant 9 (one flower) and 13 (4 flowers), spotted at the first and the second replicates respectively. The average rate of allogamy is then estimated at 2.06% over all the replicates (R1: 2.94%, R2: 3.33% and R3: 0%). The marker ‘seed size and shape’ clearly differentiates five plants of ‘Reina Mora’ collected in Sidi Allal Tazi. Plants 2 and 4 of the first plot, plant 1 of the second plot and plants 1 and 5 of the third plot (one flower each) showed a spherical shape that is very similar to that of faba bean minor ‘Zina’. On the basis of the combined markers, seeds color, size and shape, the overall allogamy rate in Sidi Allal Tazi amounts to 7.22% (R1: 8.82%, R2: 6.66% and R3: 6.06%). In terms of individual plants, outcrossing rate in Sidi Allal Tazi varied from 1.66% to 25%. This rate is closely linked to the yield, mainly the total number of pods produced per plant. Overall, ‘Zina’ expresses a much higher pod yield than ‘Reina Mora’, which creates a significant gap in terms of cross-pollination rate in individual plants. With the exception of plant 13 of ‘Zina’, from the second replicate, which produced four hybrid pods, all of the plants (‘Zina’ and ‘Reina Mora’) had only one cross-pollinated flower by plant. The lowest outcrossing rate (1.66%) was observed on plant 9 of the first replicate recording a high yield of around 60 pods. On the other hand, the highest rate of allogamy (25%) was observed on plant 1 of ‘Reina Mora’ of the third replicate, due to the very low number of pods produced (four).
In Douyet, no purple coloration was observed in the seeds collected from ‘Zina’ plants. Therefore, the outcrossing rate based on the marker ‘seed coat color’ was zero. However, four plants of ‘Reina Mora’, 5 and 12 from the first replicate and 1 and 18 from the second one produced one pod each, containing similar seeds to faba bean minor ‘Zina’ in terms of size and shape (Fig 2). The natural hybridization rate in Douyet is therefore equivalent to 3.70% (R1: 5.55%, R2: 5.55% and R3: 0%), which is half the rate recorded in Sidi Allal Tazi. The outcrossing rate of individual plants in Douyet varied from 6.25% to 10%. This rate is relatively high due to the low number of pods (10 to 16) produced by ‘Reina Mora’.
The overall outcrossing frequency observed in this experiment is low, compared to the average rate 40-50% (
Bond and Poulsen, 1983). Early studies reported quite different results. For instance,
Drayner (1959),
Rowlands (1960),
Fyfe and Bailey (1951) in England,
Picard (1963) in France,
Gottschalk (1960) in
Germany and Kambal (1969) in Sudan reported high outcrossing rates, largely exceeding 30% and, therefore, granted Vicia faba L. an intermediate position between allogamy and autogamy, whereas
Muratova (1931) admitted that intercrossing could be significantly curtailed by a simple isolation. Likewise,
Lechner (1962) reports low outcrossing rates, 8-10% in Germany and considers that isolation is sufficient for line fixation and varietal purity maintenance.
Numerous factors could limit outcrossing rates. In fact, synchrony in flowering periods was taken into account when choosing the plant material. However, the highly selfing nature of the two lines ‘Zina’ and ‘Reina Mora’, could tend to induce a gametic selection for autopollen over allopollen
(Mesquida et al., 1990). It is known that in preferential selfing species, for which inbreeding depression is weak, low levels of outcrossing are often found
(David et al., 1993). Thus, the use of self-fertile material should be preferably avoided in breeding synthetics varieties, as recommended by
Becker (1988). Another possible explanation has been put forward by
Ibarra-Perez et al. (1997) who reported that the use of parental germplasm from different geographic gene pools may give lower estimates of outcrossing rate than use of germplasm from the same gene pool. As a matter of fact, the variety ‘Zina’ is Moroccan whereas ‘Reina Mora’ is Spanish.
Weather conditions might also affect natural hybridization. It is important to note that the 2020-2021 growing season was a singular year, thanks to the heavy rainfall that spread over a long period (from October to May). The peak of precipitation, i.e. 113 mm was recorded in April at the experimental farm of Douyet which coincides with faba bean flowering season, suggesting that continuous rainfall sequences might have limited bees activity during this critical period. In addition, the variation in the abundance of flower-visiting species, either spatially or seasonally, has a major influence on cross-pollination within and among crop fields, hence the difference spotted between the two tested regions, Sidi Allal Tazi (Gharb, 7.22%) and Douyet (Saïs, 3.70%). These findings were in agreement with our primary assumption in the field, owing to the fact that the experimental farm of Douyet was strangely and unusually devoid of bees and bumblebees.
The decline in bees during the bloom period aroused our curiosity. Therefore, we launched an investigation with the Moroccan Interprofessional Federation of Beekeeping (FIMAP) to understand and supplement our results. No cases of mass death have been reported in the Gharb region apiaries, including Sidi Allal Tazi. However, the decline of honeybee populations is much more pronounced in Saïs region where massive colony losses occurred subsequent to an invasion of the varroa mite parasite, in conjunction with lax control. Fes-Meknes region beekeepers do not treat or little with varroacides or alcohol washes. Instead, they use plants consumed in a smoker, the effectiveness of which is not known
(Mohssine et al., 2020). It is also important to not overlook the repercussions of climate change that modifies plants spatial distribution and their biological cycles (with a possible desynchronization of the plant biology and the bees foraging activity) and therefore creates less favorable survival conditions for pollinators on some territories
(Soroye et al., 2020).
Our results were in agreement with those of
Mesquida et al., (1990) where outcrossing rate under natural conditions, at INRA of Rheu in France was 5.1%, using the same marker, seed color. They reported that cross-pollination, after bees introduction, barely reached 9.4%. Cages with bumblebees recorded the highest rate, 11.6%, which is still much lower than average 40-50% (
Bond and Poulsen, 1983). Contrasting results were found by
Suso et al., (2001) in Rennes, France and in Cordoba, Spain, where allogamy rates reached 33% and 65%, respectively. Similarly, higher rates (30.8%) have been published by
Hu et al., (2011) using flower color as a genetic marker, in Pullman, United States.
Comparison between the shape and size of ‘Reina Mora’ F1 hybrid seeds and the parent
The t-test results confirmed that seeds size and shape differed significantly between the parent and the F1 progeny resulting from natural hybridization (p <0.05), except for the width (Table 2). Given the relevance of the character ‘sphericity’ in the differentiation between faba bean major and minor, it is important to highlight the difference observed between ‘Reina Mora’ parental seeds and the F1 hybrid seeds resulting from the natural crossing with ‘Zina’. Indeed, the hybrid seeds are 70.8% spherical while the parental seeds exhibit a more flattened appearance with an average sphericity of 59%. This difference is illustrated in a box diagram (Fig 3).
Comparison between the shape and size of ‘Reina Mora’ F1 hybrid seeds and the self-pollinated seeds
In the same perspective, the t-Student test was carried out to check whether there are significant differences between the F1 seeds obtained from the likely natural hybridization and the seeds produced by selfing. As expected, the two types of seeds are significantly different in terms of length, width, thickness, geometric diameter, sphericity and surface area (Table 3). Seeds sphericity represents a differentiating trait. Thus, it is important to note that the sphericity of F1 hybrid seeds is around 70.8% while those derived from self-pollination show an average sphericity of 58.5%; this difference is illustrated in Fig 4.
