1. Introduction
As of 2020, the world population was over 7.8 billion and projected to increase by more than 25% to reach 9.9 billion by 2050 [
1]. This ever-growing population has led to concerns about the increased demand for food. In order to achieve sustainable agricultural development for food security, it is important to breed resilient high-yielding crops that are suited to suboptimal conditions of growth. Hybrid wheat represents a promising approach that improves yield potential, yield stability across diverse environments and consequently increases global wheat productivity [
2]. However, hybrid wheat occupies nearly 1% of the total world wheat area and is produced mainly in Europe, China and India [
3]. Central Europe, particularly France, Hungary, and Germany, contains about half of the world’s hybrid-wheat production area and is home to the two leading hybrid seed producer companies ASUR Plant Breeding SAS, (previously SAATEN UNION) and Nordsaat Saatzuchtgesellschaft mbH [
4]. All hybrids that are registered in Europe are currently produced by the application of chemical hybridization agents (CHAs), most commonly Croisor
® 100 (Sintofen; former Dupont–Hybrinova, Saaten–Union Recherche, France). In China, photoperiodic sensitivity and cytoplasmic male sterility (CMS) systems are being successfully used for grain production [
4,
5]. Wheat hybrids in India are developed using cytoplasmic male sterility (CMS) systems derived from
Triticum timopheevii and a CHA approach [
2,
6].
For the long term success of hybrid breeding programs, adequate exploitation of heterosis, cost-effective hybridization systems, high seed density and the development of high-yielding heterotic groups and patterns must be established [
4,
7,
8,
9]. Heterosis, or hybrid vigor, is the superiority of a hybrid in its performance over its corresponding parental inbred lines (mid- or best- parent heterosis). Recently, there has been significant focus on developing successful hybrids through different approaches, for more cost-effective seed production [
10,
11,
12]. Studies based on experimental data in hybrid wheat breeding have demonstrated that best-parent heterosis ranged from −70.4 to 54.3% and −26.9 to 29.2% in two successive years, respectively, using CHA Croisor
® 100 on elite winter wheat lines from the University of Nebraska–Lincoln (UNL) and Texas A and M University breeding programs; while mid-parent heterosis for grain yield varied from −15.33% to 14.13% of 112 hybrids produced using a CMS system [
8,
13].
Despite the hybrid yield advantage, development of practical hybrid seed production systems has not yet reached large-scale hybrid wheat production. This can, in part, be attributed to the high cost of hybrid wheat seed. Therefore, maximizing seed set per spike is an effective approach to tremendously improve the cost-efficiency of hybrid seed production in self-pollinating crops, such as wheat, irrespective of the hybridization system. Floral biology plays an important role in enhancing outcrossing ability. Knowledge of the genetic basis of important floral traits and exploring the genetic variability of parental pairs might offer new perspectives on methods to restore the fertility control system [
5,
9].
Numerous studies have addressed the importance of appropriate floral and flowering traits as key determinants for potential hybrid parents. Traits like anther extrusion, visual anther extrusion, pollen mass, degree and duration of flower opening, stigma receptivity, anther length, number and longevity of pollen grains show large genotypic variation and moderate to high heritabilities and thus might be integrated for a successful fertility control system [
14,
15,
16,
17,
18,
19,
20]. Considerable progress has been made in identifying proxies suitable for allowing direct and indirect selection of associated hybrid potential traits through the use of new precision phenotypic approaches and advanced high-density genomic tools (genome wide association studies and genomic prediction) [
16,
17,
18,
19,
21,
22,
23]. Consequently, anther extrusion and profuse pollen shedding have been shown to be promising traits to predict related traits such as pollen mass, openness and duration of floral opening [
16,
19]. Similarly, selection of parents with the best parental trait combinations is a key determinant of the heterosis level [
24]. However, potential male and female parents with superior floral and agronomic trait combinations may not always transmit desirable traits to their hybrid progenies. To overcome this issue, combining ability, as an important genetic parameter, has been widely adopted in plant breeding to compare the performance of lines in hybrid combinations [
13,
25,
26,
27,
28].
The concepts of general combining ability (GCA) and specific combining ability (SCA) were first established by Sprague et al. [
28]. GCA describes the average performance of a parent in different hybrid combinations, whereas SCA describes the deviation in performance of certain hybrid combinations as compared to what would be expected based on the GCA of the parents involved. GCA is an important indirect criterion in selecting inbred parents, which means that the phenotypic selection is based on the GCA [
7], SCA might be used to identify a specific cross combination for exploitation through heterosis breeding [
29].
A diallel analysis scheme was widely used to identify parental genotypes with high GCA and hybrid combinations with high SCA [
30]; and to obtain the genetic information of hybrids and their parents for further classification in heterotic patterns [
31]. To establish heterotic groups, selecting high-yielding parental lines with appropriate trait combinations could help in clustering the germplasm in different heterotic groups based on trait-per-se performance. Using this method, phenotypic and genotypic assessments of 196 genotypes for various floral and flowering traits were previously undertaken by El Hanafi et al. [
19,
32]. Our objectives in this study were to (1) investigate the efficiency of the CHA Croisor
® 100 on the selected female in crossing blocks using three different doses; (2) assess the hybrid seed set of successful hybrids produced using the appropriate rate; (3) investigate the genetic variance and heritability of the hybrid seed set and its correlation with the evaluated male floral traits; (4) evaluate the hybrid’s performance and obtain estimates of the expressed percentage of the mid-parent (MPH) and best-parent heterosis (BPH) levels; (5) and determine the patterns of GCA and SCA.
3. Discussion
Efforts to investigate the potential of hybrid breeding to further increase yield in bread wheat were carried out in this study. Understanding the genetic basis of the floral and flowering traits to enforce outcrossing ability may lead to an effective hybrid system and thus, achieve large-scale hybrid production. Successful hybrid wheat breeding programs rely on an optimized breeding strategy that combines several prerequisites for maximum effective pollination and seed set. Identification of parents with suitable hybrid traits, application of effective CHA with proper doses and timing, determination of the combining abilities and heterosis are key factors that determine the success of hybrid wheat breeding.
3.1. CHA Efficiency
In this study, we used parental lines identified previously by El Hanafi et al. [
19] with favorable floral traits to assess the impact of male floral traits on female seed set, in designed crossing blocks. First, the efficacy of the gametocide Croisor
® 100 was tested using three different doses applied at early booting stage. This CHA is widely used particularly in Central Europe and has proven its commercial value for hybrid seed production. Frequent verifications were made for well-timed application. The sterility control mechanisms applied proved the efficiency of the chemical, and adequate gapping was observed. Male sterility was induced in almost 95% of the female plants using the 13.5 L ha
−1 rate. Afterwards, seed data was collected to determine the success level of the CHA-treated female plants. The different parental combinations showed significant phenotypic variation of the hybrid seed set and other evaluated flowering and floral traits. The 13.5 L ha
−1 dose plants had an average of 35 seeds on the mother lines which supplied enough seed for hybrid performance trials.
3.2. Variation in Cross-Pollination Traits
The significant genotypic variances recorded for the important floral traits such as pollen mass, visual anther extrusion, pollen mass and anther length suggest potential for exploiting the variation present in the genetic material used. Moreover, the high heritability recorded for all traits indicates that there is sufficient variation in the population used and there is low influence from environmental factors. This also suggests that the genotypes might be efficient for hybrid seed production in several environments. Similar variation and heritability were observed in previous studies confirming that these traits can be improved by consecutive hybridization for an efficient hybrid breeding program [
16,
17,
19]. Anther extrusion, pollen mass and profuse pollen shedding were shown to be promising traits to act as proxies for predicting other harder to measure floral traits such as openness of the florets and duration of floral opening of female plants. However, little is known about the relationship with seed set and floral traits. Until now, Boeven et al. [
17] was the only study that showed a correlation between visual anther extrusion and hybrid seed set (r = 0.76,
p < 0.001). In this study, seed set was correlated with visual anther extrusion with r = 0.94 (
p < 0.001). Thus, visual anther extrusion can serve as a rapid proxy to estimate the potential of a female line in a hybrid breeding program. The present study also addresses, for the first time as far as the authors know, the effect of other floral traits on seed setting. High correlations were found between seed setting and pollen mass, pollen shedding and anther length, with r = 0.97 (
p < 0.001), r = 0.91 (
p < 0.001) and r = 0.46 (
p < 0.05), respectively. Surprisingly, the relationship between seed setting and the phenology traits, produced a non-significant correlation even though male lines were specifically chosen to flower 1–3 days earlier and to be taller than the female lines. This demonstrates the inaccuracy of using phenology traits to produce high seed set on female lines. Therefore, genomic tools might be incorporated to predict the flowering synchronization in hybrid seed production based on historical weather information [
33].
Our study showed that pollen mass had a high correlation with pollen shedding (r = 0.96) and visual anther extrusion (r = 0.95;
p < 0.001), and as was expected, the maximum extruded anthers contributed to maximum shed pollen outside the florets [
34]. This confirms the results previously found by El Hanafi et al. [
19] and Langer et al. [
16]. Moreover, anther length can, in turn, increase the quantity of pollen released from every single anther which is confirmed by the collective correlation observed between pollen mass and each of anther length (r = 0.55), pollen shedding (r = 0.60) and visual anther extrusion (r = 0.55). Similar associations were reported by [
16]. In contrast, there was no association between anther length and pollen mass or any other floral trait in a previous experiment carried out in Morocco [
19].
3.3. Hybrid Performance and Heterosis
Successful hybrid seed was produced to test the 23 F1 hybrid combinations in yield trials across two cropping seasons in Merchouch and Sidi El Aydi. The genotypic variability for days-to-heading, plant height, spike length and tillers per plant were mostly found to be non-significant. The significant G × E observed for PLH, SPS, BM, TKW and YLD indicated that for at least some of the hybrid combinations, the cross and its parents, exhibited different levels of phenotypic expression under different environmental conditions. While for the rest of the evaluated traits (DH, SPL, TLP), G × E interaction effects were non-significant indicating the stability of the evaluated hybrids and their parents across the environments.
The main target of the hybrid breeding program was to identify potential parents that can be crossed to produce F1 hybrids with a high heterosis level. Previous studies have reported important heterosis and heterobeltiosis in wheat [
13,
35,
36,
37,
38]. In this study, several hybrids exhibited significant heterosis and heterobeltiosis for the evaluated traits.
In this study, MPH for biomass ranged from −19.18 to 50.24% in the four environments. While a maximum of 13.35% was recorded for the overall data, expressed by the hybrid P9/P2. A maximum of 50.24% was recorded by the hybrid P1/P2 in the 2020 trial in Sidi El Aydi even though the parental pair involved had not shown the best biomass performance. This is not surprising considering that the cross performed better in extreme drought station (Sidi El Aydi) with supplemental irrigation as compared with a station in drought and only rainfed (Merchouch). However, negative mid- (−9.34%) and best-parent heterosis (−11.58%) was recorded for the same cross in the 2019 trial in Sidi El Aydi and attests to the instability of this cross across environments.
Stable positive heterosis over the mid-parent for biomass was recorded by six hybrids, P1/P8, P9/P2, P9/P18, P10/P5, P10/P12 and P13/P11, across each year and environment. This might be explained by the use of parental lines that were well characterized for potential floral traits combined with good agronomic performance. The wide positive range found for biomass heterosis was far better than what was found in previous studies with a maximum of 0.9% in Morgan et al. [
39] and 5.4% in Kindred and Gooding [
40]. BPH ranged from −33.53 to 39.74% in the four environments with a maximum value observed for the hybrid P10/P12 in the 2020 SEA trial. The maximum BPH for biomass for the combined data set was low (7.21%), displayed by the hybrid P13/P11.
For thousand kernel weight, a wide range of mid- and best-parent heterosis was found for the individual experiments and for the combined data. Some hybrids consistently outperformed their parents across all environments. Of the twenty-three hybrids produced, ten (P4/P17, P4/P7, P9/P2, P9/P6, P9/P8, P9/P18, P10/P5, P10/P12, P10/P14 and P10/P15) had consistently positive MPH in all environments except the cross P9/P18 in the 2020 SEA trial. This appears very promising especially because the parents were shown to have a dual parentage purpose (male and female). Many studies have reported positive and negative mid- and best-parent heterosis [
41,
42,
43]. Singh et al. [
42] reported the highest relative heterosis and heterobeltiosis of 28.14 and 24.07%, respectively, in three different environments. While two other studies reported a maximum of 16.14% MPH. Generally, greater MPH or BPH might be attributed to genetically distant parental lines that belong to different heterotic groups [
44].
Grain yield heterosis in wheat has been a trait of widespread interest to many researchers from as early as 1934 [
45]. Briggle [
46] was among the first to report heterosis in wheat and since then research efforts over the years have demonstrated predictable yield advantage and stability [
8,
13,
35,
47,
48,
49,
50,
51,
52,
53]. Easterly et al. [
35] evaluated 650 hybrids developed from a full diallel of 26 parents in experimental yield plots and reported mid- and best parent heterosis of a maximum of 24%. Adhikari et al. [
13] reported ranges from −70.4 to 54.3% and −26.9 to 29.2% grown in two cropping seasons in Lincoln, NE, and Greenville, TX, USA. In the current study, MPH ranged from −14.41 to 23.76% and from −22.66 to 22.31 in the 2019 and 2020 Merchouch trials, respectively. While, in Sidi El Aydi, MPH tended to be better with ranges of −12.01 to 33.86% and −9.47 to 36.02% in 2019 and 2020, respectively. Hybrids have been shown to out-yield their best parents by 18.36% and 23.07% as seen with the highest values found at Merchouch and Sidi El Aydi, respectively. The higher positive MPH and BPH found in Sidi El Aydi might be attributed to a greater response to irrigation applied during the critical time of vegetation growth. For the combined data across locations, the cross P9/P6, considered as the best performing hybrid, was shown to out-yield the mid and best-parent by more than 1 t ha
−1 each or 26.65% and 24.04% BPH, respectively. High yield of individual hybrids was observed in crosses involving high-yielding parents with promising parental floral traits as previously evaluated (P4, P9 and P10). In contrast, some other hybrids did not perform as expected even though both parents were reported to be high yielding, indicating that heterosis depends not only on performance per se but also combining ability.
Competitive hybrid wheat production depends on the heterosis level of yield to be sufficiently high to compensate for seed cost. It has been demonstrated in a previous study that cost-efficient hybrids would only be economically viable if the range of MPH was between 6 to 34% [
54]. Similarly, Angus [
55] estimated that 5% yield advantage over the best line-bred was required to counterbalance the higher seed cost. Thus, we can conclude that our results were very promising and the heterosis level demonstrated the potential of hybrid wheat to boost global wheat productivity and hence, show commercial hybrids to be viable.
3.4. General and Specific Combining Abilities
Several studies have estimated the GCA of each parental genotype and the SCA for each hybrid combination [
8,
13,
28,
35,
41,
52,
53,
56]. The analysis of combining ability provides an indication of the nature of gene action involved in the expression of the traits. The ratio σ
2GCA/σ
2SCA determines the preponderance of either additive or nonadditive type gene action. Higher σ
2GCA to σ
2SCA ratio was observed for TLP, BM, TKW, and YLD indicating the importance of additive gene action for the inheritance of these traits [
57,
58]. Reif et al. [
59] reported that higher σ
2GCA to σ
2SCA ratio generally indicated the genetic dissimilarity of the parents used. Similar results were observed by Gowda et al. and Adhikari et al. [
13,
52]. Similarly, we completed a cluster analysis of the initial panel from which we selected the promising genotypes for hybrid production, which showed that the parents used in this study were mostly from different genetic clusters [
19] (
Figure S3).
In this study, the magnitude of the GCA effects was relatively higher in some of the parental lines for certain traits such as BM, TKW and YLD. Seven parents (P4, P5, P9, P10, P12, P14 and P17) had significant GCA effects for yield. Interestingly, of these seven identified parents, five (P5, P9, P10, P12 and P14) were identified as good combiners, having significant GCA effects for biomass and thousand kernel weight as well. It was evident that genotypes that showed high GCA effects for yield also demonstrated high GCA for some of the yield components such as biomass and thousand kernel weight. Similarly, we have shown these two traits had high direct effect on grain yield with 0.77 and 0.75, respectively. These results were in agreement with earlier findings [
13,
53]. Parental lines with high GCA effects may be used in future crossing strategies as improved parents for F1 hybrid production.
Significant positive SCA effects on biomass were observed in two crosses, P9/P2 and P10/P5 which both involved high × high general combiners as parents. These two crosses along with six others (P4/P7, P9/P6, P9/P8, P10/P12, P10/P14 and P10/P15) exhibited significant positive SCA effects on TKW and YLD. Of these eight hybrids exhibiting significant SCA effects in a desirable direction identified for TKW, four of them (P10/P5, P10/P12, P10/P14 and P10/P15) involved high × high general combiners as parents while four hybrids (P4/P7, P9/P2, P9/P6, and P9/P8) involved high × low general combiners. The negative SCA effects observed in some of the hybrids produced may have been due to the high and contrasting specialization of the two parents involved for yield formation or due to the presence of unfavorable gene combinations in the parents. The hybrids with high and positive SCA effects are recommended for hybrid breeding.
Because best specific combiners were not particularly derived from high × high general combiners as parents but also obtained from the combination of high × low general combiners, the high GCA effects of the parents were not necessarily reliable criterion to predict high SCA effects. Rewale et al. [
60] have reported that the strong performance of the hybrids having high SCA might be attributed to additive × dominance gene action in the case of high × low GCA parental lines or to an epistatic interaction in the case of the parental pair having low GCA (a case that was not reported in this study).