Combining Ability, Heritability, and Heterosis for Seed Weight and Oil Content Traits of Castor Bean ( Ricinus communis L.)

: Hybridization is an important evolutionary force, and heterosis describes the phenomenon where hybrids exhibit superior traits compared to their parents. This study aimed to evaluate the one-hundred-seed weight and fatty acid content in F 1 generations, investigating the effects of different parental crosses using a 9 × 3 incomplete diallel design (NCII). One of the challenges faced in this study was the complexity of accurately determining the influence of both genetic and environmental factors on trait inheritance. A total of 36 F 1 crosses were analyzed for general combining ability (GCA), specific combining ability (SCA), and heritability. The results showed that the level of each index in F 1 is closely related to its parents. Significant differences in GCA and SCA were observed among parental traits in most crosses. The ratio of GCA to SCA ranged from 0 to 3, indicating the pivotal role of SCA over GCA in castor breeding efforts. High narrow-sense heritability was recorded in palmitic acid (30.98%), oleic acid (28.68%), and arachidonic acid (21.34%), suggesting that these traits are predominantly under the control of additive gene action, and hence these characters can be improved by selection. Additionally, heterosis exhibited diverse patterns across traits. Based on the evaluated combining ability, heritability, and heterosis, the inbred lines CSR181 and 20111149 were recommended for castor crossbreeding due to their potential to yield progeny with optimal oil-related traits. This research contributes valuable knowledge to the field of castor breeding, providing a foundation for developing superior castor cultivars.


Introduction
One-hundred-seed weight is an important trait which reflects seed fullness and size and has important evolutionary ecological value.Traditionally, seed weight has been considered a stable trait in species; however, several research studies have shown that there are significant differences in seed weight among species or individuals.Such differences might affect seed germination or seedling traits.Generally, larger seeds have higher germination and emergence rates than smaller seeds, and could produce more vigorous seedlings to improve survival.Vandamme et al. [1] indicated that seed size greatly affected shoot and root growth beyond the seedling stage.At the same time, larger seedlings were not sensitive to density pressure.On the other hand, smaller seeds harbored relatively faster germination that larger seeds, so they could gain a better competitive advantage [2].Recently, Zhang et al. [3] thoroughly investigated seed weight in soybean through a genomewide association study, genomic prediction, and marker-assisted selection methods.Their results suggested that soybean seed size is controlled and regulated by many genes.These findings would help us to better understand the genetic basis of soybean seed size and promote identify of gene regulation.Richardson et al. [4] used one-hundred-seed weight as a subspecies diagnosis, seed purity, and identification, and pointed out that seed size was mainly influenced by genetic factors and limited environmental factors.However, only a few studies have reported whether the role of parental combinations in offspring has considerable implications on the seed weight and oil content of oil crops [5,6].
The type of parents in hybridization and backcrossing leads to the recombination of alleles.The interaction between environment and genetic structure could cultivate offspring that are different from their parents.Hybridization is regarded as an important evolutionary force; gene transfer among species could result in more genetic material than mutants [7].
To date, at least 30-80% of species might originate from hybridization [8].In general, the hybrid is unfavorable to its ancestors, as offspring may exist in reproductive isolation, leading to hybrid weakness, lethality, or sterility.The first generation of hybrids (F 1 ), however, are the exception, harboring higher biological performance than their parents.The nature and magnitude of gene action, general combining ability (GCA), and specific combining ability (SCA) are important factors in selecting the desirable parent and crosses for the exploration of heterosis [9,10].Combining ability analysis is one of the powerful tools to assess combining ability effects and also helps in identifying the nature of gene action involved in various quantitative characters [11].This information is helpful to plant breeders for developing hybrid castor varieties with good quality.Therefore, the present investigation was undertaken for isolation of better combining parents for suitable hybrids.
Castor (Ricinus communis L.) is a special industrial oil crop.Ricinus oleic acid, one of the major contents in castor bean, is the main chemical raw material in the fatty acid of castor seed [12,13].At present, seed weight and fatty acid content in castor seed are two main indexes that have been reported to evaluate the quality of varieties [12][13][14], whereas few reports have used the ratio of ricinoleic acid in crude fat.Thus far, researchers have determined the seed weight, fatty acid composition, and content in different castor germplasms [15,16].However, little work has been conducted thus far on the heterosis and combining ability of fatty acid compositions in castor bean.Therefore, in this study, we estimated the combining ability, heritability, and heterosis of parents based on fatty acid composition.These findings will provide an in-depth understanding of the inheritance pattern of castor.

Plant Materials
A total of twelve ripe and healthy seeds of castor cultivars were collected and cultivated at the Academy of Agricultural Science in Tongliao, China, based on the their performance in terms of yield index, quality index, and stress resistance index [17].During flowering, the twelve female lines (aLmAB1, aLmAB2, aLmAB3, aLmAB4, aLmAB5, aLmAB6, aLmAB7, aLmAB8, aLmAB9, aLmAB10, aLmAB11, aLmAB12), and three male lines (20102189, CSR181, 20111149) were crossed in a 9 × 3 incomplete diallel design (NCII).Thus, 36 cross-combinations were obtained.The parental combinations are listed in Table 1.Each block was 10 m × 10 m, and the plants were transplanted with 6 × 8 per block.The design of random block method was repeated three times.After emasculation, bagging, and artificial pollination, the fruits were harvested at 60 days.About 100 seeds were randomly selected from parents and F 1 for character investigation and measurement.Husk from each seed was carefully removed.Seeds were dried in an oven at 60 • C ± 2 • C for 3 h.A total of 14 indexes were determined, including 100-seed weight, 100-seed weight (dehusked seeds), 100-seed weight (dehusked oven-dried seeds), crude fatty content (dehusked oven-dried seed), crude fatty content (100-seed), behenic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, and arachidonic acid.The fatty acid composition ratio was calculated based on the corresponding chromatographic peaks [13].By applying computer automatic and manual retrieval with NIST98 and the Wiley Registry of Mass Spectral Data, the fatty acid composition of castor oil was measured and analyzed [14].Fatty acid composition and absolute content were measured according to our previous report: for more detailed information, see [12,14].

Statistical Data Analysis
All the measurements were taken in triplicate.Analysis of variance (ANOVA) for the recorded traits, combining ability and diallel analysis, were performed using DPS v 7.05 [18].The GCA and SCA effect and genetic parameter measurements were conducted based on Model I and Method II of Griffing's method [19].The relative importance of the additive or non-additive genes (σ 2 GCA/σ 2 SCA ratio) were conducted by Baker [20].

Analysis of Variance between Parents and Hybrids
Firstly, we conducted variance analysis on the 14 characters of the hybrid combinations of castor (Table 2).The ANOVA results revealed significant differences (p < 0.05) among the 26 cross-combinations, indicating the existence of inherent variation among the 14 traits in the crosses.Therefore, the next step was to conduct variance analysis to determine combining ability.The mean performance of 9 parents and 36 F 1 crosses is listed in Tables S1 and 3. Behenic acid had the highest coefficient of variation (CV/%) (131.15), while crude fatty content (dehusked oven-dried seed) harbored the lowest, with an average of 32.85 across all crosses.aLmAB3 had the highest value of 100-seed weight (dehusked seeds) (22.55 g), 100-seed weight (dehusked oven-dried seeds) (22.11 g), crude fatty content (dehusked oven-dried seed) (67.4%), crude fatty content (100-seed) (14.99 g), palmitic acid (0.17%), linoleic acid (0.74%), linolenic acid (0.08%), and ricinoleic acid (13.18%), whereas aLmAB7 and aLmAB10 showed the lowest performances for most of the oil-related traits.For most oil-related traits, 20102189 and aLmAB3 had the higher scores.Progenies from F1-4-12 had the highest 100-seed weight, 100-seed weight (dehusked seeds), 100-seed weight (dehusked oven-dried seeds), crude fatty content (100-seed), and ricinoleic acid.F1-4-4, F1-4-6, and F1-4-7 expressed the lowest mean values for most of the traits with the exception of stearic acid.F1-5-8 yielded the highest values of palmitic acid, oleic acid, linoleic acid, and arachidic acid.Overall, the progenies from crosses involving CSR181 and 20111149 performed better for the studied traits.Moreover, the CV% in fatty acid composition was higher than that in seed oil content, indicating that hybridization is a major cause of fatty acid composition.The overall mean performance of the crosses with CSR181 and 20111149 as the male line exceeded that of the parents for the 14 traits.

Combining Ability Performance
GCA refers to the average performance of hybrid offspring in a certain trait after crossing one parent with multiple parents.It is primarily determined by the additive effects of genes and represents the stable heritable portion [15].Significant differences in GCA effects were observed between different parents and traits, indicating variations in the magnitude of additive genetic effects for different parents in relation to the trait.
Among the 12 female lines, aLmAB8 had the largest GCA value for the 100-seed weight (g), 100-seed weight (dehusked seeds), 100-seed weight (dehusked oven-dried seeds), crude fatty content (dehusked oven-dried seed), palmitic acid, oleic acid, linoleic acid, arachidic acid, and ricinoleic acid, and had positive GCA effects for all the traits (Table 4).aLmAB6 and aLmAB7 had the lowest negative GCA effects for the majority of traits with the exception of crude fatty content (100-seed) and arachidonic acid, respectively.aLmAB1, aLmAB6, and aLmAB11 exhibited the fewest positive effects for 13 traits.The highest GCAs for stearic acid, behenic acid, and linolenic acid, and arachidonic acid were observed in aLmAB4, aLmAB10, aLmAB9, and aLmAB7, respectively.
From the above conclusions, it can be observed that the general combining ability (GCA) of any trait varies with variation of the parents.Different traits of any parent show significant differences in their GCA.Therefore, it is challenging to find a universally good combination for all traits.Hence, information about parental genetic effects is crucial.

Estimation of Genetic Parameters
All GCA/SCA ratios were greater than 1.0, except for 100-seed weight, behenic acid, stearic acid, linolenic acid, and arachidic acid, as indicated in Table 5.The variance across all environments for the 14 traits was 0, suggesting that genetic factors might be so dominant in determining the trait that any minor environmental differences are not significant enough to cause detectable variability.Palmitic acid exhibited the highest broadsense heritability (100%) and the highest narrow-sense heritability (30.98%), indicating that both additive and non-additive genes influenced this trait.Most traits displayed high broad-sense heritability (100%), except for arachidic acid (45.09%), indicating that these traits are predominantly controlled by additive genes.Nonetheless, broad-sense heritability exceeded narrow-sense heritability in all traits, with narrow-sense heritability for linolenic acid being zero, suggesting a predominant or exclusive role of non-additive gene action in their inheritance.The high narrow-sense heritability was recorded in palmitic acid (30.98%), oleic acid (28.68%), and arachidonic acid (21.34%), suggesting that these traits are predominantly under the control of additive gene action, and hence these characters can be improved by selection.

Heterosis in Cross-Combination
The magnitude of heterosis (MPH and HPH) in the F 1 hybrids exhibited a wide range of variation (−100% to 1900%) (Table 6).In general, the mean HPH values were lower compared to the MPH values across all traits.Approximately 73.26% of the crosses demonstrated positive MPH, while around 21.36% showed positive HPH.F1-4-12 showed the highest positive MPH and HPH for the 100-seed weight (g), 100-seed weight (dehusked seeds), 100-seed weight (dehusked oven-dried seeds), crude fatty content (100-seed), and ricinoleic acid, whereas the lowest negative MPH and HPH for these traits were observed in F1-4-4.For the crude fatty content (dehusked oven-dried seed), 50% of MPH values were positive, while only one HPH value was positive, which was detected in F1-5-3.The largest positive MPH and HPH of behenic acid were found in F1-5-10.Palmitic acid, oleic acid, linoleic acid, and arachidic acid showed similar MPH and HPH trends, of which F1-5-8 and F1-4-7 harbored the highest and lowest values, respectively.F1-4-4 had the highest positive MPH and HPH values for stearic acid.F1-5-7 had the highest MPH and HPH of linolenic acid and arachidonic acid.

Discussion
Castor oil currently has more than 700 uses, and its market is limitless due to its diverse applications, ranging from industrial to pharmacological uses [23].The world demand for castor oil and its derivative, castor biodiesel, is continuously growing at the rate of about 3 to 5% per annum, especially as they are essential for preventing the freezing of fuels and lubricants used in aircraft and space rockets at extremely low temperatures [24].With the development of biodiesel, castor oil's primary market is expanding into the energy sector [25].
Heterosis is a general phenomenon of living nature in which heterozygote F 1 -hybrid plants are superior to their parents in one or more traits.Tang [26] analyzed heterosis using different hybridized castor combinations, and their results showed that superiority was mainly manifested in the aspects of single plant yield, soluble sugar content, and 100-seed weight.However, no information is available about heterosis in castor oil content.In this study, a comprehensive approach was used to measure castor seed weight and oil content traits, including 100-seed weight, crude fatty content, and fatty acid composition.This related method has been successfully applied in our previous studies to screen the correlation between different castor seed sizes and oil content [13].Our results indicated significant variations in seed weight and oil content traits among 15 parents and 36 F 1 hybrids (Tables 2 and S1), suggesting the possibility of selecting superior parents and hybrid varieties with high oil content.Specific hybrids, such as F1-4-4, F1-4-6, and F1-4-7, exhibited the lowest mean values for most of the traits with the exception of stearic acid, while F1-4-12 had the highest values for several traits, including 100-seed weight, crude fatty content, and ricinoleic acid.Both of the hybrids involving the parent CSR181 exhibited a variation in seed weight.Additionally, all detected traits in F1-4-12 were even higher than the average values of CSR181, indicating the presence of heterosis in F1-4-12.In contrast, the averages of the other 11 hybrids typically varied with the parents, indicating the complexity of oil content inheritance and the potential influence of factors such as non-additive genetic effects, gene interactions, and environmental influences.
The results from the analysis of different hybrids reveal a complex pattern of heterosis in castor plants.Approximately 73.26% of the hybrids exhibited positive MPH, signifying that these hybrids were superior to their parents in various traits.This phenomenon indicates the potential for increased productivity and desirable characteristics in these hybrids [27].Interestingly, around 21.36% of the hybrids showed positive HPH.Despite the negative values of HPH for crude fatty content, some specific crosses had higher values than their parents.This suggests that while overall heterosis might not favor higher crude fatty content, specific combinations have the potential to produce improved varieties in terms of crude fatty content.These crosses could be crucial for developing castor plants with enhanced oil quality.Furthermore, most of the crosses showed positive MPH for essential fatty acids such as palmitic acid, behenic acid, stearic acid, and oleic acid, indicating that these hybrids outperformed their parents in these indicators, showcasing their potential for commercial applications, particularly in the production of high-quality castor oil.However, arachidonic acid and crude fatty content exhibited mainly negative or low positive MPH and HPH, suggesting that these traits might be primarily controlled by recessive effects in the parents [21].
In castor, traditional cross-breeding is one of the most effective methods to select improved varieties by exploiting heterosis [28].This study found variations in GCA and SCA effects between the parents and crosses, with both positive and negative values for all seed weight and oil content traits.However, no evidence showed a correlation between GCA and SCA.Similar findings were reported in other studies [29].At the same time, parents with higher GCA effect were more likely to produce crosses with excellent traits [30].The hybrids F1-4-12, F1-6-2, and F1-6-3 exhibited positive SCA effects for 13 out of 14 seed weight and oil content traits, with F1-4-12 especially showing the highest SCA effect for most features.In these crosses, at least one parent has a high GCA effect for traits, for example, 20102189 and aLmAB12.Therefore, in breeding practices, while emphasizing the selection of parents with high GCA, it is also necessary to strengthen screening for SCA [31].Only by combining GCA and SCA can the heterosis of castor be effectively utilized.Additionally, F1-4-12 showed positive MPH and HPH effects for most traits, indicating its high value in castor breeding.These findings provide new insights into castor breeding and contribute to the development of new castor varieties with ideal high oil content.
Many studies have reported that the significance of GCA effects indicates that parents can pass more favorable alleles, thereby transmitting these traits onto their offspring [32].Significance of SCA effects suggests deviations in the behavior of hybrids compared to expectations based on parental GCA [33].GCA is attributed to genes with additive effects, while SCA is associated with non-additive genetic effects [34].Therefore, the presence of significant GCA and SCA effects implies the importance of both additive and non-additive genetic components in controlling the studied traits.In this study, GCA mean squares for 100-seed weight (dehusked seeds), crude fatty content, palmitic acid, oleic acid, linoleic acid, arachidonic acid, and behenic acid content were higher than the SCA mean squares, indicating the predominance of additive effects in controlling these traits.Non-additive gene action played a significant role in the 100-seed weight content, where the intensity of SCA effects significantly surpassed GCA effects.The GCA/SCA ratios for 100-seed weight (dehusked seeds) (1.90), 100-seed weight (dehusked oven-dried seeds) (1.15), crude fatty content (dehusked oven-dried seed) (1.00), crude fatty content (1.97), palmitic acid (3), oleic acid (2.19), linoleic acid (3), arachidonic acid (1), and behenic acid (1.38) were all greater than 1, indicating that these traits are primarily controlled by additive genetic effects.On the other hand, the GCA/SCA for 100-seed weight was less than 1 (0.56), indicating that non-additive gene effects predominantly control this trait.Similar results were reported by others [32,35], where substantial differences between GCA and SCA allow the improvement of specific traits by selecting parents that pass on the desired traits to their offspring with additive genetic effects.Genetic analysis of fatty acid content in seed oil and studies on yield-forming traits have demonstrated significant differences between GCA and SCA in the F 1 generation, indicating the dominance of additive variability over non-additive variability [36].
In our study, a limitation is that we did not collect environmental impact data across multiple years and locations.Temporal variability can significantly affect the consistency of our data, as environmental conditions may fluctuate seasonally and annually [37].Spatial heterogeneity further complicates our analysis, as different locations have distinct environmental characteristics and practices.Additionally, ensuring data consistency and standardization across various regions and years is challenging, potentially leading to inconsistencies in our impact estimates.We also encountered gaps in the data due to accessibility issues in certain areas.Furthermore, while our findings are valuable at a local level, they may not fully capture global environmental trends.Lastly, the influence of local context-specific factors, such as economic conditions [38], may not be fully accounted for in our analysis.These limitations should be considered when interpreting our results and drawing broader conclusions.

Conclusions
This study represents the first comprehensive report on parental general combining ability (GCA), specific combining ability (SCA), and heritability in castor for the selection of optimal oil-related traits.Both additive (GCA) and non-additive (SCA) genetic effects significantly contributed to the variation in fatty acid composition in castor.Moreover, heterosis exhibited diverse patterns across different traits, underscoring the complexity of trait inheritance in castor hybrids.Based on the evaluation of combining ability, heritability, and heterosis, the lines CSR181 and 20111149 have been identified as promising candidates for castor crossbreeding programs.These lines show potential to produce progeny with superior oil-related traits, thereby contributing to the advancement of castor breeding efforts.This research provides valuable insights and a solid foundation for future breeding strategies aimed at improving oil yield and quality in castor plants.

Table 2 .
Analysis of variance (ANOVA) for the 14 characters in castor.
* Significant at the 5% level of significance and ** significant at the 1% level of significance.

Table 3 .
Mean performance of the parents and F 1 in castor.

Table 4 .
Estimates of general combining ability (GCA) and specific combining ability (SCA) effects of parents and crosses for different traits in castor.

Table 5 .
Estimates of genetic variances and heritability for the 14 traits in castor.

Table 6 .
Heterosis of 14 traits in cross-combination in castor.