Heterosis and combining ability effect of protein content on rice ( Oryza sativa L . ) genotypes

The study comprised of a set of 56 F1s and F2s which were developed at the Teaching and Research farm of the Federal University of Technology, Owerri, Nigeria, during the early season of 2007. The parental lines consisting of eight genotypes of rice were grouped into low, medium and high protein parents and crosses were made in all possible combinations. The laboratory analysis for the protein content determination was done at the Analytical Laboratory of International Institute for Tropical Agriculture (IITA, Ibadan), in 2008. Positive heterosis was observed in all the hybrids in the F1 and 37 hybrids for the F2 over their MP indicating dominance in the positive direction. On the other hand, negative heterosis was recorded in 14 hybrids in the F1 over the HP. About 19 hybrids showed negative heterosis over the MP in the F2 while the others were positive. Only two crosses, CT 7127-49 x WITA 4(2.33) in the medium x low combination and NERICA 1 x Fofifa 16(0.76) in the high x high combination showed positive heterosis over the HP in the F2 generation while the rest of the hybrids were observed to have shown negative HP. The mean square due to GCA, SCA and reciprocal effects were highly significant for percentage protein content for the Diallel analysis, implying the presence of genetic diversity among the genotypes. About 50% of the parents showed desirable positive GCA effects and included: CT712749, EMPASC 105, WAB 96-1-1 and Max. 20 crosses showed positive SCA effects.


INTRODUCTION
Rice (Oryza sativa L.) is a staple food for over 3 billion people (Cantral and Reeves, 2002).Since protein content of rice determines its nutritional quality, it is therefore an important trait for the health of the people whose main daily food depends on rice (Chuhai et al., 1999).In order to improve the efficiency of breeding for rice nutrient quality, understanding the variation of gene expression of various characters is vital.Singh and Singh (1982) and Shenoy et al. (1991) observed that the protein content of rice is a quantitative trait.Whereas, Shenoy et al. (1991) found that heritability of rice protein content was 58.8%, Lang and Buu (2005) reported that grain protein content was predominantly controlled by partial dominance with additive gene effect and heritability of 25.9%.
For there to be success in breeding for increased protein content, it is crucial to identify the parents and crosses that possess genes for high protein contents for further genetic improvement.Diallel crosses have been widely utilized in estimating the nature and magnitude of genetic variability and interactions involved in the inheritance of desired traits in crops.When crosses are made in all possible combinations using the technique, it is possible to produce new genetic combinations that might have improved performances over the parents.Combining ability of the parents gives useful genetic information regarding the selection of parents in terms of the performance of their hybrids.The present investigation was undertaken to study the nature of genetic control and amount of heterosis available for protein and identify suitable parents to be used as donors for increased protein content in rice.

Screening of parents and the hybrids for grain protein content
The rough rice seeds harvested on individual plants during the planting season of 2008 were dehulled using Satake rice mill.The cleaned milled rice were ground and powdered by an Udy Cyclone Mill with a 1.0 mm mesh screen at the Analytical Laboratory of International Institute for Tropical Agriculture (IITA, Ibadan) in 2008 and used for the study.Total nitrogen was determined by near Infrared Reflectance (NIR) analyzer.Protein content was calculated according to Kjeldahl total nitrogen determination as: Concentration x 0.0075 x 5.95 x 100 Percentage protein = Weight of Sample Heterosis was expressed as a percentage increase or decrease over mid-parent (MP) and high-parent (HP) of percentage protein content in the F1 and F2 generations and calculated as: Heterosis of F1 over midparent (%) = Diallel cross experiment was calculated using Griffing's method 1 model 1 in which F1 hybrids and their reciprocal crosses are included according to Diallel-SAS: A SAS program for Griffing's Diallel analyses (Zhang and Kang, 1997) and analysed using Diallel-SAS05: A comprehensive program for Griffing's and Gardner-Eberhart Analyses (Zhang et al., 2005).

RESULTS AND DISCUSSION
NIR spectroscopy which is a rapid, non-destructive method was used for nitrogen determination.It has found widespread use in compositional analyses such as fat, protein, water and carbohydrates (Osborne and Fearn, 1986;Williams and Norris, 1987).The method uses the wavelength region from 700 to 2500 nm.Protein content was calculated according to Kjeldahl (1883) method.The analysis of variance showed highly significant difference for protein content.The parents vs hybrids comparison indicated significance at P= 0.05 for this same character.Significant and positive mean square and average heterosis have been suggested to be useful in determining non additive gene effects (Kirby and Atkins, 1968).When two parents involved in a cross have high x high or low x low GCA effects for the same trait, it involves additive x additive type of gene action.Manuel and Palaniswanny (1989) reported that interaction between positive x positive alleles in a cross of high x high combiners can be fixed in subsequent generation.Non additive gene effects were observed to have played major roles in the expression of protein contents in the genotypes studied.Positive heterosis was observed in all the 56 hybrids in the F 1 generation and 37 out of the same 56 hybrids for the F 2 generation over their MP indicating dominance in the positive direction.The extent of the heterosis over the MP for the F 1 s varied from 1.00 (Fofifa 16 x NERICA 1) in the high x high parent cross combination to 67.74 (IR 57689-73 x Fofifa 16) in the low x high cross combination.On the other hand, negative heterosis was recorded in 14 out of the 56 hybrids in the F 1 generation over the higher protein parent.All the 6 crosses in the high x low parent combination showed negative heterosis over the HP except the cross of NERICA 1 x IR 57689-73 that had positive heterosis (3.47).In this case where a greater number (42 out of 56) showed positive heterosis, it is indicative of over dominance of high protein content in the F 1 generation.In contrast to the observation for MP in the F 1 hybrids, the F 2 hybrids showed varied heterosis over the MP from -12.09(Max x WAB 96-1-1) in the medium x high to 5.17(IR 57689-73 x NERICA 1) in the low x high cross combination.
Out of the 56 F 2 hybrids, only 19 hybrids showed negative heterosis over the MP thus further implicating the role of dominance in expressing protein content in rice.More negative heterosis over the MP were recorded for the medium x medium crosses than in the other cross combinations, where 4 out of the 6 crosses showed negative heterosis.Similarly, half the number of hybrids in the high x low crosses showed negative MP heterosis.Only two crosses, CT 7127-49 x WITA 4(2.33) in the medium x low combination and NERICA 1 x WAB 96-1-1(0.76) in the high x high combination showed positive heterosis over the HP in the F 2 generation while the rest of the hybrids were observed to have shown negative HP (Table1).

Analysis of variance of Griffing's method 1 model 1
Analysis of variance revealed highly significant differences among the genotypes for protein content, it thus indicated that at least two samples differed from each other.To separate the differences between the different crosses and their parents, the least significant difference (LSD) test at the 5% level of probability was carried out.Results showed that protein content was under a simple additive or polygenic effect.Both GCA and SCA were highly important in determining protein content in the rice genotypes studied.Thus, not only the protein contents of the parents involved in a cross were important in expressing protein content, but also the SCA of each parents involved, since the usefulness of a particular cross in exploiting heterosis is judged by the specific combining ability effects.In the model followed, these effects indicate the interaction between the specific alleles of parents involved in the cross.
The mean square due to GCA, SCA and reciprocal effects were highly significant for percentage protein content (Table 2).This implied the presence of genetic diversity among the genotypes used for the study of this character.Computation of the components of variance revealed that SCA variance (93.4%) was much greater than GCA variance (6.6%) resulting in greater dominance variance.The proportion of additive to total genetic variance was 0.1224 while 0.8776 came from SCA variance.From Table 3, 50% of the parents showed desirable positive GCA effects of 0.6582 (CT7127-49), 0.2015 (EMPASC 105), 0.0882 (WAB 96-1-1) and 0.0190 (Max).Out of 28 cross -combinations made, 22 crosses showed positive SCA effects.However, these effects ranged from 0.132 (IR57689-73 x WAB96-1-1) to 4.612 (Fofifa 16 x WAB 96-1-1).Meanwhile, negative SCA effects were lower in magnitude and ranged from -0.1628 (WITA 4 x WAB96-1-1) to -1.337 (WITA 4 x Fofifa 16).The results in Table 3 also revealed that reciprocal effects ranged from -0.1417 (NERICA 1 x EMPASC 105) to 3.1883 (Fofifa 16 x IR57689-73).The cross WITA 4 x NERICA 1 showing the highest SCA effects alongside with IR57689-73 x Fofifa 16, IR57689-73 x NERICA 1 and Fofifa 16 x NERICA 1 involved both parents as poor general combiners, yet they developed positive SCA effects indicating the presence of non-allelic interactions at the heterozygous loci.The hybrids of CT 7127 x WAB 96-1-1 both in the direct and reciprocal crosses and CT 7127 x EMPASC 105 in the direct cross are expected to fix the genes in early generations.
When parents with high x low GCA effects showing additive x dominance gene action are involved, it will be better to exploit the F 1 if there is heterosis since this may not be fixed in the next generation.In the present study, heterosis could be exploited in the hybrids of WITA 4 x CT 7127-49, WITA 4 x EMPASC 105, Max x NERICA 1, CT 7127 x Fofifa 16, CT 7127-49 x NERICA 1, EMPASC 105 x Fofifa 16, EMPASC 105 x NERICA 1, Fofifa 16 x WAB 96-1-1 and WAB 96-1-1 x NERICA 1. Phenotypic expression of endosperm traits like protein content is under genetic control.Kuo et al. (1995) opined that it should be visualized in three perspectives: as endosperm, having a reciprocal (dosage effect) and as a seed and so can influence results of experiments because of this complexity.Kumar and Khush (1987) equally harped on this indicating that analyses from genetically heterozygous buck seed samples might provide biased information for genetic study on endosperm traits.
Manifestations of heterosis for protein content in 56 F 1 hybrids and their F 2 progenies of the rice genotypes studied was observed.Kirby and Atkins (1968) while working on the vegetative and mature plant of sorghum reported that average heterosis as well as mean square of parents versus hybrid may serve as a measure of nonadditive gene effects.If the heterotic value is zero or negative, it is an indication of lack of heterosis.Appreciable non-additive gene effects played an important role in the expression of dominance in all cross combinations in F 1 MPH as well as HPH where sizeable heterosis were observed for the F 1 hybrids.The crosses showed 100% F 1 mid-parent heterosis and 73.2% highparent heterosis.Among the crosses, IR 57689-73 x EMPASC 105, IR 57689-73 x Fofifa 16, Max x IR 57689-73, CT 7127 -49 x Max and CT 7127-49 x WAB 96-1-1 all showed very high and positive values for high-parent heterosis.50% of the parents showed negative heterosis values thus indicating that dominance effect affected the crosses more than additive effect suggesting that dominance gene action is more important than additive gene action in the inheritance of protein content.This agrees with Mohan and Ganesan (2003) who indicated that exploitation of hybrids could be informed by the magnitude of heterosis it exhibited.The present study noted the prediction of Kumar and Khush (1987) that analyses of genetically heterozygous F 2 buck seed samples on plants provided biased information for genetic study on endosperm traits while working on amylose content of rice.Although dominance gene action was primarily responsible for the inheritance of protein content in the rice genotypes studied, reciprocal and maternal effects also played major roles as both recorded highly significant mean squares for this character.Therefore, choice of parents based upon the SCA effects of their hybrids would differ from selection based upon parents' protein content.Manuel and Palaniswanny (1989) reported that there could be interaction between positive alleles in a cross involving high x high combiners which can be fixed in subsequent generations if no repulsion phase linkages are involved.In the present study, GCA effect conferred highest protein content on CT 7127-49, EMPASC 105 as well as WAB 96-1-1 which also agrees with the results of SCA effects.
Transgressive segregates is of considerable importance to the breeder as it allows for the possibility of obtaining segregates that are better (or worse) than the parents (Welsh, 1981).Transgressive segregates were observed for most of the F 1 hybrids manifested by their higher protein contents than their parents.The hybrids, WITA 4 x IR 57689-73, WITA 4 x Max and IR 57689-73 x Max which had parents with relatively lower protein contents than other hybrids, showed significant heterosis over the higher protein parents showing segregates.It is thought that complimentary epistasis or dominance x dominance epistasis could be responsible.In contrast, only one hybrid, NERICA 1 x WAB 96-1-1 with parents having high protein contents showed high-parent heterosis in the F 1 and carried it to the F 2 generation indicating co-dominance gene effect.The only seed parent which consistently produced hybrids with higher protein than other parents is CT 7127-49 which inferred that it has genes for high protein contents among the lines chosen for the study.
However, it is important to note that the type of dominance exhibited may have been influenced by the cross, interaction or environment under which they were grown.Heterotic values alone may not reveal the real identity of superior hybrids as heterosis of hybrids tend to be high when the parental means are low and vice versa.Also, high SCA effects may not always give an appropriate assessment as hybrids with low mean values may possess high SCA effects if the GCA effects of the parents were very low or negative.Gilbert (1958) had earlier suggested that parents with good mean performance would result in better genotypes and agrees with the findings of Mohan and Ganesan (2003) who based their report on GCA effects and mean performance of the parental lines they used for combining ability studies on kernel quality of rice.

Table 1 .
Estimates of Mid-Parent (MP) and High-Parent (HP) heterosis for protein content in direct and reciprocal crosses of the F1 and F2 generations of rice genotypes studied.

Table 2 .
Analysis of variance of general combining ability (GCA) and specific combining ability (SCA) for protein content in an 8 x 8 Diallel cross experiment in rice.

Table 3 .
Estimates of general combining ability effects (diagonal), specific combining ability effects(above diagonal) and reciprocal effects(below diagonal) for percentage protein content (%PC)in an 8 x 8 Diallel cross experiment.