Fe, Zn & Protein content in grain, per se performance, heterosis, combining ability of grain yield in bread wheat (Triticum aestivum) under normal & late sowing condition.

The aim of this study was to understand the inheritance of grain iron (Fe), zinc (Zn) and protein concentrations in bread wheat ( Triticum aestivum L .) under normal & late sown condition. The half diallel cross were made, using ten parents. The crosses and parents were evaluated in replicated trials for two conditions and to assess the possibility of exploiting heterosis to improve these micronutrients. The perusal of results revealed that hybrid GW 451 × GW 173 was consistent for both better parent (BH) and standard heterotic effects (SH) in all the environments. In both the sowing condition, General Combining Ability (GCA) effects of parents and Specific Combining Ability (SCA) effects of hybrids were observed to be contrasting for the micronutrient & protein content. However,  2 A /  2 D greater than unity confirmed the preponderance of additive gene action for only protein content and GW 173 was identified as good general combiners for these characters under both environments.

Wheat is one of the major staple food crops in the world, being consumed from ancient times by human beings 1 .Wheat is being cultivated on more than 17 % of cultivable land consumed by nearly 40 % of the global population occupying about 32% of the total land under cereals in the world.Wheat fulfills 21% of the protein requirements of more than 4.5 billion people in developing countries 1 .Micronutrients malnutrition is of great public health importance in several parts of the world, especially the developing and underdeveloped countries 2 .It has been estimated that about 2 billion people, about one third of the world's population, are deficient in one or more mineral elements 3 .Although required in traces, these mineral elements are involved in many vital metabolic functions 4 .It is estimated that over 60% of the world's population are anaemic due to iron (Fe) deficiency and over 30% are zinc (Zn) deficiency, which is referred as hidden hunger 2 .In India 80% of the pregnant women, 52% of the non-pregnant women and 74% of the children (6-35 months) are suffering from Fe deficiency-induced anaemia (IDA) 5 .
Fortifying such wheat's with high and essential micronutrients will be right step to eliminate "hidden hunger" and significant positive health outcomes for millions of people 6 .
Micronutrient deficiencies in humans can be remedied through food diversification, mineral supplementation, food fortification and biofortification 7 .Biofortification is one of the most effective approaches to alleviate malnutrition 8 .Biofortification is the strategy of increasing the content of bioavailable nutrients in the edible parts of staple food crops for better human nutrition 9 .However, staple crop based diets fall far short in providing the required amounts of micronutrients and heavy reliance on staple food is the root cause of micronutrient malnutrition 10 .The use of these biofortified cultivars is expected to mitigate micronutrient malnourishment in large populations especially in Africa 11 .Crop breeding, genetic manipulation, and application of mineral fertilizers are the bases of biofortification strategies and have enormous potential to address micronutrient malnourishment 12 .Biofortification is a proven strategy to combat micronutrient deficiency in large populations, particularly for those living in developing countries 6,13, 14 .However, to make it more effective, efficient, and acceptable for people, better planning, implementation, monitoring, and evaluation of biofortification programs are needed to produce cost-effective and socially acceptable biofortified food crops 6,13,14 .
Different sowing dates affect the growth of wheat mainly due to the effects of temperature and day length.Temperature and short winter growing season in central India are main reasons for low yield in wheat.High temperature increases the rate of grain filling but, the duration of grain filling is reduced considerably and grain weight is decreased 15, 16 .
Early sowing of wheat may escape from high temperature during grain filling but high temperature at the time of sowing affects germination and early vegetative growth.Therefore, identification of wheat genotypes suitable for specific sowing conditions is of great importance for achieving higher yield.
In this regard, understanding the relationship between grain yield, grain protein and grain micronutrient content under normal and late sown conditions will make a best use of heterosis breeding/hybrid wheat technology.Although wheat grain is known to have low Fe and Zn content, it seems to provide requisite micronutrient supplement to the deprived populace who solely depend on rice and who lack a balanced nutritive diet regularly 17,18 .Thus, to combat the micro-nutrient deficiencies among wheat eaters, efforts are to be made to enhance the grain micronutrient content (particularly Fe and Zn) along with sustainable crop yield potential 18,19,20 .The present investigation was executed to study the combining ability and heterosis in hybrid wheat parental lines and their derived hybrid crosses characterized for grain protein and grain Fe and Zn content under normal and late sown conditions.

Results
Performance of hybrids and parental lines.The performance of hybrids and parental lines is presented in Table 3.The results of ANOVA indicates that mean squares due to genotypic effects for the traits under study are significant (P < 0.01) in both the sowing conditions.
The per se performance of parental lines, hybrids and checks are presented in Table 4.The coefficient of variation (CV) values for the evaluated traits was < 10%, thereby indicating acceptable and effective experiment precision (Table 4).
The GYP in hybrids ranged 10.0 g (H45) to 32.9 g (H3) in E 1 and 9.4 g (H27) to Heterosis.Two categories of heterosis, viz., standard heterosis (SH) and Better parent heterosis (BH) exhibited significant variation in both the sowing methods (Table 5 and 6).In the present study, several crosses exhibited conspicuous heterotic response over better parent (heterobeltosis) and over standard check varieties (standard heterosis).i.e.MASC 6222 for normal sown and HD 2932 for late sown.
For GYP, four F 1 progenies recorded significant positive estimates for both environments.
However, the higher percentage of crosses showed positive BPH in late than normal condition for PC.Although one F 1 progenies-H30 for normal and eight F 1 progenies for late showed significant positive BPH.Specially, late condition had high significant positive crosses SH than normal.
For Fe content, H5, H9 and H36 had consistent positive significant heterosis for BPH in both condition.H5, H7, H8, H28, H30, H32 and H36 found significant positive SH for both checks in all environments.Interestingly, H5 recorded significant consistent positive BPH & SH estimates for Fe content in both environments as well as GYP in normal (SH).
For Zn content, H45 had consistent significant positive BPH in both environments and also found significant positive SH over checks except SH 1 in E 1.In addition, H36 recorded highest significant positive estimates of SH for grain Zn in normal.Although nine F 1 progenies for normal and late condition showed significant positive SH.
Combining ability.The ANOVA for the diallel design was summarized in Table 3.The analysis of variance for combining ability in (E 1 and E 2 ) all the environments revealed that mean squares due to parents and hybrids were significant (P< 0.01) for all the characters.The estimate of σ 2 GCA and σ 2 SCA revealed that both additive and non-additive gene effects were involved for inheritance of GYP and Zn content in E 1 .Whereas, the estimates of only σ SCA were significant for all the characters in both environments, which suggested importance of only non-additive gene effect for inheritance of these characters.

Normal
The value of average degree of dominance was less than one for the characters GYP in (E 1 ), protein content in (E 2 ) and zinc content in (E 1 ), it revealed partial dominance behaviour of interacting alleles for these characters.Whereas, for the character iron content in (E 1 and E 2 ) and zinc content in (E 2 ), the value of average degree of dominance was close to zero or zero, it revealed absence of dominance for both the characters.
GCA effect of parents.No any parent that concurrently had positive GCA effects for all the studied traits in both environments (Table 7).For GYP & PC, P1 had significant positive GCA effect in both environments; Whereas, P7 had highest & significant positive effects for all environments except GYP in E 1 .Hence, this parent is considered as a good general combiner.P10 & P4 was considered as poor general combiner as it possessed significant negative GCA effect in all the environments (Table 7).While for Zn content, the numbers of parents' depicted significant and positive GCA effect were 2 in E 1 and 4 in E 2 .Here, parents with negative GCA effect for yield traits had positive GCA effect for micronutrient traits in both environments.Likewise, Parents maintain the same (positive or negative) in both environments.
SCA effect of crosses.Similar to the results noticed for GCA effects of parents, there was no hybrid cross that concurrently attained positive SCA effects for all of the studied traits (Table 8).For Fe and Zn content the percentage of crosses with positive SCA effects remain constant in both environments, though the individual crosses showed differential response.
For GYP, the hybrid combinations H15 and H34 had showed positive significant SCA effect in both environments.

Discussion
Wheat is an important and most widely cultivated food crop in the world.This crop played a central role in combating hunger and improving the global food security 16 .Wheat is ranked second in total cereal production behind corn, with rice being the third 21 .The grains of this plant provide about 20% of all calories and proteins consumed by people on the globe 22 .In recent years, demand for wheat has significantly increased as a result of the global population growth, and thus wheat production has a strategic role in food security and the world economy.As a result, horizontal expansion of wheat production has arisen in recent years by moving wheat into nontraditional areas formerly considered unacceptable for production.
However, the global warming introduced various abiotic stresses such as drought, temperature extremes, and salinity that adversely affect the yield and grains quality of wheat .
The intergovernmental panel on climatic change has projected the temperature increase by the end of this century will likely to be in the range 1.8 to 4.0 o C. Global climatic change will affect agriculture through their direct and indirect effects on crops, soils, livestock and pests 24 .This will lead to heat stress at different growth stages of various crops, which can reduce crop duration, increase crop respiration rates, and alter photosynthetic partitioning to economic products For protein content, the magnitude of HB was low to high in negative and low in positive directions.While magnitude of SH was moderate in positive and low to high in negative directions.The findings were in accordance with the results of 31-33 for HB.The findings of SH were in accordance with 34 .For Fe content, the magnitude of HB was moderate in negative and low in positive directions.While magnitude of SH was moderate in both directions.The results contradicted the finding of 35 .For Zn content, the magnitude of HB and SH was high in negative and low in positive directions.
The GCA elucidates the average performance of a line in an array of hybrid crosses and it can be used to differentiate the parental lines.In our study the GCA effect of parental lines for protein content is varied with environment.The P1 had the highest significant positive GCA effect in E 2 (0.20).Another good general combiner was P7 in E 1 (0.29).P10 was considered as poor general combiner as it possessed significant negative GCA effect in all the environments.In our study, the GCA effect of parents (except P7) for grain Fe content is in contrasting manner with the both environments.In E 1 , the GCA effect of GYP and grain Fe and Zn contents were observed in contrasting manner.Negative correlation and negative GCA effect was observed between grain yield and Fe & Zn content under E 1 and E 2 indicates the need for Fe and Zn content improvement of parental materials with grain yield.These parents, therefore, may be used in combination breeding for isolating desirable types in bread wheat.
The present findings also revealed high degree of correspondence between per se performance of parents and their GCA effect for majority of the traits studied, which indicated that per se performance can be used as a reliable criterion for selection of parents for hybridization.The success in crop improvement lies in isolating superior gene combinations in terms of the genotypes with high combing ability.The genotypes with good general combining ability can be further exploited in development of new variety.
SCA is mainly associated with non-additive gene action resulting from dominance, over dominance and epistatic effects [36][37][38] .Analysis of the hybrids revealed that no specific combination obtained significant positive SCA values for all of the estimated traits simultaneously.This result is in agreement with those from previous studies in rice 39,40 as well as in maize 41 , bread wheat 42 .However, the crosses H3, H2 & H6 [significant (P < 0.01) for GYP] and H43 [significant (P < 0.01) for grain Fe and Zn content] showed positive SCA values for all the traits in normal conditions.Conversely, in late condition-H3 (significant for SPY, PC and grain Fe content), H26 for grain Fe and Zn content showed positive SCA values.These results were in agreement with previous studies in rice 39,40 , pearl millet 43,44 and maize 45,46 indicating that the grain micronutrient concentrations are mostly under additive genetic control.
The parents of cross combination with significant SCA effect for a particular trait, may not possess significant GCA effects.For instance, the cross H15 had significant positive SCA effect for yield traits in E1 and its parents had significant negative GCA effects.There were no significant SCA effects in all crosses for the estimated traits, suggesting that the values for these traits are within the limits of the parent's averages.
In Reverse case, parents with significant GCA effects did result the best hybrids with significant SCA like parents of H2, H3 and H26 showed positive GCA effects as well as hybrids were observed with positive SCA effects for GYP in normal condition.This observation could be attributed to different combination of dominant and recessive genes or alleles from one of the parents; it further substantiates the operation of non-additive gene action (additive × dominance, dominance × dominance and epistatic interactions).The hybrid H3 was observed with highest significant positive SCA effect for GYP in normal condition; GYP, PC and grain Fe content in late condition.For grain Fe and Zn content, the hybrid H43 had significant positive SCA effect in both the environments.Generally, the hybrids with high SCA effects are recommended for heterosis breeding.Thus, these crosses could be exploited in breeding programme to isolate desirable segregants in subsequent generations and hence, it would be worthwhile to use them for improvement in grain yield as well as micronutrients.
Thus, the GCA effects of parents are inadequate to predict the SCA effects of respective hybrids.Conversely, the significant correlation between SCA effects and heterosis was observed for all studied traits in both envrionments.In common, heterosis breeding targeted for particular traits on the basis of the GCA effects of parents can be taken as for the hybrid combinations.
The crosses which involved at least one good general combiner parent would produce transgressive segregants.However, for full exploitation of existing genetic variance in these crosses intermating of elite plants in early segregating generations would be profitable to built up elite population of parents with high grain yield & micronutrients.Whereas, the cross combinations with high specific combining ability estimates can be exploited in heterosis breeding.The analysis of variance for combining ability to partitioning the total genetic variance into general combining ability representing additive type of gene action and specific combining ability as measure of non-additive gene action.
Positive correlation between grain Fe and Zn concentration was observed in parents as well as in the hybrids in irrigated method.Similar associations between these grain micronutrient concentrations have been reported previously in rice 47-49 , maize 50-52 , wheat 53-55 , sorghum 56,57 pearl millet 58,59-61 and finger millet 62 .To the best of our knowledge, this is the first report on evaluation of wheat parental lines, hybrids under normal and late sown conditions for grain micronutrient content.

Conclusions
The study elucidated variation in GYP, protein content, Fe and Zn content under normal and late sown condition.Though many studies are not available in estimation of micronutrient content under normal and late sown condition, the present findings clearly indicate that the hybrids exhibited higher mean micronutrient content along with protein content under late sown condition than normal sown condition.The study paved way for further improvement of Fe and Zn content under late sown condition.However, breeding hybrids with high levels of Fe and Zn densities will require incorporating them in both parental lines, and application of genomics tools may significantly accelerate this process.
Highly significant and high positive correlation between performance per se of parental line and their general combining ability (GCA) both for Fe and Zn densities showed that parental lines of potential hybrids with high GCA can be effectively selected based on their performance per se, thus enhancing the breeding efficiency.Lack of correlation of Fe and Zn densities with grain yield in inbred lines, but significant negative (although low) correlation in hybrids merits further investigation as these results have direct bearing on the efficiency of breeding high-yielding hybrids with high levels of Fe and Zn densities.

Materials and methods
Plant materials and field performance evaluation.The experimental material consisted of a total 55 entries comprising of 45 hybrids were developed by following diallel mating design 63,64,65 by using 10 parental genotypes by excluding reciprocal crosses (Table 1).The

Table 5 . Estimates of heterobeltosis in individual environments for Grain yield per plant, protein content, iron content and zinc content.
*, ** Significant at 5 % and 1 % levels, respectively; Highest and lowest values are indicated by bold; HB = Heterobeltosis ; SH 1 = Standard heterosis over check variety MASC 6222 (TS) ; SH 2 = Standard heterosis over check variety HD 2932 (LS)

Table 6 . Estimates of standard heterosis in individual environments for grain yield per plant, protein content, iron content and zinc content.
ForZn content, H36 had highest significant and positive SCA effects in E 1 (2.66**) as well as significant positive in both environments for Fe content.In addition, the variance due to SCA (σ 2 SCA ) was greater than GCA (σ 2 GCA ) for all studied (Table3).
had highest positive significant for Fe & Zn content in E 1 .For iron content 16 each in E 1 & E 2 hybrids recorded significant positive SCA effects.For Zn content Out of 45 hybrids, the number of crosses depicted significant and positive SCA effects were 8 each in E 1 and E 2 .

Table 7 . Estimates of general combining ability (GCA) effect of parents for Grain yield per plant, protein content, iron content and zinc content.
*, ** Significant at 5 % and 1 % levels, respectively; Highest and lowest values are indicated by bold .

Table 8 . Estimates of specific combining ability (SCA) effect of F 1'S for Grain yield per plant, protein content, iron content and zinc content.
29,26.Thus need to take some remedial measures like development of hybrids, high stress tolerance and use of conventional and non-conventional approaches.Different sowing dates affect the growth of wheat mainly due to the effects of temperature and day length.Temperature and short winter growing season in central India are main reasons for low yield in wheat.High temperature increases the rate of grain filling but, the duration of grain filling is reduced considerably and grain weight is decreased 27,28 .In this regard, understanding the relationship between grain yield, grain protein and grain micronutrient content under normal and late sown conditions will make a best use of heterosis breeding/hybrid wheat technology.To combat the micro-nutrient deficiencies among wheat eaters, efforts are to be made to enhance the grain micronutrient content (particularly Fe and Zn) along with sustainable crop yield potentials29. Biofotified varieties Among the two categories of heterosis, SH is the most appropriate than BPH.SH elucidated the acceptance yield advantage of hybrid varieties over the best checks.The hybrids in the present study are observed with negative heterosis for all the studied traits in both environments.