Cottonseed oil and yield assessment via economic heterosis and heritability in intraspecific cotton populations

Due to the difficulty in producing cotton F1 hybrid seed through hand emasculation and pollination, the capture of heterosis in cotton production is limited. The purpose of this study was to evaluate the potential of 6 × 6 F1 and F2 intra-hirsutum diallel hybrids relative to heterosis, inbreeding depression, heritability and correlation. Average F1 hybrids mean values were higher than those of F2s, while F2s exceeded the parents. However, in F1s and F2s, the average mean values were 22.30 and 19.48 for seeds boll -1 , 8.87 and 8.56 g for seed index, 112.36 and 87.51 g for seedcotton yield and 30.07 and 28.30% for cottonseed oil. Heterosis over better parents was more pronounced in F1s as compared to F2s. However, in F1s and F2s, the average positive heterosis was 11.54 and 4.50%; 3.40 and 2.41%; 46.30 and 28.96% and 6.04 and 1.52%, respectively, for the above traits. Even after segregation and inbreeding depression, the F2s expressed about 50% of F1s heterosis. The inbreeding depression in F2s was -6.51 to -16.92 with low to high heritability, and significant positive correlation of cottonseed oil with other traits. The cultivar CIM-1100 derivatives performed better and exceeded all other hybrids in both generations.


INTRODUCTION
Development and utilization of high yielding and stress tolerant cultivars have a fundamental position in crop production technology package, which could be facilitated through introgressive hybridization (Khan, 2003;Khan et al., 2007a).The success of transgressive segregation depends upon the identification of genotypes with the ability to transmit high production potential into specific genotypic combinations.Heterosis is a performance of F 1 /F 2 genotypic combinations and is useful in determining the most appropriate parents for specific traits.It is an established tool for breeders to assess and understands the gene India, at least 40% of the cotton produced is derived from intra-specific hybrids of Gossypium hirsutum, and 8% from inter-specific hybrids of G. hirsutum × Gossypium barbadense (Chaudhry, 1997).The yield increase through intra-and inter-specific heterosis over the better parent or best commercial cultivar (useful heterosis) has been documented in numerous reviews (Galanopoulou and Roupakias, 1999;Wei et al., 2002;Yuan et al., 2002;Zhang et al., 2002;Khan et al. 2007a).The average increase in yield and yield components of F 1 hybrids are found to be mostly greater than heterosis in fibre quality traits.The availability of male sterile parents has opened the way for additional commercial exploitation of hybridization in cotton; hence, work continues in the study of hybrid performance by breeders (Khan et al., 1999(Khan et al., , 2000)).Basal and Turgut (2003) concluded that yield and fiber quality traits can be improved through applying 3 way crosses, or modified backcross and recurrent selection to genotypes having remarkable heterosis and combining ability.
The F 2 hybrids having greater heterogeneity and genetic variation might result in a vast range of adaptation relative to their parents and F 1 hybrids (Meredith and Brown, 1998;Wu et al., 2004).The F 2 s exhibited superiority over their better parents when grown under stress conditions and lower yielding sites.Meredith (1990) demonstrated that F 2 s can produce better combinations of yield and fiber quality than their parents.Results also indicated that F 2 s performance was highly correlated with F 1 s yield performance, and in some cases the F 2 s heterosis has been found equivalent to F 1 s heterosis.The F 2 s heterosis in cotton has also been mentioned in previous studies (Wang and Pan, 1991;Tang et al., 1993;Li et al., 2000;Xing et al., 2000;Han and Liu, 2002;Khan, 2003;Khan et al., 2007a).It is expected that F 2 s may even express only 50% of the economic heterosis shown by F 1 hybrids, and even less when heterosis is defined in terms of the higher yielding parent.Nonetheless, F 2 hybrids with lower inbreeding depression in yield and superior performance than well-adapted cultivars have been found (Meredith, 1990).The existence of such hybrids lends credence to the use of F 2 hybrids in cotton production.Khan et al. (2007b) noticed moderate to high heritability for cotton seed traits and oil content in intra-hirsutum F 2 hybrids.The higher the heritability, the simpler the selection process and greater the response to selection (Larik et al., 2000;Khan, 2003).
Numerous studies have been reported on seeds traits (seed number and index), but little work has been reported on the genetics and heterosis of cottonseed oil percentage in cotton breeding.A few reports in the literature (Kohel, 1980;Dani and Kohel, 1989;Dani, 1991;Khan, 2003;Khan et al., 2007b) have determined that cotton genotypes differ in oil percentage.The estimates of heterosis provided useful information with regard to the possibilities and extent of improvement in the Khan et al. 7419 characters of breeding material through selection.The present work provides useful information about economic heterosis of F 1 and F 2 hybrids over better parents, inbreeding depression and heritability (broad sense) in F 2 s, and correlation of cottonseed oil with seeds per boll, seed index and seed cotton yield.Though the inferences drawn here, in a strict sense, apply to the genotypes involved, the findings will provide insights into these issues in a broader sense.

Genetic material
Breeding material comprised of six different G. hirsutum genotypes and their 30 F1 and F2 hybrids (including reciprocal) generated through 6 × 6 complete diallel crossing.The parental cultivars of G. hirsutum were CIM-109, CIM-240, CIM-1100, FH-682, BH-36 and CRIS-9 having broad genetic base and were varied by date of release, pedigree, seedcotton and fiber yield as well as fiber quality traits.

Experimental design and field procedures
Experiments comprised of crossing block; F1 and F2 populations of upland cotton were conducted during 2007 -2009 at Khyber Pakhtunkhwa Agricultural University, Peshawar, Pakistan.Peshawar lies between 34°, 02′ North latitude and 71°, 37′ East longitude.Six diverse genotypes of upland cotton were hand sown during May 2007, in a non-replicated crossing block.Each cultivar having five rows, 27 m in length, with plants and rows spacing of 60 and 100 cm to facilitate hand emasculation and crossing.Cultivars were crossed in a complete diallel fashion.The half of the F1 populations was planted in 2008 for F2 seed production, produced through crossing in the previous season.During 2009, the experiments having 30 F1 and F2 hybrids with six parents were hand-sown in a randomized complete block (RCB) design.Each F1 was planted in a single row measuring 3.30 m with three replications, while in F2s the plant population was increased and were planted in four rows, 6.30 m in length, with four replications.The plants and rows spacing were 30 and 75 cm, respectively in all the populations.All the recommended cultural practices and inputs including fertilizer, hoeing, irrigation and pest control were applied same for all the entries from sowing till the harvesting and the crop was grown under uniform conditions to minimize environmental variations to the maximum possible extent.Picking was made in the months of November -December every year on single plant basis and ginning was done with eight saw-gins.

Traits measurement and statistical analyses
The data were recorded for seeds per boll, seed index seed cotton yield and cottonseed oil percentage.For seeds per boll in each genotype, 50 bolls were separated and after counting the number of seeds in each boll, the average number of seeds per boll was calculated.For seed index in each genotype, one hundred fuzzy cottonseeds free from lint, disease or any other insect pest were weighed on electronic balance.For cottonseed oil content %, the seed samples were acid-delinted and oven dried at 40°C for 24 h and oil analysis was carried out by Wide Line Nuclear Magnetic Resonance (NMR, 4000).All the data were subjected to analysis of variance and was carried out according to Gomez and Gomez (1984).Heterosis was calculated in F1 and F2 hybrids against its better parent value as suggested by Fehr (1987) and Khan et al. (2007a).The inbreeding depression in F2 hybrids was formulated as percentage decrease of F2 hybrids when compared with F1 hybrid means as outlined by Hallaner and Miranda (1986), Baloch et al. (1993) and Khan (2003).

Genetic variability in F 1 and F 2 hybrids and their parents
According to analysis of variance (ANOVA), highly significant differences (P ≤ 0.01) were recorded for seeds per boll, seed index, seed cotton yield and cottonseed oil percentage among the 30 F 1 s and F 2 s including their parents (Table 1).The average F 1 s seeds per boll, seed index, seed cotton yield and cottonseed oil % were higher than those of F 2 s, while F 2 s values for the said traits were higher than parents.Seeds per boll varied from 17.31 (CIM-240) to 22.16 (BH-36) among the parents and 18.33 (CIM-240 × CRIS-9) to 26.33 (CIM-1100 × BH-36) among the F 1 s and 17.90 (CIM-240 × CIM-109) to 23.68 (CIM-1100 × BH-36) among the F 2 s (Figure 1).Maximum F 1 seeds per boll were produced by hybrid CIM-1100 ×  and were also at par with nine other crosses that ranged from 24.00 to 25.33 seeds per boll.Six of the Seed index (g) Mean performance of F1, F2 hybrids and parents for seed index in a 6 × 6 diallel cross of upland cotton.
Seed index varied from 7.70 (BH-36) to 9.33 g (CIM-240) among parents, 7.71 (BH-36 × CRIS-9) to 10.55 g (CIM-240 × CIM-1100) among the F 1 s and 7.56 (BH-36 × CRIS-9) to 9.89 g (CIM-240 × CIM-1100) among the F 2 hybrids (Figure 2).Eleven of the F 1 hybrids had maximum seed index that ranged from 9.00 -9.88 g.In the F 2 generation, seven hybrids had remarkable seed index (9.05-9.67 g) and these hybrids can be further utilized to produce bold and higher numbers of seeds per boll.The hybrid CIM-240 × CIM-1100 has shown best performance for seed index in F 1 s (10.55 g) as well as in F 2 s (9.89 g) having more stability with less inbreeding depression.The lowest seed index was possessed by cultivar BH-36 (7.70 g) and its cross with CRIS-9 (7.56 g).
In F 1 generation, the seed cotton yield per plant varied from 54.31 -94.77 g among the parents and 59.12 -188.81 g among the hybrids (Figure 3).The highest and statistically at par seed cotton yield was recorded in F 1 hybrids CRIS-9 × CIM-1100 (188.81 g), BH-36 × CIM-1100 (183.58 g) and CIM-1100 × CIM-109 (180.92 g).These were followed by at par second top scoring hybrids i.  4).In F 1 hybrids, maximum oil percentage was obtained in the cross combination CIM-1100 × FH-682 (32.34%) and was found statistically  at par with 16 other hybrids having 30.09-31.96% oil and in which eight were CIM-1100 hybrids.In the F 2 generation, maximum oil percentage was obtained in the combination FH-682 × CIM-1100 (30.17%) which was derived from the reciprocal of top F 1 for oil percentage, and was equivalent with 15 other F 2 hybrids (eight were derivatives of CIM-1100) ranging from 28.89 -29.80% oil.
Overall, the lowest cottonseed oil percentage was found in cultivars BH-36 and CRIS-9 and two hybrids of CRIS-9 with CIM-109 and BH-36.

Heterosis over high parent and inbreeding depression
Overall, the average positive heterosis was 11.56 and 4.50%, 3.40 and 2.41%, 46.30 and 28.96%, 6.04 and 1.52%, respectively, in F 1 and F 2 hybrids for seeds per boll, seed index, seed cotton yield and cottonseed oil percentage.It seems that the mean F 2 s heterosis was low as compared to F 1 s but it still acceptable as exceeding even better parents in performance.In case of seeds per boll, the positive heterosis over the better parents was found in 23 F 1 hybrids (Figure 5) and ranged from +2.22 (CIM-240 × CIM-109 and CIM-1100 × CIM-240) to +26.92% (CIM-1100 × CIM-109).The 15 F 2 hybrids were found superior over their better parents and the heterosis varied from +1.44 (BH-36 × FH-682) to +13.48% (CIM-1100 × CIM-109) for seeds per boll.The top 10 F 1 and F 2 hybrids having maximum heterosis were also derivatives of cv.CIM-1100.The F 2 hybrids manifested inbreeding depression (-2.62 to -15.82%) for seed per boll (Figure 4).Two F 2 hybrids (BH-36 × CIM-240 and CIM-1100 × CIM-240) showed increases in seeds per boll as compared to their F 1 s and manifested even positive values for inbreeding depression due to transgressive recombination.More than 75% of the F 1 hybrids showed heterosis for seeds per boll, while in F 2 , half of the hybrids showed positive values for heterosis over better parents.Selection in these desirable F 2 hybrids can also lead to development of the best genotypes through segregating generations.
In case of seed index (Figure 6), nine F 1 hybrids attained positive heterotic effects over better parent values which ranged from +0.11 (FH-682 × CIM-109) to +13.08% (CIM-240 × CIM-1100).In F 2 generation, seven hybrids displayed heterosis over their better parent ranging from +0.55 (CRIS-9 × CIM-1100) to +6.00% (CIM-240 × CIM-1100 the F 2 hybrids displayed inbreeding depression with range of -0.43 to -6.51.As compared to F 1 s, the F 2 hybrid seed index values were decreased because of smaller seeds due to inbreeding.However, five F 2 hybrids showed increase in their mean values of seed index as compared to their F 1 hybrid means.As compared to parents, the F 2 s having bigger seeds had higher mean values.The selection in these desirable genotypes can also provide a base for further improvement in the seed index. For seed cotton yield per plant (Figure 7), 20 F 1 hybrids had positive heterosis over better parents and the increase ranged from +0.82 (CIM-109 × BH-36) to +115.22% (CRIS-9 × CIM-1100).The 16 F 1 hybrids exhibited significant heterosis over the best parent.All the CIM-1100 hybrids (except as a maternal parent with CIM-240) manifested maximum and highly significant heterosis over the better parent (+55.75 to +115.22%).In F 2 generation (Figure 6), the positive heterosis for seed cotton yield was recorded in 13 hybrids.The range of high parent heterosis was +0.73 (CRIS-9 × BH-36) to +77.03% (CIM-1100 × FH-682).The 11 hybrids were found superior as compared to better parents and had significant heterosis at P≤0.01 and P≤0.05.The F 2 genotypes also showed inbreeding depression ranged from -1.87 to -44.15%.

Heritability and correlation
In F 2 hybrids, the broad sense heritability (H 2 ) for seeds per boll was low to high (0.40 -0.90) (Figure 9).In CIM-1100 hybrids, the H 2 was moderate to high ranging from 0.78 -0.90.For seed index, H 2 was similar and ranged from 0.44 -0.93 (Figure 9), while in CIM-1100 derivatives the H 2 was moderate to high (0.66 to 0.93).For seed cotton yield (Fig. 10) the H 2 in F 2 hybrids was ranged from 0.55 -0.92, while in promising hybrids of CIM-1100 the H 2 range was 0.78 -0.92.For cottonseed oil content (Figure 10), H 2 was low to high (0.46 -0.91), while in F 2 progenies of CIM-1100 the H 2 was high (0.80 -0.91).The hybrid wise high ratio of broad sense heritability for each trait was also confirmed by desirable heterosis in the said  traits.The correlation coefficient of cottonseed oil with seed cotton yield and oil contributing traits i.e., seeds per boll, seed index and seed cotton yield was also determined (Table 2).It was also encouraging to observe that the correlation of cottonseed oil percentage was found positive with seeds per boll (r = 0.223 and 0.023), seed index (r = 0.366 and 0.376) and seed cotton yield (r = 0.272 and 0.130), respectively in both generations.

DISCUSSION
Functional heterosis was observed for seeds per boll, seed index, seed cotton yield and cottonseed oil percentage in both F 1 and F 2 generations hybrids and especially in the top 10 hybrids which also indicated remarkable heterosis in F 2 s also for all the traits.CIM-1100 hybrids showed high heritability and thus reduced environmental impact on these traits.The said results supports the idea that F 2 hybrids could work as a hybrid crop if properly managed and if the parents were selected on the basis of F 2 performance, because the F 1 hybrids cannot clarify the position of the level of stability at the F 2 level (Galanopoulou and Roupakias, 1999).It is also well known that F 2 heterosis exists in cotton and F 2 hybrids with lower inbreeding depression in yield related traits and promised superior performance than well-adapted cultivars (Dani and Kohel, 1989;Khan et al. 2007a).F 2 hybrids have being extensively grown in China demonstrating that their yields were significantly higher than commercial cultivars (Jing et al., 1995;Li et al., 1997).Results revealed that average F 1 mean values for all the traits were found greater than those of F 2 s due to inbreeding depression, while F 2 values were higher than parents which confirm the findings of Wu et al. (2004).Our conclusions are also in corroboration with those of Baloch et al. (1993); Hassan et al. (1999); Azhar and Ahmad (2000); Khan et al. (2000) and Khan (2003).
The results indicated that the best performing F 1 s also performed well in the F 2 generation with less inbreeding depression.Wu et al. (2004) noted that the performance of the F 1 and F 2 hybrids can be mostly predicted by the average performance of the parents.However, Galanopoulou and Roupakias (1999) reported that the performance of the F 2 cannot be adequately predicted by the performance of the F 1 .In general, there is a substantial reduction of heterosis from F 1 -F 2 (Zhang and Pan, 1999).In the studies reported herein, it was noted that the higher F 1 heterosis had more inbreeding depression and show abrupt decline as compared to the F 1 hybrids having moderate heterosis.The results of same pattern have also been reported by Li et al. (2000) and Wei et al. (2002).The combined performance of the hybrids in the F 1 and F 2 generations may be a good indicator of the  most promising populations to be utilized as F 2 hybrids in cotton production (Galanopoulou and Roupakias, 1999).The same confirmation was also observed in cv.CIM-1100 F 1 and F 2 hybrids and was noted as the high performers for all the traits studied.
The results also revealed that in F 2 s even after inbreeding depression, some promising population (e.g., CIM-1100 derivatives) revealed better performance for all the traits and positive selection can provide better base for further improvement.The abrupt drop in traits performance at F 2 level was due to inbreeding depression and segregation.Meredith (1990) also reported that the F 2 hybrids with lower inbreeding depression in yield had superior performance than well adapted cultivars.Khan et al. (2000), Basal and Turgut (2003) and Khan et al. (2007) also mentioned that F 1 hybrids with high heterosis were also associated with higher inbreeding depression.Average heterosis of F 2 over mid parent, based on population mean, suggested that little inbreeding depression exists for F 2 and F 3 generations and it is possible to screen and select high yielding F 2 hybrids for further use (Yuan et al., 2002).F 2 hybrids having extra ordinary performance could also be used as such for seed of F 2 crop to increase the seed cotton yield on per unit area as suggested by Baloch et al. (1993) and Khan et al. (2000) and(2009).F 2 hybrids have greater genetic variation and might result in a greater range of adaptation relative to their parents and F 1 hybrids (Meredith and Brown, 1998;Wang and Li, 2000;Wei et al. 2002;Wu et al., 2004).F 2 hybrid heterosis in cotton has been reported (Wang and Pan, 1991;Tang et al., 1993;Li et al., 2000;Xing et al., 2000;Han and Liu, 2002;Khan, 2003) and F 2 s can express at least 50% of the economic heterosis shown by F 1 hybrids, which can lead to cultivar improvement.
The cv. CIM-1100 hybrids can also be used in intensive selection for further exploitation in segregating generaions to produce new strains with better yield, oil content and seed related traits.Overall, the highest heterosis was expressed in this group of parents and hybrids for seed cotton yield, cottonseed oil followed by seeds per boll and seed index, suggesting a slight increase in cottonseed oil, seeds per boll and especially seed size and eventually the seed cotton yield will be a great achievement.Selection in these F 2 hybrids will produce better families of hybrids having higher oil percentage.CIM-1100 hybrids showed moderate to high heritability and were found more responsive for selection on the basis of phenotypic performance and heritability, while other hybrids presented low to medium heritability.Khan et al. (2007b) noticed moderate to high heritability for seed cotton yield, cotton seed traits and oil content in intra-hirsutum F 2 hybrids of CIM-1100.The higher the heritability, the simpler the selection process and the greater the response to selection (Larik et al., 2000;Khan, 2003).Positive and significant correlation between cottonseed oil, and seed cotton yield and seed traits was also encouraging and the genetic gain in yield, seed index and seeds per boll will have a direct positive impact on cottonseed oil percentage.Khan (2003) also noted positive correlation for cottonseed oil with yield and oil contributing traits.The genotypes derived from such type of F 2 hybrids will met the edible oil demand of the developing countries like Pakistan and will have a positive impact on their foreign exchange.

Conclusion
Overall, F 1 hybrid mean values for seeds per boll, seed index, seed cotton yield and cottonseed oil were higher than F 2 hybrids, while F 2 hybrids supersede their parental mean values.High parent heterosis was more pronounced in the F 1 as compared to the F 2 due to inbreeding depression.But it was encouraging to note that F 2 hybrids, even after segregation and inbreeding depression, manifested 50% or even more heterosis.The greatest heterosis was observed for seed cotton yield and cottonseed oil followed by other seed traits.An increase in cottonseed oil percentage, seed cotton yield and cottonseed traits will be a valuable addition to cotton cultivars.

Figure 9 .
Figure 9. Heritability in F2 hybrids for seed per boll and seed index in a 6×6 diallel cross of upland cotton.

Figure 10 .
Figure10.Heritability in F2 hybrids for seed cotton yield and cottonseed oil in a 6 × 6 diallel cross of upland cotton.

Table 1 .
Mean squares for F1, F2 hybrids and parents and correlation of cottonseed oil % with other traits in a 6 × 6 diallel cross of upland cotton.

1 and F 2 dialle l hybrids of upland cotton Hetersosis & inbreeding dep. (%) for seed index F 1 Heterosis F 2 Heterosis F 2 Inbre eding Depression Figure
6. Heterosis in F1s and F2s, and inbreeding depression in F2s for seed index in a 6 × 6 diallel cross of upland cotton.

1 and F 2 diallel hybrids of upland cotton Heterosis & inbreeding dep. (%) for seedcotton yield F 1 Heterosis F 2 Heterosis F 2 Inbreeding depression Figure 7.
Heterosis in F1s and F2s, and inbreeding depression in F2s for seed cotton yield in a 6 × 6 diallel cross of upland cotton.

1 and F 2 diallel hybrids of upland cotton Heterosis & inbreeding dep. (%) for cottonseed oil F 1 Heterosis F 2 Heterosis F 2 Inbreeding Depression Figure 8.
Heterosis in F1s and F2s, and inbreeding depression in F2s for cottonseed oil in a 6 × 6 diallel cross of upland cotton.

Table 2 .
Correlation of cottonseed oil percentage with other traits in a 6 × 6 diallel crosses of upland cotton.