Research Journal of Agricultural Sciences 2010, 1(3): 189-192

Evaluation and Confirmation of Hybridity on the basis of Seed Proteins and Isozymes in Sunflower Hybrids
Pallavi H M, Ramegowda, K Bhanuprakash*, Vishwanath, K and Shadakshari, Y G**
Department of Seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore-65, Karnataka, India *AICRP on Sunflower, University of Agricultural Sciences, GKVK, Bangalore-65, Karnataka, India **Section of Seed Science and Technology, IIHR, Hessaraghatta, Bangalore, India
e-mail: pallavihm@gmail.com

ABSTRACT
Maintenance of genetic uniformity of lines and hybrids is a prerequisite for successful production and placement of commercial hybrid seed in the market. Genetic purity of seed as specific seed trait is of great significance for seed science. Protein markers, seed storage proteins and isozymes, which are commonly used for the estimation of genetic purity, were used in this work to estimate genetic purity in sunflower hybrids. Esterase enzymatic system analyzed was polymorphic. A comparative analysis of genetic purity level of the sunflower hybrids was performed using electrophoretic methods. The efficiency of soluble seed protein as marker to distinguish the hybrids form its parental lines is under query due to its low polymorphism and unable to identify heterozygosity because of its dominant nature. While, hybrids distinguishing the hybrids based on the esterase isozyme banding pattern it could able to distinguish hybrids from its parental lines and hybrids exhibited bands that are present in both the parental lines their by confirming the hybridity similar to its parental lines. However, the level of polymorphism obtained by both methods was not distinct enough to be used in CMS lines identification. Key words: Sunflower, Genetic purity, Grow out test, Bio-molecular markers, Isozymes, Esterase Sunflower (Helianthus annuus L.) belongs to family Asteraceae is a diploid (2n=34) and the second most important oil crop worldwide after soybean. The increased production and productivity is credited to the release of new high yielding varieties and hybrids for commercial cultivation. Any hybrid seed production technology is successful only when the seeds supplied are of high quality and genetically pure. Identifying breeding lines and determining hybrid purity will be the major requirements in plant breeding and seed production. To test the conformity of hybrid seed, one must be able to distinguish the true hybrid resulting from cross between male and female parents and one coming from self pollination of female parent. A combination of laboratory and field plot methods may be used to determine the cultivar trueness and genetic purity of the sample. Laboratory control is based on protein markers, isozymes, seed storage proteins and molecular markers. Isozymes are the multiple molecular forms of an enzyme with similar or identical catalytic activities occurring within the same organism (Markert and Moller 1959). These can be a rapid sensitive tool for cultivar identification that can provide enough allozyme markers present in the analyzed species. Isozymes are widely used for reconstruction of phylogenetic relationships between related species. This has lead to the use of isozymes as an advanced technique to diagnose phenotype for testing genetic purity of hybrid lots (Nijenhuis 1971, Wills et al. 1979, Tanksley and Jones 1984). The aim of the present study was to compare two methods which are commonly used for estimation of genetic purity in sunflower hybrids, electrophoresis of isozymes and seed storage protein.

MATERIALS AND METHODS

In the study five sunflower hybrids were analyzed for electrophoresis banding pattern of protein and isozymes. Each seed was finely ground and protein extracted by adding 100l 0.25 M Tris-HCl buffer pH 8.8 containing 0.01% 2-mercaptoethanol for overnight. After centrifugation at 10,000rpm for 15 minutes, to the supernatant chilled acetone was added to obtain protein pellet. The protein pellet was dissolved in denaturing buffer (0.15 M Tris-HCl pH 6.8 containing 3% SDS, 5% 2-mercaptoethanol and 7% glycerol) working buffer and was used for electrophoresis. Individual seeds were tested from each sample. Polypeptides were resolved by electrophoresis of proteins under denaturing (SDS) and reducing (2-mercaptoethanol) conditions in 12.5% PAGE using the method of Laemmli (1970). Electrophoresis was performed at an initial voltage of 50 V, increased to 80 V till the tracking dye reached the gel mold; analysis time was 18 hours. The marker medium range molecular weight was used for determination of protein molecular weight in electrophoretograms. Proteins were simultaneously fixed and stained using silver nitrate. Five days old seedlings were homogenized in 0.1M TrisHCl, pH 7.5

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Plate 1 Total soluble seed protein profile of KBSH-1, KBSH-41 and KBSH-42 hybrids and their parental lines

Plate 2 Total soluble seed protein profile of KBSH-44 and KBSH53 hybrids and their parental lines

Fig 1 Electrophorograms of total soluble proteins of KBSH-1, KBSH-41 and KBSH-42 hybrids and their parental lines
KBSH-1 (1): CMS-234 A (6) RHA 6D-1 (14) KBSH-41 (2): CMS-234 A (6) RHA 95-C-1 (15) KBSH-42 (3): CMS-851 A (8) RHA 95-C-1 (15) M: Standard marker A: Male sterile B: Maintainer line R: Restorer line H: F1 hybrid

Fig 2 Electrophorograms of total soluble proteins of KBSH-44 and KBSH-53 hybrids and their parental lines
KBSH-44 (4): CMS-17 A (6) RHA 95 C-14 KBSH-53 (5): CMS-53 A (8) RHA 95-C-1 (15) M: Standard marker A: Male sterile B: Maintainer line R: Restorer line H: F1 hybrid

KBSH-1

KBSH-41

KBSH-53

Plate 3 Esterase (EST) isozyme profiles of sunflower hybrids and their parental lines
KBSH-1 (1): CMS-234 A (6) RHA 6D-1 (14) KBSH-41 (2): CMS-234 A (6) RHA 95-C-1 (15) KBSH-42 (3): CMS-851 A (8) RHA 95-C-1 (15) KBSH-44 (4): CMS-17 A (10) RHA 95 C1 (15) KBSH-53 (5): CMS-53 A (12) RHA 95-C-1 (15)

KBSH-42

KBSH-44

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Evaluation and Confirmation of Hybridity on the basis of Seed Proteins in which 1% mercaptoetanol was added to extract esterase isozyme and analyzed according to Gennady (1994). genes. Differences that found in relative mobility of protein molecules of these hybrids are due to the differences in heliathenine, which is in agreement with the results of Aksyonov (2005b) and reported that Hel4 makes the specific allelic variants in the hybrid and inbred lines. The gene controlling Heliathenin is inherited as a single co-dominant gene with independent alleles in parental lines and hybrids. These results are in conformity with other sunflower hybrids reported by Irina et al. (2001), Kumar et al. (2001), Nikolic et al. (2008). Although the electrophoretic analysis of total seed proteins or a specific protein fraction provides the most useful method for varietal discrimination and identification. The presence of too many bands revealing a magnitude of non-polymorphic polypeptides often complicate the reading of the banding patterns and interpretation, particularly in cross-pollinated crops (Chikkadevaiah and Nandini 2003). However, the level of polymorphism and variations in the banding pattern when repeated leads to confusion in the identification of polymorphism in hybrids. Same results were noticed in the study. The clarity and polymorphism of bands found to be in question because of its dominance in nature. The protein profile of hybrid KBSH-53 revealed that female line (CMS-A line) exhibited bands at Rm 0.440 and 0.464 (Region D) supposed to be appeared in hybrids but it was absent. While, in KBSH-42, band at Rm 0.314 (Region C) present both in A and R lines and absent in its hybrid. This might be due to dominant nature of protein and was not expressed in heterozygous condition of hybrid. The seed storage proteins may not be accurate to be utilizing for genetic identification of sunflower hybrids due to their low level of polymorphism. Therefore, the efficiency of the soluble seed proteins as a marker to distinguish the hybrids and parental lines is under query due to its low polymorphism, dominant in nature and unable to identify heterozygosity, similar conclusions have been also reported by Nikolic et al. (2008). However, there is alternative marker which is codominant that can be employed for distinguishing/ confirming the hybridity of sunflower. Isozymes are also a best co-dominant marker that could be utilized. Esterase isozyme profile was assesses to confirmed hybridity in sunflower hybrids. Based on the zymogram of EST, all the studied hybrids were able to distinguish form its parental lines. Female parent (CMS-234A) of KBSH-1 had medium intense band at Rm 0.814 but absent in its male parent (RHA 6D-1) (Plate 3). RHA 6D-1 had a high intense band at Rm 0.842 which was absent in CMS-234A, whereas the hybrid KBSH-1 had both the bands which were present in both the parents. This expression and clear cut differences in hybrid may be due to its co-dominant nature of EST isozyme. Similarly, in KBSH-41 (CMS-234A RHA 95-C1) the banding pattern was as that of its female parent

RESULTS AND DISCUSSION

Seed storage proteins are encoded by multigeneic loci and the production of a single locus comprises several electrophoretically separable bands. The procedure had been standardized in many crops species for different proteins fractions like albumin, globulin and glutelin. In sunflower electrophoresis of seeds storage proteins (Helianthinin) had showed promising results in genetic purity determination of hybrids and inbred lines (Aksyonov 2005a, Aksyonov 2005b). In the present study, five hybrids and its parental lines were characterized by total soluble seed proteins separated on SDS-PAGE. A wide variation was observed in the pattern of protein bands of studied hybrids and parental lines. The hybrids were differed in the number of bands, their relative mobility and intensity. The proteins separated on 12.5% acrylamide gel could be distinguished and grouped based on the standard marker (97.4 kDa). In the hybrid KBSH-1, the banding pattern of hybrid was distinct and could able to differentiate from its parental lines viz CMS-234 A, CMS-234 B and RHA 95-C-1. The protein profile of hybrid KBSH-1 differed from its female parent (CMS-234A) by a high intense band at Rm 0.462 in the region D (43.0- 29.0 kDa) and region F (20.0-14.3 kDa). These bands might be inherited from its pollen parent (RHA 95-C-1) in which same bands were noticed (Plate 1). Hence, it clearly noticed that the banding pattern of hybrid exhibited bands from its parents. Although the banding pattern of KBSH-41 is comparable with the banding pattern of its female parent line (CMS-234A), the hybrid could be distinguished by observing an extra band at Rm 0.694 at region E (29.0-20.0 kDa) and 0.834 in the region F (20.014.3 kDa). While, Hybrid KBSH-42 comprised a migrated band from male parent (95 C-1) at Rm 0.694 and 0.710 in the Region E (29.020.0 kDa) and these bands were absent in its female line (CMS-851A) thus confirming its hybridity. Protein profile of hybrid KBSH-44 differed from its parental lines by an extra band at Rm 0.763 in the region F (20.0-14.3 kDa) which was found in male parent (95 C-1) but absent in female parent (CMS-17 A). Similarly, KBSH-53 hybrid exhibited a band from male parent at Rm 0.669 in the region E (29.0-20.0 kDa) and absent in female parent. Those hybrids having marker of both male and female parents that hybrid is usually considered as genuine hybrid (Yan-Min et al. 2003). These identified bands were distinct and can be used for hybridity confirmation in these hybrids during seed production programme. The number of polymorphic zones corresponds to the number of subunits composed of the heliathenine molecule, controlled by Hel1, Hel2, Hel3, Hel4, Hel5 and Hel6

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Pallavi et al. (CMS-234A) along with a male specific medium intense band at Rm 0.930. In hybrid KBSH-42 also, the banding pattern was similar to that of its female parent along with a band at 0.546 Rm values which is specific to male parent. Further, KBSH-44 also showed comparable banding pattern as that of its female parent and a band at 0.733 Rm that reappeared, and also found in its male parent. The banding pattern of hybrid KBSH-53 was almost comparable with the banding pattern of its female line (CMS-53A), but a male (RHA 95-C-1) dominating medium intense bands observed at Rm 0.831 and 0.911 that was absent in its female parent. In general, all the hybrids exhibited bands similar to its parental lines (A line and R line) because of its codominant nature. The differences obtained in zymograms of these enzymes found useful for the determination of the seed genetic purity of sunflower hybrids, especially the esterase which was very distinct. Hybrid seeds have a heterozygote allozyme pattern; however, any seed produced by selfing should have the homozygous allozyme pattern of its female parent (Cooke 1995, Hughes et al. 1992). Therefore, Esterase isozymes (EST) that are co-dominant markers found useful in differentiating sunflower hybrids and its parental lines (Ekanayake et al. 1996, Mandal et al. 2001, Alicia et al. 2002). In conclusion, protein markers are not so useful for hybrid confirmation but EST isozyme is effective in differentiating its parental lines and hybrids on the sunflower. However, the levels of polymorphism obtained by both methods were not distinct technique to be used in distinguishing CMS lines form their maintainer lines.

LITERATURE CITED
Markert C L and Moller F. 1959. Multiple from of enzymes: tissue ontogenetic and species specific patterns. Proceedings of National Academic Sciences, USA 45: 753-758 Nijenhuis B E. 1971. Estimation of inbred seed in brussels sprouts hybrid seed by acid phosphatase isoenzyme analysis. Euphytica 20: 498-507 Wills A B, Fyfe S K and Wiseman E M. 1979. Testing F1 hybrids of Brassica oleraceae for sits by seed isoenzyme analysis. Annuals of Applied Biology 91: 263-270 Tanksley S D and Jones R A. 1984. Application of alcohol dehydrogenase allozymes in testing the genetic purity of F1 hybrids of tomato. Horticulture Science 16(2): 179-181 Laemmli U K. 1970. Cleavage of structural proteins during the assembly of the heads of bacteriophage T 4. Nature 227(5259): 680-685 Gennady P and Manchenko. 1994. Handbook of detection of enzymes on electrophoretic gels. II edition. CRC press, Inc, Boca Raton, Unites States of America Aksyonov I V. 2005a. Use of albumin markers for defining genetic purity of sunflower parent lines and hybrids. Helia 28(43): 43-48 Aksyonov I V. 2005b. Protein markers specificity of sunflower inbred lines. Helia 28(43): 49-54 Irina N, Anisimova T, Roger J F, Arthur S, Tatham and Peter R S. 1995. Genotypic variation and polymorphism of 2S albumins of sunflower. Euphytica 83: 15-23 Kumar J, Agarwal R L, Kumar A and Garg G K. 2001. Sodium dodecyl sulphate-polyacrylamide gel electrphoresis (SDS-PAGE) analysis for determination of genetic purity of sunflower hybrids. Seed Sciences and Technology 29(3): 647-652 Nikolic Z, Vujakovic M, and Jevitic A. 2008. Genetic purity of sunflower hybrids determined on the basis of isozymes and seed storage proteins. Helia 31(48): 47-54 Cooke R J. 1995. Varietal identification of crop plants. New Diagnostics in Crop Sciences 33-63 Chikkadevaiah and Nandini R. 2003. Isozymes as markers for differentiating sunflower genotypes. Helia 36(29): 51-58 Hughes J M, Mather P B and Mcdonald J G. 1992. Differentiation between sunflower and Sorghum cultivars using cellulose acetate electrophoresis. Seed Sciences and Technology 20: 15-22 Ekanayake E M D S N, Manawaprema M M C and Fernando K K S. 1996. Varietal identification using isozyme polymorphism for esterase (EST) and glutamate oxaloacetate transaminase (GOT) of some locally available big onion (Allium cepa L.) varieties. Tropical Agriculturist 151: 45-51 Mandal A B, Aparna M, Bikash C and Elanchezhian R. 2001. Isoenzyme markers in varietal identification of banana. In-Vitro Cellular and Developmental Biology Plant 37(5): 599-604 Alicia D C, Pizapro G, Poverene M, Feimgold S, Leon A J and Berry S T. 2002. Variability among inbred lines and RFLP mapping of sunflower isozymes. Genetics and Molecular Biology 25(1): 65-72

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