Prediction of Combining Ability and Heterosis Using RAPD Markers

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1 Prediction of Combining ORIGINAL SCIENTIFIC Ability and Heterosis PAPER Using RAPD Markers Prediction of Combining Ability and Heterosis Using RAPD Markers Aleksandar RADOJČIĆ, Goran DRINIĆ, Snežana DRINIĆ MLADENOVIĆ, Milomir FILIPOVIĆ Maize Research Institute, Zemun Polje, Slobodana Bajića 1, Belgrade, Serbia ( Abstract The goal of this study was to investigate the relationship between hybrid performance (combining ability and heterosis) in maize and genetic distance determined with Random Amplified Polymorphic DNA (RAPD). Obtained results showed a positive, middle and significant correlation between specific combining ability and genetic distance and between grain yield heterosis and genetic distance. Based on obtained results it could be concluded that RAPD markers can be used for genetic divergence analysis of maize inbred lines, but their use for prediction of combining ability and heterosis is still limited. Key words: combining ability, genetic similarity, heterosis, maize, RAPD markers. Predviđanje kombinacijske sposobnosti i heterozisa pomoću RAPD markera Sažetak Cilj ovoga istraživanja je bio istražiti povezanost između svojstava hibrida (kombinacijske sposobnosti i heterozisa) kod kukuruza i genetske udaljenosti utvrđene pomoću slučajno amplificirane polimorfne DNA (RAPD). Dobiveni rezultati su pokazali pozitivnu, umjerenu i signifikantnu korelaciju između specifične kombinacijske sposobnosti i genetske udaljenosti kao i heterozisa za prinos zrna i genetske udaljenosti. Na osnovi dobivenih rezultata se može zaključiti da se RAPD markeri mogu koristiti za analizu genetske divergentnosti inbred linija kukuruza, ali njihovo korištenje u predviđanju kombinacijske sposobnosti i heterozisa je još uvijek ograničeno. Ključne riječi: kombinacijska sposobnost genetska sličnost, heterozis, kukuruz, RAPD markeri Introduction The degree of heterotic effect F1 generation is correlated with genetic divergence of maize parent inbred lines, as parents are more divergent, the heterosis is higher and vice versa, (Hallauer et al., 1988). Genetic diversity can be obtained by means of physiological, morphological, agronomic, and molecular differences that exist between materials, and diversity can also be obtained by means of heterosis or through material specific combining ability, manifested in a series of crosses between varieties and hybrids (Melo et al., 2001). The standard maize breeding methods include a numerous crosses of inbred lines with different testers for estimation genetic divergence in order to their classification into heterotic groups. Testing in filed condition are very expensive and the most time consuming part of modern maize breeding. In the same time the number of inbred lines is limited for testing programs. The alternative useful techniques for classification in Proceedings. 43 rd Croatian and 3 rd International Symposium on Agriculture. Opatija. Croatia ( ) XXX) Genetics, Plant Breeding and Seed Production 335

2 Aleksandar RADOJČIĆ, Goran DRINIĆ, Snežana DRINIĆ MLADENOVIĆ, Milomir FILIPOVIĆ heterotic groups include molecular markers (Pinto et al., 2003). Methods based on these techniques allow testing a bigger number of inbred lines, and accelerate a breeding process with lower cost. The objective of this work was to analyze genetic diversity among 6 maize inbred lines by means of RAPD markers, and to estimate the correlation between genetic distance and the performance of hybrids. Material and methods Six maize inbreds were crossed to generate diallel set of progenies including reciprocals. Three inbred lines are related with BSSS (ZPL 142, ZPL 680, and ZPL 357/3) and three with non-bsss genetic background (ZPL 275/3, ZPL 17/5, and ZPL 173/3). Parent inbred lines, F1 crosses with reciprocal s, were included in a randomized complete block (RCB) split-plot experimental design with four replications at location Zemun Polje in 2003 and The trial was planted in two densities (main blocks) and plants ha -1. An analysis of variance, general combining ability (GCA), specific combining ability (SCA) and reciprocal effect (RE) for grain yield were calculated according to the Griffing s (1956) Method, Model I (fixed effects) analysis with Diallel analyses software of Burow and Coors (1994). High-parent heterosis (HPH) was calculated as HPH= [(F1-HP)/HP] x 100; where, F1 is the mean of the F1 hybrid performance and HP is the mean of the best parent. The DNA was extracted from embryo tissue using the procedure described by Sagai-Maroof et al., (1984). RAPD PCR amplification was performed with 10 decamer primers (Genosys Biotechnologies) using protocol of Williams-a (1990). Amplification products were analyzed by electrophoresis in 1,4% agarose gels containing ethidium bromide. Genetic distances (GD) were calculated between pairs of lines on the basis of the method developed by Nei and Li (1979) GD=1-[2N ij /(N i +N j )]; where, N ij is the number of bands common to lines i and j, and N i and N j are the total number of bands for lines i and j, respectively. Genetic distance may range between 0 (all bands in common) to 1 (no bands in common). The similarity matrix was submitted for hierarchical cluster analyses of unweighted pair group using arithmetical average (UPGMA) method and necessary computation were performed using NTSYS-pc program (Rohlf, 2000). The correlation between SCA and GD, as well as, HPH and GD for grain yield was calculated as Spearman s rank correlation coefficient. Results and discussion The grain yield was significant for genotypes (F1 and inbred lines per se) and environments in both plant densities. Genotypes x environment interaction were significant in higher plant densities for F1 crosses as well as for inbred lines per se, and for F1 crosses in lower plant densities (data not shown). GCA, as well as SCA was significant for grain yield in both plant densities and years. RE were significant in lower densities in both years, and in higher densities in 2003 (Table 1). SCA was more important than GCA for grain yield, indicating that yield is mainly controlled by nonadditive gene action in F1 crosses. In lower densities, inbred lines ZPL 680 had highest and significant GCA value in 2003 and ZPL 357/3 in 2004 (Table 2). In higher densities inbred line ZPL 142 had highest and significant GCA value in 2003 and ZPL 357/3 in Obviously, inbred line ZPL 357/3 shows the best GCA value in favourable conditions. In the same time, inbred line ZPL 357/3 provided the highest grain yield per se in both densities, as well as in years (Table 3). The highest HPH for grain yield is obtained in combination of inbred lines with lower grain yield per se (ZPL 680 x ZPL 17/5) and vice versa, lower HPH in combination of inbred lines with higher grain yield per se (ZPL 142 x ZPL 357/3). All inbred lines had specific RAPD phenotypes. PCR amplification of genomic DNA with 10 RAPD primers gave totally 42 RAPD fragments of different molecular weight, which of 71 % were polymorphic. The results of our study agree with other reports (Amorim et al., 2003; Subramanian and Subbaraman, 2004) that the level of polymorphism for RAPD markers is high in maize. Based of the presents/absence of amplified DNA fragments, coefficient of similarity (Nei and Li) was calculated within pairs of genotypes. The highest genetic distance was established between inbred lines ZPL 142 and ZPL 173/3 and lowest between ZPL 17/5 and ZPL 173/3, as expected due to their pedigree (Table 4). The cluster analysis based on genetic distance for RAPD data classifies six inbred lines into two principal heterotic groups (Fig 1) rd Croatian and 3rd International Symposium on Agriculture

3 Prediction of Combining Ability and Heterosis Using RAPD Markers The first cluster encompasses inbreds non related to BSSS germplasm (A) and the second consists of inbred related to BSSS germplasm (B). In the first group lines ZPL 15/5 and ZPL 173/3 form one subgroup and inbred ZP 275/3 is loosely linked to this subgroup. ZPL142 and ZPL 357/3 belong to subgroup II with ZPL 680 loosely linked to them. The correlation between SCA and GD, as well as, between HPH and GD for maize grain yield was established by Spearman s rank correlation coefficient (r s ). Genetic distance was positively correlated with SCA and HPH in all environments. Higher correlation was estimated in higher density for both years, except for correlation between HPH and GD in 2004 (Table 5). The results of our study are in agreement with results of other studies, which obtained middle strong or weak, positive correlation between SCA and HPH for grain yield and genetic distance based on molecular markers. (Melchinger, 1990; DrinićMladenović et al., 2002). Table 1. Mean squares for general (GCA) and specific (SCA) combining ability, reciprocal effects (RE) for grain yield from a diallel mating set among six maize inbreds Component 44,640 plants ha -1 64,935 plants ha GCA 10.17** 8.30** 11.19** 8.65** SCA 31.53** 66.87** 36.47** 76.16** RE 3.72** 1.63* 2.38** Error * Exceeded twice the standard error Table 2. General combining abilities (GCA) for grain yield from diallel matting set among six maize inbred lines Inbred 44,640 plants ha -1 64,935 plants ha ZPL * * ZPL 275/ * 0.286* * ZPL * ZPL 17/ * * * * ZPL 357/ * 0.315* 0.460* ZPL 173/ * Indicates significance of correlations at P < ** Indicates significance of correlations at P < Table 3. The grain yield heterosis of F1 crosses (%). Genotype Grain Yield ZPL 142 ZPL 275/3 ZPL 680 ZPL 17/5 ZPL 357/3 ZPL ZPL 275/ ZPL ZPL 17/ ZPL 357/ ZPL 173/ Table 4. Genetic distance among inbred lines based on RAPD markers. Genotype ZPL 142 ZPL 275/3 ZPL 680 ZPL 17/5 ZPL 357/3 ZPL 275/ ZPL ZPL 17/ ZPL 357/ ZPL 173/ Genetics, Plant Breeding and Seed Production 337

4 Aleksandar RADOJČIĆ, Goran DRINIĆ, Snežana DRINIĆ MLADENOVIĆ, Milomir FILIPOVIĆ Table 5. Spearman s rank correlation coefficient between SCA and GD (rs) and between HPH and GD (rs2) Years Densities rs1 rs ,640 plants ha ** 0.56* 64,935 plants ha ** 0.79** ,640 plants ha ** 0.56* 64,935 plants ha ** 0.54* * Indicates significance of correlations at P < ** Indicates significance of correlations at P < Figure 1. Dendogram based on genetic distance from RAPD data. Conclusion 1. The genetic distance revealed by RAPD markers can be used to establish consistent heterotic patterns between maize inbred lines. 2. There are middle, positive, and significant correlations between the genetic distances estimated by RAPD markers and the productivity of hybrids from the 6 maize lines under study. 3. Based on obtained results it could be concluded that RAPD markers can be used for genetic divergence analysis of maize inbred lines, but their use for prediction of heterosis is still limited. References Amorim E.P., C.C. de Souza Almeida, Melo Sereno M.J.C., Bered F., Barbosa Neto J.F. (2003): Genetic Variability in Sweet Corn Using Molecular Markers. Maydica, 48: Drinić Mladenović, S., S. Trifunović, G. Drinić, K. Konstantinov (2002): Genetic diversity and its correlation to heterosis in maize as revealed by SSR-based markers. Maydica, 47: 1-8. Griffing, B. (1956): Concept of general and specific combining ability in relation to dialel crossing sistems. Australian Journal of Biological Sciences, 9: Hallauer A.R., W.A Russel, K.R.Lamkey (1988): Corn breeding. In Sprague GF., Dudlay JW (eds). Corn and corn improvement 3 rd. Am Soc. Agron.Madison WI, pp Melchinger A.E., M. Lee, K.R. Lamkey, W.L. Woodman (1990): Genetic diversity for restriction fragment length polymorfisms: relation to estimated genetic effect in maize inbreds. Crop Sci., 30: Melo, W.M.C., Pinho, R.G.V., Santos, J.B. dos, Ferreira, D.F. (2001): Utilização de caracteres morfoagronômicos e marcadores moleculares para avaliação da divergência entre híbridos de milho. Revista Ceres, 48: Nei M., W.H. Li (1979): Mathematical model for studying genetic variation in terms of retriction endonucleases. Proc. Natl. Acad. Sci., 76: rd Croatian and 3rd International Symposium on Agriculture

5 Prediction of Combining Ability and Heterosis Using RAPD Markers Pinto, R. de M.C., C.L. de Souza Jr., L.A. Carlini-Garcia, A.A.F. Garcia, A. Pereira de Souza (2003): Comparison between molecular markers and diallel crosses in the assignment of maize lines to heterotic goups. Maydica, 48: Rohlf, F.J. (2000): NTSYS-pc. Numerical taxonomy and multivariate analysis system. Version 2.0 Exeter Software, Setaket, N.Y. Sagai-Maroof M.A., K.M. Soliman, R. Jorgenson, R.W. Allard (1984): Ribosomal DNA spacer length polimorphism in barley: Mendelian inhritance, chromosomal location and population dynamics. Proc.Natl. Acad. Sci. USA, 81: Subramanian A., Subbaraman N. (2004): Molecular Marker Based Validation of Genetic Relatedness Among Maize (Zea Mays L.) Inbred Lines and Its Influence on Heterosis. Tropical Agricultural Research, 16: Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. Tingey, S.V. (1990): DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: sa2008_0317 Genetics, Plant Breeding and Seed Production 339