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Kasetsart J. (Nat. Sci.) 46 : 515 - 521 (2012) Inheritance of some Flower Traits in Patch Petal Dendrobium Orchids Surawit Wannakrairoj* and Chakkrapong Rattamanee ABSTRACT Dendrobium is the most important tropical orchid. Patch coloration on petals is a novel trait for cultivar improvement of the genus. The trait was obtained from somaclonal variances of both cutflower and potted plant cultivars. Most of the variance is male-sterile. Hybrids between the variants and the wild-type cultivars yielded some progenies with patch petals. Genetic analysis on the inheritance of some flower traits of the variance was performed. Only a cross between both parents with patch petals could be produced from Dendrobium Anucha Flare 'Suriyon' × D. Ekapol 'Annie', the only male-fertile mutant. From the segregating populations, it was proposed that a dominant M allele controls the pinkish purple coloration and a recessive m allele controls the white coloration on sepals. For petal color, a dominant C allele was confirmed to control the pinkish purple and a recessive c allele controls the white. Importantly, dominant alleles in five loci were found to be essential for the petal patch coloration in this study. In addition, it was postulated that the El allele controls the enlarge labellum while the el allele controls the normal labellum. Keywords: genetics, color, petal, sepal, labellum INTRODUCTION Orchids are the most important floricultural crop of Thailand. Innovation is the key element for the continuous success of the industry. From the beginning, multi-gene recombination via hybridization has been proved to be the most promising tool for diversity creation (National Orchid Board, 2011). To execute an effective hybridization program, a basic knowledge of the genetics or inheritance of the traits of interest is essential. Without a good knowledge foundation on the inheritance of the traits of interest, breeding success is very difficult to attain. Flower color is one of the most important traits of orchids; a number of reports have thus been published on the inheritance of the trait (Curtis and Duncan, 1942; Mehlquist, 1958; Storey and Kamemoto, 1960; Kamemoto and Amore, 1990; Vajrabhaya and Vajrabhaya, 1996; Kamemoto et al., 1999). The genus Dendrobium is the most economically important tropical orchid (Kamemoto et al., 1999). For genetic study on dendrobium flower color, Kamemoto and Amore (1990) revealed that a single P allele controlled semi-alba while a recessive p allele controlled the alba flower in the Phalaenanthe section of the genus Dendrobium. They also reported that complementary loci, C and R, were responsible for the purple flower. The semi-alba labellum was proposed to be under the control of an independent P allele. Later, cyanidin was found to be the main anthocyanin in most Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author, e-mail: [email protected] Received date : 06/12/11 Accepted date : 28/06/12 516 Kasetsart J. (Nat. Sci.) 46(4) colored flowers of dendrobium orchids (Kuehnle et al., 1997). However, Vajrabhaya and Vajrabhaya (1996) reported that the genes controlling albinism in dendrobium orchids were similar to those in Cattleya. They also proposed a K allele for the purple keel. Interestingly, the color of sepals and petals of a dendrobium flower which belong to a separate floral whirl has not been studied independently. Besides recombinant traits from hybridization, orchid somaclonal variances that have arisen from micropropagation procedures provide many novel genetic variations (Vajrabhaya, 1977). Patch coloration on petals is a novel trait in orchids. It was first found in a Dendrobium Ekapol ‘Red’ cut-flower farm in Nakhon Pathom, Thailand. The mutant was named D. Ekapol ‘King Dragon’. The patch coloration is heritable and desirable by purchasers of potted plants. Hybridizers thus need to understand the inheritance of traits in order to set up a successful breeding program. However, only a few crosses have been made using the mutant due to its male-sterility. The sterility had also made the genetic study of the trait impossible. Recently, a partial male-fertile mutant with patch coloration on petals from D. Ekapol ‘Anna’ was obtained in a cut-flower farm of Intuwong Nursery, Nakhon Pathom, Thailand. Subsequently, the current study has used the new mutant, Dendrobium Ekapol ‘Annie’, to generate hybrid progeny with D. Ekapol ‘King Dragon’ for genetic analysis to reveal the inheritance of the patch coloration on orchid petals for the first time. In addition, alleles controlling a sepal color and an enlarged labellum were proposed. Genotypes on sepal and petal colors of the parental clones were also determined. MATERIALS AND METHODS Dendrobium Ekapol (D. Lim Hepa × D. Tomie Drake) is a tetraploid complex hybrid. D. Ekapol ‘Annie’, a mutant of D. Ekapol ‘Anna’ with dark reddish purple patch on both longitudinal sides of pinkish purple petals, was used as a pollinia parent (Figure 1a). The pollinium of this parent was kindly provided as a research gift by Intuwong Nursery. D. Anucha Flare (D. Ekapol ‘King Dragon’ x D. Suriyon Stripe) ‘Suriyon’, with a dark reddish purple patch on both longitudinal sides of light pinkish purple petals and enlarged side-lobe labellum, was used as a pod parent (Figure 1b). This pod parent was purchased from the Chatuchak Market, Bangkok. Both parents, which could be classified to be in the Phalaenanthe section, were hypothesized to be heterozygotic for patch coloration on the petals since D. Ekapol ‘Annie’ was a mutant in which the original cultivar had never produced any progeny with patch petals even from the selfing population. D. Anucha Flare ‘Suriyon’, on the other hand, was a hybrid between a normal and a patch flower. The seed pod of the cross was harvested at the mature-green age of 100 d. The seeds were sown aseptically in a Thai commercial laboratory. When the seedlings were ready to be deflasked, they were transferred (in the same shade house as the one used to cultivate the parental cultivars) Figure 1 Flower of: (a) Dendrobium Ekapol ‘Annie’; and (b) Dendrobium Anucha Flare ‘Suriyon’. Kasetsart J. (Nat. Sci.) 46(4) to thumb-pots (5 cm in diameter) containing coconut fiber, with approximately 50% shade. All seedlings had been grown in a commercial nursery near Bangkok. The hybrid seedlings were watered daily and applied with systemic pesticides as needed. Foliar fertilizer (1 g.L-1), was applied every Monday, Wednesday and Friday throughout the experiment. During the rainy months, 1818-18 fertilizer (Gaviota®) was applied every Wednesday while 13-27-27 fertilizer (Gaviota®) was applied every Monday and Friday. On the other hand, during the dry months, 18-18-18 fertilizer (Gaviota®) was applied every Wednesday while 13-27-27 fertilizer (Gaviota®) was applied every Monday and Friday. Only 105 seedlings were successfully grown to maturity. Upon blooming at the age of 18-24 mth after potting, sepal coloration, petal coloration and the labellum shape of each hybrid 517 plant were recorded twice to confirm the penetrant of the traits. A chi-square test was performed to verify all possible hypotheses on the number of genes involved, starting from the lowest number possible. RESULTS AND DISCUSSION Coloration of sepals From the 105 plants, the sepals of all plants had normal shape. Moreover, the color and shape of the sepals, petals and labella did not change over time. Thus, this indicated the qualitative fashion of inheritance. In addition, their sepal color could be categorized into two discrete groups. There were 48 plants with light pinkish purple sepal color (Figure 2a) and 57 plants with pinkish purple sepal color (Figure 2b) (Table 1). Figure 2 Flower color from Dendrobium Anucha Flare ‘Suriyon’ × D. Ekapol ‘Annie’ cross: (a) Light pinkish purple sepals and petals; and (b) Pinkish purple sepals and petals. Table 1 Color of sepals and petals of Dendrobium Anucha Flare ‘Suriyon’ × D. Ekapol ‘Annie’ progeny. Trait Number of plants Chi-square Sepal color light pinkish purple 48 pinkish purple 57 0.77 Î P = 0.25–0.5 Petal color light pinkish purple 51 pinkish purple 54 0.09 Î P = 0.75–0.9 Patch coloration absent 73 present 32 2.26 Î P = 0.25–0.5 518 Kasetsart J. (Nat. Sci.) 46(4) From the observed data, the possible hypothesis would be that the sepal colors in this cross should be under the control of a single locus since the observed ratio was 1:1 (with a statistically acceptable chi-square value of 0.61). Since the petal is the main part contributing to the color of the flower of the Phalaenanthe section and the sepal color did not co-segregate with the petal color, the locus controlling sepal color should be different from the one controlling flower color designated by Kamemoto and Amore (1990). It was proposed that a dominant M allele controls the pinkish purple coloration (presence of cyanidin pigment) on sepals and a recessive m allele controls the white coloration on sepals. The M allele was incompletely dominant over the m allele. Under the chromosome theory of inheritance or random chromosome assortment (Allard, 1960; Briggs and Knowles, 1967), the hybrid with pinkish purple sepals would be the duplex MMmm genotype while that with light pinkish purple sepals would be the simplex Mmmm genotype. Thus, D. Ekapol ‘Annie’ which had pinkish purple sepals should be the triplex MMMm genotype. On the other hand, D. Anucha Flare ‘Suriyon’ which had white sepals should be the nulliplex mmmm genotype. Coloration of petals From the progeny, it was found that the color of petals could be categorized into two groups. There were 51 plants with light pinkish purple petal color (Figure 2a) and 54 plants with pinkish purple petal color (Figure 2b, Table 1). The petal colors in this cross should be under the control of a single locus since the observed ratio was 1:1 (supported by the chi-square test result). Following the general concept of orchid color inheritance (Arditti and Fisch, 1977), which was confirmed in dendrobiums (Kamemoto and Amore, 1990), it was postulated that a dominant C allele controls the pinkish purple (or mauve) coloration on petals and a recessive c allele controls the white coloration (lack of anthocyanin pigment) on petals. The C allele was incompletely dominant over the c allele. In the same manner of random chromosome assortment as in the sepals, the hybrids with pinkish purple petals would be the duplex CCcc genotype while those with light pinkish purple petals would be the simplex Cccc genotype. The low expressitivity of the trait for color variation among simplex genotypes might indicate the involvement of modifying genes. Thus, D. Ekapol ‘Annie’ which had pinkish purple petals should then be a triplex CCCc genotype. On the other hand, D. Anucha Flare ‘Suriyon’ which had white petals should be a nulliplex cccc genotype. The dark reddish purple patch found in D. Anucha Flare ‘Suriyon’, the pod parent, was inherited from D. Ekapol ‘King Dragon’, while the dark reddish purple patch found in D. Ekapol ‘Annie’, the pollinia parent, led to an elongation of petals. For the patch coloration on the petals of progeny, it was found that there were 73 plants with normal coloration (Figure 3a) and 32 plants with dark reddish purple or yellowish green patch coloration (Figure 3b, Table 1). Since there was segregation of the patch petals in the progeny, the trait must be dominant. Under the general assumption that a mutation is a single allele change (Allard, 1960), both D. Ekapol ‘King Dragon’ and D. Ekapol ‘Annie’ thus ought to contain one mutant allele result from somaclonal variation. The P allele was then postulated to control the patch coloration while the p allele was postulated to control the non-patch coloration. D. Anucha Flare ‘Suriyon’ and D. Ekapol ‘Annie’ should then be heterozygotes of Pppp. Under this postulation, the segregation ratio of the progeny should be 3:1. However, the chi-square test result rejected this hypothesis. The phenomenon that the dominant trait presents at a lower proportion than the recessive trait in the segregating population indicated that it is an oligogenic trait. Moreover, the interallelic Kasetsart J. (Nat. Sci.) 46(4) gene interaction should be duplicate recessive epistasis. Furthermore, if a random chromosome assortment played a role in gamete production, the dominant ratio in a cross between simplex parents would be (3/4)n, where n is the number of loci controlling the trait (Allard, 1960; Briggs and Knowles, 1967). A chi-square test was conducted to determine the minimum number of loci involved. The test result of the 32:73 observed ratio (a chi-square value of 2.26) statistically conformed to a 243:781 ratio. This meant that there are five loci necessary for the petal patch coloration in this study. Thus, D. Ekapol ‘Annie’ and D. Anucha Flare ‘Suriyon’ should be of P 1p 1p 1p 1P 2p 2p 2p 2P 3p 3p 3p 3P 4p 4p 4p 4P 5p 5p 5p 5 genotypes. This is possible because D. Anucha Flare ‘Suriyon’ is a hybrid of D. Ekapol ‘King Dragon’ which is a somaclonal variant of D. Ekapol ‘Red’. D. Ekapol ‘Red’ is a sibling of D. Ekapol ‘Anna’ which is the origin of D. Ekapol ‘Annie’. Moreover, there is only one other somaclonal variant that has petal patch coloration which might indicate the rarity of the accumulation of five dominant mutant alleles in a Dendrobium plant. D. Ekapol ‘Red’ and 519 D. Ekapol ‘Anna’ would have four dominant alleles. For example, their genotypes would be P1p1p1p1P2p2p2p2P3p3p3p3P4p4p4p4 p5p5p5p5 if the mutation occurred at the P5 locus. To further verify the validity of this postulate in the future, due to the small population size in the current study, testcrossing of the progeny between the mutants and their original cultivars should be made. In order to confirm the postulate, the progeny is expected to statistically yield a ratio of 81:431 of patch to normal petals when more than 1,024 plants are observed. Labellum shape In the hybrid population, the labellum shapes could be categorized into two discrete groups. There were 88 plants with normal labella similar to that of D. Ekapol ‘Annie’ (Figure 3a) and 17 plants with enlarged labella similar to that of D. Anucha Flare ‘Suriyon’ (Figure 3b, Table 2). Since the enlarged labellum showed up only in the progeny with the patch petals, it indicated that the trait was hypostatic to the P1____P2___ P3___P4___P5___ genotypes. Moreover, the trait should be dominant due to its presence in the first Figure 3 Flower shape of the Dendrobium Anucha Flare ‘Suriyon’ × D. Ekapol ‘Annie’ cross: (a) Normal petals and labellum; and (b) Patch petals and enlarge labellum. Table 2 Labellum shape of Dendrobium Anucha Flare ‘Suriyon’ × D. Ekapol ‘Annie’ progeny. Trait Number of plants Chi-square Labellum shape normal 88 (73 + 15) enlarged 17 0.13 Î P = 0.5–0.75 520 Kasetsart J. (Nat. Sci.) 46(4) generation hybrid. Considered among the patch petal group, the ratio of normal labella to enlarged labella was 15:17 which agreed well with a theoretical ratio of 1:1 (a chi-square value of 0.125) and indicated that there is a single locus controlling the trait. With a random chromosome assortment, D. Anucha Flare ‘Suriyon’ should be a simplex assortment while D. Ekapol ‘Annie’ should be a nulliplex assortment. It was postulated that the El is the allele controlling the enlarged labellum while the el is the recessive allele controlling the normal labellum. Thus, D. Anucha Flare ‘Suriyon’ should be a simplex Elelelel genotype and D. Ekapol ‘Annie’ should be a nulliplex elelelel genotype. The enlarge labellum in Dendrobium was first observed in D. Ekapol ‘King Dragon’. The trait seems to be linked to male-sterility in the cultivar and its progeny. The enlarged labella were somewhat similar to the “normal-lip” progeny between the wild type and the pansy-lip shown by Kamemoto et al. (1999). However, the gene controlling the pansy-lip in D. D’Bush Pansy was differently reported to be a recessive allele while the “normal-lip” was proposed to be a dominant trait (Amore and Kamemoto, 1997). The enlarged labellum in this study may be considered to be a peloria, derived from a Greek word for monster (Slump, 2005). In Phalaenopsis orchids, the labellum peloria was reported to be the expression of the PeMADS4 gene in B class under the ABCDE model, a member of DEF-like MADS-box genes (Wen-Chieh, 2004; Tsai et al., 2008). The el allele in D. Ekapol ‘King Dragon’ and its blood line is thus likely to be in the B class gene of the ABCDE model since it is linked to male-sterility which is also under the control of the B class gene (Tsai et al., 2008). It should also be noted that there was some degree of variation in labellum enlargement in the current study. The expressitivity likely indicated the involvement of modifying genes. However, Rudall and Bateman (2003) reported that the genetic relationships between peloric and pseudopeloric flowers in flowering plants were ambiguous. LITERATURE CITED Allard, R.W. 1960. Principles of Plant Breeding. John Wiley & Sons. New York, USA. 485 pp. Amore, T.D. and H. Kamemoto. 1997. Inheritance of pansy-lip in Dendrobium. Lindleyana 12: 12–15. Arditti, J. and M.H. Fisch. 1977. Anthocynins of orchidaceae: Distribution, heredity, functions, synthesis, and localization, pp. 117–155. In J. Arditti, (ed.). Orchid Biology. Reviews and Perspectives, I. Cornell University Press. London, UK. Briggs, F.N. and P.F. Knowles. 1967. Introduction to Plant Breeding. Reinhold Publishing Corporation. New York, USA. 426 pp. Curtis, J.T. and R.E. Duncan. 1942. The inheritance of flower color in Cattleya. American Orchid Society Bulletin 10: 283–286. Kamemoto, H. and T.D. Amore. 1990. Inheritance of semi-alba and alba in Dendrobium, pp. 242–244. In J. Kernohan, D.G. Bonhan, N. Bonhan and L. Cobb, (eds.). Proceedings of the 13th World Orchid Conference. 13th World Orchid Conference Proceedings Trust. Auckland, New Zealand. Kamemoto, H., T.D. Amore and A.R. Kuehnle. 1999. Breeding Dendrobium Orchids in Hawaii. University of Hawai’i Press. Honolulu, USA. 166 pp. Kuehnle, A.R., D.H. Lewis, K.R. Markham, K.A. Mitchell, K.M. Davies and B.R. Jordan. 1997. Floral flavonoids and pH in Dendrobium orchid species and hybrids. Euphytica 95: 187–194. Mehlquist, G.A.L. 1958. Genetics and orchid breeding, pp. 204-207. In Proceedings of the 5th World Orchid Conference. Harvard University Printing Office. Cambridge, MA, USA. Kasetsart J. (Nat. Sci.) 46(4) National Orchid Board. 2011. Competitive Strategy for Thai Orchid in the World Market. Ministry of Agriculture and Cooperative. Thailand. 72 pp. [in Thai] Rudall, P.J. and R.M. Bateman. 2003. Evolutionary change in flowers and inflorescences: Evidence from naturally-occurring angiosperm tetra. Trends Plant Sci. 8: 76–82. Slump, K. 2005. Monstrous Orchids. Orchids 74: 334–335. Storey, W.B. and H. Kamemoto. 1960. Inheritance of semi-alba in a Cattleya pedigree. American Orchid Society Bulletin 29: 24–29. Tsai,W.-C., Y.-Y. HSIAO, Z.-J. Pan, C.-C. Hsu, Y.-P. Yang, W.-H. Chen and H.-H. Chen. 2008. Molecular biology of orchid flowers: with emphasis on Phalaenopsis. Advances in Botanical Research 47: 99–145. 521 Vajrabhaya, T. 1977. Variations in clonal propagation, pp. 177–201. In J. Arditti, (ed.). Orchid Biology. Reviews and Perspectives, I. Cornell University Press. London, UK. Vajrabhaya, M. and T. Vajrabhaya, 1996. Inheritance of albinism in Dendrobium orchids. J. Soc. Sci. Thailand 22: 173–180. Wen-Chieh, T., K. Chang-Sheng, C. MingHsiang, C. Wen-Huei and C. Hong-Hwa. 2004. Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid. Plant Cell Physiol. 45: 831–844.