Research Article

Classical and Modern Methods for Characterization of Ornamental Crops  

S.K. DATTA1 , D. Chakrabarty2
1. Retd. Scientist ‘G’CSIR-NBRI, Lucknow and Ex CSIR Emeritus Scientist, Bose Institute, Kolkata, India
2. Scientist, Plant Molecular Biology & Genetic Engineering Division, National Botanical Research Institute, Lucknow, India
Author    Correspondence author
International Journal of Horticulture, 2015, Vol. 5, No. 21   doi: 10.5376/ijh.2015.05.0021
Received: 07 Aug., 2015    Accepted: 09 Sep., 2015    Published: 19 Aug., 2016
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Datta S.K., and Chakrabarty D., 2015, Classical and modern methods for characterization of ornamental crops, International Journal of Horticulture, 5(21): 1-45 (doi: 10.5376/ijh.2015.05.0021)

Abstract

Cultivar identification and cultivar relatedness are important issues for horticultural breeders. Characterization is most important for correct identification of plants. It helps to understand the genetic diversity, to trace out the phylogenetic relationship, taxonomical status, registration, plant variety protection, farmer’s right etc. A number of classical and advanced methods comprising different parameters of cytology, morphology, physiology, phenolic compounds, DNA markers etc. are utilized. Every method has its merits and demerits. Considering the limitations of each technique one should plan the characterization program on need basis. Attempts have been made to highlight the application of all the techniques by different authors on important ornamental crops.

Keywords
Floriculture; Characterization; Genetic diversity; Classical methods; Advanced methods

Introduction          

Ornamental crops i.e. floriculture has become a very important industry in many countries as a result of science based techniques and steady supply of new, novel and improved plant materials. Ornamentals are recognized as a vital part of world’s natural heritage. The chapter will highlight the basic and applied aspects and technological advancement for characterization of ornamentals. Ornamentals are one of the most important groups of plants in the plant kingdom. Characterization is most essential for correct identification of plants in addition to other requirements. Considering the importance of characterization and identification of plants a number of classical and modern techniques have been developed and intense R&D activities are going on this subject in different areas all over the world in universities, research institutions, industrial research centers and specialized biotechnology disciplinary area on high scientific excellence. There are no universal parameters and rules for characterization. Characterization system depends mainly on the type of crop and more importantly on the objective of characterization. Cultivar identification and cultivar relatedness are important issues for horticultural breeders. One major problem in the floriculture industry is that breeders do not disclose the parentage of new hybrids. There are a number of classical methods for testing hybridity using cytological and biochemical parameters.

 

Taxonomy plays a major role to identify plants mainly on the basis of morphological characters besides cytological and chemical characters. There are crop specific both national and international societies for registration of new varieties. Each society has specified instruction and character list to be fulfilled for registration. New plant varieties can be protected under patent law (Anonymous, 1983; Anonymous, 1998; Duffett and Benten, 1995; Martin and Robert, 1995; Rick et al., 2001; Riek, 2001; Warren, 1997; Camlin, 2001). There is International Union for the Protection of New Varieties of Plants (UPOV), Geneva. According to Article 7 of the 1961/1972 and 1978 Acts and Article 12 of the 1991 Act of the UPOV Convention, protection can only be granted in respect of a new plant variety after examination of the variety has shown that it complies with the requirements for protection laid down in those Acts and, in particular, that the variety is distinct (D) from any other variety whose existence is a matter of common knowledge at the time of the filing of the application  and that it is sufficiently uniform (U) and stable (S), or “DUS” in short. The examination, or “DUS Test,” is based mainly on growing tests, carried out by the authority competent for granting plant breeders’ rights or by separateinstitutions, such as public research institutes, acting on behalf of that authority or, in some cases, on the basis of growing tests carried out by the breeder. The examination generates a description of the variety, using its relevant characteristics (e.g. plant height, leaf shape, time of flowering), by which it can be defined as a variety in terms of Article 1(vi) of the 1991 Act of the Convention. UPOV has developed internationally recognized “Guidelines for the Conduct of Tests for Distinctness, Uniformity and Stability,” or “Test Guidelines” for the harmonized examination of new crop specific varieties for protection. The individual crop specific Test Guidelines have been prepared by the appropriate Technical Working Party, which is composed of government appointed experts from each member of the Union with invited experts from other interested States and observer organizations. The protocol describes the technical procedures to be followed in order to meet the Council Regulation 2100/94 on Community Plant Variety Rights. A very eventful development in India in Indian agriculture has been the clearance of the protection of Plant Varieties and Farmer’s Rights Act 2001 by Lok Sabha on 9th August 2001. The act envisages the establishment of an effective system for the protection of plant varieties and the rights of farmers and plant breeders. The legislation was necessitated by the commitments that India made in the agreement on Trade Related Intellectual Property Rights (TRIPS) when it ratified the Uruguay GATT Round in 1994. The formulation of rules and regulations is in progress. Guidelines are being prepared for DUS testing - crop wise. Initially a list of 35 species was short-listed. Under ornamentals following species were identified-Rose (Rosa spp.), Chrysanthemum spp., Jasmine (Jasminum spp.), Marigold (Tagetes erecta), Gladiolus sp. and Orchids (Anonymous, 2002; Ganguli, 1998; Prasad and Bhatnagar, 1996; Datta, 2000; Datta, 2006; Brahmi et al., 2004) (Several genera).Author (S.K.Datta) has developed more than 100 new promising ornamental varieties through conventional breeding and induced mutagenesis. All have been registered to crop specific societies, reported to international data base at IAEA, Vienna and patented.Chrysanthemum cv. “Mother Teresa” got US Patent (Patent No. PP13678). Gamma ray induced four mutant rose varieties have been registered with the International Registration Authority For Roses (IRAR), the American Rose Society, USA (Ref. American Rose Annual, 1985, pp. 197, 201, 205).Five gamma ray induced bougainvillea mutant varieties have been registered to the International Bougainvillea Registration Society, Division of Floriculture & Landscaping, Indian Agricultural Research Institute, New Delhi, India. New Dahlia ‘NBRI’S PINKI’, developed through conventional breeding, has been registered with The Royal Horticultural Society, International Registration Authority for the genus Dahlia. All the gamma ray induced new ornamental varieties are available in Mutant Database, International Atomic Energy Agency, Vienna. All required characterization information has been documented.

 

Every technique for characterization has its advantage, disadvantage and limitations. For the last three decades one of the authors (S.K.Datta) applying a number of parameters like morphological (vegetative and floral characters), cytological (chromosome number, chromosomal behavior, karyotype etc.), anatomical (number of stomata and size, number of chloroplast per guard cell, hair structure etc.), palynological (pollen grain sterility, size, ornamentation pattern, pollen grain germination etc.), biochemical (phenolic compounds in leaves and petals, pigment composition studies using TLC, spectrophotometer etc.) and molecular (RAPD) for characterization of germplasm and new varieties of ornamentals developed through conventional breeding, sport and induced mutagenesis(Chatterjee et al., 2005; Chatterjee et al., 2006; Chatterjee et al., 2007; Chakrabarty et al., 2007; Datta, 1985, Datta, 1986, Datta, 1987; Datta, 1993; Datta, 1999; Datta, 2004; Datta and Banerji, 1995; Datta and Chatterjee, 2006; Datta and Datta, 1998; Datta and Gupta, 1981a; Datta and Gupta, 1981b; Datta and Gupta, 1983; Datta and Shome, 1994; Datta and Tandon, 1994; Datta and Singh, 1995; Datta and Singh, 1999; Datta and Singh, 2001; Datta and Singh, 2002;Datta and Singh, 2003; Datta and Singh, 2004; Datta and Singh, 2005; Datta and Singh, 2006; Gupta and Datta, 2005; Singh and Datta, 1998; Singh and Datta, 1999; Singh and Datta, 2000; Laxmi et al., 1984). Every character plays important role for proper identification of plant variety. A wide range of ornamental crops i.e. Amaryllis, Bougainvillea, Chrysanthemum, Dahlia, Gerbera, Gladiolus, Marigold, Rose, Tuberose, Lily etc. were included in the crop improvement program in general and characterization in particular. Attempt will be made to review the characterization work carried out by the authors on Amarylis, Bougainvillea, Chrysanthemum and rose. Citation of other related works on important ornamentals will be strictly restricted only on those which are most essential to understand the importance of characterization studies.

 

Morphologically most of the ornamentals display a great diversity of types. Ornamental plants include a heterogeneous and numerous groups of species with different reproducing systems, although mainly vegetative propagated. The majority of them, including the most important cut flowers and potted plants are almost unknown at the genetic level. Most of the ornamentals are landraces, cultivated in nursery, basically grown for self-consumption or sale in markets. Proper characterization of these germplasm is needed in order to be useful for breeders and farmers throughout the world. Systematic characterizations and evaluation of plant genetic resources are prerequisites for the efficient use of material through conventional methods. Correct identification of germplasm new varieties is extremely important to protect plant breeder’s rights for commercial exploitation. The existence of a large number of cultivars, maintained by vegetative propagation reinforces the need of a reliable verification of cultivar identity for nurserymen and growers. One major problem in the floriculture industry is that breeders do not disclose the parentage of new hybrids. Therefore, there is need for accurate characterization system to trace out the parents of new varieties. The identification of cultivars or breeding lines is very important in all horticultural and agricultural species in order to protect the rights of plant breeders (Wolff et al., 1995).

 

Each cultivar of a cultivated crop species is expected to be unique in one or more specific characteristics. Mostly plants are characterized and identified on the basis of qualitative or quantitative gross morphological characters. Morphological, cytological and physiological attributes are normally considered as the primary criteria for differentiating cultivars, but these characteristics alone have not always proven satisfactory. A more objective method of identifying new cultivars was needed.

 

Various principles of taxonomic procedure developed from time to time regarding utilization of morphological characters for characterization. Vegetative characters of leaves, stems, buds and growth habit of plants were considered at the beginning for characterization. Leaf form, leaf apex, leaf margin, leaf petiole, leaf texture, phylotaxy, spines or prickles, leaf coloration etc. were considered to be more reliable for characterization (Datta, 1986, Datta, 1987; Datta, 1993; Datta, 1999; Datta, 2004; Datta and Banerji, 1995; Datta and Singh, 1995; Datta and Singh, 1999; Datta and Singh, 2001; Datta and Singh, 2002; Datta and Singh, 2003; Datta and Singh, 2004; Datta and Singh, 2005; Singh and Datta, 1998; Singh and Datta, 1999; Singh and Datta, 2000). But these vegetative characters were not stable due to environmental condition. Therefore, different features of reproductive organs (flowers and fruit) were included in plant characterization and found to be more reliable. But morphological characters were always found to be very important as it permit ready determination and correlation of characters. Plant genetic resources play an important role in generating new crop varieties with the high yield potential and resistance to biotic and abiotic stresses. Morphological, biochemical and molecular procedures are currently being employed in evaluating plant genetic resources. Many works based on morphological characters, cytology and enzyme electrophoresis have been used to study the diversity and phylogeny of different ornamental species. Diversity and relationships of different taxa in various groups of crop plants have been worked out earlier primarily on the basis of crossing and cytological data.

 

In addition to morphological characterization, molecular characterization is essential for elucidating the genetic relationships among the different groups of this species. It is imperative that a set of descriptors need to be developed to define the boundaries of a plant variety. The examination of a new variety (candidate variety) for DUS generates a description of the variety, using its relevant characteristics (morphological, cytological, biochemical and molecular) by which it can be described as a new variety.

 

Biochemical markers have received more attention as the data reflect more truly the genetic variability because they are the direct products of genes (Perry and McIntosh, 1991). Chemotaxonomy, the concept of classifying plants on the basis of their chemical constituents, is not new. However, at cultivar level little has been made of chemical markers, although their importance to plant taxonomy at species and higher levels has been well demonstrated. Many of the compounds which seem to have the potential for chemical markers are products of secondary metabolism. Flavonoids and other phenolic are ubiquitous in higher plants and have been more widely used in chemotaxonomy than any other groups of plant substances. Techniques most widely used for the separation of flavonoids from plant materials have been column, paper and Thin Layer Chromatography (TLC). Although good results have been obtained with these techniques, they are not easily adopted to quantization. High Pressure Liquid Chromatography (HPLC) has been found to be applicable to the analysis of complex natural mixtures of flavonoids for rapid screening and “fingerprinting” purposes. The use of these chemical markers as an adjunct to the chemical methods presently used for plant identification will provide a more positive identification of new cultivars, particularly those protected by the plant patent law.

 

Phenolic compounds have been found to be very useful for documentation of hybrids, detection of diploid genomes constituting polyploids, detection of mutants and in solving taxonomic problems. The use of phenolic compounds in taxonomic and genetic investigations has been reviewed earlier (Bose and Frost, 1967; Datta and Basu, 1976; Datta, 1987). Different types of phenolic compounds are present in leaves, flowers, stem, root and bark of plants. Distribution pattern of some of these compounds varies not only in different species but also in cultivars of the same species. Bose (1968) reviewed the use of phenolic compounds in taxonomic and genetic investigations. Thin layer chromatographic analysis of phenolic compounds in plant tissues led Smith and Levene (1963) to detect the presence of species substances in the polyploids, indicating the presence of different diploid genomes which constitutes the polyploids. Alston and Turner (1963) reported a number of species specific phenolic compounds in Baptisia from the chromatographic pattern. F1 hybrids with new hybrid substances were reported in Betula, Collinsia, Saxifraga, Dicentra and Vinca (Clausen, 1963; Garber and Strmnaes, 1964; Jaworska and Nybom, 1967; Fahselt and Ownbey, 1968; Mukherjee and Basu, 1973; Stebbins et al., 1963; Levin, 1966). Stebbins et al. (1963) and Levin (1966; 1967) demonstrated presence of all the parental compounds in F1 interspecific hybrid. Basu and Mukherjee(1975) demonstrated species and variety specific spots in Indian Spinach and such a study in addition to cytological and morphological investigations helped them in establishing the taxonomic status of Indian Spinach. Chemotaxonomic affinities were determined among the different species of Pennisetum by thin layer chromatographic compounds (Misra and Saran, 1964). This technique has also been successfully used in mutation breeding. Mutants are often associated with changes in biochemical characteristics which can be used as markers. Morphological mutants in fruit colour and shape have been induced in Trichosanthes anguina. Chromatographic analysis of phenolic compounds in leaves showed the presence of new spots in mutant (Datta, 1976). Chromatograohic analysis of seed extracts of control and MMS induced green seed coat colour mutant ofTrigonella foenum-graecum showed quantitative differences of phenolic compounds (Laxmi et al., 1983). Changes in phenolic compounds have been reported in somatic flower colour mutations in chrysanthemum (Datta, 1987; Datta and Gupta, 1981b; Datta and Gupta, 1983a, 1983b, 1983c; Datta, 1986; Datta, 1999; Datta and Singh, 1999).

 

Electrophoresis of crude proteins and enzyme extracts has been successfully used as an additional tool to establish these relationships. Protein and isozyme polymorphism has been successfully used for demonstrating genetic variation, identifying interspecific hybrids and fingerprinting cultivars (Cardy and Kannenberg, 1982; Chaparro et al., 1987; Ellstrand, 1984) Protein electrophoresis has provided a new approach to the problems of species relationships. A crude protein extract when fractionated on a suitable gel medium produces a spectrum of bands, which is diagnostic for the species. Homology among the bands of different species based on similarity in migration velocity provides a criterion of genetic affinity from which evolutionary relationship may be inferred. The homology of electrophoretic pattern of soluble proteins has been very extensively used in the study of intra- and interspecific relationships in many crop species (Cardy and Kannenberg, 1982; Chaparro et al., 1987; Ellstrand, 1984; Johnson, 1967; Cherry et al., 1971; Nainawatyee and Das, 1972).

 

Besides biochemical markers, DNA based markers provide powerful and reliable tools for discerning variation within crop germplasm and to study evolutionary relationships (Swofford and Olsen, 1990; Gepts, 1993). The term DNA fingerprinting was first used by Alec (1985) to describe bar-code-like DNA fragment patterns generated by multilocus probes after electrophoretic separation of genomic DNA fragments. Now-a-days DNA fingerprinting is used to describe continued use of several single locus detecting systems and presently DNA fingerprinting is used to analyze the various aspects of plant genus such as taxonomy, phylogeny, ecology, genetics and breeding in inter or intraspecific level. These techniques are being widely used to detect molecular variability within and among several species. During recent decades several techniques have been introduced that detect molecular variability within and among several crop species as well as the ornamentals. PCR based techniques have been used successfully in DNA fingerprinting of plant genomes and in genetic diversity studies. These techniques include RAPD (Randomly amplified polymorphic DNA), RFLP (Restriction Fragment Length Polymorphism), SSR (Simple Sequence Repeats) or microsatellites, STS (Sequence Tagged Sites), SNP (Single Nucleotide Polymorphism), VNTR (Variable number tandem repeat), STR (Short tandem repeat), SFP (Single feature polymorphism) and AFLP (Amplified fragment Length Polymorphism)(Nei, 1987; Williams et al., 1991; Loh, 1999; Barcaccia et al., 1999; Han et al., 1999; Escaravage et al., 1998; Visser, 1997; Dubouzet et al., 1997; Han et al., 2000; Picton and Hughes, 1997; Anastassopoulos and Keil, 1996; Tsuchiya et al., 1987; Welsh and MCclelland, 1990; Gebhardt and Salamini, 1992; Vos et al., 1995; Dubouzet et al., 1998; Walker and Werner, 1997; Wol et al., 1993; Arús, 2000; Debener 2001a, 2001b; Debener, 2004; Dosda and Baril, 2001; Anand, 2000; Anderson and Fairbanks, 1990; Bernatsky and Tanksley, 1989; Rajapakse et al., 1997; Rayburn et al., 1993; Rout, 2005; Rout and Mohapatra, 2006). Molecular markers have been developed for most ornamentals to a limited extent when compared to the main food crops. Applications have been essentially studies of cultivar identification, pedigree analysis or germplasm variability using isozyme or RAPD markers. These results opened new avenues for the development of useful markers for crops, like the ornamentals. RAPD technique has also been used in plants for the construction of genetic maps (Reiter et al., 1992), genotype identification, taxonomical studies etc. (Whitkus et al., 1994; Wolfe and Liston, 1998; Guang et al., 1996; Mehmood et al., 2008). In the last few decades, these new DNA molecular markers, based on the PCR technique, such as random amplified polymorphic DNA (RAPD)(Williams et al., 1990; Welsh and McClelland, 1990) and inter simple sequence repeats (ISSRs)(Zietkiewicz et al., 1994), among others, have become excellent tools for plant breeders(Hern´andez and Mart´ın, 2003). It is being widely used to amplify a specific segment of DNA by using random primers. One can compare the genetic makeup between different species of plants and even different varieties or cultivars of a species by using RAPD markers. The genetic diversities can be understood in more details by using this molecular biological technique.

 

In RAPD method, by using a single arbitrary primer (10 mer) and amplifying DNA by polymerase chain reaction (PCR), the resulting DNA markers can easily be separated on an agarose gel by electrophoresis (Williams et al., 1990). The advantages of RAPD are its simplicity, rapidity, the requirement for only a small quantity of DNA, and the ability to generate numerous polymorphisms (Cheng et al., 1997). Therefore, it has been a powerful technique for species identification, genetic analysis(Williams et al., 1990; Cheng et al., 1997; Tzuri et al., 1991;Kiss et al., 1993; Landry et al., 1993; Wight et al., 1993; Mohan et al., 1997; Chung et al., 2002; Lim et al., 2006) and for elucidation of genetic relationships of numerous plant species, and parentage testing (Fu et al., 2008; Williams et al., 1991; Halward et al., 1992; Keil et al., 1994; Novy et al., 1994; Kindiger and Dewald, 1996; Cjuric and Smith, 1996). RAPDs have been widely used for determining the genetic relationships between different related species and identification of cultivars (Hu and Quiros, 1991) and for estimating genetic relationships and diversity among crop germplasms (Thomas et al., 1993; Hallden et al., 1996). Many crop species as well as ornamental plants have been characterized using this procedure such as Chrysanthemum, roses, Alstroemeria L., Amaranthus, Hibiscus, Pelargonium, Poinsettia, Orchid and Sunflower (Wolff et al., 1995; Martin et al., 2002; Hubbard et al., 1992; Rajapakse et al., 1992; Torres et al., 1993; Vainstein and Ben Meir, 1994; Matsumato and Fukui, 1996; Ben-Meir and  Vainstein, 1994; Debener and Mattiesch, 1996; 1998; 1999a; 1999b; Debner et al., 1996; Reynders and Bollereau, 1996; Millan et al., 1996; Esselink et al., 2003; Dubouzet et al., 1997; 1998; Mandal and Das, 2002; Faseela and Salkutty, 2007; Zhou et al., 2002; Bakker et al., 2000; James et al., 2004; Lesur et al., 2001; Ling et al., 1997; Xiang et al., 2003; Chen et al., 2006). RAPD markers have been efficiently used in the routine assessment of variety identification and hybrid seed purity in Calycanthaceae (Zhou et al., 2007), Orchid (Xiang et al., 2003) and Calla lily (Hamada and Hagimori, 1996).

 

Recently use of molecular markers has been proposed for identification of animal diversity and demonstrated on a large scale through the use of a short DNA sequence in the cytochrome c oxidase 1 (CO1) gene (Hebert et al., 2003; 2004a, 2004b). Methods for identifying species by using short orthologous DNA sequences, known as ‘‘DNA barcodes,’’ have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. Therefore it has been proposed that the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants(Paul et al., 2005; Hogg and Hebert, 2003; Kress et al., 2005; Marshall and Will, 2005; Stoeckle, 2003; Godfray, 2002; Lipscomb et al., 2003). Engineered DNA sequences also have been suggested as exact identifiers and intellectual property tags for transgenic organisms (Gressel and Ehrlich, 2002).

 

1Materialsand Methods

1.1Plant Material

Attempts were made to review the most important works carried out on characterization by different workers on different ornamental crops. Authors have done extensive work on characterization of different ornamentals (germplasm and new varieties developed through conventional breeding and induced mutagenesis) at Floriculture Laboratory, National Botanical Research Institute (NBRI), Lucknow, India (Datta and Banerji, 1995; Datta, 1997; 1998a; 1998b, 2001). They used both classical and molecular methods. Therefore, materials and methods used by the authors will be highlighted elaborately. This will give the technical details of each technology used for characterization.

 

Hippeastrum (Fam. Amaryllidaceae, popular name Amaryllis) is an excellent bulbous plant bearing beautiful flowers. It usually flowers from mid-February to April, a period when there is a real scarcity of flowers in the northern plains of India. Only two species, Hippeastrum belladonna and Hippeastrum gracillis, formerly grew in plains at NBRI. A total of three varieties, two (‘‘Snow White’’ and ‘‘Firefly’’) under H. belladonna and one (‘‘Charm’’) under H. gracillis, which produce small-sized, bell-shaped flowers with a narrow colour range, are available on the plains. A large number of Dutch hybrids, both imported and available from different regions of India, which produce giant size blooms in a much wider range of colours were collected. Using these germplasms as baseline materials for a hybridisation program, NBRI has successfully developed new varieties through selective hybridisation and selection of natural hybrids for research purposes. No detailed information is available on the genetic aspects of the genus, and factors underlying its evolution are unclear. A total of 24 varieties were selected, of which 16 were Dutch hybrids, 3 were subtropical varieties, and 5 were hybrid varieties developed at NBRI for research purposes. Details of all varieties are shown in Table 1 and Figure 1.

 

 

Table 1 Hippeastrum cultivars included in RAPD analysis

 

 

Figure 1 Amplification profiles of 24 Hippeastrum cultivars with P3 (A) and P11 (B) primer

 

Bougainvillea (Fam. Nyctaginaceae) is one of the most important perennial ornamental shrubs, sometimes climbers, in tropical and subtropical gardens. NBRI, Lucknow is maintaining a rich Bougainvillea germplasm collection of approx. 180 species/cultivars comprising both single bracted and double or multi-bracted varieties. NBRI has detected, induced, isolated, established and commercialized a series of new varieties developed through spontaneous mutation, selective hybridization, chromosome management through colchicine treatment and gamma ray-induced mutation. The existence of a very large number of cultivars and clones, maintained by vegetative propagation, reinforces the need of a reliable verification of cultivar identity for nurserymen and growers. In this study we used several primers to investigate the potential and limits of the RAPD technique for discriminating among several cultivars and species of Bougainvillea and revealing the relationships between them, regardless of their ancestral lineage, since most of the commercial cultivars are very old and their pedigree is unknown. List of the germplasms of different groups of bougainvillea used for characterization are given in Table 2.

 

 

Table 2 Bougainvillea cultivars included in RAPD analysis

 

Broadly the entire chrysanthemum has been divided into two groups-Large flowered and Small flowered. NBRI is maintaining a living germplasm of more than 250 cultivars collected from all over India and Japan comprising almost all bloom types and colour which are being used as base line material for further increase of genetic variability and improvement through indiscriminate intervarietal hybridization, induced mutation and selection. Extensive work has been done at National Botanical Research Institute, Lucknow, India for the development of new flower colour/shape mutations in chrysanthemum and has been very successful to develop more than 50 new chrysanthemum mutant varieties using colchicine and γ-radiation. The genetic constitution of the present day chrysanthemum is very complex and they carry the genes of many ancestors. It has been realized that the wild growing polyploids are not just plain duplicated diploids. It contains the characters of two, three or four diploid species according to whether they are tetraploid, hexaploid or octaploid. The genus chrysanthemum contributes a large polyploid complex ranging from 2X to 22X, besides a number of aneuploids. Maximum germplasm, maintained at NBRI, were utilized for morphological characterization. Selected original and mutant varieties were used for cytological, anatomical and biochemical characterization. For molecular characterization, 47 large flowered varieties, 48 small flowered varieties, 21 mutant varieties and 24 mini varieties were selected at random from germplasm collection. The objective was to find out the genetic diversity present in the entire chrysanthemum germplasm in general and large flowered, small flowered and mini chrysanthemum in particular. Special aim was also to find out the molecular basis of somatic flower colour mutations i.e. how gamma ray induced morphological mutants (flower colour/shape) can be identified by molecular markers. Details of materials used for characterization are given in Table 3 and Table 4.

 

 

Table 3 Morphological characters of large flowered chrysanthemum varieties included in RAPD analysis

 

 

Table 4 Morphological characters of mini chrysanthemum varieties included in RAPD analysis

 

Entire germplasm of Amaryllis, Bougainvillea, Chrysanthemum and rose, collected at NBRI, were utilized for morphological characterization. A number of gamma ray induced flower colour mutants have been developed by the authors.

 

Budwood of different rose cultivars were treated with 2, 3 and 4 Krad of gamma rays and eyes were budded on Rosa indica var. Odorata root stock. Mutations in flower color were detected in some treated plants in the form of chimera. The chimeric mutants have been established in pure form and all the mutants are being maintained at Floriculture Laboratory, National Botanical Research Institute, Lucknow, India. For the present RAPD analysis a number of somatic flower color mutants and their respective parents were selected. The details of the mutant and parent varieties have been shown in Table 5 and Figure 2.

 

 

Table 5 Original and mutant rose cultivars included in RAPD analysis

 

 

Figure 2 Amplification of parent and mutant cultivars of rose using primer P4 (A), P40 (B) and P31 (C)

 

1.2 Methods

1.2.1 Cytology

Study of mitosis was done preparing slides by squash technique. First of all fresh roots were pretreated in para dichlorobenzene and a pinch of Aesculine for 5 min in -20℃ followed by 15 min at 4℃ and then fixed in the fixative propionic acid: alcohol (1:3). Roots were hydrolysed in 1(N) HCl for 13 min and stained by usual Feulgen staining procedure (Datta, 1976; 1997).

 

1.2.2 Micromorphology

Flower petals were fixed and then mounted on SEM stubs using double sided adhesive tapes after critical point drying. Subsequently the materials were sputter coated with gold (200 Ao thickness) and scanning pohotomicrographs were taken in JEOL-JSM 35C Scanning Electron Microscope at 10 kV (Datta and Shome, 1994)..

 

1.2.3 Pollen morphology

Pollen grains were collected soon after anthesis and pollen slides were made for the study of pollen sterility, size and ornamentations both under light microscope and SEM (Datta, 1998).

 

1.2.4 Spectrophotometric Analysis of Pigments

For spectrophotometric analysis of phenolic compounds 200 mg of all above mentioned explants were extracted in 50 ml methanol containing 1% HCl. The extracts were scanned from 200-800 nm region of wave length in Utltroscope 2000, Pharmacia Biotech.  Where concentration of phenolic compounds were high, the extracts were further diluted (Datta, 1986).

 

1.2.5 TLC of Phenolic Compounds

For the study of phenolic compounds, mature leaf and petal were extracted in methanol containing 1% HCl. The chromatograms were developed on aluminium plates (6.3 X 10 cm) coated with silica gel emulsion. The plates were run 8 cm in a mixture of benzene: propionic acid: H2O (20:40:10 v/v). They were then dried in air and the spots were observed and marked under nacked eye and under UV. The plates were then sprayed with flavone reagent (diphenylboric acid ethanolamine complex) and again marked under UV. The colour reaction of each spot and their Rf values were determined from six good chromatograms.  These were then transformed into hRf (Rf X 100) values (Datta, 1987).

 

1.2.6 DNA Extraction

Total genomic DNA was extracted from young leaves of rose cultivars by CTAB procedure (Saghai-Maroof et al., 1984) with some modifications. Extraction in chloroform: isoamyl alcohol (24:1) followed by centrifugation twice at 14,000 g helped to remove polysaccharides. RNA contaminants in all the samples were digested with 100 mg/mL RNase A for 30 min at 37℃, extracted once with phenol: chloroform: isoamyl alcohol (25:24:1). After ethanol precipitation, DNAwas resuspended in 100 mL of TE (10 mM Tris-Cl + 1 mM EDTA) buffer (pH 8.0). Average yield was calculated by a spectrophotometer (Ultraspec 2000, Pharmacia Biotech) and DNA samples were stored at -20℃.

 

1.2.7 PCR Conditions

Twenty arbitrary decamer primers (Bangalore Genei, India) were used for polymerase chain reaction (PCR). PCR reaction was performed in 20 mL reaction mixture containing 5ng template DNA, 1 unit of Taq DNA polymerase, 100 μM dNTPs, 1.0 μM primer, 2.5 mM MgCl2, 10 mM Tris-HCl (pH-9.0), 50 mM KCl, 0.01% gelatin. PCR amplification was performed using a PTC-100 Peltier Thermal Cycler (MJ Research, USA) using the following conditions: preheating of 4 min at 94℃; 45 cycles of 15 sec at 94℃, 45 sec at 36℃ and 1.5 min at 72℃ and elongation was completed by a final extension of 4 min at 72℃. The final reaction mixture was cooled down to 4℃. After amplification, the PCR product was resolved by electrophoresis in 1% agarose gel with 1X TAE buffer. Bands were visualized by staining with ethidium bromide (0.5 mg/mL) under UV light and photographed. Only distinct bands were counted for data analysis, and faint bands were not considered. The size of the amplification products was estimated from a 100 bp DNA ladder (Sigma). All the reactions were repeated at least twice and only those bands reproducible on all runs were considered for analysis.

 

1.2.8 DATA Analysis

DNA fragment profiles were scored in binary fashion with ‘0’ indicating absence and ‘1’ indicating presence of band. Genetic distance was calculated by Jaccard’s coefficient (Jaccard, 1908) which is as follows:Sij=Nij/(Nii +Nij+Njj)

 

Where Sij is the similarity index between the ith and jth genotype, Nij is the number of bands present in both genotype, Nii is the number of bands present in the ith genotype but absent in the jth genotype, and Njj is the number of bands absent in the ith genotype and present in the jth genotype. The similarity matrix was converted to dissimilarity matrix (1- Sij), and a dendrogram was constructed using the Neighbor Joining Tree method using RAPDistance Package version 2.0 (Armstrong et al., 1998).

 

2 Results

Cytological investigations with special reference to the role of alteration of chromosome morphology in evolution are well known for a long time. The literature available on these aspects is voluminous. To mention a few, comparative study of karyotypes in many plants like Ornithogalum (Cleland, 1950), Crepis (Babcock, 1947), Lilium (Stewart, 1948) etc. have revealed interrelationship between species, varieties and even strains. These aspects were reviewed by Sharma and Sharma (Sharma and Sharma, 1959). Evidences were presented to show that in all possibilities, structural changes of chromosomes play a distinct role in the evolution of agricultural strains of crop plants (Hagberg and Tjio, 1950; Tijio and Hagberg, 1951; Wellhausen, 1994). Cytological investigations were mainly concentrated for understanding the genetic-evolutionary race history, inter-relationship and breeding of different species and cultivars of Alstromeria(Tsuchiya et al., 1987), Anthurium (Marutani et al., 1988; 1993; Sheffer and Croat, 1983),Bougainvillea (Sharma and Bhattacharya, 1960; Ohri and Khoshoo, 1982; Ohri and Zadoo, 1979; 1986; Zadoo et al., 1975a, 1975b, 1975c, 1975d, 1975e, 1975f) Chrysanthemum (Tanaka, 1960; Datta and Banerji, 1995; Rana, 1964; Heywood and Humphries, 1977; Nazeer, 1983; Nazeer and Khoshoo, 1982; 1983), Dianthus (Brooks, 1960), Marigold (Banerji, 1994; Jalil and Pal, 1980; Jalil and Khoshoo, 1974)and Polyanthes tuberose (Ayangar, 1963; Datta and Banerji, 1995; Laxmi et al., 1984; Sato, 1938; Schira and Lanteri, 1986; Sharma and Ghosh, 1956; Sharma and Bhattacharya, 1956; Sharma and Bal, 1956). No chromosomal changes could be detected in new ornamental varieties of chrysanthemum developed through induced mutagenesis (Datta and Banerji, 1995; Datta, 1997).

 

Heslot (1968) studied the pigments of induced mutants and that of original cultivars of rose and found that usually the nature of pigments did not alter but the mutants showed either an increase or decrease of one or several of the pigments found in the control. The anthocyanin pigments in mutant and non-mutant Coleus plants have been studied by Love and Malone (Love and Malone, 1967) and they have reported that colour differences between mutant and non-mutant plants are due to a variation in amount of one anthocyanin pigment rather than change in structure of the pigment molecule itself. Extensive spectrophotometric studies on pigment analysis in original and gamma ray induced flower colour mutants of chrysanthemum and rose clearly indicated that somatic flower colour mutations are due to qualitative and/or quantitative changes in the pigment/s as a result of mutation during pigment biosynthesis pathway (Datta, 1992, 1997,1998a, 1998b, 2001).

 

Role of phenolic compounds in solving different taxonomic problems in different crops have been reported earlier (Bose, 1975; Bose and Frost, 1967; Frost and Holm, 1971; Fahselt and Ownbey, 1968). It has been reported that species and variety specific spots for phenolic compounds occure in a large number of plants. Both naturally occurring and experimentally-produced hybrids can, therefore, be detected early through thin layer chromatographic analyais of phenolic compounds in different plant parts. Alston and Turner (Alston and Turner, 1963) have recognized a large number of species specific spots for phenolic compounds in Baptisia and numerous interspecific hybrids. Hunter (Hunter, 1967) has been able to demonstrate that Vernonia gandalupensis is really a natural hybrid between V. lindhermeri and V. interior, because all the parental compounds could be recognized in the interspecific hybrids. Species and variety specific phenolic compounds could be recognized in a number of plant species and F1 hybrids have been identified by detecting summation of parental compounds as has been done in Viola (Stebbins et al., 1963), Phlox (Levin, 1966, 1967), Saxifraga (Jaworska and Nybom, 1967), Vinca (Stebbins et al., 1963), Trichosanthes (Datta, 1987) (etc.  Significant qualitative and quantitative differences in chromatographic pattern of phenolic compounds in leaves and petals were observed in new mutant varieties of bougainvillea, chrysanthemum and rose (Datta and Basu, 1976; Datta, 2001, 1997). These changes might be due to alternation in biogenesis of certain phenolic compounds or degradation of some of the existing phenolic compounds of the original cultivars.

 

Scanning electron microscope is most commonly used for studying the leaf/seed epidermal micromorphology, morphology of the pollen taxa etc. for solving various taxonomic problems (Robinson, 1971; Sharp et al., 1978; Brisson and Peterson, 1977; Cole and Behnke, 1975; Trivedi et al., 1978; Haridassan and Mukherjee, 1987; Patricia and Clark, 1990). Petal micromorphology studies of original and mutant cultivars of chrysanthemum and rose revealed considerable variation, particularly in cell boundaries, cell surface, striations and papillae not only between the original cultivars but also between the original cultivars and their respective induced mutants. Studies indicated that flower colour change due to mutation was also associated with some changed micromorphology of petal surface. It clearly indicated that petal micromorphological characters can be utilized not only for identifying mutants but also a correlation study will help the proper identification of different present day cultivars of chrysanthemum and rose and their origin (Datta and Shome, 1994; Datta, 1997; 1992).

 

Pollen morphological features are well accepted in present days as unique stable characters which may be used as important diagnostic characters for identification even at microtaxa level.The pollen morphological characters are categorized as appertural, exine ornamentation, exine strata, shape and size in order to their importance and stability. Pollen morphological features especially aperture and exine ornamentation patterns have been used as important diagnostic characters to study the interrelationship and taxonomic and cytological status of the cultivars and even of individuals of the plant population (Lewis, 1965; Nair, 1970). A number of studies have been carried out in the field of cytopalynology to study the nature of hybridity and the effect of polyploidy on pollen morphology (Henderson, 1972; Quiros, 1975; Srivastava, 1976; Chaturvedi et al., 1990). These characters are genetically stable and any change in any of the regulating characters indicates some changes in those features. The pollen morphological characters of large number of original and their gamma ray induced mutant varieties of chrysanthemum and Lantana depressa were analysed and found variations in the nature of endocolpium and some ultra surface patterns (Datta and Datta, 1998).

 

Electrophoresis of crude proteins and enzyme extracts has been successfully used to establish phylogenetic relationships, identification of varieties, intra and inter subspecific variation of different taxa in various groups of crop plants. Protein and isozyme polymorphism has been successfully used for demonstrating genetic variation, identifying interspecific hybrids, and finger printing cultivars (Cherry et al., 1971; Nainawatee and Das, 1972; Challice, 1981; Coehen and deWET, 1981; Cardy and Kannebberg, 1982; Chaparro, 1987; Ellstrend, 1984; Oshima, 1993; Chang et al., 1995; Obara-Okeyo and Kako, 1997; Huang et al., 2000; Kuhns and Fretz, 1978). Kuhns and Fretz (1978) have shown how by combining the results for several isozyme systems, a clear separation could be achieved between the rose cultivar ‘peace’ and three of its sports ‘Chicago Peace’, ‘Flaming Peace’ and ‘Climbing Peace’, a related seedling ‘Pink Peace’ and its mutant ‘Candy Stripe’. Protein electrophoresis has provided a new approach to the problems of species relationships. Fiebich, and Henning (Fiebich and Henning, 1992) used successfully isozyme analysis in breeding of Chrysanthemum. Analysis of electrophoretic pattern of soluble proteins in the original and mutant cultivars of chrysanthemum, rose and Lantana depressa revealed the existence of variability for number and intensity of protein bands between the original and mutants (Datta, 1992, 1997).

 

RAPD markers were used to verify interspecific hybridization in Alstroemeria. Five putative inter-specific hybrids and their parents were analysed by means of four prese-lected RAPD primers (Benedetti et al., 2000) The putative parentage was confirmed in four hybrids and was excluded in one that showed completely different RAPD patterns from its putative parents and a different phenotype. Their results demonstrated that this molecular technique is a powerful tool for rapid verification of hybridity. This tool will allow screening of small immature seedlings for verification of hybridity and should improve the efficiency of breeding programme. Interspecific relationships of both vegetable and grain Anaranthus were studied using RAPD (Mandal and Das, 2002; Faseela and Salkutty, 2007).

 

Hippeastrum, popular name Amaryllis, is an excellent bulbous plant bearing beautiful flowers. National Botanical Research Institute (NBRI), Lucknow, India is one of the pioneer institutions where commendable work has been done on various ornamentals and Hippeastrum in particular. Only two species i.e. H. belladonna and H. gracillis used to grow in plains at NBRI. A total of three varieties, two under H. belladonna i.e. ‘Snow White’ and ‘Fire Fly’ and one variety i.e. Charm under H. gracillis are available in plains. A large number of Dutch hybrids which produce giant size blooms in much wider range of colours, imported and available at different regions of India were collected. These germplasms were used as base line materials for hybridization program and NBRI has successfully developed new varieties through selective hybridization and through selection of natural hybrids. No detailed information is available on the genetical aspects of the genus. The factors underlying evolution are not clear. Narain and Khoshoo (Narain and Khoshoo, 1977) on the basis of their observations and earlier authors on Hippeastrum revealed that hybridization has been an important single factor involved in the origin and evolution of garden cultivars. Next to hybridization, polyploidy has been an important factor and some of the species involved are tetraploid. They have proposed a phylogenetic tree explaining the role of different elemental species and their country of origin. Realizing the necessity of proper classification of elemental species and vast number of cultivars, Khoshoo (1971) proposed a very important concept for nomenclature of cultivated crops. Following the concept of Khoshoo (1971) and the system suggested by Traub (1958), Narain (1977) proposed a workable classification and all the cultivars have been placed under different groups on the basis of their diagnostic characters. RAPD analysis has been used to characterize genotypes of known and unknown origin and to measure genetic relationships among the hybrids of Hippeastrum. PCR amplification of total genomic DNA from 24 amaryllis cultivars using 20 random decamer primers was carried out. Of 20 primers, 7 produced 5-13 DNA bands per primer suitable for data analysis of the 24 cultivars. A total of 59 bands were produced from the 7 useful primers, ranging in size from 100 to 1500 base pairs (Figure 1A, B). The 24 cultivars were classified using the 48 polymorphic bands obtained from the 7 primers. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 24 cultivars were measured. The similarity coefficients ranged between 0.2 and 1.0. From the data obtained in the dendogram, the 7 polymorphic primers discriminated among all varieties and divided into two major clusters. The first contained 11 varieties and the second cluster contained 13 varieties. The overall results of the cluster analysis fit with the available pedigree data of the species. Hybrids with common parents (one or more) clustered together indicating their level of genetic similarity. From this study a clear genetic association of various Hippeastrum cultivars and hybrids with parents was found. Cluster analysis of RAPD profiles of Hippeastrum hybrids and/or varieties where the pedigree is not known can now be used as a tool to study ancestral relationships (Chakrabarty et al., 2007).

 

Genetic variations among 24 cut flower Anthurium andraeanum Hort. cultivars were assessed using RAPD fingerprinting (Nowbuth et al., 2005). Eight decamer primers produced a total of 98 reproducible PCR bands and genetic distance (GDNL) coefficients indicated low genetic variation among the cultivars. Dendrogram grouped the cultivars into four main clusters which did not relate to cultivar provenance or origin and were independent of floral colour and spathe category. RAPD markers fingerprinting allowed a rapid assessment of the level of genetic variation that would otherwise be difficult to evaluate using the limited number of morphological markers present among these closely related anthurium cultivars. Ranamukhaarachchi et al. (2001) utilized RAPD to determine the genetic relationships of nine morphologically similar pot plant cultivars of Anthurium sp. by developing DNA fingerprints (DFP). All cultivars tested exhibited a high degree of genetic similarity. Study showed that the nine Anthurium cultivars examined were genetically closely related indicating that RAPDs can be a useful tool to distinguish Anthurium pot plant cultivars as well as identify their genetic relationships.

 

Molecular cytogenetic characterization, genetic variability and phologeny have been investigated on different species of snapgragon (Antirrhinum subaecticum, A. majus etc.) using molecular markers (Jiménez et al., 2002, 2005; Zhang et al., 2005).

 

PCR-based DNA markers were examined on two cultivars of Asiatic Hybrid lily using random primers with various lengths (10-, 12-, 15- and 20-base). Thirty-three out of61 (54%) 15-base primers and 14 out of 21 (67%) 20-base primers generated polymorphic fragments whereas 17 out of 145 (12%) and 4 out of 24 (17%) of the 10- and 12-base primers, respectively, amplified them. The efficiency of the RAPD reaction increased with increasing primer length. They also examined inter-simple sequence repeat (ISSR) reactions using 30-anchored simple sequence repeat (ASSR) primers, and found 33 out of 63 (52%) primers that amplified polymorphic bands between the two cultivars are genetically stable (Yamagishi et al., 2002).

 

Bougainvillea (Fam. Nyctaginaceae) is one of the most important perennial ornamental shrubs, sometimes climbers, in tropical and subtropical gardens. Three species of Bougainvillea i.e. B. spectabilis, B. glabra and B. peruviana are important horticultural species, showing colourful bracts. Considering the importance and utility of genetic diversity, The National Botanical Research Institute (NBRI), Lucknow, India is maintaining a rich Bougainvillea germplasm collection of approx. 180 species/cultivars comprising both single bracted and double or multi-bracted varieties. NBRI has detected, induced, isolated, established and commercialized a series of new varieties developed through spontaneous mutation, selective hybridization, chromosome management through colchicine treatment and gamma ray-induced mutation. Studies have solved many taxonomic problems and phylogenetic relationships, and have also opened a new way to synthesize new varieties through chromosomal manipulation.

 

Chromosome numbers of horticultural varieties of Bougainvillea have been reported by a number of workers (Sen and Sen, 1954; Sharma and Bhattacharya, 1960; Ninan et al., 1959; Banerji and Banda, 1967; George and Sobhana, 1976; Begum and Datta, 1971). Basic cytogenetic studies were directed towards determination of chromosome number, mitotic and meiotic divisions, karyotype analysis, colchi-ploidy etc. DNA content has been estimated from three basal Bougainvillea species, hybrid groups, and triploid hybrid cultivars and induced tetraploids and their relationships established. These studies have solved many taxonomic problems and phylogenetic relationships, and have also opened a new way to synthesize new varieties through chromosomal manipulation. Taxonomical studies were initiated on important Bougainvillea species and cultivars to enrich information on morphological descriptions, growth habit, agro-technology, techno-economics, flowering behaviour, biochemical (TLC), affinities with coloured illustrations and their usage (Datta, 1985; Jayanyhi et al., 1999). RAPD analysis was performed to determine the genetic relationships among the most important Bougainvillea cultivars grown at National Botanical Research Institute, India. Fifty random decamer primers were screened and ten were selected for final RAPD analysis. These 10 primers used in this analysis yielded 167 scorable bands with an average of 11.3 bands per primer. Of the 167 fragments scored from these primers, 26 were monomorphic and 141 were polymorphic (84.4%). The generated similarity matrix showed that the genetic diversity within the tested genotypes was high (average similarity index=30.1%). Similarity values among the studied genotypes ranged from 6% to 89%. The resulting dendrogram divided the cultivars into two main clusters. The first contained 37 varieties and was divided into two sub-clusters at a similarity value of 0.03 with 21 and 16 varieties in each subcluster, respectively. Subcluster I included those cultivars which had affinity with buttiana group. Subcluster II contains cultivars of both spectabilis and peruviana group. The second major cluster contained 55 varieties, contain species of glabra group and cultivars whose origin are not well recorded. The similarity data obtained in this study agree, to some extent (at least for the buttiana group) with the previous classification of the tested genotypes. The information obtained from this work may be useful for better management, identification of accessions and also in avoiding duplications or mislabeling of the genotypes studied. Present RAPD analysis was found to be very helpful for the identification of cultivars, documentation, and to trace out the molecular affinity of origin of unknown group of Bougainvillea cultivars. Genetic diversity of a large number of Bougainvillea cultivars has been estimated. Origin and interaction of different species/cultivars of the four major groups were studied successfully; origin and affinity of 41 cultivars of unknown origin could be traced out up to certain level. Such study is very helpful and necessary for assessment of genetic diversity of large germplasm collections of horticultural species and their further improvement through selective breeding programme (Chatterjee et al., 2007). Srivastava et al. (2009) successfully confirmed the parentage of the hybrids and characterized 21 bougainvillea cultivars on the basis of genetic diversity through molecular markers and morphological traits. Palavras (2009) estimated genetic relationship in different bougainvillea cultivars using RAPD.

 

Identification of molecular markers linked to flower vase life is an important character to improve the efficiency of breeding programs in Carnation. 12 commercial varieties of carnation were analyzed with the RAPD technique (Benedett et al., 2005). RAPD bands significantly discriminated a population with longer vase life.

 

DNA fingerprinting using mini- and microsatellite sequences was applied to identify the genotypes and to establish the genetic distances between them in carnation and rose. The probability of two offspring from the crossing of similar genotypes having identical DNA fingerprints (DFPs) was found to be 1.8x10-6 for carnation and 2x10-8 for rose. A comparison of genetic relationships within and between categories based on known genetic history, to genetic relationships deduced from DFPs, revealed a perfect match for both flower types (Ben-Meir et al., 1997).

 

Flower doubleness as a breeding characteristic is of major importance in carnation (Dianthus caryophyllus), since flower architecture is of the utmost value in ornamentals. Based on the number of petals per flower, carnations are grouped into ‘‘single’’, ‘‘semidouble’’ and ‘‘double’’ flower types. These flower types are not easily distinguishable due to phenotypic overlaps. Scovel et al. (1998) identified a RAPD marker which was tightly linked to this recessive allele. The RAPD marker was cloned and used to generate a RFLP marker. This RFLP marker could discriminate with 100% accuracy between the semi-double and double- flower phenotypes in carnations.

 

Bacterial wilt is one of the most serious diseases of carnations (Dianthus caryophyllus) in Japan. This disease is very difficult to control with chemicals once it has occurred. Breeding of resistant cultivars is considered the optimum strategy to overcoming this disease. However, it takes over 3 months to determine the resistance of breeding materials by inoculation assays. Onozaki et al. (2003) tested to identify RAPD markers associated with genes controlling wilt resistance in a resistance-segregating population. Results suggest that at least 3 genes are concerned with resistance to bacterial wilt. In particular, 4 RAPD markers identified by bulked segregant analysis were linked to a major resistance gene. These markers should be useful for marker-assisted selection in carnation breeding programs.

 

Genetic linkage map was constructed on the basis of RAPD and SSR by using a resistance-segregating population of 134 progeny lines of carnation (Dianthus caryophyllus L.) that were derived from a cross between ‘Carnation Nou No.1’ (a carnation breeding line resistant to bacterial wilt) and ‘Pretty Favvare’ (a susceptible cultivar). Linkage analysis revealed that 124 loci could be mapped to 16 linkage groups that extended for 605.0 centiMorgans (cM) and the average interval between two loci was 4.9 cM. Quantitative trait loci (QTL) analysis to evaluate the resistance to bacterial will suggest that resistance to bacterial wilt in carnation is related to one major and at least two minor genes. This was the first report on the construction of a linkage map of the carnation (Yagi et al., 2006).

 

A RAPD-based identification system for calla lily cultivars (Zuntedeschiaspp.) was constructed after screening 60 arbitrary IO-mer primers. Using this method, two or three sequential PCR reactions enabled clear identification of 12 cultivars within several days (Hamada and Hagimori, 1996).

 

In Chrysanthemum, cultivars are identified in flowering trials, and breeders’ rights are presented by cultivar characteristics including flower, leaf and growth morphology. The application of isozyme technology could largely improve the identification of Chrysanthemum cultivars (Roxas et al., 1993). However, the level of polymorphism obtained is often insufficient to distinguish cultivars, and the growth conditions may influence the quality and quantity of isozymes (Wolff et al., 2005). Chrysanthemum cultivars are propagated vegetatively by cuttings. The cultivars that are propagated vegetatively must have the same DNA pattern, even after many years of cultivation (Wolff et al., 2005). Morphological, cytological, anatomical, biochemical and RAPD work were carried out extensively by the authors  on original chrysanthemum varieties and new varieties developed through conventional breeding and gamma ray induced mutation (Chatterjee et al., 2005, 2006a, 2006b; Datta, 1987, 1993; Datta and Chatterjee, 2006; Datta and Datta, 1998; Datta and Gupta, 1981a, 1981b, 1983; Datta and Shome, 1994; Gupta and Datta, 2005) and on rose (Datta and Gupta, 1983; Datta and Singh, 1995, 1999, 2001, 2002, 2003, 2004, 2005, 2006; Datta, 1997).

 

Genetic variation of 18 chrysanthemum cultivars was first studied by Wolff and Peter-Van Rijn (1993) using RAPD. It is known that different conditions of the RAPD reaction (thermal cycler brand, annealing temperature, primer, enzyme brand, magnesium concentration, pH, gelatin concentration etc.) influence the results. Wolff et al.(1993)made extensive studies of these factors on chrysanthemum and standardized the optimum combination of each factor to obtain reproducible patterns. Wolff et al. (1994) constructed the Pst I and Hind III genomic library for Dendranthema grandiflora Tzvelev. Probes from both libraries were tested for the presence or absence of restriction fragment length polymorphism (RFLP). The RFLP probes and primers development was very useful for further marker assisted selection in this polyploidy crop. Scott et al. (1996) used DNA amplification fingerprinting to study genetic relationships between 21 closely related chrysanthemum cultivars using 11 arbitrary octamer primers. Series of new cultivars have developed in Chrysanthemum through sporting is a well-known phenomenon. In chrysanthemum there are three different cell layers that form different parts of the plant. The samples were taken from the leaf (layers I, II and III), from florets (layers I and 11), and the epidermis of a leaf (layer I) differences at the DNA level among cultivars within one family and among the layers of one cultivar were studied by RAPD analysis. Polymorphisms between cultivars as well as between different cell layers have been observed (Wolff, 1996).  Trigiano et al. (1998) studied the relation between induced mutants of chrysanthemum ‘Charm’ family. The cultivars differ only in flower colour and were very difficult to distinguish by DAF but could be easily distinguished by ASAP (Arbitrary Signatures from Amplification Profiles). Genetic analysis of three hybrid combination of chrysanthemum was done by Huang et al. (2000). Forty five random primers were screened, of which twenty two primers were selected to detect the molecular marker in three hybrids combination of chrysanthemum by using RAPD. Jackson et al. (2000) studied the microsatellite profiling for DUS testing in chrysanthemum. DNA fingerprinting techniques based on PCR amplification of microsatellite and Copia like sequences were investigated for their utility in the chrysanthemum. Lema-Ruminska et al. (2004) characterized 10 radiomutants of chrysanthemum using RAPD. They studied and confirmed the utility of RAPD markers to identify chrysanthemum cultivars as well as to distinguish the radiomutants from the parents. Somaclonal variation of chrysanthemum was detected by RAPD markers (Martin et al., 2002). Huang et al. (2000) used RAPDs to detect different molecular markers in three hybrid populations of Chrysanthemum cultivars. They classified the patterns of molecular markers into seven types on the basis of band sharing between parents and hybrids. Kumar et al. (2006)applied RAPD technique to characterize eleven gamma ray induced radiomutants of two chrysanthemum cultivars.Their study revealed that RAPD molecular markers can be used to assess polymorphism among the radiomutants and can be a useful tool to supplement the distinctness, uniformity and stability analysis for plant variety protection in future. Minano et al. (2009) used RAPD analysis for molecular characterization of eight cut flowers and two pot plant cultivars of chrysanthemum.  The aim of this study was to analyze the genetic stability of micropropagated chrysanthemum shoots derived from selected commercial cultivars. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The analysis has been carried out with RAPD markers and different aspects of the culture, as source of explants, media composition and culture age have been considered. Xu et al. (2006) investigated the genetic diversity of 22 Chrysanthemum morifolium accessions by RAPD markers. A total of 233 bands were amplified, of which 89.7% bands were found to be polymophic. The results of cluster analysis by using UPGMA method showed that all the tested accessions showed much genetic diversity at the molecular level and could be differentiated by RAPD marks. Sehrawat et al. (2003) examined genetic variation in 13 commercial chrysanthemum (Chrysanthemum morifolium Ramat.) cultivars using RAPD markers. A total of 257 clear and reproducible bands were detected of which 239 bands were polymorphic. Genetic variation amongst cultivars was high enough to divide them into two major groups. These groupings were in consistent with their morphological differences and geographical distribution. The results indicate that RAPDs are efficient for identification of chrysanthemum cultivars and for determination of genetic relationships. Teng et al. (2006) studied the genetic variation of regenerated plantlets in chrysanthemum following in vitro mutation using RAPD methods. Results showed that genetic variation of generated plantlet was proportional to the dosage of gamma ray, while the 15 and 20 Gy treatments were not significantly different, which was consistent with the common conception that genetic variation of radiomutants was usually proportional to the dosage of mutagen within a certain range. They concluded that RAPD is a useful technique for the rapid and easy assessment of genetic variation of mutants and may become a potential tool for the quick selection of mutants with great genetic variation during early growth stages.

 

Authors (Chatterjee et al., 2006a, 2006b) selected 47 large flowered varieties, 48 small flowered varieties, 21 mutant varieties and 24 mini varieties of chrysanthemum at random from germplasm collection for molecular characterization. The objective was to find out the genetic diversity present in the entire chrysanthemum germplasm in general and large flowered, small flowered and mini chrysanthemum in particular. Special aim was also to find out the molecular basis of somatic flower colour mutations i.e. how gamma ray induced morphological mutants (flower colour/shape) can be identified by molecular markers. A total of 50 random primers were screened and only 10 gave good results which were selected for final RAPD analysis. Some primers yielded extremely different banding patterns in mutants and parents. Band generated by RAPD fragments are of low molecular weight ranging from 400 bp to 1500 bp. Bands for each primer ranged from 3-13. All the miniature varieties look like similar in vegetative stage. Though each and every miniature cultivars could not be differentiated by these selected primers but it was sufficient to estimate the genetic diversity among the twenty four cultivars. This result did not show any relation between the geographical distribution and genetic diversity of species as cultivar ‘Shizuka’ (imported from Japan) show close relation with the local cultivars ‘Little Darling’ and ‘Pancho’. The average diversity among the large flowers was found to be low whch may be due to continuous selection of superior varieties leading to extinction of some of the cultivars. Dendrogram of large cultivars indicated two major groups and dendrogram of the small group clearly indicated three groups. 

 

Jackson et al. (2000) examined how DNA fingerprinting techniques based on PCR amplification of microsatellite and Copia like sequences can be utilized in the Chrysanthemum DUS process. On the basis of their results, they have discussed the benefits of these approaches.

 

Kishimoto et al. (2003) studied the variations in chloroplast DNA of Dendranthema species by PCR-RFLP analysis to find out the maternal origin of the cultivated chrysanthemum, D. grandiflorum. Ten genes, atpH, matK, petA, perB, psaA, rbcL, rpoB, rpoC, trnK and 16S in the chloroplast DNA of 12 Japanese wild species and 1 cultivar were amplified by PCR. The amplified DNAs that were digested with each of 32 restriction endonucleases revealed 13 site changes among the 13 species in the following 9 genes plus restriction endonuclease combinations: petA-AvaII, petA-HaeIII, petA-MboII, petA-NdeII, rpoC-EcoRV, trnK-DraI, trnK-HinfI, trnK-MboII and trnK-ScrFI. No Japanese wild species showed the same PCR-RFLP pattern of chloroplast DNA as D. grandiflorum.

 

Genetic relationships among Calathea species and cultivars were studied among 34 commonly grown cultivars across 15 species using AFLP markers with near-infrared fluorescence-labeled primers. A total of 733 AFLP fragments were detected, of which 497 were polymorphic (67.8%). The 34 cultivars were separated into four clusters. Their study established the genetic relationships of commonly cultivated calatheas, provided genetic evidence supporting that C. fasciata, C. orbifolia, C. rotundifolia, C. insignis, and C. ornate were independent species, and raises a concern over genetic vulnerability of cultivars in cluster I because of their close genetic similarities (Kishimoto et al., 2003).

 

Laura (2002) applied RAPD analysis on a large number of embryogenic Cyclamen genotypes callus in order to study the variability related to the different 2,4-D concentrations and exposure times. Twenty-five random primers produced 161 reproducible bands. Six primers produced band patterns that differed among the samples. The four cell lines used in the experiments were divided into two groups showing different phenotypic and molecular behaviour. In one cell line, a different RAPD patterns observed in the callus grown at the higher 2,4-D concentration. RAPD analysis appeared to be a useful tool for the early screening of genetic variation during callus culture, and allowed precocious selection of the most stable embryogenic cell lines.

 

Silver-stained AFLP was applied to detect the genetic relationships among 83 Calycanthaceae accessions, including 76 genotypes from C. praecox and 7 accessions from the other species. Eight primer combinations yielded a total of 387 polymorphic bands, with an average of 48.4 per assay. The results obtained with the analysis of genetic similarities among accessions according to Jaccard’s similarity coefficient and the clusters constructed based on the UPGMA indicated that: (1) Chimonanthus zhejiangensis was highly differentiated from the other species and detected the closest relationship with C. salicifolius, which possibly supported that C. zhejiangensis was a distinct species rather than the synonym of C. nitens. Besides, C. praecox was much more closely related with Chimonanthus campanulatus than the other species. (2) Extensive genetic differentiation existed among C. praecox genotypes. However, the clusters did not match well with the floral morphological characters, perhaps conforming that the floral phenotypic characters (flower type and size, medium and inner tepal color, and medium tepal shape) of C. praecox were quantitative traits controlled by multi-genes rather than qualitative traits. In addition, the results also demonstrated AFLP markers were useful for evaluating the genetic relationship among species in Calycanthaceae and the genetic variation of C. praecox genotypes, and may serve as efficient fingerprinting technique for further identification and classification of germplasms (Zhou et al., 2007).

 

RAPD markers were used to assess genetic diversity in three species of toxic larkspurs (Delphinium spp). A total of 184 plants from 22 accessions were analyzed by 23 RAPD primers that amplified 188 reproducible bands. They detected 144 polymorphic bands; 10 shared by Delphinium glaucum and Delphinium occidentale, eight shared by Delphinium barbeyi and D. glaucum, and 18 shared by D. occidentale and D. barbeyi. Thirteen bands were specific for D. occidentale, 18 for D. glaucum and 19 for D. barbeyi. There were 58 bands that were specific for individual accessions and 44 bands that were common to all three species. Some of the species-specific bands were cloned and tested in Southern hybridization. Based on the presence or absence of the bands and dendrogram the genetic relationships among these three tall larkspur species was generated Li et al. (2002).

 

Lee et al. (2005) studied 55 interspecific hybrids between Dianthus giganteus and D. carthusianorum using RAPD markers. They detected 216 polymorphic RAPD bands and found that mostly the interspecific hybrids exhibited intermediate characteristics between parents. Scoevel (1998) identified RAPD marker which was linked to recessive allele of ‘single’, ‘semi double’ and ‘double’ flower type of carnation (Dianthus caryophyllus).

 

Plants regenerated from tissue culture techniques show a wide range of variability, ranging from temporary changes in the phenotype to sexually heritable mutation. Changes in DNA methylation has been hypothesized as an underlying mechanism of tissue-culture-induced mutagenesis which includes a high frequency of quantitative phenotypic variation, activation of transposable elements, heterochromatin-induced chromosome breakage events, and sequence changes due to deamination of 5-methylcytosine to thymine (Kaeppler et al., 2000). Tissue culture techniques have been widely used for mass production in superior varieties of Phalaenopsis and Doritaenopsis (Park et al., 2002; 2003); however, the occurrence of somaclonal variations among regenerants is a frequent and consistent event. Park et al. (2009) used two molecular techniques, RAPD and methylationsensitive amplification polymorphism (MSAP) analyses to characterize the somaclones. Their study demonstrated usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.

 

Gentian is one of the most important ornamental flowers in Japan and cultivars are easily proliferated vegetatively. Shimada et al. (2008) attempted to develop a reliable discrimination method to prevent the illegal propagation and distribution of various high-value cultivars. They used five (SCAR markers based on the length polymorphisms in introns of four gentian flavonoid biosynthetic genes. These SCAR markers effectively discriminated nine gentian cultivars and nine breeding lines. This method could be applied in identifying gentian cultivars/lines and therefore will aid in protecting breeders’ rights.

 

Dallavalle et al. (2002) applied RAPD experiments and isolated the specific band in Gladiolus for different sensitivities to Fusarium oxysporum Schlecht.

 

Bhatia et al. (2009) used 32 ISSR markers to study the genetic fidelity of in vitro raised 45 plants of gerbera (Gerbera jamesonii Bolus) derived from three different explants, viz., capitulum, leaf and shoot tips. Fifteen ISSR markers generated a total of 3 773 bands, out of which 3 770 were monomorphic among all the clones. The Jaccard's similarity coefficient revealed that out of 45 clones derived from different explants, 44 were grouped into a single large cluster alongwith the mother plant with a similarity coefficient value of 1.00, whereas one clone (C38) remained ungrouped. The clones derived from capitulum and shoot tip explants did not show any genetic variation, whereas, one of the leaf-derived clones exhibited some degree of variation.

 

Genetic diversity of 29 commercial and 13 wild accessions of Barberton daisy (Gerbera jamesonii) was evaluated by RAPD markers and morpho-agronomic characters. A total of 74 polymorphic bands were obtained using a set of 12 primer pairs. The genetic similarity coefficient was evaluated by Jaccard index and the genetic structure was evaluated by hierarchic classification analyses and UPGMA modeling. Shannon (H′) index analysis using the molecular markers showed higher values of genetic diversity for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster. Results indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics (Mata et al., 2009).

 

Flower color is one of the main important morphological aspects in genetic breeding programs. The use of molecular markers may serve to direct crossings, new hybrids and mutants, besides confirm and identify new genotypes for commercial purposes. The genetic divergence among six cultivars of Gerbera jamesonii was carried out with 21 primers, which amplified 37 DNA polymorphic fragments. The results showed that the RAPD is a fast, relatively inexpensive and useful technique for genetic divergence characterization between different cultivars of Gerbera jamesonii [319].

 

Sheela et al. (2006) analysed seventeen Heliconia species and varieties using RAPD markers and found a strong parallelism between genetic and morphologic/taxonomic variability of Heliconia genotypes.

 

The daylily (Hemerocallis spp.) is one of the most economically important ornamental plant species in commerce (Tomkins et al., 2001). In order to determine the effects of intensive breeding on cultivar development, and to study relationships among different species, genetic variation in the daylily was estimated using AFLP markers. Nineteen primary genotypes (species and early cultivars) and 100 modern cultivars from different time periods were evaluated using 152 unambiguous bands derived from three AFLP primer combinations. When comparing cultivar groups from different time periods (1940-1998), genetic similarity was initially increased, compared to the primary diploid genotypes, remained constant from 1940 to 1980, and then steadily increased as breeding efforts intensified and hybridizers began focusing on a limited tetraploid germplasm pool derived by colchicine conversion. Among modern (1991-1998) daylily cultivars, genetic similarity has increased by approximately 10% compared to the primary genotypes. These data were also used to evaluate recent taxonomic classifications among daylily species which, with a few minor exceptions, were generally supported by the AFLP data.

 

Taiwan lily (Lilium longiflorum Thunb. var. formosanum Baker) is distributed from lowlands to high mountains in Taiwan with large morphological variation. The genetic differentiation of seven populations from low, middle, and high altitudes was studied by evaluating seven morphological traits and 64 RAPD markers (Wen and Hsiao, 2001). RAPD analysis employing nine primers also revealed that the populations were differentiated according to the altitudinal differences. Result indicated that populations of high altitudes were more variable among individuals within populations than were populations of low altitudes. Therefore, there is a need for immediate measures to conserve the germplasms of lower altitude populations.

 

Attempts were made to develop molecular DNA markers for identification of hybrids of lily (Wiejacha et al., 2001). Plants were obtained by ovule rescue from pollinations of lily cultivars belonging to the group "oriental hybrids" with pollen of Lilium henryi, L. pumilum and L.xformolongi.  The products of amplification were detected for all the forty primers used. Eight primers generated reproducible polymorphic bands within the pair 'Marco Polo' and L. henryi, six primers within 'Alma Ata' and L. pumilum, and 'Muscadet' and L.xformolongi. They found that some bands could serve as markers to detect hybrids at the in vitro stage.

 

Limonium (fam. Plumbaginaceae) is grown in several regions of the world for use as a cut flower for both fresh and dry-flower arrangements.  In this work, thirteen wild species were tested for the study of genetic relationships and taxonomic status using RAPD analyses. The dendrogram obtained from cluster analysis showed high similarity among three species that some authors report as synonymous (L. caspia, L. bellidifolium and L. otolepis). In order to clarify the genetic relationships, further analyses were carried out on several genotypes belonging to these species. The new dendrogram showed that the genotypes did not group in clear clusters. Analysis of molecular variance (AMOVA) confirmed that the species can be considered synonymous. The use of RAPD markers was thus useful for clarifying the highly probable identity of the three Limonium species, in a plant genus that is notably of difficult interpretation (Bruna et al., 2004).

 

RAPD was used for genetic fingerprinting of Limonium spp., Alstroemeria spp., carnation and Prunus spp. for verification of hybridity in progenies of interspecific crosses and for the study of taxonomic relationships. The putative percentage was confirmed in 4 hybrids and it was excluded in one hybrid showing both a completely different phenotype and RAPD patterns from its putative parents. RAPD markers were also used for the characterization of potential parents in a breeding programme on Alstroemeria for pot plant production. In carnation, RAPD analysis was used for the identification of molecular markers associated with cut flower longevity. Regression analysis showed a positive correlation between the score of each progeny (number of RAPD markers similar to Roland) and its longevity (Chung et al., 2002). 

 

Mor et al. (2008) used nine genotypes of marigold, selected from two species, Tagetes erecta L. (3 genotypes) and Tagetes patula L. (6 genotypes) and characterized through electrophoresis of protein and using RAPD markers. The dendrogram based on protein electrophoresis grouped the nine genotypes in two clusters species-wise, whereas RAPD analysis showed clear-cut genotype and species difference, which confirms the reliability of RAPD markers over protein electrophoresis.

 

Myrtus communis L., is a wild shrub widespread throughout the Mediterranean region and the Middle East, grown for its ornamental value and aromatic properties. Bruna et al. (2007) evaluated the genetic diversity existing within wild myrtle populations for the development of breeding programs or selection of genotypes with useful traits for cultivar improvement using AFLP markers. Fifty-one individuals were analysed with three selected combination of primers. Analysis of molecular variance revealed that variability within populations represented the greatest source of variation.

 

Guo et al. (2007) used RAPD markers to estimate the genetic diversity and to test the genetic basis of the relationships between morphotypes and molecular markers of 65 lotus accessions in genus Nelumbo. Neither the UPGMA dendrogram nor the PCA analysis exhibited strict relationship with geographic distribution and morphotypes among the accessions.

 

Huge genotypic variability has been developed in Opuntia via natural hybridization associated with polyploidy and geographical isolation (Gibson and Nobel, 1986). Wang et al. (1998) did experiment with eight Opuntia accessions (five cactus fruit varieties from Mexico and Chile, two ornamental Texas accessions, and one vegetable accession from Mexico) using RAPD to determine whether polymorphism was sufficient to distinguish Opuntia accessions and to assess the patterns of genetic diversity among a selected group of accessions. Phenotypic and molecular analyses distinguished ornamental, vegetative, and fruit market accessions of Opuntia from each other, and suggested significant differences among accessions of different market classes. The experiment demonstrates the potential usefulness of molecular markers in classification of cactus accessions, and indicated the feasibility of a comprehensive effort to determine the relationships among Opuntia species using molecular markers.

 

Ko et al. (1996) studied genetic diversity of 6 most important cultivars of popular flowers of Ozothamnum diosmifolius (Vent.) DC by RAPD using 19 arbitrary primers.

 

Fajardo et al. (1998) analyzed genetic variation of 52 accessions representing 14 species of the genus Passiflora L. by RAPD using 50 random primers.

 

Palumbo et al. (2007) tested the feasibility ofTarget region amplification polymorphism (TRAP) for molecular characterization of Pelargonium collections comprising cultivars, breeding lines and wild species. On the basis of their observations, they mentioned that TRAP is an effective method for molecular characterization of ornamental collections and this will help to retain the most diverse genotypes and to use as genetic markers in future breeding programme.

 

Peltier et al. (1994) established the first linkage map for Petunia hybrida based upon both RAPD and phenotypical markers. They also applied RAPD markers on a set of ten P. hybrida, lines chosen for their diversity and on a set of seven wild species corresponding to the possible ancestors of the P. hybrida species.

 

RAPD profiles of thirteen rhododendron hybrids, species and cultivars were analyzed to study their genetic relationships. The cluster analysis grouped together varieties and/or hybrids in accordance with their known genetic relationship. The genetic relationship revealed from cluster analysis on the basis of RAPD profiles was similar to their known genetic makeup (Iqbalet et al., 1995).

 

Menziesia, a small genus in Ericaceae bear small bell-shaped flowers, which is white, yellow, pink or brick red colors in spring-summer time. Handa et al. (2003) analyzed sequences of both chloroplast matK gene and nuclear internal transcribed spacer (ITS) region from genus Rhododendron and its closely related genera including Menziesia. Results revealed that Menziesia is nested within the genus Rhododendron.They tried to make new intergeneric hybrids between Menziesia and Rhododendron by crossing. In an orchid genus Dendrobium, section Callista includes many attractive species but the flower-vase-life is relatively short. They also analyzed both matK and ITS sequence of section Callista and its related sections in Dendrobium. Results indicated that other sections such as Calyptrochilus and Pedilonum, which have long-lasting flowers, are closely related to section Callista. On the basis of this phylogenetic information, they successfully obtained intersectional hybrids of section Callistawith these other sections. Phylogenetic information is useful for the exploitation of ornamental germplasm in introducing novel resources or selecting closely related species for the introgression of horticultural interesting traits.

 

In the International Union for the Protection of New Varieties of Plants Act of 1991, mutation is mentioned as one of the mechanisms to obtain an ‘essentially derived’ variety (EDV). For the implementation of the EDV concept in the case of mutation, it is important that the level of genetic relatedness between an initial variety and derived mutant varieties can clearly be distinguished from the level of relatedness between arbitrary pairs of varieties without a derivation relation. In rose, mutants or ‘sports’ are frequently observed during multiplication, making it a suitable crop for studying the possibilities for introduction of the EDV concept in ornamentals. Vosman et al. (2009) studied genetic similarities among 83 rose varieties, including 13 mutant groups. Twelve AFLP primer combinations generated 284 polymorphic markers and 114 monomorphic (fixed) bands. Pair-wise Jaccard similarities between original varieties and derived mutants were close to 1.0 (>0.96), whereas all similarities between original varieties were below 0.80, with 75% of the non-mutant similarities even being below 0.50. On the basis of a consistent and large difference between similarities, relations between an original variety and its mutants can easily be identified and distinguished from relations between original varieties. These results open the way for implementing the essential derivation concept in rose.

 

Zhang et al. (2000) developed AFLP markers and evaluated rose varieties for identification purpose and estimated molecular diversity among modern roses using 12 prescreened primer combinations. Mahapatra and Rout (2005) identified and analyzed 34 rose cultivars using RAPD.

 

Yan et al. (2003) used 365 uni-parental AFLP and SSR markers to prepare Parental linkage maps of a segregating population of diploid rose hybrids (2n=2x=14) derived from a cross between two half-sib parents (P119 and P117).  Of the markers, 157 P119 markers (85 %) mapped on eight linkage groups and 133 P117 markers (78 %) on seven linkage groups. The resulting linkage maps of P119 and P117 spanned 463 cM and 491 cM with an average of interval between markers of 2.9 cM and 3.7 cM, respectively. The present genetic maps were used to identify quantitative trait loci (QTLs) for two growth vigour-related traits, leaf area and chlorophyll content, using the Multiple QTL Mapping approach. Three QTLs for leaf area and two QTLs for chlorophyll content were identified. The QTLs accounted, in total, for 50.8 % (range 7.0-23.1 %) and 25.8 % (range 7.6-18.2 %) of the total phenotypic variance for leaf area and chlorophyll content, respectively. The detection of highly significant major QTLs enables marker-assisted selection for growth vigour in rose.

 

Debener et al. (2000) used RAPD and AFLP fragments to infer genetic differences between the sports, the original variety and seedlings of two cut rose varieties as well as a garden rose variety. They proposed that molecular markers can be used to verify the origin of vegetatively propagated rose plants of doubtful origin, thus enabling breeders in the future to claim plant breeders rights on sports of varieties already registered.

 

A total of 305 RAPD and AFLP markers were analysed in a population of 60 F1 Rose hybrids plants based on a so-called “double-pseudotestcross” design. Of these markers 278 could be located on the 14 linkage groups of the two maps, covering total map lengths of 326 and 370 cM, respectively. The average distances between markers in the maps for 93/1-117 and 93/1-119 is 2.4 and 2.6 cM, respectively. In addition to the molecular markers, genes controlling two phenotypic characters, petal number (double versus single flowers) and flower colour (pink versus white), were mapped on linkage groups 3 and 2, respectively. The maps provide a tool for further genetic analyses of horticulturally important genes as, for example, resistance genes and a starting point for marker-assisted breeding in roses (Debener and Mattiesch, 1999).

 

Random amplified polymorphic DNA (RAF’D) markers were tested for the identification of nine rose cultivars and three clonal plants. All of the cultivars were identified by using only three primers. Moreover, individuals were also distinguished by unique RAPD marker bands (Matsumoto and Fukui, 1996).

 

RAPD markers were used to analyse genetic diversity in 25 rose cultivar and 5 species. Thirty five randomdecamer primers were employed for RAPD analysis, of which the 28 primers generated polymorphic bands. In total, 226 bands were obtained, of which 209 were polymorphic, while 17 bands monomorphic. The rose cultivars studied in the present investigation possessed some important commercial characteristics, viz. good colour Abhishek, stripe red petaled (Pusa Mansij) one of parent used for many varieties (Delhi Princess), rootstock for rose (R. bourboniana) and a source of fragrance (R. damascena) etc. having a potential for exploitation in breeding programmes. The incorporation of some of the traits to develop elite commercial cultivars could be enhanced by the application of molecular markers (Sasikumar et al., 2007). Characterization of rose cultivars and species using RAPD technique has been suggested as an efficient reliable and clonic alternative to the conventional methods those are based on morphological markers

 

Iwata et al. (2000) did RAPD analysis to clarify the origin of Damask roses. DNA analysis of the Damask varieties proved that they had an identical profile, indicating they were established from a common ancestor. They have never been allowed to reproduce sexually; their reproduction depends entirely on vegetative propagation. They identified three Rosa species, R. moschata, R. gallica and R. fedschenkoana, as parental species of the original hybridization that contributed to forming the four oldest Damask varieties by sequencing the internal transcribed spacer of ribosomal DNA.

 

AFLP markers were used to estimate the level of heterozygosity in the progrnies of two segregating populations obtained from crosses made between a parthenogenetic diploid Rosa hybrida L. used as female and two diploid botanic species (Rosa wichuraiana Crép. and Rosa rugosa Thunb.) used as pollinators. Ninety four percent and 41% of the AFLPs analysed were found heterozygous in R. wichuraiana and R. rugosa, respectively (Crespel et al., 2001).

 

The genetic diversity among 128 Iranian Rosa persica accessions was analyzed using AFLP technique. The results did not show relative agreement with the genotypes’ region of origin. Analysis revealed that Iranian R. persica genotypes are highly variable and genetically distinct from their origins (Basaki et al., 2009).

 

Authors (Datta and Chakrabarty, unpublished) studied RAPD of original rose cultivars and their gamma ray induced flower colour mutants. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. Other seven primers gave highly polymorphic banding patterns among the radiomutants (Table 2). The percentage of polymorphism varied from 50 (P31) to 100% (P4 and P34). PCR amplification with primer P4 clearly revealed that three bands (500, 700, 800 bp) were absent in ‘Contempo Stripe’ as compared to the other ‘Contempo’ mutants (Figure 2A, Lane 4). Similarly in ‘Contempo New’, a highly specific band of 900 bp was absent in comparison to other mutants when amplified with primer P40 (Figure 2B, Lane 2). In case of ‘Imperator’ (parent), two highly specific bands (400 and 2 200 bp) were noticed when genomic DNA was amplified with P4 primer (Figure 2A, Lane 7). Similarly, a polymorphic band of 600 bp was absent in parent cultivar ‘Frist Prize’ (parent) but present in its mutant, when RAPD marker P40 was used (Figure 2B, Lane 10). Amplification with primer P31 indicated a highly distinct and polymorphic band (800 bp) present only in ‘American’s junior Miss’, but absent in its mutant ‘Sukumari’, which can be used as a specific marker (Figure 2C, Lane 13-14) . A highly polymorphic band (800 bp) was present in the mutant cultivar of ‘Sylvia White’ when the RAPD marker P31 was used (Figure 2C, Lane 15). Similarly two highly distinct polymorphic bands (500 and 800 bp) were present in the parent cultivar ‘Sylvia’, which can be used to differentiate between the parent and its mutant when the RAPD marker P34 was used. The parent cultivar ‘Mrinalini’ and its mutant ‘Mrinalini lighter’ could not be distinguished by any primer tested. However, ‘Mrinalini Stripe’ could be distinguished from its parent and ‘Mrinalini lighter’ when RAPD marker P4 and P40 were used. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. Dendrogram was constructed based on the similarity matrix using Neighbor Joining Tree method showed three main clusters. Cluster A consists of ‘Mrinalini’ and its mutants. Cluster B consists of two subclusters, one subcluster consists of ‘Contempo’ and its mutants and the other contains ‘Imparator’ and its mutant. ‘Sylvia’ and its mutant have been placed in two different subclusters, indicating high genetic diversity from its parent which may be due to larger genomic rearrangements due to gamma irradiation. ‘First Prize’ and ‘American’s Junior Miss’ and their mutants were placed in cluster C. Cluster analysis separated rose mutants into different groups but genetic distance observed between them was low. In the present study most of the mutants appeared to be phenotypically same as parents, except flower colour. To clarify to what extent this phenotypical variation was related to the genetic level, present RAPD analysis showed noticeable differences between parents and their mutants. This indicated that somatic flower colour changes could have resulted due to some sort of genomic rearrangements rather than point mutations. The present RAPD analysis can be used not only for estimating genetic diversity present in different floricultural crops but also for correct identification of mutant/new varieties for their legal protection under plant variety right.

 

Bruna et al. (2006) used 150 sage genotypes of Salvia (Fam. Labiatae) coming from the African and the American continents for the valorisation of new germplasm and used RAPD analysis to characterise several sage species and to determine the genetic relationships among them. The primers used in their preliminary analysis allowed to discriminate all 17 samples. The dendrogram by UPGMA cluster analysis was characterised by two main clusters: in the first one all genotypes native to Mexico were grouped and, in a distinct sub-cluster, all cultivars of Salvia greggii examined were collected. Genotypes coming from Somalia, Morocco and the Canary islands were grouped together in the second major clade.

 

3 Discussion

Characterization is necessary for correct identification of plants. Correct identification is essential for various purposes like solving taxonomic problems, to trace out phylogenetic relation, identification of hybrid and mutant varieties, registration of new variety, plant variety protection etc. As mentioned, a wide range of classical and modern methods are utilized for characterization. Each method is based on several parameters. From survey of literature it is very clear that till date no single method and single parameter can justify correct identification of all plant materials. But each method and parameter have logistic contribution for characterization. It is not necessary that one should follow only one method for all characterization purpose. Nobody can recommend with present knowledge only one technique for all purpose characterization. For a modern and industrialized floriculture there is always demand and necessity for new varieties. Characterization and/or correct identification is most important for documentation of variety. Any technique which can clearly differentiate the new variety from others should be utilized whether it is classical or modern. Considering the limitations of each technique one should plan the characterization programme on need basis. There are specific instructions for characterization whether for taxonomic studies, registration to crop specific societies, plant variety protection etc. It has been clearly mentioned in the review how cytological, morphological, palynological, biochemical and molecular characters can be utilized for correct identification of plant. It has also been mentioned how technological advancement took place considering the limitation of each technique. The system is at rapid developmental stage.

 

Cytological studies have solved many taxonomical problems. Karyotypic studies were reported to be of little use in delineating cut-flower anthurium cultivars and hybrids (Marutani et al., 1993). The standard morphological and physiological characters used for registration or granting plant breeders’ rights are adequate for determining distinctness, uniformity and stability of the new mutant variety. Accurate identification of plants is desired for patent protection, however, it is difficult to distinguish phenotypitally similar cultivars using morphological and physiological methods or isozyme analyses. The limitation of these analyses is that they are observations of the phenotype. It was observed that morphological and protein markers in anthuriums can easily be affected by environmental factors and plant maturity (Kamemoto and Kuehnle, 1996). Moreover, such markers are known to be often similar in closely related ornamental cultivars and not effective for identification purposes (Rick et al., 2001) Isozymes have been widely used as biochemical markers, but their use is limited due to lack of polymorphism and a small number of loci and alleles available for analysis. Variation in isozymes banding patterns was found to be low among some cut flower anthurium cultivars indicating the limited utility of such protein markers for cultivar identification purposes (Kobayashi et al., 1987). Isozyme markers were applied to detect the genetic relationship among Calycanthaceae (Chang et al., 1995; Chen, 1995; Chen et al., 1999). Phylogenetic relationship among species of the Calycanthaceae and genetic diversity of natural populations were studied using DNA-based markers and more specifically using RAPD markers (Chen et al., 1999; Wen et al., 1996). Maintenance of huge germplasm and use of classical classification resulted chances of duplications (homonyms and synonyms) and therefore to prepare a correct core collection a wide range of parameters should be used to eliminate duplications. Therefore, it was very important for using a sensitive and credible biochemical and molecular technique to characterize and identify specific germplasm in addition to morphological and agronomic traits.

 

Use of molecular markers in addition to classical methods provides more positive identification of new varieties. Molecular markers are very versatile and can be used for a variety of purposes. Polymerase chain reaction (PCR) technology was introduced around 1990 (Saiki et al., 1988; Erlich et al., 1991). This technique enabled one to detect differences at the DNA sequence level in an easy and fast way, even if only small amounts of tissue are available. Williams et al. (1990) introduced the use of general purpose ten-mer primers in the RAPD technique. Wolff et al. (1993; 1995) showed that using the RAPD technique reproducible patterns were obtained in chrysanthemum and that with these patterns cultivars could be distinguished. The RAPD assay has been successfully used for studying genetic diversity of many crop species such rose (Debener and Mattiesch, 1996), chrysanthemum (Huang et al., 2000), Amaranthus (Faseela and Salkutty, 2007) etc. RAPD is most commonly used for the identification of cultivar/variety due to its simplicity, rapidity and requirement of only a small quantity of DNA to generate numerous polymorphisms. RAPD is a sensitive method of detecting genetic variation and has the advantage of being quick and easy, requiring little plant material, and having a high resolution. RAPD is a powerful technique for determining inter - and intra - specific DNA variation. Characterization of different ornamental cultivars and species using RAPD technique has been most successful. This technique has also been very suitable for confirmation of parent-hybrid relationship and to differentiate mutants from original variety.

 

It has been proved experimentally that DNA markers provide a powerful tool for identification of cultivars and species (Prevost and Wilkinson, 1999; Pasaˇkinskiene et al., 2000), phylogenetic evaluation (Wang et al., 1998; Blair et al., 1999), tagging and marker aided selection of agronomically important genes (Akagi et al., 1996; Ratnaparkhe et al., 1998; Hittalmani et al., 2000), linkage map construction and the mapping of quantitative traits loci (Debener and Mattiesch, 1999; Dunemann et al., 1998; Garcı´a et al., 2000; Takeuchi et al., 2001) (QTL). A number of DNA markers are necessary for these studies, especially for map construction, tagging and QTL mapping. RAPD markers have been developed to determine the hybridism of inter-specific hybrids in Lilium (Yamagishi et al., 1995; Obata et al., 2000). However, the number of DNA markers is still insufficient because when 10-base primers were used for RAPD analysis in Lilium species and hybrids only 16% of them amplified polymorphic bands (Yamagishi et al., 1995). Therefore, it was necessary to develop new DNA markers for genetic evaluation of horticulturally important traits in Lilium. In Cherokee rose, Rosa laevigata Minchx, accessions, RAPD analysis allowed the identification of the erroneous classification of the hybrid `Silver Moon' (Walker and Werner, 1997). Simple experimental procedures, requirement of minimal amount of plant tissue and the possibility of automation by the use of a laboratory robot (Terzi, 1997), RAPD analysis was found to be very useful in Alstroemeria breeding for rapid and early verification of hybridity in large seedling populations. RFLP analysis is hardly used to generate DNA markers in Lilium because of the genome size in Lilium species. Therefore, PCR-based markers such as RAPD and inter-simple sequence repeat (ISSR) markers were found to be useful to generate DNA markers in Lilium. Although 10-base random primers have been usually used for RAPD analysis in plant species, the usefulness of 15-and 20-base primers to amplify polymorphic bands in rose was shown by Debener and Mattiesch (Debener and Mattiesch, 1998, 1999). ISSR markers that amplify the genomic sequence between two simple sequence repeats (SSRs or microsatellite) using SSR primers have also generated many polymorphic DNA markers in several crops (Prevost et al., 1999; Wang et al., 1998; Blair et al., 1999; Akagi et al., 1996; Ratnaparkhe et al., 1998).

 

Many ornamental plant species develop mutants through sports. Most easily detectable are mutations with changed morphological traits specially flower colour. Mutants are often discovered by others than the breeder of the original variety. The discoverer can obtain plant breeders’ rights for such mutants when they are shown to be distinct from all existing varieties, including the original variety. In ornamentals there is regular development of new varieties through induced mutagenesis. To protect the interests of the breeder of the original variety the International Union for the Protection of New Varieties of Plants (UPOV) has introduced the concept of an ‘essentially derived variety’ (EDV). This concept, which is described in the UPOV (1991) Act of 1991, extends the scope of protection of the initial variety to any variety essentially derived from it. Therefore, all rights given to the breeder of the initial variety also apply to the EDV (http://www.upov.int/). Morphological characterization is sufficient for registration of new ‘sports’ or ‘mutants’ with changed phenotypic characters. However, these characters appear less suitable for relating mutants to the original variety, as they do not seem to allow an accurate determination of genetic conformity. Mutants usually are the result of just very few changes in the genetic makeup of a variety, the genetic similarity between original variety and mutant will be very high (close to 100%) as shown in roses (Debener et al., 2000). Molecular markers then constituted a viable alternative (Rick et al., 2001; Debener et al., 1996, 2000; Bredemeijer et al., 2002; Heckenberger et al., 2002; Ro¨ der et al., 2002; Esselink et al., 2003), as they provide a more accurate methodology for the determination of genetic similarity. Accurate identification of plants is desired for patent protection, however, it is difficult to distinguish phenotypically similar cultivars using morphological and physiological methods or isozyme analyses. The limitation of these analyses is that they are observations of the phenotype. In contrast, DNA polymorphisms offer direct observation of the plant genotype, and it has been shown that restriction fragment length polymorphism (RFLP) analysis was useful for cultivar identification in rose (Hubbard et al., 1992; Rajapakse et al., 1992). RAPD technique (Williams et al., 1990; Welsh and MCclelland, 1990; Caetano-Anolles et al., 1991) based on the polymerase chain reaction (PCR) has been used to detect polymorphism in five rose cultivars (Torres et al., 1993). It was also demonstrated that cultivars from one family, differing for flower colour, could not be distinguished with several DNA techniques (Wolff et al., 1995). RAPD fingerprinting techniques have been used for the identification of horticultural crop varieties, description of cultivar genotypes and for protecting breeder’s rights (Debener, 2001; Williams, 1990; Camlin, 2001). RAPD markers have been extensively used to distinguish intraspecific genetic variation in ornamental crops and detection of hybrids and clones (Arús, 2000; Debener, 2001a; Collins et al., 2003). Ranamukhaarachchi et al. (2001) showed that RAPD markers had the ability to identify pot-plant anthurium cultivars. In ornamentals, DNA markers are currently used to identify varieties and to analyze inter- and intra-speciÞc genetic relatedness (Rajapakse et al., 1997; Ben-Meir and Vainstein, 1994; Yamagishi, 1995; Torres et al., 1993).

 

The RAPD analysis is rapid, simple, and does not involve radioactive material. On the other hand, the RAPD technique is highly sensitive to reaction conditions, dominant in nature, and does not usually enable detection of a single locus (Williams et al., 1991; Mohan et al., 1997; Yang and Korban, 1996). Hence this marker is not useful, for example, in markerassisted breeding programs. In contrast to RAPD amplification, the ISSR markers are more feasible and reproducible (Godwin et al., 1997), and the distribution of ISSRs in the eukaryotic genome makes them highly informative (Tautz et al., 1984). They are also highly polymorphic and their use is cost effective, requiring no prior information of the sequence (Bornet et al., 2002).

 

Authors have very successfully utilized RAPD analysis for the identification of cultivars, documentation, estimation of genetic diversity, to trace out the molecular affinity of origin of unknown group and correct identification of induced mutants in Amaryllis,  Bougainvillea, Chrysanthemum and rose. The resolution of the molecular markers is much higher than morpho-agronomic characters to identify individual cultivars. The information obtained will facilitate choosing the appropriate breeding program to incorporate beneficial genes in desirable genotypes lacking the particular trait. Through the study, parentages of some of the hybrids of Amaryllis and Bougainvillea have been confirmed on one hand, and the groupings of the cultivars based on their diversity have been successfully carried out on the other hand.  They have suggested RAPD an efficient and reliable alternative to the conventional methods those are based on morphological markers.

 

The use of RFLPs for characterization overcomes the limitation of RAPD, since they provide substantial polymorphism and they can be unlimited in number. In general, the AFLP technique has been claimed to be suitable for molecular discrimination at the species level because of its extraordinary capacity to generate polymorphisms within individuals with narrow genetic distances (Han et al., 2000). AFLP technique (Vos et al., 1995) is highly reproducible and polymorphic, and it has been widely applied to investigate genetic relationship among species, closely related cultivars and even clones of plants (Loh et al., 1999; Zhang, 2000; Hagen et al., 2002; Steiger et al., 2002; Carr et al., 2003; Sensia et al., 2003; Lanteri et al., 2004; Owen et al., 2005; Zhao et al., 2005).

 

Recently a novel technique ‘DNA barcoding’ has been designed to provide rapid, accurate, and automatable species identifications by using short, standardized gene regions as internal species tags (Paul and Hebert, 2005; Janzen et al., 2005; Smith, 2005; Smith et al., 2005). This will facilitate the species discovery by allowing taxonomists to rapidly sort specimens and by highlighting divergent taxa that may represent new species. DNA barcoding will deliver species-level resolution in 95% to 97% of cases (Hebert et al., 2004b; Janzen et al., 2005; Ward et al., 2005). A DNA barcode is not just any DNA sequence-it is a rigorously standardized sequence of a minimum length and quality from an agreed-upon gene, deposited in a major sequence database, and attached to a voucher specimen whose origins and current status are recorded. Despite the potential benefits of DNA barcoding to both the practitioners and users of taxonomy, it has been controversial in some scientific circles (Wheeler, 2004; Will and Rubinoff, 2004; Ebach and Holdrege, 2005; Will et al., 2005). By contrast, DNA barcodes-by themselves-are never sufficient to describe new species.

 

It is clear that all techniques have positive and important role in characterization. TLC technique is very simple and it can be utilized for solving taxonomic problems, identification of hybrid at the seedling stage and also for identification of mutant varieties.

 

RAPD markers have numerous advantages. The analysis is rapid, simple, and does not involve radioactive material. Selecting the right sequence for the primer is very important because different sequences will produce different band patterns and possibly allow for a more specific recognition of individual strains. On the other hand, the RAPD technique is highly sensitive to reaction conditions, dominant in nature, and does not usually enable detection of a single locus (Williams et al., 1991; Mohan et al., 1997; Yang and Korban, 1996). Hence this marker is not useful, for example, in markerassisted breeding programs. The only disadvantage of using RFLPs for characterization of genetic resources is the cost of material and labour. However, RAPD will make the use of molecular markers more convenient and more economic. During the last several years, molecular markers are being utilized for the genetic improvement of a wide range of horticultural crops. Among the major traits targeted for improvement programs are disease resistance, fruit yield and quality, tree shape, floral characteristics, cold hardiness, and dormancy. Today, markers are being used for germplasm characterization, genetic mapping, gene tagging, and gene introgression from exotic species. DND bar coding for identifying plants is an exciting area for future.

 

Acknowledgement

Thanks are due to The Director, CSIR-National Botanical Research Institute, Lucknow, India for providing the facilities.

 

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