**1. Introduction**

456 Genetic Diversity in Plants

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The human species depends on plants. These constitute the basis for food, supply most of our needs (including clothes and shelter) and are used in industry for manufacturing fuels, medicines, fibres, rubber and other products. However, the number of plants that humans use for food is minimal, compared to the number of species existing in nature. Only 30 crops, the most outstanding of which are rice, wheat and maize, provide 95% of the calories needed in the human diet (Jaramillo & Baena, 2002). However, agricultural biodiversity is in sharp decline due to the effects of modernisation, such as concentration on a few competitive species and changes in diets. Since the beginning of agriculture, the world's farmers have developed roughly 10 000 plant species for use in food and fodder production. Today, only 150 crops feed most of the world's population, and just 12 crops provide 80% of dietary energy from plants, with rice, wheat, maize and potato providing 60%. It is estimated that about three quarters of the genetic diversity found in agricultural crops have been lost over the past century, and this genetic erosion continues (EC, 2007).

Humans need to add to their diet those crops of high yield and quality that can adapt to environmental conditions and resist pests and diseases. Advantage must be taken of native and exotic species, with nutritional or industrial potential, or new varieties must be developed. Improving crops, however, requires reserves of genetic materials whose conservation, management and use have barely begun to receive the attention that they deserve. Humans take advantage of plant genetic resources in as much as they are useful to us, which means that we must understand them, and know how to manage, maintain and use them rationally (Jaramillo & Baena, 2002). Information on genetic diversity and relationships within and among crop species and their wild relatives is essential for the efficient utilization of plant genetic resource collections for the efficient explanation of taxonomic relationships (Chan & Sun, 1997; Drzewiecki et al., 2003).

*Amaranthus* L. is a genus from *Amaranthaceae* family probably originated in America. This genus contains approximately 70 species of worldwide distribution including pigweeds, waterhemps, and grain amaranths (Sauer, 1967). The origin of various species of cultivated amaranths is not easy to trace because wild ancestors are pantropical cosmopolitan weeds (Espitia-Rangel, 1994). For human consumption there are cultivated grain amaranths – *A.caudatus, A. cruentus* and *A. hypochondriacus* and vegetable amaranths – mainly *A.dubius, A.tricolor* and *A.cruentus.* Grain amaranths are crop species of New World origin; *A. caudatus* from Andean Peru and Ecuador, *A. cruentus* and *A. hypochondriacus* from Mexico and Central America (Sauer, 1950; Drzewiecki, 2001). Nowadays, the grain amaranths are cultivated from the temperate to tropical zone and the vegetable amaranths mainly in the South Africa and South Asia (Jarošova et al., 1997).

Amaranths are very promising crops. The main reasons could be content of protein, fat and active substances. The content of seed protein is in the range 13 – 18% with very good balanced amino acids. The lysine content is relatively high in the comparison with common cereals. The content of crude proteins in leaves is from 27 to 49% in d.m. what is more than in the leaves in the spinach (Segura-Nieto, 1994). Amaranths have comparable or higher amounts of essential amino acids as whole egg protein (Drzewiecki et al., 2003). The fat content is in the range 0.8-8.0%. The linoleic acid is the predominant fatty acid, with lesser amount of oleic and palmitic acids. The oil also contains squalene, precursor of cholesterol, which is used in the cosmetics and as a penetrant and lubricant (Becker, 1994). Many compounds and extracts from amaranths possessed anti-diabetic, anti-hyperlipidemic, spermatogenic and anti-cholesterolemic effects (Sangameswaran & Jayakar, 2008; Girija et al., 2011), antioxidant and antimicrobial activity (Alvarez-Jubete et al., 2010; Tironi&Anon, 2010). Many consumers purchase amaranth because they want a wheat- and gluten-free product, like the nutritional profile of amaranth, or enjoy "exotic" foods in their diet (Brenner et al., 2000). Amaranth can be used also as a feed for pigs, hens, etc. (Pisarikova et al., 2005). From the cultivation point of view, amaranth is interesting for its heat and drought resistance and very low susceptibility to diseases and pests (Barba de la Rosa, 2009). Considering its agronomic importance, attention should be given to the cultivation, conservation, and sustainable utilization of this promising crop (Ray & Roy, 2009).

Unfortunately, amaranths are also very harmful weeds spread in all over the world. Weedy *Amaranthus* species (pigweeds) have been and continue to be a major problem in agronomic production. The weed amaranth *A. retroflexus* is considered one of the world's worst weeds. A major contributor to the noxious nature of these weedy species is their ability to efficiently adapt to the changes in agricultural management practices that are specifically designed to control and prevent colonization. For example, numerous populations of pigweeds have evolved herbicide resistance (Drzewiecki, 2001; Rayburn et al., 2005).

In the Czech Republic the cultivation of amaranth was introduced in the early 1990s (Michalova 1999; Moudry et al. 1999) and the collection of amaranth genetic resources was established in 1993 in the Czech Gene Bank. Due to the very positive effects on the human health, we try to find out genotypes suitable for the Czech conditions with utilization in the Czech cuisine. On the Czech market, there is very popular food made from amaranth flour such as chips, cookies, and breakfast cereals, etc. However, all amaranth seeds are imported into the Czech Republic from other countries. The demand for vegetable amaranth is also increasing. Presently, in the Gene Bank, there are stored 103 evaluated accessions. In the working collection (in the different stages of evaluations), there are more than 30 accessions. Seed samples of amaranth are obtained from other gene banks, universities, private subjects

waterhemps, and grain amaranths (Sauer, 1967). The origin of various species of cultivated amaranths is not easy to trace because wild ancestors are pantropical cosmopolitan weeds (Espitia-Rangel, 1994). For human consumption there are cultivated grain amaranths – *A.caudatus, A. cruentus* and *A. hypochondriacus* and vegetable amaranths – mainly *A.dubius, A.tricolor* and *A.cruentus.* Grain amaranths are crop species of New World origin; *A. caudatus* from Andean Peru and Ecuador, *A. cruentus* and *A. hypochondriacus* from Mexico and Central America (Sauer, 1950; Drzewiecki, 2001). Nowadays, the grain amaranths are cultivated from the temperate to tropical zone and the vegetable amaranths mainly in the

Amaranths are very promising crops. The main reasons could be content of protein, fat and active substances. The content of seed protein is in the range 13 – 18% with very good balanced amino acids. The lysine content is relatively high in the comparison with common cereals. The content of crude proteins in leaves is from 27 to 49% in d.m. what is more than in the leaves in the spinach (Segura-Nieto, 1994). Amaranths have comparable or higher amounts of essential amino acids as whole egg protein (Drzewiecki et al., 2003). The fat content is in the range 0.8-8.0%. The linoleic acid is the predominant fatty acid, with lesser amount of oleic and palmitic acids. The oil also contains squalene, precursor of cholesterol, which is used in the cosmetics and as a penetrant and lubricant (Becker, 1994). Many compounds and extracts from amaranths possessed anti-diabetic, anti-hyperlipidemic, spermatogenic and anti-cholesterolemic effects (Sangameswaran & Jayakar, 2008; Girija et al., 2011), antioxidant and antimicrobial activity (Alvarez-Jubete et al., 2010; Tironi&Anon, 2010). Many consumers purchase amaranth because they want a wheat- and gluten-free product, like the nutritional profile of amaranth, or enjoy "exotic" foods in their diet (Brenner et al., 2000). Amaranth can be used also as a feed for pigs, hens, etc. (Pisarikova et al., 2005). From the cultivation point of view, amaranth is interesting for its heat and drought resistance and very low susceptibility to diseases and pests (Barba de la Rosa, 2009). Considering its agronomic importance, attention should be given to the cultivation,

conservation, and sustainable utilization of this promising crop (Ray & Roy, 2009).

evolved herbicide resistance (Drzewiecki, 2001; Rayburn et al., 2005).

Unfortunately, amaranths are also very harmful weeds spread in all over the world. Weedy *Amaranthus* species (pigweeds) have been and continue to be a major problem in agronomic production. The weed amaranth *A. retroflexus* is considered one of the world's worst weeds. A major contributor to the noxious nature of these weedy species is their ability to efficiently adapt to the changes in agricultural management practices that are specifically designed to control and prevent colonization. For example, numerous populations of pigweeds have

In the Czech Republic the cultivation of amaranth was introduced in the early 1990s (Michalova 1999; Moudry et al. 1999) and the collection of amaranth genetic resources was established in 1993 in the Czech Gene Bank. Due to the very positive effects on the human health, we try to find out genotypes suitable for the Czech conditions with utilization in the Czech cuisine. On the Czech market, there is very popular food made from amaranth flour such as chips, cookies, and breakfast cereals, etc. However, all amaranth seeds are imported into the Czech Republic from other countries. The demand for vegetable amaranth is also increasing. Presently, in the Gene Bank, there are stored 103 evaluated accessions. In the working collection (in the different stages of evaluations), there are more than 30 accessions. Seed samples of amaranth are obtained from other gene banks, universities, private subjects

South Africa and South Asia (Jarošova et al., 1997).

or from collecting missions from all over the world. It corresponds with international agreements and with The Czech National Programme on Conservation and Utilization of Plant Genetic Resources and Agro-biodiversity. For maintenance and utilization of plant genetic resources of amaranths, it is very important to know them from all sides. Genetic resources studies are oriented on evaluation of the most important biological characters, with respect to the effective utilization of genetic resources in breeding and agricultural practice. Good characterization and evaluation of genetic resources under conditions similar to those of their origin can provide breeders and users with valuable information on effective utilization of genetic resources for the breeding programmes and utilization. Characterization of genetic resources is focused mainly on morphological characters. The evaluation consists of data on plant growth and development, characteristics of plant stand, analysis of yield elements, etc. (Dotlačil et al., 2001). First steps of evaluations after seed samples receiving, are field evaluations. The phenological and morphological evaluation such as length of vegetation, plant height, length of inflorescence, colour of inflorescence, type of inflorescence, etc., is performed during vegetation. The length of vegetation is very important for amaranth cultivation in the Czech Republic, because many of the amaranths genotypes are sensitive to day-length. They remain in the vegetative period for a long time and create seeds after day-shortening (NRC, 1984). In the Czech Republic, they flower in the second half of September. Because the early frost, they cannot mature their seeds.

For genetic improvement of *Amaranthus*, germplasm collections will play a key role as well. However, only limited information is available on intra- and inter-specific genetic diversity and relationships within *Amaranthus* germplasm collections (Chan & Sun, 1997). In spite of the fact that it has been the object of many studies, the genus *Amaranthus* is still poorly understood, being widely considered as a "difficult" genus. Currently, the taxonomic problems are far from being clarified especially because of the widespread nomenclatural disorder caused chiefly by repeated misapplication of names (Costea et al., 2001) which is shown in Table 1. Due to variation of morphological characters, accurate classification of amaranth genetic resources is not always possible (Transue et al., 1994).

For preliminary identification of *Amaranthus* species, the useful tool can be the number, thickness, orientation and density of branches in inflorescences. The flowers are arranged in small and very contracted cymes, which are agglomerated, axillary and additionally arranged in racemose or spiciform terminal, large and complex synflorescences. Although extremely variable, there is usually a tendency towards a morphological "type" (Costea et al., 2001).

The colour of the seeds is commonly dark-brown to blackish, or whitish-yellowish, sometimes with reddish nuances at the species cultivated as cereals. Many cultivars of *A. caudatus* have pink cotyledons visible through the seed coat. The colour may be uniform or not, in the last case usually with the marginal zone paler. Weedy species and species used as a vegetable have mostly black or dark seeds (Costea et al., 2001; Jarošová et al., 1997; Das, 2011).

Many species of the genus are greatly affected by environmental factors (nutritional elements, water availability, light conditions, injurious factors, etc. exhibiting a great morphological variability with little taxonomic significance (Costea et al., 2001). All the above mentioned characteristics are useful for the taxonomy of the genus but difficult to use for the current identification of taxa (Costea et al., 2001). Also it is dependent on the cultivation in the field conditions. In the case of a gene bank, when seed samples are received, it is necessary to sow them in the field conditions for the morphological and phenological evaluations. But in the case of weedy species, it would be better to know, if the samples are not harmful weeds. We need to exclude weeds from our collection.

Many different methods of identification have been used for evaluation of amaranth diversity. RAPD analysis was successful in the investigation of the relationships of four *A. hypochondriacus* varieties (Barba de la Rosa et al., 2009). AFLP markers were successfully used to determine species what demonstrated taxonomic ambiguity at the basic morphologic level (Costea et al., 2006). Other methods such as ITS, ISSR and isozyme profile were used to get exhaustive view of interrelationship and relative closeness among amaranth species (Das, 2011; Xu & Sun, 2001). Also other methods such as electrophoresis profiles of proteins have been successfully used to clarify the taxonomy of many families. There was published, that electrophoresis can also be used to characterize the seed protein profiles of species and cultivars, compare cultivars of different geographical origin, and provide taxonomically useful descriptors that are substantially free from environmental influence. This method is rapid, relatively cheap, largely unaffected by the growth environment and eliminate to grow plant to maturity (Juan et al., 2007; Jugran et al., 2010). Drzewiecki (2001) used SDS PAGE of urea-soluble proteins of amaranth seeds for distinguishing both – species and their cultivars. Samples of seven species were divided into three groups by protein patterns according to similarity. According to solubility, Osborne (1907) divided proteins into four classes: albumins soluble in water, globulins soluble in high salt concentration, prolamins soluble in aqueous alcohol and glutelins soluble in acid or alkaline solutions (Segura-Nieto et al., 1994). The division into four protein fractions brings the possibility to see the differences among seed samples more clearly. The first general characterization of the protein fraction spectra of amaranth species was performed by Gorinstein et al. (1991) and Drzewiecki et al. (2003). Finally, Dzunkova et al. (2011) set up the methodology for clear identification of the amaranth species using glutelin protein fraction. The washing off water, salt- and alcohol- soluble proteins in protein fraction separation process makes polymorphic peaks of amaranth glutelins to be distinguished very easily.

SDS PAGE has been the traditional method for analysing glutenin subunit composition of wheat, but the procedure is slow, laborious and non-quantitative. The chip microfluidic technology, based on capillary electrophoresis, provides new opportunities in analysis of wheat HMW-GSs. This procedure is rapid, simple to operate, enabling automatic and immediate quantitative interpretation. Other advantages over traditional gel electrophoresis are lower sample and reagent volume requirements and a reduced exposure to hazardous chemicals (Bradova & Matejova, 2008).

In this work, we focused on evaluation for precise determination of amaranth genetic resources in the Czech Gene Bank. One of our aims was to separate amaranth species according to protein patterns and to verify our hypothesis of different protein fraction pattern based on species and variety. We compared spectra of storage proteins and their fractions of wild weedy and cultivated species of amaranths and verified the suitability of this method for species identification in our collection.

for the current identification of taxa (Costea et al., 2001). Also it is dependent on the cultivation in the field conditions. In the case of a gene bank, when seed samples are received, it is necessary to sow them in the field conditions for the morphological and phenological evaluations. But in the case of weedy species, it would be better to know, if the

Many different methods of identification have been used for evaluation of amaranth diversity. RAPD analysis was successful in the investigation of the relationships of four *A. hypochondriacus* varieties (Barba de la Rosa et al., 2009). AFLP markers were successfully used to determine species what demonstrated taxonomic ambiguity at the basic morphologic level (Costea et al., 2006). Other methods such as ITS, ISSR and isozyme profile were used to get exhaustive view of interrelationship and relative closeness among amaranth species (Das, 2011; Xu & Sun, 2001). Also other methods such as electrophoresis profiles of proteins have been successfully used to clarify the taxonomy of many families. There was published, that electrophoresis can also be used to characterize the seed protein profiles of species and cultivars, compare cultivars of different geographical origin, and provide taxonomically useful descriptors that are substantially free from environmental influence. This method is rapid, relatively cheap, largely unaffected by the growth environment and eliminate to grow plant to maturity (Juan et al., 2007; Jugran et al., 2010). Drzewiecki (2001) used SDS PAGE of urea-soluble proteins of amaranth seeds for distinguishing both – species and their cultivars. Samples of seven species were divided into three groups by protein patterns according to similarity. According to solubility, Osborne (1907) divided proteins into four classes: albumins soluble in water, globulins soluble in high salt concentration, prolamins soluble in aqueous alcohol and glutelins soluble in acid or alkaline solutions (Segura-Nieto et al., 1994). The division into four protein fractions brings the possibility to see the differences among seed samples more clearly. The first general characterization of the protein fraction spectra of amaranth species was performed by Gorinstein et al. (1991) and Drzewiecki et al. (2003). Finally, Dzunkova et al. (2011) set up the methodology for clear identification of the amaranth species using glutelin protein fraction. The washing off water, salt- and alcohol- soluble proteins in protein fraction separation process makes polymorphic peaks of amaranth glutelins to be

SDS PAGE has been the traditional method for analysing glutenin subunit composition of wheat, but the procedure is slow, laborious and non-quantitative. The chip microfluidic technology, based on capillary electrophoresis, provides new opportunities in analysis of wheat HMW-GSs. This procedure is rapid, simple to operate, enabling automatic and immediate quantitative interpretation. Other advantages over traditional gel electrophoresis are lower sample and reagent volume requirements and a reduced exposure to hazardous

In this work, we focused on evaluation for precise determination of amaranth genetic resources in the Czech Gene Bank. One of our aims was to separate amaranth species according to protein patterns and to verify our hypothesis of different protein fraction pattern based on species and variety. We compared spectra of storage proteins and their fractions of wild weedy and cultivated species of amaranths and verified the suitability of

samples are not harmful weeds. We need to exclude weeds from our collection.

distinguished very easily.

chemicals (Bradova & Matejova, 2008).

this method for species identification in our collection.


1according to Mansfeld'sEncyclopedia of Agricultural and Horticultural Crops (Hanelt& IPGCPR, 2001) 2according to IPNI (2011)

Table 1. Synonyms of selected amaranth species
