Review on Use of Molecular Markers for Characterizing and Conserving of Plant Genetic Resources

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Published on International Journal of Agriculture & Agribusiness
Publication Date: January, 2020

Abdi Alemi Ketema
Department of Plant Science, Faculty of Agriculture and Veterinary Science, Ambo University
P. O. Box 19, Ambo, Ethiopia

Journal Full Text PDF: Review on Use of Molecular Markers for Characterizing and Conserving of Plant Genetic Resources.

Abstract
Molecular markers have revolutionized and modernized our ability to characterize genetic variation and to rationalize genetic selection, being effective and reliable tools for the analysis of genome architectures and gene polymorphisms in crop plants. The area of plant genomics that has shown the greatest development with respect to the use of molecular marker technology is that of population genetics. All DNA polymorphism assays have proven to be powerful tools for characterizing and investigating Germplasm resources, genetic variation and differentiation of populations, on the basis of gene diversity and gene flow estimates. As a matter of fact, the number of loci for which DNA-based assays have been generated has increased dramatically, the majority using PCR as methodology platform. The information acquired is now being exploited to transfer different traits, including biotic stress resistances and improved quality traits, to important varieties by means of marker-assisted selection (MAS) programs. The most important challenges in the near future are certainly the molecular characterization of Germplasm collections for preserving them from genetic erosion and the identification of phenotypic variants potentially useful for breeding new varieties. Knowing the presence of useful traits, genes and alleles would help in making decisions on the multiplication of plant accessions and the maintenance of seed stocks. There are no doubts that the use of molecular markers for characterization and conservation of genetic resources should be implemented so that potentially useful genes and genotypes can be added to core collections to make them exploitable by breeders.

Keywords: Molecular markers, DNA markers.

1. INTRODUCTION
Molecular markers have proven to be powerful tools for analyzing germplasm resources and assessing genetic variation within as well as genetic differentiation among populations. In fact, the area of plant genomics that has shown the greatest development with respect to the use of DNA marker technology is that of population genetics. However, both RFLP and PCR-derived markers have also been extensively applied in plant genetics and breeding for mapping Mendelian genes and QTLs. The use of molecular markers for investigating and managing genetic resources should be implemented so that useful information on genes and traits can be added to core collections to make them exploitable by breeders (Barcaccia, 2009).
Conservation of genetic resources entails several activities, many of which may greatly benefit from knowledge generated through applying molecular marker technologies. This is the case for activities related to the acquisition of germplasm (locating and describing the diversity), its conservation (using effective procedures) and evaluation for useful traits. In all, the availability of sound genetic information ensures that decisions made on conservation will be better informed and will result in improved germplasm management. Of the activities related to genetic resources, those involving germplasm evaluation and the addition of value to genetic resources are particularly important as they help identify genes and traits, and thus provide the foundation on which to enhance use of collections. ‘Characterization’ is the description of a character or quality of an individual (Marriem 1991).
The word ‘characterize’ is also a synonym of ‘distinguish’, that is, to mark as separate or different, or to separate into kinds, classes or categories. Thus, characterization of genetic resources refers to the process by which accessions are identified or differentiated. This identification may, in broad terms, refer to any difference in the appearance or make-up of an accession. In the agreed terminology of gene banks and germplasm management, the term ‘characterization’ stands for the description of characters that are usually highly heritable, easily seen by the eye and equally expressed in all environments (IPGRI/CIP. 2003). In genetic terms, characterization refers to the detection of variation as a result of differences in either DNA sequences or specific genes or modifying factors. Standard characterization and evaluation of accessions may be routinely carried out by using different methods, including traditional practices such as the use of descriptor lists of morphological characters. They may also involve evaluation of agronomic performance under various environmental conditions. In contrast, genetic characterization refers to the description of attributes that follow a Mendelian inheritance or that involve specific DNA sequences.
In this context, the application of biochemical assays such as those that detect differences between isozymes or protein profiles, the application of molecular markers and the identification of particular sequences through diverse genomic approaches all qualify as genetic characterization methods. Because of its nature, genetic characterization clearly offers an enhanced power for detecting diversity (including genotypes and genes) that exceeds that of traditional methods. Likewise, genetic characterization with molecular technologies offers greater power of detection than do phenotypic methods (e.g. isozymes). This is because molecular methods reveal differences in genotypes, that is, in the ultimate level of variation embodied by the DNA sequences of an individual and uninfluenced by environment. In contrast, differences revealed by phenotypic approaches are at the level of gene expression (proteins).

2. LITERETURES REVIEW
2.1 Genetic markers in plant breeding:
Genetic markers are the biological features that are determined by allelic forms of genes or genetic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromosome or a gene. Genetic markers used in genetics and plant breeding can be classified into two categories: classical markers and DNA markers (Xu, 2010). Classical markers include morphological markers, cytological markers and biochemical markers. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods (southern blotting – nuclear acid hybridization, PCR – polymerase chain reaction, and DNA sequencing) (Collard et al., 2005), such as RFLP, AFLP, RAPD, SSR, SNP, etc.

2.1.1. Classical markers
2.1.1.1 Morphological markers
Use of markers as an assisting tool to select the plants with desired traits had started in breeding long time ago. During the early history of plant breeding, the markers used mainly included visible traits, such as leaf shape, flower color, pubescence color, pod color, seed color, seed shape, hilum color, awn type and length, fruit shape, rind (exocarp) color and stripe, flesh color, stem length, etc. These morphological markers generally represent genetic polymorphisms which are easily identified and manipulated. Therefore, they are usually used in construction of linkage maps by classical two- and/or three-point tests. Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding. In the green revolution, selection of semi-dwarfism in rice and wheat was one of the critical factors that contributed to the success of high-yielding cultivars. This could be considered as an example for successful use of morphological markers to modern breeding. In wheat breeding, the dwarfism governed by gene Rht10 was introgressed into Taigu nuclear male-sterile wheat by backcrossing, and a tight linkage was generated between Rht10 and the male-sterility gene Ta1. Then the dwarfism was used as the marker for identification and selection of the male-sterile plants in breeding populations (Liu, 1991). This is particularly helpful for implementation of recurrent selection in wheat. However, morphological markers available are limited, and many of these markers are not associated with important economic traits (e.g. yield and quality) and even have undesirable effects on the development and growth of plants.

2.1.1.2 Cytological markers
In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin. For instance, Q bands are produced by quinacrine hydrochloride, G bands are produced by Giemsa stain, and R bands are the reversed G bands. These chromosome landmarks are used not only for characterization of normal chromosomes and detection of chromosome mutation, but also widely used in physical mapping and linkage group identification. The physical maps based on morphological and cytological markers lay a foundation for genetic linkage mapping with the aid of molecular techniques. However, direct use of cytological markers has been very limited in genetic mapping and plant breeding.

2.1.1.3 Biochemical/ protein markers
Protein markers may also be categorized into molecular markers though the latter are more referred to DNA markers. Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution (Xu, 2010).

2.1.2. DNA markers
DNA markers are defined as a fragment of DNA revealing mutations/variations, which can be used to detect polymorphism between different genotypes or alleles of a gene for a particular sequence of DNA in a population or gene pool. Such fragments are associated with a certain location within the genome and may be detected by means of certain molecular technology. Simply speaking, DNA marker is a small region of DNA sequence showing polymorphism (base deletion, insertion and substitution) between different individuals. There are two basic methods to detect the polymorphism: Southern blotting, a nuclear acid hybridization technique (Southern 1975), and PCR, a polymerase chain reaction technique (Mullis, 1990). Using PCR and/or molecular hybridization followed by electrophoresis (e.g. PAGE – polyacrylamide gel electrophoresis, AGE – agarose gel electrophoresis, CE – capillary electrophoresis), the variation in DNA samples or polymorphism for a specific region of DNA sequence can be identified based on the product features, such as band size and mobility. In addition to Sothern blotting and PCR, more detection systems have been also developed. For instance, several new array chip techniques use DNA hybridization combined with labeled nucleotides, and new sequencing techniques detect polymorphism by sequencing. DNA markers are also called molecular markers in many cases and play a major role in molecular breeding.
Since Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP) in human linkage mapping, substantial progress has been made in development and improvement of molecular techniques that help to easily find markers of interest on a largescale, resulting in extensive and successful uses of DNA markers in human genetics, animal genetics and breeding, plant genetics and breeding, and germplasm characterization and management. Among the techniques that have been extensively used and are particularly promising for application to plant breeding, are the restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), microsatellites or simple sequence repeat (SSR), and single nucleotide polymorphism (SNP). According to a causal similarity of SNPs with some of these marker systems and fundamental difference with several other marker systems, the molecular markers can also be classified into SNPs (due to sequence variation, e.g. RFLP) and non-SNPs (due to length variation, e.g. SSR) (Gupta et al., 2001).

Table 1. Comparison of most widely used DNA marker systems in plants; Adapted from Collard et al. (2005), Semagn et al. (2006a), Xu (2010), and others.

2.2 Genetic Characterization and Its Use in Decision-Making for the Conservation of Crop Germplasm
Characterization, at present is carried out either based on morphological traits or on molecular markers (biochemical and DNA markers). Morphology-based characterization has some limitations in the accurate identification of the accessions, such as limited number of traits to characterize (Rao 2004). The characterization, conservation and exploitation of crop plant germplasm maintained in gene banks propound a number of challenges to the researchers dedicated to the investigation of plant genetic resources. Common problems include the development of strategies for sampling representative individuals in natural and experimental populations, the improvement of tools and technologies for long-term conservation and for high-throughput characterization of large numbers of stored accessions. The knowledge of the genetic diversity present in a gene bank is crucial for developing sustainable conservation strategies and it is also essential for the profitable exploitation of a gene bank by specific breeding programs. As a matter of fact, germplasm characterization of plant accessions deposited in gene banks has been limited and this likely represents a major cause for the limited adoption of conserved accessions in crop breeding programs (Ferreira 2006). Consequently, the genetic characterization of accessions belonging to a given collection and the examination of genetic relationships among them should be strengthened and perpetrated not only for maintaining but also for exploiting crop genetic resources.
Conservation of the genetic resources in the agro-ecosystem in which they have evolved (in situ conservation) is now being more widely considered, as complementary to strategies based on gene banks (ex situ conservation), for limiting genetic erosion and so preserving genetic diversity. If it is true that in situ conservation has been proposed essentially for wild relatives of cultivated plants, it is also true that when considered for major crops this alternative can very often be unfeasible from a socio-economic perspective (Negri et al. 2000; Lucchin et al. 2003). Genomic DNA-based marker assays have revolutionized and modernized our ability to characterize genetic variation and to rationalize genetic selection (Lanteri and Barcaccia 2006). Molecular markers are known as particularly effective and reliable tools for the characterization of genome architectures and the investigation of gene polymorphisms in crop plants.
Besides linkage mapping, gene targeting and assisted breeding, the plant DNA polymorphism assays are powerful tools for characterizing and investigating germplasm resources and genetic relatedness. These techniques include restriction fragment length polymorphism (RFLP) markers and PCR-based molecular markers, such as simple sequence repeat (SSR) or microsatellite markers (Morgante and Olivieri 1993), amplified fragment length polymorphism (AFLP) markers (Vos et al. 1995).

2.3 Genetic Diversity and Similarity Statistics for Characterizing Plant Germplasm at the Population Level
Genetic diversity and similarity measurements are very useful for describing the genetic structure of populations. The genetic structure of natural populations of a crop plant species is strongly influenced by the reproductive system of their individuals and the union types occurring within populations. Breeding schemes that can be adopted as well as variety types that can be constituted depend on the reproductive barriers and mating systems of plants (Barcaccia 2009). Natural populations of species that reproduce by apomixis or that propagate vegetatively are polyclonal, being composed by several genetically distinct clones and usually dominated by a few well-adapted genotypes. Therefore, genetic variation within populations is distributed among clones and most populations are characterized by different levels of differentiation among genotypes.
Landraces of self-pollinated species (e.g., bean, lentil, wheat and barley) are composed of a mixture of pure lines, genetically related but reproductively independent each other. Thus, genetic as well as phenotypic variation is mainly detectable among lines due to the presence within natural populations of fixed genotypes mainly homozygous for different alleles. Spontaneous hybridization is however possible to some extent depending on the species, environmental factors and germplasm stocks. Cultivated varieties of selfing species are usually represented by pure lines obtained by repeated self-pollination of a number of hybrid individuals stemmed from two parental lines chosen for complementary morphological and commercial traits. Maize is one of the most commercially important cross-pollinated species. In many countries, existing landraces are selected by farmers for their own use and eventually sale to neighbors. Traditionally, landraces are developed by mass selection in order to obtain relatively uniform populations characterized by valuable production locally. Synthetics are also produced by intercrossing a number of phenotypically superior plants, selected on the basis of morpho-phenological and commercial traits. More rarely, plants are also evaluated genotypically by means of progeny tests. Compared to landraces, synthetics have a narrower genetic base but are equivalently represented by a heterogeneous mixture of highly heterozygous genotypes sharing a common gene pool. However, newly released varieties are exclusively represented by F1 hybrids developed by private breeders and seed companies using inbred lines belonging to distinct heterotic groups.
Genetic characterization is providing new information to guide and prioritize conservation decisions for crop plants. The most urgently required action is the effective protection of all remaining wild ancestral populations and closely related species of crop plants, most of them now endangered. They are the only remaining sources of putative alleles of economic values that might have been lost during domestication events. It is equally important to ensure that the plant genetic resources selected for conservation include populations from the geographic areas representing the different domestication centres where high estimates of genetic diversity within and differentiation among populations are expected(Barcaccia 2009).

2.4 Using molecular characterization to make informed decisions on the conservation of crop genetic resources
Information about the genetic make-up of accessions helps decision making for conservation activities, which range from collecting and managing through identifying genes to adding value to genetic resources. Well-informed sampling strategies for germplasm material destined for ex situ conservation and designation of priority sites (i.e. identifying specific areas with desirable genetic diversity) for in situ conservation are both crucial for successful conservation efforts. In turn, defining strategies is dependent on knowledge of location, distribution and extent of genetic diversity.
Molecular characterization, by itself or in conjunction with other data (phenotypic traits or geo-referenced data), provides reliable information for assessing, among other factors, the amount of genetic diversity (Perera et al.,2000), the structure of diversity in samples and populations (shim et al.,2000, Figliuolo et al 2004), rates of genetic divergence among populations (Maestri et al.,2002) and the distribution of diversity in populations found in different locations (Maestri et al ., 2004, Perera et al.,2000).
A recent study on the genetic diversity of cultivated Capsicum species in Guatemalan home gardens compared the diversity present in an array of home gardens in the Department of Alta Verapaz with a countrywide representative sample of 40 accessions conserved ex situ in the national collection (Guzmán et al., 2005). The results showed that home gardens of Alta Verapaz (H = 0.251) contained as much diversity as the entire national ex situ collection (H = 0.281). These results thus suggest that, (1) home gardens are indeed an extremely important resource for in situ conservation of Capsicum germplasm in Guatemala, and as such they should not be neglected; (2) if further collecting activities were to be undertaken, special emphasis should be given to collecting in Alta Verapaz; and (3) additional collecting in Alta Verapaz alone could disclose novel genetic diversity that is absent from the national collection. Conservation of clonally propagated crops demands more complex and expensive procedures. If these crops are maintained on-farm, their existence is endangered by several factors, one of which being the introduction of alternative improved varieties. Conservation efforts need then to be based on solid knowledge of clonal diversity. This was the case for Abyssinian banana or ensete (Ensete ventricosum (Welw.) Cheesman) from Ethiopia, which was analysed with AFLP markers (Negash, A. et al 2002) Of the 146 clones from five different regions, only 4.8% of the total genetic variation was found between regions, whereas 95.2% was found within regions. The results led to a reduced number of clones for conservation and indicated the existence of a common practice of exchange of local types between regions, which, in its turn, emphasized the need to collect further in different farming systems.
A study on taro (Colocasia esculenta (L.) Schott) genetic diversity in the Pacific, using SSR markers, showed that many of the accessions from countries of the Pacific region were identical to those of Papua New Guinea. This indicates that originally the cultivars may have been introduced throughout the region from Papua New Guinea (Mace, E.S. et al 2005) and that collection of taro genetic diversity could focus on Papua New Guinea alone. Molecular characterization also helps determine the breeding behaviour of species, individual reproductive success and the existence of gene flow, that is, the movement of alleles within and between populations of the same or related species, and its consequences (Papa, R. & Gepts P. 2003 Papa, R. & Gepts P. 2003). Molecular data improve or even allow the elucidation of phylogeny, and provide the basic knowledge for understanding taxonomy, domestication and evolution (Nwakanma, D. C., et al 2003).
As a result, information from molecular markers or DNA sequences offers a good basis for better conservation approaches. Management of germplasm established in a collection (usually a field, seed or in vitro gene bank) comprises several activities. Usually, such activities seek to ensure the identity of the individually stored and maintained samples, to ensure the safeguarding of genetic integrity and genetic diversity and to have the material available for distribution to users. These tasks are primarily a responsibility of gene bank managers and curators, and involve the control of accessions on arrival at the facilities, as well as their continuous safeguarding for the future through regeneration and multiplication. For all these routine activities, information about the genetic constitution of samples or accessions is critical and provides possibly the most important means of measuring the quality of the work being performed. Börner et al. (2000) analysed bulk seed of wheat accessions to test their genetic integrity after 24 cycles of regeneration and after more than 50 years of storage at room temperature in a gene bank. They found neither contamination nor incorrect manipulation effects such as mechanical mixtures, but did identify one case of genetic drift in one accession.
However, in the same gene bank, a study examined the genetic constitution of rye accessions that underwent frequent regeneration. Results showed that (1) a significant number of alleles present in the original sample was lacking in the newly regenerated material, and (2) new alleles in the new material were not present in the first regeneration sample (Chebotar, S., et al 2003). Thus, the use of molecular markers can quickly help check whether changes in alleles or allele frequencies are taking place. Molecular information has been used to weigh the need for decreasing the size of germplasm collections, which otherwise would add costs to the long-term conservation of germplasm. For instance, Dean et al. (1999) used microsatellite markers to analyse the genetic diversity and structure of 19 sorghum accessions known as ‘Orange’ in the USDA’s national sorghum collection. They found two redundant groups (involving five entries) among the 19 accessions evaluated. They also found that much of the total genetic variation was partitioned among accessions. As a result, the authors concluded that the number of accessions held by the US National Plant Germplasm System (NPGS) could be significantly reduced without risking the overall amount of genetic variation contained in these holdings.
Markers were also helpful in examining genetic identities and relationships of Malus accessions (Hokanson, S.C., et al 1998.). Eight primer pairs unambiguously differentiated 52 of 66 genotypes in a study that calculated the probability of any two genotypes being similar at all loci analysed as being about 1 in 1,000 million. The results not only discriminated among the genotypes, but were also shown to be useful for designing strategies for the collection and in situ conservation of wild Malus species. Selected molecular technologies render cost-effective and comprehensive genotypic profiles of accessions (‘fingerprints’) that may be used to establish the identity of the material under study. Simultaneously, these technologies can detect contaminants (and, in the case of material mixtures, contamination with introgressed genes from other accessions or commercial varieties), as well as the presence of redundant materials (or ‘duplicates’) (McGregor, et al 2002).
Moreover, molecular data provide the baseline for monitoring natural changes in the genetic structure of the accession (Chwedorzewska,et al., 2002)or those occurring as a result of human intervention (e.g. seed regeneration or sampling for replanting in the field). Whatever the case, analysis of molecular information allows the design of strategies for either purging the consequences of inappropriate procedures or amending them to prevent future inconveniences (de Vicente, M.C. 2002). A small number of potential duplicates were identified in a core collection of cassava (Manihot esculenta Crantz) when isozyme and AFLP profiles were compared (Chavarriaga-Aguirre, P., et al 1999). The core collection had been assembled with information from traditional markers, which proved to be highly effective for selecting unique genotypes. Molecular data were used for efficiently verifying the previous work on the collection and ensure minimum repetition. Thus, gene bank managers can easily realize the potential value of using molecular methods to support and possibly modify or improve a gene bank’s operations.
A special and increasingly important role of genetic characterization is that of identifying useful genes in germplasm, that is, of maximizing conservation efforts. Because the major justification for the existence of germplasm collections is use of the conserved accessions, it is important to identify those valuable genes that can help develop varieties that will be able to meet the challenges of current and future agriculture.
Characterization has benefited from several approaches resulting from advances in molecular genetics such as genetic and QTL mapping, and gene tagging (Yamada, T.,et al 2004 , Kelly, J.D.,et al 2003). Research in this field has led to the acknowledgement of the value of wild relatives, in which modern techniques have discovered useful variation that could contribute to varietal improvement (Xiao, J., et al 1996, de Vicente, M.C. et al 1993). Knowledge of molecular information in major crops and species and of the synteny of genomes, especially conservation of gene order, has also opened up prospects for identifying important genes or variants in other crop types, particularly those that receive little attention from formal research.
Until now in India, identification and classification of Hibiscus have mainly been based on morphology and according to (Wachira F, Tanaka J and Takeda Y, 2001) even if these descriptors are useful, they show limited levels of inter and intra-varietal polymorphism and hence, may not account for all the diversity in the species. Since it is difficult to identify cultivar based entirely on these morphological features, several kinds of methods which can be used to measure levels and patterns of it is important to find an effective method to accurately identify the varieties to meet research needs. The novelty of this project lies in the use of different molecular markers with increasing order of specificity to study genetic diversity which will help in development of new cultivars of Hibiscus varieties with superior properties to meet changing agronomic requirements. polymorphism and hence, may not account for all the diversity in the species. Since it is difficult to identify cultivars based entirely on these morphological features, several kinds of methods which can be used to measure levels and patterns of it is important to find an effective method to accurately identify the varieties to meet research needs.
Modern molecular techniques have been developed in order to meet the demands of the horticulture industry genetic variation, which range from morphological characterization to various DNA-based markers such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) (Crawford, D. J.2000, Newton, A. C., et al 2002, Martinez, L.,et al 2003, Fontaine, C., et al 2004, Murtaza, N.2006, Ferdousi Begum.,2013). Identification and characterization of germplasm is essential for the conservation and utilization of plant genetic resources (Suvakanta-Barik., et al 2006). Characterization of plant with the use of molecular markers is an ideal way to conserve plant genetic resources. Molecular characterization helps to determine the breeding behaviour of species, individual reproductive success and the existence of gene flow, the movement of alleles within and between populations of the same or related species, and its consequences (Papa R and Gepts P.2003).
Molecular data improves the elucidation of phylogeny, and provide the basic knowledge for understanding taxonomy, domestication and evolution of plants (Nwakanma D C., et al 2003). Random amplified polymorphic DNA (RAPD) technique has been widely used in many plant species for varieties analysis, population studies and genetic linkage mapping (Williams J. G. K., et al 1990, Yu K., et al 1993, Rout G. R., et al 2003). Optimization of the RAPD analysis depends on selection of primers. Although, the RAPD method uses arbitrary primer sequences, many of these primers must be screened in order to select primers that provide useful amplification products. By contrast, single-locus markers are usually characterized by co-dominance (i.e. both alleles identified in heterozygous individuals) and thus are more flexible and supply more robust and comparable data (Karp, 2002).
An appropriate use of molecular markers techniques requires to clearly define the issues addressed, what type of information will be needed (on genetic diversity) and to know what the different techniques can offer not only in terms of genetic information but also resource requirements, reproducibility, adaptability for automation. Furthermore, it is of pivotal importance to consider how the information will be gathered and the way in which the data will be scored and analysed. For accurate and unbiased estimates of genetic diversity adequate attention has to be devoted to:
a. sampling strategies,
b. utilization of various data sets on the basis of the understanding of their strengths and constraints,
c. choice of genetic similarity estimates or distance measures, clustering procedures and other multivariate methods in analyses of data and
d. objective determination of genetic relationships (Mohammadi and Prasanna, 2003).
For all these reasons, choosing the most appropriate technique may be difficult and often a combination of techniques is needed to gather the information one is interested in. Up to now most conservation efforts have focused on agriculturally important crops and about one third of all ex situ accessions in gene bank represents just five species: i.e. wheat (Triticum sp.), barley, rice, maize and beans (Phaseolus spp). The relative over-representation of five species does not necessarily mean that their genetic diversity has been fully covered (Graner et al. 2003) but, on the other hand, there is significant lack of knowledge about the diversity and geographic distribution of less utilized crops as well as their wild relatives (Hammer et al. 2003). Genetic studies in selected crops have demonstrated that widespread and localised alleles occurring in the entire collection are usually contained in the core subset, with only rare localized alleles excluded (van Hintum et al. 2000). Findings suggest that, although a high variability can be found among plants, most of their genotypes belong to the same landrace locally called ‘Nostrano di Storo’ (Barcaccia et al., 2003).

3. CONCLUSION
In conclusion, the most important challenges in the near future are certainly collections for preserving crops from genetic erosion, the molecular characterization of germplasm, and the identification of useful variation in germplasm, potentially useful for breeding new varieties. Knowing the presence of useful traits, genes and alleles would help in making decisions on the multiplication of accessions and the maintenance of seed stocks for responding to an expected higher demand of materials. Such information may also help in making decisions on heterogeneous accessions, where only some genotypes may possess useful alleles. Thus, the gene bank curator may have to decide to maintain the original material as it is and separate a subpopulation carrying the desirable alleles and give it new accession numbers and management protocols. This will facilitate germplasm use and add value to the collections.

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