Marcio Elias Ferreira
11.1 SUMMARY
The effective use of crop genetic resources stored in gene banks by breeding programmes is limited. The number of accessions deposited in gene banks, however, is continuously growing. Slow germplasm characterization has been pointed out as a major cause of this discrepancy. Molecular marker technology offers opportunities to increase the use of crop genetic resources deposited in gene banks. High throughput genotyping of germplasm accessions allows for the examination of genetic relationships and sampling of core collections representative of the allelic richness of the gene bank. Core collections can be used for intensive phenotypic evaluation of traits of agronomic importance and re-sequencing of candidate genes associated with their control. Single nucleotide polymorphism (SNP) variation between the accessions of the collection can be associated with phenotypic variation. The integration of genomic technology and the characterization of germplasm banks will play an important role in the sustainable conservation and increased use of crop genetic resources.
11.2 INTRODUCTION
The conservation, management and use of germplasm maintained in gene banks poses a number of challenges to the researchers dedicated to the investigation of plant genetic resources. Common problems include, for example, the development of strategies for sampling representative individuals in natural populations, the improvement of tools and technology for long-term conservation or high throughput genetic analysis of an increasingly high number of stored acessions. Central to sustainable conservation is the knowledge of the genetic diversity present in a gene bank. This is also key to the potential exploitation of gene banks by breeding programmes. Therefore, the characterization of the accessions maintained in the collection and the examination of the genetic relationship between them is important for the sustainable conservation and increased use of crop genetic resources. Germplasm characterization of plant accessions deposited in gene banks has been limited and is probably a major cause for the limited use of accessions in breeding programmes.
Germplasm characterization refers to the observation, measurement and documentation of heritable plant traits in a collection. The resulting data allows for identifying and classifying accessions, and building a catalogue of descriptors with embedded biological information that is essential for collection management or for direct use in agriculture. The characterization of plant germplasm, therefore, aims at describing and understanding the genetic diversity of the organisms under study. Today, germplasm characterization has been developed based mostly on morphological descriptors, agronomic descriptors (traits) and molecular marker technology.
The current characterization of plant germplasm collections relies strongly on morphological descriptors. Morphological descriptors are reliable, easy to study and relatively low cost to evaluate. However, the use of morphological descriptors presents some limitations:
limited polymorphism, lowering the potential success of an extended classification approach, which would require a high number of descriptors in order to compensate for the small number of morphotypes;
potential environmental influence on the phenotype, making the process of evaluation and information exchange even more complex. Here one should be careful with false positives when the environment affects specific morphotypes;
the impact of a morphological descriptor in the viability of the individual.
Germplasm characterization based on agronomic traits, on the other hand, is particularly useful in crops of economic importance. The amount of data related to agronomic traits that is available by crop germplasm evaluation is limited. Due to reasons that include relatively high costs and the difficulties of large-scale experimental trials, the use of agronomic evaluation to characterize germplasm collections is far from the actual need of uncovering the phenotypes of agronomic interest in accessions of a collection. More should definitely be done in this area to stimulate a higher use of stored germplasm in breeding programmes. A complete agronomic trait evaluation of crop germplasm in the next few years, seems to be pratically impossible to achieve, however.
Recently, germplasm characterization based on molecular methods has experienced great development. The methods that reveal sequence polymorphism in the DNA structure, known as molecular markers, fomented a revolution in the speed and quality of large-scale plant germplasm characterization. The routines of germplasm characterization have relied increasingly on the use of such methods. Among all different classes of molecular markers available for evaluating genetic diversity, microsatellites or simple sequence repeats (SSRs) (Tautz, 1989; Weber and May, 1989) are well known for their potentially high information content and versatility as molecular tools (Ferreira and Grattapaglia, 1996). Hundreds of microsatellite markers have been developed for different species, having their chromosomal location and polymorphism levels determined. The use of fluorescently labelled microsatellite marker panels greatly increased the capacity of semi-automated genotyping of a large number of accessions, allowing for a faster and highly informative characterization of accessions deposited in gene banks.
The objective of this chapter is to discuss how molecular analysis of gene banks can impact on sustainable conservation and increased use of crop genetic resources.
11.3 THE BRAZILIAN UPLAND RICE GENE BANK: A CASE STUDY
Classical attempts to directly use accessions deposited in germplasm banks in breeding programmes have been somehow limited to the identification of sources of genes of interest, such as resistance to plant pathogens or pests, and the transfer of those genes to elite material of the programme. Linkage drag has usually restrained the breeder from the initiative of using accessions from the germplasm bank since the advanced material of the breeding programme is far more attractive than the inherent risk of using germplasm of unknown pedigree, performance or phenotypic adaptation. When the risk is taken, the accessions are usually submitted to a screening condition that reveals the presence of a gene of interest and typically a backcross programme is initiated to tranfer the gene to an elite line or cultivar. This procedure, however, is usually limited to traits of monogenic control. Complex traits require more elaborate methods, such as the inbred backcross line (Wehrhahn and Allard, 1965) or the advanced backcross quantitative trait locus (AB-QTL) mapping, which integrates the classical backcross method with linkage information based on molecular markers (Tanksley and Nelson, 1996). This procedure has been effective to demonstrate how to transfer quantitative trait loci (QTL) identified in wild relatives deposited in gene banks to elite lines or cultivars of a cultivated species (Xiao et al., 1996; Brondani, Rangel and Ferreira, 2002).
More recently, genome sequencing opened the possibility of finding candidate genes for complex traits (candidate QTLs) in the genome of a crop species, and using the germplasm bank, identifying genes of agronomic importance. Marker technology allied with detailed phenotypic characterization of germplasm banks can therefore be potentially useful in gene discovery. The use of this approach is based on the integration of knowledge of genetic diversity of the germplasm bank, genomic information available for the chosen crop and intensive phenotyping of the trait of choice. A case study on how molecular analysis of gene banks can have an impact on sustainable conservation and increased use of crop genetic resources is discussed below using rice as a model.
Rice has one of the largest ex situ germplasm collections in the world, comprised of accessions of cultivated (Oryza sativa L. and O. glaberrima Steud.) and wild species. Because of its widespread use in the planet, O. sativa is considered the most important cultivated species of rice. Rice is a daily staple food in the diet of billions of people in the world, including millions of Brazilians. The production and consumption of rice in Brazil is comparable to figures observed in some Asian countries. About 60 percent of the rice production in the country is irrigated (O. sativa spp. indica), while upland rice (O. sativa spp. japonica) accounts for 40 percent of the total. A national gene bank of japonica rice is maintained by Embrapa. This gene bank contains approximately 4 000 accessions, including landraces collected in villages and isolated rural areas of the country, where rice has been cultivated since its introduction in Brazil centuries ago. This germplasm is a source of genes that control traits of economic importance, such as drought tolerance and resistance to plant pathogens. Most of these rice accessions have not yet been studied in depth. The phenotypic and genotypic characterization of the collection is just beginning.
The traits of economic interest in plants almost always involve a complex genetic control, generally determined by various genes, and a strong interaction with the environment. The genetic control of drought tolerance in plants, for instance, which is a trait of great importance for the genetic improvement of rice and other grass species, is typically quantitative. Several QTLs showing different effects on the phenotypic variation are involved in the control of drought tolerance in rice. Some QTLs might have a strong or major effect on the phenotype; others might have a small but still significant effect on the trait. The genetic and physical mapping of the genome, based on the use of molecular markers and cloned segments of bacterial artificial chromosomes (BACs), has allowed for great advances in the understanding of the genetic control of quantitative traits, at times making it possible to isolate the genes associated with quantitative trait control by the positional cloning approach (Tanksley, Ganal and Martin, 1995). This approach overcame the classic problem of lack of information regarding the gene product of loci of economic interest (usually a quantitative trait), which is very useful when reverse genetics is possible. In other words, lack of knowledge of the protein related to a trait could be circumvented in gene isolation studies using detailed genetic mapping of the region around the locus of interest, followed by sequencing selected clones of large genomic insert libraries and the identification of the desired gene by complementation studies. Genetic mapping allows for the breakdown and examination of the genetic control of a complex trait, making it possible to identify the number of genes that are involved in the expression of a certain trait, to locate these genes or gene regions on the chromosomes, measure their impact on the phenotypic variation, and understand gene interaction in the manifestation of the trait.
An alternative to positional cloning for the isolation of genes controlling quantitative traits has been advocated with the availability of massive DNA sequence data obtained for some species by whole genome sequencing projects. The approach relies on the power of association tests to verify the correlation of sequence variation at candidate genes with phenotypic variation for the trait of interest (Risch, 2000). Recent developments in the area of human genetics, particularly in the identification of factors that cause genetic diseases, have been based on genetic association tests. These tests have been used for many years in genetic studies, especially in classical medical genetics, but the genomic information gathered recently affords an opportunity for the development of new strategies of analysis. The association tests seek to identify nucleotide variation observed at a genomic region with phenotypic variation for a trait, leading to the identification of DNA regions that control the phenotypes of interest. These tests do not rely on segregating populations, but rather on the evaluation of allelic variation observed in the prospective loci in relation to the phenotype of interest in natural populations. In view of the developments of genomic technology, allelic variations are currently detected by DNA sequencing, characterized as mutations and small insertions and/or deletions (indels) known as SNPs (Collins, Guyer and Chakravarti, 1997).
The rice genome was sequenced in its entirety in 2002 (Goff et al., 2002; Yu et al., 2002), with a great part of the sequences deposited in public databases over the last few months. It is logical to assume that the use of bioinformatics and the re-sequencing of a great number of genes located on some rice chromosomes can be an effective way to correlate the allelic variability detected at these loci with the phenotypic variability for resistance to abiotic stresses. Therefore, re-sequencing of candidate genes in a set of germplasm accessions and phenotypic evaluation of the same set for drought tolerance could be an interesting approach to discover genes involved in the control of traits of economic importance in grass species. Candidate genes found in regions in the vicinity of QTLs for response to the abiotic stresses are immediate targets for drought tolerance gene isolation in this species. Genotypic and phenotypic analysis can be carried out with accessions sampled in germplasm collections, especially landraces of upland rice showing genetic variability for drought tolerance. For this purpose, the following steps should be considered:
Genetic maps making ample use of molecular markers (especially microsatellites) are constructed in order to locate genomic regions (QTLs) associated with the control of drought tolerance in grasses. Several populations of grass species (rice, maize and sorghum) segregating for alleles associated with the control of drought tolerance are then examined. Microsatellite markers are used to cover the entire rice genome by means of large-scale genotyping and mapping. Genotyping is performed using high throughput equipment such as automatic DNA sequencers. By multiplexing many polymerase chain reaction (PCR) products into a single lane on a polyacrylamide gel, a large increase in genotyping throughput is achieved. Multiplexed PCRs greatly increase the amount of collected information in a single reaction. The segregating populations are then submitted to extensive phenotypic evaluation through different bioassays and replicated experiments in the field. The genotypic and phenotypic information is used to identify the genomic regions that control the traits of interest (QTL). The QTL x environment interactions are estimated using the data obtained in different locations. In addition, anchor-markers used for mapping purposes on different grass species are employed in the construction of genetic maps of rice and other grass species, allowing the information obtained in the rice genome to be transferred to these other species via syntenic map analysis, and vice-versa (Moore et al., 1995). Synteny analysis therefore provides a comparative look at the genetic control of drought tolerance in the genome of different species.
The genomic regions delimited by QTL analysis are studied in detail using the rice genome sequence database. The microsatellite markers that flank a specific drought tolerance QTL in a chromosome are used to delimit in the physical map the BAC clones that potentially harbour the gene(s) associated with the trait. All open reading frames (ORFs) and controlling segments in the region are re-annotated and candidate genes are selected for detailed sequence analysis. Genotypic variation (SNPs) are detected after sequence alignment.
The candidate genes selected in the vicinity of a QTL are re-sequenced in a sample of rice variety accessions selected from the gene bank. This sample represents a core collection (Frankel and Brown, 1984) of the germplasm studied, i.e. it should represent the maximum possible level of the genetic diversity existing in a complete germplasm bank.
In-depth phenotypic evaluation of a core collection is undertaken to access diversity of the agronomic trait (drought tolerance) present in the upland rice gene bank. The measurement of drought tolerance is based on indirect observations of morphological, physiological, biochemical and agronomical responses of the plant variety to abiotic stress. Root morphology, for example, is usually dissected in many ways to provide clues on the ability of the plant to survive and compete under drought conditions. In this case, the root system architecture, as measured by the number and arrangement of secondary roots, dry matter weight, root abundance in different layers of soil, and root penetration ability, inter alia, are quantified as an indirect way to access drought tolerance. These measurements can be statistically treated as components of drought tolerance. It is also very common to use grain yield under drought conditions to compare genotypic reponses of different plant varieties to water-deficit stresses. Enzyme activity and metabolic bioassays can also offer biochemical means of studying drought tolerance in laboratory conditions.
It is expected that significant associations between sequence polymorphism and phenotypic variation will indicate and allow the isolation of genes that control drought tolerance in rice. This information could be readily transferred to other grass species such as maize and sorghum. The breeding programmes will certainly benefit from the data based on the molecular analysis of gene banks, stimulating an increase in use of stored genetic resources. The integration of genomic technology and characterization of germplasm banks will play an important role in the sustainable conservation of gene banks.
11.4 REFERENCES
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Collins, F.S., Guyer, M.S. & Chakravarti, A. 1997. Variations on a theme: cataloging human DNA sequence variation. Science, 278: 1580–1581.
Ferreira, M.E. & Grattapaglia, D. 1996. Introdução ao uso de marcadores moleculares em análise genética. Brasilia, Brazil, Embrapa-SPI. 220 pp.
Frankel, O.H. & Brown, A.H.D. 1984. Current plant genetic resources - a critical appraisal. In Genetics: new frontiers, 4: 1–11. New Delhi, India, Oxford & IBH Publishing Co.
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Tanksley, S.D. & Nelson, J.C. 1996. Advanced backcross QTL analysis: a method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet., 92:191–203.
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Yu, J., Hu, S., Wang, J., Wong, G.K., Li, S., Liu, B., Deng, Y., Dai, L., Zhou, Y., Zhang, X. et al. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science, 296: 79–92.
M.Carmen de Vicente, Felix Alberto Guzmán, Jan Engels and V. Ramanatha Rao
12.1 SUMMARY
This chapter first presents a brief update on the progress made in using genetic characterization to guide decision-making for several conservation activities. It will then focus on the attractive prospects offered by molecular characterization to enhance germplasm use, which is the ultimate purpose of conserving diversity of genetic resources.
12.2 INTRODUCTION
Conservation of genetic resources entails several activities, many of which may greatly benefit from knowledge generated through applying molecular marker technologies. The same applies to activities related to the acquisition of germplasm (locating and describing the diversity), its conservation (using effective procedures) and its evaluation for useful traits. The availability of sound genetic information ensures that decisions made on conservation will be better informed and 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 because they help identify genes and traits, and thus provide the foundation on which to enhance the use of collections.
“Characterization”is the description of a character or quality of an individual (Merriam-Webster, 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. This genetic connotation will be used in this chapter.
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. In addition, 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).
12.3 USING MOLECULAR CHARACTERIZATION TO MAKE INFORMED DECISIONS ON THE CONSERVATION OF CROP GENETIC RESOURCES
Information about the genetic make-up of accessions contributes towards decision-making for conservation activities, which range from collecting, managing and 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 depends 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 and Jørgensen, 2000; Figliuolo and Perrino, 2004), rates of genetic divergence among populations (Maestri et al., 2002) and the distribution of diversity in populations found in different locations (Ferguson, Bramel and Chandra, 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 (i) home gardens are indeed an extremely important resource for in situ conservation of Capsicum germplasm in Guatemala, and as such should not be neglected; (ii) if further collecting activities were to be undertaken, special emphasis should be given to collecting in Alta Verapaz; and (iii) 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, including the introduction of alternative improved varieties. Conservation efforts thus need 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 amplified fragment length polymorphism (AFLP) markers (Negash et al., 2002). Of the 146 clones from five different regions, only 4.8 percent of the total genetic variation was found between regions, whereas 95.2 percent 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 simple sequence repeats (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 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 and Gepts, 2003). Molecular data improve or even allow the elucidation of phylogeny, and provide the basic knowledge for understanding taxonomy, domestication and evolution (Nwakanma 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. These activities usually seek to ensure the identity of the individually stored and maintained samples, the safeguarding of genetic integrity and genetic diversity, and material available for distribution to users. These tasks, which are primarily the responsibility of gene bank managers and curators, involve the control of accessions on arrival at the facilities and 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 possibly provides the most important means of measuring the quality of the work being performed.
Börner, Chebotar and Korzun (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. By splitting its germplasm samples into either almost or completely pure lines, i.e. accessions, the IPK-Gatersleben gene bank (Germany) is expected to have contributed to this very positive finding (J. Engels, personal communication, 2005).
In the same gene bank, a study examined the genetic constitution of rye accessions that underwent frequent regeneration. Results showed that a significant number of alleles present in the original sample was lacking in the newly regenerated material and new alleles in the new material were not present in the first regeneration sample (Chebotar 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 national sorghum collection of the United States Department of Agriculture (USDA). 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 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 one in a thousand 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, in addition to the presence of redundant materials (or “duplicates”) (McGregor et al., 2002), these technologies can detect contaminants, and in the case of material mixtures, contamination with introgressed genes from other accessions or commercial varieties as well. Moreover, molecular data provide the baseline for monitoring natural changes in the genetic structure of the accession (Chwedorzewska, Bednarek and Puchalski, 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, 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 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. The taro core collection for the Pacific region was treated in a similar manner (Mace et al., 2005). Thus, gene bank managers can easily realize the potential value of using molecular methods to support and possibly modify or improve the operations of a gene bank.
A special and increasingly important role of genetic characterization is identifying useful genes in germplasm, that is, maximizing conservation efforts. Because the major justification for the existence of germplasm collections is for the use of the conserved accessions, it is important to identify the 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 quantitative trait locus (QTL) mapping, and gene tagging (Yamada et al., 2004; Kelly 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 et al., 1996; de Vicente and Tanksley, 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.
12.4 FUTURE TRENDS
Most marker technologies target genomic regions, which are selectively neutral; some technologies, however, target specific genes. The neutrality of markers is suitable for most uses in germplasm conservation and management. However, when the interest of conservation lies specifically in the diversity of traits of agronomic importance, some questions remain on the markers' representativeness. In such cases, the markers able to detect functional diversity are more suitable for characterizing germplasm collections.
Germplasm in collections can undergo structural molecular characterization, i.e. based on the building blocks of the DNA sequence, and functional molecular characterization, i.e. based on the identification of genes and their functions. Such characterization permits access to the raw materials - the genes - for nearly all the objectives of today's and tomorrow's breeding programmes. The information gathered from structural characterization not only provides increased clarity on existing genetic diversity and its organization in individuals, but also determines sample and population organization that may ultimately form the basis for functional characterization.
The increasing number of sequencing projects has resulted in an increased opportunity to produce expressed sequence tags (ESTs) to which gene functions may be assigned. Moreover, such projects allow for the compilation of an enormous amount of sequence data that can be used to develop markers linked to specific genes, which in turn may help identify novel functional variation (Han et al., 2004; NCBI, 2001).
In addition, the development of novel technologies continues. This usually means decreased costs - a very significant point for their application in the tasks of conserving genetic resources, which tend to involve large numbers of samples and to have difficulties in sourcing needed funds. Other improvements involve increasing the throughput, both in number of markers analysed and in number of samples, and simplifying technologies.
New developments are also taking place in designing better approaches to access new and useful genetic variation in collections, namely, allele mining and association genetics. Allele mining focuses on the detection of allelic variation in important genes and/or traits within a germplasm collection (Simko et al., 2004b). If the targeted DNA (either a gene of known function or a given sequence) is known, then the allelic variation (usually point mutations) in a collection can be identified using methods developed for the purpose (Lemieux, Aharoni and Schena, 1998).
Association studies of artificial progenies are an alternative to segregation analysis for identifying useful genes by correlation of molecular markers and a specific phenotype (Gebhardt et al., 2004). Association studies can be performed on a germplasm collection and also on other materials as long as significant linkage disequilibrium (LD) exists, for example, breeding materials. It may be especially useful for those crops where appropriate populations for genetic analysis cannot be obtained or their production is too time-consuming (Simko et al., 2004a). It is also useful for those crops for which sequence information does not exist and is unlikely to be available soon.
12.5 THE CHALLENGES AHEAD
The importance of the variation captured in genetic resources in allowing evolution and/or facilitating plant breeding has been long recognized. However, appreciating the variation held in collections is not sufficient. Conservation of genetic resources needs to go hand in hand with enhanced use of the conserved material. Identifying and making available the allelic variation that makes up the genotype and phenotype provide the groundwork on which genetic resources can be used in, for example, plant breeding.
The number of accessions held collectively by all Consultative Group on International Agricultural Research (CGIAR) gene banks is estimated at almost 600 000 (FAO, 1998). Together with the collections established by national programmes worldwide, this number reaches almost 6 million (FAO, 1998). Without doubt, these genetic resources collections, together with uncollected germplasm and that held in situ and on-farm, harbour abundant quantities of hidden allelic variants. The challenge is to unravel the mysteries of this variation so that it can be used for the benefit of humankind.
Gene banks hold large numbers of accessions, particularly of staple crops. Modern improvements in equipment and procedures allow considerable sample throughput. This can be costly. However, the more a technology develops, the lower its costs will be per data point and per sample. Nevertheless, the higher the throughput used, the higher the number of data points obtained. This requires adequate equipment for handling and storing and expertise for handling and analysing in order to draw adequate results from the investment.
One possible avenue for ensuring broader benefits from molecular characterization is the establishment of international collaboration for particular crops. Although equipment and expertise cannot currently be readily available worldwide, characterization networks are possible. In addition to carrying out the laboratory work, such networks would also facilitate access to information, thus fostering closer links between curators, breeders and molecular scientists. At the same time, countries with little expertise or equipment can make steady progress in both areas, thus making better use of the genetic resources that they hold (Hamon, Frison and Navarro, 2004).
More and more, technologies have increased throughputs, which generally means the generation of progressively larger amounts of data. Such data should not languish unused. If gene banks equip themselves with the latest technologies, then they should be able to translate such data into scientific knowledge. To do so, they need not only laboratory technical expertise, but also bioinformatics staff. This means that through their molecular work, gene banks may keep not only live plant materials, but also DNA and data. Hence, the banks may develop appropriate new roles as providers of genetic resources and their accompanying data in an array of forms. Such broadening of the gene banks' roles implies that their clientele will also expand from plant breeders to include molecular geneticists, molecular biologists and even bioinformaticists. The expanded range of roles may even lead to including activities related to phenotyping, a type of characterization beyond the traditional description of morphology and general field performance. Phenotyping is very much linked to the usefulness of good molecular characterization, together forming the basis of progress in modern genomics research (de Vicente, 2004).
12.6 CONCLUSIONS
The most important challenges in the near future are certainly the identification of useful variation (real or potential) in germplasm and its use in guiding conservation decisions. Knowing the presence of useful genes and alleles would help in making decisions on the multiplication of accessions and the maintenance of seed stocks when responding to an expected higher demand for materials. Such information may also help in making decisions on heterogeneous accessions where only some genotypes may possess useful alleles. The gene bank curator may have to decide on maintaining the original material as is and separating a subpopulation carrying the desirable alleles as well as giving it new accession numbers and management protocols. This will facilitate germplasm use and add value to the collections.
Similarly, genotypes with known and interesting genes and alleles can be added to core collections to make them more useful to the user community. To promote use of the main collection, a core collection is developed to capture 75 to 80 percent of the representative genetic diversity. From the user's perspective, such a core collection will gain value when accessions with known genes are added, even if the general genetic diversity present in these few additional accessions is already present in the core collection.
Finally, an extreme concept that could arise, based on the knowledge of the presence of valuable genes and alleles, is that of building collections based on traits. This is not a novel idea per se, but the initiative may come about once sufficient genomic results become available. Certainly, recent scientific advancements are drawing closer to this future.
12.7 ACKNOWLEDGEMENTS
The authors would like to thank Ehsan Dulloo (IPGRI, headquarters) for his critical reading of the chapter, Dimary Libreros (IPGRI Office for the Americas) for her bibliographic support and Elizabeth McAdam for copy editing.
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Prashanth S. Raghavan and Ajay Parida
13.1 SUMMARY
Mangroves are plants with the ability to tolerate a high degree of soil salinity and various other forms of abiotic stress. They are woody and generally inhabit the upper intertidal zones of estuaries within the tropical and subtropical regions. Anthropogenic influences have led to a significant reduction in its distribution worldwide and local extinction of many species and populations of the mangrove ecosystem. Our group therefore initiated a study on the molecular biology of mangroves, aiming to help in the conservation of these species. The study on mangroves in our laboratory began by analysing the genetic diversity in the various species present in the mangrove species. Twenty-four species of mangroves and mangrove associates were analysed using molecular markers. This study resulted in species-specific restriction patterns in the genera Rhizophora and Sueda, observing intra-generic variations in three genera - Avicennia, Rhizophora and Sueda. Intra/interspecific variation in the genus Avicennia and analysis of mitochondrial DNA variation in the species of Rhizophoraceae were also studied. As a first step towards characterizing genes that contribute to combating salinity stress, we constructed complementary DNA (cDNA) libraries from a mangrove species. An analysis of the expressed sequence tags (ESTs) generated from the cDNA library of one mangrove species revealed that 30 percent of the analysed ESTs showed homology to previously uncharacterized genes in the public plant databases. Several full-length genes of proteins involved in salinity tolerance and antioxidative stress pathways were isolated and submitted to the National Centre for Biotechnology Information (NCBI) gene bank database. Some of these isolated genes from the mangrove have been transferred to rice and homozygous lines of these transgenic rice plants have been generated, which are being tested for tolerance to various abiotic stresses at the laboratory and field level.
13.2 INTRODUCTION
The coastal ecosystem is one of the most productive ecosystems. The tropical and the subtropical coastlines of the world are characterized by specialized littoral plant formations, mangroves (Lakshmi, Parani and Parida, 2001). A mangrove is a plant community that inhabits the boundary between terrestrial and aquatic environments, comprising around 71 species, spread over 16 families (ibid.). All the mangrove species have the same physiognomy, physiological characteristics and structural adaptations (Yenny-Esinsine, 1980). The plant species in this particular ecosystem are constantly under varied environmental stress conditions including high saline and temperature extremes; these plants have adapted themselves to these frequent and fluctuating changes (Lakshmi et al., 1997).
However, human interference and negligence have caused rapid destruction to the mangrove forests and even caused extinction to a few of the locally existing species (Parani et al., 2000). Large areas of mangrove forests throughout the world are being converted for agriculture or exploited for wood and other forest products (Lakshmi et al., 1997). The Indian coastline covers around 7 500 km and accounts for 8 percent of the world's mangrove area, and the eastern coast of India accounts for about 82 percent of the mangrove forest cover in India (Parida et al., 1998). In the absence of any national plan for conservation and sustainable utilization, mangroves along the Indian coast have reached an alarming stage of depletion (Lakshmi et al., 1997). A 25 percent reduction in mangrove forest cover has been reported along the Indian region during the last 25 years (Parida et al., 1998).
In the coastal regions there is intense agricultural activity. Increased soil erosion and water pollution caused by intensive farm practices in the inland area gets transported through the rivers and canals and adversely affects the coastal agro-ecosystem. The seawater intrusion and the attendant soil and water quality problems caused by the groundwater depletion have already started threatening the sustainability of the agricultural ecosystem in the Saurashtra region of Gujarat and Thanjavur region of Tamil Nadu in India. This has given rise to an increase in the level of abiotic stresses such as salinity, alkalinity and drought. Climate change with its consequent rise in sea level is one of the major impending dangers affecting the coastal ecosystem. By 2025, the rise in sea level is expected to be roughly 8 to 29 cm due to global warming (Parida et al., 1998). This could cause large-scale inland flooding.
Salinization is posing an increasing problem in coastal and agricultural areas, reducing plant productivity and yield. Further, salinity is one of the major abiotic stresses decreasing plant productivity. Tolerance to salt stress is a complex trait that involves various aspects including osmotic, ionic stress and secondary stress, such as oxidative stress. Salt stress leads to dehydration and osmotic stress, with the reduced availability of water resulting in stomatal closure, and reduced supply of carbon dioxide leading to a high production of reactive oxygen species in the chloroplasts (Tanaka et al., 1999). This effect causes irreversible cellular damage. Similar effects of generation of reactive oxygen species are also seen to occur during periods of high photosynthetic activity in plants, resulting in photoinhibition (Bowler, Montagu and Inze, 1992). Photoinhibition and salinity stress together cause severe damage to the cellular processes in the plant. However, mangroves are plants that are capable of surviving in highly saline environments, having a high capacity to maintain active leaves in conditions that are expected to severely reduce the photosynthesis through photoinhibition (Cheeseman et al., 1997). In order to combat such abiotic stress effects, we have carried out studies to conserve the mangrove genetic resources, characterize and harness the genes involved in salinity/abiotic stress tolerance from mangroves, and transfer these genes to crop plants in order to generate crops with enhanced stress tolerance.
13.3 CYTOLOGICAL AND GENETIC DIVERSITY ANALYSIS OF MANGROVES
The cytological and genetic diversity present in the mangrove plants were studied as an initial part of the work. Mitotic chromosome analysis carried out on samples of different populations of Acanthus illicifolius (Lakshmi et al., 1997) revealed that the cells were characterized by 48 chromosomes resolved into 24 homomorphic pairs. There was no variation in the chromosome number seen in the plants from different populations. Chromosome analysis of ten species of the genus Rhizophoraceae (Lakshmi, Parani and Parida, 2001) revealed that there were no numerical variations in the intraspecific level and that the chromosome complements in these species were stable and underwent limited divergence during speciation.
Genetic diversity studies were carried out for intra/interspecific variations in different populations of the genus Avicennia (Parani et al., 1997). A mangrove genetic resource centre was established in the Pichavaram mangrove area, Chidambaram, India, where the endangered mangrove species are being conserved. Following the study on the genetic diversity present in Avicennia, seeds from all the populations studied were collected to represent A. marina in the mangrove genetic resources centre, and the sampling of the seeds was based on the recommendation specified by the outcome of the study. Intraspecific variation in the species Excoecaria agallocha was also studied (Lakshmi et al., 2000). The study revealed that lack of morphological variation in the plants belonging to this species was not due to lack of genetic variation.
Subsequently, 24 species of mangroves and mangrove associates were analysed using molecular markers. This study enabled the generation of species-specific restriction patterns in the genera Rhizophora and Sueda. We were able to study the intrageneric variation in three genera, Avicennia, Rhizophora and Sueda (Parani et al., 2000). Analysis of mitochondrial DNA variation in species of Rhizophoraceae (Lakshmi et al., 2002) was also studied and again resulted in species-specific profiling of different species belonging to the family of Rhizophoraceae.
13.4 ISOLATION OF SALINITY TOLERANCE GENES FROM MANGROVES
As a first step towards characterizing genes from mangroves that contribute to improving salinity stress, we constructed a cDNA library from a mangrove species Avicennia marina, using seedlings treated for 48 hours with 0.5M NaCl (Parani et al., 2002). Comparison was made between 1841 ESTs from A. marina cDNA library against sequences from the NCBI databases using the program BLASTX. Unknown genes form the largest category at 30 percent, followed by genes required for primary metabolism (13 percent). Genes involved in transcription and chromatin organization, protein synthesis and processing each represent 10 percent of the sequenced ESTs while those involved in membrane transport and intracellular trafficking represent 9 percent of the ESTs. Eight percent of the ESTs relate to signal transduction while 7 percent are similar to previously reported stress-induced genes (Preeti et al., 2005).
13.5 GENETIC ENGINEERING FOR SALINITY TOLERANCE
The mangrove genes isolated from the mangroves were transferred into crop plants using Agrobacterium tumefaciens mediated transformation (Hiei et al., 1994). Specific genes isolated from the A. marina cDNA library were cloned in binary vectors. These genes were expressed under the control of constitutive promoters. These gene constructs were then transformed into Agrobacterium tumefaciens and used for co-cultivation with rice calli and tobacco leaf explants. Rice calli were generated from seed scutella of mature rice seeds on a callus induction medium. Callus co-cultivated with Agrobaterium were washed and selected on callus induction medium containing hygromycin. The selected calli were then transferred to the regeneration medium containing shoot-inducing hormones. The regenerants were transferred to the rooting medium. The plantlets were subsequently transferred to a hardening medium. Finally, these plants were transferred to the soil in pots to raise the next generation seeds. The tillers of the rice plants were bagged before the onset of flowering in order to promote self-pollination. The seeds from the selfed plants were collected and again sown for the next generation. In a similar manner, leaf discs from tobacco plants were infected with Agrobacterium carrying the binary constructs harbouring the mangrove genes. The integration and expression of the transgenes were confirmed in these plants using various molecular analyses such as PCR, Southern hybridization, Northern hybridization, isozyme analysis and Western blot analysis.
The homozygous lines from these transgenic plants were raised and tested with various abiotic stresses such as salt stress and drought stress. Initial analyses in the laboratory have been promising. However, further analyses would need to be carried out to evaluate the performance of these transgenics. It was found that all the transgenic lines performed better than the control in stress conditions. This effort happens to be the first effort of its kind in the world in terms of transferring genes from A. marina to crop plants. This process therefore has a multiple use by conserving the genetic diversity of the mangrove species, which is being destroyed very rapidly, and by genetically engineering the crop plant with genes, which help them tolerate abiotic stress better.
13.6 REFERENCES
Bowler, C., Van Montagu, M. & Inze, D. 1992. Superoxide dismutase and stress tolerance, Annual review of plant physiology and plant molecular biology, 43:83–116.
Cheeseman, J.M., Herendeen, L.B., Cheeseman, A.T. & Clough, B.F. 1997. Photosynthesis and photoprotection in mangroves under field conditions. Plant, Cell and Environment, 20: 579–590.
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Giovanni G. Vendramin and Michele Morgante
14.1 SUMMARY
Conservation genetics of forest tree species takes advantage of the availability of molecular markers. Depending on the processes that need to be analysed, molecular markers may provide extremely useful information to monitor processes related to adaptation and migration. Markers must be carefully selected depending on the specific question. Some examples are provided and the usefulness of molecular markers in conservation genetics of forest tree species is discussed. Direct analysis of adaptive variation is now possible by analysis of quantitative trait nucleotides (QTNs), which requires that QTNs be known. A perspective on the possibilities for discovering genes involved in adaptive variation in forest trees is provided.
14.2 CONTRIBUTION
The genomics revolution of the last ten years has improved our understanding of the genetic make-up of living organisms. Together with the achievements represented by complete genomic sequences for an increasing number of species, high throughput and parallel approaches are available for the analysis of transcripts, proteins, insertional and chemically-induced mutants. All this information facilitates the understanding of the function of genes in terms of their relationship to the phenotype. Despite its great relevance, such an understanding could be of little value to population and conservation genetics because it will not elucidate the relationship between genetic variation in gene sequences and phenotypic variation in traits, but rather only that between a gene and a mutant phenotype. The relationships between complex trait variation and molecular diversity of genes can be studied based on a genomic approach, but the identification of genes responsible for the variation remains a slow and time-consuming process, especially in long-living organisms such as forest trees. Work in model plant species such as Arabidopsis and rice has, however, started to unveil an ever-increasing number of genes involved in the determination of traits of adaptive significance, such as phenology and abiotic stress tolerance/resistance. This progress will finally allow ecological and conservation genetics to directly analyse variation in genes involved in adaptive processes rather than in neutral markers. Neutral markers will, however, remain important to make inferences about stochastic processes affecting natural population evolution.
Populations may follow two main strategies to react to abiotic stresses originating from climate changes: adapt to the new climatic conditions and/or migrate to more favourable areas. Some neutral markers (e.g. highly polymorphic microsatellites and organelle markers) are very useful for monitoring past and present migration processes. Plants offer excellent models to investigate how gene flow shapes the organization of genetic diversity. Their three genomes (chloroplast, mitochondrial and nuclear) can have different modes of transmission and will hence experience varying levels of gene flow. Based on a very large data set, Petit et al. (2005) demonstrates that mode of inheritance appears to have a major effect on genetic differentiation (Gst). Gst for chloroplast DNA and mitochondrial DNA markers covary narrowly when both genomes are maternally inherited. At the range-wide scale, historical levels of pollen flow are generally at least an order of magnitude larger than levels of seed flow and pollen and seed gene flow vary independently across species (ibid.). Moreover, Petit et al. (2005) show that measures of subdivision that take into account the degree of similarity between haplotypes (Nst, or Rst) make better use of the information inherent in haplotype data than standard measures based on allele frequencies only.
Neutral organelle markers can be extremely useful for phylogeographic studies (Petit and Vendramin, 2005). The phylogeographic structure of forest tree species can be influenced by several factors, among which history during the glaciations and in the post-glacial period, life history traits of the species and human impact are assumed to have played a major role. Phylogeography can provide essential background information to disentangle current from past processes and to understand the consequences of crucial events such as colonization in the life and longevity of plant species (Petit et al., 2003). The comprehension of the past dynamics of diversity can be extremely useful to predict the possible future migrations related to the expected climate changes (Pitelka et al., 1997). In conservation and management of genetic resources, phylogeographic studies may help identify key regions deserving priority for conservation (Petit et al., 2003). A phylogeographic survey may allow tracing of wood and other plant products, providing tools to combat illegal logging or to label products originating from sustainably managed regions (Deguilloux, Pemonge and Petit, 2002). Finally, the background on seed flow to emerge from phylogeographic surveys can be used to evaluate risks associated with the use of transplastomic plants (Petit and Vendramin, 2005; Daniell et al., 1998).
Technology is also rapidly evolving in neutral marker analysis, moving from markers such as microsatellites towards single nucleotide polymorphism (SNP) markers due to cost, efficiency and automation considerations. Because of the different characteristics of the two markers systems in terms of mutation processes and rates, they will both find use in ecological studies.
14.3 REFERENCES
Daniell, H., Datta, R., Varma, S., Gray, S. & Lee, S. 1998. Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nature Biotechnology, 16: 345–348.
Deguilloux M-F., Pemonge M-H. & Petit, R.J. 2002. Novel perspectives in wood certification and forensics: dry wood as a source of DNA. Proceedings of the Royal Society of London, B, 269: 1039–1046.
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