III. Use of molecular markers for characterization and conservation of genetic resources

7. Use of molecular markers and other information for sampling germplasm to create an animal gene bank

Henner Simianer

7.1 INTRODUCTION

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the Convention of Biological Diversity (CBD, 1992). Although farm animal breeds are not well defined in general terms, they are the typical unit used for conservation. Based on an inventory of the actual breeds carried out on a global scale by FAO through the World Watch List (FAO, 2000), the specific breed characteristics and their genetic diversity, decisions need to be taken on what should be conserved.

Figure 11 gives an overview of the different conservation schemes available for farm animals. It is generally accepted that whenever possible, preference should be given to in vivo conservation schemes; in vitro schemes are often started as an additional safeguard when the population size of the endangered breeds is very small or the breed is at high risk of extinction. Semen and embryos are the first choice for cryo-conservation. It should be noted, however, that while long-term storage of deep-frozen semen is feasible in all major farm animal species, the use of cryo-conserved embryos is only established in cattle and small ruminants and under development in a number of other species (Hall, 2004). Now that Dolly has been successfully cloned, storing somatic cells to produce clones in the future may be seen as a simple and inexpensive option (Wilmut et al., 1997), but up to now practical experience with this technique is rather limited in most species.

FIGURE 11
Systematic overview of basic conservation schemes for farm animals

Table 10 shows some of the main features of in vivo and in vitro conservation schemes. It should be noted that the apparent advantage that a live population genetically adapts to changing conditions is often overemphasized since genetic change due to natural selection is not expected to be very large in only a few generations. The costs of in vitro schemes are often considered very low. If costs of reactivating the cryo-conserve at the end of the planning horizon are included, however, this may not be true. Reist-Marti (2004) pointed out that even the collecting and storing phase of a cryo-conservation scheme will be expensive if the technical infrastructure and the expertise are not available, which is the case in most developing countries.

Creating an animal gene bank may refer not only to in vitro, but also to in vivo repositories; in the latter case it might be understood as identifying a subpopulation that is actively managed in a conservation breeding programme, for example, through a sire rotation system (Kimura and Crow, 1963).

7.2 USE OF MOLECULAR MARKERS

Molecular markers are a tool to study diversity on the genetic level. The most widespread use of molecular markers in this context is the assessment of diversity within and between breeds. Although in principle all types of markers would be suitable for this purpose, microsatellites are used in 90 percent of all diversity studies (Baumung, Simianer and Hoffmann, 2004). A joint committee of FAO and the International Society for Animal Genetics (ISAG) has recommended a standard set of microsatellite markers for the major farm animal species. This recommendation was recently reviewed and extended to a larger number of species (Hoffman et al., 2004). These markers are chosen to represent neutral genetic variability in the genome.

In addition, one might also consider markers associated with so-called quantitative trait loci (QTL), i.e. markers that reflect the genetic potential of an animal for a given quantitative or qualitative trait. Farm animal research focuses very strongly on mapping QTLs and single genes so that such markers will be increasingly available in the future. Of special interest will be markers linked to disease-resistance QTL, such as trypanotolerance in cattle (Hanotte et al., 2003), nematode resistance in sheep (Coltman et al., 2001), and E.coli-resistance in pigs (Meijerink et al., 2000).

TABLE 10
Main features of in vivo and in vitro conservation schemes for farm animal biodiversity

7.3 CREATING A GENE BANK

Creating a gene bank can be considered a multi-stage decision-making process with the following stepwise decisions:

7.3.1 Which breeds to conserve?

Weitzman (1992; 1993) has presented a formal framework for the decisionmaking process on breeds to conserve. While the diversity metric suggested by Weitzman has been criticized as not accounting for within-breed diversity (see, for example, Caballero and Toro, 2002), the general framework to make decisions based on the expected conserved diversity has a strong appeal. The original proposition, which was first adopted by Thaon d'Arnoldi, Foulley and Ollivier (1998) for farm animals, is based on a diversity metric derived from a genetic distance matrix. Genetic distances can be estimated from allele frequencies at marker loci between populations: the greater the difference in these frequencies, the more distant the breeds are. Diversity is a measure for the total variability of a distance matrix for a set of breeds: the more distant the breeds in the set are, the larger the diversity will be.

Using actual allele frequencies results in the present diversity. Combining the information with extinction probabilities, i.e. the probability that a breed will go extinct over a given time horizon, for example, 50 years, results in the expected diversity at the end of the time horizon. The expected diversity will always be less than the actual diversity. The objective is to design conservation programmes in such a way that the expected diversity is maximized.

Weitzman (1993) suggests that the conservation potential is the single most informative criterion to rank breeds with respect to conservation priority. The conservation potential of a breed basically reflects the amount of expected diversity that can be conserved if a breed is made completely safe. Simianer et al. (2003) found that this criterion correctly identified the optimum subset of six breeds to be conserved in a set of 23 African cattle breeds. Pinent, Simianer, and Weigend (2005) show that the derivation of the conservation potential for a set of breeds should always take into account information on related breeds outside the set of candidate breeds for conservation (e.g. foreign breeds or commercial breeding populations) to avoid “false positives”.

Simianer (2002; 2005) suggests combining expected diversity with other criteria resulting in the expected total utility as a maximization criterion. This criteria may encompass the presence of special genetic traits (such as disease tolerance), production, and cultural or environment values of breeds, inter alia. A similar but less formal argument was made by Piyasatian and Kinghorn (2003).

7.3.2 Optimal allocation of resources

Once the decision is made as to which breeds should be sampled, it is necessary to assign appropriate shares of the conservation budget to the different breeds. In the European Union, about 40 million euros are spent per year for the conservation of farm animal genetic resources (Signorello and Pappalardo, 2003). Although this is not a centralized budget distributed in a uniform and rational process, in principle one could compare the theoretically optimal allocation with the real situation, revealing the relative efficiency of the implemented conservation programmes.

Simianer et al. (2003) have suggested a formal approach to find the optimal allocation of a given budget. This basic approach was further refined by Reist-Marti (2004). In a first application, Simianer (2002) showed that the optimal allocation of a hypothetical conservation budget to a set of 26 African cattle breeds resulted in a 60 percent increase of efficiency (in terms of conserved diversity per conservation funds) compared to uniform distribution or allocation of the total budget to the three most endangered breeds only.

7.3.3 Conservation scheme criteria for a given breed

This decision is not independent from the choice of breeds to conserve and the optimal allocation of resources. To do these first steps, costs and effects (in terms of reduced extinction probability) need to be known for the different conservation schemes. The costs can typically be subdivided into fixed costs, which are necessary to establish the conservation scheme in this breed, and variable costs, which depend on the number of animals, herds, cryo-conserved sample, inter alia. With known cost functions for different conservation schemes in the same breed, it is always possible to identify the optimum conservation scheme for a given investment level within breed.

This is demonstrated in Reist-Marti (2004) where three out of four different conservation schemes were found to be preferable in at least one out of eight African cattle breeds chosen for conservation. If such a planning process is considered on an international level, factors such as the exchange rate of currencies and relative labour costs in different countries play an important role. Labour-intensive ex situ conservation schemes may be cheaper than cryo-conservation in some countries where wages are low and the infrastructure for cryo-conservation is not available (Reist-Marti, 2004).

7.3.4 Which germplasm should be stored?

Once the aforementioned decisions are made, individual genotypes need to be identified to become part of the conservation scheme. Some general criteria can be defined concerning the desirable genetic properties of the sample:

• It should represent the genetic portfolio of the breed.

• It should have a maximum effective population size.

• Special genetic traits should be conserved.

Fulfilling these criteria may lead to a conflict of goals since maximizing the effective population size suggests collecting extreme genotypes, which may not be representative for the population.

Using parameters from population genetics, the group of animals chosen should have minimum inbreeding and a minimum relationship to each other. Note that the level of inbreeding is less critical than the average relationship, because the actual inbreeding is removable while the level of relationship inevitably determines the long-term level of inbreeding.

If reliable pedigree data are available, these parameters can be calculated for any possible sample and used to identify the optimum group of animals to be stored. If pedigree information is not available, genetic markers can be used to approximate these criteria. Eding and Meuwissen (2001) suggested estimating Malécot's (1948) kinship coefficient based on molecular marker information and deriving what they call a “core set”to conserve.

7.4 CONCLUSIONS

Molecular markers are an indispensable tool to understand the genetic structures of populations. For the sampling of germplasm to create an animal gene bank, they are necessary but in no way sufficient to make adequate decisions. In addition to diversity information derived from molecular data, there needs to be good, specific knowledge and understanding of breed characteristics and values, the risk status of breeds, and availability and cost efficiency of possible conservation programmes, among others. Considering the present situation in livestock, diversity information is often more easily accessible than information on many of the other factors listed (Ruane, 2000). It is therefore strongly recommended to concentrate co-ordinated genotyping efforts to fill in the still existing “white spots”on the global maps of farm animal diversity and to re-allocate funds to develop a better understanding of the other components of a rational decision-making process.

7.5 REFERENCES

Baumung, R., Simianer, H. & Hoffmann, I. 2004. Genetic diversity studies in farm animals - a survey. J. Anim. Breed. Genet., 121: 361–373.

Caballero, A. & Toro, M.A. 2002. Analysis of genetic diversity for the management of conserved subdivided populations. Conservation Genetics, 3: 289–299.

Coltman, D.W., Wilson, K., Pilkington, J.G., Stear, M.J. & Pemberton, J.M. 2001. A microsatellite polymorphism in the gamma interferon gene is associated with resistance to gastrointestinal nematodes in a naturally-parasitized population of Soay sheep. Parasitology, 122: 571–582.

Convention on Biological Diversity. 1992. Secretariat of the Convention on Biological Diversity. Montreal, Canada. (available at www.biodiv.org)

Eding, H. & Meuwissen, T.H.E. 2001. Marker based estimates of between and within population kinships for the conservation of genetic diversity. J. Anim. Breed. Genet., 118: 141–-159.

FAO. 2000. World watch list for domestic animal diversity. 3rd edition. (also available at dad.fao.org/en/refer/library/wwl/wwl3.pdf).

Hall, S.J.G. 2004. Livestock biodiversity. Genetic resources for the farming of the future. Oxford, England, Blackwell.

Hanotte, O., Ronin, Y., Agaba, M., Nilsson, P., Gelhaus, A., Horstmann, R., Sugimoto, Y., Kemp, S., Gibson, J., Korol, A., Soller, M. & Teale, A. 2003. Mapping of quantitative trait loci controlling trypanotolerance in a cross of tolerant West African N'Dama and susceptible East African Boran cattle. Proc. Natl. Acad. Sci. USA, 100: 7443–7448.

Hoffmann, I., Ajmone Marsan, P., Barker, J.S.F., Cothran, E.G., Hanotte, O., Lenstra, J.A., Milan, D., Weigend, S. & Simianer, H. 2004. New MoDAD marker sets to be used in diversity studies for the major farm animal species: recommendations of a joint ISAG/FAO working group. Proceedings of the 29th International Conference on Animal Genetics, Tokyo, Japan, 11–16 September, 2004: 123. (abstract)

Kimura, M. & Crow, J.F. 1963. On the maximum avoidance of inbreeding. Genet. Res. Camb., 4: 399–415.

Malécot, G. 1948. Les mathématiques de l'hérédité. Paris, France, Masson et Cie. 64 pp.

Meijerink, E., Neuenschwander, S., Fries, R., Dinter, A., Bertschinger, H.U., Stranzinger, G. & Vögeli, P. 2000. A DNA polymorphism influencing alpha (1,2) fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion. Immunogenetics, 52: 129–136.

Pinent, T., Simianer, H. & Weigend, S. 2005. Weitzman's approach and conservation of breed diversity: First application to German chicken breeds. In Proceedings of the International Workshop on the Role of Biotechnology for the Characterisation and Conservation of Crop, Forestry, Animal and Fishery Genetic Resources. (available at www.fao.org/biotech/docs/pinent.pdf).

Piyasatian, N. & Kinghorn, B.P. 2003. Balancing genetic diversity, genetic merit and population viability in conservation programmes. J. Anim. Breed. Genet., 120: 137–149.

Reist-Marti, S.B. 2004. Analysis of methods for efficient biodiversity conservation with focus on African cattle breeds. Zurich, Switzerland, Swiss Federal Institute of Technology. (PhD thesis)

Ruane, J. 2000. A framework for prioritizing domestic animal breeds for conservation purposes at the national level: a Norwegian case study. Conservation Biology, 14: 1385–1393.

Signorello, G. & Pappalardo, G. 2003. Domestic animal biodiversity conservation: a case study of rural development plans in the European Union. Ecol. Econ., 45: 487–499.

Simianer, H. 2002. Noah's dilemma: Which breeds to take aboard the ark? CD-ROM communication no. 26-02. Proceedings of the 7th World Congress on Genetics Applied to Livestock Production (WCGALP). Montpellier, France.

Simianer, H. 2005. Decision making in livestock conservation. Ecol. Econ. (in print)

Simianer, H., Marti, S.B., Gibson, J., Hanotte, O. & Rege, J.E.O. 2003. An approach to the optimal allocation of conservation funds to minimise loss of genetic diversity between livestock breeds. Ecol. Econ., 45: 377–392.

Thaon d'Arnoldi, C., Foulley, J.L. & Ollivier, L. 1998. An overview of the Weitzman approach to diversity. Genetics Selection Evolution, 30:149–161.

Weitzman, M.L. 1992. On diversity. Quarterly Journal of Economics, CVII: 363–405.

Weitzman, M.L. 1993. What to preserve? An application of diversity theory to crane conservation. Quarterly Journal of Economics, CVIII: 157–183.

Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H.S. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385: 810–813.

8.Genetic characterization of livestock populations and its use in conservation decision-making

Olivier Hanotte and Han Jianlin

8.1 SUMMARY

This chapter reviews the importance and putative impacts of molecular markers on decision-making for livestock genetic resource conservation. Livestock diversity is shrinking rapidly and there is an urgent need to define strategies to prioritize breed conservation. For most livestock species a large number of genetic markers that show different Mendelian patterns of inheritances (maternal, paternal, bi-parental) are now available. Applied at a large geographic scale to the study of livestock populations, they provide information on centres of origins and migration routes as well as identify geographic areas of admixture among populations of different genetic origins. Such information can guide the choice of breeds and geographic areas for conservation actions. Calculations within and between diversity parameters allow selection of priority breeds for conservation to maximize diversity conserved for the benefit of future human generations.

8.2 INTRODUCTION

The World Watch List for Domestic Animal Diversity (3rd ed.) documents more than 6 300 breeds of breeds of livestock belonging to 30 domesticated species (FAO, 2000). These breeds were developed following domestication and natural and human selection over the past 12 000 years. The current number of breeds is likely an underestimation since a large proportion of indigenous livestock populations of the developing world, where most animal genetic resources are found today, have yet to be described at phenotypic and genetic levels. Livestock populations have evolved a unique adaptation to their agricultural production system and agroecological environments. Their genetic diversity has provided the material for the very successful breeding improvement programmes of the developed world in the 19th and 20th century. This represents a unique resource to respond to the present and future needs of livestock production, both in developed and developing countries.

However, livestock diversity is shrinking rapidly. With the exception of the wild boar, which is the ancestor of the domestic pig, and wild red jungle fowl, which is the ancestor of the domestic chicken, the putative wild ancestors of our major livestock species, the reservoir of genetic diversity, are now either extinct (e.g. the auroch, the wild ancestor of cattle, or the ancestral species of the Old World camelids) or low in numbers and threatened by extinction (e.g. wild goat populations of the Near East, vicuña from Andean plateau, wild donkey in Africa). Among the domesticated populations, it is estimated that one to two breeds are lost every week (FAO, 2000). However, the impact of these losses on global or local diversity remains undocumented. While it is already too late for many breeds in Europe, the situation is also particularly worrying in the developing world where rapid changes in production systems are leading to the replacement of breeds or at best crossbreeding. There is therefore an urgent need to document the diversity of our livestock genetic resources and to design strategies for their sustainable conservation.

The task is enormous. It has prompted FAO and other international organizations to develop domestic animal diversity information systems and databases (ILRI's Domestic Animal Genetic Information System [DAGRIS] and FAO's DAD-IS 2.0). More recently, FAO has initiated a major country-driven documentation exercise, the State of the World's Animal Genetic Resources (SoW-AnGR). It is hoped that this book, together with the publication of a companion Strategic Priority Action Report, will lead to immediate actions for conservation on the ground at the country or regional level (SoW-AnGR, http://dad.fao.org/cgidad/$cgi_dad.dll/sow?eng).

To put this plan into practice, effective conservation of animal genetic resources (AnGR), whether in situ or ex situ, will require the mobilization of substantial economic resources over a long period of time. Financial resources are limited and they always will be. Methods are required to identify priority decisions in order to maximize the diversity conserved, both at the local and the global level, and to focus on unique genetic resources of global significance. Genetic characterization through the use of molecular markers associated to powerful statistical approaches is providing new avenues for decision-making choices for the conservation and rational management of AnGR.

8.3 GENETIC CHARACTERIZATION TOOLS: MOLECULAR MARKERS

Protein polymorphisms were the first molecular markers used in livestock. Many studies, particularly during the 1970s, have documented the characterization of blood group and allozyme systems of livestock (e.g. Baker and Manwell, 1980). However, the level of polymorphism observed in proteins is often low, which has reduced the general applicability of protein typing in diversity studies. With the development of the Polymerase Chain Reaction (PCR) and sequencing technologies associated with automatic and/or semi-automatic large-scale screening systems, DNA-based polymorphisms are now the markers of choice for molecular-based surveys of genetic variation. Importantly, polymorphic DNA markers showing different patterns of Mendelian inheritances can now be studied in nearly all of humankind's major livestock species. Typically, they include D-loop and cytochrome B mitochondrial DNA (mtDNA) sequences (maternal inheritance), Y chromosome-specific single nucleotide polymorphisms (SNPs) and microsatellites (paternal inheritance) and autosomal microsatellites bi-parental inheritance) (Avise, 1994). Interestingly, while recent developments in cytogenetic technologies should facilitate the isolation of Y chromosomes specific markers (Petit, Balloux and Excoffier, 2002), for most livestock species there are still few Y polymorphic markers. This is possibly a consequence of the demographic history of domestication and breed formation. In polygenous species, as in most livestock, we clearly expect that a small number of male lineages would have contributed to the genetic pool of the species. Polymorphic Y microsatellite markers are currently only available for cattle (Hanotte et al., 2000), yak (Xuebin, 2004), and to some extent, small ruminants (Lenstra and Econogene Consortium, 2005). To the best of the knowledge available, they have not yet been isolated in any major livestock species, e.g. the Old and New World camelids or the domestic pig. On the other hand, autosomal microsatellites have now been isolated in large numbers from most livestock species and recommended FAO/ISAG lists of autosomal microsatellite markers for genetic characterization studies are publicly available (FAO, 2004).

Important assumptions on the use of genetic markers include: (i) that the polymorphisms observed at the molecular markers are neutral; and (ii) that the use of a relatively small number of independently segregating marker loci will be a good predictor of the overall genomic diversity of a population, or in other words, that variation in allele frequencies between populations will reflect the distribution of genetic diversity within and among populations.

8.4 DIVERSITY OF GENETIC CHARACTERIZATION INFORMATION

Genetic markers will provide different types of information relevant for conservation-making decisions for livestock (Sunnucks, 2000). Autosomal microsatellite loci will be commonly used for individual genetic identification and parentage analysis, e.g. for the successful implementation and monitoring of ex situ conservation programmes, population diversity estimation, differentiation of populations, calculation of genetic distances, genetic relationships and population genetic admixture estimation. Microsatellite loci are also highly sensitive to genetic bottlenecks and are commonly used for inbreeding estimation. MtDNA sequences will be the markers of choice for domestication studies because the segregation of a mtDNA lineage within a livestock population will only have occurred through the domestication of a wild female or through the incorporation of a female into the domestic stock. More particularly, mtDNA sequences will be used to identify the putative wild progenitors, the number of maternal lineages and their geographic origins. To some extent, they may provide important information on the geographic distribution of diversity within livestock species although the usefulness of mtDNA sequences data will vary between species, depending on the demographic history of the migration from the centre(s) of domestication. Finally, the study of a diagnostic Y chromosome polymorphism is an easy and rapid way to detect and quantify male-mediated admixture.

A surprising result of the application of molecular makers in genetic characterization of livestock has been the discovery that several ancestral species, subspecies or maternal lineages have contributed to today's genetic pool of our major livestock species (Xuebin, 2004; Bruford, Bradley and Luikart, 2003; Beja-Pereira, 2004; ILRI unpublished data). It is clear from these recent results that multiple domestications and/or maternal introgression are the rule, not the exception (Table 11).

More particularly, mtDNA information supports the conclusion that there were at least five major centres of livestock domestication: the northern Andean chain (New World camelids), the Northeast African region (donkey and likely taurine cattle), the Near East (taurine cattle, sheep, goat, pigs), South Asia (Indus Valley, indicine cattle and chicken) and East Asia (pigs, chicken, horse, buffalo), to which the Hindu-Kush Himalayan region (yak) and North and Central Asia (horse) should be added. Similarly, to some extent we could expect multiple male-mediated introgression lineages. This is the case in the yak (Xuebin, 2004), with two distinct male lineages, but not the case in the domestic horse where screening for SNPs in 52 stallions from 15 different breeds did not identify a single polymorphic site (Lindgren et al. 2004).

Taking into account the history of human migration and trading, it is expected that our indigenous breeds today will often have multiple genetic signatures of origin and admixture. Available molecular data indicate that ancient genetic admixtures between livestock populations from different domestication events are common on the Asian continent and, to some extent, also in Africa, but mostly absent from Europe. This has been shown in cattle (e.g. Hanotte et al., 2002), pigs and small ruminants (Bruford, Bradley and Luikart, 2003).

TABLE 11
No. and putative centres of origin of major maternal lineage in livestock

Source: Bruford et al. (2003), Xuebin (2004) [yak], Beja-Pereira (2004) [donkey].

8.5 GENETIC CHARACTERIZATION AND CONSERVATION DECISION-MAKING

As illustrated above, genetic characterization is providing new information to guide and prioritize conservation decisions for livestock. Possibly, the most urgently required action is the effective protection of all remaining wild ancestral populations and closely related species of livestock, 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. Coordination with international wildlife conservation institutions such as the World Conservation Union (IUCN) is required. It is equally important to ensure that the breeds selected for conservation include populations from the geographic areas representing the different domestication centres where we would expect to find large genetic diversity and genetically differentiated populations. Animals and populations present at the geographic area of a centre of domestication will also be expected to be very distinct from the ones found at other centres of domestication. Also, understanding the geographic pattern of livestock migration from a centre of origin will allow the identification of populations present at the end of a migration route. It is expected that these populations will be genetically distinct from the populations present at the ends of other migration routes as a result of random genetic drift and/or the effect of local selection pressures. Importantly, knowledge of both the global diversity of the breeds and admixture events will be needed in order to be able to make sound priority decisions.

The next challenge is to make priority decisions for conservation among today's thousands of domestic breeds or populations. The primary objective is to maximize the conservation of the genetic diversity available for potential future use. At the ideal extreme, this would be achieved through the conservation of all breeds of livestock. Such a comprehensive approach would ensure complete conservation of diversity. In practice, it is unrealistic and prioritization of actions will have to be made. Two criteria (perhaps to be eventually combined) have been proposed (Gibson, Ayalew, and Hanotte, in press): priority breeds for conservation should be those with the largest within-breed diversity and/or should maximize the conservation of between breed diversity. Both within and between breed diversity parameters are classically measured using molecular genetic markers. In both cases, soundly based priority decisions for conservation at the global level will require the availability of large datasets.

The mean number of alleles (MNA) and observed (Ho) and expected (He) heterozygosity are the most commonly calculated population genetic parameters for assessing within-breed diversity. For example, in a recent study, three distinct sets of microsatellite diversity cattle data were merged to provide, for the first time, within breed diversity (He and MNA), as well as admixture information combined for Europe, Africa, the Near East and South Asia (Freeman et al., in preparation). The geographic region with the highest diversity is found between the two likely Asian centres of cattle domestication, in a broad geographic area corresponding to what are today Iran, Iraq and the Caucasian region. Global geographic analysis of admixture suggests that the region corresponds to a geographic area of around 50 percent admixture between taurine and indicine cattle. Genetic diversity and admixture information from more indigenous breeds are needed to confirm the results. If confirmed, this geographic area will undoubtedly represent a major livestock diversity hotspot, a priority region for a global plan for the conservation of the diversity of domestic cattle.

The simplest parameters for assessing the distribution of diversity between breeds using genetic markers are the genetic differentiation or fixation indices (e.g. F _ST , G _ST , θ). The most widely used is F _ST, which measures the degree of genetic variation between subpopulations through the calculation of the standardized variances in allele frequencies among populations (Weir and Basten, 1990, Mburu et al., 2003). Any set of genetic distances can also be analysed in terms of between-breed genetic diversity, and more particularly, in terms of individual breed contributions to the total diversity of a set of breeds. The most common approach used so far is a method proposed by Weitzman (1993). It involves calculating a matrix of genetic distances and generating dendrograms. Individual breed contributions are calculated by comparing the total length of the dendrogram including all breeds with the dendrogram including all breeds less the individual breed. Priority breeds for conservation would therefore be the breeds contributing most to the diversity of the set. The method can be extended further to estimate the impact of conservation decisions on the diversity of a set of breeds in the future with the calculation of the extinction probability of each breed and the marginal diversity that reflect the relative loss or gain in expected diversity of a set of breeds following a decrease or increase in the probability of survival of a breed by one unit.

The largest data set to which the Weitzman approach has been applied in livestock is on 49 African cattle breeds (Reist-Marti et al., 2003). The breeds were divided into two groups corresponding to the “taurine”and “indicine”division, and extinction probabilities were calculated for each breed. In both groups the results clearly indicate that the optimal conservation strategy is to give priority to the breeds with the highest marginal diversity rather than the most endangered ones.

8.6 FUTURE CHALLENGES AND OPPORTUNITIES

Major challenges remain for livestock conservationists. Documentation of genetic diversity is still all but lacking for some livestock species and incomplete for others (e.g. Old World camelids, chicken, buffalo, Asian small ruminants and cattle, etc.). With a few exceptions (see, for example, Hanotte et al., 2002), molecular datasets will include in a single study a limited number of breeds or populations only, and combined analysis of molecular datasets obtained in different studies will often be impossible. For example, statistical approaches are still lacking that allow the combination of genetic-distancing information obtained in separate studies. Finally, the molecular markers used to characterize diversity have little to do with the genes under selection for economically important traits (Gibson, Ayalew and Hanotte, in press).

But there are promising and exciting new avenues. We can expect that with the increased adoption of common sets of markers and common breeds of references (Freeman et al., in preparation), the combination of microsatellite datasets will be facilitated. The publication of the entire genome sequences of several livestock species will allow for the easy identification of thousands of neutral and selected genetic markers. It will open the way to detecting signatures of selection allowing researchers to trace the presence and the spread of economically important alleles (Luikart et al., 2003). A recent study in cattle milk protein genes has indirectly yet well illustrated the putative application of such selected markers in the identification of breeds and geographic areas as priorities for the conservation of specific economically important traits (Beja-Pereira et al., 2003). The new field of livestock “landscape genetics”is emerging (Manel, 2003). It will combine geo-referencing of breed distributions, spatial global genetic diversity, and climatic, ecological, epidemiological and production system information, which will facilitate and direct priority decisions for in situ breed conservation.

8.7 REFERENCES

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FAO. 2004. Secondary guidelines for development of national farm animal genetic resources management plans. Measurement of domestic animal diversity (MoDAD): recommended microsatellite markers. (available at dad.fao.org/en/refer/library/guidelin/marker.pdf).

Freeman, A.R., Bradley, D.G., Nagda, S., Gibson, J.P. & Hanotte, O. (in preparation). Combination of multiple microsatellite datasets to investigate genetic diversity and admixture.

Gibson, J.P., Ayalew, W. & Hanotte, O. (in press). Measures of diversity as inputs for decisions in conservation of livestock genetic resources. Wallingford, Oxfordshire, England, CABI.

Hanotte, O., Bradley, D.G., Ochieng, J.W., Verjee, Y., Hill, E.W. & Rege, J.E. 2002. African pastoralism: genetic imprints of origins and migrations. Science, 296: 336–339.

Hanotte, O., Tawah, C.L., Bradley, D.G., Okomo, M., Verjee, Y., Ochieng, J. & Rege, J.E. 2000. Geographic distribution and frequency of a taurine Bos taurus and an indicine Bos indicus Y specific allele amongst sub-Saharan Africa cattle breeds. Molecular Ecology, 9: 387–396.

Lenstra, J.A. & Econogene Consortium. 2005. Evolutionary and demographic history of sheep and goats suggested by nuclear, mtDNA and Y-chromosome markers. In Proceedings of the International Workshop on the role of biotechnology for the characterisation and conservation of crop, forestry, animal and fishery genetic resources. (available at www.fao.org/biotech/docs/lenstra.pdf).

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9. Genetic characterization of populations and its use in conservation decision-making in fish

Craig Primmer

9.1 SUMMARY

The conservation needs of fishes are special in many respects compared to those of terrestrial organisms. For example, aquatic organisms are the only major human food source that is primarily harvested from wild populations. This chapter will briefly outline some of the main applications in which molecular marker data are applied for conservation decision-making in fish populations, which include determining population genetic structure, estimation of effective population size and detection of population size changes.

The needs for fish conservation are special in many respects compared to those of terrestrial organisms and in particular, domestic species that have been considered in other chapters of this publication. For example, aquatic organisms are the only major human food source that are primarily harvested from wild populations (Ryman et al., 1995). Therefore, methods for the development of population management guidelines often more closely follow those commonly used for wildlife than for domestic animals and plants. This chapter will briefly outline some of the main applications for which molecular marker data are applied for preserving fish biodiversity.

9.2 DETERMINING POPULATION GENETIC STRUCTURE

One of the main applications of molecular markers related to the conservation of commercially exploited fish is to aid in the development of guidelines enabling sustainable harvesting of populations (Carvalho and Pitcher, 1994). An important first step towards this is to characterize the genetic structure of the populations being harvested, which will assist in defining the biological units that should be considered when developing a management strategy, the so-called “genetic stock concept”(e.g. Carvalho and Hauser, 1994). While not directly aimed at conserving biodiversity, but rather at maximizing sustainable harvest levels, the implementation of management strategies based on molecular data can have indirect benefits for population biodiversity since their aim is to avoid population crashes that would negatively affect harvesting. This aim also benefits the maintenance of population genetic diversity. Studies investigating the population genetic structure of fishes have been conducted for a number of decades, first using protein coding allozyme loci (e.g. Utter, Aebersold and Winans, 1987) and then starting in the mid-1980s, using mitochondrial DNA (mtDNA) polymorphisms (reviewed by Avise, 2004). More recently, tandemly repeated microsatellite DNA markers have become the molecular marker of choice for determining intraspecific population genetic relationships (for example, Koskinen et al., 2002a). In general, markers that are used today utilize the polymerase chain reaction (PCR) since they enable the analysis of archive material such as scales. (For more details regarding the different marker types, refer to Frankham, Ballou and Briscoe, 2002; Avise, 2004.)

From a fisheries management perspective, the aim of determining the intraspecific population genetic structure is to determine the units between which limited gene flow occurs: if such units are overfished, it is unlikely that population sizes will recover due to migration and hence a collapse of the fishery may occur. While understanding population genetic structure is important from an applied perspective, the same knowledge is also the basis of any biologically sound conservation strategy. For example, similar genetic criteria to those described above are the basis of several definitions of the so-called “Evolutionary Significant Unit”(ESU) (reviewed by Fraser and Bernatchez, 2001). For example, since 1978, in the United States, the Endangered Species Act (ESA) affords protection to “any subspecies of fish or wildlife or plants, and any distinct population segment of any species of vertebrate fish or wildlife which interbreeds when mature” (Endangered Species Act, Sec. 3 [15]). A distinct population segment (DPS) is not well described in the ESA, but some definitions have been developed. For example, the United States National Marine Fisheries Service (NMFS) developed two criteria for salmonid populations to be considered a DPS: it must be substantially reproductively isolated from other con-specific population units and it must represent an important component in the evolutionary legacy of the species.

Molecular marker information can therefore assist in defining ESUs, which in turn can be used for developing conservation strategies of populations. A related technique that has been useful for delineating the population of origin of individuals in mixed stock fisheries is individual assignment, where individuals are assigned to the population from which their multi-locus microsatellite genotype has the highest probability of occurring (see, for example, Cornuet et al., 1999 and the program “GeneClass”, available at www.montpellier.inra.fr/URLB). From a conservation perspective, assignment tests can be useful for distinguishing native born individuals from those that are hatchery-reared, or for identifying individuals whose genetic make-up most closely resembles the original stock, based on, for example, archived scale data (see the review in Hansen, Kenchington and Nielsen, 2001).

9.3 UNDERSTANDING THE GENERAL EFFECTS OF DIFFERENT AQUATIC ENVIRONMENTS ON FISH GENETIC DIVERSITY AND DIFFERENTIATION

Based on the wealth of population genetic research already conducted in fish, several studies summarizing data based on allozymes (Gyllensten 1985; Ward, Woodwark and Skibinski, 1994) and microsatellites (DeWoody and Avise, 2000) have been published that demonstrate some general trends in genetic structuring for species occupying different habitats during their life cycle. These studies have revealed marked differences in the level of genetic differentiation and genetic diversity between populations of marine and freshwater species, with marine species generally exhibiting lower levels of inter-population differentiation (Gyllensten, 1985; Ward, Woodwark and Skibinski, 1994) and higher genetic diversity (Gyllensten, 1985; Ward, Woodwark and Skibinski, 1994; DeWoody and Avise, 2000). This general observation has generally been hypothesized as a result of higher effective population sizes and/or higher inter-population migration rates in marine than in freshwater environments and has implications for the conservation of genetic diversity. Lower effective population sizes and/or lower inter-population migration rates in the freshwater environment predict that populations of freshwater species will be more prone to extinction than marine species and thus worthy of particular conservation concern. This does not imply, however, that marine populations are immune to such effects (see below).

9.4 ESTIMATION OF EFFECTIVE POPULATION SIZE

Accurate estimates of effective population size (Ne) are central to the development of appropriate conservation strategies in any species because Ne predicts the rate of loss of neutral genetic variation, the fixation rate of deleterious and favourable alleles, and the rate of increase of inbreeding experienced by a population, etc. (Frankham, Ballou and Briscoe, 2002). Importantly, the Ne is often many times smaller than the census size (N) of the population; the Ne/N ratio averages just 0.11 in a survey of vertebrate species (Frankham, 1995). In fish, Ne/N ratios may be expected to be even more extreme due to the high female fecundity of many species enabling large census numbers to be obtained from minimal numbers of breeding individuals. For example, the winter chinook salmon run in the Sacramento River of California consists of around 2 000 mature individuals but the effective size of the population has been estimated to be only 85 (Ne/N= 0.04: Bartley et al., 1992).

While estimates of Ne can be gained using direct methods based on field data (estimates of sex ratio bias, offspring production, variation in family size, etc.), obtaining such data can be very cumbersome in many wild populations, especially in aquatic species. Hence, indirect methods for Ne estimation based on molecular marker data have also been developed. From a practical viewpoint, these methods can be broken down into two categories: those that require data from a single population sample (single generation methods, for example, Hill 1981; Pudovkin, Zaykin and Hedgecock, 1996; Beaumont, 1999; Luikart and Cornuet, 1999) and those requiring samples from the same population collected at least one generation apart (temporal methods: Waples, 1989; Anderson, Williamson and Thompson, 2000; Wang, 2001; Berthier et al., 2002). The temporal methods generally utilize variation in temporal allele frequencies to estimate the level of genetic drift and hence the effective population size. This group of methods tends to give more reliable results than the single generation methods. An important recent advance has been the development of methods that take into consideration the effects of migration on Ne estimation (Wang and Whitlock 2003; the MLNE program is available from www.zoo.cam.ac.uk/ioz/software.htm). The major limitation in the use of these methods is that double the sampling effort is required. This can be a particularly big problem for late breeding species since collection of samples at least one generation apart can fall outside the time frame of a funded study. Nevertheless, due to the existence of historical scale samples in a number of commercially important species from which sufficient DNA can be extracted, originally collected for understanding the age structure of populations, temporal methods for Ne estimation have been applied relatively frequently in fish. In a high profile example, Hauser et al. (2002) demonstrated through microsatellite analyses of a time series of historical scale samples that New Zealand snapper (Pagrus auratus) has undergone a decline in genetic diversity during their exploitation history. In addition, effective population sizes were estimated at five orders of magnitude lower than estimated census sizes. This study provided one of the first indications that genetic problems can potentially exist in marine species for which individual numbers are in their millions.

9.5 DETECTION OF POPULATION SIZE CHANGES

From a conservation perspective, detection of recent dramatic changes in population size (population bottlenecks) is another important aspect of any population monitoring programme (Frankham, Ballou and Briscoe, 2002). Signals of past population bottlenecks can be detected using molecular genetic analyses. For example, one commonly applied method makes use of the assumption that populations that have experienced a recent reduction in Ne will show a reduction in both heterozygosity and allele number at polymorphic loci. However, the reduction in allele number is faster than the reduction in heterozygosity. Therefore, in a recently bottlenecked population, the observed heterozygosity is higher than the expected equilibrium heterozygosity when calculated from the observed number of alleles, under the assumption of a constant-size population (Luikart and Cornuet 1997). Several statistical tests have been developed to determine whether a population exhibits a significant number of loci with heterozygosity excess, hence indicating the occurrence of a population bottleneck (Cornuet and Luikart 1996; implemented in the program Bottleneck available at www.montpellier.inra.fr/URLB). Additional tests for identifying reductions or expansions in population size based on DNA sequence data (Rogers and Harpending, 1992; Templeton, 1998) or allele frequency data (e.g. Beaumont, 1999) have also been developed. The choice of method depends on a number of factors including the samples available, the molecular marker data available and the time frame of any potential bottlenecks. An important feature of all the molecular data applications described above is that temporal information i.e. data collected for the same populations over an extended time period, can be extremely valuable. Fortunately, due to the existence of historical scale samples in a number of commercially important species, the collection of such data is often more feasible in fish than in other taxonomic groups. This has been utilized to good effect by fish conservation geneticists (e.g. Nielsen, Hansen and Loeschcke, 1997; Nielsen, Hansen and Loeschcke, 1999; Hansen et al., 2002, Hansen et al., 2002; Koskinen et al., 2002b). In addition, regular monitoring of populations is important for enabling a distinction between normal population size fluctuations and those severe enough to warrant conservation measures (Laikre, 1999).

9.6 PRIORITIZING POPULATIONS FOR CONSERVATION

Given that resources for preserving biodiversity are limited, there is an important need for biologically sensible criteria that can be applied simply to prioritize fish genetic diversity conservation efforts. Currently, however, such methods are rarely applied (for an exception, however, see Allendorf et al., 1997), although decisions are often made in a rather ad hoc manner or based on solely non-biological criteria such as monetary value. While such criteria should not necessarily be ignored, they should not completely replace biological criteria. Although methods for identifying populations harbouring a higher proportion of a species genetic diversity exist (e.g. Crozier, 1997; Caballero and Toro, 2002; Reist-Marti et al., 2003; Simianer et al., 2003), such methods are yet to be applied in a fish biodiversity preservation context. One model outlining a method for population conservation prioritization, based on experiences in brown trout is outlined in Laikre (1999). This model combines molecular genetic, phenotypic and socio-economic/cultural criteria to prioritize populations for conservation.

9.7 CONFLICTING NEEDS OF DIFFERENT SECTORS OF SOCIETY

As noted above, due to the commercial and cultural importance (e.g. recreational angling) of many fish populations, conservation guidelines of commercially and/or culturally important populations potentially conflict with the needs of other interest groups such as commercial and recreational anglers. Therefore, a challenge when developing a conservation programme for such populations is to find a balance that satisfies all groups, including researchers, decision-makers and end-users. While considerable effort will undoubtedly be put into improving the analytical methodologies described above, it should also be recognized that efforts aimed at decreasing this imbalance are likely to be equally important for fish biodiversity preservation and increased communication between these groups should be encouraged.

9.8 REFERENCES

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10. Molecular marker based analysis for crop germplasm preservation

Sergio Lanteri and Gianni Barcaccia

10.1 SUMMARY

This chapter aims to highlight the potential impact and possible drawbacks of the application of DNA marker technology to crop germplasm preservation. Some of the issues related to crop management and use, which can be addressed through information derived from DNA markers, are discussed with reference to case studies.

10.2 INTRODUCTION

DNA-based assays have revolutionized and modernized our ability to characterize genetic variation. The first advantage of molecular techniques is their capacity to detect genetic diversity at a higher level of resolution than other methods. Furthermore, DNA-based assays are robust and speedy, and information may be obtained from small amounts of plant material at any stage of development. In addition, they are not affected by environmental conditions.

However, managing biodiversity requires more than genetic characterization through DNA polymorphism detection, because information is needed to address key issues of both ex situ and in situ plant germplasm management and to assist in the process of decision-making. For ex situ crop germplasm maintenance, molecular tools may contribute to the sampling, management and development of “core”collections as well as the utilization of genetic diversity. For the in situ and on-farm preservation strategies of genetic resources, molecular markers might help in recognizing the most representative populations within the gene pool of a landrace and identifying of the most suitable strategies for their managing and use.

Many different molecular techniques are presently available and each differs in informational content. Multi-locus approaches may be convenient but have some technical and/or analytical drawbacks, such as dominance (i.e. only one allele identified, no possibility to discriminate between homozygous and heterozygous individuals). As a consequence of simultaneous visualization of many marker alleles, multi-locus data typically are analysed as pair-wise comparison of complex patterns that only have meaning relative to others in the same study; thus results are to a limited extent comparable among studies. By contrast, single-locus markers are usually characterized by co-dominance (i.e. both alleles identified in heterozygous individuals) and are therefore more flexible and supply more robust and comparable data (Karp, 2002).

An appropriate use of molecular markers techniques requires clearly defining the issues addressed and the type of information that will be needed (on genetic diversity), and knowing what the different techniques can offer, not only in terms of genetic information, but also resource requirements, reproducibility and 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: (i) sampling strategies; (ii) the utilization of various data sets on the basis of understanding their strengths and constraints; (iii) the choice of genetic similarity estimates or distance measures, clustering procedures and other multivariate methods in analyses of data; and (iv) objective determination of genetic relationships (Mohammadi and Prasanna, 2003). For all these reasons, choosing the most appropriate technique may be difficult, hence often a combination of techniques is needed to gather the information of interest.

Up to now most conservation efforts have focused on agriculturally important crops; around one-third of all ex situ accessions in gene bank represents just five species: wheat (Triticum sp.), barley, rice, maize and beans (Phaseolus spp.). The relative over-representation of the five species does not necessarily mean that their genetic diversity has been fully covered (Graner et al., 2004); in fact there is a significant lack of knowledge on the diversity and geographic distribution of less utilized crops as well as their wild relatives (Hammer, Arrowsmith and Gladis, 2003).

This chapter presents two case studies addressed to assess, through molecular tools, the genetic diversity in a minor crop, such as Cynara cardunculus, and in landraces of a major crop such as Zea mays.

10.3 CASE STUDY 1: ASSESSMENT OF THE GENETIC DIVERSITY IN CYNARA CARDUNCULUS

Cynara cardunculus L. is a diploid (2n=2x=34), predominantly cross-pollinated species native to the Mediterranean Basin. It includes two crops: globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC) as well as wild cardoon (var. sylvestris[Lamk] Fiori), a non-domesticated perennial that has been recognized as the ancestor of both cultivated forms. Globe artichoke represents an important component of the South European agricultural economy but is grown all over the world for its large immature inflorescences (capitula); its commercial production is mainly based on perennial cultivation of vegetatively propagated clones. Cultivated cardoon is grown for its fleshy stems and roots and is of some regional importance in Italy, Spain and the south of France; its propagation is carried out through seed.

Previous studies have shown that all the forms within Cynara cardunculus have the following characteristics: (i) they are a promising source of seed oil, both with respect to quantity and quality; (ii) they can be exploited for the production of lingo-cellulosic biomass for energy or paper pulp; and (iii) they are a source of biopharmaceuticals. The roots contain inulin, a known improver of human intestinal flora, while the leaves are a source of antioxidant compounds, such as luteolin and di-caffeoylquinic acids (cynarin). Notwithstanding its wide possibilities of exploitation, little is known on the amount and distribution of genetic diversity. In order to assess this, the following questions had to be addressed:

• Which area should be included in the survey?

• How can diversity of natural and cultivated populations growing in situ be assessed rapidly and efficiently to quantify the distribution of genetic variation and gather information for both identification of populations representative of the genetic variation and application of sampling or in situ preservation strategies?

• How much diversity is present in the Cynara cardunculus cultivated forms of different growing regions and what criteria should be adopted for the development of a core collection?

• Can individual plants be adequately identified for future application of plant breeding programmes?

Of course, DNA-based methods can efficiently contribute to answering the above questions, but which are the most suitable for obtaining results that are reproducible by different laboratories, capable of being scored and analysed using standardized methods, and/or suitable for entry into database? Since techniques detecting heterozygotes (i.e. co-dominant markers) and providing data on allelic differences were desirable, microsatellites (simple sequence repeats [SSR]) were considered the most suitable markers although not available at the time of starting the work. Our first objective was thus to develop highly informative microsatellite markers by following different strategies, as reported by Acquadro et al. (2003; 2005a; 2005b). Fifty primer pairs were designed and 32 were chosen as being highly polymorphic. We also felt it necessary to complement the microsatellite analysis with amplified fragment length polymorphism markers (AFLPs) due to their high reproducibility and their high information content.

Two spatially isolated areas were chosen for our survey: Sardinia and Sicily, the latter implicated as the origin of artichoke domestication. In both islands, wild cardoon extensively colonizes dry and undisturbed areas while autochthonous globe artichoke germplasm is still the most commonly grown. The first problem to face was how many populations within each area and how many plants within each population had to be sampled. Indeed, in studies aimed at analysis of population structure, it is necessary to balance the need to collect as large a sample size as possible and to get allele frequencies from as many loci as possible, with the need to screen as many populations as possible.

10.3.1 Wild cardoon genetic variation

On the basis of the different pedo-climatic conditions of the growing areas, we identified three populations in Sardinia and four in Sicily (Figure 12) (Portis et al., 2004; 2005a).

FIGURE 12
Geographic location of wild cardoon (C. cardunculus var.sylvestris) and globe articoke populations

It is well known that the minimum sample size for detecting alleles at a given frequency is greater in inbreeding than in outbreeding species (Gregorius, 1980); being Cynara cardunculus, an allogamous species, 30 individuals per population were randomly sampled, which ensured (P< 0.95) the detection of alleles present at relative frequencies between 0.08 and 0.09 (Gillet, 1999). Plants were genotyped using the developed SSRs and seven AFLP primer combinations, which generated more than 400 polymorphic bands. Genetic divergence between populations was found to be consistent between the two marker systems.

As a result of the geographical isolation, the Sardinian and Sicilian populations were clearly differentiated, forming two distinct gene pools (Figure 13D). Both marker systems show that the Sardinian and Sicilian populations possess a remarkable number of unique (private) alleles (Table 12) showing that the two gene pools must have evolved independently.

Several criteria have been suggested to identify priority population for sampling the maximum possible genetic variation (Maguire, Peakall and Saenger, 2002): (i) allelic richness, or the number of alleles per locus; (ii) evaluated as locally common alleles, that is, those that are common in one to several populations but not in the species as a whole; and (iii) identified as unique (private) alleles. Based on any one of these criteria and for both AFLP or SSR data sets (Table 12), the same priority populations were identified: Palazzolo and Bronte in Sicily, and Oristano in Sardinia.

FIGURE 13
PCO plot of first three principal coordinates depicting the genetic relationship among accessions from populations of globe artichoke: “Spinoso sardo” (A), “ Violetto di Sicilia” (B), “Spinoso di Palermo” (C), and wild cardoon (D)

TABLE 12
Number of bands, and common and rare alleles within a location, detected with SSR and AFLP. Roccella (ROC), Bronte (BRO), Piano Tavola (TAV), Palazzolo (PAL), Sassari (SAS), Nuoro (NUO), Oristano (ORI)

In both islands, most of the AFLP and SSR genetic variation was present within rather than between populations, which is consistent with data reported by Gaudel, Taberlet and Till-Bottraud (2000) for outbreeding species. Nevertheless, a remarkable extent of within-population clustering was detected. It was not possible to identify any overlap between populations and thus, despite the high ratio of within-to-between population genetic variance, the AFLP banding pattern of each genotype was found to be a relatively reliable predictor of its parental population.

10.3.2 Globe artichoke genetic variation

An analogous approach was applied for the analysis of genetic variation in autochthonous Sicilian and Sardinian globe artichoke germplasm, which is at risk of genetic erosion due to the recent introduction of varieties selected abroad or germplasm from other regions which best fit market demand (Lanteri et al., 2001; Portis et al., 2005b). In Sicily seven populations were identified, of which three were of the spiny type, “Spinoso di Palermo”(SP), which is generally cultivated on the western side of the island, and four of the non-spiny type “Violetto di Sicilia”(VS), which is confined to its eastern side. In Sardinia, five populations of the autochthonous spiny type “Spinoso sardo”(SS) (Figure 12) were identified. Due to the limited polymorphism of SSR markers in globe artichoke, AFLP and random amplified polymorphic DNA (RAPD) markers were applied.

In Sicily a significant genetic differentiation between spiny and non-spiny types was found as principal coordinate (PCO) analysis identified two main clusters, one of which groups the three representative SP populations, while the other groups the four VS populations (data not reported). Around one-third of the AFLPs scored were found specific to one or other of these two types.

In both Sicilian and Sardinian populations, most the genetic variation was present within, rather than between populations. As the three varietal types are vegetatively propagated, the within-genetic variation presumably reflects their multiclonal composition, a direct consequence of the limited selection criteria adopted by farmers. An additional source of variation may be via spontaneous mutations, which are maintained as they are not subject to any meiotic sieve and might not be detectable.

Despite the high level of within-population genetic variation present, most of the populations could be genetically differentiated from one another due to farm fragmentation or adaptation to local pedo-climatic conditions. In Sicily the between-population differentiation was more evident in VS, allowing the PCO to define four clusters with minimal overlap (Figure 13B). The opposite was true in Sicilian SP and in Sardinian SS: two of the populations were partially overlapping, presumably as a consequence of some exchange of material between farmers (Figure 13A-C). Our data resulted very informative for the implementation of on-farm germplasm preservation strategies. On the basis of the criteria reported above, the Rosolini (VS) and the Buonfornello (SP) populations in Sicily and the Oristano population in Sardinia are the most representative of the gene pool of the varietal types, and priority should be given to them for application of on-farm preservation strategies.

10.3.3 Globe artichoke and cultivated cardoon germplasm characterization

The use of molecular markers that quantify the genetic diversity within and between accession may significantly increase the efficiency of the assessment and management of germplasm collection by reducing redundancy. Several works have detailed the philosophy behind optimizing collections to ensure diverse genetic representation through either the creation of “core”collections or some form of hierarchical sampling. We applied eight AFLP primer combinations for characterizing a living collection of globe artichoke germplasm maintained at CRAS (Oristano, Sardinia).

We also included in the analysis accessions collected in the field in Spain, Turkey and the United States for a total of 89 varietal types; for three of them different provenances were assessed for a total of 118 accessions (Lanteri et al., 2004a). Figure 14 shows the dendrogram from unweighted pair-group method arithmetic average (UPGMA) cluster analysis of AFLP data. Our results suggest that traits selected by humans play an important role in understanding variation and differentiation within cultivated artichoke germplasm. Two main clusters were found within branch A: A1 and A2. Cluster A1 mainly contained Catanesi types with small elongated heads, while cluster A2 included the Romaneschi types, with big spherical or sub-spherical non-spiny heads, mainly cultivated in central Italy together with the American accessions of Green Globe, which accounts for more than 85 percent of the artichoke production in the United States. Branch B contained two additional clusters: B1 and B2, both of which included Spinosi and Violetti types with medium-small heads. Cluster B1 also included all the Turkish accessions. AFLP data furnished important information for assembling a core collection of globe artichoke germplasm, taking into account the hierarchical structure of the gene pool. Our results showed that the genetic variation detected within the same varietal type was in some cases higher than that found between varietal types. The Jaccard's similarity index among clones of the same varietal type might thus be considered a threshold value that identifies material sharing the same genetic background. On the basis of this threshold a limited number of core subset could be identified. Genetic studies in selected crops have demonstrated that widespread and localized alleles occurring in the entire collection are usually contained in the core subset, with only rare localized alleles excluded (van Hintum et al., 2000). The core subset can thus provide an introduction to further studies on biodiversity of the entire collection or to identify suitable material for future breeding efforts. Furthermore, the results obtained in this study, as well as in another study aimed at genotyping selected clones of the varietal type “Spinoso sardo”(Lanteri et al., 2004b), showed that AFLP markers are useful to identify duplicates and provide evidence for the uniqueness of a particular genotype.

FIGURE 14
Dendrogram obtained from UPGMA cluster analysis of AFLP data of 118 globe artichoke accessions

Wild and cultivated cardoon were also included in the analysis Co-phenetic correlation coefficient = 0.92

An analogous study was carried out in cultivated cardoon (Portis et al., 2005c), whose genetic variation of the material in cultivation in Spain and Italy was assessed by DNA profiling at five microsatellite loci and with eight AFLP primer combinations. The analysis of genetic similarities showed that the Spanish and Italian accessions represent two distinct gene pools. This study also demonstrated that a fruitful method in characterizing germplasm collections with AFLP markers is to use a two-tiered approach: first, using low-density profiles to compare all samples and resolve the main cluster, and then using high-density profiles to resolve samples within clusters.

10.4 CASE STUDY 2: ITALIAN LANDRACES OF MAIZE AND MOLECULAR MARKERS FOR THEIR CHARACTERIZATION AND CONSERVATION

Maize (Zea mays L.) is one of the most important crops in Italian agriculture. The species was introduced in the national cultivation system approximately four centuries ago and is grown mainly for human consumption. Since then a number of landraces have been developed to meet specific needs of cultivation and utilization and to overcome environmental constraints of different areas. As a consequence, new landraces originated from the original populations introduced, through adaptation to local conditions as well as hybridization brought about by continuous exchange and trade. These landraces were locally maintained by farmers as open-pollinated populations, thus each represented a collection of highly heterozygous and heterogeneous plants. Although a considerable range of variation within each population was present, a between-population differentiation was detectable for several distinctive traits as a consequence of both natural and human selection pressure.

Within the last few decades, the Italian agricultural scenery has profoundly changed and the subsistence mixed farming unit is now transformed into an intensive monoculture (Bertolini et al., 1998). At present, a small number of populations of flint maize (Z. mays var. indurata) can be found under very peculiar agricultural situations or in marginal areas, such as alpine valleys, and on small fields traditionally managed according to low-input agronomic practices, and with production exclusively addressed to human consumption (Lucchin, Barcaccia and Parrini, 2003). The agricultural environment together with the traditional diet of these regions allows preservation of some landraces and limits diffusion of modern hybrids. Unfortunately, many locally cultivated populations were lost before it was realized that they were important sources of germplasm. Maize breeders have recently become more aware of the need for both maintaining genetic diversity among hybrid varieties and improving the management of genetic resources through the conservation of landraces. Consequently, there was renewed interest for in situ conservation of the landraces, not only to preserve important sources of genetic material for breeding, but also to allow their valorization as essential components of sustainable agriculture. Landraces are the cultivated maize material with the highest genetic variation as well as with the best adaptation to the natural and anthropological environment where they have evolved. In addition, they contain locally adapted alleles and likely represent an irreplaceable bank of highly co-adapted genotypes. Knowledge of genetic diversity among local populations and breeding stocks is expected to have a significant impact on the improvement of this crop. In maize, information on both qualitative and quantitative morphological traits of existing landraces may be useful in maintaining their genetic variability and preserving them from genetic erosion. Nowadays, after years of lack of interest in the so-called “old local varieties”, this valuable source of maize germplasm has been rediscovered and exploited as a niche crop suitable for the cultivation of marginal lands.

The development of molecular markers has greatly facilitated basic and applied research programmes of maize genetics and breeding. DNA polymorphism assays are also known as powerful tools for characterizing gene pools and investigating germplasm resources.

10.4.1 Molecular characterization of field populations belonging to an Italian landrace of flint maize ( Zea mays var. indurata )

A comparative characterization of farmer populations of the flint maize landrace “Nostrano di Storo”was recently carried out by Barcaccia, Lucchin and Parrini (2003) using different types of PCR-based markers. The inbred line B37 and three synthetics (VA143, VA154 and VA157) selected from as many landraces were used as reference standards. Genetic diversity and relatedness were evaluated with SSR and Inter-SSR markers. Nei's total genetic diversity as assessed with SSR markers was HT=0.851 while the average diversity within populations was HS=0.795. The overall Wright's fixation index FST was as low as 0.066. Thus, more than 93 percent of the total variation was found within population. Dice's genetic similarity coefficients within and between populations on the basis of Inter-SSR fingerprints were 0.591 and 0.564, respectively. The UPGMA dendrogram displayed all populations except for one clustered into a distinct group, in which a synthetic variety selected from the landrace “Marano Vicentino”was also included (Figure 15).

One population and the other two synthetics, “Spino Bresciano”and “Dente di Cane Piemontese”, were clustered separately. 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, Lucchin and Parrini, 2003). This result was also confirmed by a further molecular investigation carried out using AFLP and RAPD markers to fingerprint pooled DNA samples from all farmer populations.

FIGURE 15
UPGMA dendrogram of the maize farmer populations

Although gene flow from commercial hybrids might have occurred, the large number of polymorphisms and the presence of both unique alleles and alleles unshared with B37 and synthetics are the main factors underlying the value of this flint maize landrace as a source of genetic variation and peculiar germplasm traits. Because of its exclusive utilization for human consumption, such a molecular marker characterization will be a key step to promoting the in situ conservation and protection of the landrace.

10.4.2 Construction of a linkage map for a maize landrace based on a pseudo-testcross strategy using multi-locus PCR-based markers

Genetic linkage maps based on molecular markers represent basic tools for investigating and characterizing local germplasm resources. A linkage map of the flint maize landrace “Nostrano di Storo”based on dominant multi-locus PCR-derived markers (RAPD, I-SSR, AFLP and SAMPL), was constructed according to a one-way pseudo-testcross mapping strategy (Barcaccia et al., 2000; 2005). The feasibility of such a study depended on the presence of high levels of heterozygosity in the landrace parent mapped and on the informativeness of the PCR-based marker systems used. Co-dominant single-locus SSR markers were adopted to assign each linkage group to a specific chromosome and all marker loci to specific chromosome arms. The final genetic map includes 282 marker loci and covers 1.826 cM (Table 13).

This genetic map based on multi-locus marker systems and on easily detectable molecular markers will find application and prove useful for rapidly characterizing the genetic diversity within and relatedness among farmer populations belonging to the “Nostrano di Storo”landrace maintained according to different conservation strategies.

TABLE 13
Summary of the map statistics, including length, number of marker loci per chromosome and total

10.4.3 Assessment of the optimal plant and molecular marker sample size to estimate genetic diversity in maize landraces

The genetic characterization of landraces represents an essential step for their conservation. This requires establishing the most appropriate system and type of markers (i.e. random or mapped) and the minimum number of markers and plants required to describe the genetic structure of a given population. In spite of their importance for the success of any germplasm conservation programme, little information is available on landraces because almost all studies were performed on inbred lines. A sample of plants were chosen as representative of the landrace in terms of morpho-phenological and agronomic traits, and then assayed at hundreds of marker loci, either mapped or random. Genetic similarity and diversity coefficients computed using mapped markers proved to be significantly higher than estimates based on total markers. Moreover, no significant changes of marker allele frequency and polymorphism information content were scored when the number of sampled ears was progressively reduced from 50 to 15, even if a steady increase of standard errors was observed. The influence of the number and type of molecular markers on genetic similarity and diversity measurements was also investigated: no significant changes in terms of mean polymorphic index content (PIC) values were observed, whereas the coefficient of variation (CV) of standard deviations raised proportionally to the reduction of sample size. A total of 120 random markers and 80 mapped markers were needed to get CVs of standard deviations lower than 5 percent. Also, when the number of ears and markers were evaluated together, no significant changes of the mean PIC values were observed and an increase of the molecular data variability was confirmed (Pallottini, 2002).

Influence of marker and plant sampling on statistics used to measure genetic diversity should be useful to investigate the genetic consequences of different modes of conservation of maize landraces (on-farm, in situ and ex situ). In conclusion, reliable and effective investigations of landrace population genetic structure with AFLP markers can be performed using at least 30 ears per population and one plant per ear, and require at least 80 mapped marker loci.

10.4.4 Effects of different conservation strategies (on-farm, in situ and ex situ ) on the population genetic structure of maize landraces as assessed with molecular markers

The on-farm, in situ and ex situ conservation methods may exert a different influence on the genetic structure of populations grown by farmers. This influence should be accurately evaluated to avoid genetic erosion and conservation programme failure. In fact, the loss of genetic diversity could be due to inbreeding that can result from drift and migration to natural and human selection and gene flow. Each of these factors has a different relative importance on the types of conservation methods. Molecular markers were used to investigate the influence of the conservation strategy on the genetic structure of farmer populations grown for two years with three different methods: (i) on-farm conservation by farmers, using own seeds and traditional agronomic practices; (ii) in situ conservation in the original area but taking into account the spatial isolation from other fields cultivated with hybrid varieties; and (iii) ex situ conservation far from the original area with no gene flow due to the total absence of fields grown with the same crop (Pallottini, 2002). Statistical tests failed to reveal any significant difference in terms of diversity/similarity absolute values among the populations conserved according to the three distinct strategies (Figure 16).

However, about 10 percent of the comparisons performed for the marker allele frequency parameter at the total assayed loci showed significant differences. Even the differences between genetic variation parameters computed for mapped and random marker loci were significant. In particular, some marker loci were more affected than others by changes of the marker allele frequency depending on the conservation method. These markers, distributed throughout the genome, could be related to important genes involved in the adaptation to environmental conditions or responsible for traits evaluated in the selection by farmers.

In sum, although all conservation methods studied have determined the significant changes to the genetic structure of the farmer populations, the genetic variation and diversification that occurred with ex situ conservation was much stronger than that observed for in situ and on-farm conservation. It is worth mentioning that to monitor these changes, the level at which the investigation is performed is essential. When the mean values of the more common genetic diversity and/or similarity indexes are taken into account, no significant differences are highlighted because of the large set of molecular data and the occurrence of bidirectional changes of marker allele frequencies over all marker loci. Consequently, variation of the marker allele frequency has to be computed and interpreted at each single marker locus or between pairs of marker loci, but not on the whole molecular marker data set.

FIGURE 16
PIC values and standard errors computed in the original population (or) and in the populations obtained from on-farm (of),

10.5 CONCLUSIONS

These case studies show how molecular marker techniques may help in characterizing and managing genetic diversity. However, other questions have to be answered. Molecular tools are very informative but are generally employed anonymously. They are able to detect high levels of DNA polymorphisms, but are they really providing the kind of information required to make effective and sound judgments on diversity? What is the functional relevance of the polymorphism detected? Indeed, understanding the significance or assessing the value of the diversity is still a difficult challenge.

New approaches have been recently developed for adapting the current PCR-based techniques to target functional diversity. As stated by Tanksley and McCouch (1997), “new findings from genome research indicate that there is a tremendous genetic potential locked up in germplasm collections that can be released only by shifting the paradigm from searching for phenotypes to searching for superior genes with the aid of molecular linkage map.”At present, the increasing information available from genome mapping means that markers known to be very closely linked to traits of interest can be better addressed for characterizing genetic diversity and help in identifying variation of use to breeders. Furthermore, the identification of genes controlling a trait and the availability of their DNA sequences may facilitate the classification of variation in germplasm pools. High-resolution genetic maps enable closely linked markers to be used and the increasing numbers of expressed sequence tags (ESTs) and single nucleotide polymorphism (SNP) markers provide routes for more targeted sequence-based approaches. Classification of the sequence variants at a targeted locus would substantially reduce the amount of work needed to assess their potential for breeding and lead to the identification of superior alleles (Sorrels and Wilson, 1997).

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