**4. Seed dessimination and ownership**

tential to show enhanced resolution of QTLs. Success depends on population size, control of population structure and the degree of LD in the population. LD levels vary both within and between species [73]. With this approach, marker–trait association is only expected when a QTL is tightly linked to the marker, because the accumulated recombination events occur‐ ring during the development of the lines will prevent the detection of any marker–trait loose association. In maize, the application of this approach has demonstrated the association be‐ tween several candidate genes and kernel composition traits, starch pasting properties and

Using SSR markers, the genetic diversity among inbred lines derived from the Portuguese germplasm collection was evaluated and compared with worldwide maize inbreds repre‐ sentatives [36]. The Portuguese inbred lines have maintained a level of genetic diversity sim‐ ilar to the foreign lines. Moreover, it was concluded that they are derivatives of miscellaneous populations, showing high genetic diversity and consequently representing a potential valuable source of interesting genes to introduce into modern cultivars [36], and a

Until now, no LD analysis or association studies were undertaken on the group of inbred lines of Portuguese origin, neither the identification of genes/QTLs controlling bread mak‐ ing ability. Presently, in order to address this gap, the collection of Portuguese maize inbred lines, derived mainly from Portuguese landraces, is being genotyped using microsatellites to

Currently, the national efforts are focused on the study of the genetic control and the envi‐ ronmental effect on the antioxidant and aroma compounds as well as the bread making abil‐ ity. This study applies an association mapping approach using the previously characterized inbred lines that differ for endosperm types and colors. QTL associated candidate genes will be identify on the basis of positional information of the recently maize cloned genes (re‐ viewed by [75]). Candidate genes will be validated on the enduring landraces [7] and mod‐ ern improved OPVs (VASO project) that are also being characterized at genetic, nutritional, organoleptic (aroma volatiles) composition and antioxidants bioactivity. Specific molecular markers tightly linked to the identified QTLs will be identified or developed to provide breeders and farmers user-friendly markers to select for superior genotypes for quality maize bread. Additionally, it will allow the exploration of maize local resources and natural quality diversity in the reinvention of traditional maize to produce modern high quality

Nowadays, the most promising maize populations at agronomic, molecular and quality lev‐ el, collected during the 2005 expedition, are being evaluated and selected under a participa‐ tory approach in 13 different locations. This field research has been done in articulation with the original VASO project locations and improved populations, and now is under the super‐ vision of the ESAC researchers. The association of the farmers' perception with the newly available molecular and quality data can be extremely valuable to aggregate or separate populations, creating possible pools with heterosis that will be very useful to generate new

amylose levels [74].

276 Plant Breeding from Laboratories to Fields

valuable germplasm for association studies.

detect population structure and to study LD.

bread with potential health benefits.

**3.4. Testing of higher quality experimental cultivars**

The potential advantages of PPB, such as the faster dissemination of new varieties, higher adoption and increased biodiversity within the crop, can only be achieved if the seed of the new varieties is available in sufficient amounts to all the farmers' community [19]. Although the varieties developed through PPB will have specific adaptation to certain environmental conditions, it is likely that they will also perform well on farms that share similar climates and soil types. It is unlikely that they will spread as far as varieties specifically targeted to have wide adaptation in higher input systems [76], but it is possible that they will benefit many farmers in neighboring areas. Genetically variable materials, such as OPV and syn‐ thetics, make more likely their usefulness to farmers in environments that differ from the original selection environment [77].

The global community, through the Convention on Biological Diversity and the Internation‐ al Treaty on Plant Genetic Resources for Food and Agriculture, has recognized the contribu‐ tion of farmers to the maintenance of genetic resources. Given the actual and potential future impact of PPB, this contribution will increasingly include new PPB' varieties. [78]. Such varieties need recognition and protection.

According to European Union regulations, farmers are allowed to reproduce non-certified seeds for themselves, but they are not able to sell them. Generally, only varieties that are of‐ ficially registered and listed, after meeting DUS and VCU requirements, can be multiplied by the formal seed system [79]. Formal seed systems were put in place in Europe, in the mid-19th century, as a result of the development of specialized plant breeding products and to create transparency in a seed market where variety names were rapidly proliferating [80]. Current variety registration for commercial purposes requires that the new variety be dis‐ tinct from all the varieties of common knowledge, uniform in its essential characteristics and highly stable after repeated multiplication (DUS= Distinctness, Uniformity and Stability, [80]). In addition, testing for cultivation and use values (VCU) was introduced as a require‐ ment for commercial release, in order for farmers to have an independent assessment of the yield, quality and value of the grain [6]. These last ones are the real concerns for farmers, but in practice this evaluation is based mostly on quantitative Weld criteria such as grain yield, maturity time, standing ability and disease resistance, which are easy to measure. Less at‐ tention is given to aspects such as storability, cooking quality and by-product use, which may determine the overall value of the variety, especially for small farmers. To this extent, standard VCU tests do not easily reflect the more complex requirements of small-scale farm‐ ers and this has been one of the problems of official variety release in meeting the needs of such farmers [79].

This formal system is unfriendly for farmers' varieties such as landraces and new varieties developed through PPB, leaving these varieties outside the legal market of seeds [81]. These varieties are less likely to meet the stringent DUS and VCU criteria because they lack uni‐ formity and rarely perform well across the majority of test sites. Nevertheless, the European Union has recently approved a special treatment for the so called "Conservation Varieties" by which landraces and varieties adapted to local and regional conditions and threatened by genetic erosion can be registered for commercialization under certain conditions (Directive 2008/62/EC from 20 June 2008). The special treatment consists, of


The varieties obtained in our project can be registered as conservation varieties. In the regis‐ tration process, the varieties have to be characterized for a minimum number of morpholog‐ ical traits and only less than 10% of the plants can be out of type. Attention is given to the region where the variety is traditionally used as a crop and to where it is naturally adapted. The registration is obtained if description of the varieties, denomination, results from nonofficial trials, knowledge associated with sow, multiplications and use, and other informa‐ tion is provided to the genetic resources authorities. This information is then evaluated by the national entity (DGADR in the Portuguese case). In the national catalogue of varieties (CNV) the respective location of origin is indicated, and the regions of seed production can be identified besides the seed origin. Seeds are submitted to sampling and quality standards. Storage must be done in close packages and producer labels are required. The conservation varieties may be marketed only in their regions of origin or in additional regions, as long as these regions are comparable, regarding semi-natural and natural habitats, to the region of origin of this variety. The maximum quantity of seed per specie, allowed for commercialization purposes, is 10% of the seeds used annually in the country, if this condition does not exceed the total amount of seed needed to sow 100 ha. In the case of the Portuguese maize varieties, the maximum allowed is 0.3% of the seeds used in the country during a growing season, but limited to 100 ha. This means that conservation varieties, if we consider the maximum of 100 ha, represent 0,073% of the Portuguese maize area (100 ha/137 413 ha) and can represent 10 000€ (20 kg of seed/ha x 5 €/Kg of seed x 100 ha) in Portugal. This data indicate that this germplasm should be used preferably in marginal areas under PPB project. It also indicates that maize PPB projects should be integrated in the food market preferably in those where the direct output is not the seed itself, but for example, the bread that has a higher market value. Nevertheless, in the process of registration of conservation varieties, the PPB farmer alone will not be able to provide all types of unofficial tests and information needed. So supportive associations should step forward and help on this registration process.

[80]). In addition, testing for cultivation and use values (VCU) was introduced as a require‐ ment for commercial release, in order for farmers to have an independent assessment of the yield, quality and value of the grain [6]. These last ones are the real concerns for farmers, but in practice this evaluation is based mostly on quantitative Weld criteria such as grain yield, maturity time, standing ability and disease resistance, which are easy to measure. Less at‐ tention is given to aspects such as storability, cooking quality and by-product use, which may determine the overall value of the variety, especially for small farmers. To this extent, standard VCU tests do not easily reflect the more complex requirements of small-scale farm‐ ers and this has been one of the problems of official variety release in meeting the needs of

This formal system is unfriendly for farmers' varieties such as landraces and new varieties developed through PPB, leaving these varieties outside the legal market of seeds [81]. These varieties are less likely to meet the stringent DUS and VCU criteria because they lack uni‐ formity and rarely perform well across the majority of test sites. Nevertheless, the European Union has recently approved a special treatment for the so called "Conservation Varieties" by which landraces and varieties adapted to local and regional conditions and threatened by genetic erosion can be registered for commercialization under certain conditions (Directive

**2.** an exemption from official examination if the applicant can provide sufficient informa‐ tion about the variety through other means such as unofficial tests and knowledge from

The varieties obtained in our project can be registered as conservation varieties. In the regis‐ tration process, the varieties have to be characterized for a minimum number of morpholog‐ ical traits and only less than 10% of the plants can be out of type. Attention is given to the region where the variety is traditionally used as a crop and to where it is naturally adapted. The registration is obtained if description of the varieties, denomination, results from nonofficial trials, knowledge associated with sow, multiplications and use, and other informa‐ tion is provided to the genetic resources authorities. This information is then evaluated by the national entity (DGADR in the Portuguese case). In the national catalogue of varieties (CNV) the respective location of origin is indicated, and the regions of seed production can be identified besides the seed origin. Seeds are submitted to sampling and quality standards. Storage must be done in close packages and producer labels are required. The conservation varieties may be marketed only in their regions of origin or in additional regions, as long as these regions are comparable, regarding semi-natural and natural habitats, to the region of origin of this variety. The maximum quantity of seed per specie, allowed for commercialization purposes, is 10% of the seeds used annually in the country, if this condition does not exceed the total amount of seed needed to sow 100 ha. In the case of the Portuguese maize varieties, the maximum allowed is 0.3% of the seeds used in the country during a growing season, but limited to 100 ha. This means that conservation varieties, if we consider the maximum of 100 ha, represent 0,073% of the Portuguese maize area (100 ha/137 413 ha) and can represent 10 000€ (20 kg of seed/ha x 5 €/Kg of seed x 100 ha) in Portugal. This data indicate that this

2008/62/EC from 20 June 2008). The special treatment consists, of

**1.** a certain degree of flexibility in the level of uniformity that is required, and

such farmers [79].

278 Plant Breeding from Laboratories to Fields

practical experiences [6].

In Portugal, such an association has been created in 2010, the ZEA+ association, where a cluster of maize researchers and participatory breeding farmers are joined together. This as‐ sociation was the logical step to fill in the gap at the logistical level to deal with all the infor‐ mation that had been collected at etnobotanical, agronomical, genetic, and molecular and food quality by the national maize cluster of research. This association main objective is the study and promotion of the conservation and valuation of agricultural genetic resources in a perspective of rural development, emphasizing the link with the urban communities. In this way, this association is dedicated to traditional landraces, including conservation and au‐ tochthones varieties. It can provide logistical or managing support to germplasm improve‐ ment through participatory plant breeding where its associates collaborate. The Zea+ association could contribute to successfully market conservation varieties and to help estab‐ lish some kind of small seed enterprise for farmers, in order to have a clean source of seed from those varieties. It should also support the registration of improved varieties already validated with field and molecular marker data. This would allow the reinvestment of the potential royalties in science, to provide more information to farmers and researchers.

Besides seed dissemination, it is also necessary to consider the maintenance of the genetic gains achieved. If the improved material is not managed in a systematic way, it may be di‐ luted by physical contamination or out-crossing and thus dissolve back into the local popu‐ lation. The benefits achieved by PPB may then be lost, leaving no secure point of reference to return to in the future [79]. Consequently, this responsibility should be vested by a farm‐ ers/researchers association established to produce and market the seed.

This registration possibility does not mean that the process of selection cannot continue, rather than at certain intervals a reasonably defined 'milestone' is set up along the road of improvement [79]. Further enhancement of productivity and stability is achieved through practicing "non-stop selection" within landraces across the marginal production environ‐ ments, to exploit the useful adaptive variation constantly released by the genome.

## **5. Future prospectives and market development**

At a time when a team of young scientists is taking care of this PPB project, which is reach‐ ing it maturation, we can foresee a new future for the Portuguese small farming. Its quality oriented purpose for food, its sustainability and environment friend signature, will be an important piece to bring our sustainable small farming system back to its feet.

In the medium/long term, we expect that the abandoned northern agricultural systems will survive due to the local abundance of water. In these environments maize will play an im‐ portant role in the production of quality food. In this case, the national germplasm will play an important role in the recovery of our small farming system. Participatory plant breeding is the tool necessary to take advantage of our rich maize germplasm collected in the 70's and preserved in our Portuguese Plant Gene Bank (BPGV), or in same cases still present at our farmers fields. Without such an investment in breeding, to raise their yielding capability to reasonable levels, our genetic resources will remain in a useless tomb or vanish definitely from our farm land.

In scientific terms, we foresee that with this new multidisciplinary team of young scientists new findings will came along, especially at understanding the genetics of important agro‐ nomical and quality traits that will translate into improved high quality varieties. The role of this quality oriented varieties, either under open pollination or hybrid form, will fit in a new agricultural system oriented to quality tourism, where maize will represent only a piece, as important as it may be, within this system. Entertainment, like the traditional "desfolhadas" (harvest festivities), historical, architecture, archeological and cultural attractions, together with the combined restoration of old water mills and cob stores, all of this complemented with folkloric music will complete the system.

The potential fixation of our farmers, consequence of the economic recovery, will benefit the most the environment (water management, soil conservation, genetic resources preser‐ vation, and the control of forest fires). Farmers will always be the breeder's best allies and the best curators of our genetic resources. New genetic and analytical tools are now avail‐ able that can help the traditional plant breeding methodologies. Sustainable, quality orient‐ ed, and environmental friendly agriculture still has a role to play in countries like Portugal and beyond.

Nevertheless this extended PPB project was only possible due to funding obtained through several national (from Fundação para a Ciência e a Tecnologia, Portugal, POCI/AGR/ 57994/2004, PTDC/AGR-AAM/70845/2006, PTDC/AGR-ALI/099285/2008) and international (FP7 program, SOLIBAM project) research projects of limited duration. Its survival depends on finding sustainable ways of self-support that may be obtained by higher marketing of im‐ prove quality varieties, maintaining genetic diversity, with increased market value, more at‐ tractive to the final consumer.

So, further supportive actions to market creation and market promotion should be taken. As adapted from [6] to our national reality, partnerships should be built or strengthen through the organization of meetings involving market-chain stakeholders to discuss how to change market potential. Niche markets for traditional landraces raw materials or traditional land‐ races food products (maize bread) should be further exploit. This is the case of the gluten intolerant market, to which the 100% maize bread can be an attractive alternative. Also, the general market should be aimed with media advertisement campaigns to improve consumer awareness of important nutritional or ecological-friendly traits from traditional landraces, for example, using the summary of the research project activities. On the same level, an ecolabeling of products (such as the maize bread) obtained from traditional landraces as a com‐ mitment to the preservation of biodiversity, could call the attention of consumers. As already highlighted above, the ecological practices of traditional production systems, where traditional landraces are maintained, should be promoted. Agrobiodiversity ecotourism could be one way of doing so, because it publicizes the diversity of cultivated plants and the associated cultural practices by involving activities as farm and market visits, participation in agricultural activities and food (bread) preparation, food tasting and attending feasts or celebrations associated with agricultural practices. Finally and as a last resource, farmers who provide environmental services, such as conservation functions should be compensat‐ ed. A governmental direct support could be provided to farmers who cultivated traditional varieties targeted for protection.
