PROINPA FOUNDATION BRIGHAM YOUNG UNIVERSITY THE McKNIGHT FOUNDATION

SUSTAINABLE PRODUCTION OF QUINOA (Chepopodium quinoa, Willd.) A NEGLECTED FOOD

CROP IN THE ANDEAN REGION

ANNUAL REPORT 2003 – 2004

PROINPA Investigators:

A. Bonifacio G. Plata R. Saravia M. Pinto W. Rojas R. Quispe JL. Soto JL. Marconi G. Aroni A. Vargas E. Carrasco A. Carmargo J. Rojas JC. Aroni

BYU Investigators:

D.J. Fairbanks P.J. Maughan E.N. Jellen C.E. Coleman B. Geary

Sustainable Production of Quinoa Project Directors: Alejandro Bonifacio and Daniel Fairbanks Participating Scientists: Raúl Saravia, Wilfredo Rojas, José Luis Soto, Genaro Aroni, Giovana Plata, Jorge Rojas, Milton Pinto, Amalia Vargas, Reinaldo Quispe, Juan Carlos Aroni, José Luis Marconi, Adelaida Camargo, Carolina Alanoca, Juana Flores, Jeff Maughan, Eric Jellen, Craig Coleman, Mikel Stevens, Brad Geary, and Von Jolley Executive Summary The studies conducted major categories: 1) conservation and use of enetics, 3) integrated management of pests an s, 5) harvest and post-harvest technologies, 6) so The McKnight Foundatio ive support from other sources as well. The follo ments during the 2003–2004 year in the seven maj 1) Conservation and use Initial viability the 2002–2003 growing season was stu ssions evaluated (2250 accessions) had germinat required for gene banks; 2% of the accessions (5 Studies were initiated at PROINPA laboratories in Bolivia, based on microsatellite markers developed at BYU, to evaluate genetic diversity in a selected set of 2557 accessions from the Bolivian germplasm collection. Descriptive statistics and multivariate analysis were used to define a core collection of 282 accessions, which represent 11% of the selected set of accessions. The statistical analyses and selection of accessions were based molecular marker analysis and 15 quantitative variables. The analysis identified six major groups. To constitute the core collection, 27 accessions were selected from the first group, 63 from the second, 98 from the third, 79 from the fourth, 8 from the fifth, and 7 from the sixth. Two activities were conducted to determine the factors that contribute to genetic erosion of quinoa in the Northern and Southern Altiplano regions. Data were collected from six case studies in three zones in the Southern Altiplano (Zone 1: Provinces of L. Cabrera and Oruro; Zone 2: Provinces of A. Quijarro and Potosí, and Zone 3: Provinces of Nor Lípez y D. Campos and Potosí). A total of 32 farmers participated (81% men and 19% women). The studies collected information about social, cultural, economic, biological, and environmental factors that influence farmers’ decisions regarding ex situ conservation of genetic resources of the ecotype ‘Quinua Real’. We made use of audiovisual information about traditional customs, permitting us to understand the significance of Andean mythology, rescue traditional knowledge, and understand gender roles in rural societies. Eight case studies were conducted in two zones in the area surrounding Lake Titicaca (Zone 1: Province of Ingavi, and Zone 2: Province of Omasuyus). The families of Ingavi by custom plant 1–3 quinoa ecotypes, whereas in Omasuyus they plant 2–5 ecotypes. In both zones, quinoa cultivation is not the most important economic activity when compared to cattle and potatoes. The period to which families devote the most time to quinoa production is the time of harvest and post-harvest, when compared to other activities that influence crop yield. Most families prefer to consume quinoa at lunch instead of breakfast or dinner. The dishes ‘kispiña’ and ‘pesq’e’ are preferred over tortillas, soup, allpi, and lagua de quinua.

during the 2003–2004 year fall into the followinggenetic resources (ex situ and in situ), 2) breeding and g

d diseases, 4) resistance to adverse abiotic factorcioeconomics, and 7) training and distribution of information.

n finances this project, and many of the activities recewing information briefly summarizes the accomplishor categories:

of genetic resources

of the portion of the quinoa germplasm collection regenerated during died. The results showed that 98% of the acceion rates greater than 85%, meeting the standard

6 accessions) failed to meet the standard.

2) Genetics and breeding During the most recent growing season, we selected 1,118 outstanding plants and lines in generations F2 through F6. The selected progenies include types with maturity ranging from very early (120 days) to late (150 days), which permits subsequent evaluation in different ecological zones. Seed characteristics include seed color (white, coffee, black, pale yellow, pink, and red) and saponin content (present or absent). We have emphasized mildew resistance as a selection criterion: the majority of selected lines display severity of infection less than 25 and 30%. We also have observed hypersusceptibility to mildew in some lines, which are also part of the selected material. Replicated yield trials display highly significant differences among selected lines for severity of mildew infection, harvest index, and hundred-seed weight. Through statistical analysis for yield and mildew resistance, we have identified promising lines that will enter trials in the next growing season. Seed-increase efforts did not produce the desired results due to adverse factors, including drought during early growth stages, flooding during the vegetative-growth stage, and hail during seed set. Nonetheless, we were able to obtain 1,107 kg of seed for varieties and advanced lines, which will permit planting of 138.5 hectares. We have analyzed the results obtained in the breeding program for the past 30 years and from this analysis we have determined that the best strategy for the breeding program is a central base, two or three subcenters for replicated yield trials, and participatory evaluation. Aspects included in this strategy for evaluation include the breeding program, conservation of varietal purity, seed increase, certification, and distribution of varieties to farmers. In the area of molecular genetics and cytogenetics, we have sequenced-characterized and genetically mapped numerous DNA markers in quinoa (SNPs: 51 characterized, 10 mapped; SSRs: 208 characterized, 90 mapped; AFLPs 238 mapped; RAPDs 10 mapped). Two new SSR libraries with 1250 sequenced clones were produced, and three recombinant inbred populations are available for genetic mapping, with maps made on two of these populations. We have completed a high-quality BAC library of the quinoa genome with approximately 70,000 clones and 10X genomic coverage. The library has been arrayed and deposited at the Arizona Genomics Institute (AGI). We have generated three cDNA libraries (from immature developing seeds, developing floral tissue, and whole seedlings at seven days post-germination). A total of 424 ESTs from the floral and developing-seed libraries have been characterized and annotated (GenBank accessions CN781906-CN782329). Forty-five ESTs were highly abundant and 27 of these had putative plant-defense functions (based on homologies with known genes). Twenty EST sequences were used for primer design and amplification of genomic DNA from five geographically diverse accessions in the quinoa germplasm bank. A total of 51 SNPs were identified with an average of one SNP per 462 nucleotide pairs, indicating that ESTs can be efficiently mined for polymorphic DNA markers in quinoa. Two distinct, full-length 11S seed-storage protein cDNAs (GenBank accessions AY562549 and AY562450) and one partial 7S seed-storage protein cDNA were identified in the ESTs from the developing-seed cDNA library. Southern blots indicate the presence of at least two 11S genes in the quinoa genome. A BAC containing one of these genes has been definitively identified and another clone has been obtained that tentatively contains the second gene. The haploid genome size of quinoa was determined through flow cytometry and is approximately 967 million nucleotide pairs. We analyzed the metaphase karyotypes of 24 Chenopodium species (and three chromosome races of C. album) and the results of this analysis confirm the tetraploid nature of the quinoa genome. We have also identified BAC clones containing the 18S-5.8S-26S and 5S rDNA regions. BYU and PROINPA submitted a joint proposal to the National Science Foundation of the USA under the Plant Genome Research Program (PGRP) and Developing Country Collaborations in Plant Genome Research (DCC-PGR) program for funding of quinoa genomics.

3) Integrated management of pests and diseases Research was conducted in the Central and Southern Altiplano. We studied identification of a new bioinsecticide for quinoa moth control, the population dynamics of the larval and adult stages of quinoa moth and armyworm complex, the level of economic damage of the quinoa moth, morphological differences for the larval and adult stages of the armyworm complex, the efficiencies of seven bioinsecticides, the percentage of loss due to mildew, and standardization of a method for evaluating the degree of susceptibility and tolerance of quinoa genotypes to mildew. The new bioinsecticide is MVP, which displayed efficiencies of 44 and 47% in the Central and Southern Altiplano regions, respectively. We found that adults of the armyworm complex are present in small numbers (less than six adults per sample) during the entire quinoa growth cycle. Larvae of the quinoa moth and armyworms appeared late due to late planting because of drought during the planting season and heavy rains during December and February. The level of quinoa-moth larval presence for the genotype ‘Línea Púrpura’ in the Central Altiplano was two larvae per plant, and for the ecotype Mañiqueña in the Southern Altiplano it was three larvae per plant. We classified the different species of armyworms into four groups based on morphological differences, principally form and color of dorsal and subdorsal longitudinal lines. Tests of seven bioinsecticides for organic quinoa production in the Southern Altiplano showed that none of the products for armyworm control exceeded 30% efficiency; the product Baculovirus phthorimaea was the best with 28.6% efficiency. For quinoa moth control, the products Piretro and Dipel 2X displayed the highest efficiencies with 51.2 and 48.5%, respectively. For mildew resistance, the genotypes considered as resistant (advanced breeding lines línea 320 and línea púrpura) display significantly higher yields under mildew infection than the genotypes considered as susceptible (the varieties Chucapaca and Sayaña). We standardized a greenhouse evaluation method for identifying resistant and susceptible genotypes for mildew infection, determining that the best stage for inoculation is at greater than 35 days when the plant has attained a height of 20–25 cm. 4) Resistance to adverse abiotic factors Studies in this category attempt to explain the mechanisms for tolerance to drought and salinity in quinoa. In the first activity, we researched nastic movements in quinoa in response to water stress. In the second, we evaluated drought tolerance. In the third, we studied tolerance to salinity in the germination and seed-filling stages. The fourth was devoted to plant sources of nitrogen and their relationship to soil properties. The results show that quinoa reacts with a complex set of strategies to water stress. The first is leaf movement to a more oblique position relative to the sun as an immediate response, which permits the plant to reduce its area of evapotranspiration. Soon after this the apical part of the stem bends, and the leaves and succulent part of the inflorescence begin to droop. Accessions from the core collection show differences in the timing and level of water deficit for wilting. We also observed differences in recovery rates when irrigation was applied. We also found that the core collection is an important source of diversity for tolerance to different levels of soil salinity, thus offering an opportunity for selection. Lastly, we studied nitrogen-fixing plant species in the Altiplano, which can be used as green manure for organic nitrogen fertilization (Medicago sp, Lupinus agustifolia and Lupinus sp, Vicia faba, Lupinus mutabilis). Some wild species are also forages, allowing for multiple uses. Wild species require studies to identify methods to break seed dormancy and increase germination.

5) Harvest and post-harvest technologies Activities developed in this area show that direct seeding with a prototype of a manual seed drill produced the highest germination percentage and plant height under drought conditions. We also promoted use of the direct thresher in seven communities with the participation of 105 farmers, to reduce seed losses and contamination in harvested grain. We also promoted technologies for sickle cutting (93 farmers), cutting with a mower (44 farmers), a mechanized thresher (143 farmers), use of a manual forced-air seed cleaner (73 farmers), and a mechanical forced-air seed cleaner in quinoa processing plants. The results showed that if the mechanical forced-air seed cleaner is used before processing, the amounts of saponin, contaminating sand, and water required for washing are substantially reduced. 6) Socioeconomics Socioeconomic characterization of communities where the project has intervened allowed us to categorize families into three economic strata (high, medium, and low), taking into account local criteria. Characterization of the demand for whole-grain and processed quinoa destined for the national and export markets allowed us to determine that the total demand in the businesses studied is 5,300 metric tons per year. To this, we add the demand identified by eight businesses studied in 2003 and easily conclude that the annual national demand for quinoa commercialization can exceed 7,000 metric tons. Characterization of the quinoa processing systems employed by agroindustries reveals differences in the systems, the quality and quantity of processed raw material, and the mechanized technology utilized. Small, family-based agroindustries, as well as medium and large businesses confront some of the same problems, the greatest bottleneck being seed drying and elimination of contaminating pebbles. The activity devoted to diverse uses of quinoa has been well accepted by families with small processing businesses who frequently enroll in courses on diverse uses of quinoa, and in some cases have opted to adopt new uses to increase family income. Our study on economic impact and adoption of the variety ‘Chucapaca’ has given us a preliminary idea of the current state of this variety in the Central and Northern Altiplano. This study is in the first phase of compilation of secondary information, development of tools for evaluation of information, and identification of areas where this variety is cultivated and potential areas for its cultivation. 7) Training and distribution of information We have sponsored events for training one scientist in breeding and genetics, one in disease resistance, five in learning English, two in participatory methods, and 15 in exchange programs for experience in participatory methods. We also have trained 331 farmers in techniques for integrated crop management and diverse uses of quinoa, employing methods of the Escuelas de Campo (Field Schools), training courses, and Local Agricultural Research Committees in the Central and Southern Altiplano regions. PROINPA published a quinoa calendar with important agricultural dates in Bolivia. Publications in professional journals coauthored by BYU and PROINPA scientists include two articles published in 2004, two submitted and accepted, and two recently submitted and currently in review. A total of 448 fully annotated quinoa sequences were submitted from the project and are currently catalogued in the GenBank DNA database.

Product 1. Collections of the National Bank of Andean Grains conserved under adequate conditions Activity 1.1: Molecular characterization of the germplasm collection utilizing microsatellite DNA markers To quantify genetic diversity in the quinoa germplasm collection, identify genetically similar groups, determine molecular standards, and identify duplicate accessions, we characterized the genetic diversity using microsatellite DNA markers. The material was grown in the field and in pots. We sampled 10 leaves per accession at the same physiological stage to represent the “average genome” of each accession. DNA extraction was conducted following the protocol described by Dellaporta et al. (1983), as adapted for quinoa by P.J. Maughan at BYU. PCR amplification of microsatellite segments was conducted with two PCR programs: touchdown PCR and hot-start PCR. The microsatellite markers selected for our studies were developed at BYU and identified as having easily distinguishable electrophoretic alleles and high levels of polymorphism among quinoa accessions. They were designated as QATG19, QCA6, and QCA58. We compared two methods for marker visualization and scoring: 1.8% agarose gels and high-resolution polyacrylamide gels with silver staining. We decided to use polyacrylamide gels for evaluation of the germplasm collection because discrimination among different alleles is much clearer when compared to agarose gels. Since July 2004, we have extracted and quantified DNA from 2,075 accessions and quantified the DNA in 1,962 accessions. We have amplified three microsatellite markers in DNA from 800 accessions. Separation of the amplified fragments in polyacrylamide or agarose gels has been conducted with 572 accessions and analysis of the results has been done for 468 accessions. Activity 1.2: Construction of the core collection based on agro-morphological characteristics. Through statistical methods, we identified a subset of accessions from the germplasm collection to constitute a core collection on the basis of agro-morphological characters. The analysis was

conducted with 2,557 accessions and 15 quantitative variables: emergence of the floral bud, 50% flowering, milky stage of developing seeds, physiological maturity, leaf length, leaf width, length of the petiole, plant height, length of the seed head, stem diameter, seed diameter, hundred-seed weight, grain yield, and harvest index. Through principal component and cluster analysis we identified six distinct groups. Discriminate functions had a high degree of precision for classification of the six groups. In all cases, more than 80% of accessions were grouped into the same six groups. Later we applied the same statistical

procedures to the accessions within each group to identify and select the most dissimilar accessions according to procedures based on group size and proportional method (Brown, 1989). The first group contributed 27 accessions to the core collection, the second 63 accessions, the third 98 accessions, the fourth 79 accessions, the fifth 8 accessions, and the sixth 7 accessions, for a total of 282 accessions in the core collection representing 11% of the complete collection. Representation of genetic variation was measured with Pearson’s coefficient, which revealed that the most important correlations in the complete collection were maintained in the core collection. The origins of accessions in the core collection were as follows: 230 accessions from Bolivia, 34 from Perú, 5 from Ecuador, 4 from Argentina, 1 from México, and 8 from the Organization of American States (passport data are in the process of being recovered).

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Product 2. A proposal for in situ quinoa conservation in Bolivia

Activity 2.1: Evaluation of information from case studies regarding genetic variability in the ecotype ‘Quinua Real’ in the Southern Altiplano

Evaluation was conducted in three zones of the Southern Altiplano (Zone 1: Provinces of L. Cabrera and Oruro; Zone 2: Provinces of A. Quijarro and Potosí, and Zone 3: Provinces of Nor Lípez y D. Campos and Potosí). A total of 32 farmers participated (81% men and 19% women) and the project was carried out in two phases.

Phase 1. Through on-site verification visits and interviews, we added to and confirmed information about quinoa crop management with each of the collaborating families. This work was conducted with the assistance of neighboring farmers from the same communities and nearby communities. We gathered information about where they planted quinoa, different planting methods (on both sloping and level terrain), cultural practices, methods for pest control, and harvest and post-harvest methods, as well as aspects related to commercialization in communities and regional markets. In parallel with these visits, we compiled information about festivities in the community, customs and rituals associated with quinoa cultivation, and traditional and medicinal uses of varieties conserved in the communities. We also produced “talking maps” of the places where quinoa is grown, crop rotation systems, and agricultural calendars that detail activities practice by family members during the quinoa growing season, with a focus on gender. Phase 2. We worked on evaluation with focus groups in the three zones. Each focus group consisted of 8–12 men and women with whom we evaluated information from the case studies. Farmers’ opinions, in some cases, allowed us to make adjustments to the information to form a consensus on terminology. We determined that there is greater quinoa consumption during the months with the greatest activity for quinoa cultivation, including September (planting), March, April, and May (harvest and post-harvest period). The results were agreed upon by the farmers who assisted with the evaluation, pointing out that quinoa consumption in its various forms gives them greater energy and more strength to continue with their activities. Regarding the time spent on different activities, farmers devote the most time to soil preparation, planting, harvest, and post-harvest activities. Participation by women was considered to be a primary source of information regarding the use of quinoa in nutrition and the manner for food preparation according to ecotype. Testimonials from elderly people were a source for confirmation of traditional knowledge, varieties, and tools. Opinions regarding gender indicated that in zone 1 the man and the woman participate in specific activities for quinoa production, whereas in zone 2, both the man and the woman participate together in production. In zone 3, there is complete participation by the family. This work helped to ratify and add to information about social, cultural, economic, biological, and environmental factors that influence farmers’ decisions regarding in situ conservation of “Quinua Real’ genetic resources. We have available audio-visual information about traditional customs, which allows us to understand the significance of Andean mythology, to rescue traditional knowledge, and to determine the role of gender in rural societies. Activity 2.2: Case studies on genetic variability in the area surrounding Lake Titicaca

We conducted eight case studies with four families in the Province of Ingavi (Zone 1) and four families in the Province of Omasuyos (Zone 2) to determine the factors responsible for the reduction in quinoa genetic variability in communities surrounding Lake Titicaca. These studies also permitted us to describe crop management practices and other agricultural activities of rural families in the Northern Altiplano.

Zone 1: Collaborating families in the community of Patarani own their own homes and take advantage of basic services. Their principal economic activity is raising dairy cattle with most of their agricultural

production destined for subsistence consumption. They cultivate two varieties of quinoa (Blanco o Chimi juira and Churo Juira) on small plots. According to the information we collected, the time devoted to quinoa cultivation is minimal compared to other crops, such as potato, forages, and raising animals. For quinoa production, these families devote most of their efforts to harvest, threshing, cleaning (6, 4, and 4 hours respectively). These families consume more quinoa at lunch than at breakfast or dinner, with Kispiña being the recipe most preferred. Zone 2: in general, these families own their own

homes, which are built in a rustic manner, and they have available 10 hectares of land, four for potatoes, quinoa, oats, and barley, and six for animal pasture. The principal economic activity is raising beef and dairy cattle. The time devoted to quinoa production is minimal when compared to the time devoted to cultivating potato, oca, and barley, and raising cattle. Families cultivate two ecotypes of quinoa (Janko jupa and Wila Jupa) in a space no larger than 1000 m2. For quinoa production, the families spend the greatest time in weeding (5 hours), harvest (4.5 hours), and drying (8 hours). Families consume the greatest amount of quinoa at lunch (860 g/week) compared to dinner (290 g/week) and breakfast (260 g/week). At lunch they prefer Pesq’e and quinoa soup compared to other recipes. In both zones, families contribute to in situ conservation by planting 1–5 ecotypes. Quinoa production is not the most important economic activity, when compared to raising cattle and cultivating potato. Families spend most time in harvest and post-harvest activities. Families prefer to consume quinoa at lunch with kispiña and pesq’e being the most important recipes, with recipes of lesser importance being tortillas, sopa, allpi, and lagua de quinua. Product 3. BNGA collections conserved under adequate conditions Activity 3.1: Evaluation of initial variation in the quinoa collection for storage

To quantify the percentage of initial viability in the part of the germplasm collection regenerated in the 2002–2003 growing season, and to create a database for viability in this collection, we conducted a germination study of 2,306 accessions. We did not study accession with a small amount of seed or germplasm consisting of wild quinoa. The results of this study showed that 1,425 accessions had germination percentages exceeding 95%, 612 had percentages between 90.1 and 95%, 203 between 85.1 and 90%, and 56 with less than 85%. Thus, 98% of accessions (2,250) met the standard for gene banks of greater than 85%, indicating that this material is being maintained under adequate storage conditions. Nonetheless, 2% of the material examined (56 accessions) did not meet the standard, largely because the seeds had not reached full physiological maturity due to the late maturity of the accessions. This material must be planted under controlled conditions to ensure that we obtain seed of high biological quality. Product 4. Quinoa breeding conducted with modern techniques and adequate equipment Activity 4.1a. Crossing diverse parents to obtain F1 plants A total of 22 different parents, which had characteristics of interest for breeding (large seed size, early maturity, and resistance to mildew) were crossed. The morphological marker most useful for confirmation of true hybrids was plant color, which permits such identification in seedlings. Leaf-axil color can be used at the branching stage, and the type of seed head is also useful but only after it has formed. Seed color can be used when the seeds have fully formed. Presence or absence of saponins

and seed type are characteristics useful only after the seeds have matured and dried. During the most recent growing season, we made 60 crosses and obtained 251 hybrid plants. Activity 4.1 a. Production of F2 to F6 segregating populations and selection for favorable characteristics The objectives of this activity were to generate segregating populations, evaluate progenies, and select plants that are resistant to mildew, early maturing, and have large seeds. During the most recent growing season, drought during germination, floods during the vegetative period, and hail as plants were reaching maturity, seriously damaged the crop. We were able to partially salvage plants through irrigation, propping up plants with soil, construction of soil drains, and application of foliar fertilizers. In the F2 generation, we selected 248 individual plants from 23 crosses. The severity of mildew in the selected plants varied between 3 and 40%, with both hypersensitivity and partial resistance evident. In the F3 generation we selected 225 outstanding plants from 15 crosses. The severity of mildew in these plants varied between 10 and 25%. In the F4 generation we selected plants with severity of mildew between 7 and 25%, with 440 plants from 30 families. In the F5 generation we selected 59 individual plants and 146 plants from material in advanced generations. White seed color predominates, as do large- and medium-sized seeds within the selected progenies and families. Coffee, black, and chullpi seeds were rare. We selected phenotypes that can be used for extraction of natural dyes, also types with the highest quantities of saponins for possible industrial uses. Figure 1 . Examples of sheep wool dyed with quinoa pigments Activity 4.1c. Yield trial of early-maturing lines Considering that early-maturing material could be an option for reducing irrigation, we evaluated the yields of 10 early lines in 6 locations in the Bolivian Altiplano. In spite of efforts dedicated to field work, we lost the materials in two locations due to drought and flooding. The results from the other four locations showed highly significant differences for resistance to mildew. The coefficient of variation was in the range of 15.5 to 26.5%, values acceptable for field work. Differences for harvest index, grain yield, and hundred-seed weight were highly significant for lines tested at Letanias as well as at Tiahuanacu, with the exception of harvest index, which was not significant at Letanias. These results indicate that there are significant genetic differences for grain yield among the lines. At Letanias, grain yield was highest in L-118 Bl rr at 1,835 kg/ha and lowest for the variety Mañiqueña (Real), which was 678.3 kg/ha. At Tiahuanacu the highest yielding genotype was L-10(90) at 2,2288.9 kg/ha, and the lowest yield was in Uyuni 1 dd with 1,100.9 kg/ha.

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Figure 2. Severity of mildew infection (%) at four dates and yield (Tn/ha) at Letanías. In the Southern Altiplano (Chacala) we observed highly significant differences among lines. Table 1. Mean squares in the ANOVA for harvest index, yield and hundred-seed weight (Hichuraya and Chacala). Hichuraya Chacala Source df MS(HI) MS(Yield) MS(100sw) df MS (HI) MS(Yield) Lines 9 0.007568** 12924** 20.726** 9 3072895 341433** Blocks 3 0.002460ns 2505 ns 20.053 * 2 286 143 ns Error 27 0.002366 3006 6.025 27 313995 17444 Total 39 39 CV % 20.7 27.6 CV% 22.6 At Chacala, according to Duncan’s comparison for Uyuni 1 (Sweet), control genotype (Mañiqueña) and Uyuni 1 (Bitter) were the highest yielding genotypes with 1004.0, 984.6 and 984.1 kg/ha respectively. Línea Dulce 10(90) and Say/14/ yielded 779.0 and 711.6 kg/ha.

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Figure 3. Mean yields of 10 genotypes evaluated at Chacala Figure 3 shows substantial variation for yield among lines varying from very low (146 kg/ha) to acceptable (1,004.0 kg/ha). Table 2. Early material and days to maturity at Letanías (Northern Altiplano) and Chacala (Southern Altiplano)

Genetic Material Color Seeds Saponin Letanías Chacala Difference L- dulce 10(90) Purple Chullpi Sweet 127 148 21 L- amargo 10(90) Purple Chullpi Bitter 127 153 26 L- 118 grano blanco Green White Sweet 130 155 25 L- 118 grano café Green Coffee Sweet 130 154 24 L- 118 grano café, planta roja Red Coffee Sweet 147 156 9 Sayaña /3/, grano café Purple Coffee Sweet 130 156 26 Sayaña/14/, café planta verde Purple Coffee Sweet 130 146 16 Mañiqueña (Control) Green White Bitter 120 134 14 Uyuni 1 (Sweet) Green White Sweet 131 148 17 Uynui 1 (Bitter) Green White Bitter 131 148 17

Differences for severity of mildew infection in certain periods were highly significant. Variation for levels of infection is very evident in short periods, such as a week, which indicates high virulence of the pathogen under conditions favorable for its dissemination. Yield, hundred-seed weight, and harvest index were highly significant, indicating good potential for selection. Early lines showed different numbers of days to maturity at Letanías (Northern Altiplano) and Chacala (Southern Altiplano), with differences varying from 9 to 26. Activity 4.1. Yield test of semi-early lines This activity refers to evaluation of yield and other agronomic characters in 18 selected lines. The trial was replicated in three locations: Tiahuanacu, Letanías, and Belén. The results were obtained under adverse climatic conditions such as delayed rains, drought during seedling emergence and seed set, flooding during the flowering period, and hail at maturity. The analysis of variance for severity of mildew infection shows highly significant differences but blocking displayed significant differences only on a few dates when readings were taken. The analysis of variance for harvest index, yield, and hundred-seed weight at Letanías, Tiahuanacu, and Belén shows highly significant differences for lines. On the other hand, there are no significant differences for blocks. Severity of mildew infection during the period of evaluation (January) was very high within a few days, which shows that when conditions are favorable for infection the disease can advance very rapidly. Activity 4.1e. Seed increase for varieties and advanced lines Because there is no organization dedicated to seed production, the PROINPA Foundation has taken on the responsibilities for initial seed increase of varieties and advanced lines, testing the viability of increased seed, and promotion of seed of improved varieties.

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Figure 4. Monthly precipitation during the growing season. Figure 4 shows the amounts of rainfall received during the months of the 2003–2004 growing season. There was very little precipitation during the planting season (October–November) and very high precipitation during January and February. This unfavorable pattern of precipitation caused deficiencies in germination and emergence. Later the high amounts of rain and humidity caused flooding and high severity of mildew infection. Nonetheless, the results we were able to obtain were acceptable given this pattern of precipitation. In spite of these adverse conditions, we obtained 500.7 kg of seed from improved varieties and 322.4 kg of seed from advanced lines, for a total of 823 kg. This amount of seed is sufficient to plant 103 hectares. Activity 4.1f. Seed increase for the early varieties Mañiqueña and Jach’a Grano The Southern Altiplano has suffered from drought and frost that affected seed filling. In the Central Altiplano adverse factors included drought, which delayed planting, and later hail compacted the soil reducing germination. Another important aspect is the photoperiod for normal quinoa development, which was disrupted by the late planting and low germination. All of these factors contributed to loss of some plots and substantial reduction in yields in other plots. The number of days to maturity were fewer at Letanías when compared to Chacala, with a difference of 14 to 17 days. This difference is attributable to adverse environmental conditions and abiotic stress. A total of 284 kg of seed was obtained, which does not meet the current demand. This amount of seed is sufficient for planting only 35.5 hectares. Seed purity and germination percentages were high. Activity 4.2. Development of a quinoa breeding strategy We took a retrospective and self-critical look at the breeding program, conservation of improved varieties, and seed distribution to develop a quinoa breeding strategy for the future. The strategy should be aimed at meeting the demands of farmers and the market. It should include new beneficial plant characteristics and new criteria for selection, such as earliness, seed types, less sensitivity to photoperiod, and short vegetative phase. Maintenance of varietal purity is important and very critical in quinoa because mechanical mixtures happen very easily. Transposable element activity and paramutation are other factors that cause genetic changes in varieties. Maintenance of varieties should take place at centers with the appropriate technical and scientific expertise so that seed production can be coordinated between seed specialists and research centers. Distribution of varieties should facilitated through participatory methods and organized groups. Seed distribution should be well timed, in the appropriate place, and with the correct varieties for the location. In summary, the technology must be taken to farmers and the cost of seed should be low enough to make seed accessible, or perhaps with graduated prices based on farmers’ economic abilities to pay. Seed certification should be brought up to date to meet current needs and legal requirements. Activity 4.3. Genomic, molecular, and cytogenetic studies of quinoa We have sequenced-characterized and genetically mapped numerous DNA markers in quinoa (SNPs: 51 characterized, 10 mapped; SSRs: 208 characterized, 90 mapped; AFLPs 238 mapped; RAPDs 10 mapped). Two new SSR libraries with 1250 sequenced clones were produced, and three recombinant inbred populations are available for genetic mapping, with maps made on two of these populations. We have completed a high-quality BAC library of the quinoa genome with approximately 70,000 clones and 10X genomic coverage. The library has been arrayed and deposited at the Arizona

Genomics Institute (AGI). We have generated three cDNA libraries (from immature developing seeds, developing floral tissue, and whole seedlings at seven days post-germination). A total of 424 ESTs from the floral and developing-seed libraries have been characterized and annotated (GenBank accessions CN781906-CN782329). Forty-five ESTs were highly abundant and 27 of these had putative plant-defense functions (based on homologies with known genes). Twenty EST sequences were used for primer design and amplification of genomic DNA from five geographically diverse accessions in the quinoa germplasm bank. A total of 51 SNPs were identified with an average of one SNP per 462 nucleotide pairs, indicating that ESTs can be efficiently mined for polymorphic DNA markers in quinoa. Implementation of molecular-marker analysis in the germplasm characterization program is underway with markers that were specifically identified at BYU as being well suited to PROINPA’s laboratory (see Activity 1.1). Additional markers have been identified and are in the process of being added to this program. The genetic map has revealed linkage of a molecular marker to the gene that governs the presence or absence of saponins and can be used in marker-assisted selection for this characteristic in seedlings. Identification of markers linked to genes conferring resistance to mildew is awaiting characterization of mildew resistance in mapping populations, a study that is currently underway. Two distinct, full-length 11S seed-storage protein cDNAs (GenBank accessions AY562549 and AY562450) and one partial 7S seed-storage protein cDNA were identified in the ESTs from the developing-seed cDNA library. Southern blots indicate the presence of at least two 11S genes in the quinoa genome. A BAC containing one of these genes has been definitively identified and another clone has been obtained that tentatively contains the second gene. The haploid genome size of quinoa was determined through flow cytometry and is approximately 967 million nucleotide pairs. We analyzed the metaphase karyotypes of 24 Chenopodium species (and three chromosome races of C. album) and the results of this analysis confirm the tetraploid nature of the quinoa genome. We have also identified BAC clones containing the 18S-5.8S-26S and 5S rDNA regions. Quinoa genomics is now well established with an initial genetic map in place and expansion of this map underway, a high-quality BAC library of the quinoa genome, and cytogenetic studies completed. Based on this research, BYU and PROINPA submitted a joint proposal to the National Science Foundation of the USA under the Plant Genome Research Program (PGRP) and Developing Country Collaborations in Plant Genome Research (DCC-PGR) program for funding of quinoa genomics. Information on funding for this proposal is expected from NSF in June 2005. Product 5. Techniques for integrated management of pest and diseases Activity 5.1. Evaluation of the efficiency of two new bioinsecticides for quinoa moth control in organic and conventional quinoa in the communities of Jalsuri (Central Altiplano) and Chacala (Southern Altiplano)

For this activity we evaluated the effectiveness of two new bioinsecticides: MVP (Bacillus thurigiensis var. Kurstaki) and Micosplag (Metarhizium anisopliae, Paecilomyces lillacynus and Beauveria bassiana) for control of quinoa moth larvae in the Central and Southern Altiplano regions. The results showed that MVP was the most effective at both locations, with 44% effectiveness in the Central Altiplano and 47% effectiveness in the Southern Altiplano.

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Figure 5. Effectiveness of different bioinsecticides at 7, 13 and 19 days after application at Jalsuri.

Figure 6. Percentage effectiveness of different bioinsecticides for quinoa moth control at Chacala.

Activity 5.2. Population dynamics of adults and larvae for quinoa pests and their natural enemies at Jalsuri (Central Altiplano) and Chacala (Southern Altiplano)

We evaluated the population dynamics of adult and larval armyworms and quinoa moth and their natural enemies at Jalsuri (Central Altiplano) and Chacala (Southern Altiplano). The results showed that adults of the armyworm complex are present during the entire growing season but in small numbers, never exceeding six individuals per sample. On the other hand, adult quinoa moths appeared after 8 January 2004 and the population gradually increased until it reached 15 adults per trap on 16 February 2004. From this point on, the number of captured insects progressively decreased until at harvest time the number of individuals trapped was zero, as shown in Figure 7. Armyworm and quinoa-moth larvae appeared later that usual, probably due to late planting because of drought during the planting season and heavy rains during December and February (Figure 8).

Figure 7. Population dynamics of armyworm (ticona) and quinoa moth (polilla) adults captured at Chacala.

Figure 8. Population dynamics of armyworm (ticona) and quinoa moth (k’cacos) larvae captured at Chacala.

At Chacala we identified four natural enemies. Three (Copidosoma sp., Venturia sp., and an unidentified wasp species) attack the quinoa moth, and the wasp Nina nina attacks armyworms. The largest number of individuals belonged to Copidosoma sp. (54); the next most prevalent was the unidentified wasp (43). We captured 40 individuals of Venturia sp. Activity 5.3 Evaluation of Helicoverpa armigera virulence for control of armyworm larvae in the laboratory

Activity 5.4. Evaluation of Helicoverpa armigera virulence for control of armyworm larvae in the field

We were unable to complete these activities due to drought during the planting season and heavy rains in December, January, and February, resulting in too few adults in the quinoa plots.

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r)Activity 5.5. Determination of the level of economic damage due to the quinoa moth

We evaluated the level of economic damage by constructing cages to isolate 12 plots, each with 10 quinoa plants. We introduced 0, 5, 10, and 15 larvae per plant during the milky-seed growth stage. We determined that economically significant damage on línea púrpura in the Central Altiplano was 2 larvae per plant and for the ecotype Mañiqueña in the Southern Altiplano it was 3 larvae per plant.

Activity 5.6. Study of the morphological differences of different species of armyworms for identification

We collected armyworm larvae in the field and raised them in the laboratory. We classified them into four groups based on overall form and color of longitudinal, dorsal, and subdorsal lines (Figure 9).

Figure 9 Characteristics of the four groups of armyworm larvae Activity 5.7. Evaluation of seven bioinsecticides for organic control of armyworms and quinoa moth in the Southern Altiplano

The results of this activity showed that the bioinsecticides tested provided less than 30% effectiveness for armyworm control, with Baculovirus phthorimaea performing best at 28.6% effectiveness. The products Piretro and Dipel 2X were the best for quinoa moth control with 51.2 and 48.5% effectiveness respectively.

Activity 5.8. Evaluation of components for integrated management of mildew

We tested the yield of two mildew-resistant and two susceptible genotypes and determined that the resistant types (línea 320 and línea púrpura) yielded approximately 50% more than susceptible types (Chucapaca and Sayaña).

Activity 5.9. Characterization of virulence genes and mating types of different isolates of Peronospora farinosa

We standardized the inoculation method to characterize virulence genes and mating types in Peronospora farinosa. In this first phase we determined that the best inoculation method was placing drops of inoculum covered with small pieces of tissue paper on the leaves of plants at 35 days after planting (height of 20 to 25 cm). At this age there is a good correlation between field and laboratory results. Resistance increases with the age of the plants. Activity 5.10. Characterization of accessions from the germplasm bank and breeding material for resistance to mildew The results of this activity showed that testing plants at a height of 25 cm allowed us to distinguish resistant, moderately resistant, and susceptible genotypes. Product 6. Resistance to adverse abiotic factors Activity 6.1. Study of nastic movements of quinoa plants in response to water deprivation The core germplasm collection was constructed to represent the maximum genetic diversity of the overall collection of quinoa. For this activity, we proposed evaluating the core collection for response to water deficit based on nastic movements.

Figure 10. Evaluating the core collection for resistance to water deficit.

Quinoa plants have several responses to water deficit, with movements of leaves and parts of the inflorescence being the most notable. The first response is a change in leaf position from erect to inclined, avoiding the sun’s rays. The most susceptible leaves are the basal leaves and those on the middle third of the plant. The second response is folding of the young leaves near the meristematic zone or the primordial seed head. Other responses include wilting of the apical portion of the stem, and drooping of leaves and the inflorescence.

Variation for responses to drought in the core collection is evident. However we need to develop an appropriate scale for evaluation to differentiate the accessions and breeding lines.

Activity 6.2. Influence of two salt concentrations on germination and growth

We conducted germination tests with salt solutions on 200 accessions from the core collection. Later we conducted a test with the same accessions, submitting them to salt stress during the vegetative stage. The results of the germination test were not satisfactory due to the high heterogeneity of germination among the control (no salt) samples, which displayed high levels of abnormal or defective germination. Likewise, days to germination and the length of the germination period were highly variable in controls. This situation may have strongly influenced the results of the results of the test with salt solutions due to a possible interaction between salt concentrations and the old age of the seeds. The results of application of salt solutions at two concentrations showed a negative effect of salt concentration on plant growth. A concentration of 0.2 M had a lower effect on growth and lower lethality than 0.4 M, which was lethal to 50% of accessions and partially lethal to 30%. Only 18% showed a high level of tolerance. These preliminary results show that genetic differences for tolerance to salinity exist. Figure 11. Variability of accessions for salt tolerance. Activity 6.2. Plant sources of nitrogen and their influence on soil characteristics In the Altiplano one of the most important limits for quinoa production is low soil fertility. Organic material improves soil physical properties and water retention. Furthermore, the nitrogen provided by organic matter helps improve drought tolerance in quinoa. With these factors in mind, we determined to collect seed from nitrogen-fixing species and determine their germination capacity for potential use of these species for green manure. We collected seed of three wild legume species that could be used as both green manure and forage. Seed of Medicago spp. displayed no problems with germination. However, seed of Lupinus angustifolia displayed dormancy: fresh seed failed to germination whereas seed stored for three years was capable of germination. The biomass yield of fava bean and Tarwi (native lupine) was 43 and 24.7 metric tons per hectare, and thus these species are good options for green manure. Although green manure is an excellent option for organic nitrogen fertilization, we face an obstacle in that farmers are reluctant to use green manures because many of them believe that God will punish them for plowing under the plants instead of harvesting them or allowing animals to eat them.

Figure 12. Tarwi (Lupinus mutabilis) before incorporation into the soil as a green manure.

Product 7. Technologies for integrated management of improved varieties

Activity 7.1. Effect of four planting methods on agronomic characteristics and yield

This work was conducted in the Community of Jalsuri and evaluated the effect of four planting methods: 1) planting on the tops of rows with a prototype of a manual seed drill after making rows with a tractor-driven cultivator, 2) direct planting with the same manual seed drill, 3) hand-scattering of seeds followed by making rows with a tractor-driven cultivator, and 4) hand-scattering of seeds followed by disc harrowing of the field with a tractor. The best results were obtained by direct seeding with the manual seed drill, based on higher germination rates and plant height under conditions of low soil water content.

Activity 7.2. Demonstration of the direct thresher in two communities of the Central Altiplano to improve the quality of commercial grain

This work was done with farmers participating in Field Schools and Training Groups in the vicinity of Jalsuri, where we promoted use of the direct thresher. This activity involved seven communities and 105 farmers who threshed the production from 8.7 hectares. This thresher reduced the degree of grain contamination. Farmers interviewed after participation pointed out the following positive aspects of the thresher: it is easy to operate, barley and wheat as well as quinoa could be threshed in the same machine, and it saves time and labor. The farmers showed interest in purchasing a thresher as a cooperative group.

Activity 7.3a: Promotion and use of technologies for harvesting plants with a scythe and a mechanical mower, and use of a manual forced-air seed cleaner in the Central Altiplano

We conducted this work with farmers participating in the Field Schools and Training Groups in the vicinity of Irpa Chico. A total of 93 farmers from 5 communities participated with scythe harvesting, 105 farmers from 7 communities with a mechanized thresher, and 73 farmers from 4 communities with a manual seed cleaner. This activity stimulated interest in adopting these technologies. Activity 7.3b Promotion of cutting with a mower, threshing, and seed cleaning with a mechanical forced-air seed cleaner in the Southern

Altiplano

This work was conducted in the vicinity of San Pedro de Quemes, Llica, and Caquena. Cutting with a mower involved 4 communities with the

participation of 44 farmers, use of the mechanical thresher involved 2 communities with 38 farmers, and use of the mechanical forced-air seed cleaner involved 111 farmers from 5 communities.

Activity 7.4 Demonstration of the advantages of the mechanical forced-air seed cleaner in processing plants

Evaluation of the mechanical forced-air seed cleaner was conducted at the SOPPROQUI (Sociedad Provincial de Productores de Quinua) processing plant. The results showed that utilization of this seed cleaner prior to desaponification reduced the amount of water required for seed washing, the amount of saponin on the seeds, and the amount of contaminating sand in the processed product, producing grain of better quality. Product 8. Socioeconomic characterization of communities and demands from agroindustries that work with quinoa for the local and export markets Activity 8.1: Socioeconomic characterization of communities in which the project has intervened a) Socioeconomic files We produced two socioeconomic files that contain information related to 1) Social Aspects (community and municipal organization, demographic indicators, family information, population, authorities, and gender), 2) Economic Aspects (income, commercialization channels, and land tenure), and 3) Environmental Aspects (agroecological characteristics and production systems). Through this activity we identified rural development aid organizations that provide credit, associations, and cooperatives. b) Levels of Well Being Stratification for levels of well being exist in the impact area of Jalsuri (members of the Cooperativa Jalsuri Irpa Chico and the Community of Callisaya). The criteria that key informants decided to use to group families were land tenure, technological level, sources of income, educational level of children, use of day workers, and amount of agricultural land planted. The results indicated that of the 27 active members of the Cooperativa Jalsuri Irpa Chico, 52% are in the high category, 18% in the medium category, and 30% in the low category. For the Community of Callizaya, 50% and in the high category, 15% in the medium category, and 35% in the low category, which is below the poverty level. Activity 8.2: Complete characterization of the demand for quinoa by new businesses and agroindustries with production for local and export markets We characterized five Bolivian quinoa-exporting businesses: QuinuaBol, SAITE, Quinoa Food Cia, CECAOT, and Jatary srl. The annual demand for quinoa from these businesses exceeds 2800 metric tons. The principal countries to which the export product is destined are: the USA, France, Germany, the Netherlands, the UK, Japan, Canada, Switzerland, Austria, Belgium, and Spain, among others. The principal businesses that market quinoa on a world scale are: Quinua Corporation (USA), PRIMEAL Y MARKAL (EU), and GEPA (Germany). This latter company is an NGO that promotes unionized markets and distributes quinoa and its processed products to markets in France, Belgium, the Netherlands, England, and Switzerland. Buyers distribute the product to a network of stores in Europe and North America. Our study of businesses that distribute quinoa nationally in Bolivia showed a demand between 900 and 1200 metric tons per year. There are other potential markets, such as school-lunch programs and supplements to breast milk for infants, which demand approximately 1250 metric tons per year.

Activity 8.3: Characterize the quinoa processing system in agroindustries Taking into account the assembly line for processing and the operating capacity of processing plants, we were able to classify businesses into small (family-owned and operated), medium, and large. Small businesses focus on the national market, whereas medium and large business focus on both the national and international markets. For this activity, we received assistance from Procesadora de Cereales Andina, Grano de Oro, and Urkupiña (all small businesses administered by families), and medium and large businesses, such as IRUPANA, SAITE srl, SOPPROQUI, QUINUA BOL, and Quinua Food.

Small plants typically have inadequate facilities and specialization of tasks, and generally do not keep records of raw material processed. Medium and large businesses have areas and equipment assigned to specific processes, and keep detailed records of purchases and sales.

We found that more than 90% of businesses have problems with grain drying, equipment maintenance, and an average of 15% product loss during processing. According to the perceptions of business owners, they need to devote attention to the quality of the processed commercial product (whole quinoa, cakes, and flour). They are aware that the national market does regulate product quality. The requirement for high-quality raw material with a lower content of impurities is ongoing, and business owners recognize the need to work with farmers to improve harvest and post-harvest practices to produce a high-quality product that can demand a good price. Activity 8.4: Promotion and distribution of new products in rural and urban areas to increase consumption and value added to processed productions We developed eight training courses on diverse uses of quinoa in six Central Altiplano communities attended by 198 people (65% women and 35% men). The best-accepted recipes were torta bizcochuelo, jugo de quinua con piña o manzana, galletas de quinua, and el api de quinua. Sweet k’ispiña, which is a traditional dish prepared by rural families, also was well accepted.

The courses encouraged people to adopt new ways of preparing quinoa and the possibility of including these in daily diets, especially for children. Currently, groups that have participated in the training courses are serving a drink made of quinoa and apple juice at meetings and community work projects. Testimonials from participants are positive regarding the benefits of training, such as “we can use the recipes we learned in our houses for our children,” and “we need to grow more quinoa to make these recipes.”

Activity 8.5: Study on the economic impact and adoption of the improved variety ‘Chucapaca’ in the Central Altiplano The objective of this study was to quantify the economic impact and degree of adoption of the improved variety ‘Chucapaca,’ and to identify the characteristics of this variety that have contributed to its acceptance.

Currently we have secondary information, which was compiled at documentation centers. This information includes technical reports on evaluation, distribution of improved varieties, technical bulletins, and reports from workers at the now defunct IBTA in communities of the Central and Northern Altiplano regions from 1995 to 1998.

We have a semi-structured survey available. We also have identified key informants and communities in the Central and Northern Altiplano regions where people frequently plant this variety. We have found zones where fields planted with this variety vary between 0.25 and 2 hectares. It is important for us to point out that farmers easily identify and describe the specific characteristics of the variety Chucapaca. Nonetheless, we have observed that for the most part varietal purity is not well maintained; fields planted with this variety often contain 60% mixtures of local and native varieties with Chucapaca. Product 9. Strengthening the capacity of students, technicians, scientists, and farmers Activity 9.1.Strengthening the capacity of students, technicians, and scientists

The number technicians trained, their specialties, and where they were trained are summarized in Table 3.

Specialty Number of technicians

Place

Plant Breeding (Ph.D. Degree) 1 BYU, Utah, USA Disease Resistance 1 Ecuador English 5 La Paz, Bolivia Participatory Methods 2 Cochabamba,

Bolivia Exchange Programs for Participatory Methods

15 La Paz, Bolivia

Table 3. Information regarding training of technicians

Activity 9.2. Training farmers in integrated crop management in the Central Altiplano

Training sessions were conducted through the Field Schools Program in the communities of Hichuraya Baja, Callisaya, Villa Remedios, Hichuraya Alta, and Sangramaya. We trained 331 farmers in various areas, including mildew control, the life cycle of the quinoa moth, seed selection, quinoa harvest methods, production-cost management, and diverse uses of quinoa.

Activity 9.3. Training farmers in integrated crop management through the Field School Program and Local Agricultural Research Committees in the Southern Altiplano

The Field School Program was implemented in the Community of Villa Candelaria in the Province of Nor Lípez. We also organized a Local Agricultural Research Community (CIAL) in the Community of Chacala in the Province of A. Guijarro. We trained 17 farmers in integrated crop management through the Field School Program in eight sessions, where 39% of the participants were women.

The CIAL-Chacala decided to research selection of early quinoa accessions. Members of the committee selected four accessions, which they will multiply through seed increase during the next growing season.

Product 10. Activity 10.1 Publications

PROINPA published the following document: 1 Quinoa Calendar with Agricultural Dates PROINPA and BYU scientists published the following peer-reviewed scientific articles: P.J. Maughan, A. Bonifacio, E.N. Jellen, M.R. Stevens, C.E. Coleman, M. Ricks, S.L. Mason, D.E. Jarvis, B.W. Gardunia, and D.J. Fairbanks. 2004. A genetic linkage map of quinoa (Chenopodium quinoa) based on AFLP, RAPD, and SSR markers. Theoretical and Applied Genetics 109:1188–1195. Coles, N.D., C.E. Coleman, S.A. Christensen, E.N. Jellen, M.R. Stevens, A. Bonifacio, J.A. Rojas-Beltran, D.J. Fairbanks, and P.J. Maughan. 2004. Development and use of an expressed sequenced tag library in quinoa (Chenopodium quinoa Willd.) for the discovery of single nucleotide polymorphisms. Plant Science 168:439–447. Results from research were also presented at several international conferences, including the RedBio Conference in the Dominican Republic, the American Society of Agronomy Annual Meetings in the USA, the European Society of Agronomy in Denmark, the Fourth International Chromosome Conference in England, the Plant and Animal Genome Conference in San Diego, the International Congress of Andean Crops in Argentina, and the McKnight Grantees Conference in the Netherlands.