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Fingerprinting, assessment of genetic diversity of cassava (manihot esculenta crantz) cultivated by small farmers in the colombian atlantic coast
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Fingerprinting, assessment of genetic diversity of cassava (manihot esculenta crantz) cultivated by small farmers in the colombian atlantic coast

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  • 1. In many countries of Africa, Asia and Latin America, cassava is an important product and food security crop. It is the fourth most important product after rice, the wheat and the maize. Both the root and leaves are used as a source of calories and protein for human and animals: the roots are an important raw material for the starch, animal feed, and ethanol industries. (Ceballos, 2002). The cassava is cultivated great majority for small farmers and identified as a cultivation that can avoid the hunger and to give security for its good behavior low marginal conditions of climate and floor in areas where other cultivations fail (Iglesias, et. al, 1994). In Colombia, the Atlantic coast is the area of more cassava production with 42% of the national production (Gottret et. al. 2002). In this zone 70% of cassava producers are small holder farmers with farm sizes ranging from 0.5 to 2Ha, although there are producers with 2 to 5 Ha of cassava and occasionally more than 5 Ha (Gottret et al, 2002). As in other countries, the cassava cultivated by small farmers has not very identified varieties that can be formed by cassava landraces or improved varieties, existing an ignorance of the genetic variability of the cassava in this region. The knowledge of the genetic variability, including the cassava, in any region of the world, it contributes basic information for works of genetic improvement and conservation of the species. The conservation of the germplasm, avoids the loss of wild species and cultivated by problems of genetic erosion (Jaramillo, 2002). The genetic variability can be considered through the direct evaluation of the genome, using molecular markers. When the variability is calculated by differences alélicas in several loci through electroforesis, the used parameters are the alelos number and the heterozygosity . (Nei, 1987). The Simple Sequence Repeat (SSR) markers has been used for studies of genetic variability of cassava in countries like Sierra Leona (Dixon, 2003), Uganda (Kizito, 2003), Nigeria (Dixón, 2002), Ghana (Okay, 2003), Perú (Alcántara, 2001), Guatemala (Montes, 2003) and Cuba (Beovides, 2004), finding high values of diversity and evidenced substructure in African populations, positive aspects for the improvement of the cultivation. Although in some of these works colombian varieties coming from the Germplasm Banks of CIAT were included, to determine differences between these and genotypes of other countries, in Colombia they have not been carried out studies of genetic variability that include cassava genotype collected in situ, in the country or some region of this. The objective of this work was to analyze the genetic variability of cassava cultivated by small farmers from Colombian Atlantic Cost, by Simple Sequence Repeat (SSR). It was collected asexual seed of cassava cultivated by small farmers in Colombian Atlantic Cost in the departments of Córdoba, Sucre, Atlantic and Magdalena. We visit 400 farmes and 1045 accessions were collected and planted in pots in the greenhouse. Later on young leaves were obtained and they were dried in an oven at 40 °C during three days. Later on, DNA was extracted using the method of Dellaporta (Dellaporta et. al, 1983). The AND obtained was quantified by fluorometry and they were carried out dilutions to a concentration of 10 ng/µl of DNA. The amplifications were carried out in termocycle PTC-100 via PCR using the program 30 cycles to: 94°C for 30”; 55°C for 30”; 72°C for 1 min, and an extension of 5 min at 72°C. The PCR product was denatured and electrophoresced on 6% polyacrylamide gels. The revealed was made with tint with silver and the data were extracted for the statistical analysis. Simple Sequence Repeat (SSR) markers were used for DNA fingerprinting and assessment genetic variability. Nine SSR markers with the highest polymorphism information content (PIC), as determined from an evaluation of 30 genotypes from the collection and analysis using the CERVUS program, were selected from a set of 36 molecular markers routinely used for cassava genetic diversity studies at CIAT (Mba, 2001). The selected markers are: SSRY12, SSRY51, SSRY63, SSRY69, SRY82, SSRY100, SSRY151, SSRY155 and SSRY179. Of the 1045 collected accessions, we worked with 717 as they displayed complete molecular information with the nine marking selected microsatellites. In this study 14 genotypes were also included from the Germplasm Bank of CIAT that correspond to landraces and varieties improved originals from the program of Cassava Breeding of CIAT and liberated in the region. These varieties were used as control in this study. The varieties were: Corpoica Gines (CM 4843-1), Corpoica Colombiana (CM 3306-19), Corpoica Sucreña (CM 3555-6), Corpoica Verónica (CM 4919-1), ICA Costeña (CG 1141-1), ICA Negrita (CM 3306-4), SM 1433-4, ICA P-12 (MCOL 1505), Cubana (CM 4574-7), Venezolana (MCOL 2215), Blanca Mona (MCOL 2253), Secundina (MCOL 2063), MTAI-8, MVEN-25. The genetic relations between the genotypes were elaborated with base in the coordinates obtained for the individuals with an analysis of multiple correspondence (ACM), using programs elaborated in SAS (CIAT, SAS: Version 9.1.3). The parameters of genetic diversity: percentage of polymorphic loci, to number means of alelos by locus polymorphic, observed heterozygous average (Ho), and genetic diversity average (Nei, 1978) were considered with the use of the SAS program. Genetic Relationships among accessions: Cluster analysis of the raw SSR data using ACM resulted in 6 groups denominated Group 1, Group 2, Group 3, Group 4, Group 5 and Group 6 (figure 1).
    • Alcántara, J., Guarino, L., Fregene, M. (2001). Análisis de la diversidad genética en Manihot esculenta “Yuca” mediante el uso de marcadores microsatélites–SSR. CIAT. Cali, Colombia. p. 9
    • Beovides Y. Barrera E., Gutiérrez J. P., Buitrago C., Marin J., Fregene M.. (2004). Characterization of genetic diversity: Simple Sequence Repeat (SSR) Assessment of Genetic Diversity of Local Cassava Varieties from Cuba. Funding: Cassava Biotechnology Network (CBN). CIAT. Cali. Annual Report: Output: 12-34
    • Ceballos H., Morante H., Calle F., Lenis J.I., Jaramillo G., Pérez J.C. (2002). Mejoramiento genético de la yuca. En: La yuca en el tercer milenio: Sistemas modernos de producción, procesamiento, utilización y comercialización. Publicación CIAT. p 295.
    • Dellaporta SL, Wood J, Hicks JR (1983). A plant DNA minipreparation: version II. Plant Mol Biol Rep 1: 19-21
    • Dixon A., Raji B., Marin J., Ospina C., Buitrago C., Fregene M. (2003). Characterization of genetic diversity: Simple Sequence Repeat (SSR) Assessment of Genetic Diversity of Local Cassava Varieties from Sierra Leone. Annual Report: Development and use of biotechnology tools for cassava improvement. CIAT, Cali – Colombia. Output 8-43.
    • Dixon, et al. (2002). Simple Sequence Repeat (SSR) Marker Diversity in Cassava ( Manihot esculenta Crantz) Landraces form Nigeria. Annual Report: Development and use of biotechnology tools for cassava improvement. CIAT. Cali. Annual Report: Output: 8-43.
    • Gottret, M. V.; Escobar, Z.; Perez S., Salomón (2002). El sector yuquero en Colombia: Desarrollo y competitividad. En: La yuca en el tercer milenio. Publicación CIAT.
    • Iglesias C., Hernández L. A. (1994). Introducción de diversidad genética mejorada a nivel de campos de agricultores. En: Interfase entre los programas de mejoramiento, los campos de los agricultores y los mercados de la yuca en Latinoamérica. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia. p. 151-157.
    • Jaramillo G. (2002). Recursos geneticos de Manihot en el centro Internacional de Agricultura Tropical CIAT. En: La yuca en el tercer milenio: Sistemas modernos de producción, procesamiento, utilización y comercialización. Publicación CIAT. Cali, Colombia. P. 271-294.
    • Kizito E., Gullberg U., Bua A. Omara J., Egwang M., Castelblanco W., Gutierrez J., Buitrago C., Fregene M. (2003). Simple Sequence Repeat (SSR) Assessment of Genetic Diversity of Local Cassava Varieties from Uganda. Annual Report: Development and use of biotechnology tools for cassava improvement. CIAT, Cali – Colombia. Annual Report: Output: 8-36.
    • Mba R.E.C., Stephenson P., Edwards K., Melzer S., Nkumbira J., Gullberg U., Apel K., Gale M., Tohme J., Fregene M. (2001). Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculentaCrantz) genome: towards an SSR-based molecular genetic map of cassava. Theoretical and Applied Genetics, 102:21–31
    • Montes Luis et al. (2003). Simple Sequence Repeat (SSR) Assessment of Genetic Diversity of Local Cassava Varieties from Guatemala. Annual Report: Development and use of biotechnology tools for cassava improvement. CIAT, Cali – Colombia. Annual Report: Output: 8-21
    • Nei M. (1987). Molecular evolutionary genetics. Columbia University Press, New York, p. 512.
    • Okay E., Otoo J., Buitrago C., Fregene M., Dixon A. (2003). Characterization of genetic diversity: Simple Sequence Repeat (SSR) Assessment of Genetic Diversity of Local Cassava Varieties from Ghana and Predictability of Heterosis. Annual Report: Development and use of biotechnology tools for cassava improvement. CIAT, Cali – Colombia. Annual Report: Output: 8-28
    ADRIANA MERCEDES ALZATE G. 1 , FRANCO ALIRIO VALLEJO 2 , HERNÁN CEBALLOS 3 , JUAN CARLOS PÉREZ 4 , MARTÍN FREGENE 5 1 Centro Internacional de Agricultura Tropical CIAT, adrimeral@hotmail.com; 2 Professor at University Nacional of Colombia - Palmira; 3 Projet Cassava Breeding Manager at CIAT; 4 Projet Cassava Breeding Investigator associate at CIAT; 5 Product Development Manager at Donald Danforth Plant Science Center
    • In ACM six groups were identified genetically different that explain 81% of the variation, with a low differentiation coefficient (Gst 0.18), indicating that the diversity not is this given by the differences between the groups but inside groups.
    • Of the six differentiated groups, four of them (Grupo1, Grupo3, Grupo5 and Grupo6), they were represented in a high percentage by a variety. The groups 2 and, presented the biggest number of genotypes and the genetic biggest variability, for that reason what was not possible identified a specific variety that represented them.
    • The measures of genetic variation demonstrated that, in the cultivations of cassava of small farmers from Atlantic Cost, observed high heterozygosity (0.56087), that which confirms the entrecruzamiento highly in the cassava and its nature heterocigota, its condition alógama makes that the population this continually segregating and generating genetic variability new. It was also total high heterozygosity (HT: 0.61692) and high level of prospective heterozygosity (HS: 0.50280).
    In the analysis of each group it is observed that In the Group 1, 85.5% of the genotypes corresponds to the Venezuela variety; in Group 3, Ica Costeña has a frequency of 85%; in Group 5 Ica P-12 appears 43.3% of the time; and Group 6, the variety Blanca Mona is shown 71.3% of the time. Groups 2 and 4, has the largest number of accessions but none of the varieties is predominant in these groups (figure 2) . In the figure 3, it is observed that the varieties more sowed by the small farmer in Atlantic Cost, they are: Venezolana (26%), ICA Costeña (9%) and Blanca Mona (7%). The varieties P-12, MTAI-8, Cubana and Secundina, they have smaller percentages that 5%. Other cassava genotypes sowed by the farmers are equal at the 46%. Genetic variability Total heterozygosity (Ht) in all the agreements was high with a value of 0,61692, 18% must to the differentiation between groups (Gst: 0.18498), indicating that the diversity not is this given by the differences between the groups but inside groups. The pondered average of the awaited heterozygosity (Hs) was 0,50280, which indicates the probability that two alelos selected randomly in a given genotype are different. The observed heterozygous (Ho) with a value of 0,56087 was high, which confirms cassava´s outcrossing and highly heterozygote nature (table 1). Fondo Conmemorativo de Becas Ginés-Mera para Estudios de Postgrado en Diversidad Biológica. Dr. Hernán Ceballos. Dr. Martín Fregene. D r. Franco Alirio Vallejo. Myriam Cristina Duque. Juan Bosco Cuasquer. Martha Cecilia del Pilar Alzate. G roup of work of Cassava Breeding and Cassava Genetics at CIAT. The results of ACM analyzed by groups (figure 2) are related with the obtained He in each group; the groups 2 and 4, present the high values of He, and indeed it is where it is the biggest number of genotypes compared with the other groups, for example, Grupo1, presents the value low of He, and it is where it is the biggest percentage in the Venezuela variety (table 2). Tabla 1. Genetic diversity of cassava varieties cultivated by small farmer from Colombian Atlantic Coast . Table 2. Genetic diversity average (He) according to the genetic groups defined in ACM of cassava varieties cultivated by small farmer from Colombian Atlantic Coast FINGERPRINTING, ASSESSMENT OF GENETIC DIVERSITY OF CASSAVA ( MANIHOT ESCULENTA CRANTZ ) CULTIVATED BY SMALL FARMERS IN THE COLOMBIAN ATLANTIC COAST INTRODUCTION MATERIALS AND METHODS ANALYSIS AND RESULTS CONCLUSIONS REFERENCES ACKNOWLEDGMENTS n : Number of genotypes He : Heterozygosity inside each group Gst : Coefficient of genetic differentiation Ho: Observed heterozygous He: Prospective Heterozygous K : Number half of alelos for locus HI: Average out observed heterozygous Hs: Average pondered of prospective heterozygous HT: Total Genetic diversity Figure 2. Distribution of genotypes in the groups: Group1, Group 3, Group 5, Group 6, Group 2 and Group 4 according to results of ACM Figure 3. Varieties more cultivated by the small farmer in Atlantic Cost Colombian Figure 1. Analysis of multiple correspondence (ACM) of varieties from the Colombian Atlantic Coast bases on 9 SSR markers Grupo1 Grupo2 Grupo3 Grupo4 Grupo5 Grupo6 Grupo 6 Grupo 5 Grupo 1 Grupo 2 Grupo 3 Grupo 4 Gst: 0,18498 0.52690 68 Grupo6 0.51645 68 Grupo5 0.60670 142 Grupo4 0.50052 75 Grupo3 0.56988 159 Grupo2 0.37751 222 Grupo1 He n Población 0,44987 0,4087 3 y155 0,55458 0,8529 4 y151 0,5365 0,4986 7 y100 0,57631 0,6172 5 y51 0,46355 0,4183 4 y63 0,40652 0,4755 4 y12 0,56336 0,6744 4 y135 0,43596 0,3542 5 y179 0,53856 0,748 6 y82 He Ho No. Allele for locus Loci 0,61692 0.50280 0.56087 4.6 H T H S H I K