1. Biodiversity in Agroecosystems Milano, 24-25 February 2011 UNIVERSITY of FLORENCE Department of Plant, Soil and Environmental Science EVALUATION OF THE GENETIC VARIABILITY USING MOLECULAR MARKERS IN POPULATIONS OF CULTIVATED SPECIES AND ITS UTILIZATION IN THE GENETIC IMPROVEMENT OF THE ZOLFINO BEAN Lisetta Ghiselli and Stefano Benedettelli lisetta.ghiselli@unifi.it

2. The Zolfino cv. is a typical Tuscan common bean WHAT ARE THE PROBLEMS FOR THE CULTIVATION OF “ZOLFINO” ? Pratomagno landscapeTraditional food GENETIC EROSION LOW PRODUCTION PROBLEMS The actual breeding systems in practice could produce varieties not suitable for this cultivation It is cultivated in the hilly and mountainous region of Pratomagno

3. “GREEN REVOLUTION” Caused a loss of GENETIC DIVERSITY Contributed to world famine reduction LOSS of numerous heterogeneous traditional farmers’ varieties The breeding systems were changed. This resulted in the selection of cv with: High production, Uniform crops, Introduction of standards, Homogeneous plants

4. 150 species of food crops are cultivated NOW Mankind lives off no more than 12 plant species

5. DANGERS OF GENETIC EROSION GENETIC EROSION Green revolution (intensive agricultural systems, environmental pollution, ground erosion) High degree of genetic similarity of new varieties Modern Breeding systems in practice Problems of adaptability of species to environmental change (climate change), Increase in the vulnerability of agricultural crops to abiotic and biotic stress (pests and diseases) Loss of local species and varieties usually results in an irreversible loss of genetic diversity. This has dangerously reduced the genetic pool that is available for natural selection

6. DANGERS OF GENETIC EROSION 1845potatoPhytoftora infestans (UK) 1860vitis europeaPhylloxera vastatrix 1890coffeeHemileia vastatrix (rust) Sri Lanka 1917wheatPuccinia graminis (stem rust) 1943riceCochliobolus myabeanus 1946oatCochliobolus victoriae (America) 1960tobaccoPeronospora tabacina (Italy) 1970coffeeHemileia Vastatrix (Brasile) 1971maizeHelminthosporium maydis (America) 1950bananaFungal diseases History has provided some important examples of these dangers

7. EXPERIMENT ON THE ZOLFINO BEAN To address the problem of genetic uniformity, an experiment was carried out on the Zolfino bean. The experiment was conducted over five years. The OBJECTIVE was to select genotypes with different properties that could be used for a multi-line variety constitution.

8. EXAMPLE: “ZOLFINO” BREEDING GERMPLASM Pure lines combination MULTI-LINE VARIETY CONSTITUTION Field trials evaluation Seed production Genetic evaluation of the gene pool COLLECTION Pure lines Varietal trial evaluations DNA extraction

9. GENOTYPE FIELD EVALUATION Morphological Production Tolerance to biotic and abiotic stress Quality characteristics In collaboration with the farmers LABORATORY ANALYSIS Genetic characterization with SSR primers Genetic variability with Storage Proteins Diseases monitoring and characterization VARIABLES CONSIDERED

10. 2 m 1,8 m LINE EVALUATION AND SEED PRODUCTION For the production of seed, the material was isolated from insects in tunnels. This was done to promote self- impollination

11. Plot Data % Emergence % Flowering Production t/ha Plant data date of flowering estimation of fruit development plant height n. of side-branches viral incidence Post-harvest parameters in the field n. of pods pod length pod width n. of seeds per pod weight of 1000 seeds MORPHOLOGICAL CHARACTERISTICS

12. 12 different SSR Primer Combinations 20.1 kD ► 30 kD ► 43 kD ► 67 kD ► ► 94 kD ► F1F1 F2F3F4F1 F1F1 F1F1 F5F6F7 6 4 3 2 5 10 9 7 8 12 11 1 4 different STORAGE PROTEINS Evaluation of Genetic Variability To evaluate the genetic variation in the selected lines two methods were used

13. STATISTICAL DATA ANALYSIS Quantitative characters Univariate analysis of variance (ANOVA): years and locality were considered random effect factors genotypes were considered fixed effect factors Multivariate analysis of variance (MANOVA) included the Principle Component Analysis PCA and cluster identification (k-means clustering) Molecular data Jaccard index Sahn clustering method The results were shown using Dendrograms

14. RESULTS: MORPHOLOGICAL AND YIELD CHARACTERISTICS The PCA showed the distribution of the lines on the basis of the morphological and yield data. We obtained an initial phenotypic classification, where it was possible to observe homogeneity in each group. These genotypes, respected the varietal standards of the modern varieties.

15. RESULTS: GENETIC CHARACTERISTICS This dendrogram shows the distribution of the lines representative of the populations of Zolfino on the basis of the genetic variability obtained from using the SSR primers

16. G 46 G 36 G 31 G 28 G 27 G 25 G 19 G 17 G 15 G 1 G 13 G 40 G 22 G 16 G 24 G 23 G 38 G 14 By combining the genetic and morphological characteristics, it was possible to obtain pure lines for the multi-line variety constitution. The aim was to have the maximum variability in each homogeneous class

17. MULTI-LINE VARIETY CONSTITUTION Variety nameGenotypeVariety Characteristics Variety nameGenotypeVariety Characteristics Variety 1G36Less productive genotypes. Elevated genetic variability Variety 4G13Morphological and productive characteristics variable. Less genetic variability. G27G14 G17G24 G1G31 Variety 2G19Average production genotypes. Elevated genetic variability Variety 5G27Average morphological and productive characteristics. Less genetic variability. G15 G28 G22 G36 G16 G40 Variety 3G25Highly productive genotypes. Elevated genetic variability G23 G46 G22 On the basis of the results, after the fourth year, we constituted five multi-line varieties. Each variety was produced from the combination of four different genotypes extracted from 18 genotypes.

18. EXPERIMENTAL FIELD EVALUATION It is very important to evaluate the behaviour of the plants under field conditions for many years The photo shows the experimental field trials to evaluate the morphological and productive characteristics in the final year. The samples were cultivated in a randomized block design with three replicates in four localities in Tuscany

19. GENERAL COMBINING ABILITY AND SPECIFIC COMBINING ABILITY (SCA) GENOTYPE G1G1 G 17 G 19 G 27 G 36 G 1 G 17 G 1 G 19 G 1 G 27 G 1 G 36 G 17 G 19 G 17 G 27 G 17 G 36 G 19 G 27 G 19 G 36 G 27 G 36 It is also important to evaluate the combining ability of each single genotype with all the other genotypes For example combining 5 genotypes, 2 at a time, it is possible to have 10 different combinations These formula (Partial Diallelic Cross) are used to evaluate the Combining Ability (general and specific) of each single genotype (by Griffing 1956) The method allows the identification of those genotypes that combine better with others

20. CONCLUSIONS The combination of Multivariate analysis with Genetic variability data are useful: To define new varieties that are suitable as food crops and that are adapted to different environments To obtain multi-line varieties which contain elevated genetic variability, and consequently an elevated stability in production To monitor the genetic changes of frequencies of genotype within both populations and varieties, useful for germplasm conservation and for variety stability

21. Tank you very much for your attention Tank you very much for your attention