Biodiversity in AgroecosystemsMilano, 24-25 February 2011

UNIVERSITY of FLORENCEUNIVERSITY of FLORENCEDepartment of Plant, Soil and Environmental ScienceDepartment of Plant, Soil and Environmental Science

EVALUATION OF THE GENETIC VARIABILITY EVALUATION OF THE GENETIC VARIABILITY USING MOLECULAR MARKERS IN POPULATIONS USING MOLECULAR MARKERS IN POPULATIONS OF CULTIVATED SPECIES AND ITS UTILIZATION OF CULTIVATED SPECIES AND ITS UTILIZATION

IN THE GENETIC IMPROVEMENT OF THE IN THE GENETIC IMPROVEMENT OF THE ZOLFINO BEANZOLFINO BEAN

Lisetta Ghiselli and Stefano BenedettelliLisetta Ghiselli and Stefano Benedettellilisetta.ghiselli@unifi.it

The Zolfino cv. is a typical

Tuscan common bean

WHAT ARE THE PROBLEMS FOR THE CULTIVATION OF “ZOLFINO” ?

Pratomagno landscape Traditional 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

“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

150 species of food crops are cultivated

NOWMankind lives off no

more than 12 plant species

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

DANGERS OF GENETIC EROSION

1845 potato Phytoftora infestans (UK)

1860 vitis europea Phylloxera vastatrix

1890 coffee Hemileia vastatrix (rust) Sri Lanka

1917 wheat Puccinia graminis (stem rust)

1943 rice Cochliobolus myabeanus

1946 oat Cochliobolus victoriae (America)

1960 tobacco Peronospora tabacina (Italy)

1970 coffee Hemileia Vastatrix (Brasile)

1971 maize Helminthosporium maydis (America)

1950 banana Fungal diseases

History has provided some important examples of these dangers

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.

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

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

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

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

mar

ker

mar

ker

mar

ker

mar

ker

mar

ker

mar

ker

primer 12 primer 13

primer 4 primer 6

altezza attesa 163 bp

1000bp

1000bp

500bp

500bp

100bp

100bp

12 different SSR Primer Combinations

20.1 kD ►

30 kD ►

43 kD ►

67 kD ►

94 kD ► ►

F1

F2 F3

F4 F1

F1

F1

F1

F5

F6

F7

•6

•4•3 •2

•5

•10

•97•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

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

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.

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

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 24G 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

MULTI-LINE VARIETY CONSTITUTION

Variety name Genotype Variety Characteristics

Variety name Genotype Variety Characteristics

Variety 1 G36 Less productive genotypes.

Elevated genetic variability

Variety 4 G13 Morphological and productive characteristics variable. Less

genetic variability.

G27 G14

G17 G24

G1 G31

Variety 2 G19 Average production genotypes.

Elevated genetic variability

Variety 5 G27 Average morphological and productive characteristics.

Less genetic variability.

G15 G28

G22 G36

G16 G40

Variety 3 G25 Highly 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.

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

GENERAL COMBINING ABILITY AND SPECIFIC COMBINING ABILITY (SCA)

GENOTYPE

G1

G17

G19

G27

G36

G1G17 G1G19 G1G27 G1G36

G17G19 G17G27 G17G36

G19G27 G19G36

G27G36

5

2 2

1

5 5 52 2 2

1 1

1 4

2 2

1 2

2 ( 1)( 2)

GCA ii

SCA ij ii j j i i

S X Xp p p

S x X Xp p p

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

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

Tank you very much for your attention

Tank you very much for your attention