Activity 1: Conservation of plant genetic resources

ACTIVITY DESCRIPTION

(1) The establishment of a base collection of traditional common bean landraces: accessions of traditional common bean landraces from Croatia, neighbouring countries (Serbia, Bosnia and Herzegovina, Macedonia) and European Union countries in which common bean represents a t valuable  agricultural crop (Slovenia, Spain, Austria) will be collected and these accessions will represent the base collection.

(2) Field trial: the collected seed will be multiplied and regenerated in a field trial during which the accessions will be morphologically described using the descriptor list to form a working collection that will represent bush morphotypes. The seed coat colour of the accessions from the working collection will be analysed with phenotyping platform equipped with different cameras and sensors (eg. VIS, NIR, IR etc.).

(3) Sample preparation: during the field trial, samples of plants for DNA extraction will be collected as well as seeds samples for further analyses of the mineral composition.

(4) Determination of the origin: for the purpose of determining the origin of the bean accessions, the phaseolin type will be determined.

The activity is related to Objective 1 in the part concerning the establishment of a collection of plant genetic resources for the purpose of describing and evaluating the morphological characteristics of common beans, determining the origin of the accessions as well as plant genetic resources conservation.

Activity 2: Phenotyping

ACTIVITY DESCRIPTION

(1) Determination of the nutritive quality of the traditional common bean landraces: seeds of the accessions from the working collection will be harvested at the stage of technological maturity, and their fresh and dry weight will be determined. Seed mineral composition (N, P, K, Ca, Mg, Fe, Zn, Mn i Cu) will be determined in dried, grinded, and homogenized samples. Total nitrogen content will be analyzed by micro Kjeldhal procedure, whereas content of all other mineral nutrients will be determined by ICP (Inductively Coupled Plasma Analysis).

(2) Phenotypic variability of the root system: -root system traits will be determined on plants grown in pouches. Plant root system will be photographed using phenotyping platform equipped with different cameras and sensors (eg. VIS, NIR, IR etc.) and analyzed using WinRhizo (Regent Instruments, Inc).  For the computation of ionic activities of the nutrient solution Geochem-EZ software will be used.

Digital image acquisition and image analysis using WinRhizo software enables fast, uniform, and repeatable analysis of root system traits for all genotypes. Described characteristics make this method very interesting for the scientific research as well as for plant breeding industry. Plant phenotypic characteristics (architecture) are tightly connected with the morphology and function of plant organs. Thus, root system architecture is of fundamental importance for water and nutrient acquisition (especially P and N). Therefore, based on root traits such as root width, length, surface area, volume, root angles, ratio between fine and coarse roots, root branching, etc. genotypes with potentially enhanced water, nitrogen and phosphorus acquisition efficiency will be selected.

(3) Determination of phenotypic and physiologic reaction of selected common bean genotypes under water, nitrogen, and phosphorus deficiency:

Common bean genotypes selected based on root architectural traits will be grown under experimental conditions of nitrogen, phosphorus and water deficit. For the calculation of the ionic activity within nutrient solutions Geochem-EZ will be used. Plants will be grown in greenhouses till the period of bloom (until the blooming stage??). During the growth period phenotypic plant characteristics such as leaf area, plant height, shoot biomass, chlorophyll content, stomatal conductivity, leaf temperature, growth rate, etc. will be measured by phenotyping platform using different VIS and NIR sensors. For the estimation of stress intensity chlorophyll fluorescence measurements will be performed using Plant stress kit (Opti-sciences). Based on those measurements maximal quantum yield of PS 2 (Fv/Fm), actual quantum yield of PS 2 (YII), apparent electron transport rate (ETR), proportion of open PS 2 (qP), and non-photochemical quenching (NPQ) will be calculated. At the end of the experiment shoot and root fresh and dry weight will be determined, as well as nitrogen and phosphorus content in the plant.

The activity is related to Objective 1 concerning phenotyping of the plant collection in order to: determine and describe traits related to enhanced nutritive value of common bean seeds; determine and describe common bean root traits and to select genotypes with potentially enhanced phosphorus and nitrogen acquisition efficiency, and drought tolerant genotypes; and to determine phenotypic plasticity of the selected common bean genotypes under stressful conditions of water, nitrogen, and phosphorus deficit.

Activity 3: Genotyping 

ACTIVITY DESCRIPTION

(1) Genotyping by microsatellite markers: developed microsatellite markers will be used in the analysis of genetic structure and diversity of traditional common bean landraces as well as in the association mapping analyses for the purpose of determining gene-related markers controlling the amount of bioactive nutrients and desirable phenotypic characteristics of the root system.

(2) Analysis using the DarTSeq platform: with the help of DarTSeq sequencing technology, SNP markers will be developed which will facilitate the detection of associations between the markers and phenotypes related with the amount of minerals and the particular phenotypic characteristic of the root system.

(3) Association mapping: a whole genome association mapping analysis will be carried out in order to identify quantitative trait loci (QTLs) related to the selected phenotypic characteristics; the increased efficiency of phosphorus and nitrogen utilization, increased tolerance to the drought and concentration of mineral elements in the seed.

The activity is related to Objective 1 in the part concerning the implementation of the genotyping of the collection in order to estimate the genetic structure and diversity of traditional common bean landraces, and the identification of quantitative trait loci (QTL) for desirable properties of root system as well as the amount of bioactive nutrient content.