Proposals for integration with the 7 th Framework Programme
The Second Call 2007 CALL 2A: FP7-KBBE-2007-2A
KBBE-2007-1-2-15 Reducing the utilisation of mineral fertilisers by improving the efficiency of nutrient use in European crops
QTL ANALYSIS OF MINERAL NUTRIENT USE EFFICIENCY IN TOMATO
(Lycopersicon)
Institute of Genetics and Cytology of the National Academy of Sciences of Belarus
Phone: (37517) 2841946
Fax: (37517) 2841917
E-mail: A.Kilchevsky@igc.bas-net.by
Address: Akademicheskaya St. 27, 220072 Minsk, Republic of Belarus
Scientific adviser:
Prof. Dr.Sc. A.V. Kilchevsky
Members of team:
Scientific researcher S.V. Malyshev
Ph.D. O.G. Babak
Ph.D. M.M. Dobrodkin
Summary
The inclusion of mineral nutrient use efficiency as traits within breeding programmes will require the use of modern quantitative and molecular genetic tools. Nutrient use efficiency has proved to be a difficult trait to assess and improve in plants. Functional genomics, proteomics and gene mapping will identify candidate genes and QTLs for efficient plants. Differences in gene expression within high and low, mineral nutrient use efficient cultivars with and without stress will be assessed. Progress will also be made towards development of a medium resolution gene map an important tool for the future improvement of tomato cultivars. The results from the quantitative genetic and molecular data will be modeled to inform on the optimum design of future practical breeding programmes.
Introduction
Plants have evolved sophisticated mechanisms to deal with levels of mineral fertiliser (e.g. N, P,
K) in the soil. Because significant differences in mineral nutrient use efficiency are known to occur between plant varieties, molecular identification of mechanisms governing this process could result in the development of useful markers, or of crops with reduced the need for fertiliser. Recent research has revealed that plant possess several classes of constitutive and inducible high- and low-affinity transport systems that must be involved in mineral uptake and homeostasis in general, and thus could play a key role in nutrient use efficiency. These include the high (NRT2) and low (NRT1) affinity nitrate transporters, NR, NiR, and the enzymes of
GOGAT pathway for ammonia assimilation. The other group of factors responsible for the phosphate nutrient is encoded by genes (LePT1 and LePT2) of high-affinity inorganic phosphate
(Pi) transporters. However, despite on the great progress in understudying of molecular mechanisms of mineral nutrient use efficiency, many genes have not been placed in genetic maps and remain unexplored.
Main part
Objective: The aim of subproject is to identify those tomato genes whose expression varies in response to the levels of mineral fertilisers (N, P), and which might be candidates for involvement in mineral nutrient use efficiency of tomato.
Work plan:
Task 1. Production of resource material for gene-linkage analysis.

The collection of cultivars and lines of tomato will be assessed on the efficiency of mineral fertilisers using. The contrast forms will be crossed to develop RIL population as future tool for QTL mapping analysis. The individual plants of mapping population will be scored for such efficiency using quantitative scale.
Task 2. Genotyping of parent lines (male and female) by microsatellite and AFLP markers. Finding markers presented by different alleles in the parents and suitable for genotyping of RIL for mapping.
Set of known tomato microsatellite markers will be used for genotyping. Polymorphic AFLP bands with
EcoRI/TaqI and EcoRI/MseI pairs will be scored from both parents. Loci, different in male and female lines will be chosen for subsequent RIL genotyping.
Task 3. Development of SNP markers from mineral uptake specific gene sequence data. Testing for polymorphisms in parent lines.
Sequences of the mineral uptake specific genes (both existing and generated by the project) will be assessed to reveal high frequency allelic variation. SNP markers will be developed and tested for allelic polymorphism (suitability for linkage mapping) in parents. Taking in account these SNP markers, the total number of genetic markers for linkage map construction is estimated to be over 300.
Task 4. Genotyping of RIL progeny with genetic markers obtained in Task 2 and 3.
DNA of 100-150 individual RIL tomato plants from a single cross will be extracted. All genetic markers developed in Task 2 and 3 and suitable for linkage mapping (possessing different alleles in both parents) will be scored for all offspring tested. Genotyping data will be documented for further statistical analysis.
Task 5. Statistical analysis and characterization of linkage groups.
The gene marker alleles data set will be analyzed by statistical software (e.g. Mapmaker). Gene markers
(anonymous, such as microsatellites and AFLP, as well as SNPs derived from annotated genes) coinherited in progeny will constitute linkage groups.
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Deliverables
DNA from parental lines and RIL progeny produced and archived.
Assessment of microsatellite and AFLP variation in parents.
Identification of SNPs in existing and newly generated mineral uptake specific sequences.
Genotyping of 100 RIL offspring for at least 300 markers.
Generation of draft linkage map using DNA markers and QTLs.
Main publications
Kilchevsky A , Khotylyova L, Peshich V, Kogotko L, Schoor A, Gavrilov A, Kruk A (1999)
Breeding of vegetables with minimum pollutant accumulation. In: Scarascia Mugnozza GT Porceddu E,
Pagnotta MA (eds) Genetics and Breeding for Crop Quality and Resistance, Kluwer, Dordrecht, Boston,
London, pp 313-322
Korzun V, Malyshev S
, Voylokov AV, Börner A (2001) A genetic map of rye (
Secale cereale
L.) combining RFLP, isozyme, protein, microsatellite and gene loci. Theor Appl Genet 102: 709-717
Malyshev S
, Korzun V, Voylokov A, Smirnov V, Börner A (2001) Linkage mapping of mutant loci in rye ( Secale cereale L.). Theor Appl Genet 103: 70-74
Malyshev S
, Korzun V, Efremova TT, Börner A (2001) Inheritance and molecular mapping of a gene determining vernalisation response in the Siberian spring rye variety 'Onokhoyskaya'. Cereal Res
Comm 3-4: 259-265
Börner A, Buch-Sorlin GH, Hayes PM,
Malyshev S , Korzun V (2002) Molecular mapping of major genes and quantitative trait loci determining flowering time in response to photoperiod in barley.
Plant Breeding 121: 129-133
Lukaszewski AJ, Rybka K, Korzun V, Malyshev SV , Lapinski B, Whitkus R (2004)Genetic and physical mapping of homoeologous recombination points involving wheat chromosome 2B and rye chromosome 2R. Genome 47: 36–45
Khlestkina EK, Than MHM., Pestsova EG, Röder MS, Malyshev SV
, Korzun K, Börner A
(2004) Mapping of 99 new microsatellite-derived loci in rye ( Secale cereale L.) including 39 expressed sequence tags. Theor Appl Genet 109: 725–732
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Kilchevsky AB , Babak OG (2004) Breeding of energy effective tomato varieties. 2004
EUCARPIA Congr.: Genetic Variation for Plant Breeding, Vienna, Austria, pp 459-462
Kartel NA, Kilchevsky AB (2005) Biotechnology in crop production, Minsk, 2005, 310 pp
Malyshev SV , Grushetskaya ZE, Kartel NA, Kilchevsky AV SSR-genotyping of tomato lines and varieties from Belarus collections. PAA/Solanaceae 2006: Genomics Meets Biodiversity, Monona
Terrace, Madison, Wisconsin, July 23-27, 2006