BG-01
Evaluation of transgenic carrot expressing potato snakin-1 polypeptide
Magdalena Klimek, Ewa Grzebelus, Rafal Baranski*
Department of Genetics, Plant Breeding and Seed Science, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Krakow, Poland
Correspondence to: baranski@ogr.ar.krakow.pl
We developed carrot plants with potato StSn1 gene coding for snakin-1, a cysteine-rich polypeptide. It was found previously that snakin-1 exhibits antimicrobial activity to some pathogenic bacteria and fungi challenged in vitro. Agrobacterium tumefaciens EHA105 strain containing a binary plasmid with the T-DNA region comprising the StSn1 gene and the nptII selection gene, both under the control of CaMV 35S promoter, was used for delivery of the transgenes to carrot cell suspension. The obtained calli and embryos were stimulated to plant development and further cultivated in a growing chamber. Transgenic tissue was selected on kanamycin enriched media and then their status was verified based on molecular analyses including gene specific PCR and reverse transcribed PCR, and Southern hybridization. The well developed plants are evaluated in laboratory assays for their resistance response to Alternaria radicina and Botrytis cinerea. The assays are performed using detached leaves and petioles inoculated with agar plugs overgrown by the fungus and then the disease symptoms are recorded in time course of the disease progress. Comparison of resistance response and molecular data will be presented.
The support of the Polish Ministry of Science and Higher Education (grant No. 3089/P01/2007/32) is gratefully acknowledged.
BG-02
Cultivated carrot germplasm resources-attempt to evaluation of genetic diversity using molecular tools
Anna Maksylewicz-Kaul1, Alicja Macko1, Rafal Baranski1, Thomas Nothangel2,
Dariusz Grzebelus1*
1 Department of Genetics, Plant Breeding and Seed Science, University of Agriculture in Krakow, Al. 29 Listopada 54, 31425 Krakow, Poland
2 Institute for Breeding Research on Horticultural and Fruit Crops, Federal Research Centre for Cultivated Plants - Julius Kühn-Institut, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany
Correspondence to: dgrzebel@ogr.ar.krakow.pl
A binational Polish-German research program has been initiated in 2008 to evaluate genetic resources of carrot with regard to their nutritional value. In spite of filed trials, morphological description and chemical analyses, the research aims in evaluation of genetic diversity at molecular level. We collected one hundred cultivars and landraces cultivated in all continents. The accessions were grown in pots in a glasshouse and fully characterized according to IPGRI descriptors. A great variation in both canopy and root morphology was observed. The roots were also preserved for further chemical analyses. Leaf material was collected during vegetation, freeze dried, and then total DNA was extracted using a CTAB protocol. Several molecular techniques will be applied to reveal polymorphism of the collection including arbitrary markers, RAPDs and AFLPs, microsatellites, transposon display, and gene specific markers. The first, preliminary results showing polymorphism of microsatelite loci and transposon insertions will be presented.
The research is supported by the binational Polish-German co-operative program (German Research Foundation, DFG, and Polish Ministry of Science and Higher Education, MNiSW, grant No. 97/N-DFG/2008/0).
BG-03
Mapping Vrn, the gene controlling vernalization requirement in carrot
María S. Alessandro1, Claudio R. Galmarini1,2, Aamir Ali 3,4 and Philipp W. Simon4,5
1 Estación Experimental Agropecuaria La Consulta, Instituto Nacional de Tecnología Agropecuaria, La Consulta, Mendoza, Argentina
2 Facultad de Ciencias Agrarias, UNCuyo y CONICET
3 Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan
4 Dept. of Horticulture, University of Wisconsin-Madison, Madison, USA
5 USDA-ARS, Vegetable Crops Unit, Dept. of Horticulture, University of Wisconsin-Madison, Madison, USA
Carrot (Daucus carota L.) is a cool-season vegetable normally classified as a biennial or winter annual species, requiring vernalization to induce flowering. Nevertheless, some cultivars adapted to warmer climates require less vernalization and can be classified as early-flowering or annual. Previous progeny evaluation of crosses between early- (Criolla INTA) and late-flowering (biennial lines from INTA breeding program) cultivars showed that F1 were completely annual and observed segregation ratios in F2 and BC1 families were not significantly different from expected segregation ratios under the hypothesis of a single dominant gene conditioning the annual habit, which we call Vrn. The objective of the present work was to localize the genomic region that controls vernalization response in carrot. We evaluated an F2 progeny, derived from the intercross between 'Criolla INTA' and a biennial carrot, in a two year trial at La Consulta. 280 and 297 plants were sown each year, once per week plants were evaluated. Individual plants were scored as being vegetative until the first floral internode elongated. Percentages of flowering F2 plants were 73.0% and 72.7% each year. DNA from 125 F2 plants was extracted. To construct the linkage map 310 SSR, 145 RAPD and 8 SCAR markers were screened in the population. Also the AFLP technique was used with EcoRI and MseI as digestion enzymes. We obtained 293 polymorphic markers for the population: 27 SSR (22 codominant), 30 RAPD, 1 SCAR (codominant) and 212 AFLP. Molecular and phenotypic data were analyzed with the JoinMap3.0 program to map the flowering gene.
BG-04
Transgenic carrot plants accumulating ketocarotenoids show tolerance to UV and oxidative stresses
Jayaraman Jayaraj and Zamir K. Punja*.
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada. (*Presenter and author for correspondence. E-mail : punja@sfu.ca ).
Ketocarotenoids are strong antioxidant compounds which accumulate in salmon, shrimp, crustaceans and algae, but are rarely found naturally in higher plants. In this study, we engineered constitutive expression of an algal b-carotene ketolase gene (bkt) in carrot plants to produce a number of ketocarotenoids from b-carotene. These included astaxanthin, adonirubin, canthaxanthin, echinenone, adonixanthin and b-cryptoxanthin. Leaves accumulated up to 56 mg/g total ketocarotenoids and contained higher b-carotene levels but lower levels of a-carotene and lutein. The photosynthetic capacity of transgenic plants was not significantly altered by these changes. However, when high-expressing transgenic plants were exposed to UV-B irradiation, they grew significantly better than the wild-type controls. Similarly, leaf tissues exposed to various oxidative stresses, including treatment with hydrogen peroxide and methyl viologen, showed less injury and retained higher levels of chlorophyll a and b. Total carotenoid extracts from transgenic leaves had higher antioxidant and free-radical scavenging activity in vitro compared to control leaves. Transgenic tissues also accumulated lower amounts of hydrogen peroxide following exposure to oxidative stresses, suggesting that free radical and reactive oxygen species were quenched by the ketocarotenoids.
BG-05
Analysis of carotenoids and polyacetylenes in carrot roots by means of Raman spectroscopy
Malgorzata Baranska1, Rafal Baranski2, Agnieszka Kaczor1, Maciej Roman1, Katarzyna Obal1, Hartwig Schulz3, Jonathan Schulz-Witte3
1 Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland, e-mail:baranska@chemia.uj.edu.pl
2 Dept. of Genetics, Plant Breeding and Seed Science, Faculty of Horticulture, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Krakow, Poland
3 Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany
Raman spectroscopy has been applied for in situ studies of carotenoids and polyacetylenes in carrot (Daucus carota L.) roots. The components are measured directly in plant tissue without any preliminary sample preparation. The analysis is based on the identification of intensive and characteristic key bands observed in the Raman spectrum of a carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar but their Raman spectra exhibit some specific differences, which can be used for their discrimination. Both compounds contain a -C?C-C?C- group, and its characteristic, strong and polarized, symmetric stretch mode can be seen at 2257-2251 cm-1 in the Raman spectrum. Carotenoids can be identified by -C=C- stretching vibrations (at about 1520 and 1155 cm-1) of the conjugated system occurring in their polyene chain. The assignment of vibrational bands and some structural study were supported by theoretical calculations. The Raman mapping technique revealed detailed information regarding the relative distribution of polyacetylenes and/or carotenoids in the investigated plant tissues. It has been found that the distribution of these components varies between carrot cultivars and especially a significant difference can be seen between cultivated carrot and wild relatives.
Acknowledgments
This work was supported by the grant from the Polish State Committee for Scientific Research. The authors are also grateful to the Academic Computer Center "Cyfronet" in Krakow.
H. Schulz, M. Baranska, R. Baranski, Biopolymers, 2005, 77, 212.
R. Baranski, M. Baranska, H. Schulz, P.W. Simon, T. Nothnagel, Biopolymers, 2006, 81, 497-505.
R. Baranski, M. Baranska, H. Schulz, Planta, 2005, 222, 448-457.
M. Baranska, H. Schulz, R. Baranski, T. Nothnagel, L. Christensen, J. Agric. Food Chem., 2005, 53, 6565-6571.
M. Baranska, H. Schulz, Analyst, 2005, 130, 855-859.
BG-06
Molecular mapping of AOX2 genes in carrot–an approach for breeding application on stress tolerance
Hélia G. Cardoso1, Maria Doroteia Campos1, Thomas Nothnagel2 and Birgit Arnholdt-Schmitt1*
1 EU Marie Curie Chair, ICAM, University of Évora, Apartado 94, 7002-554 Évora, Portugal
2 Federal Center of Breeding Research on Cultivated Plants, Institute of Horticultural Crops, Neuer Weg 22/23, D-06484 Quedlinburg, Germany
Correspondence to: eu_chair@uevora.pt
Plant mitochondria play an important role in diverse metabolic pathways and are involved in pathogen stress responses and in the process of programmed cell death [1]. Under diverse biotic as well as abiotic stress conditions, plant mitochondrial can control the reactive oxygen species (ROS) generation by means of energy-dissipating systems [2]. One of the systems involved is the alternative pathway mediated by alternative oxidase (AOX), located in the inner membrane of mitochondria. Until now, all plant species tested possess the genetic capacity to express the alternative pathway under any kind of developmental and environmental conditions [3-6]. Additionally, AOX was recently suggested to be related to efficient cell reprogramming under stress [7].
In higher plants, AOX is codified by a multigene family, which can be separated into two subfamilies, AOX1 and AOX2. In Daucus carota L. four AOX genes have been identified, two belonging to the AOX1 subfamily (AOX1a and AOX1b) and two belonging to the AOX2 subfamily (AOX2a and AOX2b) [8]. Variations previously identified at the DNA level in both AOX2 genes were exploited as molecular markers. This work provided a useful tool for genetic research leading us to the mapping of both genes. The use of two carrot segregation populations allowed us to localize both genes in the linkage map of carrot.
Further linkage of AOX2 candidate genes to important agronomical traits, such as stress tolerance, could allow us to consider these genes as functional molecular markers and apply them to carrot plant breeding as MAS.
[1] Clifton et al. (2005) Plant Mol Biol 58:193–212; [2] Pastore et al. (2007) J Exp Bot 58:195-2103; [3] Considine et al. (2001) Plant Physiol 126:1619-1629; [4] Costa et al. (2007) J Plant Physiol 164:718-727; [5] Giraud et al. (2008) Plant Physiol 147:595-610; [6] McCabe et al. (1998) Plant Physiol 118:675-682; [7] Arnholdt-Schmitt et al. (2006) Trends in Plant Sci 11:281-287; [8] Costa et al. (Accepted for revision in Plant Physiology and Biochemistry).
BG-07
ILP in AOX2 genes–a useful tool to investigate genetic diversity in Daucus carota L.
Maria Doroteia Campos, Hélia G. Cardoso, Rita Costa, M. Catarina Campos and Birgit Arnholdt-Schmitt1*
EU Marie Curie Chair, ICAM, University of Évora, Apartado 94, 7002-554 Évora, Portugal
Correspondence to: eu_chair@uevora.pt
Changes of the length of introns may be a source for DNA polymorphisms defining a new molecular marker for genetic diversity that is important for the study of biodiversity, population dynamics and ecological relationships. Knowledge of available plant genetic research is fundamental to support programs for the development of new cultivars, as well as to protect existing natural resources.
Here we report the development of a technique to investigate genetic diversity in D. carota L. that is based on polymorphism resulting from the PCR- mediated amplification of the specific introns in the coding region of the alternative oxidase genes AOX2a and AOX2b. Primers designed on the boundaries of AOX2a intron 3 and AOX2b intron 1 were able to amplify bands of different sizes within any given carrot genotype. These bands were isolated and sequenced to show that they were indeed DNA sequences corresponding to introns of the carrot AOX2 genes.
Polymorphic bands could be detected comparing band patterns obtained from 12 plants of the cv. ‘Rotin’ and 14 inbreed lines.
Analysis of the AOX2a DNA amplification pattern revealed the existence of a single band in 9 of 12 individual plants of cv. ‘Rotin’, and a 3 band pattern in the remaining 3 individual plants. In the same genotypes, AOX2b appeared to be more polymorphic, being identified six different band patterns.
Comparing the PCR result of the inbred lines, AOX2b presented a high homogeneity, with 13 inbred lines showing a single band pattern and only one line presenting 3 polymorphic bands, with both patterns already identified in cv ‘Rotin’. However, AOX2a DNA amplification revealed high variability and a new band pattern.
This work was supported by the European Commission through the Marie Curie Chair and FCT for scholarships to M. Doroteia Campos (SFRH/BI/15991/2006) and Hélia Cardoso (SFRH/BPD/27016/2006). We would like to thank Vilmorin & Cie, France, for financial support and providing us carrot breeding material.
BG-08
Polymorphism of carotenoid biosynthesis pathway genes in different coloured carrots
J. Clotault1, M. Lecomte1, D. Peltier2, M. Briard1, E. Geoffriau*1
1 Agrocampus Ouest – INHP, UMR GenHort, F-49045 Angers, France
2 Université Angers, UMR GenHort, F-49045 Angers, France
Correspondence to: Emmanuel.Geoffriau@agrocampus-ouest.fr
Carrot germplasm exhibits a wide variation of root colour depending on pigment composition: carotenoids (yellow, orange, red), anthocyanins (purple) and no pigment (white). Molecular selective events which led to this colour variability are unclear. Our work aimed at measuring the diversity between colour types and to study possible impact of selection on carotenoid biosynthesis pathway genes in carrot. The haplotypes were determined for 48 individuals spread in 6 colour-based groups containing 8 cultivars each: white, yellow, orange open-pollinated, orange hybrid, red and purple. Partial sequences have been obtained for phytoene desaturase (PDS), lycopene ε-cyclase (LCYE), lycopene β-cyclase 1 (LCYB1) and zeaxanthin epoxidase (ZEP) genes. Diversity was measured through several indexes (nucleotide diversity π, index of genetic differentiation FST). The red and purple groups exhibited the highest π for LCYB1 and ZEP. FSTindex was often the highest between red or purple groups and other groups, whereas FSTbetween these two groups was generally low. Red, purple and yellow groups are mainly composed by Asian cultivars. Our results would suggest a higher genetic diversity in material of Asian origin. Results were also analyzed regarding selective effects according to gene position in the metabolic pathway. Polymorphism observed would be coherent with a hypothesis of stabilizing selection on genes acting early in the pathway and diversifying selection on genes acting at branch points. The influence of gene position in the pathway regarding selective constraints and the effect of selection for colour trait and geographic origin on carrot genetic diversity structure are currently studied
.