Genetic drift in perennial ryegrass populations: Analysis of phenotypic data, Illumina genotyping assays and candidate genes
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1 Genetic drift in perennial ryegrass populations: Analysis of phenotypic data, Illumina genotyping assays and candidate genes Gräserabbildungen: COMPO GmbH & Co.KG
2 Outline Introduction Phenotypic data Illumina SNP genotyping assays Candidate gene approach Outlook soilcropandmore.info soilcropandmore.info Page 2/23 soilcropandmore.info
3 Introduction PhD project is dealing with: Monitoring the effects of natural selection in populations of Lolium perenne L. and development of markers for the trait persistence cooperation with Institute of Crop Science and Plant Breeding (IPZ) of the Bavarian State Research Center for Agriculture (LfL) fussballrasen.jpg Page 3/23
4 Introduction - Material experimental design: five locations, four years of cultivation, 19 forage varieties DNA extraction, phenotyping of the plots at the five sites and of the single plants in Steinach ( ), Bavaria Page 4/23
5 Introduction - Material Phenotyping of the plots: data from sites Hötzelsdorf, Spitalhof and Schmalenbeck (no data from Detern) e.g. traits: growth after winter, defects (diseases a.s.o.), denseness, were phenotyped each plot in four repetitions B Page 5/23 A Genotype mixtures and winter survival of variety `Guru from sites Hötzelsdorf (A) and Schmalenbeck (B) after four years of cultivation, with drastic changes being visible at Schmalenbeck.
6 Introduction - Material Phenotyping of the single plants: 96 single plants of each of the five sites were transplanted to Steinach, Bavaria with 12 plants per row in 2008 per variety and site eight rows with 12 plants each side by side arranged traits: e.g. diseases (rust, foliar diseases, Fusarium a.s.o.), heading date, mass production and others were phenotyped phenotyping from 2008 until 2011, this data available Page 6/23 horstmannrasen.de
7 Introduction - Phenotyping scheme of the Bundessortenamt (BSA), Germany used for phenotyping each trait was scored with a number (one to nine), 1 trait is not formed, 9 trait is formed clearly rasengesellschaft.de rasengesellschaft.de; Gray Leaf Spot infected Lolium leaf (Foto: Vincelli) Page 7/23
8 Results - Analysis of phenotypic data two types of sets: plots and single plants both data sets evaluated and analyzed, trends observed, hypotheses set and discussed in cooperation with Bavarian Institute, group of LfL/Dr. Hartmann rasengesellschaft.de; Gray Leaf Spot infected Lolium leaf (Foto: Vincelli) Page 8/23
9 heading date (days after 1st of April, mean value) Guru Guru Guru Guru Ivana Ivana Ivana Ivana Moronda Moronda Moronda Moronda Sambin Sambin Sambin Sambin Results - Analysis of phenotypic data single plants data sets showing trends, but: phenotyping in 2010 and 2011 completely different compared to 2009, even with respect to standards of Bundessortenamt 70,00 60,00 50,00 40,00 30,00 20,00 10,00 0,00 Heading date (mean value) of 2009 vs 2010 vs BSL MW AESCH09 MW AESCH10 BSL Page 9/23 De Hö Schm Sp De Hö Schm Sp De Hö Schm Sp De Hö Schm Sp Variety and site
10 Results - Analysis of phenotypic data single plants unexpected effects have to be clarified hypotheses: climatic effects mistakes in field practice (randomization ) phenotyping suboptimal keepbanderabeautiful.org amnh.org Page 10/23
11 Results - Analysis of phenotypic data plots data only for three sites available phenotyping at those sites done very differently not every trait phenotyped at every site and with the same number of repetitions possible to observe trends and to evaluate the available data statistically, but comparison between north and south Germany will be not appropriate Page 11/23 locomnet.de dominika.twoday.net
12 Rating for "deficits after winter", mean value 10 Results - Analysis of phenotypic data plots Phenotyping plots - deficits after winter M_NWIN 2005 MW M_NWIN 2006 MW M_NWIN 2007 MW M_NWIN 2008 MW 1 0 Sp Hö Schm Sp Hö Schm Sp Hö Schm Sp Hö Schm Guru Guru Guru Ivana Ivana Ivana Moronda Moronda Moronda Sambin Sambin Sambin Variety and site Page 12/23
13 Results - Analysis of phenotypic data climate average temperature 20cm above soil rain sum average temperature 20cm above soil rain sum Page 13/
14 Results - Analysis of phenotypic data climate average temperature 20cm above soil rain sum average temperature 20cm above soil rain sum Page 14/
15 Results - Analysis of Illumina GoldenGate genotyping assay analyses of bulked samples conducted by TraitGenetics method: results: Page 15/23 Lewis et al., 2007; 2007 Illumina, Inc.; Pub. No Oct07
16 Results - Analysis of Illumina GoldenGate genotyping assay normalized theta value nearest zero homozygous for allele A normalized theta value nearest one homozygous for allele B Page 16/23 GenomeStudio SNP Graph of an individual marker showing allele compositions of samples from four varieties from four trial sites; yellow dots: variety Guru, sampled at Hötzelsdorf (Hö) in 2004 and 2008 (two/three bulks analyzed; no shift/differentiation); green dots: Guru sampled at Schmalenbeck (Sc) in 2004 and 2008 (two bulks analyzed each), with indicated shift in allele frequency between the two years.
17 Results - Analysis of Illumina GoldenGate genotyping assay Page 17 results of Illumina GoldenGate assay checked with function hierarchical clustering of DiSTo2 four different varieties clustered together; some outliers expectation that genotype mixtures could cluster according to the site of origin not observed interesting: seed lots are clustering closely; Moronda Spitalhof and Hötzelsdorf different compared to rest of Moronda entries
18 Results - Candidate gene approach in 2007 Tamura & Yamada published in Theor Appl Genet: A perennial ryegrass CBF gene cluster is located in a region predicted by conserved synteny between Poaceae species CBF genes = C-repeat binding factor CBF/dehydration-responsive element binding protein 1 (DREB1) regulon Page 18/23
19 Results - Candidate gene approach CBF/DREB 1 regulon most important transcription unit involved in cold acclimation sequences available; new primer design etc. Page 19/23
20 Results - Candidate gene approach problem: amplified PCR fragments too small compared to the fragment sizes calculated work in progress LpCBF Ia ~ 763 bp LpCBF Ib ~ 857 bp LpCBF II ~ 881 bp LpCBF IIIb ~ 899 bp LpCBF IVb ~ 943 bp LpCBF Va ~ 721 bp Fermentas Page 20/23
21 Results - conclusion lab work: Candidate gene approach is not working yet work in progress de.fotolia.com Illumina GoldenGate genotyping assay correlation with phenotyping data started Page 21/23 pinkmonkey.de
22 Outlook Analysis phenotypic data of the plots and of the single plants Correlation Illumina GoldenGate genotyping assay data with phenotypic data Page 22/23 kunst-fuer-alle.de
23 Acknowledgements funding: Bayerisches Staatsministerium für Ernährung, Landwirtschaft und Forsten. AG GED, AG PBP, AG EXT Dr. Klaus Dehmer, Dr. Stephan Hartmann (LfL, Bavaria) Page 23/23
24 eide/ ger-de/schafe_auf_der_weide_imagelarge.jpg primolo.de Thank you for your attention! soilcropandmore.info Page 24 cutcaster.com
25 Page 25 Illumina, Inc. Customer Solutions, 9885 Towne Centre Drive, San Diego, CA , (toll free), (outside the U.S.),
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