Introduction to the use of molecular genotyping techniques

Transcription

1 Introduction to the use of molecular genotyping techniques Gregorio López-Ortega, Almudena Bayo-Canha, Emma Skipper and Felicidad Fernández Budapest 3 rd -5 th of March

2 STSM (Spain to UK) Pomological characterization and introduction to the use of molecular genotyping techniques Pomological characterization Fingerprinting of 10 varieties S-alleles of 10 varieties Checking two molecular markers for fruit size Felicidad Fernández Fernández East Malling Research, United Kingdom COST STSM Reference Number: ECOST-STSM-FA

3 ! "# $%&'( )'* +$&,-. ' Pomological characterization C210-7 X Colney 300 individuals Phenotypical and Pomological Evaluation of the Variation Present in a Sweet Cherry Mapping Population. E. S. Skipper 1, G. Lopez-Ortega 2, A. Bayo-Canha 2, and F. Fernández-Fernández 1 1 East Malling Research, New Road, East Malling ME19 6BJ UK Sweet September C210-7 X X Lapins Colney 2 IMIDA, C/ Mayor, Murcia Spain gregorio.lopez2@carm.es Introduction: A group of 125 individuals of the F1 progeny Cherry (Colney x C210-7 [Lapins x Sweet September]) has been evaluated at East Malling Research (EMR). This contribution shows the natural variation in flowering, harvest date and fruit quality traits. Harvesting date: Harvesting date records: Estimate ripening date each two-three days. Quality assessments: Standard quality assessments: good, rots, cracks, soluble solids, acidity, colour, fruit size, firmness, weight. Aims: Characterise phenotypical variation in the progeny to enable QTL analysis. Identify individuals with extreme phenotypes with a potential to extend harvest season due to delayed ripening/maturity. Results: The harvest period was between 16 th of July and 21 th of August. Half of the population had white flesh while the other half had black flesh. The average weight of the fruits were between 6-10 grams. There were only three hybrids with an average weight higher than ten. The 80% of the progeny had sugar content between ºbrix and a 60% of the hybrids firmness was among (g/mm). Period: 16th of July and 21st of August Average weight: 6-10 gr 80% ºbrix 60% g/mm2 50% white flesh/50 black flesh '! " &#" &! " %#" %! " $#" $! " #"! " %!! (%#! " %#! (&!! " &!! (&#! " &#! ('!! " '!! (' #! " ' #! (#!! " #!! (##! " Future Work 3,&# 4%.. ' /01# # 2 ' Develop a linkage map using the Illumina 6K SNP chip in collaboration with IASMA Record phenotype data over subsequent years and in different environments for the mapping of QTLs relating to flowering characteristics and quality traits. This work was conducted within the framework of COST Action 1104

4 Fingerprinting Objective: Fingerprinting ten varieties Methodology: DNA extraction Dneasy kit Quiagen PCR amplification in two multiplex Sizing with ABI

5 Molecular Markers

6 Results

7 S-Alleles Objective: S-alleles determination of ten varieties Methodology: PCR with two primer pairs Sizing with ABI Analyse the results

8 S-allele Markers *6-FAM-F-BOX5 A (Vaughan 2006): TTK SCH ATT RYC AAC CKC AAA AG *F-Boxintron R: CWG GTA GTC TTD SYA GGA TG *VIC-PaConsI-F (Sonneveld 2003): MCT TGT TCT TGS TTT YGC TTT CTT C *PaConsI-R2 (Sonneveld 2006): GCC ATT GTT GCA CAA ATT GA

9 Results Control Cultivars Determined References Summit S1S2 S1S2 Colney S5S6 S5S6 Inge S4S9 S4S9 Schneider S3S12 S3S12 Cryall-seeding S2S7 S2S7 Sumburst S3 S3S4' Noble - S6S13 Cristobalina - S3S6

10 Results for our ten varieties Cultivars Crystal Champaing Ronde grosse Gemelle Summerland Carly O'Lory Determined S3S5 S3S6 S2S9 S5S17 S1S9 7-91C - Big Lory - Mister Early - Riaño - Walter C -

11 Markers for Fruit size

12 Markers for fruit size Colors indicate whether the haplotype is associated with large, small, or presumed small fruit size LARGE PRESUMED SMALL SMALL NO EFFECT

13 Conclusions The goals of the STSM were achieved A set of markers for fingerprinting Markers for hybrid selection Proposal Consensus a set of markers for fingerprinting Protocol for S-alleles MAS

14 Acknowledgements THANKS FOR YOUR ATTENTION

15 References Aranzana MJ, Pineda A, Cosson P, Dirlewanger E, Ascasibar J, Cipriani G, Ryder CD, Testolin R, Abbott A, King GJ, Iezzoni AF, Arús P A set of simple-sequence repeat (SSR) markers covering the Prunus genome. Theor Appl Genet 106: Clarke JB, Tobutt KR Development and characterization of polymorphic microsatellites from Prunus avium Napoleon. Molecular Ecology 3(4): Dirlewanger E, Cosson P, Tavaud M, Aranzana MJ, Pizat C, Zanetto A, Arús P, Laigret F Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.) Theor Appl Genet 105: Mnejja M, Garcia-Mas J, Howad W, Badenes ML, Arús P Simple-sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl.) are highly polymorphic and transferable to peach and almond. Molecular Ecology 4(2): Sonneveld T, Tobutt KR, Robbins TP Allele-specific PCR detection of sweet cherry self incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers. Theor Appl Genet 107: Sonneveld T, Tobbins TP, Tobutt KR Improved discrimination of self-incompatibility S-RNase alleles in cherry and high throughput genotyping by automated sizing of first intron polymerase chain reaction products. Plant Breeding 125: Testolin R, Marrazzo T, Cipirani G, Quarta R, Verde I, Dettori MT, Pancaldi M, Sansavini S Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome 43(3): Vaughan SP, Russell K Characterization of novel microsatellites and development of multiplex PCR for large-scale population studies in wild cherry, Prunus avium. Molecular Ecology 4(3): Vaughan SP, Russell K, Sargent DJ, Tobutt KR Isolation of S-locus F-box alleles in Prunus avium and their application in a novel method to determine self-incompatibility genotype. Thero Appl Genet 112: Zang G, Sebolt AM, Sooriyapathirana SS, Wang D, Bink MC, Olmstead JW, Iezzoni AF Fruit size QTL analysis of an F1 population derived from a cross between a domesticated sweet cherry cultivar and a wild forest sweet cherry. Tree Genetics & Genomes 6: STSM Scientific Report