1 Biometris, Wageningen UR, Droevendaalsesteeg 1, P.O. Box 16, 6700 AA Wageningen, The

TITLE

Bayesian QTL analyses using pedigreed families of an outcrossing species, with application to fruit firmness in apple

AUTHORS

MCAM Bink1, J Jansen1, M Madduri2, RE Voorrips2, C-E Durel3,4,5, AB Kouassi3,4,5*, F Laurens3,4,5, F Mathis3**, C Gessler6, D Gobbin6***, F Rezzonico6,7, A Patocchi6,7, M Kellerhals7,A Boudichevskaia8****, F Dunemann8$, A Peil8, A Nowicka9,B Lata10, M Stankiewicz-Kosyl10,K Jeziorek11,E Pitera11, A Soska11,K Tomala11, KM Evans12,$*, F Fernández- Fernández 12,W Guerra13, M Korbin14, S Keller14, M Lewandowski14, W Plocharski14, K Rutkowski14, E Zurawicz14, F Costa15,16, S Sansavini15, S Tartarini15, M Komjanc16, D Mott16, A Antofie17$**,M Lateur17A Rondia17, L Gianfranceschi18, and WE van de Weg2

1 Biometris, Wageningen UR, Droevendaalsesteeg 1, P.O. Box 16, 6700 AA Wageningen, The Netherlands

2 Plant Breeding, Wageningen UR, Droevendaalsesteeg 1, P.O. Box 16, 6700 AA Wageningen, The Netherlands

3 INRA, UMR1345 Institut de Recherche en Horticulture et Semences, SFR 4207 Quasav, Pres L’UNAM, F-49071 Beaucouzé, France

4 Université d’Angers, UMR1345 Institut de Recherche en Horticulture et Semences, F-49045 Angers, France.

5AgroCampus-Ouest, UMR1345 Institut de Recherche en Horticulture et Semences, F-49045 Angers, France.

6 Plant Pathology, Institute of Integrative Biology (IBZ), ETH Zurich, CH-8092 Zurich, Switzerland

7 Research StationAgroscopeChangins-Wädenswil, ACW, Schloss 1, P.O. Box CH-8820, Wädenswil, Switzerland

8Institute for Breeding Research on Horticultural and FruitCrops, Julius Kühn-Institut, Pillnitzer Platz 3a, 01326 Dresden, Germany.

9Department of Experimental Design and Bioinformatics, Warsaw University of Life Sciences – SGGW, 02-776 Warsaw, Poland

10 Laboratory of Basic Research in Horticulture, Faculty of Horticulture, Biotechnology, and Landscape Architecture, Warsaw University of Life Sciences SGGW, 02-776 Warsaw, Poland

11Department of Pomology, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences – SGGW, 02-776 Warsaw, Poland

12 East Malling Research, New Road, East Malling, Kent ME19 6BJ, United Kingdom

13 Research Centre for Agriculture and Forestry Laimburg, Posta Ora – 39040 Vadena (BZ), Italy

14 Fruit Breeding Department, Research Institute of Horticulture, 96100 Skierniewice, Poland

15Department of Fruit and Woody Plant Science, currentDepartment of Agricultural Sciences, University of Bologna, Via Fanin 46, 40127 Bologna, Italy

16 Department of Genetics and Biology of Fruit Crops, IASMA Research and Innovation Centre, FoundationEdmundMach, ViaMach1, 38010Trento,Italy

17WalloonAgricultural Research Centre (CRA-W), Gembloux, Liroux 9, B-5030, Belgium

18 Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milan, Italy

Currentaddresses:

* Université Félix Houphhoët-Boigny, Unité de Formation et de Recherche (UFR) 'Biosciences', Laboratoire de Génétique, 22BP 582 Abidjan 22, Côte d’Ivoire

** Fabienne Mathis, VEGEPOLYS - Pôle de compétitivité , 7 rue Dixmeras - 49044 Angers cedex 01 France

*** Tecan Group Ltd., CH-8708 Männedorf, Switzerland

**** Leibniz-Institut fürPflanzengenetikundKulturpflanzenforschung (IPK), Corrensstr. 3, D-06466 SatdtSeelandOt. Gatersleben

$ Julius Kühn-Institut, Institute for Breeding Research on Horticultural and Fruit Crops, Erwin Baur Str. 27, D-06484 Quedlinburg, Germany

$* Washington State University (WSU-TFREC), 1100 N. Western Avenue, Wenatchee, 98801- WA, USA

$** Direction Générale Qualité et Sécurité - Métrologie Légale SPF Economie, P.M.E., Classes Moyennes et Energie, NorthGate, Bd du Roi Albert II, 16, 1000Bruxelles, Belgium

CORRESPONDING AUTHOR

Marco Bink

Postal address

Biometris,Wageningen University & Research centre

PO Box 100, 6700 AC Wageningen

The Netherlands

Email

Electronic Supplementary Materials 4: Extrapolation of QTL results

Sources for +QTL-alleles & stabillity of marker-trait associations

Introduction

Assessment of the markers that are linked to the QTL +alleles across the mapping parents gives indications of the robustness of these linkages, the potential presence of different sources of +alleles, the tightness of the linkage and provides the information needed to apply these markers for MAB. Sources for +alleles are traced and distinguished by the flow of marker alleles throughthe pedigree of the parents of mapping poplations up to the founder level. In the case of multiple sources, additional research on the presence of +alleles of different sizes may be useful.

LG1b

Fruitfirmness QTL-LG1b is located around a single marker locus (SSR-CHVf1) , thightly associated to the apple scab resistance gene Vf) andsegregated in 17 parents (Figure 6) of which all but one had the same linked marker allele (SSR-CHVf1-159),which allele is also associated to Vf). This marker could be traced over four to seven generations to a common source, the wild M. floribunda 821 (also the source of the Vfresistance; Figure 1), thus confirming results of Vinatzer et al (2004) on the origin of this marker. One parent (‘Discovery’) showed a different marker (SRR-CHvf1-163) which could be traced back for one generation to ‘Beauty of Bath’, which is not related to M.floribunda 821 and thus represents a second, independent source for a +QTL-allele which may or may not be based on a different gene or allele of different size. Examination of wider germplasm of cultivated apple showed the -159 marker to be unique for the M.floribunda 821 lineage. The stability of the marker-trait association makes it amenable for application in marker-assisted breeding. This stability may be due to a tight linkage to the QTL but may also be the combinedresult of selection for two highly desired traits: fruit firmness and Vf resistance to apple scab (Venturia inaequalis), SSR-CHVf1 is in fact at about maximal 200 kb from HcrVf2 (Patocchi et al 1999, Vinatzer et al 2004) , the gene found to be responsible for the Vf resistance (Belfanti et al 2004). For the second source (-163), nothing is yet known on the stability of the marker-trait association asit occurred at low frequency within the wider HiDRAS germplasm (including an additional series of modern and old cultivars), being present in just four other cultivars which did not occur in the pedigree of any of the mapping populations.

Figure ESM4.1 Pedigree of 13 families that segregate for the fruit firmness QTL LG1b. Lineages are presented that trace to the founders M.. floribunda 821 and ‘Beauty of Bath’, the sources for +QTL-alleles. Red and blue lines indicate a maternal and paternal relation respectively

LG10b

Fruitfirmness QTL 10b spans a region of 24cMthat includes three SSR markers (Figure 4). Thirteen parents were heterozygous for the QTLand 12 had the +allele in the homozygous condition (Figure 6). Their +allele came from 16 to 17founders and was represented by 14 different marker-haplotypes (Table ESM4.1).In addition, a series of founders and cultivars were identified that lack any+allele, and their marker-hapltypes werealso assessed (data not shown). QTL genotypes can now be predicted for genetically-linked germplasm by combining marker haplotype information and Identity By Descent (IBD) probabilities. This is examplified for the cultivar‘Rubens’ (Figure ESM4.2), which could candidate as crossing parent. Its mother ‘Gala’ was a mapping parent that was heterozygous for the QTL. It is uncertain whether ‘Gala’ passed its +allele astheegg cell from which ‘Rubens’ derived contains a recombination for this region. Thefavourable haplotype of ‘Gala’ was derived from ‘Golden Delicious’ (Figure ESM4.2). ‘Golden Delicious’ passed this same favourable haplotype to ‘Elstar’, which in turn passed it to ‘Rubens’. ‘Rubens’ thus carries at least a single dose of the favourable +allele.

Figure ESM4.2. Flow of marker alleles of for fruit firmness QTL10b across the pedigree of cultivar ‘Rubens’ for successive SSR loci. ‘Gala’ is a parent of two of the mapping families and showed to be heterozygous for the QTL, which+/ – is marked by a dark/light grey vertical bar. The font colour of the marker alleles indicates the founder from which they derive (e.g. dark and light red for alleles from ‘Cox’) .

The presence of multiple sources of +alleles raises the question of the presence of alleles of different size. To examine this (Jansen et al. 2009), marker densities must first be increased to adequately account for other segregating QTL. For this specific QTL, the responsible gene has been identified as the polygalacturonase gene MdPG1 (Costa et al. 2010, Longi et a.l 2012) which was shown to contain several polymorphisms within the coding region across a set of 77 cultivars (Longhi et al. 2013). Whether these polymorphisms led to +alleles of different size is not known yet.

References

Belfanti E, Silfverberg-Dilworth E, Tartarini S, Patocchi A, Barbieri M, et al. 2004. The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc Natl Acad Sci USA 101:886–90

Costa F, Peace CP, Stella S, Serra S, Musacchi S, Bazzani M, Sansavini S, Van de Weg WE (2010) QTL dynamics for fruit firmness and softening around an ethylene-dependent polygalacturonase gene in apple (Malusxdomestica Borkh.). J Exp Bot 61:3029-3039

Longhi S, Moretto M, Viola R, Velasco R, Costa F (2012) Comprehensive QTL mapping survey dissects the complex fruit texture physiology in apple (Malus x domestica Borkh). J Exp Bot 63:1107–1121.

Longhi S, Hamblin MT, Trainotti L, Peace CP, Velasco R, Costa F (2013) A candidate gene based approach validates Md-PG1 as the main responsible for a QTL impacting fruit texture in apple (Malus x domestica Borkh) BMC Pl Biol 13:37

Patocchi A, Vinatzer BA, Gianfranceschi L, TartariniS, ZhangHB, Sansavini S, GesslerC (1999)Construction of a 550 kb BAC contig spanning the genomic region containing the apple resistance gene Vf. Mol. Gen. Genet. 262:884-891

Vinatzer BA, Patocchi A, Tartarini S, Gianfranceschi L, Sansavini S, Gessler C (2004) Plant Breeding Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab-resistant accessions in Malus germplasm. Plant Breeding 123:321—326

.Vinatzer BA, Patocchi A, Gianfranceschi L, Tartarini S, Zhang HB, Gessler C, Sansavini S,(2001): Apple (Malus sp.) contains receptor-like genes homologous to the Cf resistance gene family of tomato with a cluster of such genes co-segregating with Vf apple scab resistance. MPMI 14:508—515

• Pedigree contains a recombination event for the QTL-region.