Mol Breeding (2015) 35:45 DOI 10.1007/s11032-015-0242-4
Identification of SNPs linked to eight apple disease resistance loci Melanie Ja¨nsch • Giovanni A. L. Broggini • Juliane Weger • Vincent G. M. Bus • Susan E. Gardiner • Heather Bassett • Andrea Patocchi
Received: 4 July 2014 / Accepted: 2 September 2014 Ó Springer Science+Business Media Dordrecht 2015
Abstract Although genetic and genomic studies have progressed to a very advanced level in apple, the application of this acquired knowledge for markerassisted breeding (MAB) remains limited mainly to pyramiding monogenetically inherited resistances against apple scab, powdery mildew and fire blight. Crucial contributing reasons are the uncertainty in map position of some genes and the lack of tightly linked markers suitable for high-throughput analysis (HTA) that reduces the costs of MAB. Single-nucleotide polymorphism (SNP) markers have the potential to resolve these major issues. Here we present the refined map positions of the apple scab resistance genes Rvi2, Rvi4 and Rvi11, and the systematic search for SNPs associated with apple scab (Rvi2, Rvi4, Rvi6,
Rvi11, Rvi15), powdery mildew (Pl2) and fire blight (FB_E and FB_MR5) resistances. With the aid of the ‘Golden Delicious’ sequence, several SNPs linked to each of the eight resistances were identified in the genomic regions around the resistance loci previously delimited by simple sequence repeat markers. The specificity of the alleles in coupling with the resistances was determined by screening eight apple genotypes, six of them being founding clones of modern apple cultivars. These SNPs can now be used to develop SNP-based HTA assays for MAB.
Melanie Ja¨nsch and Giovanni A. L. Broggini have equally contributed to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0242-4) contains supplementary material, which is available to authorized users. M. Ja¨nsch G. A. L. Broggini J. Weger A. Patocchi (&) Agroscope, Institute for Plant Production Sciences, Phytopathology, 8820, Wa¨denswil, Switzerland e-mail:
[email protected] G. A. L. Broggini J. Weger Plant Pathology Group, Swiss Federal Institute of Technology, Universita¨tstrasse 2, 8092 Zurich, Switzerland
Keywords Venturia inaequalis Podosphaera leucotricha Erwinia amylovora Malus 9 domestica Marker-Assisted Selection
While genetic and genomic studies have been developed to a very high level in many crops, the application of the findings in marker-assisted breeding V. G. M. Bus The New Zealand Institute for Plant and Food Research Limited, Private Bag 1401, Havelock North 4157, New Zealand S. E. Gardiner H. Bassett The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand
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(MAB) is still limited and has been successfully demonstrated for very few traits only (Collard and Mackill 2008). In apple breeding, programmes have focused on increasing resistances to the most important orchard diseases: scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha) and fire blight (Erwinia amylovora). When Bassil and Lewers (2009) assessed the use of MAB by Rosaceae breeders worldwide, they showed that MAB was employed in a small number of programmes only, mainly for disease resistance and fruit quality traits exhibiting Mendelian inheritance. Apple is one of the Rosaceae crops for which most of the genetic markers for resistance have been published and are available for MAB. During the course of MAB development in apple, molecular markers have evolved from restricted fragment length polymorphism (RFLP) (Roche et al. 1997) to a variety of polymerase chain reaction (PCR)based markers (e.g., Koller et al. 1994; Gianfranceschi et al. 1996; Tartarini et al. 1999; Gardiner et al. 2007; Bus et al. 2008), while achieving increased reproducibility and ease of use. Today, a large number of SSRbased genetic framework maps are available for apple (Guilford et al. 1997; Liebhard et al. 2002; Vinatzer et al. 2004; Silfverberg-Dilworth et al. 2006; Celton et al. 2009; Han et al. 2011; Ferna´ndez-Ferna´ndez et al. 2012), many of which indicate SSRs that are linked to major resistance genes for scab, powdery mildew and fire blight. Frey et al. (2004), who generated a multiplex of six SSR and two SCAR markers for scab and mildew resistance genes, demonstrated that this type of marker is well suited for a low level of multiplexing. However, identification of the SSR alleles associated with the resistance may be challenging without the appropriate controls and standardization as proposed by Patocchi et al. (2009). Collard and Mackill (2008) suggested a series of bottlenecks that may explain the generally low uptake of MAB by breeding programmes, and the following five apply to apple: (1) the uncertainty in map position of some resistance loci; (2) the lack of markers tightly linked to resistance; (3) the lack of marker polymorphism exhibited in some breeding populations; (4) many traits controlled by quantitative trait loci (QTLs) have not been validated over different backgrounds and environments, and many QTLs have a too-wide interval of confidence for use in MAB; and (5) the lack of markers suitable for high-throughput analysis
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Mol Breeding (2015) 35:45
(HTA) that are needed to increase the cost efficiency of MAB. One of the aims of the FruitBreedomics project (Laurens et al. 2010; www.fruitbreedomics.com) is to improve MAB for resistance gene pyramiding, and priority was given to those resistances most used in apple breeding: Rvi2 (Vh2), Rvi4 (Vh4), Rvi6 (Vf), Rvi11 (Vbj), Rvi15 (Vr2) for scab; Pl2 for powdery mildew; and FB_E and FB_MR5 from the genotypes ‘Evereste’ and ‘Malus xrobusta 5’ for fire blight, respectively. Research was initiated to define the position of resistances that had not been fine-mapped satisfactorily (Rvi2, Rvi4 and Rvi11), and to develop cost-effective genetic markers for high-throughput screening of all the resistances listed. For this last purpose, SNP markers seemed most suitable because of their high frequency (between 4.5 and 20 markers/ kbp) in the apple genome (Velasco et al. 2010; Micheletti et al. 2011) and coupled with the availability of technologies enabling the simultaneous genotyping from a few to thousands of markers per DNA sample. The resistance genes Rvi6, Rvi15, Pl2 and FB_MR5 have been cloned in the last decade (Belfanti et al. 2004; Schouten et al. 2014; Rikkerink et al. 2007; Broggini et al. 2014), while for FB_E, positional cloning is in progress (Parravicini et al. 2011). Hence, for these last-mentioned genes, the map position is correct and information available on the sequence of the resistance gene itself, markers developed during the positional cloning and/or the sequence of the surrounding genomic regions are available and can be used to facilitate the identification of SNPs closely associated with these genes. For the apple scab resistances Rvi2, Rvi4 and Rvi11, this information is not yet available, but since they have been mapped (Bus et al. 2005; Gygax et al. 2004) and introgressed into cultivars (‘Regia’, ‘Reka’) or advanced selections (M. Kellerhals, pers. comm.; R. Volz, pers. comm.), they are of considerable interest to breeders. In this paper, we present: (1) the verification of the reported map positions of the apple scab resistances Rvi2, Rvi4 and Rvi11; (2) the identification of SNP markers for all eight resistances mentioned above, and (3) the validation of the specificity of the alleles of the SNPs in coupling with the resistance by determining the allele composition of these SNPs in eight apple genotypes, six of them being founding clones of modern apple cultivars.
Mol Breeding (2015) 35:45
Page 3 of 21
Materials and methods
inaequalis isolates at 2 9 105 spores/ml. Incubation conditions were as previously described by Bus et al. (2005). If the resistance phenotype did not change and hence was still producing a double recombination, the progeny plant was considered a ‘‘genotype–phenotype incongruent’’ (GPI) plant (Gygax et al. 2004) and excluded from the map construction. If the progeny plant was not available for re-inoculation, it was excluded from the mapping. The second step consisted of reducing the region delimited by the markers flanking the resistance locus by adding published or new SSR markers. New SSR markers were identified by searching in the ‘Golden Delicious’ genomic interval delimited by the flanking markers. The sequences of the flanking markers were blasted against the ‘Golden Delicious’ genomic sequence (Velasco et al. 2010) using BLAST (Altschul et al. 1990), and SSR repeats were identified using the microsatellite finder webtool ‘‘YourLabData’’ (2011). SSR-specific primers were designed using the Primer 3 webtool v.0.4.0 (Rozen and Skaletsky 2000), labeled primers were purchased from Microsynth AG (Balgach, Switzerland), and the SSR marker analysis conducted according to Ja¨nsch et al. (2012). The parents of the cross and a total of 10–25 resistant and susceptible progeny plants as well as recombinants around the resistance locus (4–11 genotypes) were screened to identify and map the SSR markers that were polymorphic in the resistant parent. The third step involved the search for SNPs. The source of the sequence information required to design primer pairs to amplify and sequence amplicons of the parents of the relevant cross varied according to the state of knowledge about the resistance. Where the resistance had been cloned (Rvi6, FB_MR5, Rvi15 and Pl2) or was in the process of being cloned (FB_E), either the sequence of the gene (promoter and terminator region included) itself, or the sequence of the Bacterial Artificial Chromosome (BAC) clone on which the gene was located, or previously published markers (SCAR/CAPS) tightly linked to the gene, were used to identify SNPs (Table 1). For the resistance genes Rvi2, Rvi4 and Rvi11, the ‘Golden Delicious’ genomic sequence (Velasco et al. 2010) was used to identify SNPs in the interval between the flanking markers. Using Primer3 v.0.4.0 (Rozen and Skaletsky 2000), primer pairs producing amplicons in the range of 1,200–1,300 base pairs (bp) were designed, together with an additional internal primer
Plant material and DNA extraction To develop new markers and confirm the association with a specific resistance, DNA extracted in the course of previous studies was used where available (Table 1). For those genotypes for which DNA had to be (re-)extracted, DNeasy mini kits (Qiagen, USA) were used. For all mapping populations, resistant and susceptible genotypes (randomly selected, in total 10–25 depending on the populations) and genotypes exhibiting recombination close to the resistance locus in previous studies (4–11 genotypes) were investigated. In addition, eight genotypes, five apple cultivars and one selection reported to be founder clones of the majority of modern apple cultivars, namely ‘Golden Delicious’, ‘Delicious’, ‘Cox’s Orange Pippin’, F226829-2-2 (derived from M. floribunda 821 and founder of most Rvi6 apple cultivars), ‘Jonathan’ and ‘McIntosh’, as well as two cultivars that are also of great interest for breeding (‘Braeburn’, ‘Granny Smith’; Noiton and Shelbourne 1992, van de Weg, pers. comm.), were used to verify the degree of specificity of the allele in coupling with the resistance of the identified SNPs. For simplicity, we refer to these eight genotypes as the ‘‘eight founders’’. Procedure used to identify and map the SNPs Figure 1 shows the procedure that was used to refine the map position of certain loci and then to identify SNP markers for all the resistance loci (Rvi2, Rvi4, Rvi6, Rvi11 and Rvi15 for scab; Pl2 for powdery mildew; and FB_E and FB_MR5 for fire blight). The first step of the procedure consisted of evaluating the quality of previous mapping. If refining of the map position was required, datasets were completed to ascertain missing SSR and SCAR marker data around the resistance. A genetic map of the region was then calculated and the location of the resistance compared with the published map position. Markers causing tensions (double recombinants among flanking markers) in the maps were removed. For the Rvi2 and Rvi4 families, when the double recombination was attributed to the phenotype and the plant was still available, the plants were re-phenotyped. Three replicate trees of the selected accessions were sprayed with a ‘‘breeding mix’’ suspension of conidia from unspecified V.
45
123
123
Pathogen
Venturia inaequalis
V. inaequalis
V. inaequalis
V. inaequalis
V. inaequalis
Erwinia amylovora
E. amylovora
Podosphaera leuchotricha
R gene (syn.)
Rvi2 (Vh2)
Rvi4 (Vh4)
Rvi6 (Vf)
Rv11 (Vbj)
Rvi15 (Vr2)
FB_E
FB_MR5
Pl2
M. zumi
11
3
12
2
2
1
2
2
LG
‘Royal Gala’ 9 A689-24
‘Idared’ 9 ‘M. 9 robusta 5’; ? Varia
‘MM106’ 9 ‘Evereste’; ’Evereste’ 9 ’MM106’
‘Golden Delicious’ 9 GMAL2473
‘Golden Delicious’ 9 A722-7
Varia
‘Gala’ 9 TSR33T239
‘Golden Delicious’ 9 TSR34T15
Mapping population
473
2,133
2,703
989
148
2,071
242
158
Original size of mapping population Bus et al. (2002)
Reference for mapping
U2SCAR, N18SCAR
FEM14 or FEM17 and rp16k15
CHFbE01 and CHFbE08
ARGH17 and 77G20RP
CH05e03 and T6_SCAR
M2S and M8S
Rikkerink et al. (2007)
Fahrentrapp et al. (2013)
Parravicini et al. (2011)
Galli et al. (2010a)
Gygax et al. (2004)
Patocchi et al. (1999b)
S22_1300 Bus et al. and OPB10(2005) [2000
Z13_900 and OPL19_433
Flanking markers (original publication)
gene sequence with promoter/terminator sequences
BAC 16K15 Fahrentrapp et al. (2013)
BAC 44A20 Parravicini et al. (2011)
BAC 32A4 (Galli et al. 2010b)
None
None
None
None
Sequence used for SNP identification (reference)
None
rp16k15 (Fahrentrapp et al. 2013) EH034548 (Norelli et al. 2009)
M45TA (Parravicini et al. 2011)
9c10T7 and 21k14T7 (Galli et al. 2010b)
M2S, M7T, M8S, AM5T and M18 (Patocchi et al. 1999a) None
ARGH37, GmTNL1 (Galli et al. 2010a, b)
None
Marker sequences searched for SNP (reference)
Page 4 of 21
M. 9 robusta 5
M. ‘Evereste’
GMAL2473
M. baccata jackii
Malus floribunda 821
Russian apple R12740-7A
Russian apple R12740-7A
Donor
Table 1 Summary of the material and information used to identify the single-nucleotide polymorphisms (SNPs)
45 Mol Breeding (2015) 35:45
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Page 5 of 21
45
Fig. 1 Flowchart of the process used to refine the map position of Rvi2, Rvi4 and Rvi11, and the development/identification of single-nucleotide polymorphism (SNP) markers for all the resistance loci. WGS whole genome sequence, GPI genotype– phenotype incongruence (Gygax et al. 2004)
at about 500 bp from one end to be used for sequencing. PCR was performed using DNA of the parents and the original resistance donor. The PCRs contained 5 ll of Hot Star PCR Master Mix (Qiagen, Hombrechtikon, Switzerland), 3 ll ddH2O and 1 lM of each primer (Supplementary Table 1) and 1 ll DNA (1 ng/ll) in a total reaction volume of 10 ll. Amplifications were performed in a SensoQuest labcycler (SensoQuest, Germany) using the following
programme: 95 °C for 15 min, followed by 38 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and a final elongation cycle of 72 °C for 10 min. PCR products were electrophoresed in 1.5 % agarose gels, followed by staining with ethidium bromide, and visualized under UV light. The amplicons from primer pairs producing a single band from the two parents and from the donor of the resistance gene were sequenced according to
123
45
Page 6 of 21
Rezzonico et al. (2009). Sequences were assembled and searched for SNPs using Geneious Pro 5.5.3 and 5.5.4 (Biomatters, New Zealand). When a specific allele of a SNP was found in the resistant parent and in the donor of the resistance, but was absent in the susceptible parent, a subset of resistant and susceptible progeny plants, as well as the recombinants, was screened to map the SNP. JoinMap 4 (van Ooijen 2006) was used to calculate the genetic maps. As only a subset of the whole population (recombinants and some resistant and susceptible plants) was screened for the SNP, the genotypes of the remaining progeny plants were inferred, with the presence of the allele in coupling assumed for resistant plants and considered absent in susceptible plants. In order to estimate the degree of informativity of each SNP allele in coupling with the resistance of the identified SNPs, the amplicons containing the SNP were amplified from the eight founders and sequenced. The sequences of the founders were aligned with the sequence of the parents of the cross using Geneious Pro 6.0.6. An example is shown in supplementary Figure 1.
Results Refined map positions of Rvi2, Rvi4 and Rvi11 As the resistance genes Rvi6, Rvi15, FB_MR5 and Pl2 have been cloned, and FB_E is in the process of being positionally cloned and has been found to be located in a genetic window of 78 kb, the map position of all these genes can be considered as precisely determined. Therefore, only the map positions for Rvi2, Rvi4 and Rvi11 needed further refining. Bus et al. (2005) mapped Rvi2 on linkage group 2 (LG2) in one progeny above the SSR CH05e03 and in a second progeny below this marker. The genetic and phenotypic information of the progeny derived from the cross between ‘Golden Delicious’ 9 TSR34T15 (Bus et al. 2002) was analyzed as described in Fig. 1. Out of the 158 plants in the family, 12 exhibited recombination events in the region where the gene was mapped. Nine out of twelve recombinants had to be excluded: two were found to be outcrosses; three of the re-inoculated plants were found to be resistant, but did not show the typical Rvi2 resistance reaction of stellate
123
Mol Breeding (2015) 35:45
necrosis; for another three, repeating the phenotyping demonstrated they were not recombinants; and one was a plant with uncertain phenotype that was no longer available. Revisiting the mapping with the completed and verified dataset resulted in Rvi2 being mapped onto the central part of LG2 below CH05e03 and above OPL19SCAR (Fig. 2). The map position of Rvi4 at the top of LG2 also changed slightly following the checking of the original molecular data that reduced the number of true recombinants from 17 to eight and the addition of two SSR markers to the dataset, Hi22d06 and Hi02a07 (Silfverberg-Dilworth et al. 2006). Rvi4 is now flanked by the SSRs Hi22d06 and CH02c02a, at 2.1 and 0.4 cM, respectively, from Rvi4 (Fig. 2). When the genotypic dataset of Rvi11 was completed to ascertain the previous missing data, two GPI and one outcross plant were identified and removed from the dataset. Eight SSR markers were developed from the ‘Golden Delicious’ genome sequence in the interval CH05e03–CH03d01 in an attempt to reduce the length of the distance between flanking markers for Rvi11. Two of these new markers, SSRs CH-Vbj1 and CH-Vbj3 (Supplementary Table 1), were polymorphic in the progeny population and could be mapped. Since CH-Vbj1 co-segregates with SSR CH02c06 above CH05e03, this SSR does not derive from the targeted interval. The Rvi11 gene is now bracketed by the SSR markers CH05e03 (0.6 cM) and CH-Vbj3 (1.5 cM) (Fig. 2). The SSR alleles in coupling (and in repulsion, respectively) with the resistance were as follows: 221 bp (193 bp) for CH-Vbj1, 130 bp (166 bp) for CH-Vbj3, and null allele (199 bp) for Ch05e03. SNP identification Rvi2 Sixty-one SNPs were identified in eight out of 44 amplicons deriving from the region between CH05e03 and OPL19SCAR (Supplementary Table 2). Sequences from the eight founders indicated that only two SNPs identified in the amplicons, FBsnRvi2-7 and FBsnRvi28 (both at 1.2 cM from Rvi2, Fig. 2), are specific to the resistance allele and therefore can be expected to be useful for MAB in most crosses. The alleles associated with Rvi2 for all other SNPs were present in at least one founder (Table 2).
Mol Breeding (2015) 35:45
Page 7 of 21
45
Fig. 2 Refined genetic maps of Rvi2, Rvi4 and Rvi11. Names of the amplicons containing the single-nucleotide polymorphisms (SNPs) comprise the name of the resistance gene followed by a number, and in brackets, the number of SNPs found in the amplicon. Genetic distances are indicated in cM
Rvi4 Together with the 22 amplicons located between SSRs Hi22d06 and CH02a02a, 10 amplicons originally developed for Rvi15 were searched for SNPs, because the new map position of Rvi4 now corresponded with that of Rvi15. Fifty-three SNPs were detected in three amplicons, two originally developed for Rvi15 (ARGH37 and TNL1) and one developed for Rvi4 (FBsnRvi4-1) (Supplementary Table 2). All the SNPs identified co-segregate with Rvi4 (Fig. 2), and twelve are fully informative on the basis of the eight founders sequenced (Table 2). Rvi6 Out of the five previously developed markers for Rvi6, only amplicons from M8S and M18 revealed three and
25 SNPs, respectively (Supplementary Table 2). M18 and M8S flank the Rvi6 resistance and were mapped by Patocchi et al. (1999b) at three and seven recombination events, respectively, in 2,071 plants analyzed. All SNPs are located within a maximum distance of 0.9 cM from the resistance gene. None of the founders except F2-26829-2-2, which carries Rvi6, carry the allele from M8S in coupling with Rvi6 (Table 2), while this applies for 18 out of 25 SNPs from M18. Rvi11 Of fifty-five pairs of primers designed for the Rvi11 region, approximately half of them (29) amplified fragments from both parents and progenitor M. baccata jackii and were subsequently sequenced. In two amplicons flanking Rvi11, 17 SNPs were identified in A722-7 (the parent carrying Rvi11): eight in
123
1.6 (upstream)
1.6 (upstream)
1.6 (upstream)
0.9 (upstream)
0.9 (upstream)
FBsnRvi2-1
FBsnRvi2-2
FBsnRvi2-3
FBsnRvi2-4
FBsnRvi2-5
Rvi2
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Resistance
123 R Y R M Y
204 220 239 417 496
R R R R Y Y Y
461 489 531 590 693 732 805
R
Y
287
K
Y
282
515
W
220
497
M
215
R
K
192
468
R
187
R
Y
72
366
K
K
31
58
M
Y
150
341
Y Y
34
SNP type
53
Position of the SNP in the alignment of the sequences of the founders
A
T
A
A
C
T
C
A
A
A
A
T
T
A
C
T
A
T
T
T
C
T
A
G
T
G
C
C
C
Allele in coupling
Br, Co, De, GS
Br, Co, De, GS
Br, De, GS
Br, De, GS
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De, Jo, Mc
Br, De, Jo, Mc
Br, De
Br, De
Br, De
Br, De
Br, De
F2, Jo
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2
Co, De, F2
Br
Co, De, F2
Br, Co, De, F2
Co, De, F2
Co, De, F2
Co, De, F2
Founder(s) with allele in coupling*
KM104993–KM105002
KM104983–KM104992
KM105126–KM105135
KM104973–KM104982
KM104963–KM104972
Accession no alignment sequence of the founders
Page 8 of 21
KM104994
KM104984
KM105127
KM104974
KM104964
Accession no. sequence R parent
Table 2 Detailled information on the SNPs identified in this work
45 Mol Breeding (2015) 35:45
Resistance
Distance from the R-locus (cM or kbp) and position relative to R-locus
0.6 (downstream)
0.6 (downstream)
1.2 (downstream)
1.2 (downstream)
Amplicon name
FBsnRvi2-6.1
FBsnRvi2-6.2
FBsnRvi2-7
FBsnRvi2-8
Table 2 continued
R
488
9
R
Y
Y
460
661
Y
439
Y
R
436
536
R
421
M
R
404
Y
M
403
390
R
367
292
Y
365
W
R
332
242
M
232
R
M
70
K
218
Y
122
217
M
121
Y
M
95
169
K
87
M
W
70
133
R
W
555 43
SNP type
Position of the SNP in the alignment of the sequences of the founders
A
C
T
A
C
A
G
A
C
C
A
A
A
A
A
C
G
C
A
T
T
C
T
C
A
T
T
G
A
Allele in coupling
GD, GS, Jo, Mc
Co, De, F2, GS
Co
Co
Co
None
Co
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, De
Br, Co, De, GS
Founder(s) with allele in coupling*
KM105023–KM105032
KM105013–KM105022
KM105003–KM105012
KM105136–KM105145
Accession no alignment sequence of the founders
Page 9 of 21
KM105023
KM105014
KM105004
KM105137
Accession no. sequence R parent
Mol Breeding (2015) 35:45 45
123
0 cM (cosegregates)
0 cM (cosegregates)
0 cM (cosegregates)
FBsnRvi4-1
ARGH37
TNL1
Rvi4
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Resistance
Table 2 continued
123 KM105054
KM105034
KM105043
Accession no. sequence R parent
R Y Y W M M Y S K R S Y S W K R Y Y Y Y Y K Y Y R K
49 58 74 92 93 96 99 101 105 120 121 123 124 127 131 148 157 168 171 181 189 190 202 229 237
Y
29
57
K K
135 146
R
304
G
A
T
C
T
T
C
T
C
T
G
G
T
None
None
None
Br, Co, De, F2, GS, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Jo
Br, Co, De, F2, GS, Jo, Mc
None
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
None
Br, Co, De, F2, GS, Jo, Mc
None
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, De, F2
None
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De
None
Br, De
Founder(s) with allele in coupling*
KM105053–KM105062
KM105033–KM105042
KM105043–KM105052
Accession no alignment sequence of the founders
Page 10 of 21
C
C
G
A
G
G
T
A
C
T
T
T
G
T
T
G
A
G
G
R R
77
Allele in coupling
SNP type
243
Position of the SNP in the alignment of the sequences of the founders
45 Mol Breeding (2015) 35:45
B 1.1 cM (7 rec/2,071) (downstream)
3 rec/2,071 (upstream)
M8S
M18
Rvi6
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Resistance
Table 2 continued
Y M Y M Y Y Y W Y R K R Y S R S R R M
339 396 399 440 451 453 456 514 521 539 566 569 575 621 623 635 657 663 671
T
Y R
32 100
A
C
Y
18
G
R
G
A
T
A
G
G
G
G
G
T
G
T
A
T
T
T
T
T
A
C
C
C
C
C
T
A
C
F2
F2
F2
F2
F2
F2
F2
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo, Mc
None
Jo, Mc
Br, Co, De, F2, GS
None
Br, Co, De, F2, GS, Jo, Mc
Br, Co, De, F2, GS, Jo
Br, Co, De, F2, GS, Mc
Br, Co, De, F2, GS, Mc
Br, Co, De, F2, GS, Mc
Br, Co, De, F2, GS, Mc
None
None
None
Br, Co, De, F2, GS, Jo, Mc
GD, Jo
Br, Co, De, F2, GS, Jo, Mc
Jo
Br, Co, De, F2, GS, Jo, Mc
Allele in Founder(s) with coupling allele in coupling*
2
R
Y
330
193
Y
286
R
W
263
156
R
260
Y
M
248
124
SNP type
Position of the SNP in the alignment of the sequences of the founders
KM105063–KM105073
KM105074–KM105082
Accession no alignment sequence of the founders
Page 11 of 21
KM105064
KM105074
Accession no. sequence R parent
Mol Breeding (2015) 35:45 45
123
2.8 cM (upstream)
0.8 cM (downstream)
FBsnRvi11-1
FBsnRvi11-2
Rvi11
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Resistance
Table 2 continued
123 KM105092
M M K W M M
357 381 434 461 482 501
R R
60 357
Y
W
318
406
R
285
R
R
264
S
Y
262
395
R
239
341
R
236
R
M
228
293
R
225
Y
K
219
Y
R
209
292
R
205
268
Y
181
Y
W
160
111
M
156
Y
Y
150
60
SNP type
Position of the SNP in the alignment of the sequences of the founders
G
A
C
C
G
G
T
T
C
T
A
A
T
G
A
C
A
G
G
C
G
G
C
G
T
G
A
T
A
A
T
Allele in coupling
None
None
None
None
None
None
None
None
None
None
F2
F2
F2
F2
F2
F2, Br, De, GD, Jo, Mc
F2
F2, Co, GS
F2
F2
F2, Br, De, GD, Jo, Mc
F2
F2
F2, Co, GS
F2, Co, GS
F2, Br, De, GD, Jo, Mc
F2, Co, GS
F2
F2
F2
F2
Founder(s) with allele in coupling*
KM105092–KM105100
KM105083–KM105091
Accession no alignment sequence of the founders
Page 12 of 21
KM105083
Accession no. sequence R parent
45 Mol Breeding (2015) 35:45
KM104871 KM104881
0.2 cM (downstream)
[0.11; \0.19 cM (upstream)
[0.29; \0.31 cM (downstream)
0 cM (cosegregates)
21k14T7
FBsnFBE-1
FBsnFBE-2
FBsnFBE-3
FB-E
KM105120
70 kbp (upstream)
FBsnRvi15-1
KM104891
KM105120
KM105101
0.7 cM (7 recs/989) (upstream)
9C10T7
Accession no. sequence R parent
Rvi15
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Resistance
Table 2 continued
S
188
S S R R S M W S R Y S
115 130 132 167 168 193 195 196 197 205
Y
551 67
Y
495
R Y
422 192
Y
230
R
M
75 153
S
S
296 49
M
Y
874
Y
R
775
264
Y
733
224
Y
621
Y
W
618
Y
R
494
111
Y
492
117
SNP type
Position of the SNP in the alignment of the sequences of the founders
G
T
A
C
A
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
GS, Mc
None
None
None
Br, De, F2
None
Br, Co
None
None
None
None
None
None
None
None
None
None
None
Br, Co, De, GS, Mc
None
None
None
Founder(s) with allele in coupling*
KM104891–KM104900
KM104881–KM104890
KM104871–KM104880
KM105110–KM105119
KM105120–KM105125
KM105101–KM105109
Accession no alignment sequence of the founders
Page 13 of 21
A
G
A
G
C
C
C
C
T
A
C
G
G
A
C
G
C
T
T
T
C
G
T
C
T
A
T
Allele in coupling
Mol Breeding (2015) 35:45 45
123
123
FB-MR5
Resistance
0.17 cM (downstream)
0.19 cM (upstream)
FBsnFBMr5-1
0.31 cM (downsream)
M45TA
rp16k15
0 cM (cosegregates)
FBsnFBE-5
0.22 cM (downstream)
0 cM (cosegregates)
FBsnFBE-4
NZsnEH034548
Distance from the R-locus (cM or kbp) and position relative to R-locus
Amplicon name
Table 2 continued
W Y R
389 585 713
R Y K R Y Y Y R R R M S R
51 65 95 111 127 175 181 209 215 776 805 924
W
113 46
M
R
249 106
R
240
Y
191 K
Y
35
Y
57 144
W R
86 190
Y
M
388
Y
Y
286
49
W
207
195
SNP type
Position of the SNP in the alignment of the sequences of the founders
G
C
A
A
A
A
T
C
T
A
G
T
A
T
A
A
A
T
T
T
T
A
A
C
T
A
T
A
C
T
A
Allele in coupling
Br
Br
Br
None
None
None
None
None
None
GS
None
None
None
Mc
None
None
None
None
None
None
None
Br, De, F2, GD
None
None
None
None
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Br, De, F2, GD, GS, Mc
Founder(s) with allele in coupling*
KM104921–KM104931
KM105146–KM105157
KM104932–KM104942
KM104911–KM104920
KM105158–KM105167
KM104901–KM104910
Accession no alignment sequence of the founders
Page 14 of 21
KM104921
KM105149
KM104932
KM104911
KM105158
KM104901
Accession no. sequence R parent
45 Mol Breeding (2015) 35:45
Amplicon name
FBsnPl2-1
Resistance
Pl2
Table 2 continued
0 cM (cosegregates)
Distance from the R-locus (cM or kbp) and position relative to R-locus
KM104943
Accession no. sequence R parent
K R R K R R K M Y M R Y Y R R Y R Y W K Y R W K S Y M
93 99 101 102 104 106 114 127 142 159 245 425 531 699 785 808 913 918 944 1,060 1,095 1,113 1,136 1,218 1,445 1,458
R
1,025 53
K
1,004 Y
R
48
R
992
SNP type
929
Position of the SNP in the alignment of the sequences of the founders
A
C
G
T
T
GD
GD
None
GD
GD
None
GD
None
None
De
None
None
None
None
GD
None
None
None
GD
None
GD
GD
GD
GD
None
None
GD
None
None
None
None
Br
Founder(s) with allele in coupling*
KM104943–KM104952
Accession no alignment sequence of the founders
Page 15 of 21
A
T
T
T
T
A
C
G
G
C
C
A
C
C
A
G
G
G
T
A
A
T
C
G
T
G
G
Allele in coupling
Mol Breeding (2015) 35:45 45
123
Mol Breeding (2015) 35:45
FBsnRvi11-1 and nine in FBsnRvi11-2 (Supplementary Table 2). SNPs from FBsnRvi11-1 were mapped at 2.8 cM above Rvi11, while those from FBsnRvi11-2 were located at 0.8 cM distance below the resistance (Fig. 2). The mapping of the SNPs demonstrated that two of the 10 SNPs of FBsnRvi11-1 (Y7 and R8 at position 343 and 375 bp, respectively) are not derived from the Rvi11 region, indicating that the primers for amplicon FBsnRvi11-1 amplify DNA fragments from two different loci. All eight SNPs from Rvi11 in FBsnRvi11-1, and eight out of nine from FBsnRvi112, are fully informative based on the sequence from the eight founders (Table 2). Rvi15 When eight amplicons were analyzed for the occurrence of SNPs, nine SNPs were detected in three of them: 9c10T7 and 21k14T7 developed both by Galli et al. (2010a, b), and FBsnRvi15-1 (Table 2; Supplementary Table 2). Markers 9c10T7 and 21k14T7 bracket Rvi15 with seven and two recombination events, respectively, between each marker and Rvi15 out of 989 individuals of the mapping population (Galli et al. 2010a, b). FBsnRvi15-1 is located on Rvi15 BAC clone 32A4 (Galli et al. 2010c) at approximately 70 kbp from the gene and is on the same side as, but closer to Rvi15 than 9c10T7. No amplification was obtained for fragment FBsnRvi15-1 from ‘Braeburn’, ‘Delicious’ and F226829-2-2. All eight SNPs of 9c10T7 and FBsnRvi151 were 100 % informative in the founders from which an amplicon could be amplified, while the allele of 21k14T7 in coupling with the resistance was detected in ‘Cox’s Orange Pippin’ and ‘Braeburn’ (Table 2). ** Partial sequence of the amplicon FBsnRvi2-6
KM104953–KM104962
None G
None T
R 1,548
211 0 cM (cosegregates) FBsnPl2-2
KM104953
Y
Founder(s) with allele in coupling* Allele in coupling SNP type Position of the SNP in the alignment of the sequences of the founders Accession no. sequence R parent Distance from the R-locus (cM or kbp) and position relative to R-locus Amplicon name Resistance
Table 2 continued
123
* Genotypes not amplifying the amplicon are considered as not carrying the allele in coupling. Br ‘Braeburn’, Co ‘Cox’s Orange Pippin’, De ‘Delicious’, F2 F2-26829-2-2, GD ‘Golden Delicious’, GS ‘Granny Smith’, Jo ‘Jonathan’, Mc ‘McIntosh’
Page 16 of 21 Accession no alignment sequence of the founders
45
FB_E On searching for SNPs in nine amplicons developed from the 78-kbp sequence of the BAC clone 44A20 (Parravicini et al. 2011) spanning the FB_E locus and delimited by the markers ChFbE01 and ChFbE08, as well as the amplicon from marker M45TA (Parravicini et al. 2011), four amplicons were rejected because of multiple allele amplifications. In the remaining six amplicons, 29 SNPs were detected (Supplementary Table 2). The maximum distance to the FB_E locus was that from M45TA (0.3 cM), while all other markers were located between the FB_E flanking
Mol Breeding (2015) 35:45
markers ChFbE01 and ChFbE08, which are located at 0.11 and 0.07 cM from the FB_E locus, respectively. The alignment of the sequences obtained from the eight founders showed that amplicon FBsnFBE-2 of the genotype F2-26829-2-2 contained an insert of 429 bp, which was not present in the other genotypes. Eleven SNPs were informative in all the founders (Table 2).
Page 17 of 21
45
sequences of the parents of the cross. This indicates preferential amplification of the resistant allele in plants heterozygous for Pl2. All SNPs co-segregated with the resistance, with 16 of them in FBsnPl2-1 and one in FBsnPl2-2 being fully informative in the eight founders sequenced (Table 2).
Discussion FB_MR5 Twenty-two SNPs were identified in three out of fourteen amplicons analyzed for the presence of SNPs: FBsnFBMr5-1, rp16k15 (Fahrentrapp et al. 2013) and NZsnEH034548 (Norelli et al. 2009; Gardiner et al. 2012; Table 2, Supplementary Table 2). Marker rp16k15, developed on one BAC 16k15 insert-end, and NZsnEH034548 were mapped by Fahrentrapp et al. (2013) at 0.17 cM resp. 0.22 cM downstream from FB_MR5. FBsnFBMr5-1 was developed about 29 kbp from the other BAC insert-end (t15k16) upstream from FB_MR5. Therefore, markers FBsnFBMr5-1 and rp16k15 flank FB_MR5. For FBsnFBMr5-1, no SNPs could be identified in the sequences of either the resistant or the susceptible parent. However, different point mutations (SNPs) were observed when the sequences of the parents were compared. In the progeny plants, only one or the other allele was observed, but no SNPs, indicating a possible preferential amplification of the allele on the resistant chromosome in heterozygous resistant plants. A very different sequence was obtained from the founder F226829-2-2 and therefore excluded from the alignment with the other founders, while for ‘Braeburn,’ only a partial sequence could be assembled. Sixteen of the 22 SNPs were fully informative in seven founders (Table 2). Pl2 SNP identification for Pl2 was based on the sequence of the cloned gene. Twenty-eight pairs of primers were evaluated, and SNPs were detected in two amplicons, FBsnPl2-1 and FBsnPl2-2 (Supplementary Table 2). Twenty-nine SNPs were identified in FBsnPl2-1 deriving from the promoter sequence, while one SNP was identified in FBsnPl2-2 deriving from the open reading frame region. As for FB_MR5, SNPs in both amplicons could be detected only by comparing the
We have first refined the map position of three apple scab resistances (Rvi2, Rvi4 and Rvi11) and then identified closely linked SNP markers for eight major resistance loci that are widely used in apple breeding programmes around the world. Depending on the information available and the complexity of the locus, we used different strategies for the identification of SNPs, according to the state of knowledge about the specific locus. For the resistance genes that have been cloned or are in the process of being cloned, essential information, such as BAC clone sequences with tightly linked SCAR or CAPS markers produced during their positional cloning, was available. The aim was to identify markers that have a 100 % success rate when used in MAB (Bus et al. 2009). When we refined the map positions of Rvi2, Rvi4 and Rvi11, we found that the map positions of Rvi2 and Rvi4 changed slightly compared with those of the first report (Bus et al. 2005), demonstrating that repeated phenotyping of individuals whose phenotype is uncertain, and verification of ‘‘suspect’’ genotyping data such as double recombination events, improves the precision of mapping. Rvi2 and Rvi11 now map to the same interval between Ch05e03 and Ch03d01 (Fig. 2). Both Rvi2 and Rvi11 induce a stellate resistance reaction after inoculation with scab: Rvi2 a stellate necrosis (Bus et al. 2005) and Rvi11 both stellate necrosis and chlorosis, sometimes with limited sporulation (Gygax et al. 2004). As both resistance reactions are similar and the two resistance genes map to the same LG2 region, they may belong to the same resistance gene cluster, or even be allelic. Fine mapping in enlarged populations of precisely phenotyped seedlings and subsequent cloning of both genes will enable clarification of this question. The same issue applies to the scab resistance genes Rvi4 and Rvi15. Our redefinition of the map position of Rvi4 moved this locus from below to above the SSR CH02c02a (Fig. 2), where Rvi15 was previously
123
45
Page 18 of 21
mapped (Galli et al. 2010a). Moreover, it was possible to identify SNPs associated with Rvi4 in two markers (TNL1 and ARGH37) developed for Rvi15, indicating that the loci are closely associated (Fig. 2, Table 2). Both Rvi4 and Rvi15 condition a hypersensitive response upon inoculation with scab and pin-point pits can be observed in hosts carrying either gene from 6 days post-inoculation (Galli et al. 2010c). Recently, transformation of ‘Gala’ independently with the three Rvi15 candidate genes belonging to the Drosophila Toll and mammalian interleukin-1 receptor nucleotide-binding site leucine-rich repeat family of resistance genes (TIR-NBS-LRRs) (Galli et al. 2010b) by Schouten et al. (2014) proved that Vr2-C is the gene of the Rvi15 cluster that induces apple scab resistance. As both genes condition a hypersensitive response, we suggest that Rvi4 may be a TIR-NBS-LRR encoding gene, too. Our use of the ‘Golden Delicious’ reference sequence for identifying SNPs linked to the Rvi2, Rvi4 and Rvi11 loci proved to be successful, as several were detected for each gene. However, issues regarding the presence of paralogs were encountered during their identification and it was difficult to identify primers that amplified only the two alleles of a specific region without co-amplifying both homolog and paralog sequences (Supplementary Table 2). This work was further hampered by the genetic distance between the wild apple, donors of the resistances, and the domesticated apple. When the ‘Golden Delicious’ reference sequence was used as a template for the design of the primers, we often observed failure of the amplification of the allele derived from the resistant parent or from the resistance donor. For example, in the case of Rvi11, only about half (29) of the 55 primer pairs screened amplified fragments from both parents of the cross, as well as the Rvi11 donor M. baccata jackii. The subsequent sequencing was then complicated by the co-amplification of homologs and paralogs, which resulted in SNPs being finally identified in only two amplicons out of 55 primer pairs designed (Supplementary Table 2). Of note is also the finding that in one of these two amplicons, FBsnRvi11-1, two SNPs were identified that derived from a different locus. A similar but opposite effect was found when sequences derived from the chromosome carrying the resistance were used as templates for primer design (i.e., BAC clone sequences or sequence of the
123
Mol Breeding (2015) 35:45
resistance gene). In these cases, the primers often failed to amplify the marker allele from the chromosome not carrying the resistance. Failure of amplification of the ‘‘susceptible’’ allele could be total (no amplicon produced from cultivars not carrying the resistance gene), or when this allele was in competition with the resistance allele (genotypes heterozygous for the resistance gene), only the ‘‘resistant’’ allele was amplified. This was the case for both the amplicons containing SNPs for Pl2 and for the FBsnFBMr5-1 amplicon of FB_MR5. In the latter case, the amplification of a sequence very different from all other amplicons (F2-26829-2-2) was also observed (data not shown). Although the identification of functional markers for the Rvi6 resistance, the currently most-used scab resistance in apple breeding, would have been very useful, we did not attempt to identify SNPs within Rvi6 itself. ‘Florina,’ the accession used for cloning, contains several paralogs of Rvi6 (Broggini et al. 2009), and from the alignment of Rvi6 and its paralogs, it was concluded that it would be very difficult to amplify both Rvi6 and its susceptible homologs without amplifying its numerous paralogs, or without obtaining only amplification from the resistant allele (presence/absence polymorphism). Therefore, SNPs were searched for in previously mapped markers only. To assess the specificity of the SNP allele in coupling with the resistance, the presence of this allele was evaluated in eight founders. The results provided a first indication of the chances of a SNP being informative in any given cross. SNPs not exhibiting the resistance allele in the founders lacking the relevant resistance should be preferred to SNPs that are amplified in such genotypes. However, the latter SNPs should not be discarded a priori, as they may still be 100 % informative in specific crosses, while in the worst case, such as crosses between two cultivars heterozygous for the SNP, they will be informative in half of the progeny plants. This point demonstrates the requirement for parental testing before crosses are made, or at least before initiating MAB on the whole progeny. The alignment of the sequences of the eight founders with the parents of mapping populations is important for the development of SNP-based assays. This information can be employed to avoid designing primers in regions of the sequences of the amplicons containing SNPs present in the founders and absent in
Mol Breeding (2015) 35:45
the resistant parent. This will reduce the risk of developing assays that will fail in amplifying the ‘‘susceptible’’ allele of the SNP leading to a ‘‘null’’ allele. In summary, the research presented here provides the basis for the increased use of SNP markers in MAB for resistance loci in apple. Few such markers have been publicly available to date, an exception being the medium-throughput, high-resolution melting point PCR marker previously developed from the sequence of a candidate gene for FB_MR5 and designated NZsnEB140229 (Gardiner et al. 2012). The sequences containing our newly identified SNPs linked closely to eight resistance loci used by apple breeders internationally are now available for the development of SNP-based assays, which will enable HTA for foreground selection of seedlings with multiple resistance gene pyramids in large breeding populations of thousands of seedlings. Once these assays are made widely available by commercial genotyping companies, MAB will be available even to breeders who do not have access to a DNA laboratory. When combined with the use of SNP-based genome-wide selection that has already been developed for, and applied to apple, further major efficiencies will be gained (Kumar et al. 2012), especially when combined with other strategies for reducing the length and number of breeding cycles (Le Roux et al. 2012; van Nocker and Gardiner 2014). Acknowledgments The authors thank Dr. Elena Zini for the support in the development of the Rvi11 SSR markers. This work has been (partly) funded under the EU Seventh Framework Programme by the FruitBreedomics Project No. 265582: Integrated Approach for Increasing Breeding Efficiency in Fruit Tree Crop. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The New Zealand component of the research was funded by the Plant and Food Research Pipfruit Core programme 1433.
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C, Sansavini S (2004) The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proc Natl Acad Sci USA 101:886–890 Broggini GAL, Galli P, Parravicini G, Gianfranceschi L, Gessler C, Patocchi A (2009) HcrVf paralogs are present on linkage groups 1 and 6 of Malus. Genome 52:129–138 Broggini GAL, Wo¨hner T, Fahrentrapp J, Kost TD, Flachowsky H, Peil A, Hanke V, Richter K, Patocchi A, Gessler C (2014) Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC-NBS-LRR resistance gene of Malus 9 robusta 5. Plant Biotech J 12:728–733 Bus VGM, White A, Gardiner S, Weskett R, Ranatunga C, Samy A, Cook M, Rikkerink E (2002) An update on apple scab resistance breeding in New Zealand. Acta Hort 595:43–47 Bus VGM, Rikkerink EHA, van de Weg WE, Rusholme RL, Gardiner SE, Basset HCM, Kodde LP, Parisi L, Laurens FND, Meulenbroek EJ, Plummer KM (2005) The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple. Mol Breed 15:103–116 Bus VGM, Chagne´ D, Bassett H, Bowatte D, Calenge F, Celton J-M, Durel C, Malone M, Patocchi A, Ranatunga A, Rikkerink E, Tustin D, Zhou J, Gardiner S (2008) Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.). Tree Genet Genome 4:233–236 Bus VGM, Esmenjaud D, Buck E, Laurens F (2009) Application of genetic markers in rosaceous crop. In: Folta KM, Gardiner SE (eds), Genetics and genomics of rosaceae, plant genetics and genomics: crops and models, 6:563–599 Celton J-M, Tustin DS, Chagne´ D, Gardiner SE (2009) Construction of a dense genetic linkage map for apple rootstocks using SSRs developed from Malus ESTs and Pyrus genomic sequences. Tree Genet Genome 5:93–107 Collard BC, Mackill DJ (2008) Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos Trans R Soc B Biol Sci 363:557–572 Fahrentrapp J, Broggini GAL, Kellerhals M, Peil A, Richter K, Zini E, Gessler C (2013) A candidate gene for fire blight resistance in Malus 9 robusta 5 is coding for a CC–NBS– LRR. Tree Genet Genome 9:237–251 Ferna´ndez-Ferna´ndez F, Antanaviciute L, van Dyk MM, Tobutt KR, Evans KM, Rees DJG, Dunwell JM, Sargent DJ (2012) A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence. Tree Genet Genome 8:991–1002 Frey JE, Frey B, Sauer C, Kellerhals M (2004) Efficient low-cost DNA extraction and multiplex fluorescent PCR method for high-throughput marker-assisted selection (MAS) in apple breeding. Plant Breed 123:554–557 Galli P, Broggini GAL, Gessler C, Patocchi A (2010a) Phenotypic characterization of the Rvi15 (Vr2) apple scab resistance. J Plant Pathol 92:219–226 Galli P, Broggini GAL, Kellerhals M, Gessler C, Patocchi A (2010b) High-resolution genetic map of the Rvi15 (Vr2) apple scab resistance locus. Mol Breed 26:561–572 Galli P, Patocchi A, Broggini GAL, Gessler C (2010c) The Rvi15 (Vr2) apple scab resistance locus contains three TIR–NBS–LRR genes. Mol Plant Microb Interact 23:608–617
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