DOI 10.1007/s00122-007-0558-5
O R I G I N A L P A P E R
Inheritance of Phytophthora root rot resistance in red raspberry
determined by generation means and molecular linkage analysis
Jeremy A. Pattison · Suren K. Samuelian ·Courtney A. Weber
Received: 22 November 2006 / Accepted: 11 April 2007 / Published online: 22 May 2007 © Springer-Verlag 2007
Abstract Classical and molecular methodologies were used to determine the inheritance of Phytophthora root rot (PRR) resistance in red raspberry. The varieties ‘Latham’ and ‘Titan,’ resistant and susceptible, respectively, were
used to create F1, F2, B1, B2, and S1 populations for
analy-sis. Generational means analysis was used to calculate the components of genetic variation and estimates of narrow and broad sense heritability for the plant disease index and the incidence of petiole lesions. The plant disease index showed additive genetic variation with additional signiW-cant interactions, but the incidence of petiole lesions was non-additive. A dominant, two-gene model was shown to
be the best Wt for the observed segregation ratios when
clas-siWcation for resistance was based on a combination of all
criteria measured. Molecular linkage maps were generated
from the segregating B2 population. Linkage maps of both
parents were constructed from ampliWed fragment length
polymorphism (AFLP), Random ampliWed polymorphic DNA (RAPD), and uncharacterized resistant gene analog polymorphism (RGAP) markers with seven linkage groups each totaling 440 and 370 cM of genetic distance,
respec-tively. An analysis of the distributional extremes of the B2
population identiWed several RAPD markers clustered on
two linkage groups associated with PRR resistance. QTL
analysis identiWed two similar genomic regions on each
map that explained signiWcant percentages of phenotypic variation observed for the disease assessment criteria. Genetic mapping supports the dominant two-gene model developed from generational means analysis. The results reconcile conXicting reports on inheritance of PRR resis-tance, provide a basis for further investigation of durable resistance to Phytophthora caused diseases, and indicates that recurrent selection is the appropriate approach for the development of new resistant cultivars.
Introduction
Root rot in red raspberries, caused by Phytophthora
fraga-riae var. rubi Wilcox and Duncan, reduces crop
productiv-ity and is a major production constraint in all
raspberry-growing regions of the world (Wilcox et al. 1993; Wilcox
and Latorre 2002). An integrative management approach
utilizing resistant cultivars, registered fungicides, and avoidance or amelioration of wet soils to reduce the eVects of the disease is recommended for control (Wilcox et al.
1999; Heiberg 1995; Maloney et al. 1993). Typical
symp-toms include severe root rot, leaf chlorosis, stem and
peti-ole lesions and necrosis, and wilting (Wilcox 1989).
Resistance to Phytophthora root rot (PRR) is found par-ticularly in cultivars derived from Rubus idaeus strigosus Michx., the native North American red raspberry and less so in those derived from R. idaeus vulgatus Arrhen., the
native European red raspberry (Bristow et al. 1988;
Daub-eny and Anderson 1993; Knight 1991; Levesque and
Daub-eny 1999; Pattison and Weber 2005). Nestby and Heiberg
(1995) found that several cultivars, including ‘Asker’
which is of European origin, could confer resistance to
Communicated by H. Nybom.
J. A. Pattison
Virginia Polytechnic Institute and State University,
Southern Piedmont Agricultural Research and Extension Center, 2375 Darvills Road, Blackstone, VA 23824, USA
S. K. Samuelian · C. A. Weber (&) Department of Horticultural Sciences,
New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA e-mail: caw34@nysaes.cornell.edu
some progeny populations. Other early products of North American breeding programs such as ‘Latham,’ ‘New-burgh,’ ‘Durham,’ and ‘Chief’ are also highly resistant to
PRR (Barritt et al. 1981) and have been used as parents to
produce newer resistant varieties (Nestby and Heiberg
1995). Studies to date have not determined if the same
genes control the North American and the European source for resistance. More productive cultivars with improved fruit quality have dominated the recent market, but many
are susceptible to PRR (Pattison and Weber 2005).
Inheritance in a parent-oVspring regression study of Weld screened progenies showed that resistance to PRR is herita-ble with consideraherita-ble additive genetic variation and that moderate progress toward resistance could be achieved
through recurrent selection (Barritt et al. 1979). However,
Nestby and Heiberg (1995) concluded that resistance is
more inXuenced by non-additive genetic variation (i.e., dominance and epistasis) and/or environmental eVects and calculated low estimates of narrow sense heritability and thus a poor prospect for recurrent genetic improvement. The experimental conditions were quite diVerent between the two studies and direct comparisons of data are not appropriate as heritability is population speciWc. Neverthe-less, these studies reveal that resistance to PRR is heritable and may be conditioned by major genetic factors. However, there is no consensus for the mode of inheritance for resis-tance to PRR or for the most appropriate approach to breed-ing cultivars with improved resistance.
Molecular linkage maps have been constructed for many
agricultural crop species (Collins et al. 1998; Graham et al.
2004; Kanazin et al. 1996; Liebhard et al. 2003; Sargent
et al. 2004) with a diversity of marker types providing
researchers Xexibility in choosing the most appropriate sys-tem for the crop species of interest based on available sequence information and speciWc study objectives. Ran-dom ampliWed polymorphic DNA (RAPD) and ampliWed fragment length polymorphisms (AFLPs) are frequently used in generating molecular linkage maps because they require no host genome sequence information, can produce moderate to high levels of polymorphisms, and are
techni-cally simple and very cost eVective for the amount of
infor-mation gained relative to the time and money invested. Other marker systems are available but require sequence information speciWc to the target crop’s genome. There is, however, much conservation among certain classes of genes with disease resistant genes in particular showing shared sequence motifs across a wide range of host plants
and pathogens. Leister et al. (1996) designed degenerate
primers based on the conserved LRR (leucine rich repeat) regions of RPS2 from Arabidopsis thaliana L. and the N gene of tobacco (Nicotiana tabacum L). AmpliWcation products obtained from potato (Solanum tuberosum L.) were shown to be homologous to known resistance genes
and linked to the nematode resistance gene Grol and the
late blight resistance locus R7 (Leister et al. 1996). Chen
et al. (1998) used degenerate and speciWc primers designed
from conserved motifs of R genes targeted to bacteria, fungi, and viruses, and found that many ampliWcation prod-ucts could be resolved using polyacrylamide gel electro-phoresis. They proposed that this method could be useful for generating markers linked to disease resistance loci. This conserved marker approach has been used to isolate and characterize resistant gene analog polymorphisms (RGAPs) linked to disease resistance traits in cassava
(Manihot esculenta Crantz) (Lopez et al. 2003), citrus
(Cit-rus grandis Osbeck. x Ponci(Cit-rus trifoliata Raf. hybrid)
(Deng et al. 2000), grape (Vitis vinifera L.) (Di Gaspero
and Cipriani 2002, 2003), lettuce (Lactuca spp.) (Shen
et al. 1998), maize (Zea mays L.) (Collins et al. 1998), and
soybean (Glycine max Merr.) (Kanazin et al. 1996).
The objectives of this study were to determine the mode of inheritance of resistance to PRR, develop a molecular genetic linkage map to identify markers associated with the resistant phenotype, and test the usefulness of degenerate primers designed from conserved disease resistant gene motifs for producing markers linked to PRR resistance.
Materials and methods
Plant material
A single PRR resistant F1 individual, NY00-34
(‘Titan’ £ ’Latham’) (Maloney 2001), was used as the
female parent to generate B1, B2, and F2 populations. These
populations, an additional F1 population and the S2
popula-tion were screened for PRR resistance (Pattison et al. 2004)
as well as replicated clones of ‘Latham,’ ‘Titan’ and
NY00-34 (Table1) using the method described by Pattison et al.
(2004). Tissue culture plugs were used for screening clonal
material of ‘Latham’ and ‘Titan’ and root cuttings for NY00-34.
Disease screening and assessment
The root systems of seedling plants with two to four true leaves were washed free of planting media and soaked in a dilute solution of antibacterial soap for 15 min and then transplanted into a hydroponic basin as described by
Patti-son et al. (2004). The seedlings’ crowns were placed into a
rock wool cube and placed into planting holes evenly distributed across the lid of the hydroponic basin. The roots were submerged and grown with aeration in half strength Peter’s Professional Hydro-Sol 5-11-26 nutrient solution (W.R. Grace & Co., Fogelsville, PA), supplemented with
nutrient solution to the basin and the pH was adjusted to 6.5. The basins were kept in a growth chamber with 14-h days at 20°C. Tissue culture plug plants of the resistant (‘Latham’) and susceptible (‘Titan’) parents were included as controls in all hydroponic basins.
Inoculation of the basins was done with two pathogenic isolates of P. fragariae var. rubi, ATCC 16184 (M14) and NY588. Inoculum was produced by growing the isolates separately in clariWed V-8 broth for 14–21 days as
described by Bristow et al. (1988). Mycelium was collected
using a Buchner funnel and 2 g of each isolate were placed in 500 ml of Wlter sterilized deionized water and frag-mented in a blender for two consecutive 5-s pulses. Each hydroponic basin was inoculated with the mycelial suspen-sion when plants reached 15–20 cm in height. Aeration was withheld for 48 h after inoculation. The nutrient solution was sampled weekly for the presence of zoospores by trans-ferring 5 ml aliquots into 15 ml centrifuge tubes, vortexing for 1 min and examined in a hemocytometer under the microscope. Disease screening and assessments were per-formed at 30–40 days post-inoculation as previously
described (Pattison et al. 2004) and consisted of a plant
dis-ease index (Table2), a root regeneration score (Table3),
stem lesion length, and the percent incidence of petiole lesions.
Inheritance analysis
Chi-square distributions were used to test goodness-of-Wt
for proposed inheritance models. The components of genetic variation for the plant disease index and incidence of petiole lesions were calculated using generation means
analysis according to Mather and Jinks (1982) based on the
means and standard errors of the three clonal parents and
four populations (F1, F2, B1, and B2). The analysis was
per-formed using a spreadsheet program (Ng 1990) that
esti-mated broad sense (H2) (Allard 1960) and narrow sense
(h2) (Warner 1952) heritability. The adequacy of the
addi-tive-dominance model was evaluated by using a joint scal-ing procedure, which uses weighted least squares estimates
based on the generation means (Cavalli 1952) and tested for
Wt using a chi-square distribution. Lack of Wt suggests the presence of non-additive gene eVects other than dominance such as, additive £ additive, additive £ dominance and/or dominance £ dominance. In such instances, further analy-sis can be performed to determine which interactions are signiWcant using a t-test.
Separate goodness-of-Wt tests were also performed using simple inheritance models to test the hypothesis that the action of a few genes is involved in PRR resistance in red raspberry. For this analysis a forced classiWcation of individuals was
Table 1 Goodness of Wt
analy-sis for the proposed inheritance model based on the observed segregation ratios of the P1, P2,
P3, F1, F2, B1, B2, and S2
genera-tions after exposure to
Phytoph-thora fragariae var. rubi
Cross/genotype Code n Mean Observed Expected 2 P
R S R S Latham P1 18 1.50 18 0 – – – – Titan P2 18 4.73 0 18 – – – – Latham £ Titan F1 51 3.00 29 22 1 1 0.96 0.35 NY00-34 P3 18 1.67 18 0 – – – – NY00-34? F2 80 3.36 44 36 9 7 0.05 0.84 NY00-34 £ Latham B1 50 1.74 50 0 3 1 15.36a <0.001 NY00-34 £ Titan B2 159 3.85 52 107 3 5 1.52 0.23 Titan? S2 30 4.77 0 30 – – – –
a Chi-square value was
calcu-lated using the Yates correction factor for small expected class sizes (Strickberger 1985)
Table 2 Plant disease index for assessing the susceptibility of red
raspberry genotypes following inoculation with Phytophthora
fraga-riae var. rubi
Score Symptoms
1 Slight root rot. No shoot symptoms 2 Slight root rot. Slight shoot symptoms 3 Moderate root rot. Moderate shoot symptoms 4 Severe root rot. Severe shoot
symptoms with living crown tissue
5 Perennial crown dead
Table 3 Root regeneration score for assessing susceptibility of red
raspberry genotypes following inoculation with Phytophthora
fraga-riae var. rubi
Score Symptoms
0 No new root tissue. Old roots and crown necrotic 1 Healthy crown and older root tissue with little
to no new root tissue
2 Healthy crown and older root tissue with moderate new root production 3 Healthy crown and older root tissue with
done where all individuals that maintained crown and older root tissue free from necrosis (plant disease index values of 1, 2, or 3) were classiWed as resistant genotypes and those in which the disease moved into the crown (plant disease index scores of 4 or 5) were classiWed as susceptible. Multiple per-mutations were tested for Wt including a single dominant-recessive gene model up to a three-gene model.
Marker generation
Newly expanded leaf tissue was collected from the B2
pop-ulation and parents and DNA isolated as described by Lodhi
et al. (1994) for all PCR procedures. AFLP markers were
generated using the procedure of Vos et al. (1995) with
small modiWcations. All adapters and primers were obtained from MWG-Biotech, Ebersberg, Germany. Digestions, ligations, and ampliWcations were performed on a PTC-100 thermocycler (MJ Research, Watertown, MA) with a heated lid. DNA template was prepared by digesting 200–300 ng genomic DNA with 2 U each of EcoRI and MseI restriction enzyme in a Wnal volume of 40 l for 2 h at 37°C following the manufactures directions (NEB, Beverly, MA). Next,
15l of a solution containing 5 pM EcoRI adapter
(EcoRI_ad1 CTCGTAGACTGCGTACC and EcoRI_ad2 AATTGGTACGCAGTC) and 50 pM MseI adapter (MseI_ad1 GACGATGAGTCCTGAG and MseI_Ad2 TACTCAGGACTCAT), 1 U T4 Ligase (NEB) and
3.7 £ NEB Ligase buVer were added, and the ligation was
performed at 37°C for 2 h. The same procedure was per-formed to generate EcoRI/PstI template using 5 pM EcoRI adapter and 5 pM PstI adapter (PstI_ad1 CTCGTAGACT-GCGTACATGCA and PstI_ad2 TGTACGCAGTCTAC).
Pre-ampliWcation was conducted with no selective nucleotides (EcoRI + 0–GACTGCGTACCAATTC, MseI + 0—GATGAGTCCTGAGTAA, and PstI + 0—GACTGC GTACATGCAG) followed by ampliWcation with two
selective nucleotides. AmpliWcation with three selective
nucleotides was preceded by pre-ampliWcation with one selective nucleotide, e.g., MseI + C and MseI + G.
Pre-ampliWcation was carried out with 4 l of tenfold-diluted
ligated template, 49.3 mM Tris–HCL (pH 8.3), 2.5 mM
MgCl2, 1 mM tartrazine, 1.5% Wcoll, 125 M of each
dNTP, 20 pmol each primer, and 1 U Taq DNA polymerase in a Wnal volume of 40 l. Primer combinations
EcoRI + NN⬘/MseI + NN⬘, EcoRI + NN⬘/MseI + NN⬘N⬙, EcoRI + N/PstI + N, EcoRI + NN⬘/PstI + N, and EcoRI +
NN⬘/PstI + NN⬘, where N are the selective nucleotides, were analyzed in order to Wnd the optimum number of selective nucleotides that would generate clearly scorable AFLP fragments. AmpliWcations were performed with ten-fold-diluted pre-ampliWcation mixture as template.
Optimal separation of AFLP fragments was achieved
with the primer combinations EcoRI + NN⬘/MseI + NN⬘N⬙
and EcoRI + NN⬘/PstI + NN⬘, which were further used in
this study. After diluting the ampliWcation products 1:1 with Stop/Loading buVer (Li-Cor, Lincoln, NE), the AFLP fragments were denatured at 94°C for 3 min and separated by electrophoresis through 6.5% denaturing polyacryl-amide gel (Gel Matrix, Li-Cor) on a LI-COR Gene ReadIR 4200 automated DNA sequencing system (Li-Cor). Visual-ization of the fragments was achieved by end labeling of the EcoRI primers with IR800 dye. Calculation of the DNA sizes was performed based on 50–700 Size-Standard IR800 Dye (Li-Cor). The AFLP markers were labeled according
to the Standard List for AFLP Primer Nomenclature (http://
www.wheat.pw.usda.gov/ggpages/keygeneAFLPs.html). For example, marker EcoRI + CC/MseI + CTA with a size of 366 bp is labeled as E16M59_366.
DNA ampliWcation for RAPD marker generation was
based on the procedure of Cai et al. (1994). PCR reactions
were done in a total volume of 25l containing 49.3 mM
Tris–HCL (pH 8.3), 2.5 mM MgCl2, 1 mM tartrazine, 1.5%
Wcoll, 125 M of each dNTP, 0.4 M primer, 1 U Taq poly-merase, and 60 ng of genomic DNA. DNA was ampliWed in a MJ Research PTC-100 thermocycler with a heated lid for 45 cycles. Each cycle was programmed for 1 min at 94°C, 1.5 min at 35°C, 2 min at 72°C, followed by a 6 min exten-sion at 72°C at the end of the last cycle before cooling to 6°C. AmpliWed DNA fragments were separated on a 2% TAE agarose gel and visualized by ethidium bromide
stain-ing. RAPD marker nomenclature is as follows: the Wrst
let-ters designate primer origin (BC = University of British Columbia, Vancouver, BC, Canada; OP = Operon Technol-ogies, Alameda, CA). The next three numbers indicate the primer number followed by three numbers indicating the fragment size in base pairs.
The RGAP reactions were done with the same procedure as for RAPDs with the following modiWcations. In each
reaction, 0.4M of both a forward and reverse primer was
added. Each cycle was programmed for 1 min at 94°C, 1 min at 45°C, 2 min at 72°C, followed by a 6 min exten-sion at 72°C at the end of the last cycle before cooling to
6°C. AmpliWed DNA fragments were separated on a 2.5–
3% TAE Metaphor™ agarose gel (BioWhittaker Molecular Applications, Rockland, ME) and visualized by ethidium bromide staining. Sequence speciWc primers designed from
conserved motifs of resistance genes (Chen et al. 1998)
were used to amplify resistant gene like sequences
(Table4). A total of 79 primer combinations were tested on
the parents and combinations that produced polymorphisms were used on the mapping population.
Analysis of distributional extremes
Based on the theory used in bulked segregant analysis,
were analyzed for signiWcant interactions with speciWc RAPD markers. DNA from nine resistant and susceptible phenotypes from the distributional extremes of the popula-tion was analyzed separately with each RAPD primer instead of as pooled DNA samples used for bulked segre-gant analysis. The nine resistant individuals had a plant dis-ease index score of 1 or 2 and the nine susceptible plants had a plant disease index score of 5. A total of 225 RAPD primers were screened for polymorphisms between the two groups of individuals. These primers were previously
shown to produce ampliWed products in an unrelated
raspberry population (data not shown). Each primer that produced a signiWcantly linked fragment, as determined by
a t-test at = 0.05, was used on the 68 individuals of the
mapping population to determine map location of linked markers.
Molecular genetic mapping and QTL analysis
IdentiWed polymorphisms were scored in the mapping
pop-ulation and mapped using JoinMap 3.0® (Van Oijen and
Voorips 2001). The population design and high level of
polymorphism in red raspberry allowed for the develop-ment of separate parental linkage maps. Each map con-tained both heterozygous markers (present in both parents and segregating 3:1 in the population) and backcross mark-ers (present/heterozygous in one parent and homozygous recessive/absent in the other and segregating 1:1 in the pop-ulation). Markers deviating signiWcantly from expected segregation ratios were removed from the data set after observing many to be unlinked or clustering to a separate
small linkage group (data not shown). Linkage groups were assigned using a LOD score of 5.0.
QTL analysis was performed using MapQTL 4.0® (Van
Oijen et al. 2002). LOD thresholds were calculated by
per-mutation analysis for each trait and linkage group individu-ally. Kruskal–Wallis analysis was used to detect markers
linked to the phenotypic data and signiWcant QTL were
declared by interval mapping when the sample data exceeded the LOD threshold.
Results
Clonally replicated plants of ‘Latham’ and ‘Titan’ displayed the resistant and susceptible phenotype, respectively, when exposed to P. fragariae var. rubi using the hydroponic
sys-tem (Pattison et al. 2004), and NY00-34 was classiWed as
resistant as ‘Latham’ based on all assessment criteria
(Tables5, 6, 7, 8). The F1 and F2 populations segregated
widely for all assessment criteria with recovery of both parental phenotypes except for stem lesion length, which did not reach the size observed in ‘Titan’ and was skewed
toward no lesions (Tables5, 6, 7, 8). Little segregation was
observed in the B1 population for all resistance
measure-ments with no individuals expressing susceptibility similar
to the susceptible parent, ‘Titan’ (Tables5, 6, 7, 8). The B2
population generated segregants of both parental phenotypes for all traits except stem lesion length again where the
sever-ity of ‘Titan’ was not observed (Tables5, 6, 7, 8).
Simple segregation for the presence of stem lesions was observed where individuals with low plant disease index
Table 4 Sequences of the primers used to amplify resistance gene analogs (RGAs) in red raspberry designed from conserved domains of
resistance genes from multiple species
a Pto and RPS2 genes from tomato and Arabidopsis, respectively, confer resistance against Pseudomonas syringae pv. tomato. The N gene
from tobacco confers resistance against tobacco mosaic virus. The Cf9 gene from tomato confers resistance against Cladosporium fulvum. The
Xa21 gene from rice confers resistance against Xanthomonas campestris pv. Oryzae
Primer Sequence (5⬘–3⬘) R genea/domain References
PtoKin 1 GCATTGGACAAGGTGAA Pto/protein kinase Chen et al. (1998)
PtoKin 2 AGGGGGACCACCACGTAG
AS1 CAACGCTAGTGGCAATCC RPS2 and N/P-loop Leister et al. (1996)
RLRR-f CGCAACCACTAGAGTAAC RPS2/LRR Chen et al. (1998)
RLRR-r ACACTGGTCCATGAGGTT
XLRR-f CCGTTGGACAGGAAGGAG Xa21/LRR Chen et al. (1998)
XLRR-r CCCATAGACCGGACTGTT
NLRR-f TAGGGCCTCTTGCATCGT N/LRR Chen et al. (1998)
NLRR-r TATAAAAAGTGCCGGACT
CLRR-f TTTTCGTGTTCAACGACG Cf9/LRR Chen et al. (1998)
CLRR-r TAACGTCTATCGACTTCT
NBS-F1 GGAATGGGNGGNGTNGGNAARAC RPS2 and N/P-loop and Kinase 3a Yu et al. (1996)
(·3) scores had no stem lesions (Tables5, 8). However, plants with high plant disease index scores (¸4) did not always display a stem lesion, despite severe symptom expression for all other traits. The incidence of petiole lesions exhibited complex segregation with both parental
phenotypes identiWed in all generations except B1 where
individuals with no petiole lesions were not observed
(Table8).
Generational means analysis of the plant disease index revealed that the additive-dominance model could not
ade-quately account for all of the variation observed in these
populations as indicated by the joint scaling test [2
(df = 3) = 30.303, P < 0.001]. Additional analysis revealed signiWcant additive £ additive, additive £ dominance and dominance £ dominance interactions. However the addi-tive variance was the largest component of the total vari-ance, producing high narrow and broad sense heritability
estimates of h2= 0.86 and H2= 0.88, respectively.
Most of the variance in the incidence of petiole lesions was observed to be dominant with little additive genetic
Table 5 Relative frequency
distributions (percentage) of plant disease index scores in parental clones and red rasp-berry populations segregating for resistance to root rot caused by Phytophthora fragariae var.
rubi
Genotype/population Family n Plant disease index score Mean § SE
1 2 3 4 5 Latham P1 18 44 56 0 0 0 1.50 § 0.11 Titan P2 18 0 0 0 28 72 4.72 § 0.11 NY00-34 P3 18 33 67 0 0 0 1.67 § 0.11 Latham £ Titan F1 51 20 24 14 24 20 3.0 § 0.20 NY00-34 £ NY00-34 F2 80 9 19 27 17 27 3.36 § 0.15 Latham £ NY00-34 B1 50 38 50 12 0 0 1.74 § 0.09 Titan £ NY00-34 B2 159 6 11 16 26 41 3.85 § 0.10 Titan £ Titan S2 30 0 0 3 17 80 4.77 § 0.09
Table 6 Relative frequency
distributions (percentage) of root regeneration scores in parental clones and red raspberry popula-tions segregating for resistance to root rot caused by
Phytoph-thora fragariae var. rubi
Genotype/population Family n Root regeneration score Mean § SE
0 1 2 3 Latham P1 18 0 0 67 33 2.33 § 0.11 Titan P2 18 100 0 0 0 0 NY00-34 P3 18 0 0 50 50 2.50 § 0.12 Latham £ Titan F1 51 43 16 29 12 1.10 § 0.15 NY00-34 £ NY00-34 F2 80 44 34 19 4 0.83 § 0.10 Latham £ NY00-34 B1 50 0 20 60 20 2.00 § 0.10 Titan £ NY00-34 B2 159 67 18 11 5 0.54 § 0.07 Titan £ Titan S2 30 100 0 0 0 0
Table 7 Relative frequency distributions (percentage) of stem lesion length in parental clones and red raspberry populations segregating for
resis-tance to root rot caused by Phytophthora fragariae var. rubi
Genotype/population Family n Stem lesion size (cm) Mean § SE
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Latham P1 18 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Titan P2 18 0 0 0 0 0 11 11 6 6 17 11 6 0 11 17 6 9.83 § 0.80 NY00-34 P3 18 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Latham £ Titan F1 51 69 6 10 0 4 4 2 4 2 0 0 0 0 0 0 0 1.13 § 0.30 NY00-34 £ NY00-34 F2 80 70 10 0 4 5 1 1 0 3 1 5 0 0 0 0 0 1.36 § 0.31 Latham £ NY00-34 B1 50 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Titan £ NY00-34 B2 159 48 8 6 7 4 9 6 3 1 4 2 0 0 0 0 1 2.35 § 0.24 Titan £ Titan S2 30 10 17 7 7 3 0 7 3 7 3 10 0 3 0 0 20 7.17 § 1.09
variance. For this trait the joint scaling test revealed that an additive-dominance model explains most of the variability
observed without signiWcant interactions [2 (df = 3) =
6.145, P > 0.1]. Narrow and broad sense heritability were
estimated to be h2= 0.14 and H2= 0.95, respectively.
Esti-mates of components of genetic variance for stem lesion length and root regeneration score were not made due to the lack of parental variances (i.e., the resistant parent scored 0 for stem lesion length, and the susceptible parent scored 0 for root regeneration score).
When the plant disease index data was used to force the classiWcation of individuals into resistant and susceptible
classes, the best-Wt genetic model for resistance contained
two dominant genes (Fig.1). Deviation from the expected
segregation ratios was observed only in the B1 generation
(Table1).
Two parental linkage maps (one utilizing markers origi-nating from NY 00-34 and one with markers from ‘Titan’)
were constructed (Fig.2). The NY 00-34 map contained
seven linkage groups with 138 AFLP, 68 RAPD, and 20 RGAP markers covering 440 cM of genetic distance. Link-age group length ranged from 35 to 86 cM. A total of 67 (29%) markers were removed from this data set due to sig-niWcant deviations from expected segregation ratios. The ‘Titan’ map also had seven linkage groups constructed from 153 AFLPs, 47 RAPDs, and 11 RGAPs covering 370 cM of genetic distance. Linkage group length ranged from 26 to 70 cM. Nineteen (8.8%) markers were skewed and removed from this data set for map construction. Link-age group homology between the maps was identiWed for four of the seven groups using co-dominant markers
(Fig.2).
A total of 79 resistant gene homology approach (RGA) primer combinations were tested, and 66 (84%) of the com-binations produced good quality ampliWcation products, averaging 6.5 bands/combination. The remaining 13 (16%) did not visibly amplify DNA. Of the 66 successful combi-nations, 17 (26%) produced polymorphic bands among the
parents with an average of 1.9 polymorphic
bands/combi-nation. All of the segregating markers ampliWed from the
RGA primers behaved as dominant markers with most Wtting the expected segregation ratios of either 1:1 or 3:1. An even distribution of RGA markers was observed across most linkage groups.
RAPD markers (ten total) putatively associated with plant disease index scores were identiWed by analyzing the
Table 8 Relative frequency distributions (percentage) of the frequency of the incidence of petiole lesions in parental clones and red raspberry
populations segregating for resistance to root rot caused by Phytophthora fragariae var. rubi
Genoytpe/population Family n Frequency of incidence of petiole lesions Mean § SE
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Latham P1 18 17 33 33 17 0 0 0 0 0 0 0 0.15 § 0.02 Titan P2 18 0 0 0 0 0 17 0 0 17 11 56 0.86 § 0.04 NY00-34 P3 18 17 44 28 11 0 0 0 0 0 0 0 0.13 § 0.02 Latham £ Titan F1 51 47 0 8 6 10 10 4 0 10 2 4 0.28 § 0.05 NY00-34 £ NY00-34 F2 80 26 15 7 16 5 4 7 3 1 0 15 0.33 § 0.04 Latham £ NY00-34 B1 50 0 22 26 28 16 6 2 0 0 0 0 0.25 § 0.02 Titan £ NY00-34 B2 159 15 9 4 9 7 10 7 6 7 7 18 0.50 § 0.03 Titan £ Titan S2 30 0 3 0 0 0 10 0 0 17 3 67 0.88 § 0.04
Fig. 1 Proposed genotypes for a dominant two-gene hypothesis for
Phytophthora root rot resistance in red raspberry based on best-Wt
modeling and generational means analysis. Parentheses indicate ob-served number of segregants in each population
Latham (P1) x Titan (P2) AABb Aabb F1 population AB Ab Ab AABb AAbb ab AaBb Aabb 1 Resistant : 1 Susceptible (22:29) NY00-34 (P3) x NY00-34 (P3) AaBb AaBb F2 population AB Ab aB ab
AB AABB AABb AaBB AaBb
Ab AABb AAbb AaBb Aabb
aB AaBB AaBb aaBB aaBb
ab AaBb Aabb aaBb aabb
9 Resistant : 7 Suceptible
(44:36) NY00-34 x Latham NY00-34 x Titan
AaBb AABb AaBb Aabb
B1 B2
AB Ab AB ab AB Ab aB ab
AB AABB AABb AABB AaBb Ab AABb Aabb AaBb Aabb Ab AABb AAbb AABb Aabb ab AaBb Aabb aaBb aabb
3 Resistant : 1 Susceptible 3 Resistant : 5 Susceptible
Fig. 2 Genetic linkage maps for
NY 00-34 and Titan red rasp-berry. Linkage groups that could be aligned with heterozygous 3:1 RAPDs and AFLPs are indi-cated with connecting dotted
lines. Vertical bars represent
QTL for plant disease index (pdi), stem lesion length (sl), incidence of petiole lesions (pl), and root regeneration score (rrs). RAPD marker nomenclature is made up of the Wrst letters desig-nating primer origin (BC Uni-versity of British Columbia, OP Operon Technologies). The next three numbers indicate the
prim-er numbprim-er followed by three
numbers indicating the fragment size in base pairs. The AFLP markers were labeled according to the Standard List for AFLP Primer Nomenclature (http:// wheat.pw.usda.gov/ggpages/ keygeneAFLPs.html), e.g.,
EcoRI+AT/MseI + GTC is
la-beled E14M76. The marker sizes are given in base pairs. For example, marker EcoRI + CC/
MseI + CTA with a size of
366 bp is labeled as E16M59_366 36 _32 0 0 36 _31 9 h B C 0 0 3 1 5 00 C C 1 _ 13 9 B C 5 92 06 0 0 BC 4 6 4 0 8 0 0 2 9 _ 1 4 1 B C 109 05 00 11 BC 4 2 9 2 4 0 0 18 29 _17 9 29 BC 6 4 2 1 1 0 0 36 CC 1 _ 1 7 0 39 PTO K IN 8 5 0 44 30 _26 9 C G 1 0 _2 51 52 X L RR r+ N L RR f3 0 0 58 CG1 0 _ 2 7 2 59 R L R R f+ A S 124 0 60 BC 4 9 0 0 8 0 0 61 BC 5 3 3 0 5 5 0 64 36 _22 5 0 P4 _ 4 3 0 L 2 C G 9_ 63 L 2 3_ 1 6 8 h CG2 _ 1 1 2 h 14 35 _19 0 15 3 5 _ 0 50 L B C 6 22 08 5 0 34 CG4 _ 2 2 0 h 40 45 _80 h 44 C G 1 _0 9 9h C C 2 _ 19 2h 45 CG1 _ 1 8 6 C G 1 _ 2 5 0 B C 622 06 00 47 35 _25 5h C G 2 _ 3 45 h P T O K in 1+R L R R f2 60 C C 2 _29 8h CG3 _ 1 7 8 h 48 XL R R 2 5 3 51 B C 574 07 00 52 B C 640 06 50 53 45 _29 0 h 57 B C 571 08 00 61 45 _21 1 0 29 _13 5 5 CG9 _ 5 2 0 8 B C 0 0 21 05 0 10 CG1 _ 1 1 1 B C3 4 7 0 8 0 0 CG5 _ 2 4 6 P 1 _ 3 0 4 P3 _ 1 6 8 11 B C 5 9 21 60 0 P 1 _ 23 5 B C 0 3 40 62 5 13 C G 1 0 _ 1 26 4 7_2 09 35 _44 0 48 _47 3 14 PTO K IN 3 7 5 15 C G 1 0_ 2 76 B C 1 1 90 475 C G 2_ 15 9 16 23 _12 8 18 36 _31 4 19 C C 1 _ 14 5 P T O K 2 + R L R R f20 0 20 B C 4 2 90 35 0 22 B C 3 5 30 50 0 34 B C 2 8 50 70 0 B C 5 71 0 7 5 0 B C 2 6 41 05 0 37 3 5_ 2 40 C G 3_ 1 4 5 29 _06 0 38 45 _23 7 40 23 _24 1 42 PTO K IN 2 6 0 48 B C 2 7 01 20 0 C G 3 _ 12 4 49 B C 0 2 50 90 0 51 C G 9_ 29 6 54 B C 0 1 71 10 0 58 op v-08 07 00 61 B C 2 7 30 65 0 B C 3 10 0 4 0 0 CG1 _ 2 9 0 C G 2 _ 3 6 9 63 30 _21 3 64 R L R R f+ A S 130 0 65 B C 5 9 21 40 0 0 CG4 _ 2 0 1 L 12 CG1 0 _ 2 6 8 13 P3 _ 4 6 1 L 31 29 _22 6 32 35 _14 0 34 CG1 _ 1 9 6 h 35 P2 _ 2 6 8 h 36 35 _36 1 P 1 _27 2 48 _22 7L 4 8_1 4 8 37 40 _22 4 h 38 P2 _ 2 7 6 h 39 CG2 _ 3 4 8 _ 3 5 2 40 48 _20 7 P 2 _31 3 41 C C 2_0 9 7h B C 0 0 3 0 75 0 23 _10 2 h 42 CG3 _ 1 6 3 4 0 _ 2 2 3 C G 3 _ 0 7 8L 2 4 _1 80 L 43 CG5 _ 3 6 3 B C6 2 4 1 3 0 0 44 B C 6 4 01 30 0 45 45 _22 0 h 46 O P O 0 8 1 10 0 O P 008 140 0 48 B C 1 1 90 62 5 49 B C 2 6 12 00 0 51 45 _27 1 52 CC 2 _ 1 3 4 h 55 Ti ta n 2 P1 _ 2 67 L 0 BC 5 0 4 0 6 5 0 12 C G 2_ 40 7 L 15 C G 10 _2 9 3 h 18 30 _25 4 h 21 BC 0 4 2 1 0 0 0 22 C G 2_ 15 2 h 23 C G 1_ 13 3 25 23 _17 0 26 BC 6 3 9 0 6 0 0 28 CG9 _7 0L C C 2_ 167 h 35 _1 10 L 29 47 _26 3 C G 2 _ 15 4L 30 C G 9_ 15 5 h C G 5 _ 2 6 5L 32 N L R R 3 00 B C 59 30 650 33 CG5 _2 61h 34 C G 9_ 87 L 3 0_ 3 6 3 L 36 BC 5 9 2 0 7 2 5 38 35 _3 64 L 39 C C 2_2 57L B C 0 1 2 0 30 0 40 P 1 _31 5L C G 2_ 266 h 41 B C 577 11 50 29 _40 2h BC 4 2 9 1 4 5 0 42 P 3 _29 2h C G 4_ 374 h 43 X L R R r+ N L R R r6 50 40 _12 0h 45 30 _26 5 h 47 BC 6 4 8 1 3 0 0 49 BC 5 7 1 1 8 0 0 51 B C 618 10 00 29 _10 6h 53 B C 615 10 50 40 _19 5h 55 BC 6 1 0 0 9 0 0 61 BC 6 0 1 0 8 0 0 62 C G 4 _ 2 18 P 4_1 79 0 CG1 _ 1 3 9 5 BC 6 4 3 1 7 0 0 11 BC 1 1 9 1 5 2 0 16 P2 _ 4 7 5 25 BC 5 8 9 0 7 0 0 31 P4 _ 1 4 0 2 9 _ 2 69 RL RRr + NL R Rf 2 4 0 4 8 _ 1 5 0 38 P2 _ 2 6 8 h 40 C G 1_ 19 6 h P 2 _2 7 6 h 41 40 _22 4 h 46 23 _10 2 h 51 CG3 _ 1 6 0 52 BC 3 8 3 1 4 0 0 54 C C 2_0 9 7h 56 BC 6 2 4 1 3 0 0 57 C G 3 _ 1 97 P 4_3 26 58 B C 261 0 9 5 0 P 2_3 0 7 59 45 _22 0 h 60 P 4 _36 7 47 _06 0 62 BC 2 9 6 0 2 5 0 64 BC 3 2 9 1 2 0 0 66 CG9 _ 1 5 4 68 CC 2 _ 0 8 8 75 30 _13 0 81 BC 6 1 1 0 8 0 0 86 NY 0 0 -3 4 2 48 _51 3 0 CC 1 _ 1 8 5 1 CL RRf + A S 1 3 5 0 9 C G 2_ 11 3 14 23 _16 8h C G 2 _ 1 12 h 16 BC 5 7 4 1 9 0 0 17 XL R R r+ N L R R f7 2 5 20 32 _13 5 22 23 _22 3 24 P T OK 2 + RL RRf 6 0 0 31 BC 6 2 2 0 8 5 0 36 BC 6 0 1 0 9 0 0 37 36 _29 0 38 C G 4_ 22 0 h 23 _ 2 1 8 P T OK 2 + NL RRf 5 0 0 39 PTO K IN 7 0 0 40 3 0 _ 1 7 9 P T Ok 1 + NL RRr 6 2 5 CG1 0 _ 3 8 5 41 C G 1_ 09 9 h C C 2_ 1 92 h 42 35 _25 5 h 43 23 _23 7 C G 1 0 _3 22 C G 2_ 34 2 44 45 _80 h C G 1 0 _3 82 C G 2 _ 3 4 5h C G 3_ 178 h CC 2 _ 2 9 8 h B C 4 4 5 0 6 2 5 45 P 1 _29 0 46 AF L P2 3 _ 0 9 5 47 C G 1_ 35 0 48 BC 0 3 3 1 0 0 0 49 BC 7 5 1 0 7 0 0 50 BC 5 7 4 0 7 0 0 51 BC 6 4 0 0 6 5 0 53 45 _29 0 h 56 BC 2 6 5 0 6 5 0 57 XL R R f+ A S1 7 0 0 60 P 3 _26 7 62 P2 _ 3 5 0 h 0 X L RR r+ N L RR r2 5 0 C C 2 _ 1 4 4 h 5 23 _26 8 h 11 35 _04 9 h C C 2 _ 305 h 16 BC 6 4 8 1 2 5 0 24 P2 _ 2 1 9 h 26 C C 2_1 14h 23_ 153 h C G 1_ 12 6 h C G 1 _ 2 2 6h 28 C G 4_ 07 3 h 35 B C 2 6 50 45 0 0 B C 4 8 90 90 0 C G 1 0 _ 1 6 7 1 B C 0 3 41 30 0 4 24 _25 6 7 CG1 _ 2 8 1 8 C G 3_ 21 0 P 4 _ 25 7 C G 5_ 06 6 B C 5 93 1 0 0 0 9 CG9 _ 1 8 4 h 13 P 2 _32 5h 14 B C 6 4 60 27 5 16 CG4 _ 2 5 0 C G 1 0 _ 3 7 4 CG3 _ 1 6 7 17 CG4 _ 0 6 7 B C1 3 2 0 8 5 0 18 40 _10 1 21 36 _31 0 26 P T O K 2+ A S 19 00 28 B C 2 9 61 00 0 30 CG1 0 _ 2 3 5 C G 2 _ 1 1 7 34 CG1 _ 0 7 8 36 B C 6 4 31 15 0 37 B C 6 4 32 55 0 47 B C 5 7 40 60 0 48 29 _05 7 53 B C 3 1 01 05 0 57 CG3 _ 1 3 9 0 32 _29 8 2 BC 2 5 9 0 7 0 0 15 CG4 _ 1 1 0 L 19 24 _35 5 22 B C 622 07 00 28 CG5 _ 2 6 9 51 NY 0 0 -3 4 3 NY 0 0 -3 4 4 Ti ta n 4 NY 0 0 -3 4 5 NY 0 0 -3 4 6 BC 6 3 8 0 8 0 0 0 29 _17 6 4 30 _14 6 C G 5 _ 11 1 7 BC 5 8 9 0 4 0 0 9 BC 1 1 9 0 7 0 0 11 P to k in 1 + NL RR r1 1 0 0 12 bc4 6 50 550 13 P 4 _37 5 C G 9_1 71h 14 48 _23 3 B C 639 175 0 BC 4 8 5 1 1 5 0 3 6 _ 1 8 3 15 30 _08 7 30 _31 4 C G 9_ 31 5 4 7_ 1 1 0 h 48 _18 3 32 _18 2 23 _10 8L C G 1 _ 2 21 NL RR3 5 0 16 36 _14 7 36 _10 1 C G 1_ 28 9 18 C G 2 _ 2 1 6 C G 3_0 57 19 C C 2_1 77L C G 1 _ 12 2h 21 BC 0 0 2 0 7 0 0 24 CG9 _ 4 7 9 25 BC 3 2 9 0 7 0 0 26 B C 04 4090 0 0 P T O K 2+ C L R R f250 B C 34 1080 0 10 B C 23 1130 0 16 29 _ 3 66 17 C C 2_1 67h 20 B C 63 9060 0 21 B C 11 9105 0 22 C G 10_ 293h 28 30 _ 2 54 h 29 B C 26 4200 0 32 3 0 _11 1 36_ 0 98 34 C G 2_1 52h 36 48 _ 4 97 37 XL RRr + RL RR r5 1 0 39 B C 45 7110 0 40 C G 9_1 55h 41 24 _ 2 24 42 3 6 _25 0 C G 5_ 261 h 43 B C 79 2150 0 45 C G 1_1 3 7 B C 6 48 1300 47 C G 10_ 342 C G 4_07 0 49 40 _ 2 61 50 30 _ 2 65 h 51 B C 13 2070 0 40_ 120 h XL RRr + N L R R r6 5 0 52 B C 57 7115 0 C G 2 _ 266 h B C 42 9145 0 29_ 402 h 54 C G 4_3 74h 55 P 3 _2 92h 56 B C 57 1180 0 58 B C 61 8100 0 62 29 _ 1 06 h P 2_ 3 6 9 64 40 _ 1 95 h 65 B C 61 0090 0 71 P 2 _35 0h 0 CC 1 _ 0 6 7 5 CC 2 _ 1 4 0 L 6 35 _04 9h C C 2_ 305 h 7 X L R R r+ NL R R r2 5 0 CC 2 _ 1 4 4 h 8 P 1 _20 6L 3 0 _1 51L 9 23 _26 8h C G 5_ 283 10 B C 615 0 6 00 3 2 _ 05 7L BC 6 3 8 1 0 0 0 B C 1 1 9 2 4 9 0 11 C G 4_ 07 3 h 14 29 _10 4 3 2 _ 17 5 C G 1_ 17 2 17 P 2 _21 9h 3 2_1 50 C G 1_ 10 2 L 18 BC 6 4 8 1 2 5 0 19 C C 2_1 1 4h 23_ 1 53 h C G 1_ 22 6 h C G 1 _ 1 2 6h 21 CC 2 _ 1 0 6 3 0 _ 0 6 3 23 XL R R r+ R L R R r5 0 0 32 C G 9_ 18 2 33 BC 0 0 2 0 9 0 0 36 P 1 _19 4L 53 XL R R r+ N L R R r9 0 0 67 24 _21 1 69 C G 4_ 16 2 L 70 Ti ta n 1 NY 0 0 -3 4 1 35 _21 2 L 0 C G 10 _1 4 5 35 _ 2 0 5 h 2 P4 _ 1 5 2 PTO K in 1 + R L R R f2 5 0 3 47 _26 6 5 B C 5 9 31 70 0 7 P1 _ 1 7 2 10 CG4 _ 1 0 4 11 B C 5 9 40 90 0 12 CC 2 _ 1 5 4 13 CG2 + 2 7 6 14 XL R R r+ NL R R r3 5 0 15 32 _21 0 36 _11 4 16 C G 4 _ 1 16 C G 3 _0 60 17 B C 1 1 90 55 0 4 8_ 2 2 2 C G 5_ 17 3 3 0_ 3 0 8 C G 3_ 09 1 B C 2 70 1 5 5 0 19 B C 2 6 41 80 0 20 CG3 _ 1 8 0 22 P3 _ 1 0 3 2 9 _ 0 9 8 23 B C 3 2 90 80 0 24 29 _18 5 26 P2 _ 2 4 6 29 P3 _ 1 9 5 3 6 _ 0 6 7 L 40 _24 7 45 Ti ta n 3 Ti ta n 5 Ti ta n 6 NY 0 0 -3 4 7 Ti ta n 7 pdi pl rrs pdi pl rrs pdi sl pl rrs pd i sl pl rrs
distributional extremes of the B2 population (Table9). Markers associated with resistance originated from ‘Latham.’ Conversely, all markers associated with suscepti-bility were heterozygous among the parents and segregated 3:1 in the population. No markers of strictly ‘Titan’ origin were found to be associated with either resistance or sus-ceptibility. The linked RAPD markers clustered on two linkage groups of NY 00-34 (groups 1 and 5) and to linkage
group 1 on the ‘Titan’ map (Fig.2).
QTL analysis was performed on both parental maps for each of the disease assessment criteria. QTL were identiWed on linkage group 1 of both parental maps for the plant dis-ease index, the incidence of petiole lesions and the root regeneration score. This QTL corresponded to one of the regions of BSA marker clustering. On linkage group 1 of NY 00-34, the QTL accounted for »30, 61, and 25% of the phenotypic variation for plant disease index, incidence of petiole lesions and root regeneration score, respectively. On linkage group 1 of the ‘Titan’ map, the QTL accounted for »26, 33, and 29% of the phenotypic variation for plant dis-ease index, incidence of petiole lesions, and root regenera-tion score, respectively.
QTL for plant disease index, stem lesion size, incidence of petiole lesions, and root regeneration score were found to be located around the other BSA marker cluster on link-age group 5 of NY 00-34 and accounted for »28, 10, 28,
and 15%, respectively, of the observed variation. A second region on the ‘Titan’ map (group 7) yielded signiWcant QTL for plant disease index, stem lesion size, incidence of petiole lesions and root regeneration score and accounted for »15, 18, 14, and 16%, respectively, of the observed variation.
Discussion
Overall control of resistance to root rot caused by P.
fraga-riae var. rubi is neither monogenic nor extensively
quanti-tative based on the observed segregation in these red raspberry populations. The segregation of plant disease index scores over all populations revealed that an additive-dominance model could not account adequately for the var-iation present for root rot resistance. While additive vari-ance was the largest portion of the observed varivari-ance, accounting for high heritability estimates for plant disease index, other interactions were also signiWcant. The complex nature of symptoms in this disease can account for this mixed inheritance model. This is demonstrated by the data on incidence of petiole lesions, which was calculated to fol-low a dominance model with a fol-low narrow sense heritabil-ity. Inferences can be made from the segregation observed
in the F1 and B2 population that depending on the trait
mea-sured, dominance eVects and/or heterozygosity within the parents have variable importance based on the genetic make up of the population studied.
This data also suggests that NY00-34 is more heterozy-gous for resistance than ‘Latham’ and that more than one gene is conditioning resistance. Goodness of Wt tests using forced classiWcation of resistance show that a dominant two-gene model accounts for most of the variability in overall resistance.
The B1 population did not Wt our two-gene model due to
skewing toward resistance. This population was very di
Y-cult to establish in the greenhouse and the hydroponic screening system due to powdery mildew to which ‘Latham’ is extremely susceptible. This problem was not encountered in the other populations. Six individuals in the
B1 population were classiWed as intermediate for plant
dis-ease index (score of 3), which was not observed in the clonal test of ‘Latham’ and NY00-34 and would not expected in this population if the dominant two-gene model were correct. While there is no evidence of a direct genetic linkage between susceptibility to powdery mildew and sus-ceptibility to PRR, it is conceivable that a physiological
linkage exists. Vigor plays a diYcult to quantify role in
resistance to many diseases. Many diseases infect and spread in rapidly growing tissues that provide more possi-ble infection sites. While developing the hydroponic system it was observed that with little root growth there were fewer
Table 9 RAPD primer and fragment size (base pairs) linked to
Phytophthora root rot resistance in red raspberry as determined by
plant disease index score and identiWed by analysis of the distributional extremes of the B2 population
a
Fragment size in nucleotides estimated from gel with 1 kb ladder
b
Parental origin of linked marker (both indicates a heterozygous marker segregating 3:1)
c SigniWcance of diVerence determined by t-test
*P = 0.05 **P = 0.01 ***P = 0.001
Primer Fragment Linkage group
(NY 00-34/Titan)
Mean pdi score for marker present/absentc Sizea Originb BC003 1,500 NY 00-34 5 1.86/4** BC109 500 NY 00-34 5 1.33/4.08** BC592 600 NY 00-34 5 1.5/3.9*** BC464 800 NY 00-34 5 1.5/3.9*** BC571 1,800 NY 00-34 1 4.33/2.11** BC429 1,450 Both 1/1 4.22/2.11** BC437 850 Both Skewed 4.22/2.11** BC577 1,150 Both 1/1 4.25/2.4* BC610 900 Both 1/1 4/2.25* BC648 1,300 Both 1/1 4.63/2.1**
PRR symptoms even in known susceptible genotypes. Such plants entered a semi-dormant state and failed to either
grow or display disease progression (Pattison et al. 2004).
Severe infection with powdery mildew also produces a general lack of vigor with reduced root growth in the
hydroponic system. There were six individuals in the B1
population with a plant disease index rating of 3. There were none in either parent clonal test and none would be
expected in the B1 population if the two-gene model was
accurate, but 12 susceptible individuals (four or Wve rating) would be expected. The six intermediate genotypes all had low root regeneration scores (1) and a high incidence of petiole lesions (40–60%) and therefore, we believe they are susceptible. Classifying those six (44:6) as susceptible gives a chi-square of 3.84 and thus P > 0.05, which is much more likely due to chance with the small population tested.
However, other genetic factors cannot be ruled out with the data observed especially considering the results of the generational means analysis. The experimental design and resulting genetic model proposed in this study can only adequately account for major sources of genetic variation and given the complexity of plant pathogen interactions it is
likely that other minor genes are involved. In spite of the B1
population not Wtting our genetic model without reclassiW-cation of the intermediate response, the presence of large amounts of additive, and interaction genetic variation observed in the other populations suggests that the recovery of high percentages of resistant individuals is possible using either recurrent selection or backcross procedures.
As was observed by Nestby and Heiberg (1995) with
both positive and negative speciWc combining abilities in multiple half-sibling families, high levels of heterozygosity in the parents can provide conXicting inheritance patterns. This combined with suggested oligogenic control of resis-tance to PRR makes inheriresis-tance of resisresis-tance hard to pre-dict. The subterranean habit of the disease and the complex symptom expression are also complicating factors when trying to study this plant–pathogen interaction because monitoring the eVectiveness of inoculation procedures and the progression of root symptoms in soil-based assays is
diYcult. The hydroponic system makes it possible to score
the disease reaction in the pathogen’s target tissue
repeat-edly in a non-destructive manner (Pattison et al. 2004).
Strong correlations between the plant disease index, root regeneration score and incidence of petiole lesions have
been reported earlier (Pattison and Weber 2005). In the
cur-rent work, we have observed molecular evidence for this based on the co-localization of the identiWed QTL for those traits on the molecular genetic linkage map. QTL for stem lesion size, which was shown to possess a weaker correla-tion with plant disease index, was present on only two of the four declared QTL regions on both parental maps. Therefore, if the goal of selection were for absolute
resis-tance then the qualitative criteria would be appropriate. Alternatively, if population improvement with recurrent selection is the objective, quantitative data such as inci-dence of petiole lesions may help to identify those individu-als in breeding populations that possess intermediate levels of resistance with more conWdence.
Even with all the challenges in evaluating resistance to PRR, the results of this study favor the Nestby and Heiberg
(1995) conclusion that variation for resistance to PRR in
red raspberry has both signiWcant non-additive and additive components. In our study, the importance of the two genetic components varied depending on how resistance was assessed. The complex expression of secondary symp-toms (leaf chlorosis, petiole lesions, necrosis, and scorch-ing) makes an additive model more likely to account for the observed variability for the plant disease index, which eval-uates the whole plant response to infection. Multiple genetic factors likely inXuence traits such as the environ-mental stress response (i.e., nutrient and water stress) and their segregation in these populations may be responsible for the predominating additive model proposed. However, our data show that by evaluating individual symptoms of PRR, most of the variability in PRR resistance can be explained by dominance eVects in a two-gene system.
A genetic linkage map of red raspberry was recently constructed using AFLPs, SSRs, and EST-SSR markers using a pseudo-testcross mapping strategy (Graham et al.
2004). Our mapping strategy varied in that a segregating B2
population was used to generate two linkage maps, one for each parent, and used dominant markers. Our genetic map was approximately half the size of the published map of
Graham et al. (2004) and therefore, likely does not have
full genome coverage.
The RGA used in this study did not investigate the homology of ampliWed fragments to known resistance genes. Rather, the goal at this stage was to generate markers
segregating in a Mendelian fashion from sequence speciWc
primers designed from resistance genes, observe map placement and test for association with the resistant
pheno-types. AmpliWcation products were easily separated using
Metaphor agarose gels, which allowed for eYcient marker
generation. Few RGAP markers were found in close prox-imity to the identiWed QTL. This is the Wrst documentation of using this method as strictly a marker generation system for molecular mapping in red raspberry.
The two genetic analyses support the conclusion that resistance to PRR is conditioned by major genes in these red raspberry populations. Our experimental design for molecular mapping and subsequent QTL analysis did not allow for the Wne scale dissection and identiWcation of minor genes inXuencing resistance as the population size was appropriate only for identifying regions that produced
best suited for utilizing this type of genetic variation Wt well into contemporary raspberry programs, which already use recurrent selection and modiWed backcrossing for maximiz-ing the recovery of resistant progeny. Future work will focus on producing sequence speciWc markers in close proximity to the identiWed QTL to test the robustness of the association with the resistant phenotype in the cultivated germplasm of red raspberry.
Acknowledgments This project was funded in part by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education and Extension Service (NRI, USDA-CSREES), grant number #NYG-632526.
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