2024 Volume 93 Issue 3 Pages 232-241
Alternaria blotch, a major apple (Malus × domestica Borkh.) fungal disease in Japan, is caused by the Alternaria alternata apple pathotype that produces a host-selective toxin called AM-toxin. Although control of Alternaria blotch currently relies on spring-to-summer fungicide use, there is a growing need for sustainable agriculture practices that reduce chemical inputs in orchards. Therefore, breeding cultivars for resistance to Alternaria blotch is of particular interest. Given that ‘Golden Delicious’ (GD) and several of its offspring cultivars are moderately susceptible to the disease, a genetic analysis of their susceptibility was performed. Quantitative trait locus (QTL) analysis of the ‘Fuji’ × ‘GD’ population identified a single QTL on chromosome 11 in ‘GD’, which explained 48.7% of the phenotypic variation. This QTL was located in the same region as the previously identified susceptibility gene Alt derived from ‘Starking Delicious’. Therefore, we named it QTL Alt2, a putative allele of Alt (later renamed Alt1). Interaction analysis revealed that Alt1 was dominant over Alt2. A DNA marker set that simultaneously detects Alt1 and Alt2 was developed for breeding use. This marker shed light on the inheritance of the Alt locus in modern Japanese cultivars and selections. Moreover, Alt2 was less common than Alt1 in heirloom cultivars. These findings offer new insights into apple breeding for Alternaria blotch resistance and the interaction mechanism between apple and host-selective toxin-producing A. alternata.
Alternaria blotch, a fungal disease affecting apples (Malus × domestica Borkh.), is caused by the Alternaria alternata apple pathotype that produces a host-selective toxin (HST) known as AM-toxin (Tsuge et al., 2013). The disease is particularly endemic in East Asia (Sawamura and Yukita, 2014). Lesions caused by Alternaria blotch occur mainly on the leaves and sometimes on the fruit. In severe cases, leaves fall in summer and fruit damage can occur (Sawamura and Yukita, 2014). Additionally, early-season leaf drop negatively impacts flower-bud formation. The control of Alternaria blotch, which currently relies on the use of fungicides from spring to summer (Sawamura and Yukita, 2014), is economically important due to market fruit quality requirements. However, reducing chemical inputs in orchards is essential for sustainable apple production. One solution is to plant resistant cultivars in place of susceptible ones. Therefore, developing cultivars that are resistant to Alternaria blotch is a crucial priority in breeding programs (Abe et al., 2010).
The genetics of the interaction between apples and HST-producing A. alternata differs from other major diseases, such as apple scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotricha). In contrast to these other diseases, susceptibility dominates over resistance in HST-producing A. alternata interactions, and resistant cultivars are more common in genetic resources. Previous reports have studied the susceptibility derived from ‘Delicious’, revealing that susceptibility is dominant over resistance and is controlled by a single gene, Alt (Saito and Takeda, 1984). Alt was mapped on the top of chromosome 11, and sequencing of bacterial artificial chromosome library clones revealed a promising candidate encoding a coiled-coil–nucleotide-binding site–leucine-rich repeat (CC–NBS–LRR) type disease-resistant gene (Moriya et al., 2019). A DNA marker was developed to detect a 12-bp insertion in the promoter region of this gene, unique to Alt. Among the eight breeding founders, the susceptible cultivar ‘Indo’ carries Alt in addition to ‘Delicious’, but the others do not, namely, ‘Golden Delicious’ (GD), ‘Ralls Janet’, ‘Jonathan’, ‘Cox’s Orange Pippin’, ‘Worcester Pearmain’, or ‘McIntosh’ (Moriya et al., 2019).
Besides in-depth study of ‘Delicious’-derived Alt, the responses of apple cultivars and Malus species to inoculation with the A. alternata apple pathotype have also been investigated (Abe et al., 2010). While cultivars and materials from segregating populations carrying Alt show a highly severe reaction to A. alternata inoculation (Moriya et al., 2019), some cultivars, such as ‘Fuji’, ‘GD’, ‘Sekaiichi’, ‘Mutsu’, and ‘Orin’ exhibit susceptible or moderately susceptible responses (Abe et al., 2010). It is unclear whether they carry Alt1. Because the susceptibility level of these cultivars is distinct from Alt1 carriers, other major genetic factors besides Alt could control susceptibility. Elucidating the cause of susceptibility in these cultivars is essential to fully understand the interaction mechanisms between apples and HST-producing A. alternata. This knowledge will accelerate the breeding of Alternaria blotch-resistant cultivars.
In this study, we conducted quantitative trait locus (QTL) analysis to identify the causal locus (termed Alt2 of the Alt locus) conferring moderate susceptibility to Alternaria blotch. The most significant single nucleotide polymorphism (SNP) associated with susceptibility was used to develop a DNA marker for use in breeding. The interaction between Alt2, a newly identified susceptibility gene, and Alt1, a previously identified susceptibility gene, was investigated. We also clarified the inheritance of the Alt locus in modern Japanese apple cultivars and selections.
Eight founders of the modern apple breeding programs, 19 of their offspring mainly bred in Japan (modern cultivars/selections), and 35 heirloom cultivars from European and North American countries were used for screening of susceptibility genes and genealogical analysis (Table S1).
A segregating F1 population of 63 individuals derived from the ‘Fuji’ × ‘GD’ cross, which was previously used to construct genetic linkage maps (Kunihisa et al., 2016; Minamikawa et al., 2021), was used for QTL analysis of Alternaria blotch symptoms. In addition to the previous population, a segregating F1 population of 50 individuals derived from the cross ‘Orin’ × ‘Starking Delicious’ was used to analyze the interactions among susceptibility genes.
Plant materials were grown in the orchard of the Institute of Fruit Tree and Tea Science, NARO, Morioka, Japan. Cultivars and selections were grafted on dwarfing rootstocks and planted at least 1 m apart. Plants from segregating populations were planted in a nursery at 15 cm spacing with their own roots. Fungicide was sprayed every two weeks until late summer. Genomic DNA was extracted from young leaves using a robotic DNA extractor PI-50α (Kurabo), following the method described by Moriya et al. (2019).
Alt1 detection using a DNA markerFounders and modern cultivars were genotyped for Alt1 using the DNA marker developed by Moriya et al. (2019). PCR was performed in a 10 μL solution containing 1 × GoTaq G2 Hot Start Master Mix (Promega), 0.2 μM each of the three primers (Alt-F, Alt-R, and Alt-specific; Table S2), and 10 ng of genomic DNA. The amplification was performed using an initial denaturation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 10 min. PCR products were separated by gel electrophoresis, then visualized by staining with ethidium bromide.
Evaluation of Alternaria blotch symptomsThe AKI-3 monoconidial isolate of the highly virulent A. alternata apple pathotype, isolated in Akita, Japan in 1959 (Sawamura, 1962), was used for the inoculation test. The conidia from the media were stored at −80°C until use. Inoculation was performed using the detached leaf method (Abe et al., 2010). Briefly, the five, second, or third leaves were removed from the growing shoot and washed with distilled water. Leaves were inoculated with a conidial suspension (2 × 105 conidia·mL−1) diluted in 0.01% Tween 20 and placed in a Petri dish at 20°C and 100% relative humidity under dark conditions. At 48 h post-inoculation, each leaf was visually scored for disease severity on a scale of 0 (no symptoms) to 5 (near-complete necrosis of the entire leaf), as previously described (Abe et al., 2010). The mean symptom score of a single test was considered the score of the material. The ‘Fuji’ × ‘GD’ population was assessed twice in two years, while the ‘Orin’ × ‘Starking Delicious’ population was evaluated only once. The evaluation scores of the cultivars were adopted from a previous study by Abe et al. (2010), which used the inoculation test procedure described above. The resistance level was classified according to the scale of resistant (0–0.5), moderately susceptible (0.5–2.5), and susceptible (2.5–5) (Moriya et al., 2019).
Broad-sense heritability (h2)The disease score of the ‘Fuji’ × ‘GD’ population was analyzed using two-way analysis of variance (ANOVA). The following model was used:
Pij = μ + Gi + Yj + Eij,
where Pij denotes the disease score in the ith individual at the jth year, μ is the grand mean, Gi indicates the effect of the ith individual, Yj is the effect at the jth year, and Eij denotes the residual effect in the ith individual at the jth year.
ANOVA analysis partitioned the total variance into three components: variance among individuals (σg2), variance among years (σy2), and variance due to residuals (σe2). Broad-sense heritability (h2) was calculated using the following equation:
h2 = σg2/(σg2 + σy2/y + σe2/y),
where y indicates the number of years.
Genetic linkage map construction of the ‘Fuji’ × ‘GD’ populationGenotype data for simple sequence repeat (SSR) and SNP markers, obtained using the Apple 20K Infinium genotyping array (Bianco et al., 2014), were previously collected from 63 individuals of the ‘Fuji’ × ‘GD’ population (Kunihisa et al., 2016; Minamikawa et al., 2021) and used to construct a genetic linkage map. The linkage map was constructed using JoinMap 4.1 (van Ooijen, 2006), following the double pseudo-testcross strategy (Grattapaglia and Sederoff, 1994). The Haldane’s map function was applied. When more than two markers showed the same segregation pattern, only two markers were retained in the dataset, and the others were removed from the map construction.
QTL analysisQTL analysis was performed using MapQTL 6 software (van Ooijen, 2009), employing Kruskal-Wallis and interval mapping procedures. A significance level of P < 0.001 for the K* statistic and a logarithm of odds (LOD) value of P < 0.05, determined through a permutation test with 3,000 iterations, were used as the threshold for identifying QTL presence in Kruskal-Wallis and interval mapping, respectively.
Genotype database of founder cultivarsA genotype database comprising 197 apple cultivars and selections, constructed by Minamikawa et al. (2021), was used to identify polymorphism among the founder cultivars of the Japanese apple breeding program (i.e., ‘GD’, ‘Starking Delicious’, ‘Jonathan’, ‘Ralls Janet’, ‘Worcester Pearmain’, ‘Indo’, ‘Cox’s Orange Pippin’, and ‘McIntosh’). This database incorporates genotypes obtained using the Apple 20K Infinium genotyping array (Bianco et al., 2014).
DNA marker for simultaneous detection of Alt1 and Alt2A primer pair was designed to specifically amplify the Alt2-associated allele (Alt2-specific-F and Alt2-R; Table S2). The primer design was based on the flanking sequence of SNP ss475882335 (GDsnp01167), obtained from the Genome Database for Rosaceae (GDR) (Jung et al., 2019). A multiplex PCR assay, termed the Alt locus test (ALT) marker, was designed by combining the newly designed primers (Alt2-specific-F and Alt2-R; Table S2) with the Alt1-specific primer set (Alt-F, Alt-R, and Alt-specific; Table S2). PCR amplification was performed in a 10 μL solution containing 1 × GoTaq G2 Hot Start Master Mix (Promega), 0.2 μM each of 5 primers, and 10 ng of genomic DNA. The PCR conditions were same as for the Alt1-specific marker described above.
Because the ALT marker is a dominant marker, an SSR marker, Mdo.chr11.34 (Terakami et al., 2016), was also used to discriminate between homozygotes and heterozygotes in modern and heirloom cultivars. Mdo.chr11.34, located at 2.824 Mb of chromosome 11 in the GD double haploid 13 (GDDH13) genome version 1.1 (Daccord et al., 2017). PCR and capillary electrophoresis were performed as previously described (Moriya et al., 2012).
Interaction between Alt1 and Alt2One-way ANOVA was used to test the interaction between Alt1 and Alt2 in terms of susceptibility in the ‘Orin’ (Alt2 carrier) × ‘Starking Delicious’ (Alt1 carrier) population, with disease score and Alt genotypes as the response and explanatory variables, respectively. The Alt genotype was determined using the ALT marker described above. The Tukey-HSD test was used for multiple comparisons between Alt genotype groups. All statistical analyses were performed using R 4.2.2 software (R Core Team, 2022).
DNA marker analysis for Alt1 revealed the presence of this allele in nine cultivars/selections, namely, ‘Starking Delicious’, ‘Indo’, ‘Kinsei’, ‘Orei’, ‘RedGold’, Rero 11, ‘Hokuto’, ‘Kitakami’, and Morioka 61 (Table S1). Previous studies showed that all these apples, except Rero 11, are susceptible to Alternaria blotch (Abe et al., 2010; Suzuki et al., 1985). Disease score data for Rero 11 was not available and Alt1 was not detected in seven moderately susceptible cultivars: ‘GD’, ‘Ralls Janet’, ‘Mutsu’, ‘Orin’, ‘Fuji’, ‘Sekaiichi’, and ‘Kio’ (Table S1). This suggests that the susceptibility of these cultivars is governed by mechanisms distinct from Alt1. These cultivars can be divided into two groups: ‘GD’ derivatives (i.e., ‘Mutsu’, ‘Orin’, ‘Sekaiichi’, and ‘Kio’) and ‘Ralls Janet’ derivatives (i.e., ‘Fuji’). Consequently, we designed the ‘Fuji’ × ‘GD’ population to identify genetic factors contributing to their susceptibilities.
Alternaria blotch susceptibility in the ‘Fuji’ × ‘GD’ populationInoculation of the ‘Fuji’ × ‘GD’ population with the A. alternata apple pathotype revealed segregation in mean disease symptom severity between the two inoculation tests (Fig. S1A). The population exhibited two distinct groups: one with disease scores ranging from 0 to 0.5 and the other with scores between 0.5 and 2.5. A significant correlation coefficient of 0.65 (P < 0.001) was observed between the two inoculation tests (Fig. S1B), and the broad-sense heritability (h2) was determined to be 0.775 (Table S3).
Genetic map construction for the ‘Fuji’ × ‘GD’ populationThe genetic linkage maps for ‘Fuji’ and ‘GD’ comprised 17 linkage groups, spanning 1,154 and 1,391 centimorgans (cM), respectively (Table 1; Fig. S2). The numbers of loci were 878 and 784 for ‘Fuji’ and ‘GD’, respectively, suggesting that the maps provided comprehensive coverage of the entire apple genome.
Summary of genetic linkage maps for ‘Fuji’ and ‘Golden Delicious’.
Kruskal-Wallis QTL analysis of the disease score in the ‘Fuji’ × ‘GD’ population identified a single significant peak (K* = 35.6) on chromosome 11 from ‘GD’ (G11; Fig. 1A). No other significant peaks were detected on the remaining chromosomes (Fig. S3). Interval mapping analysis identified a QTL (LOD = 8.98) at the same genomic position on G11 (Figs. S3 and S4). This QTL explained 48.7% of the phenotypic variation that accounted for 62.8% of the genetic variance (h2). Two SNP markers, ss475882335, and ss475883590, were identified at the Alt2 peak on G11. These markers are physically located at 2.830 Mb and 2.695 Mb, respectively, on chromosome 11 of the GDDH13 genome version 1.1 (Daccord et al., 2017). Ss475882335 and ss475883590 lie close to the predicted gene MD11G1032000 (2.805–2.809 Mb), which is an allele of a promising candidate gene for Alt1 identified in ‘Delicious’. Consequently, this QTL was designated as Alt2. The mean disease scores for individuals carrying (cytosine [C]) and non-carrying (adenine [A]) Alt2 ranged from 0 to 2.4 and 0 to 0.5, respectively (Fig. 1B).
Quantitative trait locus (QTL) analysis of Alternaria blotch symptoms in the ‘Fuji’ × ‘Golden Delicious’ (GD) population. (A) Kruskal-Wallis analysis of mean disease score. The single nucleotide polymorphism markers ss475882335 and ss475883590, exhibiting the highest significance, are displayed. Significant markers (P < 0.001) are indicated by triangular dots. G11 denotes the linkage group (chromosome) 11 of ‘GD’. (B) Distribution of mean disease score based on the genotype of ss475882335 (GDsnp01167). Individuals possessing cytosine (C) and adenine (A) are Alt2-carriers and non-carriers, respectively. Two asterisks (**) indicate statistical significance at P < 0.01 using the t-test.
Genotype data for ss475882335 and ss475883590 were obtained from the genotype database (Table 2), showing that only ‘GD’ was heterozygous for both loci. The Alt2-associated allele cytosine (C) was only detected in ‘GD’ at ss475882335, demonstrating linkage disequilibrium between Alt2 and ss475882335. However, the Alt2-associated allele thymine (T) was detected in all cultivars at ss475883590, suggesting that ss475883590 is not useful for further study. A multiplex PCR was performed using Alt1- and Alt2-associated primer pairs (ALT marker), simultaneously amplifying the target alleles (Fig. 2). The amplified lengths of Alt1- and Alt2-associated products were 194 bp and 115 bp, respectively.
Founder genotypes for single nucleotide polymorphism (SNP) markers ss475882335 and ss475883590 associated with a significant quantitative trait locus (QTL) for Alternaria blotch susceptibility.
Gel electrophoresis image of the DNA marker (Alt locus test [ALT] marker) for simultaneous detection of Alt1 and Alt2. PC: Positive control amplified between the Alt-F and Alt-R primers. M: 100 bp ladder, S.D.: ‘Starking Delicious’, G.D.: ‘Golden Delicious’, W.P.: ‘Worcester Pearmain’, C.O.P.: ‘Cox’s Orange Pippin’, OSD06: the individual from the ‘Orin’ × ‘Starking Delicious’ population.
To elucidate the interaction of Alt1 and Alt2 on disease severity, the relationship between disease symptoms and Alt locus genotypes was investigated in the F1 population ‘Orin’ (Alt2 carrier) × ‘Starking Delicious’ (Alt1 carrier) (Fig. 3). The most severe disease symptoms were observed in the groups carrying the Alt1 susceptibility allele, either alone or in combination with Alt2. The group carrying only the Alt2 susceptibility allele exhibited less severe symptoms, followed by the group with no susceptibility alleles (alt homozygotes). There was no significant difference in mean disease scores between individuals carrying Alt1 and those with both Alt1 and Alt2. One individual with a score of 3 may be an outlier because it was assessed only once or because it was very susceptible.
Alternaria blotch symptom scores for genotypes at the Alt locus in the ‘Orin’ × ‘Starking Delicious’ population. Different letters above the plot indicate statistically significant differences at P < 0.05 based on the Tukey-HSD test.
Analysis of the ALT marker on cultivars identified Alt1 and Alt2 carriers (Fig. 4; Table S1). Among modern cultivars, seven and six cultivars carried Alt1 and Alt2, respectively. Of these, ‘Orei’ carried both Alt1 and Alt2. ‘Jonagold’ carried Alt2, but it was resistant to Alternaria blotch. Screening heirloom cultivars identified an American cultivar ‘White Winter Pearmain’ as an Alt2 carrier, which was also an Alt1 carrier (Table S1). The 288-bp allele of Mdo.chr11.34 was the only allele associated with both Alt1 and Alt2, and no cultivars were homozygous for either Alt1 or Alt2.
Pedigrees and inheritance of the Alt locus in modern apple cultivars and selections primarily bred in Japan. Alt1 and Alt2 are hypothesized to be alleles of the same Alt locus in ‘Delicious’ including its sports. N/A: not applicable.
In this study, we identified Alt2, a QTL on chromosome 11 of ‘GD’ that confers moderate susceptibility to Alternaria blotch. This QTL is likely to be allelic to Alt1, a susceptibility gene previously mapped in ‘Starking Delicious’. A DNA marker for simultaneous testing of Alt1 and Alt2 was developed. The combination of this DNA marker and genealogy analyses revealed the genetic causes of susceptibility to Alternaria blotch in cultivars developed by modern Japanese breeding programs. However, the genetic basis of susceptibility in ‘Fuji’ remains unclear.
Phenotypic evaluationCompared to the disease score conferred by Alt1 (> 2.5), the disease score conferred by Alt2 was less severe (Fig. 1B; Table S1), suggesting that Alt2 induces a milder susceptibility than Alt1. Year had no significant effect on disease score in the phenotypic evaluation of the ‘Fuji’ × ‘GD’ population, and the correlation coefficient between the two separate years was significant (Table S3; Fig. S1), indicating the accuracy of phenotypic evaluation. However, some individuals showed inconsistent results between the two tests, with a difference in disease score greater than 2. This fluctuation likely occurred due to the uneven leaf condition used in the study and the limitations of visual inspection for disease scoring. Maintaining uniform conditions for multiple plant materials in high-density plantings is difficult due to disease and pest infestations, leading to uneven leaf conditions between years. Leaf condition is a major factor for scoring the variability of Alternaria blotch susceptibility in ‘Orin’ (Abe et al., 2010). Additionally, unknown minor effect(s) from ‘Fuji’ could contribute to this fluctuation.
The disease scale proposed by Abe et al. (2010) set the boundary between scores 1 and 2 at a symptom diameter of 1 mm. This could cause a mischaracterization of the difference in disease severity at this boundary compared to the existing lesion area. Moreover, visual inspection is less precise than machine-based methods in measuring the exact area of leaf necrosis, confusing disease scoring and leading to a scattered score in some individuals. In future experiments, it would be preferable to use a machine-based method, such as the Leaf Doctor app (Pethybridge and Nelson, 2015), to accurately assess the disease area for QTL analysis.
QTL analysisQTL analysis identified Alt2 at the top of chromosome 11 of ‘GD’, accounting for 48.7% of the phenotypic variation (Figs. 1A and S4). The overlap in disease score distributions between Alt2 carriers and non-carriers in the 0–0.5 range indicates the possibility of a resistant phenotype even if they carry Alt2 (Fig. 1B). One example of this distribution in modern cultivars is ‘Jonagold’ (Fig. 4). This indicates that map-based positional cloning of Alt2 is challenging, as the genotypes at the Alt locus cannot be accurately inferred from individuals with these scores. However, given that the Alt2 peak is close to the Alt1 position and they share a common inheritance pattern (dominant susceptibility) for the phenotype, Alt1 and Alt2 are likely alleles of the same gene. Therefore, a candidate gene approach is a promising strategy to identify the causal gene for Alt2. Of the previously listed candidate genes, a disease-resistance gene is the most likely the cause (Moriya et al., 2019). Assuming that one allele of this gene is Alt2, positional cloning can be omitted, and a molecular biology approach such as gene editing can be used to confirm the causal gene of Alternaria susceptibility and generate breeding material for Alternaria blotch-resistant apple cultivars.
Conversely, no QTLs were identified on the ‘Fuji’ linkage map. When ‘Fuji’ was crossed with the resistant cultivar ‘Jonathan’, 8% of the progeny were susceptible (Abe et al., 2014). In addition, ‘Fuji’ carried neither Alt1 nor Alt2. These results suggest that the susceptibility of ‘Fuji’ is controlled by a mechanism other than Alt1 and Alt2, possibly a QTL with a small effect. QTL analysis power for detecting small-effect QTLs increases with population size (Beavis et al., 1994), suggesting that a larger segregating population is needed to identify QTL from ‘Fuji’.
DNA marker developmentThe ALT marker that simultaneously detected the Alt2-associated C allele of ss475882335 and the Alt1-specific 12-bp insertion (Moriya et al., 2019) is a valuable tool for marker-assisted parental and seedling selection and screening of genetic resources (Fig. 2). Although the ALT marker is a dominant marker, distinguishing between homozygotes and heterozygotes is important when screening genetic resources of unknown pedigree. For this objective, the Mdo.chr11.34 SSR marker can be used to evaluate the homozygosity of the Alt locus as the 288-bp allele of this marker co-segregates with both Alt1 and Alt2 in the materials listed in Table S1. The combination of the ALT and Mdo.chr11.34 SSR markers can accurately identify the Alt locus genotype in any material.
Interaction between Alt1 and Alt2Individuals carrying both Alt1 and Alt2 had the same disease score as individuals carrying only Alt1 (Fig. 3), suggesting that Alt1 is dominant over Alt2. However, since the phenotypic evaluation was only performed once in the ‘Orin’ × ‘Starking Delicious’ population, further evidence is needed to confirm the interaction. The predicted causal gene for Alt1 is a CC–NBS–LRR type disease-resistance gene, which may act as a receptor for AM-toxin or a component of the host protein complex for AM-toxin (Moriya et al., 2019). Due to its saprotrophic nature, A. alternata may exploit a particular step in the gene-for-gene interaction-mediated hypersensitive response between the candidate resistance gene and AM-toxin to invade apples (Moriya et al., 2019). Under this hypothesis, the functional difference between the translated proteins or the expression patterns of Alt1 and Alt2 may lead to different responses to A. alternata infection. This difference may be due to distinct affinities of Alt1 and Alt2 for AM-toxin or the AM-toxin complex, or in the strength of signal transduction toward the downstream response.
Because the C nucleotide base at ss475882335 (2,830,820 bp) in GDDH13 ver1.1 is consistent with the Alt2-associated allele, the flanking sequence of ss475882335 in GDDH13 ver1.1 is likely derived from the Alt2 coupling chromosome. Moriya et al. (2019) compared the deduced amino acid sequences of candidate genes, including Alt1 from ‘Starking Delicious’, the resistant allele (alt) from ‘Starking Delicious’, and GDDH13 (Alt2), which were hypothesized to encode proteins involved in susceptibility to Alternaria blotch disease. Interestingly, the CC and NBS domains of these three sequences contained two and one Alt1-unique amino acid substitutions, respectively, while the LRR domain contained six alt-unique amino acid substitutions (i.e., Alt1 and Alt2 had common amino acids). It remains to be determined whether these amino acid differences contribute to the phenotypic polymorphisms observed following infection with the A. alternata apple pathotype. Additionally, sequencing and comparative analysis of resistance alleles from other cultivars is warranted.
Genealogical implications of Alt1 and Alt2In Japan, apple crossbreeding activities began before the first documented case of Alternaria blotch in 1956 (Sawamura and Yukita, 2014). Therefore, early apple breeding programs prioritized fruit quality and adaptability to the humid and temperate Japanese climate rather than Alternaria blotch resistance. Consequently, susceptible cultivars such as ‘GD’, ‘Delicious’ (including its sports), and ‘Indo’ were frequently used as parents in these breeding programs without considering Alternaria blotch resistance, unintentionally incorporating susceptibility genes into renowned Japanese apple cultivars. Our study revealed their causative susceptibility genes (Fig. 4). Some apple cultivars, including ‘Kio’, are still used as parents in crossbreeding, and Morioka 61 is a valuable resource for mutation breeding through self-compatibility induction (Abe et al., 2023). Information on the Alt locus genotype will be a valuable asset for future breeding strategies to prevent the transmission of susceptibility genes to the offspring of breeding materials.
Alt1 and Alt2 frequency in heirloom apple cultivarsWe primarily used heirloom cultivars from USA (Table S1). Consequently, while the genetic diversity of domestic apples has been preserved over the last eight centuries (Gross et al., 2014), our sampling may be biased. Although Alt2 was less common than Alt1 in heirloom apple cultivars in this study, it would be interesting to determine the frequency of Alt2 in a diverse germplasm collection of cultivated apples, especially from Europe. Additionally, pedigree construction studies could help to elucidate the origin of susceptibility genes (Howard et al., 2023; Muranty et al., 2020).
An Alt2 carrier ‘GD’ and an Alt1 carrier ‘Delicious’ are widely used as parents in worldwide crossbreeding (Noiton and Alspach, 1996). This suggests that some modern cultivars may carry either Alt1 or Alt2, especially those from European and North American countries, where Alternaria blotch is not a major concern. Unlike countries that have not been affected by Alternaria blotch, Japanese apple breeders should be aware of whether the parents used in their breeding programs have either Alt1 or Alt2, especially when introducing cultivars from these countries.
Unveiling the molecular mechanisms underlying apple–HST-producing A. alternata interactionsThe interaction mechanisms between apple–HST-producing A. alternata and susceptible cultivars other than Alt1 and Alt2 remain unknown. ‘Ralls Janet’ and its offspring ‘Fuji’ are the first examples of these unidentified mechanisms, as neither Alt1 nor Alt2 were detected in these cultivars. In addition, ‘Cogswell’, ‘Wagener’, and ‘York Imperial’ represent further examples of unidentified interaction mechanisms, as they were moderately susceptible to A. alternata despite not carrying Alt1 or Alt2 (Table S1). Further research is warranted to elucidate the susceptibility of these cultivars.
Concerning the location of the causal locus for Alternaria blotch susceptibility, a marker development study in China identified the SSR marker CH05g07, located on chromosomes 12 and 14, as a flanking marker near the causal susceptibility gene in ‘GD’ (Li et al., 2011). Furthermore, CH05g07 was generally associated with resistance to Alternaria blotch in the USA (Li et al., 2019). However, some cultivars, such as ‘Jonathan’, exhibit inconsistent resistance levels in different reports; susceptible by Li et al. (2019) and resistant by Abe et al. (2010), suggesting that the mechanisms of interaction between apple–HST-producing A. alternata and apple cultivars may vary depending on the location from where the data were collected. To understand the mechanisms for these inconsistencies, future studies should investigate the characteristics of the fungi and molecular details of the responses in plants.
Terakami et al. (2021) reported that susceptibility to black spot disease in Japanese and Chinese pears (Pyrus pyrifolia and P. ussuriensis, respectively) was controlled by an orthologous locus of Alt, suggesting that the subtribe Pyrinae–HST-producing A. alternata shares common interaction mechanisms. These findings advance our understanding of the interaction mechanisms of apple–HST-producing A. alternata and contribute to the development of resistance breeding strategies.
In conclusion, the Alt2 gene, which confers moderate susceptibility to the A. alternata apple pathotype, was identified on the top of chromosome 11 of ‘GD’, corresponding to the position at which Alt1 was detected. Alt1 and Alt2 are likely alleles of the same gene, and the severe susceptibility caused by Alt1 is dominant over the moderate susceptibility caused by Alt2. We developed a DNA marker to detect both the Alt1 and Alt2 alleles, which removed Alternaria blotch susceptibility from apple breeding programs through marker-assisted selection. Our findings offer new insights into apple breeding for Alternaria blotch resistance and plant–HST-producing A. alternata interaction mechanisms.
