Abstract
The organic acid concentration in apple (Malus ×domestica) juice is a major component of hard cider flavor. The goal of this study was to determine if the malic acid markers, Ma1 and Q8, could classify the titratable acidity concentration in cider apple accessions from the United States Department of Agriculture Malus germplasm collection into descriptive classifications. Our results indicate that for diploid genotypes, the Ma1 marker alone and the Ma1 and Q8 markers analyzed together could be used to predict cider apple acidity (P < 0.0001). Alone, the Ma1 marker categorized acidity into low (<2.4 g⋅L−1), medium (2.4–5.8 g⋅L−1), and high (>5.8 g⋅L−1) groups. The combination of Ma1 and Q8 markers provided more specificity, which would be useful for plant breeding applications. This work also identified a significant difference (P = 0.0132) in acidity associated with ploidy. On average, the triploids accessions had 0.33 g⋅L−1 higher titratable acidity than the diploid accessions. Based on the results of this work, we propose a genetics-based classification system for cider apples with the acidity component defined by the Ma1 and Q8 markers.
Although fresh market apples comprise the majority of global apple (Malus ×domestica) production, cider apples have been of particular interest recently because their higher acidity and tannin content (a group of polyphenols with bitterness and astringency) can potentially produce a hard cider with a depth of flavor similar to that of wine. Cider-specific apples can have five-times to 10-times more tannins than dessert apples and a wide range of organic acid concentrations (perceived as sourness and often described as sharpness) (Thompson-Witrick et al., 2014). In the United States, there is currently more demand than supply for cider-specific apple cultivars (Pashow, 2018). In response to this supply chain imbalance, cider-specific cultivars, mostly of European origin, are being planted throughout the country (Miles et al., 2020). However, there is limited information regarding the juice quality characteristics of these apple cultivars.
The emerging U.S. cider industry has adopted a method of classifying cider apples that was originally developed at the Long Ashton Research Station (LARS) near Bristol, UK, over a century ago (Barker and Ettle, 1910). The LARS system classifies apple cultivars into one of four categories, sweet, bittersweet, sharp, or bittersharp, based on tannin and titratable acidity (TA) concentrations. Tannins were originally measured using the Löwenthal permanganate titration method (Snyder, 1893). A tannic acid concentration of 2 g⋅L−1 is used to separate nonbitter from bitter apples. Acidity was, and still is, measured using an acid–base titration at a pH endpoint of 8.1. Apples with a malic acid equivalent concentration less than 4.5 g⋅L−1 are classified as sweet, and those with a malic acid equivalent concentration greater than 4.5 g⋅L−1 are classified as sharp. Although plant genetics and sensory science have progressed greatly since the early 20th century, the LARS system has remained unchanged.
A French classification system is used to divide cider apples into the following six categories (English translation in parenthesis): amère (bitter); douce amère (bittersweet); douce (sweet); acidulée (subacid); aigre (sharp); and aigre amère (bittersharp) (Institut Français Des Productions Cidricoles, 2009). The acidity component of the French cider apple classification method has three categories, douce, acidulée, and aigre. The douce category is defined as having less than 4.5 g⋅L−1 TA, which is the same threshold used by the LARS classification system. The acidulée category is defined as TA values between 4.5 and 6.75 g⋅L−1 TA. The aigre category includes apples with greater than 6.75 g⋅L−1 TA.
A Spanish cider classification system has undergone changes more recently than the LARS classification system and now includes the following six technical groups: sweet (<1.45 g⋅L−1 tannic acid and <4.85 g⋅L−1 TA); bittersweet (>1.45 g⋅L−1 tannic acid and <4.85 g⋅L−1 TA); semi-acid (<1.45 g⋅L−1 tannic acid and 4.85–6.56 g⋅L−1 TA); semi-acid-bitter (>1.45 g⋅L−1 tannic acid and 4.85–6.56 g⋅L−1 TA); acid (<1.45 g⋅L−1 tannic acid and 6.56 g⋅L−1 TA); and acid-bitter (>1.45 g⋅L−1 tannic acid and >6.56 g⋅L−1 TA) (Ministerio De Agricultura, Pesca y Alimentacion, 2003).
The major organic acid in mature apple fruit is malic (≈90% to 95%, 3–5 g⋅L−1), followed by quinic (≈4%; 0.2–0.5 mg⋅L−1), citric (≈1.5%; 0.05–0.07 mg⋅L−1), and trace amounts (<0.05 mg⋅L−1) of ascorbic, shikimic, succinic formic, maleic, and tartaric acid (Wu et al., 2007; Zhang et al., 2010). The genetic underpinnings of apple acidity were first described in 1959, and subsequent studies have led to the identification and characterization of the malic acid (Ma) locus on linkage group 16 (Maliepaard et al., 1998; Nybom, 1959; Visser and Verhaegh, 1978; Xu et al., 2012; Yao et al., 2008). This locus has been reported to control 17% to 42.3% of the variation in acid concentration in apple fruit (Xu et al., 2012). The gene underlying Ma, named Ma1, has been identified to encode an aluminum-activated malate transporter-like protein (Bai et al., 2012; Khan et al., 2013). A single nucleotide mutation from the guanine (G) to adenine (A) at position 1455 in the coding sequence of Ma1 results in a premature stop codon that truncates 84 amino acids at the C-terminus, causing low acidity (Bai et al., 2012; Li et al., 2020). Therefore, the Ma1 allele, with “G” at position 1455, is associated with high acid (Ma), and the Ma1 allele with “A” at position 1455 is associated with low acid (ma). This distinction defines the difference between the dominant Ma and recessive ma alleles. However, the dominance of the Ma1 allele is incomplete, which was indicated by the wide range of TA values for the heterozygous Mama allele, suggesting that both additive and dominant effects of the Ma1 allele exist (Verma et al., 2019; Xu et al., 2012). Recently, it was found that in response to excess nitrate accumulation, the MdBT2 protein modulated and downregulated the expression of MdCIbHLH1 and MdMYB73, which regulate malate-related genes, thus reducing acidity in apples (Zhang et al., 2020).
Linkage group 8 also contains an important quantitative trait locus (QTL), named Ma3, that regulates apple acidity (Kumar et al., 2013; Liebhard et al., 2003; Ma et al., 2015; Sun et al., 2015; Verma et al., 2019). Recently, the Ma3 locus has been shown to have an incomplete dominance effect on apple acidity (Rymenants et al., 2020). Jia et al. (2018) identified two natural variations in hierarchical epistatic genes, MdSAUR37 and MdPP2CH, that affect fruit acidity in the Ma3 region. To genotype the Ma3 locus, a sequence tagged site (STS) marker, named Q8, was developed, which is physically located at 11.1 Mb between genes MdPP2CH (8.7 Mb) and MdSAUR37 (11.6 Mb) on chromosome 8 in the GDDH13 apple reference genome (Daccord et al., 2017; Jia et al., 2018). Additionally, three more QTLs, Ma4, Ma5, and Ma6 located on chromosomes 6, 1, and 4, respectively, were found to be relevant for fruit acidity levels (Ban and Xu, 2020; Rymenants et al., 2020). These QTLs appeared to explain more variation in the background of MaMa and Mama with relatively high acidity levels (>10 g⋅L−1) (Ban and Xu, 2020).
Because of the major effects of Ma1 and Ma3 loci on fruit acidity levels less than 10 g⋅L−1, we hypothesized that they could be used to predict acidity in apples, thereby allowing for classification into acidity ranges that would aid cider apple breeding, cultivar selection, and cider production. The goal of this study was to develop a genetic system for classifying M. ×domestica cider apple acidity using the Ma1 and Q8 markers.
Materials and Methods
Study location and accession selection.
Apples were harvested in 2017, 2018, and/or 2019 from the Malus germplasm collection maintained by the U.S. Department of Agriculture (USDA) National Plant Germplasm System in Geneva, NY (lat. 42°53′40.3″N, long. 77°00′23.8″W). We compiled a list of 330 M. ×domestica cultivars within the Malus germplasm collection that are mentioned in historic European and American texts and/or are currently being used for hard cider (Supplemental Fig. 1). Additional M. ×domestica accessions were identified by searching the USDA Germplasm Resources Information Network (GRIN) Global database (USDA, 2020) for accessions with astringent, aromatic, and/or acidic fruit that were greater than 50 g at harvest. Trees that appeared unhealthy or had insufficient fruit for analyses were excluded. Ploidy data were obtained from a 20,000 single-nucleotide polymorphism (SNP) array as part of an ongoing collaborative apple pedigree reconstruction project (Denancé et al., 2020; Howard et al., 2018; Muranty et al., 2020). Accessions without associated ploidy data were removed from the dataset. These steps led to the selection of 217 M. ×domestica accessions (Supplemental Table 1).
DNA extraction and accession genotyping.
Young leaves of the selected accessions were collected between 2017 and 2019. Leaf tissue (15–20 mg) was ground for 1 min using a tissue lyser (TissueLyserII; Qiagen, Venio, the Netherlands). Samples were incubated for 1 h in a hexadecyltrimethylammonium bromide (CTAB) extraction buffer containing polyvinylpyrrolidone (catalogue number BP431-500; Thermo Fisher Scientific, Waltham, MA) and β-mercaptoethanol (catalogue number BP176-100; Thermo Fisher Scientific) (Doyle and Doyle, 1987). A spectrophotometer (NanoDrop 1000; Thermo Fisher Scientific) was used for DNA quantification.
A cleaved amplified polymorphic sequence marker (CAPS1455) targeting base 1455 in the open reading frame of the Ma1 gene was used to distinguish the SNP between the Ma1 alleles, as previously described (Bai et al., 2012). Briefly, polymerase chain reaction (PCR)-amplified products were digested overnight with BspHI (New England Bio Laboratories, Ipswich, MA) in a 37 °C water bath overnight. Digested products were visualized on a 1.5% agarose gel, and Ma1 genotypes were determined based on band patterning. The PCR program included 2 min at 98 °C, 35 cycles of 10 s at 98 °C, 15 s at 55 °C, and 90 s at 72 °C, and then a final 5 min at 72 °C. The reactions were conducted in 20-μL volumes containing 1 × PrimeSTAR MAX DNA Polymerase (R045A; Takara/Clontech, Mountain View, CA), 0.5 mm of each primer, and 30 ng of genomic DNA in a gradient thermal cycler (Mastercycler® EP; Eppendorf, Hamburg, Germany). Restriction digestion was performed for 12 h at 37 °C in 20-μL reactions that contained 10 μL PCR products, 2 units of BspHI restriction enzyme (New England Biolabs, Ipswich, MA), and 1 × NEBuffer 4 (New England Biolabs). After sample incubation, 7 μL of sample and 3 μL of loading dye were injected into each well of a 1.5% (w/v) agarose gel. The samples were suspended in a 1-N Tris/Acetate/EDTA (TAE) buffer solution. After 1 h of electrophoresis, the gels were stained with ethidium bromide at a concentration of 2 μL to 100 mg of gel. Then, the banding patterns in the gel were illuminated with a 110-V ultraviolet light transilluminator (Thermo Fisher Scientific). The banding patterns in the gel images were visually scored and the Ma1 alleles for each accession were recorded.
The Q8 marker primer sequences were 5′-AAAAATTGAAACTTGTGGATCGTT-3′ (forward primer) and 5′-AAATCAAAAGCATACCACCACA-3′ (reverse primer). The marker was PCR-amplified using 1× OneTaqDNA Polymerase (New England BioLabs) with the following conditions: 2 min at 98 °C, 35 cycles of 30 s at 94 °C, 30 s at 54 °C, and 45 s at 68 °C. The PCR products were visualized on 1.5% agarose gel and scored for Q8 genotypes.
Fruit sampling and processing procedures.
Of the 217 M. ×domestica accessions, 32 accessions were collected for 3 years, 114 accessions were collected for 2 years, and 71 accessions were collected for 1 year (Supplemental Table 1). The biennial bearing habit of many of these accessions made fruit unavailable during every year. Fruit was sampled from mid-August to mid-November. Before harvest, two fruit per accession that were near the reported harvest date in GRIN-Global were field-tested in situ for maturity using the cortex starch pattern index (SPI) (Blanpied and Silsby, 1992). An iodine solution (0.22% w/v iodine, 0.88% w/v potassium iodine) (EMD Millipore Corp., Billerica, MA) was applied to the stem-side of an equatorial cross-section of the apple. A visual rating of the stained cortex flesh (hypanthium and mesocarp) was conducted and recorded using a scale ranging from 1 to 8, where 1 = 100% staining (no starch degradation) and 8 = 0% staining (complete starch degradation). As much as possible, fruit was harvested when they were rated to have a SPI of 6 or more.
Fifteen fruit were randomly harvested from different regions of the tree canopy while avoiding selecting two or more fruit from the same branch. The unique identifying plant introduction (PI) number given to each accession in the USDA-PGRU collection was recorded and used to track the fruit throughout the phenotyping process. After harvest, the 15 fruit were randomly divided into three groups of five apples to allow for three subsamples of five fruit per sampled accession (Evans et al., 2012). The fruit were stored at 4 °C in a commercial storage room under ambient atmospheric gases for 1 to 4 weeks before fruit processing analysis at the Cornell University Agricultural Experiment Station Research Orchards in Ithaca, NY.
The SPI was determined for all sample apples as described. The calyx half of each pooled subsample of five apples was milled and pressed in a juicer (model 280; Norwalk Juicers, Bentonville, AR). Juice from each subsample replication was stirred and aliquoted into 50-mL tubes. All juice-extracting equipment was rinsed with water between samples to minimize cross-contamination. Juice samples were stored at −80 °C until titrations were performed.
Juice titratable acidity and pH.
Samples were thawed to room temperature and then vortexed for 10 s. Juice TA and pH were measured with an automatic titrator (Unitrode pH meter, 778 sample processor, and 800 Dosino dosing device; Metrohm, Herisau, Switzerland). Juice acidity was measured by titrating a 5-mL juice aliquot against a standardized 0.1 N NaOH solution to an endpoint of pH 8.1 and expressed in grams per liter of malic acid equivalents.
Statistical analysis.
Linear models were developed using Ma1, Q8, or both markers as predictor variables and TA as the response variable with RStudio (version 1.1.442; RStudio, Boston, MA). The linear models were used to predict 95% confidence intervals (CIs) with the Estimated Marginal Means data analysis package. The data were not transformed before analysis. P ≤ 0.05 was considered statistically significant.
Results
Fruit maturity, titratable acidity, and pH.
The SPI for the 217 evaluated accessions ranged from 5 to 8 (mean, 6.96; se ± 0.06) (Supplemental Table 1). More than 85% of the accessions had an average SPI more than 6. The TA ranged from 1.09 to 11.53 g⋅L−1 (mean, 4.48 ± 0.16 g⋅L−1) (Fig. 1A). The pH ranged from 2.3 to 5.1 (mean, 3.84 ± 0.03) (Fig. 1B). The sample population had 113 (52%) accessions with TA less than the mean and 104 (48%) accessions with TA greater than the mean. There were two peaks in the TA distribution, one in the lower range representing most of the accessions with very low acidity with a peak at 1.5 g⋅L−1, and the other between 4.6 and 6.6 g⋅L−1, suggesting a bimodal distribution for fruit TA within our sample population.
The distribution of (A) titratable acidity and (B) pH of cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The distribution of (A) titratable acidity and (B) pH of cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The distribution of (A) titratable acidity and (B) pH of cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Allelic demographics.
Of the 217 genotyped accessions, 181 (83%) were diploid accessions and 36 (17%) were triploid accessions (Supplemental Table 1). For the Ma1 marker, 17 (9%) of the diploid accessions were homozygous dominant (MaMa), 107 (59%) were heterozygous (Mama), and 57 (32%) were homozygous recessive (mama) (Table 1). For the Q8 marker, which is a likely marker for the Ma3 gene, 129 (71%) of the diploid accessions were homozygous dominant (Q8Q8), 48 (27%) were heterozygous (Q8q8), and 4 (2%) were homozygous recessive (q8q8). The most common genotype combinations were Mama-Q8Q8 (66 accessions; 37%), mama-Q8Q8 (49 accessions; 27%), and Mama-Q8q8 (37 accessions; 20%) (Table 1). The other allelic combinations each represented less than 10% of the total sample population, including three MaMa-Q8q8 accessions, four Mama-q8q8 accessions, and eight mama-Q8q8 accessions. There were no MaMa-q8q8 or mama-q8q8 combinations among the accessions.
The number of cider apple accessions for each allelic combination of the Ma1 and Q8 markers. Leaves from the sample population (N = 217) were collected from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY.
Allele diversity was less robust for the triploid accessions. The mamama_Q8Q8Q8 combination comprised 39% of our triploid samples, Mama−_Q8Q8Q8 comprised 28% of our triploid samples, and Mama−_Q8q8- comprised 33% of our triploid samples (Table 1). This was partly because of a smaller sample size (N = 36) and the inability to identify the third allele in triploid heterozygous accessions.
Relationships among the genotypes and phenotypes.
Ploidy significantly influenced the TA concentration (P = 0.0132), with diploid accessions having 0.33 g⋅L−1 lower TA than triploids (4.43 and 4.76 g⋅L−1, respectively) (Table 2). The pH was not significantly affected by the ploidy level. The interactions between Ma1 and Q8 and ploidy were not significant, indicating that ploidy is independent of the Ma1 and Q8 allele composition. Because of ploidy being a significant factor for TA, diploid and triploid accessions were analyzed separately for each Ma1 and Q8 allelic combination using estimated marginal means analyses and CIs (Tables 3 and 4).
Fixed effects from a simple linear model of the Ma1 and Q8 markers and ploidy in a study of cider apple accessions (N = 217) harvested between 2017 and 2019 from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY.
Estimated marginal means and the lower confidence intervals (LCIs) and upper confidence intervals (UCIs) for juice titratable acidity and pH for the Ma1 alleles alone and Ma1 + Q8 allelic combinations among diploid (N = 181) cider apple accessions harvested between 2017 and 2019 from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. Some of the Ma1 and Q8 allele combinations were omitted because of nonavailability of accessions with those genotypes.
The estimated marginal means and the lower confidence intervals (LCIs) and upper confidence intervals (UCIs) for juice titratable acidity and pH within each of the Ma1 allelic combinations among triploid cider apple accessions (N = 36) harvested between 2017 and 2019 from the United States Department of Agriculture Malus germplasm collection in Geneva, NY. Some of the allelic combinations were omitted because of very low or no availability of accessions with those genotypes. The third allele in heterozygous triploid accessions was not identified; therefore, “−” represents an unknown allele.
For the diploids, the Ma1 marker had a significant effect on TA (P < 0.0001) and pH (P < 0.0001), but the Q8 marker did not show a significant effect for either TA or pH (Table 2). However, a detailed analysis of each Ma genotype group indicated that there was a significant difference between Q8Q8 and q8q8 within the Mama genotype, where all the q8q8 accessions were found. This suggests that the insignificant effect of Q8 on acidity of the 181 diploid accessions may have been related to the limited number of accessions (N = 4) that possessed the recessive q8q8 in our sample population (Tables 1 and 3, Fig. 2A). The analysis also revealed that for Mama and MaMa, the Q8Q8 genotype exhibited higher TA than Q8q8, although that difference was not significant (Fig. 2A, Table 3). In the mama group, the difference in acidity between the Q8Q8 and Q8q8 genotypes were negligible (Fig. 2A, Table 3). Consequently, when the Ma1 and Q8 markers were considered in combination, both TA (P < 0.0001) and pH (P < 0.0001) were correlated with the genotype (Table 2).
The variation in titratable acidity for the Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers, as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variation in titratable acidity for the Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers, as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variation in titratable acidity for the Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers, as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Classifying cider apples by phenotype, genotype, and region of origin.
The 217 cider apple accessions genotyped during this study were classified under the different existing regional classification systems based on acidity (Fig. 3). Using the 4.5 g⋅L−1 threshold from the LARS classification system, there was an approximately equal distribution of sweet (52%) and sharp (48%) cider apples. The French and Spanish classification systems showed a very similar spread of cider apple accessions, with 52% and 57% in the sweet category, 30% and 24% in the semi-acid category, and 18% and 19% in the acidic category, respectively (Fig. 3).
Comparison of cider apple acidity classification systems for cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. For the Long Ashton Research Station (LARS), French, and Spanish classification systems, the percentages of accessions were calculated by titratable acidity. For the marker-based classification systems, the percentages of accessions were calculated according to the Ma1 and Q8 allelic combinations for diploid (N = 181) and triploid (N = 36) accessions. The third allele in heterozygous triploid accessions was not identified; therefore, “−” represents an unknown allele.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Comparison of cider apple acidity classification systems for cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. For the Long Ashton Research Station (LARS), French, and Spanish classification systems, the percentages of accessions were calculated by titratable acidity. For the marker-based classification systems, the percentages of accessions were calculated according to the Ma1 and Q8 allelic combinations for diploid (N = 181) and triploid (N = 36) accessions. The third allele in heterozygous triploid accessions was not identified; therefore, “−” represents an unknown allele.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Comparison of cider apple acidity classification systems for cider apple accessions (N = 217) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. For the Long Ashton Research Station (LARS), French, and Spanish classification systems, the percentages of accessions were calculated by titratable acidity. For the marker-based classification systems, the percentages of accessions were calculated according to the Ma1 and Q8 allelic combinations for diploid (N = 181) and triploid (N = 36) accessions. The third allele in heterozygous triploid accessions was not identified; therefore, “−” represents an unknown allele.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The 95% CI range (lower to upper CI) from the estimated marginal means test for each Ma1 allele alone and Ma1 and Q8 allele combinations were used to develop classification thresholds for the diploid and triploid accessions separately (Table 3, Fig. 3). The Ma1 marker-based classification system discretely categorized the cider apple accessions with minimal overlap among the genotypes (Fig. 3). For the diploid accessions, the Ma1 marker could be classified into low [mama (<2.4 g⋅L−1)], medium [Mama (2.4 to 5.8 g⋅L−1)], and high [MaMa (>5.8 g⋅L−1)] acidity levels. There were only two accessions that overlapped between the upper threshold for diploid Mama (5.78 g⋅L−1) and the lower threshold for MaMa (5.60 g⋅L−1). The diploid Ma1 and Q8 allelic combinations categorized cider apples with multiple category overlaps; however, they provided more specificity. There was a significant difference between the Mama_q8q8 (1.62–4.81 g⋅L−1) and Mama_Q8Q8 (5.5–6.8 g⋅L−1) alleles.
The triploid accessions were classified into two categories solely using the Ma1 allele (mamama and Mama−). Homozygous dominant triploid Ma1 alleles were absent in the sample population. Using both the Ma1 and Q8 alleles for the triploid accessions, there were three identified categories of Ma1 and Q8 alleles (mamama_Q8Q8Q8, Mama−_Q8q8−, and Mama−_Q8Q8Q8). The triploid homozygous recessive ma1 alleles and homozygous dominant Q8 alleles (mamama_Q8Q8Q8) had an upper 95% CI value of 3.11 g⋅L−1, which was 0.69 g⋅L−1 higher than their diploid counterpart (mama_Q8Q8). There were six accessions that overlapped in TA between the upper CI of the Mama-_Q8q8- (6.88 g⋅L−1) and lower CI of MaMa−_Q8Q8Q8 (6.17 g⋅L−1) allele combinations (Table 4, Supplemental Table 1).
When the accessions were separated into broad geographic regions of origin, they exhibited a similar distribution, with accessions representing a TA range between 1.09 and 11.53 g⋅L−1 and pH between 2.3 and 5.1 (Fig. 4). Five accessions were removed because of unknown regions of origin and seven accessions were removed due to low representation (≤ 3 accessions). Triploid accessions were represented in all major regions of origin. Among the 80 French accessions, there was a noticeable skew toward low to medium acid accessions, with 55 accessions with a LARS acidity threshold less than 4.5 g⋅L−1 and only 25 accessions with a LARS acidity threshold more than 4.5 g⋅L−1. Among the 55 accessions with low acid, 41 had the mama_Q8Q8 genotype. Within accessions that originated in England (N = 48), approximately one-quarter (N = 13) also possessed the mama_Q8Q8 genotype. Twenty-two accessions originated in Central Europe; 19 of which possessed the heterozygous Mama alleles, indicating a strong skew toward medium acid accessions. All 14 Spanish accessions in the dataset had the homozygous dominant Q8Q8 allele and possessed all three types of the Ma1 alleles (MaMa, Mama, and mama). Three of the four accessions that possessed the homozygous recessive q8q8 genotype originated in North America.
The variations in titratable acidity and pH among cider apple accessions (N = 205) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY, sorted according to their country or region of origin.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variations in titratable acidity and pH among cider apple accessions (N = 205) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY, sorted according to their country or region of origin.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variations in titratable acidity and pH among cider apple accessions (N = 205) harvested between 2017 and 2019, from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY, sorted according to their country or region of origin.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Discussion
Marker-based system for categorizing cider apples.
This study lays the groundwork for using the Ma1 and Q8 (Ma3) acidity markers to categorize cider apple germplasm. Both the diploid and triploid Malus accessions had statistically significant correlations between the TA concentration and the Ma1 marker alone or Ma1 + Q8 markers analyzed together, but not the Q8 marker alone. Based on the Ma1 marker and the estimated marginal mean model with a 95% CI, the diploid accessions could be grouped into ranges of low [mama (<2.4 g⋅L−1)], medium [Mama (2.4 to 5.8 g⋅L−1)], and high [MaMa (>5.8 g⋅L−1)] acidity. These three categories had negligible overlap in TA concentrations among accessions in our sample population. However, using only the Ma1 marker resulted in less precision than using both the Ma1 and Q8 markers because of the larger range for each acidity category. Using both the Ma1 and Q8 markers resulted in more specificity (smaller ranges), but also extensive overlap among the seven allelic combinations. For example, there were 16 accessions that overlapped between the upper CI of the heterozygous Mama allele and the lower CI of the homozygous dominant MaMa when the homozygous dominant Q8 allele was constant between both Ma1 allele types. Therefore, the utility of using one or both markers would depend on the end-user’s goals. Using both the Ma1 and Q8 markers would be very beneficial to plant breeders in terms of precision breeding and making marker-assisted selections, whereas using the Ma1 marker alone would be useful for establishing a broad marker-based cider apple acidity classification system. The Ma1 marker is presently used in SNP arrays by breeders; however, there are no SNPs that are currently associated with the Q8 marker.
Even though we analyzed a robust list of cider apple accessions, there were only four diploid and no triploid homozygous recessive q8q8 genotypes. The lack of some allelic groups limited our ability to make conclusions about this allelic combination for identifying fruit acidity. It is unclear why there are very few cider apples with the homozygous recessive q8q8 alleles; however, the reason may be related to human selection of apples with higher acid for cider production.
There were 128 accessions with a TA concentration for the Mama genotype group that spanned both sides of the LARS 4.5 g⋅L−1 threshold. Therefore, the Ma1 marker cannot be used as a predictor of the acidity component of the LARS cider apple classification system. With three acid categories, the French and Spanish systems were somewhat more closely matched to the Ma1 acidity markers. For example, the Ma1 marker categorized accessions with less than 2.4 g⋅L−1 as low acidity, and the French and Spanish systems classified apples with less than 4.5 and 4.85 g⋅L−1 as douce and sweet, respectively. Medium acidity ranged from 2.4 to 5.8 g⋅L−1 for the marker-based system, whereas the medium ranges were 4.5 to 6.75 g⋅L−1 and 4.85 to 6.56 g⋅L−1 for the French and Spanish systems, respectively. In general, there was a 1- to 2-g⋅L−1 difference among the French, Spanish, and Ma1 marker-based classification systems. Although apple classification systems are widely used by apple growers and cider producers, we have not found documentation that explains how the specific TA thresholds were chosen. The variations among the English, French, Spanish, and our proposed marker-based systems could be attributable to the perception of other flavor components in the apples, such as astringency, bitterness, and sweetness, which affect the way that acidity is perceived (Hampson et al., 2000). More sensory studies must be conducted to analyze the interdependency and perception thresholds among these important flavor components.
Similar to other studies that investigated apple fruit acidity genetics, our analyses indicate that the Ma1 gene is a reliable predictor (P < 0.0001) of TA (Bai et al., 2012; Khan et al., 2013). Therefore, our study adds to the body of literature indicating that the Ma1 gene largely determines apple acidity (Bai et al., 2015; Brown and Harvey, 1971; Kouassi et al., 2009; Nybom, 1959; Visser and Verhaegh, 1978; Xu et al., 2012). Although apple fruit acidity is affected by geographic, horticultural, and seasonal variations, the controlling genes are static and can assist breeders in making selections and/or crosses and apple growers in choosing cultivars to plant.
For the mama genotype, Xu et al. (2012) reported a mean TA concentration of 2.06 g⋅L−1, which is similar to the concentration of 1.92 g⋅L−1 obtained during our study. However, for the Mama and MaMa genotypes, Xu et al. (2012) reported mean TA values of 8.46 and 10.38 g⋅L−1, respectively, whereas our data indicate mean TA concentrations of 5.45 and 6.42 g⋅L−1, respectively. The discrepancies between these studies may be attributed to the sample populations and differences in maturity at harvest. The study by Xu et al. (2012) used two mapping populations containing a maternal ‘Royal Gala’ and two paternal M. sieversii parents, whereas the sample population in our dataset consisted almost entirely of M. ×domestica cider cultivars. Duan et al. (2017) proposed that cultivated apples M. ×domestica possess two distinct genetic regions of substantially reduced genetic diversity near the Ma1 gene in comparison with progenitor species M. sieversii. Therefore, increased genetic diversity in M. sieversii could have resulted in the higher TA for the Mama genotype in the Xu et al. (2012) study.
The Ma1 and Q8 markers were the focus of our study because of their large genetic effect on fruit acidity that has been commonly detected in M. ×domestica accessions with acidity levels lower than 10 g⋅L−1 TA. We propose that the Ma4, Ma5, and Ma6 markers should be used in future studies to further delineate cider apples into more precise classes, particularly those with acidity levels higher than 10 g⋅L−1 (Ban and Xu, 2020; Rymenants et al., 2020).
Because organic acids are metabolized during the ripening process, pH usually decreases (Ma et al., 2015). It should be noted that pH is in a logarithmic scale, and small differences can result in sensorially detectable perceptions of the overall flavor and taste of the fruit (Hampson et al., 2000). The low-acid mama genotypes had a pH range between 4 and 5, which is greater than the ideal pH range (3.2–3.8) for microbial control during cider fermentation (Kosseva et al., 2016). Higher pH (>3.8) could result in microbial contamination and the development of off flavors during cider fermentation. The pH values of diploid and triploid accessions are presented in Supplemental Fig. 2A and B.
Within and Among year variability.
Apple fruit acidity can be affected by within year (for example, harvest maturity and fruit location within the canopy) and year-to-year variations (for example, environmental conditions, crop load, and other biotic factors such as disease pressure) (Brown and Harvey, 1971). The variation in TA has been found to be dependent on cultivar and year more so than horticultural practices in the orchard (Bourvellec et al., 2015). Multiple years of acidity data collection for many of the accessions in our study minimized those effects. Additionally, fruit from different parts of the tree were mixed to obtain a representative sample. Furthermore, to reduce variability attributable to the ripening stage, all accessions in this study were harvested at a similar maturity, as measured using the SPI.
Previous studies examining the relationship between the Ma1 gene and TA evaluated the fruit at a mean SPI between 4 and 6 (Bai et al., 2012, 2015; Xu et al., 2012). However, the current study was focused on apple accessions for the hard cider industry; therefore, the goal was to test the fruit when the SPI was between 6 and 8, a later stage in the maturity process. This late-stage timing was unique to this study and helped to gain a cider-specific focus on developing a genetic marker–based classification system for cider apples. Because TA fluctuates because of biotic and abiotic factors, a marker-based system provides a general range of how much acidity to expect; however, exact concentrations vary based on the aforementioned factors.
Diploid and triploid accessions.
This study is one of the first to elucidate a significant difference in acidity concentrations in cider apples attributable to ploidy. On average, the diploid accessions had a TA concentration 0.33 g⋅L−1 less than that of triploid accessions, despite the lack of the dominant MaMaMa triploid genotype in our sample population. Therefore, the mean triploid acidity values were skewed toward the heterozygous and homozygous recessive Ma1 genotypes. This study corroborates the evidence from other fruit crops, such as citrus (Citrus sp.) and blueberry (Vaccinium sp.), that ploidy increases acidity levels (Ahmed et al., 2020; Mengist et al., 2020). The effect of ploidy on increasing acidity levels in cider apples could be attributable to the quantitative and additive natures of acid biosynthesis controlled by the major Ma1 gene (Brown and Harvey, 1971). Therefore, more copies of the dominant Ma1 allele would increase acidity concentrations.
Evaluated germplasm.
The USDA Malus germplasm collection contains the world’s largest and most diverse catalog of Malus accessions, including a large collection of European and historic American cider apple cultivars, which was ideal for our analyses (Volk and Henk, 2016). The European cider cultivars in our study also included recently acquired traditional Spanish cider cultivars and traditional English and French bittersweet and bittersharp cultivars (Supplemental Table 1). A limitation to our study was that the USDA Malus germplasm collection consists of a single tree per accession and, thus, no field replication. Nonetheless, the sheer diversity of the collection has often been used to study the genetics of other important crop traits, such as dihydrochalcone content, volatile profiles, and anthocyanin production (Gutierrez et al., 2018; Sugimoto et al., 2015). Additionally, the geographically and genetically diverse accessions used for our study captured wide ranges of juice TA and pH that allowed us to identify potential genetic signatures to categorize cider apple genotypes. This diversity was further exploited to analyze accessions according to their country or region of origin.
Every country or region of origin had representative samples of low, medium, and high TA. There were some interesting trends observed during the analyses of acidity and region of origin. For example, 41 of the 80 analyzed French accessions possessed the homozygous recessive mama alleles and homozygous dominant Q8Q8 alleles, indicating a strong selection preference for low-acid accessions. The dominant Q8Q8 allele was observed in all the Spanish accessions, indicating that this genotype could be fixed in most Spanish accessions. Future pedigree and genetic linkage studies will uncover the history, movement, and introgression of progenitor species and lineages among the cider apple cultivars.
Future cider apple classification recommendations.
This is the first study to use a marker-based classification system to identify cider apples. Although we concluded that the Ma1 and Q8 markers could usefully segregate accessions in our sample pool, more germplasm must be evaluated to encompasses a wider range of allelic variability, particularly for triploids and accessions with the recessive q8 alleles. Identification of additional acidity genes in apples would also help to account for more variability. Furthermore, sensory analyses would help to understand the thresholds for the human perception of acidity at different TA concentrations, as well as elucidate how other factors, such as sugar and polyphenol content, could affect the perception of acid in apples and cider (Hampson et al., 2000; Rymenants et al., 2020). Finally, adding genetic markers for sugar and polyphenol content would create a robust suite of markers for plant breeders, horticulturists, and commercial cider producers to use to rapidly identify potential cider apple cultivars in germplasm collections and breeding populations.
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Flowchart illustrating the selection process for the cider apple accessions (N = 217) from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY that were used in this study.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Flowchart illustrating the selection process for the cider apple accessions (N = 217) from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY that were used in this study.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
Flowchart illustrating the selection process for the cider apple accessions (N = 217) from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY that were used in this study.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variation in pH for each of the combinations of Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019 from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. There were no mama-q8q8 or MaMa-q8q8 gene combinations. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variation in pH for each of the combinations of Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019 from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. There were no mama-q8q8 or MaMa-q8q8 gene combinations. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The variation in pH for each of the combinations of Ma1 and Q8 alleles among (A) diploid accessions (N = 181) and (B) triploid accessions (N = 36) between 2017 and 2019 from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY. There were no mama-q8q8 or MaMa-q8q8 gene combinations. The blue dots represent the estimated marginal means for each accession. The red plus signs represent outliers as defined by the estimated marginal means test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 146, 4; 10.21273/JASHS05056-21
The cider apple accessions (N = 217) from the U.S. Department of Agriculture Malus germplasm collection in Geneva, NY used in this study. The Plant Introduction (PI) number is a unique inventory number assigned by the U.S. Department of Agriculture. The Malus UNiQue genotype (MUNQ) code is based on single sequence repeat (SSR) data (Denancé et al., 2020). The accessions were genotyped for the Ma1 and Q8 alleles and phenotyped for titratable acidity (TA) and pH. The Ma1 and Q8 markers are unable to distinguish the third allele in heterozygous triploid accessions, thus “-” represents a missing allele. Titratable acidity, pH, and the starch pattern index (SPI) values are a mean of three biological replicates for each year the accession was harvested. The table is sorted by ploidy, followed by ascending TA concentration.
