Abstract
Persimmon (Diospyros kaki Thunb.) is generally hexaploid, and a single AST locus controls the pollination-constant non-astringency trait on each of six corresponding chromosomes. The pollination-constant non-astringent (PCNA) genotype is nulliplex and requires homozygous recessive alleles (ast) at the AST locus. There are several non-PCNA cultivars/selections that could be cross parents; however, the probability of yielding nulliplex offspring depends on the number of recessive alleles (ast). In genotyping for the AST locus in hexaploid persimmon, in contrast to the situation in diploid plants, we need to detect the AST/ast allele dosage; this cannot be detected by common codominant markers. In this study, we detected the allele dosage of Mast, which is a marker allele strongly linked to the ast allele among cultivars, by quantitative real-time polymerase chain reaction (qPCR) using three reference sites, actin (DkAct), anthocyanin reductase (DkANR), and L5R, whose sequences are conserved in the genome of persimmon cultivars. Based on the allele dosage of the Mast, AST/ast genotypes were estimated for 63 non-astringent cultivars/selections, of which only five cultivars/selections were estimated to be simplex or duplex. The quantitative genotyping method using qPCR may be generally effective for polyploid plants.
Oriental persimmon, known simply as persimmon, is an important fruit crop in East Asia. Normally, persimmon accumulates abundant condensed tannins (proanthocyanidins) in fruit, which cause strong astringency. One of the main goals of the persimmon industry is to breed superior non-astringent cultivars. The most valuable and preferred cultivar is the pollination-constant non-astringent phenotype, which does not accumulate high amounts of tannins (Akagi et al., 2009a; Yonemori et al., 2000).
The trait of natural astringency loss in pollination-constant non-astringent (PCNA) is controlled by a single gene (AST) (Yonemori et al., 2000). Previous reports have suggested that expression of the PCNA genotype requires the AST locus to be nulliplex. Indeed, plants that are heterozygous for the AST locus will express the astringency trait in fruit flesh (non-PCNA type; Ikeda et al., 1985; Yamada and Sato, 2002). Cultivated persimmon is hexaploid (2n = 6x = 90) (Namikawa and Higashi, 1928; Tamura et al., 1998; Zhuang et al., 1990b), and Kanzaki et al. (2001, 2008) suggested the polysomic inheritance of the AST locus from the segregation ratio of AST-linked marker polymorphisms. Recently, Akagi et al. (2009b) determined the number of marker alleles linked to the AST locus using qPCR analysis and showed that six alleles are responsible for the astringency trait in persimmon, demonstrating that PCNA genotype is nulliplex at the AST locus.
In the actual breeding of PCNA-type persimmons, the hexaploid inheritance of the AST/ast allele restricts the breeder in intercrossing PCNA-type cultivars, selections, or both. When some non-PCNA-type cultivars were used as parents, PCNA offspring could only be obtained by a backcross of non-PCNA offspring obtained by a PCNA × non-PCNA cross to a PCNA genotype, and the percentage of PCNA-type offspring was low (Ikeda et al., 1985; Yamada and Sato, 2002). The ineffectiveness of such a cross is the main reason for the avoidance of non-PCNA genotypes in previous breeding programs. At present, the number of available PCNA-type cultivars is limited, and repeated crosses using these cultivars have resulted in inbreeding depression in progeny (Yamada et al., 1994). Hence, a good breeding strategy requires the inclusion of non-PCNA genotypes to widen the genetic pool and to avoid inbreeding depression.
In diploid plants, amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs), or simple sequence repeats (SSRs) are commonly used as dominant or codominant markers to detect polymorphisms (D'Surney et al., 2001). Dominant markers only detect the presence or absence of one of the two alleles. Codominant markers can detect the allelic state of both alleles, but are not usually used to quantify the allelic dosage in polyploid plants. In hexaploid persimmon, although a few restricted fragment length polymorphism (RFLP) or sequence-amplified characterized region (SCAR) markers linked to the AST/ast genes were identified by Kanzaki et al. (2008, 2009); these markers cannot detect the allelic state at the AST locus. In contrast to the situation in diploid plants, the relative ratio of two or more alleles must be determined to infer the genotype and the resulting phenotype of polyploid plants. Quantitative PCR has been used to measure chromosomal dosage for yeast (Bond et al., 2004) and to determine allele dosage in the tetraploid arabidopsis [Arabidopsis thaliana (L.) Heynh.] (Henry et al., 2006). In hexaploid persimmon, Akagi et al. (2009b) demonstrated the possibility of determining the AST/ast allele dosage by qPCR in a well-characterized selection. Information on the AST/ast allele dosage at the AST locus of a non-PCNA parent will greatly contribute to breeding superior PCNAs. If the ast allele of a non-PCNA parent is single dose (AAAAAa genotype) or double dose (AAAAaa), no hybridization can yield any PCNA offspring in crosses between non-PCNA and PCNA types because the ratio of the PCNA offspring depends on the ratio of a gamete whose genotype is “aaa,” which is derived from a non-PCNA parent (Table 1). When the ast allele of a non-PCNA parent is more than triple dose (AAAaaa, AAaaaa, or Aaaaaa), it is possible that crosses between the non-PCNA and PCNA yield PCNA offspring at ≥5% under autoploid or autoalloploid inheritance (Table 1). To date, a few reports and recent unpublished results on some persimmon cultivars suggested the possibility that hexaploid persimmon is basically autohexaploid, but a few cultivars show autoallohexaploid and the details are not fully understood (Akagi et al., 2009b; Akagi and Yonemori, 2008; T. Akagi, unpublished data). The ambiguous inheritance mode is suggested in general polyploidy plants (Comai, 2005). For instance, allotetraploid sour cherry (Prunus cerasus L.) derived from hybridization between the diploid sweet cherry (Prunus avium L.) and the tetraploid ground cherry (Prunus fruticosa L.) shows a complex mode of inheritance in interspecific reciprocal crosses between sweet cherry and ground cherry (Hauck et al., 2006).
Each genotype at the AST locus in non-pollination-constant, nonastringent (non-PCNA) persimmon parents and corresponding ratio of the pollination-constant, nonastringent (PCNA) phenotype in F1 progeny, in crosses with PCNA types.
Kanzaki et al. (2001, 2008, 2009) identified a polymorphic marker linked to the AST locus in two breeding selections. The polymorphisms linked to the AST or ast alleles in this marker locus were well-characterized and showed four large-scale indels (Fig. 1; Kanzaki et al., 2008; T. Akagi, unpublished data). One of them, the retroposon-like ≈2.5-kb insertion on this marker locus was strongly linked to the ast allele (Akagi et al., 2009b; Kanzaki et al., 2009). This marker allele is denoted as Mast, and the other three markers, which are linked to the AST allele, are denoted as MAST1–3 in this study (see Fig. 1). The objective of the present study is to demonstrate that qPCR analyses to determine the allele dosage of Mast can be used to show the various genotypes of the AST/ast locus in the 60 non-PCNA cultivars. Information on the ast allele dosage or frequency among the cultivars will also provide an insight into the progress of ast allele accumulation. Furthermore, we will discuss further application of quantitative genotyping with qPCR not only for the AST locus, but also for other loci with polysomic inheritance.
Large-scale polymorphisms in Mast and MAST1–3 (Kanzaki et al., 2009; T. Akagi, unpublished data), and the specific primer sets targeting each polymorphism shown in Table 3 (arrows indicate the primers). Primers with an asterisk were used in quantitative real-time PCR analysis for Mast (Table 3).
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
Large-scale polymorphisms in Mast and MAST1–3 (Kanzaki et al., 2009; T. Akagi, unpublished data), and the specific primer sets targeting each polymorphism shown in Table 3 (arrows indicate the primers). Primers with an asterisk were used in quantitative real-time PCR analysis for Mast (Table 3).
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
Large-scale polymorphisms in Mast and MAST1–3 (Kanzaki et al., 2009; T. Akagi, unpublished data), and the specific primer sets targeting each polymorphism shown in Table 3 (arrows indicate the primers). Primers with an asterisk were used in quantitative real-time PCR analysis for Mast (Table 3).
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
Materials and Methods
Identification of an ast-linked marker allele conserved among cultivars.
To confirm the validity of our hypothesis that Mast is linked to the ast gene but not to the AST gene among cultivars, we tested whether non-PCNA cultivars have at least one marker allele for MAST, while PCNA cultivars only have Mast. We used the 60 non-PCNA cultivars shown in Table 2 and the following 23 PCNA cultivars: Fujiwara-gosho, Fukuro-gosho, Fuyu, Gosho, Ikutomi, Izu, Izushi-ogosho, Jiro, Hana-gosho, Kanshu, Maekawa-jiro, Matsumotowase-fuyu, Midai, Mikado, Mushiroda-gosho, Ogosho, Okugosho, Suruga, Soshu, Taishu, Tenjin-gosho, Tokuda-gosho, and Yoshimoto-gosho.
Detection of the Mast allele dosage and estimation of the quantitative genotype in the AST locus by quantitative real-time PCR analysis in non-pollination-constant nonastringent (non-PCNA) persimmon cultivars.
Genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987) and was subjected to PCR analyses using the four PCR primers sets specific to the four marker alleles shown in Fig. 1. Briefly, we used primer sets (PCNA-F, E9R) for Mast, (E4, E9R) for MAST1, (A2F, E9R) for MAST2, and (A3F, A3R) for MAST3 (see Fig. 1; Kanzaki et al., 2009; T. Akagi, unpublished data). Primer sequences are given in Table 3. We analyzed the sequences of the PCR products amplifying the Mast, which is the 2.5-kb inserted fragment ast-linked marker allele, using ExoSAP-IT (GE Healthcare Bio-Sciences, Piscataway, NJ) and CEQ8000 (version 7.0; Beckman Coulter, Tokyo).
Primer information for general use in PCR analysis and for quantitative real-time PCR analysis. Each polymorphism of Mast and MAST1–3 is shown in Fig. 1.
Examination of the reference sites for qPCR in the persimmon genome.
In qPCR analysis to determine the allele dose in the genome, a reference site where the number in the genome and sequences are conserved among the samples is necessary (Henry et al., 2006). Transposable sites, such as transposon- or retroposon-like, or high-copy genes are not suitable because the detected number of the sites could vary among persimmon cultivars. Considering the possibility that there are null or mutated alleles in a reference site locus, we performed three independent qPCR analyses using three reference sites in this study. For the qPCR reference site, we selected the genomic sequence of the two genes encoding actin (DkAct) (accession no. AB473616; Akagi et al., 2009a) and anthocyanidin reductase (DkANR) (accession no. AB195284; Akagi et al., 2009a; Ikegami et al., 2007), and one genomic marker locus named L5R (accession no. AB537429; T. Akagi, unpublished data), the sequence of which is conserved in the genus Diospyros L. and is linked to the AST locus. To identify the conserved sequences of DkAct, DkANR, and L5R, their PCR products were directly analyzed using ExoSAP-IT and CEQ8000 (version 7.0). The conserved sequences were used as reference sites for qPCR. The primers for qPCR were designed using Primer Express Software (version 2.1; Applied Biosystems, Tokyo).
qPCR analysis for determination of the Mast allelic dosage.
We used the 60 non-PCNA cultivars shown in Table 2 to determine the Mast allele dosage using qPCR analysis. In addition to these cultivars, we used three breeding selections, 170–26, 275–13, and 325–22, whose genotypes at the AST locus have been suggested from the segregation ratio of their offspring (Kanzaki et al., 2001, 2007, 2008; Yamada and Sato, 2002). DNA samples were thoroughly purified with phenol and chloroform extraction and were diluted to a concentration of 60 ng·μL−1 to keep a constant amplification efficiency among samples in the following qPCR analysis. Samples were analyzed using an ABI PRISM 7900HT sequence detection system (Applied Biosystems) with the SYBR Green system using SYBR Premix Ex Taq (TaKaRa, Tokyo). All reactions were carried out in a total volume of 25 μL/well, consisting of 12.5 μL of SYBR Premix, 9 μL of sterilized distilled water, 1 μL of each of forward and reverse detection primers (5 μm), 0.5 μL of Rox dye, and 1 μL of template DNA. All DNA samples were completely denatured at 95 °C in a heating block for 10 min and were then cooled on ice before each reaction. The standard amplification protocol consisted of an initial denaturing step at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 57 °C for 5 s, and 72 °C for 15 s. The primer sequences used for detection of Mast, DkANR, and L5R are shown in Table 3.
Standard curves for the Mast and two reference sites were generated using two replications of eight serial 2-fold dilutions of genomic DNA of ‘Jiro’ (PCNA), which has six Mast in its genome (Akagi et al., 2009b). We set the maximum DNA concentration of the standard sample at approximately 120 ng·μL−1. In the qPCR analysis, the average threshold cycle (Ct) was automatically determined by ABI PRISM 7900HT as the default state. Ct is defined as the point at which fluorescence rises appreciably above the background. For each measurement, independent standard curves were constructed and three replications of each sample were analyzed. The mean Ct of three replications was used for each sample. The detected value of the Mast relative to that of the reference site represents the Mast allelic dosage in the genome relative to that of the standard sample ‘Jiro’.
Results
Mast is strongly linked to the ast gene in persimmon cultivars.
All 25 PCNA cultivars had only Mast among the four marker alleles shown in Fig. 1 (data not shown). In contrast, all 60 non-PCNA cultivars had at least one of the other three marker alleles, MAST1, MAST2, and MAST3 putatively linked to the AST gene (Fig. 1), and most of them also had Mast. These results indicate that only Mast is linked to the ast gene among the four alleles in the cultivars, and that non-PCNA cultivars possibly have many ast genes. The sequence analysis for Mast showed single nucleotide polymorphisms among the cultivars. We designed the primer sets (forward: PCNA-A1F and reverse: Co-A1R) by amplifying the Mast fragment specifically using the conserved sequences among the cultivars (Table 3).
qPCR analysis estimation of the ast allele dosage.
We designed three specific primer sets for reference site of qPCR analysis, (DkActRTF and DkActRTR) for DkAct, (DkANR-RTF1 and DkANR-RTR1) for DkANR, and (Consv-5F and Consv-5R) for L5R, using the conserved sequences among the cultivars (Table 3). In qPCR analysis, the amplification efficiency is an important factor affecting the results. The ideal value of the amplification efficiency is 100%, which means the quantity of amplified fragments is doubled after one thermal cycle of PCR reaction. In this qPCR analysis, the amplification efficiencies of the DkAct, DkANR, L5R, and Mast markers were on average 95.1% ± 1.2%, 96.4% ± 0.6%, 96.6% ± 1.3%, and 97.6% ± 2.7%, respectively, and all standard curves were significantly linear (P > 0.995) (Fig. 2, the standard curves generated from genomic DNA of ‘Jiro’). These were valid enough to allow analysis of the allelic dosage in the genome.
Standard curves for Mast (round points), L5R (square points), DkANR (triangular points), and DkAct (diamond-shaped points) in quantitative real-time PCR analyses using eight serial two-fold dilutions of the genomic DNA of ‘Jiro’ (PCNA). The x-axis indicates threshold cycle (Ct), automatically determined by ABI7900HT as the default state. The y-axis indicates relative DNA dosage expressed as logarithms. The minimum relative DNA dosage at the highest dilution of the genomic DNA of ‘Jiro’ was defined as “1.”
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
Standard curves for Mast (round points), L5R (square points), DkANR (triangular points), and DkAct (diamond-shaped points) in quantitative real-time PCR analyses using eight serial two-fold dilutions of the genomic DNA of ‘Jiro’ (PCNA). The x-axis indicates threshold cycle (Ct), automatically determined by ABI7900HT as the default state. The y-axis indicates relative DNA dosage expressed as logarithms. The minimum relative DNA dosage at the highest dilution of the genomic DNA of ‘Jiro’ was defined as “1.”
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
Standard curves for Mast (round points), L5R (square points), DkANR (triangular points), and DkAct (diamond-shaped points) in quantitative real-time PCR analyses using eight serial two-fold dilutions of the genomic DNA of ‘Jiro’ (PCNA). The x-axis indicates threshold cycle (Ct), automatically determined by ABI7900HT as the default state. The y-axis indicates relative DNA dosage expressed as logarithms. The minimum relative DNA dosage at the highest dilution of the genomic DNA of ‘Jiro’ was defined as “1.”
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 135, 1; 10.21273/JASHS.135.1.59
The Mast allelic dosage relative to that of the standard sample ‘Jiro’ was determined using qPCR analyses. We calculated the number of Mast alleles in the genome based on the fact that ‘Jiro’ has six Mast alleles (Akagi et al., 2009b). Our qPCR results accurately showed the number of Mast alleles in the genome and the estimated genotypes at the AST locus in the 60 non-PCNA cultivars and three breeding selections (Table 2). The number of Mast or the estimated genotype at the AST locus was generally consistent in the three independent calculations using DkAct, DkANR, or L5R as reference sites. This result supported the validity of the three reference sites for qPCR analysis of the persimmon genome. Only ‘Kuramitsu’ showed strikingly different Mast dosage or estimated genotypes, and a few cultivars had slighter inconsistencies, with the three reference sites (Table 2). This is possibly due to some mutated or null alleles in the reference sites that reduce amplification in qPCR analysis. To avoid this problem, several reference sites are needed for qPCR analysis when genotyping persimmon cultivars.
Most non-PCNA cultivars had zero to three Mast, indicating an estimated genotype of AAAAAA, AAAAAa, AAAAaa, or AAAaaa at the AST locus (Table 2). The ratio of each estimated genotype at the AST locus among the 60 non-PCNA cultivars was as follows: 14.3% (AAAAAA), 33.9% (AAAAAa), 32.1% (AAAAaa), 21.4% (AAAaaa), and 5.4% (AAaaaa or Aaaaaa). The average of the Mast allele dosage in a cultivar ranged from 1.72 to 1.77. The estimated ast allelic frequency in the 60 cultivars was 29%. Previous reports by Kanzaki et al. (2001, 2007, 2008) and Yamada and Sato (2002) suggested the AST/ast genotypes of three breeding selections, 170–26, 275–13, and 325–22, from the segregation ratio of their offspring in the AST/ast (non-PCNA/PCNA) phenotypes. The dominant AST allele dosage of 170–26, 275–13, and 325–22 was suggested to be triple, double, and single dose, which indicate the AST/ast genotypes of AAAaaa, AAaaaa, and Aaaaaa, respectively. Our results showed that the estimated genotypes of 170–26, 275–13, and 325–22 are AAAaaa, AAaaaa, and Aaaaaa, respectively (Table 2), which are consistent with the results from the segregation analyses.
The AST/ast genotypes of ‘Busshigano’, ‘Egosho’, and ‘Yoshino’ were estimated to be Aaaaaa or AAaaaa (Table 2). In addition, 12 cultivars were estimated to have the triple-dose ast alleles (Table 2). At present, when a non-PCNA-type cultivar is used as a parent, PCNA offspring are obtained only by backcrossing the second generation, and the ratio of obtainable PCNA offspring is low (Ikeda et al., 1985; Yamada and Sato, 2002). However, our results suggest that some non-PCNA cultivars have a triple dose or more of the ast allele, meaning that crosses between them and a PCNA genotype would yield about 5% to 50% PCNA offspring under autohexaploid inheritance in a single generation.
Discussion
Examination and application of the methodology of qPCR analysis for quantitative genotyping.
Quantitative PCR to determine the gene or allele dosage was first used in the field of medicine (Ochshorn et al., 2006; Zimmermann et al., 2002) because changes in gene dosage cause significant damage to humans. Plant molecular biologists applied qPCR to determine the copy number of a transgene (Song et al., 2002) and to study karyotypes in arabidopsis (Henry et al., 2006). Henry et al. (2006) also proposed the novel strategy of genotyping for polyploid plants by quantitative analysis of allele dosage with qPCR. In our study, we applied this qPCR methodology to quantitative genotyping of the AST locus in several hexaploid persimmon cultivars. We accurately estimated the AST/ast genotype in almost all of the non-PCNA cultivars studied (Table 2).
However, our methodology for the quantitative AST/ast genotyping by qPCR still has a few problems. There were significant calculation errors in the measurements for cultivars that had a high ast allele dosage, such as Busshigano, Egosho, and Yoshino (Table 2, see se). This is presumably due to the methodology of the qPCR analysis. When we compare the single-dose allele to double-dose allele by qPCR analysis, a 2-fold difference is detected. When we compare the quadrivalent-dose allele to pentavalent-dose allele, the ability to detect a 1.25-fold difference is required. That is difficult and accompanies some errors in analysis because the detection of a 1.25-fold difference means detection of a difference of about 0.322 (= log21.25) cycles in real-time PCR analysis. A solution to this problem is to measure the number of marker alleles linked to the dominant AST gene because the more the recessive ast alleles are, the less the dominant AST alleles are in a genome. The detection of dominant and recessive alleles by qPCR will enable exact quantitative genotyping. However, in this study, we could not detect the dosage of MAST linked to the dominant AST allele because of the MAST polymorphisms shown in Fig. 1. It is necessary to identify the conserved sequences linked to the AST allele or the AST gene itself to detect the dosage of the AST allele.
Quantitative genotyping by qPCR is effective in two respects, marker-assisted selection (MAS) and the genome mapping of polyploid plants. Quantitative genotyping by qPCR could contribute to MAS, especially for recessive traits in polyploid plants, such as the PCNA trait in persimmon, because the probability of obtaining nulliplex offspring greatly depends on the dosage of the recessive allele in the parents, an example of which is shown in Table 1. We can also apply quantitative genotyping for loci whose allele balances involve quantitative traits in polyploid plants. Furthermore, genome-wide mapping of autohexaploid sweetpotato [Ipomoea batatas (L.) Lam.] has been reported recently (Cervantes-Flores et al., 2008a, 2008b; Kriegner et al., 2003). In these reports, the segregation ratio of offspring was modeled corresponding to quantitative genotype in each marker locus. Allele dosage or quantitative genotyping in each marker locus is important in this mapping. Hence, the qPCR approach for quantitative genotyping would be possibly applicable to this genome-wide mapping in autoploid plants. To date, a number of maps in well-characterized autopolyploid plants, such as sugarcane (Saccharum L.) or potato (Solanum tuberosum L.), have been developed using various approaches (e.g., interspecific crossing or comparative genomic analysis) (Garcia et al., 2006; Le Cunff et al., 2008; Ming et al., 2002), but genome-wide mapping of other many autopolyploid plants has not progressed yet mainly due to various technical difficulties derived from genomic complexity (Henry et al., 2006; Luo et al., 2006). The qPCR method for quantitative genotyping could be one of the simple solutions to this problem and would facilitate the genome mapping in many autopolyploid plants, including persimmon.
Transition of ast allelic frequency possibly elucidates the origin of PCNA type.
Our results suggested that quantitative AST/ast genotypes vary among the 60 non-PCNA cultivars (Table 2). The ast allelic frequency was 29%. The cultivar with the highest ast allele dosage, Yoshino, originates from the same area as a PCNA cultivar, Gosho. In addition, ‘Egosho’ and ‘Busshigano’, which also contain a high ast allele dosage (Table 2), originate in areas near to that of ‘Gosho’ or other major PCNA cultivars such as Fuyu. The existence of ‘Gosho’ is first mentioned in the seventeenth century (Kikuchi, 1948), and ‘Gosho’ was the only PCNA cultivar to be grown over a wide area 200 years ago (Yamada, 2005). This suggests that ‘Gosho’ is the original PCNA, as inferred by Yamada (2005), and that the accumulation of the ast allele and the resultant generation of the PCNA phenotype occurred around the region where ‘Gosho’ arose. There are more than 1500 cultivars of persimmon in China, Japan, Korea, and Europe (Wang et al., 1997; Yamada, 2005). The ast allelic frequency is presumed to differ among countries. Therefore, a detailed detection of the ast allelic frequency among the cultivar groups in these areas could contribute to understanding how the ast allele has accumulated.
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