bitterness inheritance in apricot (p. armeniaca l.) seeds

ORIGINAL PAPER

Bitterness inheritance in apricot (P. armeniaca L.) seeds

P. Negri & D. Bassi & E. Magnanini & M. Rizzo &

F. Bartolozzi

Received: 12 February 2007 /Revised: 13 January 2008 /Accepted: 2 March 2008 / Published online: 14 May 2008# Springer-Verlag 2008

Abstract Seed bitterness, due to cyanogenic glucosides, hasbeen reported in apricot as a recessive trait, being determinedby a single gene. In this study, 21 F1 and 10 F2 populationsfrom parents with either bitter or non-bitter (‘sweet’)phenotype were tested by seed tasting. Both the ‘bitter’ andthe ‘sweet’ phenotypes were represented in populations from‘bitter×bitter’ and ‘sweet×sweet’ crosses, as well as fromself-pollination of either bitter- or sweet-seeded trees,providing evidence that more than one gene is involved inthis trait. Ten populations showed segregation ratios incon-sistent with a monofactorial inheritance of seed taste with the‘sweet’ trait dominant over the ‘bitter’. On the other hand,data from spectrophotometric assays indicate that seedcyanoglucoside content cannot be regarded as a quantitativetrait. All the observed segregation ratios can be explained byan inheritance mechanism based on five, non-linked genes,involved in two distinct biochemical pathways. Three geneswould control different steps in an ‘additive’ pathway (eitherthe biosynthesis of cyanoglucosides, or their transport, orboth) leading to accumulation of these metabolites in seeds:

homozygosis of recessive alleles of at least one of themwould result in the sweet phenotype. Two more genes wouldprovide a cleaving activity, participating to cyanoglucosidecatabolism; heterozygosis or homozygosis of dominantalleles at these loci would produce the ‘sweet’ phenotype,while homozygosis for recessive alleles of at least one ofthem would interrupt the catabolic pathway, leading to the‘bitter’ trait, if associated with the anabolic function.

Keywords Apricot . Inheritance . Cyanogenic glucosides .

Complementary genes . Epistasis

Introduction

In various Prunus species, seed bitterness is due tocyanogenic glucosides (McCarty et al. 1952). In seeds,cyanoglucosides have been considered not only as allelo-chemicals (Nahrsted 1985; Jones 1988; Patton et al. 1997;Prates et al. 1998), but also as storage forms for reducednitrogen and carbonyl compounds that are catabolized upongermination for early plantlet growth (Clegg et al. 1979;Selmar et al. 1988; Swain and Poulton 1994).

In developing Prunus spp. seeds, these compounds aredetectable as the monoglucoside (R)-prunasin, which issubsequently converted to the diglucoside (R)-amygdalin,the prominent form in mature embryos (Frehner et al. 1990;Swain et al. 1992a; Swain and Poulton 1994). As shown inP. serotina (Wu and Poulton 1991; Swain et al. 1992b;Poulton and Li 1994), the release of HCN in undamagedseeds is prevented by different compartmentation of thecyanogenic glucosides, which are stored in cotyledonaryparenchyma, and their catabolic hydrolases, either confinedto cell walls and protein bodies at a subcellular level(mandelonitrile lyase) or restricted to specific procambial

Tree Genetics & Genomes (2008) 4:767–776DOI 10.1007/s11295-008-0149-x

Communicated by A. Abbott

P. Negri (*) : E. Magnanini :M. RizzoDipartimento di Colture Arboree, University of Bologna,via Fanin 46,40127 Bologna, Italye-mail: [email protected]

D. BassiDipartimento di Produzione Vegetale, University of Milan,via Celoria 2,20133 Milan, Italy

F. BartolozziIl sole 24 ore, Editoria specializzata,via Goito 13,40126 Bologna, Italy

cells (amygdalin and prunasin hydrolase). Moreover, in P.serotina, each of these catabolic enzymes occurs as severalisoforms, and the existence of multigene families of,respectively, eight and five members has been inferred formandelonitrile lyase (Hu and Poulton 1999) and prunasinhydrolase (Zhou et al. 2002).

In different plant species, compartmentation of cyano-genic glucosides other than amygdaline has been alsoobserved as an evolutionary strategy to prevent accidentalHCN release (Gruhnert et al. 1994).

Cyanogenic glucosides are synthesized by the maternaltissues and transported to the developing seeds. Indeed, inapricot as well as in almond (Kester and Asay 1975), all theseeds produced by a single tree show the same phenotype,i.e., either ‘bitter’ or ‘sweet’ (non-bitter) or, as reported foralmond and apricot populations, ‘slightly bitter’; thus, thepresence of cyanoglucosides in seeds has to be considered asa seed-parent trait. How these compounds can move in planttissues without being cleaved by catabolic enzymes (whichwould result in HCN production) remains an unansweredquestion. The assumption—derived from the linustatin path-way studied in Hevea brasiliensis cyanophoric seedlings—that the diglucoside amygdalin may represent the transportform in Rosaceae (Selmar et al. 1988) has not been confirmedby studies on germinating seeds of black cherry (Swain andPoulton 1994). Mostly unknown, too, are the underlyingmechanisms of differential accumulation of cyanoglucosidesin plant organs: this may be attributed either to their tissue-specific synthesis or, in the acyanogenic organs, to theirenzymatic cleavage without HCN production, immediatelyconverted into non-cyanogenic compounds by detoxifyingenzymes such as β-cyanoalanine synthase (Miller and Conn1980; Selmar et al. 1988; Swain and Poulton 1994).

The inheritance of cyanoglucosides has been studied inTrifolium repens, where two independent loci (Corkill1942) responsible for cyanoglucoside biosynthesis (Ac)and catabolism (Li) have been investigated at the biochem-ical and molecular levels (Hughes et al. 1988, 1990;Hughes 1991).

Cyanoglucoside inheritance has been studied in Prunusspp. in relation to seed bitterness. In peach, the ‘sweetkernel’ behaves as a recessive trait, controlled by a singlegene (sk), linked to the fuzzless skin (nectarine) trait(Werner and Creller 1997), and therefore assigned to thelinkage group 5 of a genetic map based on an interspecificalmond×peach cross (Bliss et al. 2002).

In almond, ‘sweet’ has been reported as a dominant traitinherited as a simple Mendelian factor (Heppner 1923,1926; Dicenta and García 1993); Spiegel-Roy and Kochba(1974) first suggested that the trait may be controlled by atleast three genes, but later discarded this hypothesis,accepting the monofactorial inheritance (Spiegel-Roy andKochba 1981).

In apricot, the monofactorial inheritance of kernel tasteshould correspond to the recessive allele being responsiblefor ‘bitter’ (Kostina 1977).

Bitter almond and apricot seeds are currently used forthe extraction of commercial amygdaline. The availabilityof ‘sweet’ apricot seeds could eventually increase theirmarketability, as by-products of the food processingindustry for the production of flavoring pastes and forother applications (Stoewsand et al. 1975). As a part of anongoing apricot breeding program, we have assessed the‘kernel taste’ trait for more than 10 years in cultivars andpopulations from controlled crosses, and the results arereported in this paper.

Materials and methods

Controlled crosses and trait evaluation

Precautions were taken to avoid outcrossing events, frompollen collection, flower emasculation (apricot has hermaph-rodite flowers), isolation of emasculated trees, seed collection,and tree planting. In the present study, F1 populations from 21apricot controlled-crosses were evaluated (Table 1). Male and/or female parents were cultivars with either bitter (‘Aurora’,‘Cricot’, ‘Farmingdale’, ‘Goldrich’, ‘Mono’, ‘NJA 54’,‘Ouardi’, ‘Pelese di Giovanniello’, ‘San Castrese’, ‘SH 47’,‘Tirynthos’) or sweet seeds (‘Boreale’, ‘Harcot’, ‘Harogem’,‘NJA 1’ and ‘Reale d’Imola’). ‘Cricot’, ‘Goldrich’, ‘Harcot’,‘NJA 1’, ‘Ouardi’, ‘Reale d’Imola’, ‘San Castrese’, and‘Tirynthos’ recurred in more than one cross, either as male orfemale parents. The F2 generation of 10 self-pollinated,either ‘bitter’ or ‘sweet’ accessions was also evaluated.Among the F1 and F2 populations, half-sib groups wereavailable, sharing one of the parents (for instance, ‘Ouardi’×‘Reale d’Imola’, ‘Ouardi’×‘Tirynthos’, ‘Reale d’Imola’×‘Tirynthos’, ‘Tirynthos’ self-pollinated). Molecular markerassays were run through the populations, and the fewseedlings from outcrossing (less than 2%: data not shown)were discarded. Out of the 31 populations, six were made upof more than 100 seedlings (trees on their own roots raisedfrom the original seeds); six included 55–86 and thirteen28–48 individuals, while the six smallest ranged from 14to 23 seedlings.

Seeds of single F1 or F2 individuals were controlled forbitterness in at least two different years. In preliminaryobservations bitter taste intensity, depending on sensoryevaluation, was difficult to assay; because of this, only twophenotypic classes were considered: ‘bitter’ and ‘sweet’. Eachseedling was classified by tasting samples of mature peeledseeds. The observed segregation ratios were tested based onsimple or complex inheritance hypotheses (from two to fivegenes) by the χ2 test calculated with the Yates correction for

768 Tree Genetics & Genomes (2008) 4:767–776

continuity (Zar 1984). Hypotheses either disagreeing with atleast one of the observed segregation ratios, or yieldingincompatible results for at least one of the half-sib populationgroups (i.e., a×b, b×c, a×c), were discarded.

Detection of cyanogenic glucosides

To assess whether the difference between ‘bitter’ and ‘non-bitter’ taste, sporophyte-dependant trait clearly perceived at

the sensory level, did correspond, respectively, to eitherpresence or absence of cyanogenic glucosides, four matureseeds were collected in a single year from individual treesand analyzed in 21 cultivars and in a variable number (14 to31) of F1 seedlings from ‘sweet’×‘sweet’ (‘NJA 1’×‘Realed’Imola’), ‘bitter’× ‘sweet’ (‘Cricot’× ‘Harcot’, ‘NJA54’×‘Reale d’Imola’, ‘Mono’×‘NJA 1’), and ‘bitter’×‘bitter’populations (‘Goldrich’×‘Cricot’, ‘Goldrich’×‘Pelese diGiovanniello’). Seeds were air-dried, individually ground

Table 1 Seed bitterness segregations observed in 31 apricot populations (21 as F1 populations and 10 as F2 populations) interpreted on the basisof two contrasting hypotheses (1 or 5 genes)

Population Size (no.) Observedfrequencies (%)

Single gene with ‘sweet’dominant

Five genes (B1 B2 B3+S1 S2)

Expectedfrequencies (%)

χ2a Expectedfrequencies (%)

χ2a

Bb Sc Bb Sc Bb Sc

‘BO 81604331’(‘San Castrese’×‘Reale d’Imola’)×‘Cricot’ 23 39.13 60.87 50.00 50.00 0.69 (ns) 50.00 50.00 0.69 (ns)‘BO 82604010’ (‘Palummella’×‘Reale d’Imola’) s.p.d 44 13.64 86.36 25.00 75.00 2.45 (ns) 14.06 85.94 0.02 (ns)‘BO 82604011’ (‘Palummella’×‘Reale d’Imola’) s.p.d 45 100.00 0.00 100.00 0.00 – 100.00 0.00 –

‘BO 82607002’ (‘Reale d’Imola’×‘Monaco Bello’) s.p.d 28 39.28 60.72 25.00 75.00 2.33 (ns) 43.75 56.25 0.08 (ns)‘BO 83604013’ (‘Monaco Bello’×‘Reale d’Imola’) s.p.d 14 7.14 92.86 25.00 75.00 – 10.55 89.45 –

‘BO 83604014’ (‘Monaco Bello’×‘Reale d’Imola’) s.p.d 110 100.00 0.00 100.00 0.00 – 100.00 0.00 –

‘Boreale’×‘Harcot’ 28 25.00 75.00 25.00 75.00 0.00 (ns) 25.00 75.00 0.00 (ns)‘Goldrich’×‘Cricot’ 122 100.00 0.00 100.00 0.00 – 100.00 0.00 –

‘Goldrich’×‘Pelese di Giovanniello’ 43 100.00 0.00 100.00 0.00 – 100.00 0.00 –

‘Goldrich’×‘Reale d’Imola’ 48 64.58 35.42 50.00 50.00 3.52 (ns) 62.50 37.50 0.02 (ns)‘Harcot’×‘Aurora’ 63 49.21 50.79 50.00 50.00 0.00 (ns) 50.00 50.00 0.00 (ns)‘Harcot’×‘BO 81604133’ (‘San Castrese’×‘Reale d’Imola’) 45 35.55 64.45 25.00 75.00 2.10 (ns) 37.50 62.50 0.01 (ns)‘NJA 1’×‘Mono’ 81 49.38 50.62 50.00 50.00 0.00 (ns) 50.00 50.00 0.00 (ns)‘NJA 54’×‘Reale d’Imola’ 23 34.78 65.22 50.00 50.00 1.56 (ns) 37.50 62.50 0.00 (ns)‘Ouardi’×‘Reale d’Imola’ 39 66.67 33.33 50.00 50.00 3.69 (ns) 62.50 37.50 0.14 (ns)‘Reale d’Imola’×‘Harogem’ 18 38.89 61.11 25.00 75.00 1.18 (ns) 43.75 56.25 0.03 (ns)‘Reale d’Imola’×‘San Francesco’ 140 30.00 70.00 25.00 75.00 1.61 (ns) 32.81 67.19 0.38 (ns)‘Reale d’Imola’×‘Tirynthos’ 55 45.45 54.55 50.00 50.00 0.29 (ns) 46.87 53.13 0.01 (ns)‘San Castrese’×‘Reale d’Imola’ 319 49.85 50.15 50.00 50.00 0.00 (ns) 50.00 50.00 0.00 (ns)‘San Castrese’×‘Tirynthos’ 28 100.00 0.00 100.00 0.00 – 100.00 0.00 –

‘SH 47’×‘Reale d’Imola’ 29 48.27 51.73 50.00 50.00 0.00 (ns) 50.00 50.00 0.00 (ns)

‘BO 82605005’ (‘Portici’×‘Reale d’Imola’) s.p.d 47 6.38 93.62 25.00 75.00 7.70 (**) 10.55 89.45 0.48 (ns)‘BO 82607001’ (‘Reale d’Imola’×‘Monaco Bello’) s.p.d 35 42.85 57.15 25.00 75.00 5.04 (*) 43.75 56.25 0.00 (ns)‘BO 86611014’ (‘NJA 1’×‘Reale d’Imola’) s.p.d 21 76.19 23.81 100.00 0.00 – 75.00 25.00 0.02 (ns)‘Cricot’×‘Harcot’ 59 35.59 64.41 50.00 50.00 4.34 (*) 37.50 62.50 0.03 (ns)‘Cricot’×‘Reale d’Imola’ 252 69.84 30.16 50.00 50.00 38.89 (**) 75.00 25.00 3.57 (ns)‘Farmingdale’ s.p.d 15 33.33 66.67 100.00 0.00 – 42.19 57.81 0.19 (ns)‘Harcot’×‘Reale d’Imola’ 77 37.66 62.34 25.00 75.00 5.92 (*) 37.50 62.50 0.02 (ns)‘NJA 1’×‘Reale d’Imola’ 151 42.38 57.62 25.00 75.00 23.41 (**) 43.75 56.25 0.06 (ns)‘Ouardi’×‘Tirynthos’ 86 60.46 39.54 100.00 0.00 – 56.25 43.75 0.46 (ns)‘Tirynthos’ s.p.d 34 32.35 67.65 100.00 0.00 – 42.19 57.81 0.97 (ns)

In bold: bitter-seeded.Upper part: populations compatible with the monofactorial inheritance of ‘sweet’ as a dominant trait.Lower part: populations whose segregation ratios do not match the single-gene inheritance hypothesis.a Calculated with Yates chi-square correction for continuity (critical χ2

1df; P=0.05=3.841); (ns): not significant (p>0.05); (*): significant (0.01<p<0.05); (**): highly significant (p<0.01); (–):χ2 test can not be appliedb Frequency of the ‘bitter’ phenotype (%).c Frequency of the ‘sweet’ (non-bitter) phenotype (%).d s. p. = self-pollinated (individuals examined at the F2 level)

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after removing the integuments, then analyzed for theircyanogenic glucoside content. The methodology is based onthe spectrophotometric detection of CN− released fromground tissues after enzymatic digestion with β-glucosidases(Masia and Cabrini 1994). In addition, to check thereliability of tasting as a method to estimate cyanogenicglucoside levels in apricot kernels (Stoewsand et al. 1975),some of the samples were classified into six groups ofbitterness by taste scoring from 0 (no bitter) to 5 (extremelybitter). Other than in seeds, the cultivars were also tested forcyanogenic glucosides in the bark of 1-year-old shoots (four,collected the same year in late summer, from single fieldgrown trees).

Results

Segregation ratios

Out of the 31 tested populations, 21 showed segregationratios of seed bitterness compatible with the monofactorialinheritance of ‘sweet’ as a dominant trait (Table 1, upper

part). However, the remaining ten populations (Table 1,lower part) were inconsistent with this hypothesis. Withineach population, seedlings evaluated for two or more yearsalways showed either the ‘bitter’ or the ‘sweet’ phenotype(data not shown), as previously noted for almond (Spiegel-Roy and Kochba 1974).

Cyanogenic glucosides

The results of cyanoglucoside detection for some apricotaccessions are reported in Table 2. The seed-released CN−

ranged from 1,000 to 3,000 ppm in most of the ‘bitter’cultivars, corresponding to amygdaline concentrations of1.5–5%, comparable to previous findings in apricot(Stoewsand et al. 1975; Femenia et al. 1995). The highvariability recorded in some cases (such as ‘Massa’ and ‘PA7005-2’) could be due to the small sample size; neverthe-less, variability is largely expected for cyanogenic gluco-sides, as their transport could be affected by seed position(topography) within the tree, like for other storagemetabolites (e.g., proteins, starch, sugars) in seeds or fruits.Although it has been reported that in apricot seeds acyanide content as low as 180 ppm is not perceivable bytaste as ‘bitter’ (Stoewsand et al. 1975), in our assays theseeds classified as ‘sweet’ (such as those of ‘Reale di

Table 2 CN− released by individual seeds and bark samples afterenzymatic digestion with β-glucosidases, measured spectrophotometri-cally (ppm)

Cultivara CN− (ppm)

Seedb Barkb

‘A 2478’ 3,929.9 (564.0) 284.0 (100.7)‘Bebeco’ 7,082.7 (410.1) 59.3 (41.7)‘Canino’ 2,386.2 (624.8) 672.3 (109.5)‘Cricot’ 2,224.4 (430.1) –‘Farmingdale’ 1,404.7 (77.5) –‘Goldrich’ 2,084.0 (107.3) –‘Harcot’ – 408.3 (83.9)‘Helena de Roussillon’ 920.0 (420.1) 137.5 (26.8)‘Hellin’ 1,353.8 (625.1) –‘Ivresse’ – –‘Massa’ 1,959.6 (1,006.2) –‘Mono’ 1,393.4 (299.2) 73.4 (22.5)‘NJA 1’ – 14.7 (5.7)‘NJA 53’ 4,296.2 (751.7) 57.5 (15.0)‘Ouardi’ 1,553.8 (523.0) 1,120.1 (321.7)‘PA 7005-2’ 1,961.3 (744.9) –‘Pelese di Giovanniello’ 1,835.1 (33.8) –‘Reale d’Imola’ – 20.6 (4.4)‘San Castrese’ 1,785.9 (538.3) 4.0 (2.1)‘Tirynthos’ 1,181.6 (127.8) –‘Venturina’ 3,388.3 (1,091.8) –

– Values below the assay sensitivity threshold of 1 ppma In bold: bitter-seeded genotypesb In brackets: standard deviation

Fig. 1 CN− seed content in apricot F1 seedling populations derivedfrom controlled crosses (ppm on dry weight)


Imola’, ‘NJA 1’, ‘Harcot’ and ‘Ivresse’) did not developHCN at all, as previously found in other ‘sweet’ apricotgenotypes (Femenia et al. 1995). However, as regards thebark samples, an appreciable level of cyanogenic gluco-sides was detected in the ‘sweet’ cultivars ‘Harcot’, ‘Realedi Imola’, and ‘NJA 1’, but not in all the ‘bitter’ genotypes.

In six populations, all the 28 seedlings classified as‘sweet’ were negative for seed cyanoglucosides (thesensitivity of the assays is below 0.002%), while the 86bitter-seed seedlings showed from 190 to 2,500 ppm ofCN− content, corresponding to 0.3–4.2% amygdaline(Fig. 1). The bitter intensity scored by taste in the lattergroup did not correlate to seed cyanoglucoside level (r=0.005: data not shown), possibly due to the interference ofother compounds with the perception of the sole amygda-line taste. In this respect, seed tasting can be considered a

method reliable only to qualitatively detect the presence ofthe metabolite.

Discussion

The size of the populations was regarded as generallyreliable in term of driving sound inheritance hypotheses,and the six smallest progenies with less than 25 individuals(Table 1) were considered only as a confirmation of theoverall results.

Out of the 31 tested populations, ten (including two largeprogenies with 151 and 252 individuals) showed segrega-tion rates inconsistent with the monofactorial inheritance ofseed bitterness as a recessive trait in apricot (Kostina 1977).Indeed, the high frequencies of the sweet phenotype

Fig. 2 Flow chart assemblingall the gene interactions deducedfrom inheritance hypothesessharing the assumption that theeither bitter (a) or non-bitterphenotype (b) results from theaccomplishment (+) or break-down (−) of two metabolicpathways: cyanoglucosideanabolism (one to three genes)and catabolism (one to threegenes), with the eventual inter-ference of an inhibitor gene ateither one or both. The 23checked hypotheses (Table 3)involve a maximum of fivegenes (those not always includ-ed are framed in light)


produced by the ‘Ouardi’×‘Tirynthos’ cross (‘bitter’×‘bitter’) and by self-pollinated ‘Tirynthos’ prove that the‘sweet’ seed taste cannot be considered a dominant trait.Similar considerations can be driven, yet taking intoaccount their small size, from the F2 populations of both‘Farmingdale’ and the ‘bitter’ accession ‘BO 86611014’(from the ‘sweet’×‘sweet’ cross ‘NJA 1’×‘Reale d’Imola’).Moreover, frequencies expected on the basis of a singlegene hypothesis differ significantly (Table 1) from thoseobserved in both ‘bitter’× ‘sweet’ (‘Cricot’× ‘Harcot’,‘Cricot’× ‘Reale d’Imola’) and ‘sweet’×‘sweet’ populations(‘Harcot’×‘Reale d’Imola’, ‘NJA 1’×‘Reale d’Imola’), aswell as in the self-pollination offspring of two ‘sweet’accessions: ‘BO 82605005’ (issued from the ‘bitter’×‘sweet’ cross ‘Portici’×‘Reale d’Imola’) and ‘BO 82607001’(from the ‘sweet’×‘bitter’ cross ‘Reale d’Imola’×‘MonacoBello’).

The single gene inheritance is not consistent even whenassuming the dominance of the ‘bitter’ trait: in this case, itcould not explain the non-negligible yield of ‘bitter’individuals in populations from ‘sweet’ parents, both inF1 populations like ‘Boreale× ‘Harcot’, ‘Harcot’× ‘BO81604133’ (‘San Castrese’×‘Reale d’Imola’), ‘Harcot’×‘Reale d’Imola’, ‘NJA 1’× ‘Reale d’Imola’, ‘Reale diImola’×‘Harogem’, ‘Reale d’Imola’×‘S. Francesco’, and inF2 populations like those from ‘BO 82607001’ and ‘BO82607002’ (both from ‘Reale d’Imola’×‘Monaco Bello’).

On the other hand, if seed bitterness resulted from atypical polygenic, quantitative system, a continuous fre-quency distribution of seed cyanoglucoside content shouldbe observed in a given population, and the discriminationbetween sweet and bitter seeds would be due only to thetaste sensitivity threshold. Our data, in accordance withpreviously published results (Gomez et al. 1998), do notsupport the quantitative inheritance model, as all of the‘sweet’ seeds tested showed amygdaline levels at least 190-fold lower than the minimum value analytically recordedfor the bitter seeds (Fig. 1). Thus, the findings fromindividual seeds showed the qualitative nature of thedifference between bitter and sweet phenotypes, while thewithin-tree variability of CN− content recorded in bitteraccessions (Table 2) is probably due to the influence of thefruit position, affecting secondary metabolite accumulation.

Taking into account the evidence against both thequantitative and the monofactorial inheritance, we assumedthat several genes (two or more) may be involved in seedbitterness. Furthermore, it had to be explained the contem-porary presence of ‘sweet’ seedlings from self-pollinationof bitter-seed accessions like ‘BO 86611014’ (‘NJA1’×‘Reale d’Imola’), ‘Farmingdale’ and ‘Tirynthos’, and‘bitter’ individuals from selfed ‘sweet’ phenotypes like ‘BO82604010’ (‘Palummella’× ‘Reale d’Imola’), ‘BO82605005’ (‘Portici’×‘Reale d’Imola’), ‘BO 83604013’

(‘Monaco Bello’×‘Reale d’Imola’), ‘BO 82607001’ and‘BO 82607002’ (both from ‘Reale d’Imola’×‘MonacoBello’); because of this evidence, we postulated the actionof epistatic mechanisms for seed bitterness.

We considered that seed bitterness could result from twodistinct biochemical pathways: an ‘additive’ pathway(either the biosynthesis of cyanoglucosides or their trans-port or both) leading to the storage of these metabolites inseeds, and a catabolic pathway of cyanoglucosides, possiblyassociated with their simultaneous conversion into non-cyanogenic compounds. Moreover, each of these pathwayscould be made up of one or possibly more steps, controlledby genes postulated to be unlinked. The functioning of boththe anabolic and the catabolic pathways would be ensuredby the dominant alleles at each one of the involved loci. Asa breakdown mechanism, one inhibitor gene could eventu-ally interfere with either cyanoglucoside anabolism (“IA”),or catabolism (“IC”), or both (see Fig. 2 for all the possiblegene interactions).

The ‘bitter’ phenotype would result from a completeanabolic pathway, due to the presence of at least onefunctional allele at each one of the involved loci (here named“B”) in the absence of any inhibitor gene product, associated

Table 3 Summary of the 23 inheritance hypotheses postulated forapricot seed bitterness: number of genes involved in cyanoglucosideanabolism/catabolism and the possible interactions of an inhibitorgene with either one or both pathways

Number of genesa involved in cyanoglucogenesis

Catabolism Anabolism

Inhibitorsb 1 2 3

1 Absent (2)c (3)c (4)c

Present IA (3)c (4)c (5)c

IC (3)c (4)c (5)c

IA+IC (4)c (5)c (6)e

2 Absent (3)c (4)c (5)d

Present IA (4)c (5)c (6)e

IC (4)c (5)c (6)e

IA+IC (5)c (6)e (7)e

3 Absent (4)c (5)c (6)e

Present IA (5)c (6)e (7)e

IC (5)c (6)e (7)e

IA+IC (6)e (7)e (8)e

a Number of complementary genes considered for each metabolicprocess; in brackets: total number of loci, inhibitor(s) included.b Inhibitor gene(s) have been eventually included, supposed tointerfere with either cyanoglucoside anabolism (IA) or catabolism(IC) or both (IA+IC).c Tested hypothesesd Hypothesis fully matching the resultse Hypotheses not tested


with the breakdown of the catabolic function—attributableeither to recessive homozygosis at one or more of thecorresponding loci (here called “S”) or to inhibitor genedominant allele(s). Conversely, the ‘sweet’ phenotype couldbe due either to the breakdown of the anabolic pathway, or tothe triggering of the catabolic function, or to both.

In the proposed model, the ‘sweet’ seedlings issued fromself-pollinated ‘bitter’ genotypes (supposedly lacking the‘subtractive’ function) can be explained by the segregationof recessive alleles (either at any of the loci responsiblefor the anabolic pathway, or at the catabolism inhibitorgene). At the same time, this hypothesis accounts for the‘bitter’ seedlings present in a self-pollination populationof a ‘sweet’ tree. Given the latter phenotype can be dueto either the catabolism or the inhibition of anabolic en-zymes, the ‘bitter’ seedlings would result from the segrega-tion of recessive alleles at one or more of the correspondingloci.

Having our data available, in an effort to find aninheritance hypothesis consistent with all of the observedsegregations, we developed systematically all possible

models involving up to five non-linked genes (Table 3).According to the hypothesis being challenged, a range ofpossible genotypes were ascribed to each tested cultivar:those bearing contradictions among the segregation ofdifferent tested populations were rejected.

Out of the 23 challenged models, only one matchedfully the segregation observed in all populations, while allthe others could only partially account for the results:either they conflicted with the observed frequencies ofsome populations, or generated contradictions amongdifferent crosses once a genotype was ascribed to a givencultivar.

The simplest inheritance hypothesis explaining allobserved frequencies does not include any inhibitor gene,but requires at least five genes: three complementarygenes (B1, B2, B3) for as many steps of the anabolicpathway and two complementary genes (S1, S2) for thecatabolic pathway (Fig. 3). According to this model, the‘bitter’ phenotype would correspond to 40 (B1- B2- B3-s1s1--, or B1- B2- B3- S1- s2s2) out of 243 possible geno-types (Table 4).

Fig. 3 Flow chart of thesimplest inheritance hypothesisfully matching the observed fre-quencies. The either bitter (a) ornon-bitter phenotype (b) resultsfrom the accomplishment (+) orbreakdown (−) of two metabolicpathways: cyanoglucosideanabolism (three complementarygenes) and catabolism (twocomplementary genes)


For each of the ten crosses whose segregation areclearly inconsistent with the single-gene theory (Kostina1977), the observed bitter/sweet segregation matches to atleast one of those postulated from the proposed five-genes

inheritance (Tables 1 and 4). In all remaining populations,the observed frequencies, although compatible withKostina’s (1977) theory, fit, generally better, to one or moreof the segregation ratios predicted by the suggestedinheritance. The theoretical segregations listed in Table 1correspond to those obtained from the genotype(s) ascribedto each cultivar in Fig. 4, which minimize(s) the χ2 valuewithout generating discrepancies among half-sib populationresults.

The model of inheritance suggested would also accountfor the different distribution frequencies of ‘bitter’ seedlingsobserved among populations in relation to the seedcyanoglucoside content (Fig. 1), assuming that an incom-plete dominance and/or the existence of different functionalalleles at each of B1, B2, and B3 loci influence cyanogluco-side accumulation. These considerations recall thosereported for the Ac locus in clover (Hughes and Stirling1982; Hughes et al. 1984), which is supposed to be acomplex of linked genes coding for at least three of theenzymes in the cyanoglucoside biosynthetic pathway(Hughes et al. 1988, 1990).

Also in sorghum and cassava, the cyanoglucosidebiosynthetic pathway consists of three enzymes: twoCytochrome P450s, converting the amino acids into α-hydroxynitriles via acetaldoxime, and the glucosyltrans-ferases, able to couple the nitrile to one or two sugarmoieties (Bak et al. 1998; Andersen et al. 2000). Theentire biosynthetic pathway for dhurrin, the sorghumcyanoglucoside, has been recently engineered in Arabidopsis(Tattersaal et al. 2001; Kristensen et al. 2005). The resultingcyanoglucosides are accumulated in the vacuole, presumablyby a specific transport mechanism.

As regards the two loci suggested for a cyanoglucosidecatabolic activity with a role in seed bitterness, they couldcorrespond to those coding for amygdalin hydrolase andprunasin hydrolase, i.e. the first two enzymes required forthe three-step cyanoglucoside catabolism, deeply investi-gated in P. serotina as a model species (Zhou et al. 2002).Indeed, both the cyanogenic diglucoside (R)-amygdalin andmonoglucoside (R)-prunasin taste strongly bitter.

The assays of cyanoglucoside bark content, although notdesigned for monitoring the seasonal and even diurnalturnover of these metabolites (Poulton 1990), detectedappreciable levels even in ‘sweet’ genotypes (‘Harcot’,‘NJA 1’, and ‘Reale d’Imola’). If the biosynthetic pathwayin apricot consists of three genes each with a singleisoform, then the absence of cyanoglucosides in the seedsof these cultivars suggest that the sweet phenotype is due toeither catabolism in seed or inability to transport them.Otherwise, it could be supposed that the genes controllingseed bitterness are either different from those responsiblefor cyanoglucosides in somatic tissues or differentiallyexpressed at tissue level. Further studies are planned on

Table 4 ‘Bitter’ and ‘sweet’ seed expected frequencies in apricotpopulations derived from cross and self-pollination, according to theproposed model of inheritance based on five genes

Frequency (%) Crossa Self-pollinationa

‘Bitter’ ‘Sweet’ Bitter×Bitter

Bitter×Sweet (andreverse)

Sweet×Sweet

‘Bitter’ ‘Sweet’

100.000 0.000 • • • •75.000 25.000 • • • •62.500 37.500 • •56.250 43.750 • • • •50.000 50.000 • • •46.875 53.125 • •43.750 56.250 • •42.187 57.813 • • • •37.500 62.500 • • •35.156 64.844 • •32.812 67.188 • •31.640 68.359 • •31.250 68.750 • •28.125 71.875 • • •26.367 73.633 •25.000 75.000 • •24.609 75.391 • •23.437 76.563 • •21.875 78.125 •21.094 78.906 • • •18.750 81.250 • • •18.457 81.543 • •17.578 82.422 • •16.406 83.594 •15.625 84.375 • •14.062 85.938 • • •12.500 87.500 • •12.305 87.695 •11.719 88.281 • •10.937 89.063 •10.547 89.453 • • •9.375 90.625 • •8.203 91.797 •7.812 92.188 • •7.031 92.969 • •6.250 93.750 • •5.469 94.531 •4.687 95.313 • •3.125 96.875 • •0.000 100.000 • • • •

a Dots: only possible expected frequencies for a given crossb ‘Bitter’ phenotype (40 possible genotypes: B1- B2- B3- s1s1 -- or B1-B2- B3- S1- s2s2)c ‘Sweet’ phenotype (203 possible genotypes: b1 b1 -- -- -- -- or B1- b2b2 -- -- -- or B1- B2- b3 b3 -- -- or B1- B2- B3- S1- S2-)


somatic tissues and seeds collected from anthesis tomaturity to gain more insights about the biochemicalprocesses leading to seed bitterness in apricot.

The five-gene model could also explain seed bitternessinheritance in almond, especially as to the low frequency of‘bitter’ seedlings from ‘sweet’×‘sweet’ and ‘bitter’×‘sweet’(or reciprocal) crosses (Table 4), as reported in severalstudies (Dicenta and García 1993; Heppner 1923, 1926;Spiegel-Roy and Kochba 1974), but always attributed topossible outcrossing. Although this trait has been proposedas monofactorial with the ‘sweet’ allele dominant (Heppner

1923, 1926; Dicenta and García 1993), it should be notedthat most segregation data on almond (bred and selected,unlike apricot, for sweet kernel) have been collected from‘sweet’×‘sweet’ crosses and, due to self-incompatibility ofmost cultivars, self-pollination data are lacking.

Acknowledgments We would like to thank A. Masia (BolognaUniversity, Italy), who provided the cyanoglucoside detection method,P. Morandini and C. Soave (Milan University, Italy) for criticalreading of the manuscript, and L. Cabrini and L. Proni for theirassistance in field data collection. Work partially funded by the C.N.R.(National Research Council, Rome).

Fig. 4 Tentative genotypes(in italics) assigned to cultivarsand consistent with all of theobserved ‘bitter’ and ‘sweet’seed segregation (listed inTable 1). Typed in bold ‘bitter’parents. Framed in bold self-pollinations. Arrow (pointingto the pollen donor) crossevaluated


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bitterness inheritance in apricot (p. armeniaca l.) seeds

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