Euphytica (2007) 153:221–225 DOI 10.1007/s10681-006-9257-6
Use of recessive homozygous genotypes to assess genetic control of kernel bitterness in almond Federico Dicenta Æ Encarnacio´n Ortega Æ Pedro Martı´nez-Go´mez
Received: 25 November 2005 / Accepted: 11 August 2006 / Published online: 5 October 2006 Springer Science+Business Media B.V. 2006
Abstract Bitter flavour of the almond kernel is due to the cyanoglucoside amygdalin and has been the first characteristic considered in breeding programmes. In such programmes, the seedlings from sweet-kernelled parents were used to study the transmission of bitterness, which was shown to be a monogenic characteristic, the sweet flavour being dominant. The aim of this work was to investigate more deeply the inheritance of bitterness in almond, by studying for 2 consecutive years the bitter flavour in 169 seedlings of 9 families (obtained exclusively for this purpose), one or both parents being bitter-kernelled. With the exception of the presence of slightly bitter seedlings, the results support the hypothesis of monogenic inheritance of this trait, the bitter flavour being recessive, although other factors could have a slight influence on the expression of this characteristic. Heterozygous individuals showed sweet, slightly bitter or year-changeable (sweet-slightly bitter) phenotypes. Our results demonstrated the possibility of using as parents bitter-kernelled individuals with some outstanding characteristic desirable for transmission to the progeny, always in combination with a homozygous sweet progenitor. F. Dicenta (&) Æ E. Ortega Æ P. Martı´nez-Go´mez Departamento de Mejora Vegetal, CEBAS-CSIC, Apdo. 164, E-30100, Espinardo, Murcia, Spain e-mail:
[email protected]
Keywords Almond Æ Amygdalin Æ Bitterness Æ Fruit breeding Æ Prunasin Æ Prunus dulcis
Introduction In almond [Prunus dulcis (Mill.) D.A. Webb], as in other Prunus species, bitter flavour of the kernel is due to the cyanoglucoside amygdalin (McCarty et al. 1952; Conn 1980; Frehner et al. 1990; Arra´zola 2002). In bitter kernels, amygdalin is synthesised from prunasin, which comes from the mother plant (Frehner et al. 1990), i.e. the parent with bitter genotype (Dicenta et al. 2000). The ancestors of cultivated almonds are mostly bitter-kernelled (Grasselly 1976a, b), and the sweet flavour seems to be the consequence of a mutation of an originally bitter almond (Heppner 1923). ´ lvarez (1798), in the book entitled According to A ‘‘Natural History’’, Pliny indicated that the Romans were proud of making bitterness disappear from almond kernels. Since then, sweet flavour against bitter flavour of the seed has been considered the first characteristic for selection in almond breeding programmes. Later, the undesired presence of bitter seedlings in almond breeding programmes was used by breeders to study its inheritance. Earlier studies of the inheritance of bitterness in almond kernels were conducted by Heppner (1923, 1926), who indicated that it was a monogenic characteristic, being recessive, while the heterozygote
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was the most common genotype. However, some authors suggested a more complex mechanism of inheritance. When studying intervarietal crosses, Spiegel-Roy and Kochba (1974), determined that sweet flavour was dominant, and rejected the hypothesis of Heppner, indicating the possibility of three genes being involved. On the other hand, Grasselly and Crossa Raynaud (1980) accepted the theory of Heppner, although they found highly variable sweet/bitter ratios. In general, studies following those of Heppner confirmed his hypothesis and provided new information regarding the genotype (homozygous or heterozygous) for this characteristic in many sweet cultivars (Grasselly and Crossa-Raynaud 1980; Dicenta and Garcı´a 1993; Vargas et al. 2001). Two of these studies supporting Heppner’s hypothesis must be high lighted. Dicenta and Garcı´a (1993), studying 51 families and 1969 individuals, classified seven cultivars as homozygous sweet and five as heterozygous sweet, and indicated the possibility of using bitter selections, with some outstanding characteristic that it is desirable to transmit to the progeny, in combination with homozygous sweet selections. Later, Vargas et al. (2001), in a study of 152 families with 4919 individuals, classified 18 almond cultivars as heterozygous sweet and 20 as homozygous sweet. To date, all the results obtained have come from the study of progenies that were obtained for breeding purposes, and thus from crosses between sweet-kernelled cultivars. As far as we know, there has been no study of the inheritance of this characteristic using homozygous bitter parents, which would definitively confirm the hypothesis of Heppner. The aim of this work was to make deeper our knowledge of the genetic control and transmission of bitter flavour of almond kernels, by studying progenies from crosses in which one or both parents were bitter-kernelled.
(homozygous sweet, SS), ‘Garrigues’ (heterozygous slightly bitter, Ss), ‘S3062’, ‘S3064’ and ‘S3067’ (homozygous bitter, ss) selections, obtained in CEBAS-CSIC from the cross ‘Garrigues’ · ‘Tuono’. In 1997, bitter selections were crossed with ‘Peraleja’ and ‘Garrigues’ and also self-pollinated (with the exception of ‘S3062,’ which is self-incompatible), to obtain three different types of crosses (Table 1): (a) Homozygous bitter (ss) · Homozygous sweet (SS); (b) Homozygous bitter (ss) · Heterozygous slightly bitter (Ss); (c) Homozygous bitter (ss) · Homozygous bitter (ss). After collecting pollen from the male parents, crosses were carried out by hand pollination with a paintbrush. The seeds obtained were germinated by traditional stratification and the descendants planted in the experimental field, in 1998. Once the trees had started in bearing, the study of kernel flavour was carried out over two consecutive years (2001 and 2002), when the trees were 4 and 5 years old respectively.
Materials and methods
Results and discussion
Plant materials
Most of the progenies from crosses between the bitter selections (‘S3062’, ‘S3064’ and ‘S3067’) and the sweet cultivar ‘Peraleja’ were non-bitter (sweet or slightly bitter), and only three individuals
To study inheritance of bitter flavour, five almond genotypes were used as parents: ‘Peraleja’
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Kernel bitterness evaluation The fruits were collected when fully ripe (with mesocarp opened), their mesocarp was removed and the nuts were kept at room temperature. After some weeks, the nuts were cracked and each kernel sample was tasted by two persons, who classified its flavour as sweet, slightly bitter or bitter. Statistical analysis The number of individuals of each flavour per family and type of cross was analysed by the test of goodness-of-fit chi-square (v2), against the expected frequencies for one, two or three genes with dominance of sweet flavour.
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Table 1 Number of individuals with sweet, slightly bitter or bitter kernels from the crosses indicated Parentage
Total Observed values
Expected values
Non-bitter
20 45 23 88 49 4 18 71 4 6 10
12 32 15 59 8 1 6 15 0 0 0
5 2 3 10 12 0 1 13 0 0 0
Significance
0.050b 0.089b 0.000b 0.102b 0.184 1.000 0.222 0.014 0.000c 0.000c 0.000c
0.823 0.765 1.000 0.749 0.668 0.317 0.637 0.905 1.000 1.000 1.000
Bitter Non-bitter Bitter
Sweet Slightly bitter Sweet–slightly bitter ‘S3062’ · ‘Peraleja’ ‘S3064’ · ‘Peraleja’ ‘S3067’ · ‘Peraleja’ Total (ss · SS) ‘S3062’ ‘Garrigues’ ‘S3064’ · ‘Garrigues’ ‘S3067’ · ‘Garrigues’ Total (ss · Ss) ‘S3064’ · ‘S3064’ ‘S3067’ · ‘S3067’ Total(ss · ss)
v2
2 9 5 16 3 2 3 8 0 0 0
a
1 2 0 3 26 1 8 35 4 6 10
20.0 45.0 23.0 88.0 24.5 2.0 9.0 35.5 0.0 0.0 0.0
0.0 0.0 0.0 0.0 24.5 2.0 9.0 35.5 4.0 6.0 10.0
Expected frequencies for a monogenic trait, with the sweet allele being dominant, and goodness-of-fit chi-square (v2 ) a
Genotypes with a different evaluation, sweet or slightly bitter flavour, in the 2 years studied
b
v2 calculated only for the number of observed and expected non-bitter (sweet or slightly bitter) genotypes
c
v2 calculated only for the number of observed and expected bitter genotypes
were bitter. This fits the expected frequencies according to the assumed hypothesis of the v2 test (Table 1) and confirms earlier studies (Heppner 1923, 1926; Dicenta and Garcı´a 1993; Vargas et al. 2001) that sweetness is dominant over bitterness. Almost 70% of these seedlings were sweet, 11% were slightly bitter and the remaining 18% were sweet or slightly bitter, depending on the year. Sweet genotypes generally have amygdaline concentrations of 0–10 mg/100 g, which increases to 15–30 mg/100 g in the slightly bitter genotypes. In marked contrast, in bitter genotypes the amygdaline concentrations reach 3000–6000 mg/100 g (Arra´zola 2002). For this reason we have pooled the data from sweet and slightly bitter genotypes when v2 testing genetic hypotheses. The three bitter seedlings found in progenies from bitter · ‘Peraleja’ crosses are presumably the consequence of a mistake during the pollination process (self-cross fertilisation of the bitter selection) or, and which is less likely, during the establishment of the assay in the field. In crosses between the bitter selections and the slightly bitter cultivar ‘Garrigues’, around half of the seedlings were non-bitter and the other half were bitter (Table 1), which also fits Heppner’s (1923, 1926) hypothesis. More than 20% of these
seedlings were sweet and the remainder were slightly bitter (18%) or sweet-slightly bitter (11%). All seedlings from self-pollination of the bitter selections were bitter (Table 1), in accordance with the established hypothesis. With the exception of the presence of slightly bitter seedlings, in general, our segregation results (Table 1) fit the monogenic hypothesis of Heppner (1923, 1926). Chi-square tests for the two and three gene hypotheses failed to provide better explanations of the segregation (data not presented). Our results verify the hypothesis established by Dicenta and Garcı´a (1993), concerning the possibility of using bitter genotypes as parents, always in combination with a sweet homozygous genotype, since bitter seedlings are not expected. This could be interesting when the bitter parents have some outstanding characteristic, which it is desirable to transmit to the progeny, such as high productivity (Spiegel-Roy and Weinbaum 1985) or resistance to nematodes (Kochba and Spiegel-Roy 1976). The more or less important presence of slightly bitter individuals cannot be explained by the established hypothesis. All the seedlings from crosses between homozygous bitter and homozygous sweet are expected to be heterozygous sweet, yet slightly bitter seedlings were found. In the same way, in
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the case of seedlings from bitter selections · ‘Garrigues’ (ss · Ss), half of the offspring is expected to be heterozygous sweet, but again we found sweet and slightly bitter individuals. In fact, the selfsame genitor ‘Garrigues’ and other cultivars such as ‘Genco’ or ‘Tuono’, are slightly bitter. So, these results confirm the previous results obtained by Dicenta and Garcı´a (1993), who indicated that the slightly bitter genotypes must be heterozygous, but that not all the heterozygous individuals show the slightly bitter flavour. Furthermore they observed that when heterozygous slightly bitter cultivars (in this case ‘Garrigues’) are crossed, a higher proportion of slightly bitter seedlings is obtained than in the case of heterozygous sweet cultivars. On the other hand, Arra´zola (2002), when studying different cultivars, determined that levels of amygdalin were high in the bitter and, in general, low or null in the sweet ones, always with some variation within each group. The slightly bitter cultivars showed, in general, higher concentrations of amygdalin than the sweet cultivars, although not in all cases, so it seems that the amygdalin content is not a perfect trait to distinguish clearly sweet and slight bitter cultivars. This makes it more difficult to detect slightly bitter individuals, whose identification would depend on amygdalin contents and perception of the slight bitter flavour by the tester, in which many factors are involved (Rouseff and Matthews 1984; Dura´n and Costell 1998). There are few references regarding the inheritance of amygdalin content in almond progenies. McCarty et al. (1952) studied benzaldehyde from amygdalin hydrolysis in a progeny from the cross sweet almond · bitter peach. These authors found that the descendants had benzaldehyde levels, which varied in a continuous way between those of the parents, although some produced even less benzaldehyde than the sweet parent. Despite being an interspecific cross, this study called into question the simplicity of the Hopper’s hypothesis of transmission. Arra´zola (2002) also found a highly variable amygdalin content within an offspring from ‘Garrigues’ · ‘Tuono’ (both heterozygous) the contents being very high for bitter,
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intermediate for slight bitter and low for sweetkernelled seedlings. It is important to point out that from the 24 seedlings showing a different flavour in the 2 studied years, 23 were slightly bitter in 2001 and sweet in 2002 (data not shown) and only one the opposite. So, it seems that some slightly bitter genotypes could vary their flavour, depending on environmental conditions. In conclusion, our results support the previously proposed view that the bitter kernel flavour is a recessive characteristic in almond, with the exception that in this study, for the first time, homozygous bitter parents have been used. Acknowledgements This study was financed by projects AGF98-0211-C03-02 and AGL2001-1054-C03-01 of the Spanish Ministry of Science and Technology. We wish to thank Dr. David Walker for revision of the English in the manuscript.
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