Short communication
On the origin of the plum cultivars `Cacaks Beauty',
`Cacaks Best', `Cacaks Early' and `Cacaks Fruitful' as
investigated by the inheritance of random amplified
polymorphic DNA (RAPD) fragments
R. Heinkel, W. Hartmann, R. StoÈsser
*UniversitaÈt Hohenheim, Institut fuÈr Obst-, GemuÈse- und Weinbau 370, D-70593 Stuttgart, Germany
Accepted 25 February 1999
Abstract
Parentage analysis was performed on the plum cultivars `Cacaks Beauty', `Cacaks Best', `Cacaks Early' and `Cacaks Fruitful' using the RAPD-technique with 26 decamer primers. The cross combination `Stanley'`Pozegaca' of the progeny `Cacaks Fruitful' could be confirmed and the data were in agreement with those of the breeders Paunovic et al. (1974). For the cultivars `Cacaks Beauty', `Cacaks Best', and `Cacaks Early' the analysis showed a high conformity with the mother `Wangenheim' and the supposed father `Stanley', whereas the questionable father `Pozegaca' indicated a low similarity.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Parentage analysis; RAPDs;Prunus domestica
1. Introduction
The four investigated cultivars originated from the research station in Cacak (former Yugoslavia). The cultivars `Cacaks Beauty' (`Cacanska lepotica'), `Cacaks Best' (`Cacanska najbolja') and `Cacaks Early' (`Cacanska rana') are a cross combination of `Wangenheim'`Pozegaca' and `Cacaks Fruitful'
Scientia Horticulturae 83 (2000) 149±155
* Corresponding author. Tel.: +49-711-459-2350; fax: +49-711-459-2351.
E-mail address:stoesser@uni-hohenheim.de (R. StoÈsser).
(`Cacanska rodna') is derived from `Stanley'`Pozegaca', according to the data of the breeders Paunovic et al. (1974).
However, our observations and also the published data of the breeders Paunovic et al. (1978) on fruit size and chemotaxonomy between parents and progenies have cast doubt on this. Data from Paunovic et al. (1978) showed also that progenies from other cross combinations with `Stanley' have in general big-sized fruit, while progenies from `Pozegaca' are middle-sized fruit. Fruits from `Cacaks Fruitful' are described as middle-sized whereas `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' are big-sized. The questionable father `Pozegaca' for `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' gave rise to further investigations at the DNA-level.
Previous reports, using biochemical and molecular markers for parentage studies in plants include the following: isozymes (Chyi and Weeden, 1984), RFLPs (Nybom, 1990) and RAPD-markers (Harada et al., 1993). Random amplification of polymorphic DNA (RAPD) analysis has emerged as a powerful technique for the detection of sequence polymorphisms.
2. Material and methods
2.1. Plant material
Young, expanding leaves of the plum cultivars `Cacaks Beauty', `Cacaks Best', `Cacaks Early', `Cacaks Fruitful', `Pozegaca P25', `Stanley', and `Wangenheim' were collected in May from trees maintained in the field at the Versuchsstation fuÈr Gartenbau (UniversitaÈt Hohenheim). The samples were freeze dried and stored atÿ208C.
2.2. DNA isolation
Total DNA from leaves of each cultivar was extracted according to the procedure described by Gregor, 1995 for plums.
2.3. RAPD analysis
2.4. Data scoring and analysis
The banding patterns were scored visually, and assisted by the computer program WINCAM 2.2 (CYBERTECH). Fragments appearing on the same gel position in banding patterns from different cultivars which have been generated with a certain primer were supposed to be homologous. Reproducible bands of consistent intensity were scored as present or absent for each individual. All amplification runs were repeated two times. In general bands were not scored if they were faint or diffuse. The presence/absence data from each study were used to calculate a similarity matrix based on Jaccard's coefficient of similarity (Link et al., 1995), Sijnij/(nijninj), with nij the number of bands common to individuals iand j, ni and nj the number of bands unique to individual i and j, respectively. Only polymorphic bands were considered for the calculation.
3. Results
The 26 primers amplified a total of 158, 144 and 160 scorable fragments for the parental cultivars `Wangenheim', `Pozegaca' and `Stanley', respectively (Table 1). A total of 102 fragments were found to be monomorphic. Cultivar specific bands could be detected in `Wangenheim' 18, `Pozegaca' 12 and `Stanley' 37 bands.
Fig. 1. RAPD-fingerprint (inverted) for the cultivars `Pozegaca' (P), `Wangenheim' (W), `Stanley' (S), `Cacaks Best' (CB), `Cacaks Beauty' (CS), `Cacaks Early' (CE) and `Cacaks Fruitful' (CF) using primer UBC 18 (Molecular standard M 100 bp-Ladder). The 800 bp fragment indicates a polymorphic fragment from `Stanley'.
The number of cultivar specific fragments scored from the progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' ranged from 34 to 38 and was lower for `Cacaks Fruitful' with 30 bands (Table 2). The parental cultivar `Wangen-heim' showed a heredity of cultivar specific fragments to the first named progenies of 7±9 bands and the putative parental cultivar `Stanley' 21±30 bands. For the progeny `Cacaks Fruitful' the analysis demonstrated a heredity for the father `Pozegaca' of 9 and the mother `Stanley' of 21 RAPD-markers. Only in one case was a cultivar specific fragment of `Pozegaca' detected in `Cacaks Beauty'.
Another criterion for the determination of paternity is the number of nonmaternal fragments. There was good conformity between the progenies `Cacaks Beauty', `Cacaks Best', `Cacaks Early' and the putative father `Stanley' when considering nonmaternal fragments and the calculation of cultivar specific and novel bands. However, this agreement could not be reached for `Pozegaca'.
Table 1
Evaluation of bands in RAPD profiles for the cultivars `Wangenheim', `Pozegaca' and `Stanley'
Wangenheim Pozegaca Stanley
Total fragments 158 144 160
Monomorphic fragments 102 (64.6%) 102 (70.8%) 102 (63.7%) Polymorphic fragments 56 (35.4%) 42 (29.2%) 58 (36.3%) Cultivar specific fragments 18 (11.4%) 12 (8.3%) 37 (23.1%)
Table 2
Evaluation of the inheritance of fragments from the parental cultivars `Wangenheim', `Pozegaca' and `Stanley' by the progenies `Cacaks Beauty', `Cacaks Best', `Cacaks Early' and `Cacaks Fruitful'
Cacaks Best Cacaks Beauty Cacaks Early Cacaks Fruitful
Fragments
Total fragments 169 165 172 163
Polymorphic fragments 67 (39.6%) 63 (38.2%) 70 (40.7%) 61 (37.4%) Cultivar specific fragments 34 (20.1%) 38 (23.0%) 38 (22.1%) 30 (18.5%)
Inheritance of parental cultivar specific fragments
Wangenheim 7 (38.9%) 9 (50.0%) 8 (44.4%) 0 (0.0%)
Pozegaca 0 (0.0%) 1 (8.3%) 0 (0.0%) 9 (75.0%)
Stanley 27 (73.0%) 28 (75.7%) 30 (81.1%) 21 (56.8%)
Exclusion criteria: nonparental fragments
Nonmaternal fragments 32 31 35 21
Nonparental fragments
Two nonparental bands were detected in `Cacaks Beauty' and five markers in `Cacaks Best' and `Cacaks Early'. No new fragments could be found in `Cacaks Fruitful'.
3.1. Similarity according to Jaccard
The calculation of the similarity between the parental cultivars and the progenies confirmed the results obtained by the comparison of the fragment patterns (Table 3). The genetic similarity between the putative parental cultivar `Stanley' and the progenies varied from 0.532 to 0.603. Lower values showed the comparison between the mother `Wangenheim' and the progeny `Cacaks Best' with 0.398, `Cacaks Beauty' with 0.368 and `Cacaks Early' with 0.385. The genetic similarity between the father `Pozegaca' and the progeny `Cacaks Fruitful' was 0.493. However, for the other questionable parental cultivars the similarity reached very low values between 0.179 and 0.225.
The comparison between the parental cultivars `Wangenheim'±`Stanley' and `Pozegaca'±`Stanley' showed low similarity values of 0.152 and 0.112. In contrast, the cultivars `Wangenheim' and `Pozegaca' were found to be very similar with a value of 0.614. Here the data demonstrated a higher conformity between the latter parental cultivars than between their supposed progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early'.
The full-sibs `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' indicated between themselves high conformity in genetic similarity from 0.612±0.646. On the other hand the similarity values ranged from 0.365 to 0.422 for the half-sib `Cacaks Fruitful' and was thereby distinctly lower.
4. Discussion
According to the data published by the breeders Paunovic et al. (1974), the progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' arose from the cross combination `Wangenheim'`Pozegaca' and the progeny `Cacaks Fruitful'
Table 3
Similarity matrix between parents, progenies and full-/half-sibs, calculated according to Jaccard
Cacaks
Cacaks Fruitful 0.422 0.404 0.365
Wangenheim 0.398 0.368 0.385 0.315
Stanley 0.603 0.532 0.580 0.582 0.152
Pozegaca 0.225 0.180 0.179 0.493 0.614 0.112
from `Stanley'`Pozegaca'. The putative father `Stanley' showed high conformity in the number of cultivar specific fragments and the genetic similarity with his progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early' in the analysis. From the mother `Wangenheim' a lower number of cultivar specific bands was inherited to the progenies. The reason for the low inheritance of cultivar specific fragments from the mother could be seen in the choice of primers which essentially showed polymorphic fragments for `Stanley' and the lower heredity frequency of the markers. The data of the analysis for the cross combination of the progenies from `Cacaks Fruitful' were in agreement with the breeder data.
Only in one case did a cultivar specific fragment from `Pozegaca' appear in `Cacaks Beauty', while in `Cacaks Best' and `Cacaks Early' no appropriate band could be found. With the used method it was impossible to verify if this fragment of the same length is also in accordance with the sequence of `Pozegaca'. We have also to take into consideration a possible mutation in primer sites (Bachmann, 1994), heteroduplex formation (Hunt and Page, 1992) and recombination.
The proportion of nonparental fragments ranged from 1.2% to 3.0% for the cultivars `Cacaks Beauty', `Cacaks Best' and `Cacaks Early'. The appearance of novel bands is in accordance with data published in the literature for grapes (BuÈscher et al., 1994) and eucalyptus (Nesbitt et al., 1997). Novel bands can arise, possibly due to mutation on a primer-binding site, insertion, deletion (Williams et al., 1990), heteroduplex formation (Davis et al., 1995) or a recombination event at the genomic level. Contamination of the DNA samples can be excluded. If nonparental fragments are reproducible, they cannot confuse the determination of paternity (Scott et al., 1992).
The results of this study for the progeny `Cacaks Fruitful' are in good accordance with the breeders Paunovic et al. (1974). For the progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early', the investigation indicates the same parents. Besides phenotypic observations (Paunovic et al., 1978), the analysis of the genotypes with the RAPD-technique points to `Stanley' as the father. According to these results there could be no confusion of the mother during the experiment and the harvest. Most likely there seems to be a mistake of different pollinizers during collection of pollen, preparation and pollination. A mistake during pollination of pollen of `Pozegaca' with `Stanley' is probable. However with the methods used it is impossible to elucidate with certainty whether `Stanley' is the pollinizer of the progenies `Cacaks Beauty', `Cacaks Best' and `Cacaks Early'. Further analysis with other methods, e.g. RFLPs, could be useful.
Acknowledgements
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