Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen
Introduction
Cultivation of strawberry (Fragaria × ananassa Duchesne ex Rozier, 2n = 8x = 56) began in Europe more than 250 years ago with an accidental hybridization event between the octoploid wild species of Fragaria chiloensis and Fragaria virginiana (Darrow, 1966). It has become an extremely important fruit crop. With expansion of strawberry production into Europe and USA, various genetic studies were conducted using wild and cultivated strawberries from the early twentieth century. In those days, important findings of two kinds were performed to understand the evolution of the species in the genus Fragaria. The first finding was the existence of a euploid series of the wild species. Ichijima (1926) and Longley (1926) examined chromosomes of Fragaria and demonstrated the existence of diploid (2n = 2x = 14), hexaploid (2n = 6x = 42) and octoploid (2n = 8x = 56) wild species, and determined that the basic chromosome number was x = 7. About 20 wild species of 2x, 4x, 5x, 6x, 8x, and 10x have been identified (Staudt, 1989, Hancock, 1999, Hummer et al., 2009). Another finding was formation of the irregular polyploidy plants obtainable by hybridization between different species with different polyploidy levels. Yarnell (1931) reported that an enneaploid, i.e., nonaploid, plant (2n = 9x = 63) was obtained from Fragaria vesca crossed with F. chiloensis. Fedorova (1934) described two pentaploids obtained from the diploid F. vesca × Fragaria moschata L. (synonym = Fragaria elatior hexaploid (2n = 6x = 42)). Other researchers reported similar phenomena (Ichijima, 1926, Longley, 1926, Scott, 1951, Darrow, 1966, Morishita et al., 1996, Bors and Sullivan, 2005). Scott (1951) suggested that the emergence of polyploidy strawberries was associated with the production of unreduced gametes. These gametes are significant in producing high polyploidy levels of wild strawberry plants (Bringhurst and Senanayake, 1966, Darrow, 1966, Senanayake and Bringhurst, 1967, Bringhurst and Gill, 1970). Actually, Staudt (1984) observed single and double restitution in microsporogenesis of a F1 hybrid between F. virginiana and F. chiloensis. In addition, Shi et al. (2002) confirmed 2n and 4n unreduced gamete formation in F. vesca and F. pentaphylla, and that the 2n gamete was formed by abnormal division at metaphase II, in other words; second division restitution (SDR). Although it is important for evolution, genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation, few papers related to examination of genetic background in artificially produced irregular polyploid strawberries.
Four types of unreduced gamete formation occur in plants, depending on the meiotic stage at which the nuclear restitution occurs: first division restitution (FDR), intermediate meiotic restitution (IMR), post-meiotic restitution (PMR) and second division restitution (Ramanna and Jacobsen, 2003). The first and fourth types apply to strawberries. In FDR, the spindles fail to form; homologous chromosomes do not separate, and are encased by the nuclear membrane. The chromosomes duplicate and separate in the second meiotic division. Offspring retain parental heterogeneity. For SDR, chromosome separation occurs during the first meiotic division and the subsequent duplication produces highly homozygous gametes. DNA analysis of the backgrounds of the parents and polyploid offspring can determine whether FDR or SDR is involved (Veilleux, 1985, Werner et al., 1992, Bretagnolle and Thompson, 1995, Bastiaanssen et al., 1998).
DNA analysis can determine genetic relations between parents and their progenies. Kunihisa et al., 2003, Kunihisa et al., 2005, Kunihisa et al., 2009 have developed CAPS markers to identify octoploid strawberry cultivars. These markers are single-genome specific and sufficient to visualize single allelic pair conditions of homozygotes and heterozygotes. CAPS markers include a pair of forward and reverse primers and a restriction enzyme with which the primers are able to clip single allelic pairs. In a homozygote A type (HMZ-A) DNA segments have specific base sequences that cannot be cut by the restriction enzyme, and are represented as a single electrophoretic band. In homozygote B-type (HMZ-B) DNA segments can be cut, and two smaller bands are produced. If the larger and the two smaller bands are identifiable, the DNA segments are regarded as heterozygote type (HTZ). CAPS marker analysis can thus detect genetic background of parents and their irregular polyploidy progeny of unreduced gamete origin, If the electrophoretic banding patterns obtained by the CAPS markers indicates heterozygocity and coincides with the 8x parent and the progeny, then the progeny must be derived from an unreduced gamete produced by FDR. If the banding pattern is heterozygous in the 8x parent but homozygote in the progeny, then the progeny must have been derived from the unreduced gamete by SDR.
For this reason, our objective was to examine unusual polyploid progeny of F. vesca × F. × ananassa to determine whether FDR or SDR was involved in the unreduced gamete production.
Section snippets
Plant growth and measurement
Five diploid everbearing strawberry cultivars, F. vesca ‘Alba’, ‘Alexandria’, ‘Baron Solemacher’, ‘Mignonette’ and ‘Reugen’ were used as maternal and six June-bearing type octoploid Japanese cultivars, F. × ananassa ‘Ai-berry’, ‘Asuka ruby’, ‘Hokowase’, ‘Nyoho’, ‘Sachinoka’ and ‘Toyonoka’ were used as paternal parents. Cross-pollination was conducted using at least five flowers of each combination from late autumn 2007 to late winter 2008 in a greenhouse at the Faculty of Agriculture, Kagawa
Results
Based on reproductive behavior (Fig. 1), the 627 seedlings were grouped into 204 plants of A type and 423 plants of B type (Table 1). The A-type plants produced inflorescences during the three-month period following sowing but the B-type plants did not. In contrast, the B-type plants produced stolons, whereas the A-type plants did not. The 204 A-type plants appeared to be typical F. vesca by the morphological characteristics of their flowers and by flow cytometry analyses (Fig. 2A). A
Flow cytometry analysis
Some researchers have used propidium iodide (PI) staining for flow cytometry (Nehra et al., 1991, Akiyama et al., 2001, Brandizzi et al., 2001), and mithramycin staining (Nyman and Wallin, 1992) to measure the cell nuclear DNA content. However, for strawberry plants, a few papers have reported the use of DAPI staining which is easier than either the PI or mithramycin staining.
The relative DNA contents of the cells using DAPI staining were sufficient to distinguish the polyploidy levels in the
Acknowledgement
The study was conducted for “Research on hybridisation between octoploid cultivated strawberry plants and Japanese wild strawberry plants” of the Japanese Ministry of Agriculture, Forestry and Fisheries.
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