Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Abstract

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.

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.