Pollinizer efficacy of several ‘Ingeborg’ pear pollinizers in Hardanger, Norway, examined using microsatellite markers

‘Ingeborg’ is currently the main commercial pear cultivar grown in Norway. However, fruit set and subsequent yields of this cultivar have proven to be variable and overall low averaging 10–20 t·ha. Pear seeds found in ‘Ingeborg’ fruits are often underdeveloped, suggesting that incomplete fertilization might be a major cause of poor fruit set. In some years, sporadically unfavorable environmental conditions during and immediately after pollination inHardanger district, westernNorway, have resulted in poor fruit set of ‘Ingeborg’. In this study, the pollinizer efficacy of several pollinizers, namely ‘Clara Frijs’, ‘Herzogin Elsa’, ‘Anna’, ‘Color ee de Juillet’, and ‘Belle lucrative’, from several orchards located in the Hardanger district was investigated using 12 microsatellite markers for two growing seasons (2014 and 2016). Pollinizer efficacy was estimated by genotyping ‘Ingeborg’, each individual pollinizer, as well as normally developed seeds from ‘Ingeborg’ fruit, and conducting gene assignment analyses to identify the pollen contribution from each of the pollinizer cultivars. In addition, S-allele genotyping was conducted, and only one pollinizer, ‘Anna’, was identified as being semicompatible with ‘Ingeborg’, whereas all other pollinizers were fully compatible. ‘Clara Frijs’ and ‘Belle lucrative’ were identified as the most efficient pollinizers probably because these cultivars were abundant compared with all other pollinizers within all, but one of the examined orchards. Higher yields could not be attributed to a particular pollinizer, and genetic effects associated with the triploid nature of ‘Ingeborg’ are most likely implicated as a cause behind the low and variable yield of this cultivar. BP 10273 pear (‘Conference’ · ‘Bonne Louise’) was bred at SLU Balsg ard (Swedish University of Agricultural Sciences), and after evaluation in Western Norway, it was named ‘Ingeborg’ in 1994 (Hjeltnes and Ystaas, 1993). This cultivar has become the most widely planted commercial pear variety grown in Norway, including the Hardanger district, western Norway. ‘Ingeborg’ is a triploid (3x) and is believed to be the result of fertilization of an unreduced diploid (2n) egg cell from ‘Conference’ with a haploid (n) pollen cell from ‘Bonne Louise’ (Sehic et al., 2012). Although ‘Ingeborg’ possesses good pomological traits that are highly desirable to Norwegians, fruit set and subsequent yields of this cultivar tend to be erratic and significantly lower than other pear varieties grown in Norway (Meland and Frøynes, 2014). Yields vary significantly between different orchards in the Hardanger region within any one growing season and parthenocarpy may play a role. Seeds extracted from ‘Ingeborg’ fruits are frequently underdeveloped. Triploids typically have low fertility due to a reproductive barrier whereby three sets of chromosomes cannot be divided evenly during meiosis yielding unbalanced segregation of chromosomes (Phillips et al., 2016). Triploids are typically highly infertile; however, limited fertility and seed production can result from the formation of apomictic embryos or through the union of aneuploid or unreduced gametes (Ramsey and Schemske, 1998). It should be noted that pears, which are auto-incompatible, may have seeds even if they are self-pollinated. Ny eki et al. (1998) found that even during self-pollination, pears can bear fruits, which are a) entirely seedless (parthenocarpic), or b) the seeds were empty or flat without any viable germination, or c) some viable seeds developed at a low rate (0.5% to 2%) in addition to empty seeds. Incompletely formed seeds, low seed number per fruit, or both have reduced sink strength (Weinbaum et al., 2001), which results in lower fruit weight and decreased yields. Self-fertilization in European pears (Pyrus communis L.), similar to other fruit species of the Rosaceae family, is prevented by gametophytic self-incompatibility (Crane and Lewis, 1942). Consequently, interplanting of suitable pollinizer genotypes in pear orchards is essential for fertilization of the ovules, which in itself is necessary for a successful set of an optimum crop load (Webster, 2002). Identifying cross-compatible pear cultivars is traditionally accomplished with testcrosses and more recently using polymerase chain reaction (PCR) based S-genotyping (Mota et al., 2007; Quinet et al., 2014; Sanzol, 2009). However, planting cross-compatible pollinizers, which have coincidental flowering time overlap with the main commercial cultivar, does not always guarantee consistently high yields. In addition, environmental variables, such as rainfall, temperature, and cloud cover, may also negatively affect pollinators and the effective pollination period (EPP) (Sanzol and Herrero, 2001). EPP is defined as the difference between the ovule longevity minus the time between pollination and fertilization (Williams, 1965). Because of generally unfavorable environmental conditions for pear pollination during the Nordic spring, ‘Ingeborg’ orchards have been established using multiple pollinizer cultivars. Despite this, yields are often low and erratic in some ‘Ingeborg’ orchards in Hardanger, Norway, and this requires further investigation to identifywhich of the pollinizers is the most effective, both in high and poor yielding orchards. Determining pollen compatibility of individual pollinizers may be accomplished by 1722 HORTSCIENCE VOL. 52(12) DECEMBER 2017 genotyping progeny plants produced by germinating seeds extracted from pear fruits of the main commercial cultivar and using the obtained molecular data to identify the male parent. However, the occurrence of aneuploid seedlings with poor viability, frequent among triploids (Zhang and Park, 2009) such as ‘Ingeborg’, makes the above procedure impractical. Consequently, genetic analyses should be performed on the pear seeds themselves instead of the progeny plants. The seedlings that are produced from seeds will be primarily aneuploids because of unbalanced chromosome segregation in meiosis (Brownfield and Kohler, 2011) with poor viability due to the triploid nature of ‘Ingeborg’. However, limited number of progeny frommaternal triploids could also be diploids (generational reversion) and tetraploids (fertilization from unreduced gametes from one or both parents) (Phillips et al., 2016). Microsatellite markers or simple sequence repeats (SSRs) have proven efficient in parent-offspring analyses on pear (Kimura et al., 2003). Although a comparative study has shown that the identification of a highly informative set of single-nucleotide polymorphism (SNP) from a large panel showed significantly more accurate individual genetic assignment compared with the combination of SSR loci (Glover et al., 2010), Moore et al. (2014) found that microsatellite markers are accurate genetic markers for genetic assignment, especially in combination with informative SNPs. In the case of plant parentage, pollination and dispersal analyses, and microsatellites with their various limitations remain an important genetic marker (Ashley, 2010). In addition, there are several readily available microsatellite markers at present, developed from either apple (Gianfranceschi et al., 1998; Liebhard et al., 2002) or pear (Fern andezFern andez et al., 2006), that can be used in the genetic analyses of European pear genotypes. In this study, pollination efficacy of several commonly used ‘Ingeborg’ pollinizers in the Hardanger region was investigated using microsatellite markers. To examine the causes of fertilization between Ingeborg vs. all pollinizer cultivars, molecular analyses of S alleles were performed. Materials and Methods The environmental conditions in Ullensvang, a municipality of Hardanger, Norway’s biggest fruit producing region, during flowering were conducive to pollination of ‘Ingeborg’ in 2014 and 2016 (Table 1). Because of the unfavorable climatic conditions in 2015 [low minimum temperatures (<7.3 C) and prolonged heavy rainfall during bloom] that contributed to the low fruit set and insufficient even for research sampling, this year was excluded. Dates of first bloom (BBCH 60), full bloom (80% of blossoms open), and petal fall (80%) (Jackson and Looney, 1999) for ‘Ingeborg’ in six commercial orchards as well as for five pollinizer cultivars during 2014 and 2016 are presented in Table 2, confirming that there was sufficient overlap between all pollinizers and ‘Ingeborg’ in both 2014 and 2016. At harvest, 50 randomly sampled ‘Ingeborg’ fruit were gathered from each of the six commercial orchards. Fruits were cut open and all pear seedswere extracted. Orchard size, yield, and age of the six different orchards are presented in Table 3. Pear producers did not provide beehives for pollination, but neighboring farms that are growing cherries and plums are renting beehives for pollination. The distance between some pear orchards and these beehives was 100 m and more. Molecular and phenology analyses. Tissue samples (leaves) for DNA analyses were collected in the Spring of 2014 from a single tree of the main commercial cultivar (Ingeborg) and from pollinizer genotypes (‘Clara Frijs’, ‘Herzogin Elsa’, ‘Anna’, ‘Color ee de Juillet’, and ‘Belle lucrative’) present in the analyzed orchards. The genomic DNA was isolated from 70 to 80 mg of leaf powder using the CTAB method (Cullings, 1992; Doyle and Doyle, 1987). Extraction and isolation of genomic DNA from pear seeds were conducted according to Padmalatha et al. (2008). As it was impossible to obtain enough high-quality DNA from a single seed for the genetic characterization, well developed seeds collected from each individual orchard were mixed and ground together to obtain a single homogenous sample. Twelve SSR primer pairs (Table 4) were chosen based on their polymorphism observed in a previous study on European pears (Gasi et al., 2013). All PCR reactions were carried out in accordance with the protocol described by Gasi et al. (2013