Pears

Chapter 8 Pyrus Richard L. Bell and Akihiro Itai 8.1 Basic Botany of Pyrus Pear species belong to the genus Pyrus, the subtribe Pyrinae, the subfamily Maloideae (Pomoideae) in the family Rosaceae. There are at least 26 widely recognized primary species and 10 naturally occurring interspecific hybrid taxa (Table 8.1), which are distributed in Europe, temperate Asia, and the mountainous areas of northern Africa (Bell et al. 1996). Many of the known species are native to Asia. All species of Pyrus are intercrossable, and there are no major incompatibility barriers to interspecific hybridization in spite of the wide geographic distribution of this genus (Westwood and Bjornstad 1971). Dispersal is believed to have followed the mountain chains both east and west (Bell et al. 1996). Speciation probably involved geographic isolation and adaptation to colder and drier environments (Rubzov 1944). Kikuchi (1946) classified Pyrus species into three groups, small fruited species with two carpels, large fruited species with five carpels, and their hybrids with 3–4 carpels. Small fruited species, sometimes known as Asian pea pears, P. calleryana Decne., P. fauriei Schneid., P. betulifolia Bunge, and others are used for ornamental purpose or rootstocks. Of large fruited species with five carpels, there are four major cultivated species, P. communis L. (European pear), P. bretschneideri Rehd. (syn. P. pyrifolia (Burm.) Nakai var. sinensis Kikuchi), P. ussuriensis Maxim. and P. pyrifolia, which are R.L. Bell (*) United States Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA e-mail: richard.bell@ars.usda.gov commercially cultivated in the temperate zone. P. communis is native to Europe and is the main commercial species in Europe, North America, South America, Africa, New Zealand and Australia. P. bretschneideri is the main species in northern and central China. P. pyrifolia is the main species in Japan, southern and central China, Taiwan, and Korea. P. nivalis Jacq., the snow pear, is cultivated in Europe for making perry, an alcoholic pear cider. P. pashia P. Don. is cultivated in northern India, Nepal, and southern China. The genome size of P. communis has been estimated by using flow cytometry (Arumuganathan and Earle 1991). According to their report, DNA content of P. communis is 1.03 pg/2C, compared to 0.54 pg/ 2C in peach, and the genome size is approximately 496 Mbp/haploid nucleus. Asian pears are thought to have been domesticated in pre-historic times and to have been cultivated in China for at least 3,000 years (Lombard and Westwood 1987). European pears are thought to have been cultivated in Europe as early as 1000 BC. Homer referred to a large orchard with pears in the Odyssey, written in between 900 and 800 BC. The earliest written records of Japanese pears date back to the ancient manuscript of the Emperor Jito in AD 693 (Kajiura 1994). Pear is the third most important temperate fruit species after grape and apple, with a 2009 world production estimated at 21.9 metric tons (FAO 2010). Asia produced the most (16.4 million t), followed by Europe (3.9 million t), North and Central America (885,321 t), and South America (744,032 t). The European pear (P. communis) production is concentrated in five production areas: Europe, North America, South America, South Africa, and Oceania, while the production of Asian pears (P. bretschneideri, P. pashia, and P. pyrifolia) is concentrated in Asia. C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_8, # Springer-Verlag Berlin Heidelberg 2011 147 148 R.L. Bell and A. Itai Table 8.1 Pyrus species, interspecific hybrids, and geographic distributiona Geographic group and species Centers of diversity European European P. communis L.b Western and Southeast Europe, Turkey P. cordata Desv. France, Spain, Turkey P. nivalis Jacq. South central Europe, Ukraine, France P. canescens Sprach P. complexa Rubtzov Caucasus P. salvifolia DC Europe (Crimea) Circum-Mediterranean P. elaeagrifolia Pall. P. syriaca Boiss. P. cossonii Redher P. gharbiana Trab. P. mamorensis Trab. P. spinosa Forssk.(syn. P. amygdaliformis Vill.) Southeast Europe, Ukraine, Turkey Tunisia, Libya, Middle East, Armenia Algeria Morocco, Algeria Morocco Southeast Europe, Turkey Mid-Asian P. glabra Boiss. P. korshinskyi Litv. P.longipes Coss. & Dur. P. pashia D. Don. P. regelii Rehd. P. salicifolia Pall. Iran Afghanistan, Kyrgyzstan, Tajikistan, Uzbekistan Algeria Pakistan, India, Nepal, Bhutan, Afghanistan, China, Indochina Afghanistan, central Asia Northern Iran, Turkey, Caucasus East Asian P. armeniacifolia Yu P. betulifolia Bunge P. bretschneideri Rehd. P. calleryana Decne. P. dimorphophylla Makino P. fauriei Schneid. P. hondoensis Nak. & Kik. P. hopeiensis T. T. Yu P. koehnei C. K. Schneid. P. phaeocarpa Rehd. P. pseudopashia T. T. Yu P. pyrifolia (Burm.) Nak. P. serrulata Rehd. P. sinkiangensis T. T. Yu P. taiwanensis Iket. & Ohashi P. ussuriensis Maxim. P. uyematsuana Makino P. xerophila T. T. Yu Northwestern China, Kazakhstan Central and northern China, Laos southern Manchuria China Central and southern China, Japan, Korea, Taiwan, Vietnam Japan Korea Japan China (Hebei, Shandong) South China, Taiwan Northern China China (Guizhou, Yunnan, Kansu) China, Japan, Korea, Taiwan Central China Northwestern China Taiwan Siberia, Manchuria, northern China, Korea Northwestern China a Source: USDA (2009b) Includes subspecies and several taxa listed as valid species names for which species status is uncertain. Also includes several taxa not recognized as valid species b 8.2 Conservation Initiatives 8.2.1 Evaluation of Genetic Erosion Information on Pyrus is limited. The International Union for the Conservation of Nature (IUCN 2008) Red List currently contains only nine Pyrus taxa (Table 8.2). Included in the “Low risk/near threatened” category are P. salicifolia Pall., and four taxa of uncertain taxonomic status: P. anatolica Browicz, P. asia-mediae (Popov) Maleev, P. oxyprion Woronow, and P. serikensis G€uner & Duman. Two taxa, P. asia-mediae and P. hakkiarica Browicz, are listed as “data deficient” but are included because of concern. G€uner and Zielinski (1996), in their account of the status of Turkish woody 8 Pyrus 149 Table 8.2 Conservation status of Pyrus taxa listed on the IUCN red lista Taxon Native range Status Pyrus anatolica Browicz Turkey Lower risk/near threatened Pyrus asia-mediae (Popov) Maleev Kazakhstan Data deficient Kyrgyzstan Uzbekistan Pyrus cajon Zapryagaeva Tajikistan Endangered Pyrus hakkiarica Browicz Pyrus korshinskyi Litv. Turkey Kyrgyzstan Data deficient Critically endangered Pyrus oxyprion Woronow Pyrus salicifolia Pall. Pyrus serikensis G€uner & Duman Pyrus tadshihkistanica Zapryagaeva Tajikistan Uzbekistan Turkey Turkey Turkey Tajikistan Lower risk/near threatened Lower risk/near threatened Vulnerable Critically endangered Comment Probably synonymous with P. communis Not seen since originally described. Taxonomic status not clear Rare endemic, declining due to agricultural expansion. May be synonymous with P. lindleyi Probably P. syriaca Threatened by overgrazing and overharvesting Taxonomic status not clear Rare endemic population reduced by cutting a Citation: IUCN (2008) flora, also list P. elaeagrifolia ssp. bulgarica and P. yaltirikii as low risk/near threatened and P. elaeagrifolia Pall. ssp. elaeagrifolia and spp. kotschyana, P. pyraster ssp. caucasica (syn. P. caucasica Fed.) and ssp. pyraster (syn. P. communis ssp. pyraster), P. spinosa (syn. P. amygdaliformis Vill.), and P. syriaca Boiss. as “low risk/least concern”. According to Browicz (1972), P. anatolica is probably P. elaeagrifolia subsp. kotschyanae or an interspecific hybrid of P. elaeagrifolia, P. commuuner and Zielinski nis, and P. amygdaliformis. G€ (1996) state that P. serikensis is “known until recently as P. boissieriana subsp. crenulata”, which Aldasoro et al. (1996) recognize as a synonym of P. cordata. The taxon P. hakkiarica is probably P. syriaca (Davis 1972). The International Dendrological Society also lists P. magyarica (probably a synonym of P. communis ssp. pyraster, and only described in Hungary) as either endangered, vulnerable, or rare (Lear and Hunt 1996). In Germany and the Czech Republic, wild populations of P. pyraster are threatened (Endtmann 1999; Sindelář 2002), although there are efforts to maintain those genetic resources by in situ and ex situ preservation (Kleinschmit et al. 1998; Wagner 1999; Paprštein et al. 2002). Other such efforts at in situ conservation have been planned for the Middle East (Amri et al. 2002). Wild populations in Kyrgyzstan (Blaser 1998), the Kopet Dag woodlands of Turkmenistan (World Wildlife Fund 2001), and elsewhere in central Asia are threatened by logging, fuelwood gathering, and other activities in previously protected forests. Among Asian species, P. calleryana is listed as a vulnerable endemic species in Japan (Ohba 1996), and P. kawakamii (syn. P. calleryana) is listed as vulnerable in Taiwan (Lear and Hunt 1996). Throughout the world, there are most likely wild populations of various other species, which are also threatened by deforestation. In addition, indigenous landrace cultivars are being replaced by more modern cultivars. 8.2.2 In Situ and Ex Situ Conservation Ex situ conservation of wild crop relatives of the major cultivated Pyrus species has received much more actual work than in situ conservation. A 1989 survey listed 34 collections in 22 countries that probably contained at least one crop relative species (IBPGR 1989). Major ex situ germplasm collections resulting from plant exploration and exchange have been established in several countries, including the USA (Postman 2008; USDA 2009a), the United Kingdom (University of Reading 2009), and the People’s Republic of China (Aldwinckle et al. 1986; Xu et al. 1988; Zhang 2002). Smaller collections exist in many countries with native Pyrus populations. The European Cooperative Program for Plant Genetic Resources lists 32 noncultivated taxa in 11 European collections in eight 150 R.L. Bell and A. Itai countries (EUCPPGR 2009a). The EURISCO database has accession level information for close to 1,000 accessions of 48 non-cultivated taxa in numerous national collections (EUCPPGR 2009b). dehydration (Reed and Chang 1997). Pre-treatment with cold-acclimation and abscisic acid has been shown to be very important for pear genotypes (Bell and Reed 2002). 8.2.3 Modes of Preservation and Maintenance 8.3 Origin and Evolution of Pyrus Most ex situ preservation is in the form of clonally propagated trees. Clones preserved for their edible fruit are usually propagated by budding or grafting onto seedling or clonal pear rootstocks of various species or clonal quince rootstocks. The rootstocks can be also propagated onto specific seedling or clonal rootstocks, maintained in stool beds as self-rooted plants, or planted as self-rooted trees. In many cases, clones are indexed for viruses and phytoplasmas, treated by thermotherapy if needed, and maintained as “disease tested” or certified disease-free germplasm (Postman and Hummer 1988). The genetic diversity of wild populations is best preserved by systematic collection of seed. Preservation of these seed collections of Pyrus can be accomplished by drying to a relative humidity of 20% and storing in a desiccator at the temperature of 4–6 C for long-term storage (2–10 years or more), or even at 20 C. For subfreezing temperatures, drying to lower a moisture content of 2–5% may be preferred (Roberts 1975). In vitro methods of preservation are intended as secondary or back-up collections, established as a safeguard against loss of trees in nursery or orchard plantings due to disease, insect, or climatic hazards, e.g., due to low winter temperature. Medium-term storage of clonal propagules has been attained through in vitro culture of shoots, and long-term storage has been achieved through cryopreservation of in vitro cultured apical meristems in liquid nitrogen. Medium-term storage involves slowing growth through low temperature and medium manipulations. Viable cultures can be maintained for 12–18 months at 4 C with a 16-h photoperiod, and storage for over 2 years can be achieved using gas-permeable bags instead of glass culture tubes. Defoliated shoots have been stored for up to 4 years at 2 C on an agar medium without growth regulators (Druart 1985). The three major techniques of cryopreservation are slow freezing, vitrification, and encapsulation– Maloideae (Pyrinae) contains the 25 genera, including Pyrus (pear), Malus (apple), Eriobotrya (loquat), Cydonia (quince), and Chaenomeles (Chinese quince). The basic chromosome number of the Maloideae (x ¼ 17) is high compared to other Rosaceae subfamilies (x ¼ 7–9), indicating a polyploid origin. Classical biochemical studies on leaf phenolic compounds, isozyme studies, and botanical data support the hypothesis of an allopolyploid origin (Chevreau et al. 1997). It has been suggested that the Maloideae arose as an amphidiploid of two primitive forms of Rosaceae, crossing a basic chromosome number of 8 and 9 (Sax 1931; Zielinski and Thompson 1967). These were possibly primitive members of the Prunoideae (x ¼ 8) and Spiraeodeae (x ¼ 9). A molecular study of the chloroplast gene rbcL suggests that Spiraeodeae is the maternal ancestor of Maloideae (Morgan et al. 1994). But phylogenetic analyses of granule-bound starch synthase gene (GBSSI) sequences for a broad sampling of taxa with the different chromosome numbers across the Rosaceae family did not support the wide hybridization between basic chromosome number of 8 and 9 (Evans and Campbell 2002). Instead, sequences of GBSSI genes resolved Gillenia (x ¼ 9) to the clade including taxa with x ¼ 15 or 17. Evans and Campbell (2002) suggest that the higher chromosome number of the Maloideae arose via hybridization and polyploidization among closely related species of an ancestral lineage (the lineage that also gave rise to Gillenia), followed by aneuploid reduction. The majority of cultivated pears are functional diploids (2n ¼ 34). A few polyploid (triploid and tetraploid) cultivars of P. communis and P. bretschneideri exist. Speciation in Pyrus has proceeded without a change in chromosome number (Zielinski and Thompson 1967). This genus is considered to have originated in the mountainous area of western and southwestern China during the Tertiary period (65–55 million years ago) and to spread to the east and west. Three subcenters of diversity for the genus have been identified (Vavilov 1951): (1) the Chinese center, where forms of 8 Pyrus P. pyrifolia and P. ussuriensis are grown; (2) the Central Asiatic center, consisting of Tajikistan, Uzbekistan, India, Afghanistan, and western Tian-Shan mountains, where forms intermediate between P. communis and P. bretschneideri occur, and where P. communis is thought to have hybridized with P. heterophylla, and the putative species, P. korshinski and P. boisseriana; (3) the Near Eastern center comprising the Caucasus Mountains and Asia Minor, where domesticated forms of P. communis occur. Using sequences of non-coding regions of chloroplast DNA, Kimura et al. (2003) conducted a phylogenetic analysis of 24 cultivars belonging to P.  bretschnedieri, P. calleryana, P. communis, P. pyrifolia, and nine interspecific hybrids. A cladogram based on mutations defined one European group and three Asian pear groups. Analysis of trnL–trnF divided Asian pears into six groups, while all European pears had identical sequences. These chloroplast DNA genotypes were useful for verifying the species ancestry of the interspecific hybrids. In a study of the nuclear ribosomal DNA internal transcribed spacer region of 44 accession representing 19 mostly Asian Pyrus species, Zheng et al. (2008) concluded that certain types of pseudogenes were more useful than functional ITS copies in resolving phylogeny, especially among Asian species. 8.4 Role in Development of Cytogenetic Stocks and Their Utility Cytogenetic stocks, other than polyploids and haploids, have not been developed in Pyrus, and species related to the major cultivated species have played no role in their development. Haploids of ‘Doyenné du Comice’, ‘Bartlett’ (syn. ‘William’s’), and ‘Harrow Sweet’ pear have been obtained by selection of seedlings based on morphological traits as well as pollination with irradiated P. communis pollen, followed by in vitro culture of the resulting embryos (Bouvier et al. 1993), and doubled haploids were subsequently obtained by either spontaneous doubling or treatment with oryzalin (Bouvier et al. 2002). Triploids have been obtained by anther culture of diploid P. pyrifolia cultivars (Kadota and Niimi 2004). In a series of crosses involving diploid, triploid, and tetraploid 151 P. pyrifolia and P. communis pear cultivars, Cao et al. (2002) reported aneuploid seedlings at a frequency of 6%, mostly from intraspecific crosses of tetraploids by triploids and diploids by triploids. The only reports of the generation of polyploids and anueploids by wide crosses involve intergeneric hybridization of Pyrus with Cydonia, Malus, or Sorbus (Rudenko 1974) (see Sect. 8.6.2). 8.5 Role in Classical and Molecular Genetic Studies 8.5.1 Classical Genetic Studies Most classical genetic studies have been performed within single cultivated species, specifically P. communis, P. pyrifolia, and P. bretschneideri. Most of the interspecific studies are also between improved genotypes of these cultivated species rather than wild clones or other Pyrus species. 8.5.1.1 Productivity of Scions Studies in China indicated that clones and seedlings of P. pyrifolia were more precocious in fruit bearing than those of P. bretschneideri and P. communis, although no conclusions on heritability or mode of inheritance were made. 8.5.1.2 Fruit Quality Crosses between the European pear and the Asian pear, P. ussuriensis, were initially made either to improve fire blight resistance or for cold-hardiness for the northern regions of North America and Europe. Lantz (1929) characterized the mode of inheritance of fruit flavor as quantitative, with “dominance” of poor flavor. Pu et al. (1963) reported that the aromatic quality of some P. ussuriensis cultivars appeared dominant to the sweet, bland flavor of P. pyrifolia and P. bretschneideri. Subjective sensory scores for flavor in P. communis  P. pyrifolia hybrid progenies and intercross progenies were found to have low narrowsense heritability (Bell and Janick 1990). 152 In crosses between European and Asian species, perhaps the most striking differences among parents arise in flesh texture. Modern European cultivars are characterized by melting or buttery flesh, while P. pyrifolia and P. bretschneideri are characterized by finely grained, crisp flesh. Very little is known about the inheritance of flesh texture in pears. The differences between crisp flesh and melting flesh are likely due to a combination of enzymes affecting cell wall adhesion. Graininess is often difficult to determine independently from grit cell content. In some populations, however, grit may be confined primarily to the skin and core, and grain can be rated relatively independent of grit. Westwood and Bjornstad (1971) reported that in interspecific hybrids of wild forms of Pyrus, gritless flesh was dominant to gritty flesh. Golisz et al. (1971), on the other hand, found that in crosses of cultivars of P. communis with P. ussuriensis, the majority of offspring had numerous stone cells typical of the P. ussuriensis cultivars used as parents. Pu et al. (1963) also reported that the coarse texture of P. ussuriensis is dominant to fine grained texture of P. pyrifolia or P. bretschneideri. Zielinski et al. (1965) presented data indicating that presence of stone cells is dominant to their absence among certain crosses of P. communis cultivars. Thompson et al. (1974) concluded that in crosses involving species that contain grit, the content was controlled by a minimum of four loci, acting in an independent and additive fashion. Bell and Janick (1990) computed narrow-sense heritabilities of 0.14 and 0.48 for texture and grit scores in P. communis progenies, respectively. Heritability of texture within backcrosses of P. pyrifolia  P. communis to P. communis as well as intercrosses of the hybrids was low. Heritability of grit within backcrosses of P. pyrifolia  P. communis to P. communis as well as intercrosses of the hybrids was moderate (0.48 and 0.55, respectively). These values and the relatively large ratios of general to specific combining ability variance indicate that moderate genetic gains in grit, but not texture, in interspecific backcrosses should be possible in these traits through mass selection. Some researchers report that it is difficult to recover pure crisp-flesh selections in crosses between Asian species and P. communis, and fruit grain tends to be coarse (Wang 1990; Hough personal communication). However, White et al. (2000b) reported high narrow-sense heritabilities for both firmness (0.62) and crispness (0.89) in five crosses involving R.L. Bell and A. Itai P. pyrifolia, P. bretschneideri, and P. communis. They also found that skin grit was highly heritable, russet and aroma were moderately heritable, and juiciness, sweetness, sourness, astringency, and attractiveness were of low heritability (0.05–0.21). White and Selby (1994) concluded that all of these traits, plus skin thickness, are inherited in an additive fashion in P. pyrifolia  P. communis progenies, based on segregation patterns. Flesh firmness was also highly heritable in breeding populations of P. pyrifolia (Machida and Kozaki 1976; Kajiura and Sato 1990). In crosses between major Japanese P. pyrifolia cultivars with ‘Max Red Bartlett’, Sansavini et al. (2002) noted a higher frequency of nashi-type texture, fruit shape, and sugar content. Various fruit shape parameters in crosses of P. pyrifolia or P. bretschneideri with P. communis had narrow-sense heritabilities ranging from 0.55 to 0.75, indicating that breeding for specific fruit shape in this interspecific hybridization project was possible (White et al. 2000a). Corking of the skin produces a brown russet, which is seen on many cultivars. Many people find the appearance attractive when the russet is smooth, uniform, and light tan, and for the fresh market trade, russeted cultivars such as ‘Beurré Bosc’, ‘Angelys’, and ‘Taylor’s Gold’ are acceptable. But when the fruit is processed whole for puree, russeting results in a flecked or brown colored product, which is unacceptable. The presence of large and dark colored lenticels on the skin is also objectionable for the same reason. Wellington (1913) proposed that russeting in P. communis was controlled by a single gene, with smooth skin incompletely dominant to russet. Kikuchi (1930), however, concluded that in P. pyrifolia two loci, R and I, are involved. Genotypes that are RR are entirely russeted, while Rrii genotypes are partially russeted. I partially inhibits cork formation so that russet does not extend over the entire fruit, but under humid conditions, RrM- genotypes are more russeted than under dry conditions. Wang and Wei (1987) also found that russeting was recessive to non-russeted skin. All of these workers based their conclusion on the segregation within progenies that involved P. pyrifolia or hybrids of P. pyrifolia and P. communis. In the P. communis progenies studied by Zielinski et al. (1965) and those studied by Crane and Lewis (1949), the inheritance of russeting appeared to be more complex, suggesting control by several genes. Narrow-sense heritability in 8 Pyrus P. communis progenies was moderate (0.51), and similar (0.57) in progenies of P. communis  P. pyrifolia backcrossed to P. communis, but very high (0.94) in progenies derived from intercrossing P. communis  P. pyrifolia hybrids (Bell and Janick 1990). 8.5.1.3 Host Resistance to Disease Fire blight, incited by the bacterium Erwinia amylovora (Burrill) Winslow et al., is the most serious disease affecting pears in North America, Europe, and the Middle East (van der Zwet and Beer 1999). It is difficult to control even when recommended prophylactic measures are followed. No true immunity to fire blight in the seedlings or clones has been encountered, although high levels of resistance exist. Resistance in P. communis is relatively rare, with only 5–10% being rated as at least moderately resistant (Oitto et al. 1970; van der Zwet and Oitto 1972; Thibault et al. 1987; van der Zwet and Bell 1990). All other European, circum-Mediterranean, and midAsian species are generally susceptible. A high level of resistance occurs in greater frequency among the East Asian species, especially P. calleryana and P. ussuriensis. The evergreen pear, P. kawakamii, is susceptible. With the exception of a few seedlings selected by Reimer, P. betulifolia is also susceptible. Other species are variable, with a range of levels present. Hartman (1957) listed resistant clones within P. calleryana, P. betulifolia, P. phaeocarpa, P. fauriei, and P. variolosa (syn. P. pashia). The inheritance of resistance/susceptibility to fire blight is complex. Layne et al. (1968) found that when most of the factors affecting phenotypic expression of fire blight resistance were controlled, segregation for resistance in artificially inoculated seedling progenies was continuous, regardless of the resistance phenotypes of the parents or the species sources of resistance being tested. The proportion of offspring in each class was significantly influenced by parental phenotypes, however, and to a lesser extent by the species source of resistance. Because most segregation distributions were continuous and a number of them showed an approximately normal distribution, fire blight resistance was concluded to be polygenically inherited. Because a specific type of inheritance pattern characteristic of any of the three species studied (P. communis, P. ussuriensis, and P. pyrifolia) was not detected, it 153 was concluded that the same or similar genes for resistance may be present in each species. In a few cases, with each species source of resistance, they found some seedling progenies with a segregation pattern that suggested major gene inheritance, with dominance of resistance. Thompson et al. (1962) also found evidence for major gene and polygenic inheritance in studies with the same Pyrus species, although they thought that monogenic resistance was present only in P. ussuriensis. Thompson et al. (1975) postulated the presence of a dominant gene conferring sensitivity in some genotypes. This conclusion, however, was based upon classifying ratings of natural blight into discrete classes; no bimodal distribution was demonstrated. The consensus developed from all other studies is that host response is inherited in a quantitative fashion, and is due to predominantly additive genetic effects, with dominance or epistasis playing only a minor role in differences among parents in the ability to transmit resistance to their offspring. Heritability within pure P. communis populations estimated from offspringmidparent regression was 0.52 for epiphytotic fire blight severity in mature trees, and was similar in populations involving P. communis  P. pyrifolia hybrids (Bell et al. 1977). Heritability ranged from 0.30 to 0.51 when based upon artificial inoculation of parents in the orchard and of 6-month old seedlings in the greenhouse (Quamme 1981). General combining ability has been found to be significant, while specific combining ability was not significant (Quamme et al. 1990). More recent studies involving molecular markers have further elucidated the inheritance of resistance and suggest the involvement of a few quantitative traits loci (QTLs) (See Sect. 8.6.4.1.1 Disease Resistance). Pear leaf spot, caused by the fungus Fabraea maculata Atk. (anamorph: Entomosporium mespili (DC.) Sacc.), occurs in most areas of the world where pears are grown under warm, humid conditions. Susceptible cultivars are often defoliated by mid-summer, resulting in weak trees and a reduction in fruit buds. Infected fruits are disfigured, cracked, misshapen, and unmarketable as fresh fruit. In the nursery, the disease can be serious, causing defoliation and stunted growth. Most major cultivars of the European pear, P. communis, for which data are available, are considered susceptible (Bell 1990). Genotypes of a number of East Asian species, P. ussuriensis, P. pyrifolia, P. calleryana, P. fauriei, and P. dimorphophylla, have been reported as resistant (Wisker 1916; Tukey and Braese 1934; 154 Kovalev 1940; Beck 1958). Zalaski et al. (1959) reported that seedlings of P. caucasica were more resistant than interspecific hybrids of P. salicifolia or P. amygdaliformis with P. communis. A multi-year study of 207 Pyrus genotypes (Bell and van der Zwet 1988) demonstrated variability within and among species and interspecific hybrids. Pure species or interspecific hybrids involving P. calleryana, P. pyrifolia, and P. ussuriensis were generally more resistant than P. communis genotypes. In a later study, Bell and van der Zwet (2005) found that the least susceptible genotypes were the P. communis cultivars ‘Beurre Fouqueray’ and ‘Bartlett’, the P. pyrifolia cultivar ‘Imamura Aki’, and the P. ussuriensis  P. pyrifolia hybrid NJ 477643275. As species groups, the P. ussuriensis  P. pyrifolia hybrids and the pure P. pyrifolia cultivars were most resistant. Drain (1954) noted resistance in the P. communis  P. pyrifolia hybrids, ‘Mooers’ and ‘Hoskins’. Lombard and Westwood (1987) listed seedlings of P. caucasia and P. cordata Desv. as moderately tolerant, those of the CircumMediterranean species P. amygdaliformis, P. elaeagrifolia, and P. syriaca Boiss., and seedlings and clones of P. betulifolia as having high tolerance, P. calleryana seedlings and clones as very tolerant, and P. pashia D. Don. as susceptible. In contrast to some of the other studies, Lombard and Westwood (1987) list P. pyrifolia and P. ussuriensis as having only low tolerance. Since sources of resistance to this disease have been identified, breeding for resistance should be possible. The inheritance of resistance in crosses involving P. communis, P. pyrifolia, and P. ussuriensis indicates that the high level of resistance in P. calleryana is transmitted in an additive manner to its offspring (Bell and van der Zwet 1988). The principal fungal pathogen of P. pyrifolia and P. bretschneideri cultivars in Asia is Alternaria alternata (Fr.) Keissler pv. kikuchiana (formerly A. kikuchiana Tanaka), which causes black spot. Although the major Japanese cultivar, ‘Nijisseiki’, is susceptible, many others are resistant (Hiroe et al. 1958; Kanato et al. 1982). Many of the European pear cultivars are resistant. Susceptibility is controlled by a single dominant gene, and most of the susceptible cultivars are heterozygous at the locus (Kozaki 1973). Pear decline is caused by a phytoplasma transmitted by the pear psylla, Cacopyslla spp. (Hibino and Schneider 1970). Infection results in sieve-tube necro- R.L. Bell and A. Itai sis in susceptible trees. The severity of symptoms depends on the age and vigor of trees, the species of scion and rootstock, and environmental conditions, and can vary from leaf curl of young trees on tolerant rootstocks to premature reddening of foliage and reduced growth, or sudden wilting and death. The disease is particularly severe when cultivars of a generally tolerant species such as P. communis are grafted onto the sensitive species, P. pyrifolia, P. ussuriensis, and to a lesser extent, P. calleryana (Lombard and Westwood 1987). Most P. communis rootstocks are tolerant, and seedlings of P. betulifolia are the most tolerant. Resistance was found to be inherited in a quantitative fashion, with seedlings intermediate between the resistant and susceptible parents (Westwood 1976). Seedlings of intraspecific crosses of P. communis, P. betulifolia, and P. calleryana exhibited a low frequency of severe decline symptoms, and 75% of the seedlings of P. ussuriensis cv. Chieh Li and P. pyrifolia cv. Japan Golden Russet were resistant in spite of the fact that unselected clones of these species are usually susceptible. European pear scab, incited by the fungus Venturia pirina Aderh., is a serious disease of European pear. Host resistance of European pears is somewhat confusing because V. pirina has at least five biotypes, which appear to have a fairly narrow range of distribution (Shabi et al. 1973). Thus, cultivars that are reported resistant in one region may be susceptible in another where they are exposed to another biotype. Vondracek (1982) points out that differences among various reports in the degree of resistance could be due to differential races, environmental influences, and differences in the definition of “resistance”. Several European cultivars, including ‘Bartlett’, ‘Conference’, and ‘Dr. Jules Guyot’, are at least moderately resistant (Bell 1990). Kovalev (1963) reported that P. pyrifolia and P. ovoidae (P. ussuriensis var. ovoidae Rehder) are generally resistant, while genotypes of P. ussuriensis observed were susceptible. Westwood (1982) indicated that clones or seedling populations of P. caucasica and P. communis were susceptible, while P. pyrifolia and P. nivalis were variable. Postman et al. (2005) found that 27 of 31 P. pyrifolia, P. ussuriensis, or P. bretschneideri cultivars were highly resistant to leaf infection, and 20 of 30 were resistant to fruit infection. Species rated as resistant to fruit infection included P. betulifolia, P. calleryana, P. dimorphophylla, P. fauriei, P. pashia, P. pseudopashia, and P. regelii, 8 Pyrus while P. salicifolia, P. syriaca, and P. nivalis rated as having low susceptibility. Only the single clone of P. cordata was highly susceptible. Resistance to fruit infection in interspecific Asian  European progenies was moderately heritable (White et al. 2000a, b). 8.5.1.4 Host Resistance to Arthropod Pests Pear psylla is the major insect pest of pears in North America and western Europe. While Cacopsylla pyricola Föerster is the only species that exists in North America, C. pyri L. and C. pyrisuga Föerster are also endemic to Europe, and C. bidens is endemic to the Middle East. Other species or putative species exist in Asia. Damage occurs directly as a result of nymphal feeding in the phloem of leaves, which can cause leaf necrosis and defoliation, and indirectly can result in poor fruit size and reduced flower bud development. The nymphs excrete large amounts of honeydew which support the growth of sooty mold, both on leaves and fruit. Honeydew on young developing fruit can lead to russeting, and sooty mold on the fruit can further reduce crop value. Foliage feeding also suppresses root growth and reduces tree vigor. In addition to the damage pear psylla causes by itself, it also transmits the pear decline phytoplasma (Jensen et al. 1964). Pear species vary considerably in their resistance to pear psylla (Bell 1990). The East Asiatic species are generally more resistant than those from Asia Minor or Europe. Resistance to pear psylla was first discovered in the East Asian species, P. betulifolia, P. calleryana, P. fauriei, P. ussuriensis, and P. bretschneideri (Westigard et al. 1970; Quamme 1984) and in hybrids of P. communis and P. ussuriensis (Harris 1973; Harris and Lamb 1973; Quamme 1984). Among European species, resistance has been reported in some genotypes of P. nivalis and Sorbopyrus (Westigard et al. 1970; Bell and Stuart 1990; Bell 1992). Within P. communis, moderate to high degrees of resistance has been reported in genotypes of relatively poor fruit quality, specifically an old Italian cultivar, ‘Spina Carpi’ (Quarta and Puggioni 1985; Briolini et al. 1988), and in 15 cultivars from Yugoslavia and Hungary (Bell and Stuart 1990; Bell 1992, 2003). Harris and Lamb (1973) reported the use of P. ussuriensis as a source of psylla resistance. They 155 found that 60% of one progeny of P. communis  P. ussuriensis was resistant to psylla, concluding that resistance was quantitative and controlled in an additive genetic fashion. The narrow-sense heritability of resistance to pear slug, the larva of the sawfly Caliroa cerasi (L.), in two populations of interspecific crosses involving interspecific hybrid parents (P. pyrifolia  P. communis and P. communis  P. bretschneideri) were found to be high (0.79 and 0.87) (Brewer et al. 2002). Particularly low frequencies and severities of infestation were found in progenies of the P. bretschneideri cultivars ‘Yali’, ‘Shiyueli’, and ‘Huobali’. 8.5.2 Molecular Genetic Studies Linkage maps and molecular markers would be useful in traditional cross-breeding programs for perennial crops such as fruit tree species. However, genetic studies in pear, as in many fruit trees, have been rare. There is little information on genetic linkage maps and development of molecular markers on pears despite much research on mapping and molecular markers in apple. The long juvenile periods, the space necessary to manage large number of progenies, and the high level of heterozygosity due to a gametophytic incompatibility have limited inheritance studies to a few morphological characters (Chevreau et al. 1997). 8.5.2.1 Development of Molecular Markers Isozymes The first report on the use of isozymes in pears was in 1980 by Santamour and Demuth to identify six ornamental cultivars of P. calleryana by peroxidase patterns. Peroxidase diversity has also been studied in several species of Pyrus (Merendez and Daley 1986) and in 172 cultivars of P. pyrifolia (Jang et al. 1991, 1992). Isozymes variability in pollen was reported by Cerezo and Socias y Company R (1989). However, these approaches are used for cultivar identification and to differentiate genetic sports. Chevreau et al. (1997) examined the inheritance and linkage of isozyme loci in P. communis cultivars. They analyzed the polymorphisms of 11 enzymes (AAT: 156 aspartateaminitransferase, ENP: endopeptidase, EST: esterase, LAP: leucineaminopeptidase, PRX: peroxidase, SOD: superoxide dismutase, ADH: alcohol dehydrogenase, DIA: diaphorase, PGD: 6-phosphogluconate dehydrogenase, PGI: phosphoglucoisomerase, PGM: phosphoglucomutase) in 11 progenies from controlled crosses. According to their report, 22 loci were identified and segregation was scored for 20 loci. Three pairs of duplicated loci-forming intergenic hybrid bands were detected and these were found to correspond to equivalent duplicated genes in apple. They identified 49 active alleles and one null allele and revealed three linkage groups, which could all be related to existing groups on the apple map. Conservation of isozyme patterns, duplicated genes, and linkage groups indicates a high degree of synteny between apple and pear. No linkage map for pears was constructed based on isozyme analysis. R.L. Bell and A. Itai restriction and PCR amplification. AFLP has several advantages over the RAPD technique, including a higher number of loci analyzed and a higher reproducibility of banding patterns. Monte-Corvo et al. (2000) investigated the genetic relationships among 39 cultivars including 35 P. communis and four P. pyrifolia cultivars using AFLP and RAPD markers. They confirmed that AFLP markers were five times more efficient in detecting polymorphism per reaction. Although some differences can be noticed between the dendrograms resulting from AFLP and RAPD analyses, both techniques produced similar results. Yamamoto et al. (2002b) also made 184 and 115 polymorphic AFLP fragments using 40 primer combinations in the F1 population originating from ‘Bartlett’, and ‘Hosui’, respectively. They reported that the average number of polymorphic fragments per primer combination was 4.6 in ‘Bartlett’ and 2.9 in ‘Hosui’. Random Amplified Polymorphic DNAs Intersimple Sequence Repeats Random amplified polymorphic DNAs (RAPDs) have been widely used in pear genetic studies because RAPDs have the advantages of being readily employed and requiring small amount of genomic DNA. RAPD markers have been successfully used for identification and genetic relationships of pear. Oliveira et al. (1999) investigated molecular characterization and phenetic similarities between several cultivars of P. communis and P. pyrifolia and several wild species by RAPD markers. A total of 118 Pyrus spp. and cultivars native mainly to East Asia were analyzed by RAPD markers to evaluate genetic variation and relationships among the accessions (Teng et al. 2001, 2002). According to their reports, RAPD markers specific to species were identified, and the grouping of the species and cultivars by RAPD largely agrees with morphological taxonomy. RAPD markers have also been used to identify parentage (Banno et al. 2000). Banno et al. (1999) also identified an RAPD marker linked to the gene conferring susceptibility to black spot disease (A. alternata Japanese pear pathotype). Amplified Fragment Length Polymorphism Amplified fragment length polymorphism (AFLP) technology is a powerful tool that combines DNA Intersimple sequence repeat (ISSR) means a genomic region between SSR loci. The complementary sequences to two neighboring simple sequence repeats (SSRs) are used for PCR primers. Polymorphism diversity is lower than in SSR, but ISSR has the advantages of being readily employed and no knowledge of the DNA sequence for the targeted gene is required. Monte-Corvo et al. (2001) reported that ISSR analysis was used for cultivar identification and the determination of phylogenetic relationship in P. communis. Simple Sequence Repeats Simple sequence repeats (SSRs) or microsatellites are excellent sources of polymorphisms in eukaryotic genomes. The development of SSRs is labor-intensive. However, SSRs have been very useful in studying diversity in Pyrus. Yamamoto et al. (2002a) constructed a genome library enriched with (AG/TC) sequences from ‘Hosui’ Japanese pear using the magnetic bead method. They obtained 85 independent sequences containing 8–36 microsatellite repeats. Out of the 85 sequences, 59 contained complete (AG/TC) repeats. Thirteen primer pairs could 8 Pyrus successfully amplify the target fragments and showed a high degree of polymorphisms in the Japanese pear. Kimura et al. (2002) identified 58 Asian pear accessions from six Pyrus species using these nine SSR markers with a total of 133 putative alleles. They obtained a phenogram based on the SSR genotypes, showing three major groups corresponding to the Japanese, Chinese, and European groups. Moreover, nine apple SSRs were intergenetically applied to the characterization of 36 pear accessions (Yamamoto et al. 2001). All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear species and accessions. The differences in fragment size are mostly due to the differences in repeat number. A total of 79 alleles were detected from seven SSR loci, and thus pear and apple varieties could be differentiated (Yamamoto et al. 2001). This data show that Pyrus has a close genetic relationship with Malus. Wunsch and Hormaza (2007) also reported that the use of seven SSRs developed in apple could distinguish 61 of 63 European pear cultivars and revealed the usefulness of this set of SSR primers for cultivar identification. They found that the variability detected with SSRs in European pear varieties was low when compared with the variability detected in other fruit crops in the Rosaceae (Wunsch and Hormaza 2007). To date, more than 100 SSRs have been developed from European and Japanese pears (Yamamoto et al. 2002a, b; Bassil et al. 2005; Fernandez-Fernadez et al. 2006; Inoue et al. 2007; Terakami et al. 2007). These SSR markers have been used for cultivar identification, the evaluation of genetic diversity, and the construction of genetic linkage maps. Restriction Fragment Length Polymorphisms and Other Markers Restriction fragment length polymorhisms (RFLPs) have been used to identify Japanese pears, including the parentage of 10 cultivars, with two minisatellite probes from human myoglobin DNA (Teramoto et al. 1994). Similar attempts have been made to distinguish Pyrus species with RFLPs of chloroplast DNA (Iketani et al. 1998; Katayama and Uematsu 2003). However, these markers were used for cultivar identification and investigating genetic relationships among Pyrus 157 species. Sequence characterized amplified region (SCAR) markers were developed from RAPDs to evaluate and identify P. communis and P. pyrifolia cultivars (Lee et al. 2004). Another unique marker, copia-like retrotransposons, have been identified in pears by Shi et al. (2002). They suggest that the transposition of retrotransposons takes place during evolution, leading to diversification. However, no data on the inheritance of these markers have yet been reported. 8.5.3 Constructing Linkage Maps The first linkage maps in Pyrus species were developed for ‘Kinchaku’ and ‘Kousui’ Japanese pears using RAPD markers (Iketani et al. 2001). Black spot and pear scab are the most severe diseases of Japanese pear. Only a few cultivars are susceptible to black spot. On the other hand, most cultivars of Japanese pear are susceptible to pear scab. A survey of P. pyrifolia germplasm has identified ‘Kinchaku’ as the only cultivar having resistance. Iketani et al. (2001) used the pseudo-testcross method (Grattapaglia and Sederoff 1994) and constructed two separate maps from segregation data of 82 F1 individuals. The reason for using the pseudo-testcross method is that it is very difficult to make F2 or backcross populations in pears because of self-incompatibility and the long juvenility period of seedlings. The linkage map for ‘Kinchaku’ consisted of 120 loci in 18 linkage groups (LG) spanning 768 cM, while that for ‘Kousui’ contained 78 loci in 22 linkage groups extending over 508 cM. This was the first report of a linkage map of pear species. The resistance allele of Asian pear scab (Vn) and the susceptibility allele of black spot were mapped in different linkage groups in ‘Kinchaku’. However, in both maps, the number of linkage groups did not converge into a basic chromosome number (x ¼ 17). Therefore, the total map length is still not sufficient for covering the complete genome. The length of the apple genome was reported to be 1,200 cM or a little more (Conner et al. 1997). Pear has the same basic chromosome number as apple. In addition, the nuclear DNA content of pear species is estimated at 75 or 80% of that of apple (Dickson et al. 1992). These two pear maps are estimated to cover at least about a half of the total genome (Iketani et al. 2001). 158 The second linkage map was constructed using 63 F1 individuals obtained from an interspecific cross between the European pear ‘Bartlett’ and the Japanese pear ‘Hosui’ by Yamamoto et al. (2002b, 2004). They constructed maps based on AFLP and SSR markers from pear, apple, and Prunus; isozymes; and phenotypic traits (leaf color and S-genotype). The map of ‘Bartlett’ consisted of 256 loci including 178 AFLPs, 76 SSRs (32 pear, 39 apple, 5 Prunus), one isozyme, and a self-incompatibility locus on 19 linkage groups over a total length of 1,020 cM. The average distance between each pair of loci was 4.0 cM. The size of linkage groups ranged from 88 cM (LG 4) to 11 cM (LG 18). The segregation of many markers on LG 14 was largely distorted. The self-incompatibility locus (S-locus) was in the bottom of LG 17. The map of ‘Hosui’ contained 180 loci including 110 AFLPs, 64 SSRs (29 pear, 29 apple, 6 Prunus), two phenotypic traits, and four other markers on 20 linkage groups encompassing a genetic distance of 995 cM. Genetic linkage maps of these cultivars were aligned using 37 co-dominant markers that showed segregating alleles in both the cultivars (Yamamoto et al. 2002b, 2004). They also found that of the 80 SSRs obtained from apple, more than four-fifths could produce discrete PCR bands in pear. Similar findings were observed in European pears by another research group (Pierantoni et al. 2004). Yamamoto et al. (2004) reported that 38 apple SSR markers showed 39 segregating loci on the linkage map of ‘Bartlett’, and that 27 SSRs produced 29 loci on that of ‘Hosui’. Moreover, the authors considered synteny between pear and apple linkage maps. A total of 36 SSRs originating from apple were mapped on the genetic linkage maps of ‘Bartlett’ and apple. Only two SSR loci were aligned to different linkage groups of pear and apple. Another 34 apple SSR loci were positioned in presumably homologous linkage groups of pear. All pear linkage groups were successfully aligned to the apple consensus map by at least one apple SSR, indicating that positions and linkages of SSR loci were well-conserved between pear and apple. Their trials were the first major effort in comparing maps of apple and pear. Other maps were developed for two European pear cultivars ‘Passe Crassane’ and ‘Harrow Sweet’ using SSRs, MFLPs, AFLPs, resistance gene analogs (RGAs), and AFLP-RGAs markers in 99 F1 individuals (Dondini et al. 2004). Different levels of susceptibility to fire blight, one of the most destructive diseases, exist among European pear culti- R.L. Bell and A. Itai vars. This suggests that it is possible to identify quantitative trait loci (QTL) related to fire blight resistance in pear germplasm. ‘Passe Crassane’ is susceptible to fire blight, and ‘Harrow Sweet’ is resistant. The ‘Passe Crassane’ map consists of 155 loci including 98 AFLPs, 37 SSRs, 6 MFLPs, 4 RGAs, and 10 AFLPRGAs for a total length of 912 cM organized in 18 linkage groups. The average distance between each pair of loci is 5.8 cM. The size of each linkage group ranges from 7.0 to 92.9 cM. The ‘Harrow Sweet’ map consists of 156 loci including 101 AFLPs, 35 SSRs, 3 MFLPs, 3 RGAs, and 14 AFLPs-RGA for a total length of 930 cM organized in 19 linkage groups. Pierantoni et al. (2007) also reported the genetic linkage maps using 99 seedlings derived from the cross ‘Abbe Fetel’  ‘Max Red Bartlett’. The total length of the two maps is 908.1 cM (with 123 loci) for ‘Abbe Fetel’ and 897.8 cM (with 110 loci) for ‘Max Red Bartlett’, divided into 18 and 19 linkage groups with an average marker density of 7.4 cM and 8.0 cM, respectively. However, four linkage groups in both the maps were not denominated because SSR markers originating from apple were not mapped on these linkage groups. More recently, Yamamoto et al. (2007) constructed integrated high density genetic linkage maps of the European pear cultivars ‘Bartlett’ and ‘La France’ based on more SSRs, AFLPs, isozymes, and phenotypic traits. The map of ‘Bartlett’, constructed by using an F1 population derived from cross between ‘Bartlett’ and ‘Hosui’, consisted of 447 loci, including 58 loci by pear SSRs, 60 by apple SSRs, and 322 by AFLPs. This map covered 17 linkage groups over a total length of 1,000 cM with an average distance of 2.3 cM between markers. The map of ‘La France’, which was constructed using an F1 population derived from a cross between ‘Shinsei’ and selection 282-12 (‘Hosui’  ‘La France’), consisted of 414 loci, including 66 loci of pear SSRs, 68 of apple SSRs, and 279 of AFLPs on 17 linkage groups encompassing a genetic distance of 1,156 cM. These are the first maps that converged into the basic chromosome number (n ¼ 17) of pear. A total of 66 SSR markers derived from apple were mapped on pear maps and showed genome synteny with the saturated reference map of apple. These maps could cover the entire genome of pear and should be useful as pear reference maps. In the future, more SSRs and other molecular markers for agronomically important characters could be 8 Pyrus developed to construct the fine linkage maps useful for marker-assisted selection. 8.6 Role in Crop Improvement Through Traditional and Advanced Tools 8.6.1 Traditional Breeding Efforts Using Interspecific Hybridization Although interspecific hybridization has a limited role in scion and rootstock cultivar development, there have been some traits for which it has become increasingly important and novel breeding efforts are underway. These efforts involve crosses between the major cultivated species and target a variety of traits. Breeding for many traits has been reviewed by Bell et al. (1996) and Hancock and Lobos (2008). In general, there are no barriers to interspecific hybridization within Pyrus (Bell and Hough 1986). Sources of many economically important traits exist within the genus, although not all within the major cultivated species. General responses of Pyrus species to various diseases are given in Table 8.3, responses to arthropod pests and nematodes are given in Table 8.4, and responses to abiotic stresses are furnished in Table 8.5. 8.6.1.1 Adaptation to Low Chilling Hours Adaptation to low-chilling regions utilizing hybrids of the high chilling hour requiring P. communis and low chilling hour requiring P. pyrifolia has been a goal of the pear breeding program at the University of Florida in the USA and has resulted in the release of “Flordahome” pear (Sherman et al. 1982). Similar efforts have begun in Mexico (Rumayor et al. 2005) and Brazil (Barbosa et al. 2007; Instituto Agronomico Campinas Brazil 1987; Rasseira et al. 1992). In India and Pakistan, “semi-soft” interspecific hybrids or hybrids with P. pashia are also being grown (Sandhu et al. 2005). 8.6.1.2 Novel Fruit Texture and Flavor Perhaps the most ambitious program is being conducted in New Zealand, which seeks to combine the flavor of European pears with the crisp flesh of the best 159 Japanese P. pyrifolia cultivars (i.e., Nashi) and genotypes of P. bretschneideri with long storage potential (White and Brewer 2002). Red-skinned cultivars of the latter species are being employed as parents. The program maintains intraspecific populations of P. communis, P. pyrifolia, and P. bretschneideri, as well as interspecific populations. From the P. communis  P. pyrifolia populations, ‘Maxie’ and ‘Crispie’ have been released. Similar efforts with European  Nashi progenies have been reported from Germany (Kaim et al. 2006) and Italy (Sansavini et al. 2002; Musacchi et al. 2005) and have been undertaken in the USA (Bell unpublished data). Additional goals of the latter two programs include improved resistance to fire blight and pear psylla. 8.6.1.3 Precocity Studies in China indicated that clones and seedlings of P. pyrifolia were more precocious than P. bretschneideri and P. communis (Department of Horticulture, Zhejiang Agricultural University 1978). 8.6.1.4 Cold Hardiness Pears are grown in many parts of the world where the winter temperatures are sufficiently severe to cause cold injury to shoots, fruit spurs, trunks, and roots, and may even cause death of whole trees. In northern parts of Europe, Asia, Canada, and the United States, the need for cold hardy cultivars is especially important. Spring frost during bloom is also a constant threat in many pear growing regions, even in those regions where winter injury is not a problem. Thus, resistance of blossoms to frost injury is also an important consideration in the adaptability of pear cultivars to particular regions. The greatest efforts in breeding and selecting for cold hardiness in pears were made in the former Soviet Union and North America, especially in the steppe and prairie regions where the winters are severe. Stushnoff and Garley (1982) reviewed the early work in the United States and Canada to improve cold hardiness of pears, especially the work in Minnesota, North Dakota, and Iowa where P. ussuriensis seedlings imported from northern Russia and the hybrids of P. ussuriensis  P. communis were tested. Several pear cultivars, including ‘Patten’, ‘Harbin’, and ‘Bantam’, 160 Table 8.3 Response of Pyrus species to diseasesa,b Bacterial Species Fire blight Blossom blast Crown gall European P. caucasica S MS S P. communis VS-R S-MR S-MS P. cordata VS – – P. nivalis S MS – Collar rot Fungal Fabrea spot White spot European pear scab Asian pear scab Powdery mildew Phytoplasma Pear decline MS MS – – MS MS-MR MS – – S-R – – MS S-R MR MS-MR – R – – MS MS MR MR MS MR-R MR MR Circum-Mediterranean P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca S-MS S-MS – VS VS S MR MR – MR – MR – – – – – – – MR – – – MS MR MR – – – MR – – – MR – – MR MR – – MR – – – – – – – MR MR – MS – – MR MR – – – – S S S MR S S – – – MS – – VS – – – MR MR – – – MR – – MS – – MR – – VS-MS S-MR R MS MR-R MR-R S MR MS-MR R MR – MS MS MS – MS – MS MS MR – MR MR – – – – VS – MR – MR – MS – – – – – MR – R MR – – – – S S-R – – – – – – – MR – – MR – – MR MR MR MR – S-R S-R – S-MR – – – – – – S S MR – MR R MR – – – MS MR MR S MS-MR MR MS – – – S S Mid-Asian P. pashia P. regelii P. salicifolia East Asian a References: Bell and van der Zwet (1988), Hancock and Lobos (2008), Lombard and Westwood (1987), van der Zwet and Keil (1979), Westwood (1982) Degrees of response: VS very susceptible, S susceptible, MS moderately susceptible, MR moderately resistant, R resistant b R.L. Bell and A. Itai P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis 8 Pyrus 161 Table 8.4 Response of Pyrus species to arthropod pests and nematodesa,b Species Pear psylla Codling moth Blister mite European Wooly pear aphid Root lesion nematode P. caucasica P. communis P. cordata P. nivalis MS S-R MS MS-R S S S-R MS MS MS MR – MS-MR MS-MR VS MR VS VS MS-MR – MS MS-MR S MS – MS-MR S-R S-R – MR – – MR MR MR MR – MR MS-MR MR VS – S MR S S – – – MR MS-MR R MS MR – MR MR MR MR MS-MR VS – S – – R R R R R MR MR MR MS MS-R R MS R R R MS R S-R MS MS MR MR MR MR MR MR MR MR MR MR MR – R VS MS-MR – R – MS-MR MS-MR S – R R MS-MR – MS-MR – – – Circum-Mediterranean P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca Mid-Asian P. pashia P. regelii P. salicifolia East Asian P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis a References: Bell (1990), Bell et al. (1996), Hancock and Lobos (2008), Lombard and Westwood (1987), Westwood (1982) Degrees of response: VS very susceptible, S susceptible, MS moderately susceptible, MR moderately resistant, R resistant, – no data b have been developed that are sufficiently cold hardy to survive in the harsh prairie winter, but none of them are grown elsewhere to any extent. Recently, the cultivars ‘Luscious’ (Peterson et al. 1973) and ‘Gourmet’ (Peterson and Waples 1988) were released from the South Dakota Agricultural Experiment Station, and ‘Summercrisp’, from the Minnesota State Agricultural Experiment Station (Luby et al. 1987). These cultivars are recommended for northern regions and have higher quality than cultivars developed earlier at those stations. In Canada, breeding and testing for cold hardiness in pear has been done by public and private plant breeders in Ontario, Manitoba, Saskatchewan, and Alberta. The most promising cultivars that combine cold hardiness with size and quality are hybrids of P. communis  P. ussuriensis. The most promising cultivars for the prairies include ‘Golden Spice’, ‘Olia’, ‘David’, ‘John’, ‘Peter’, ‘Philip’, ‘Pioneer 3’, ‘Tait Dropmore’, and ‘Tioma’ (Morrison 1965). The cultivar ‘Ure’ was released from the Research Station at Morden (Ronald and Temmerson 1982). None of these cultivars is as high quality as the P. communis cultivars grown in the main pear growing regions. Nevertheless, they are valuable sources of germplasm for improving cold hardiness in pear, because they are able to withstand winter temperatures, which are often as low as 30 to 40 C, and their fruit quality is superior to their P. ussuriensis parents. Cold hardiness of pear cultivars, species, and interspecific hybrids has been assessed in various European countries, especially after unusually severe winters (Ludin 1942; Anjou 1954; Enikeev 1959; Zavoronkov 1960; Matjunin 1960; Sansavini 1967). In general, the cultivars of P. communis are less hardy than those of P. ussuriensis. Greatest progress in breeding for cold 162 R.L. Bell and A. Itai Table 8.5 Adaptation of Pyrus species to abiotic stressa,b Species Climatic factors Edaphic factor Low chill Cold hardiness Low pH High pH European P. caucasica P. communis P. cordata P. nivalis Wet soils Dry soils Sandy soils Clay soils L M-H H L H M-H L H H M L – H M H – H M-H L – H M H – H H H M-H H L-H L M-H VH L M M M M L H L H L L L M – M H L VH VH M – – M L M – H – – H H H H – H H H H – – M M H M – – – VH – – S M M VH – – S M M – – – – – M-H M-H M M H – – H – VH L H S VH L S L M-H – L M H M S H M VH H – H H H M M – M M L – L L S-L M – – L L H-VH – H-VH H H-VH – M – L L VH – H L L – – – – M H – M-VH – M – M – H M VH – H-MH – H-VH – M – L M Circum-Mediterranean P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca Mid-Asian P. pashia P. regelii P. salicifolia East Asian P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis a References: Bell (1990), Bell et al. (1996), Hancock and Lobos (2008), Lombard and Westwood (1987), Westwood (1982) Degrees of response: S susceptible, L low, M moderate, H high, VH very high, – no data b hardiness has been made by crossing hardy selections of P. ussuriensis with good quality cultivars of P. communis. 8.6.1.5 Fire Blight Several pear breeding programs have used Asian species to breed for fire blight resistance. These programs have been reviewed by van der Zwet and Keil (1979). Breeding for fire blight resistance in pear began in the nineteenth century after the introduction of Chinese sand pears (P. pyrifolia) to the eastern United States (Hedrick et al. 1921). The cultivars ‘Le Conte’, ‘Kieffer’, and ‘Garber’ were derived from interspecific hybridization of P. pyrifolia with P. communis and were grown because they were substantially more resistant to fire blight than the European cultivars of P. communis, but they were inferior in terms of fruit quality. During 1925–1960, a major pear breeding program was conducted in Tennessee. About 1933, McClintock discovered a resistant pear seedling and later named it ‘Late Faulkner’ (Drain 1943). Since 1925, the work consisted mainly of crossing resistant species, primarily P. pyrifolia, and also included P. calleryana and P. ussuriensis, with the more resistant cultivars of P. communis. From 1945 to 1966, several pear cultivars were introduced from this program, including ‘Ayres’, ‘Dabney’, ‘Carrick’, ‘Hoskins’, ‘Mericourt’, ‘Mooers’, ‘Morgan’, and ‘Orient’. A review of this breeding program was prepared by Deyton and Cummins (1991). Between 1942 and 1968, another large pear breeding program was conducted at the University of Illinois at Urbana. Pear species and cultivars had been 8 Pyrus tested since 1919 (Anderson 1920). Hough (1944) used primarily cultivars and selections of Chinese species, such as P. bretschneideri cv. Pai Li and P. ussuriensis 76, and crossed them with several P. communis cultivars. The pear breeding program at Rutgers University was initiated by Bailey and Hough (1961, 1962) in 1948. It provided some very interesting and important blight-resistant selections, many with P. pyrifolia and P. ussuriensis parentage. An apparently blight-resistant P. pyrifolia seedling, NJ 1, was used extensively in this breeding program. In 1968, Hough and Bailey (1968) introduced three new blight-resistant pear cultivars for the fresh market, named ‘Star’ and ‘Lee’ (both from a cross of ‘Beierschmitt’  NJ 1) and ‘Mac’ (‘Gorham’  NJ 1). A few years later in Maryland, ‘Star’ and ‘Lee’ were found to be susceptible, whereas ‘Mac’ was confirmed to be moderately resistant (Oitto et al. 1970; van der Zwet et al. 1974). The program at Cornell University utilized another selection from the Illinois program, P. ussuriensis 65, as a source of fire blight resistance. No cultivars have been derived from those crosses, but many selections have proven to have resistance to fire blight and pear psylla and have been used in other programs for that combined purpose. Like most breeding programs for fire blight resistance, the program of the US Department of Agriculture (USDA) initially emphasized on P. communis sources of resistance, with less use of other species. For the past 20 years, advanced backcross selections derived from NJ1, ‘Pai Li’, and P. ussuriensis 76, and more recently P. bretschneideri cultivars have been employed as parents. In 2006, ‘Sunrise’, an early maturing cultivar, which has both ‘Seckel’ and NJ1 in its pedigree as sources of resistance, was released. The program at Purdue University has also used P. ussuriensis 76 as a source of resistance, but no cultivars derived from this selection have been released. The only cultivar derived from an interspecific (P. pyrifolia) ancestry is ‘Green Jade’ (Janick 2004). The Romanian pear breeding program at ClujNapoca, Pitesti-Maracineni, and Voinesti have utilized a P. pyrifolia clone as a source of moderate tolerance to fire blight (Andreieş 2002; Branişte et al. 2008). The program in Cluj-Napoca has identified sources of resistance in P. pollveria, P. lindeyi, P. malifolia, P. persica, P. ussuriensis, and P. variolosa (Sestras et al. 2008). 163 8.6.1.6 Fungal Leaf and Fruit Pathogens Resistance to Fabraea leaf and fruit spot has been at least a secondary objective of a few breeding programs. Drain (1954) noticed resistance in ‘Mooers’ and ‘Hoskins’, and resistance to this disease was an added benefit of the effort to breed for fire blight resistance. The USDA program has surveyed and studied resistance to Fabraea in its germplasm and breeding populations, identified sources of resistance in P. communis and hybrids with P. ussuriensis (Bell and van der Zwet 2005), and incorporated some of these genotypes into its main program for fire blight and pear psylla resistance. It is unfortunate that one of the most promising selections for pear psylla resistance, NY 10353, is very susceptible to Fabraea. Resistance to both Fabraea and Mycosphaerella sentina (Fckl.) Schroet from P. pyrifolia has been incorporated into the Romanian pear breeding program (Andreieş 1983). Resistance to European pear scab has been a primary or secondary objective of European pear breeding programs (Bellini and Nin 2002), including those in Germany and Australia, although most programs use P. communis as source of resistance, One exception is the Romanian program at Voinesti-Dimbovita, which has utilized resistance from P. pyrifolia (Andreieş 1983). Pear leaf spot, caused by M. sentina, is a minor disease of pear. Resistance has been reported in at least 15 P. communis cultivars (Bell et al. 1996), based on epiphytotic conditions, and resistance of other species has apparently not been reported. 8.6.1.7 Pear Psylla Resistance to pear psylla has been a major objective of the USDA program, which has utilized P. ussuriensisderived resistant selections obtained from the Cornell and Rutgers University programs as well as P. bretschneideri in addition to P. communis cultivars (Bell and van der Zwet 1998). The programs of the University of Bologna, Agriculture and Agri-Food Canada and the Institut National de la Recherché Agronomique (INRA) have also utilized the Cornell selections. The Romanian program has utilized its P. pyrifolia selection as a source of resistance as well as local cultivars or P. communis (Branişte et al. 2008). 164 8.6.1.8 Pear Rootstocks Lombard and Westwood (1987) reviewed the utilization of Pyrus species as rootstocks throughout the world. In addition to P. communis, they list P. betulifolia, P. calleryana, P. caucasica, P. communis var. pyraster, P. elaeagrifolia, P. kawakamii, P. pashia, P. pyrifolia, and P. xerophila as used for rootstocks. In Syria, P. syriaca is also used as a rootstock, where it is adapted for low pH soils and drought (Al Maarri et al. 2007). Most breeders of rootstocks for European pear have utilized P. communis or Cydonia oblonga L. germplasm. However, the INRA program in France has utilized P. nivalis and a clone listed as P. heterofolia (Simard et al. 2004). The latter species name is not a recognized taxon, and the clone appears to be an interspecific hybrid with characteristics of P. pyrifolia or P. bretschneideri and P. betulifolia. In addition, a joint program of INRA and the Spanish agency IRTA is breeding for low pH soils by using P. amygdaliformis, P. cordata, and P. elaeagrifolia as parents (Bonany et al. 2005). Low pH soils cause limeinduced chlorosis in pear trees on Cydonia rootstocks favored for their ability to induce reduced stature and precocious bearing. 8.6.2 Intergeneric Hybrids Karpov (1966) and Rudneko (1978) reviewed works on intergeneric hybridization at the I. V. Micurin Central Genetical Laboratory that included crosses between Malus baccata and pear and crosses between mountain ash (Sorbus spp.) and European pear. In general, such wide crosses are incompatible, for example, due to the degeneration of pollen tubes in the upper third of the style of the Sorbus parent (Panfilkina 1976). Putative intergeneric hybrids were produced by pollinating the apples ‘Kassel Reinette’ and ‘Golden Winter Pearmain’ with mixtures of European pear, quince, and Amelanchier pollen (Nikolenko 1962). Although most seedlings resembled the apple parent, three with intermediate morphological features bore pear-like fruit. A few seedlings derived from hybridizing a tetraploid clone of ‘Fertility’ pear with the tetraploid apple selection BM2812 have also been obtained in Sweden (Nybom 1957). In China, the intergeneric cultivar ‘Ganjin’ was produced from a cross of ‘Red Delicious’ apple and the R.L. Bell and A. Itai pear ‘Pingouli’ (P. pyrifolia) (Zhang et al. 1991). In Korea, compatible mentor pollen was used to produce apple  pear hybrids, with a 75% germination rate with apple as the seed parent and a 17% germination rate for the reciprocal cross (Shin et al. 1989). Experiments by Kim et al. (2004) indicated that using P. pyrifolia as the seed parent resulted in higher seed set and germination than the reciprocal cross. Three S-RNase alleles were screened by PCR-RFLP to confirm hybrid status. In addition to true hybrids, intergeneric crosses of Pyrus communis with Chaenomeles japonica (Thunb.) Lindl. ex Spach (Japanese quince) and Malus pumila have produced apomictic seedlings (Dolmatov et al. 1998), as have crosses between P. pyrifolia and Malus pumila (Inoue et al. 2002). Although intergeneric hybrids have generally been artificially produced, a naturally occurring apple  European pear hybrid has been reported (Dimitrov and Delipavlov 1976) and Sorbopyrus auricularis is apparently naturally occurring. Hybridization of Japanese pear (P. pyrifolia) and Malus pumila resulted in production of some small seeds, which germinated at the reduced frequency of 71%, but all seedlings died within 6 months (Shimura et al. 1980). Sokolova (1970) reported that using either young (presumable immature) or old pistils should be pollinated rather than mature pistils to increase the chances of overcoming incompatibility in intergeneric crosses. Methods of enhancing the success of intergeneric hybridization include pollen irradiation (Jakovlev et al. 1968), and in vitro culture of seeds or embryos (Jakovlev et al. 1971; Banno et al. 2003; Papikhin et al. 2007; Sun and Leng 2008). The treatment of stigmas with a low concentration of boric acid, gibberellin, or succinic acid prior to pollination has improved hybridization of mountain ash with pear (Shcherbenev 1973, 1975). Growing hybrid seedlings at high temperature (34 C) overcame symptoms of hybrid lethality, but the high temperature eventually killed the seedlings (Inoue et al. 2003). Intergeneric hybrids between Sorbus aria and P. communis are at least partially sterile, and meiosis is irregular (Sax and Sax 1947). Pakhomova (1971) noted a failure of pairing of most chromosomes, formation of univalents and multivalents, unequal and asynchronous separation, and irregular tetrads and polyads in Malus baccata  P. communis hybrids. However, gamma-irradiation of a hybrid induced chromosome doubling in the microspores, albeit with 8 Pyrus some chromosome abnormalities (Pakhomova 1974). Gonai et al. (2006) also used gamma-irradiation of shoots of putative hybrids to overcome hybrid lethality. Marker analysis using SSRs confirmed the hybrid nature of the lone survivor. Rudenko (1974) reported that apple  pear F2 hybrids had either diploid, triploid, or tetraploid chromosome complements. Pistil cutting has been reported to improve hybridization success between apple and P. pyrifolia (Li et al. 1997). Fruits from intergeneric apple  European pear crosses have been small and irregular in shape with few seeds per fruit (Inozemtsev 1972). The seeds were generally of low viability. The structure of the pericarp of hybrids between Malus baccata and the pear ‘Michurin’s Winter Beurre’ (‘Bere Zimnyaya Michuriina’) was intermediate between the apple and pear parents, and grit cell content was much reduced (Gorshkova 1980). Hybrids of Malus baccata  P. communis have been shown to be highly resistant to apple scab, Venturia inaequalis (Cooke) G. Wint. (Gorshkova and Vanin 1973). Hybrids between ‘Fuji’ apple and ‘Oharabeni’ pear generally resembled the pear parent. The five hybrids showed resistance to apple blotch, apple scab, pear scab, and pear rust. Rudenko (1985) reported that Pyrus  Cydonia hybrids were produced as early as 1916. Later work in Moldvia resulted in additional hybrids. The chromosome number was diploid (2n ¼ 34), and inflorescences were intermediate between Pyrus and Cydonia in that they had 2–3 flowers. An artificial hybrid of the European pear Pyrus pyrifolia and Cydonia oblonga,  Pyronia veitchii, has been produced (Shimura et al. 1983). The clone of Pyronia veitchii var. luxemburgiana was backcrossed to pear in an attempt to produce a rootstock for pears (Rogers 1955). In addition to the production of novel hybrids, intergeneric crosses have been used to develop maternal pear haploids using irradiated apple pollen (Inoue et al. 2004). The authors also attempted to obtain apomictic seedlings through crossing ‘Gold Nijisseiki’ Japanese pear and apple using non-irradiated pollen. Only one of the 53 seedlings survived. 8.6.3 Sources of Other Desirable Traits Westwood (1982) reviewed the general ratings of Pyrus species for a large number of biotic and abiotic 165 stresses and adaptations. Additional extensive literature reviews have been compiled by Bell (1990), Bell et al. (1996), and Hancock and Lobos (2008). Levels of resistance of Pyrus species to diseases and arthropod pests and adaptation to various climatic and edaphic conditions are given in Tables 8.3–8.5, respectively. 8.6.4 Role in Crop Improvement Through Advanced Tools 8.6.4.1 Pear Breeding Through MarkerAssisted Breeding A long juvenile period and high level of heterozygosity due to a strict gametophytic incompatibility have limited the parental combinations in pear breeding programs. Marker-assisted selection (MAS) is considered to be a powerful tool for increasing selection efficiency by identifying favorable genetic combinations in fruit trees as documented in other crops. The major advantage of MAS is the ability to evaluate many traits at the seedling stage in fruit trees that have a long juvenile phase. Especially, MAS in pear breeding programs can be particularly important for traits that are difficult to evaluate. However, available markers for MAS are limited in Pyrus. Disease Resistance Fire blight caused by Erwinia amylovora is the most harmful disease in North America and Europe. Fire blight continues to spread throughout western, central and southern Europe despite quarantine measures adopted (Jock et al. 2002). Different levels of susceptibility to fire blight exist in European pear cultivars. Fire blight resistance in pear is known as a quantitative trait (Dondini et al. 2002). Dondini et al. (2004) constructed two genetic linkage maps of the parental lines ‘Passe Crassane’ (susceptible) and ‘Harrow Sweet’ (resistant) using SSRs, MFLPs, AFLPs, RGAs, and AFLP-RGAs markers and conducted QTL analysis for fire blight resistance. QTL analysis identified four regions (LGs 2, 4 and 9: 2 QTLs in LG2) of ‘Harrow Sweet’ associated with fire blight resistance, while no 166 QTLs related to resistance were found in susceptible ‘Passe Crassane’. About 50% of the total variance was due to four QTLs, and two QTLs on LG2 showed large LOD values (Dondini et al. 2004). Pear scab, caused by two species of Venturia, Venturia nashicola and Venturia pirina, is one of the most important diseases of Asian and European pears. V. nashicola is pathogenic only on Asian pears, not on European pears, while V. pirina is pathogenic only on European pears, not on Asian pears (Bell et al. 1996; Ishii et al. 2002). The positions and linkage groups (LGs) of the genes for resistance to scab were identified in Asian and European pears (Terakami et al. 2006; Pierantoni et al. 2007). The major resistance gene Vnk of the Japanese pear cultivar ‘Kinchaku’ against V. nashicola was identified in the central region of LG1 (Terakami et al. 2006). Six markers (one SSR: Hi02c07 and five STSs derived from AFLP and RAPDs: STS-OPW2, STS-OPAW13, STS-OPO9, STS-CT/CTA, and STS-OPAQ11) showed tight linkages to Vnk, being mapped with distance ranging from 2.4 to 12.4 cM. Gonai et al. (2009) reported that STS-OPW2 and STS-OPO9 could be useful for MAS for pear scab resistance by introducing Vnk from ‘Kinchaku’. In contrast, while a single dominant gene for V. pirina resistance has not been found in European pear cultivars, there is evidence of polygenic resistance (Chevalier et al. 2004). Pierantoni et al. (2007) reported the position of two putative QTLs related to scab resistance in two F1 population maps (‘Abbe Fetel’  ‘Max Red Bartlett’) being located on LG3 and 7. The two QTLs explained 88% of the phenotypic variance and the LOD values were higher than 10, suggesting the involvement of these two major genes in V. pirina resistance (Pierantoni et al. 2007). Black spot disease, which is caused by Alternaria alternata Japanese pear pathotype, is one of the most serious diseases in Japanese pear cultivation. The susceptibility to black spot disease is controlled by single dominant gene designated as A (Kozaki 1973). Banno et al. (1999) tested 250 RAPD primers to screen a pair of bulked DNA samples derived from open-pollinated progenies of Japanese pear ‘Osa Nijisseiki’ to identify markers linked to the susceptible A gene of black spot disease. The CMNB41 primer generated a 2,350 bp fragment, which was present in the susceptible bulk but not in the resistant one. This RAPD marker, CMNB41/2350, was at a distance of about 3.1 cM R.L. Bell and A. Itai from the susceptible A gene. They found that the frequency of occurrence of the CMNB41/2350 marker was 96% in susceptible cultivars and progenies of ‘Osa Nijisseiki’  ‘Oharabeni’. More recently, the exact positions and linkage groups of the genes for susceptibility to black spot were identified in two Japanese pear cultivars, ‘Osa Nijisseiki’ (designated as Ani) and ‘Nansui’ (Ana) (Terakami et al. 2007). Ani and Ana were located at the top region of LG11 and linked to two SSR markers CH04hO2 and CH03d02. Insect Resistance Dysaphis pyri is an important aphid pest of P. communis, and no cultivaris are currently reported to be resistant. Evans et al. (2008) screened microsatellite markers with a progeny of ‘Doyenne du Comice’  an accession of P. nivalis to identify markers linked to the major gene (Dp-1) for resistance to D. pyri. They found that Dp-1 is flanked by NII006b and NII014 on linkage group 17, 2.3 and 3.6 cM away, respectively. Fruit Quality Ethylene production by cultivated Japanese pear fruits varies from 0.1 to 300 nl g 1 h 1 during fruit ripening, suggesting that there are both climacteric and non-climacteric cultivars. Climacteric-type fruits exhibit a rapid increase in ethylene production and have a low storage potential, while non-climacteric fruits show no detectable ethylene production and fruit quality maintained for over a month in storage. Fruit storage potential is closely related to the maximum level of ethylene production in Japanese pear. Itai et al. (1999, 2003b) have cloned three ACC (1-aminocyclopropane-1-carboxylate) synthase genes (PpACS1, 2, 3), and studied their expression during fruit ripening. PpACS1 was specifically expressed in cultivars of high ethylene production, while PpACS2 was specifically expressed in cultivars of moderate ethylene production. Moreover, they have identified RFLP markers linked to the ethylene evolution rate of ripening fruit using RFLP analysis with two ACC synthase genes (PpACS1 and PpACS2). RFLPs were designated as A (2.8 kb of PpACS1) linked to high levels of ethylene (>10 nl g 1 h 1) and B (0.8 kb of 8 Pyrus PpACS2), linked to moderate levels of ethylene (0.5–10 nl g 1 h 1), when the total DNA was digested by HindIII. These markers (A and B) are useful for the selection of Japanese pear cultivars with enhanced post-harvest keeping ability. These markers were converted to more convenient and easier PCR-based CAPS markers (Itai et al. 2003a). Using this CAPS system, a total of 152 cultivars were categorized into four marker types (AB, Ab, aB, ab); types AB and Ab show high levels, aB a moderate level, and ab a low level of ethylene production during fruit ripening (Itai and Fujita 2008). Furthermore, linkage analysis of these two markers were conducted in the F2 populations derived from selfpollinated OT16, an F1 of ‘Osa Nijisseiki’ (a selfcompatible mutant of ‘Nijisseiki’)’  ‘Cili’, which revealed that the recombination frequency between the two markers was 20.8  3.6%. F2 populations in Pyrus have not been reported so far because of a strict gametophytic self-incompatibility. These are the first populations of self-pollinated F2 in Pyrus species. Pears are mainly marketed and served as fresh fruit and must have an attractive appearance. The fruit color is the most important factor for the fruit appearance. There are wide variations in skin colors. In Japan, yellow-green and brown russet pears are preferred. Inoue et al. (2006) reported the RAPD markers linked to major genes controlling the fruit skin color in Japanese pear. Two F1 progenies from the cross of ‘Kousui’  ‘Kinchaku’ and ‘Niitaka’  ‘Chikusui’ segregated by fruit skin color were used for segregant analysis. They tested 200 RAPD primers against four bulks and the OPH-19 primer generated a 425 bp fragment, which is present in the green-skin bulk. This RAPD marker (OPH-19425) had a recombination frequency of 7.3% from the green skin phenotype. This marker could select green fruit with probability as high as approximately 92%. In European pears, red-colored fruits have considerable eye appeal for consumers. Efforts have been made to select red-skinned sports and seedlings. The inheritance of the red color character was studied in seven progeny obtained by using cultivars with red skin and non-red skin (Dondini et al. 2008). One of these progenies (derived from the cross ‘Abbe Fetel’  ‘Max Red Bartlett’, a red bud sport of ‘Bartlett’) was used to construct two linkage maps and red color was confirmed as a monogenic dominant trait, being located on LG 4 of ‘Max Red Bartlett’ (Dondini et al. 2008). The Red gene is 167 positioned between two AFLP markers, E31M56-7 and E33M48-5, at a distance of 13.5 cM and 18.2 cM, respectively. Self-incompatibility Most pear cultivars have been classified as selfincompatible. Therefore, the presence of pollinizers interplanted in the orchard is a requirement to get an economic crop from most of the cultivars (Sanzol and Herrero 2002). The progression of our understanding of incompatibility in Pyrus has accelerated greatly since the mid-1990s. In Pyrus, gametophytic selfincompatibility is controlled by a single polymorphic gene locus, represented by the S-locus. The S-locus harbors a multi-allelic gene, which encodes for S-RNase that blocks incompatible-tube growth through the style (Ushijima et al. 1998). In Japanese pear, cDNAs encoding S1- to S10-RNase have been isolated and sequenced (Sassa et al. 1997; Ishimizu et al. 1998; Takasaki et al. 2004; Kim et al. 2006). Based on the nucleotide sequences, Ishimizu et al. (1999) established a PCR-RFLP (S1- to S7-) system for S-genotype assignments in Japanese pear. Takasaki et al. (2004) modified this system and established the system for discriminating S1- to S9-allele in Japanese pear. Kim et al. (2007) also established a new PCR-RFLP system for the determination of S-genotypes (S1- to S10-) of Japanese pear. In Japanese pear, a naturally occurring self-compatible mutant cultivar, ‘Osa Nijisseiki’, was found as a bud sport mutant of an self-incompatible cultivar ‘Nijisseiki’. The S-genotype of ‘Osa Nijisseiki’ was referred to as S2S4sm, with compared to S2S4 of ‘Nijisseiki’ (Sassa et al. 1997). Recent molecular analysis suggested that the mutation of ‘Osa Nijisseiki’ was due to the lack of S4-RNase gene expression in the style and was caused by a large deletion of a 236-kb region around S4-RNase (Okada et al. 2008). The pollen-S determinant was a long-standing puzzle of the S-RNase-mediated self-incompatibility in the Rosaceae. Recently, the pollen determinant of S-specificity in the Rosaceae was found to be an F-box protein (Ushijima et al. 2003). Sassa et al. (2007) isolated multiple F-box genes (PpSFBBs) in a genome region in ‘Osa Nijisseiki’ pear. These PpSFBBs are good candidates for the pollen-S determinant in pear. Based on the S-allele-specific sequence polymorphism of PpSFBB-genes, the most conserved SFBB in Japanese 168 pear, a new S-genotyping system, has been constructed (Kakui et al. 2007). Both S-alleleic constitution and cross-incompatibility groups have been determined for many Japanese pear cultivars, although the situation contrasts with the scarce information available in European pear. In Asian countries, artificial pollination is often used for stable production, therefore knowing Sgenotype of commercial cultivars is very important, in comparison with open-pollination in Europe. Recently, molecular techniques have been developed for the identification of S-genotypes in European pears (Sanzol and Herrero 2002; Zuccherelli et al. 2002; Zisovich et al. 2004; Takasaki et al. 2006; Moriya et al. 2007). Six S-alleles (Sa- to Sh-) were identified using 10 cultivars by Zuccherelli et al. (2002), four S-alleles (S1- to S4-) were identified using seven cultivars by Sanzol and Herrero (2002), and seven S-alleles (Si- to So) were identified by Zisovich et al. (2004). Takasaki et al. (2006) isolated the full length cDNAs of nine S-RNases (Sa- Sb-, Sd-, Se-, Sh-, Sk-, Sl-, Sq-, and Sr-) and established a CAPS marker system for genotyping European pear cultivars harboring these nine alleles. Moriya et al. (2007) found new S-alleles (Sg- Ss-, and St-) and modified the CAPS marker system for S-genotyping of cultivars harboring 17 S-alleles. Using the CAPS analysis, they have assigned a total of 95 cultivars to 48 genotypes. Both the methods and the determination of S-genotypes will facilitate stable pear production. 8.6.4.2 Genetic Engineering Agrobacterium-mediated pear transformation was first demonstrated by Mourgues et al. (1996) and since then has been demonstrated by several groups around the world using several different pear cultivars (Reynoird et al. 1999; Malnoy et al. 2000, 2003, 2005a, b; Lebedev et al. 2002a, b, c; Bell et al. 1999; Gao et al. 2007; Tang et al. 2007; Wen et al. 2008). This research dealt mostly with European pears. Agrobacterium-mediated transformation was used to introduce various transgenes largely aimed at providing in fire blight resistance, including attacin E in ‘Passe Crassane’ (Reynoird et al. 1999), T4 lysozyme in ‘Passe Crassane’ (Malnoy et al. 2000), defensin Rs-AFP2 in ‘Burakovka’ (Lebedev et al. 2002a), phosphinotricine acetyl transferase (PAT) in the rootstock GP217 (Lebedev et al. 2002b), lactoferrin in ‘Passe Crassane’ R.L. Bell and A. Itai (Malnoy et al. 2003), EPS-depolymerase in ‘Passe Crassane’ (Malnoy et al. 2005a), and Harpin-Nea in ‘Passe Crassane’ (Malony et al. 2005b). Other studies have focused on plant architecture by introducing rolB and rolC genes from Agrobacterium rhizogenes (Bell et al. 1999; Zhu et al. 2003), insect resistance (Tang et al. 2007), taste improvement by the thaumatin II gene (Lebedev et al. 2002c), fruit ripening by the ACC oxidase gene (Gao et al. 2007), abiotic stress tolerance by spermine synthase (Wen et al. 2008), and human health benefit by stilbene synthase (Flaishman et al. 2005). While potentially improved forms of existing elite cultivars have been produced, years of field trials, product testing, and public acceptance are still required before genetically engineered pears reach the marketplace. In spite of many obstacles, transgenic studies will bring useful tools to assist the creation of cultivars better adapted to the future requirements for stable production in this species. 8.7 Genomic Resources Developed Information on genomic resources of Pyrus is limited when compared to Malus and Prunus. To date, over 1,200 sequencing data entries have been recorded on GenBank, EMBL, and RefSeq. These include a collection of expressed sequence tags (ESTs), expressed genes, and molecular markers. GenBank currently lists 1,215 nucleotide sequences derived from 22 taxa and one Malus domestica  P. communis hybrid (National Center for Biotechnology Information 2009). The most entries are found for P. communis and P. pyrifolia. Entries for ESTs currently number 888, with most derived from P. pyrifolia (606), followed by P. communis (244). There are 132 probe sequences published in GenBank. Recently, the National Institute of Fruit Tree Science in Japan has initiated an EST project with the goal of developing the unique expressed gene set for Japanese pear. Current efforts have centered on sequencing over 25,000 cDNAs from libraries of developing and ripening fruit, flowers, leaves, buds, and shoots. These efforts have resolved into 10,350 unigenes (Nishitani et al. 2009). Di-nucleotide and tri-nucleotide repeats, which should be useful as genetic markers, were found. As an additional potential marker type, singlenucleotide polymorphisms (SNPs) among P. communis, 8 Pyrus P. bretschneideri, and P. pyrifolia were discovered. Sequencing of other sources is in progress. This research will provide the potential to speed up the process of gene discovery and characterization. Most of these genomic resources have been developed in P. communis or P. pyrifolia. Most studies of gene expression have dealt with fruit physiology. In addition, a cDNA library of genes expressed during infestation of pear psylla-susceptible (P. communis cv. Bartlett) and pear psylla-resistant (P. ussuriensis  P. communis BC1 hybrid NY10355) pears was constructed by suppression subtractive hybridization (Salvianti et al. 2008). Fourteen genes were differentially expressed in ‘Bartlett’ and 27 were differentially expressed in NY10355, many of which are known to be involved in response to biotic and abiotic stresses. Further research is needed to determine whether any of these can be used to develop markers for resistance. 8.8 Scope for Domestication and Commercialization Aside from the major species cultivated for fruit – P. communis, P. pyrifolia, P. bretschneideri – and perhaps P. pashia, other Pyrus species will likely only be used as sources of specific donor traits in breeding rather than as cultivated crops in their own right. One major exception to that statement is that other species may have direct utility as rootstocks for the cultivated species or as sources of specific traits in breeding rootstocks. Another exception is the use of selected clones and seedlings of P. calleryana, P. fauriei, and P. salicifolia as ornamental tree cultivars. The importance of Pyrus species as sources of important dietary and therapeutic compounds has not been thoroughly investigated. A few studies of the level of phenols and total antioxidant activity of P. communis, including variation in total phenolics among cultivars, have been reported (Imeh and Khokhar 2002; Sun et al. 2002), but comprehensive data on other species is apparently lacking. Pear ranks lower than apple, but higher than orange, in total antioxidant activity. Hou et al. (2003) reported that Pyrus taiwanensis was relatively low in free radical scavenging activity. 169 8.9 Some Disadvantages of Related Species The invasive potential of P. calleryana, either through intraspecific (Culley and Hardiman 2009) or interspecific (Vincent 2005) hybridization is becoming welldocumented. The use of clones of P. calleryana as ornamental trees in the United States has lead to escape from cultivation through open-pollination between cultivars with differing S-allele genotypes and with other species such as P. communis. Presumably, use of P. fauriei, the Korean pea pear, as a dwarf ornamental could lead to similar invasiveness for that species. Even though these species are not used in breeding for fruit bearing scion cultivars, their extensive use in the landscape poses a problem. The potential for invasiveness may be alleviated by genetic engineering with gene systems, which result in pollen or seed sterility. Pears can cause allergic responses in sensitive people. This response has been documented for fruit of European pear, P. communis, and the gene responsible for the allergen, Pyr c 1, has been cloned (Karamloo et al. 2001). However, there are, to date, no reports of other Pyrus species having been assayed for the occurrence of this gene and allergen. However, pollen of Pyrus pyrifolia has been shown to cause an allergic reaction, known as pollenosis, in Japanese pear growers, especially those workers involved in handpollination (Teranishi et al. 1988). 8.10 Some Recommendations for Future Action More extensive information on the current distribution, status, and genetic diversity of wild populations of Pyrus species is needed to assess the risk of genetic erosion in the potential gene pool for pear improvement. Greater efforts for in situ and ex situ preservation must follow, as must efforts to characterize and evaluate collected and native germplasm for economically important traits. Greater international cooperation and access to genetic resources would be desirable, while taking into consideration the perspectives and needs of both germplasmrich and germplasm-deficient countries. 170 Studies of the mode of inheritance to define narrowand broad-sense heritabilities for these traits, as well as to determine breeding values for prospective parents, should be undertaken for those traits for which such information is lacking. Interdisciplinary studies of the underlying physiological mechanisms of important traits are also needed. This is especially true of rootstock breeding, in which the mechanism(s) controlling tree size, precocity, yield, and tree architecture are not known. The foregoing types of studies should be combined with developing genomic technologies and resources within Pyrus to define the genetic architecture of traits and generally advance genetic improvement within the domesticated species. 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