依頼総説
DNA Markers and Molecular Breeding in Pear and Other Rosaceae Fruit Trees
2021 年 90 巻 1 号 p. 1-13
詳細
2021 年 90 巻 1 号 p. 1-13
Pear (Pyrus spp.) is one of the most important edible fruits belonging to the family Rosaceae. DNA markers, molecular genetics and genomics, and molecular breeding of pear have greatly progressed over the last few decades. The development of reliable DNA markers, such as simple sequence repeats and single nucleotide polymorphisms, has allowed DNA profiling of pear accessions, assessment of the genetic diversity within pear species, and analyses of phylogenetic relationships among pear species. Reference genetic linkage maps and genome-wide molecular markers have enabled practical marker-assisted selection for resistance to black spot and/or pear scab diseases, self-compatibility, harvest time, and fruit skin color in Japanese pear breeding programs. Molecular breeding has been shown to more than triple the selection efficiency of practical breeding compared with conventional breeding. Furthermore, breeding programs using two novel genomics-based approaches—genome-wide association studies and genomic selection—focusing on fruit quality and texture, and quantitative traits for breeding, are in progress. Co-linearity and functional synteny have been identified between pear and apple (Malus × domestica Borkh), and have been used to efficiently predict the function of a gene of interest and develop selection markers in related species.
The family Rosaceae includes many economically important crops that produce edible fruits (e.g., apple, apricot, cherry, loquat, peach, pear, plum, quince, raspberry, and strawberry) and nuts (e.g., almond), and ornamental flowers (e.g., rose) (Hummer and Janick, 2009). The family includes 2,500 to 3,000 diverse species from 90 genera, which are primarily native to temperate climate regions (Hummer and Janick, 2009; Potter et al., 2007). Traditionally, the family has been classified into several subfamilies: e.g., Amygdaloideae, Maloideae, Rosoideae, Spiraeoideae (Hummer and Janick, 2009). However, it has been suggested that the family comprises three subfamilies—Dryadoideae, Rosoideae, and Spiraeoideae—based on nucleotide sequence data from six nuclear and four chloroplast regions (Potter et al., 2007); all genera previously assigned to Amygdaloideae and Maloideae are included in the subfamily Spiraeoideae in this classification system.
The most economically important members of the Rosaceae are pear (Pyrus spp.) and apple (Malus × domestica Borkh.), both of which belong to the subfamily Spiraeoideae, tribe Pyreae (or Maleae). Another economically important fruit tree, loquat (Eriobotrya japonica (Thunb.) Lindl.), also belongs to this tribe. Several Prunus species that bear “stone fruit” are important fruit trees; these include peaches and nectarines (Prunus persica (L.) Batsch), cherries (P. avium L., P. cerasus L.), plums (P. domestica L., P. salicina Lindl.), and apricots (P. armeniaca L., P. mume Siebold et Zucc.), which all belong to the subfamily Spiraeoideae, tribe Amygdaleae (Potter et al., 2007).
Global annual fruit production of apples exceeds 86 million tons (FAOSTAT, 2018), making it the third most important fruit after bananas and citrus. Annual fruit production of peaches and nectarines is 24.5 million tons, and that of pears is 23.7 million tons (FAOSTAT, 2018). Four important Pyrus species, i.e., European pear (P. communis L.), Japanese pear (P. pyrifolia Nakai), and Chinese pear (P. bretschneideri Rehd. and P. ussuriensis Maxim.), have been commercially grown for edible fruit production for at least two to three thousand years (Bell, 1990; Bell et al., 1996). The major edible pear species, European pear, is cultivated in Europe, North America, South America, Africa, and Australia (Bell et al., 1996), whereas Japanese pear and Chinese pear are cultivated in East Asian countries (Bell et al., 1996).
Breeding of perennial Rosaceae fruit trees is often hampered by many disadvantages compared with annual crops, such as lengthy breeding cycles, a long juvenile period, high cost of raising individuals to maturity in the field, and high heterozygosity. The advent of genomics and biotechnology has opened new opportunities to overcome these disadvantages in fruit breeding. Here, recent progress in genetics and genomics is reviewed for pear and other Rosaceae fruit trees, focusing on highly reliable molecular markers, diversity of genetic resources, DNA profiling systems, genetic linkage maps, marker-assisted selection in breeding programs, functional synteny, whole-genome sequences, transcriptome analysis, and data-driven breeding. Subsequently, attractive new breeding approaches are discussed in terms of future perspectives including genome-wide association studies, genomic selection, omics studies, and new breeding techniques.
Simple sequence repeat (SSR, also referred to as microsatellite or short tandem repeat) markers are genomic polymorphic loci that consist of repeating DNA motifs that are usually 1–6 bp in length. They are typically co-dominant and show high polymorphism and suitability for automated use (Weber and May, 1989). Compared with other molecular markers, SSR markers provide a more reliable method for DNA fingerprinting, constructing genetic maps, and evaluating genetic diversity because of their co-dominant inheritance and the large number of alleles per locus. Since SSR analysis is based on a polymerase chain reaction (PCR), the procedure is simple and requires only a small amount of DNA. In the last couple of decades, over 1000 SSR markers in Japanese and European pears have been developed from genomic DNA sequences (Fernandez-Fernandez et al., 2006; Inoue et al., 2007; Sawamura et al., 2004; Yamamoto et al., 2002a, b, c), expressed sequence tags (ESTs) (Nishitani et al., 2009; Zhang et al., 2014), and next-generation sequencing (NGS) data (Yamamoto et al., 2013). Numerous SSR markers have been developed from the whole-genome sequence of the Chinese pear ‘Dangshansuli’ (Chen et al., 2015; Fan et al., 2013; Wu et al., 2013). SSR markers developed in several Pyrus species have been used as anchor loci for pear reference genetic linkage maps (Chen et al., 2015; Yamamoto et al., 2007).
Although SSR markers seem to be the best choice for studies of genetics and genomics, single nucleotide polymorphisms (SNPs) derived from whole-genome sequencing data or EST data can be used as a high-throughput marker system. An SNP is a substitution of a single nucleotide at a specific position in the genome. Montanari et al. (2013) developed 1096 SNPs for three European pear cultivars. Among them, 857 SNP markers showed polymorphism and were mapped in the segregating populations of European pear and interspecific families. Data from a large-scale EST analysis of the Japanese pear ‘Hosui’ (synonym ‘Housui’) was used to generate a 1536 SNP array (Terakami et al., 2014). By analyzing progeny of an interspecific cross, a total of 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups (LGs) on a genetic linkage map of ‘Hosui’ (Terakami et al., 2014). Using restriction-associated DNA sequencing (RADseq), Wu et al. (2014) genotyped and mapped 3143 SNPs derived from Chinese pear. Montanari et al. (2019) recently identified the most robust and informative SNPs to include on the Axiom Pear 70 K Genotyping Array. Evaluation of this array in 1416 diverse pear accessions from the USDA repository identified 66,616 SNPs (more than 90% of all SNPs) as being high quality and polymorphic. Li et al. (2019) developed a large-scale SNP genotyping array, 200K Axiom PyrSNP, based on a diverse panel of 113 re-sequenced pear genotypes; 83% of the 200,000 SNPs on this array were of high quality. The high density and uniform distribution of the SNPs on this array facilitated prediction of the centromeric regions on all 17 pear chromosomes.
In apple, in the related genus Malus, thousands of SSR markers have been developed (Celton et al., 2009; Gianfranceschi et al., 1998; Guilford et al., 1997; Liebhard et al., 2002, 2003; Moriya et al., 2012; Silfverberg-Dilworth et al., 2006; van Dyk et al., 2010). These have been used for genetic maps, genetic diversity analyses, and DNA fingerprinting in both apple and pear. The 8K apple Infinium SNP array has been developed by an international research program, RosBREED (Chagné et al., 2012), and a 20K SNP array has been developed by a European research program, FruitBreedomics (Bianco et al., 2014); both these programs focus on bridging the gap between genomics and breeding. More recently, the Axiom apple 480K SNP genotyping array has been developed and validated (Bianco et al., 2016).
The genus Pyrus contains at least 22 widely recognized primary species, all of which are native to mildly temperate regions of Europe, North Africa, and Asia. Some Pyrus species are commercially cultivated in more than 50 countries around the world (Bell, 1990; Bell et al., 1996). Despite the wide geographic distribution, all Pyrus species are intercrossable, and there seems to be no incompatibility with regards to interspecific hybridization (Westwood and Bjornstad, 1971). Genetic resources have not been fully identified due to the low morphological diversity, lack of non-morphological characteristics that can be used to differentiate species, and widespread crossability. During the past few decades, genetic diversity of Asian pears, European pears, and other Pyrus has been evaluated by using several types of DNA markers: i.e., random amplified polymorphic DNA (RAPD; Williams et al., 1990), amplified fragment length polymorphisms (AFLP; Vos et al., 1995), SSRs, and inter-simple sequence repeats (ISSRs; Zietkiewicz et al., 1994).
These DNA marker systems have been used to examine the genetic diversity and genetic relatedness of Asian pears. RAPD analysis allowed successful evaluation of 19 Japanese pear cultivars (Kim et al., 2000a, b), 33 Asian pear accessions (Kim and Ko, 2004), and l18 Pyrus accessions that are native mainly to East Asia (Teng et al., 2001, 2002). Species-specific RAPD markers were identified, and the grouping of the species and cultivars by RAPD agreed with morphological taxonomy (Teng et al., 2001, 2002). Kimura et al. (2002) identified 58 Asian pear accessions from six Pyrus species by using nine SSR markers, and Bao et al. (2007) identified 98 pear cultivars that are native mainly to East Asia by using six SSR markers.
The genetic diversity of a total of 145 wild relatives of European pear and cultivated individuals of European pear maintained in the National Plant Germplasm System (USA) was evaluated at 13 SSR loci (Volk et al., 2006) by Bayesian cluster analysis. The cultivated pears were closely related to each other and were most closely related to wild relatives that showed a genotype intermediate between the P. communis ssp. pyraster and P. communis ssp. caucasica groups. Many studies on genetic diversity of European pears have been reported; e.g., wild and semi-wild pears (P. pyraster) in Poland were evaluated by using AFLP markers (Dolatowski et al., 2004); several cultivars of European pear and Japanese pear and several wild species were evaluated by using RAPD markers (Oliveira et al., 1999); 24 European pear cultivars were evaluated by using several markers (Monte-Corvo et al., 2001); 25 cultivars of European pear and Japanese pear were evaluated by using RAPD and 18S rDNA markers (Lee et al., 2004); 31 Tunisian pear accessions (P. communis L.) were evaluated by using 7 SSR markers (Brini et al., 2008); 95 Italian pear landraces were evaluated by using 9 SSR markers and chloroplast DNA (cpDNA) (Ferradini et al., 2017); 48 pear accessions native to the Indian Himalayan region were evaluated by using 20 SSR markers and 23 morphological traits (Rana et al., 2015); and 94 Slovenian pear accessions were evaluated by using SSR and AFLP markers (Sisko et al., 2009).
cpDNA usually shows maternal inheritance in angiosperms. Although cpDNA evolves very slowly relative to nuclear and mitochondrial DNA, structural alterations in cpDNA, such as insertions, deletions, inversions, and translocations, have been found in related plants (Palmer et al., 1985). Structural mutational events in cpDNA provide useful tools for reconstructing the plant phylogeny and thereby tracing the course of evolution (Downie and Palmer, 1992). Iketani et al. (1998) examined cpDNA polymorphisms in 106 East Asian Pyrus accessions; they observed four haplotypes with a combination of three independent restriction site mutations. Kimura et al. (2003a) identified nucleotide sequences at six noncoding regions of cpDNA (5.7 kbp in total) that were polymorphic among eight pear accessions from five species: a total of 38 nucleotide substitutions, deletions, and insertion mutations were found. The complete sequence (159,922 bp) of the Japanese pear chloroplast genome has been reported (Terakami et al., 2012); the genome includes a pair of inverted repeats separated by a small single-copy region and a large single-copy region and a total of 130 predicted genes including 79 protein-coding genes, four ribosomal RNA genes, and 30 tRNA genes.
SSR markers have been widely used in forensic investigations of human parentage (Roewer, 2013), and have been shown to display high reliability and high discriminative ability. In plants, DNA identification techniques have played important roles in protecting breeders’ rights: e.g., in preventing the false labeling of fruit tree cultivars, preventing illegal fruit imports from foreign countries, and solving problems relevant to cultivar registration. The large number of SSR markers developed in pear and other Rosaceae fruit species could lead to the identification of true pedigrees and exact parentages. The parentage of the Japanese pear ‘Hosui’, the second-most common pear produced in Japan, was successfully ascertained by DNA analysis: ‘Kosui’ (synonym ‘Kousui’) and ‘I-33’ were found to be the female parent and the male parent, respectively, about 50 years after the original cross (Sawamura et al., 2004). The pedigree of ‘Hosui’ [‘Kosui’ ♀ (‘Kikusui’ ♀ × ‘Wasekouzou’ ♂) × ‘I-33’ ♂ (‘Ishiiwase’ ♀ × ‘Nijisseiki’ ♂)] was identified (Sawamura et al., 2004) (Fig. 1). The parentage of 14 pear cultivars, comprising eight and six cultivars derived from intraspecific and interspecific crosses, respectively, has been analyzed using 20 SSR markers. In 10 out of 14 cultivars, the parent–offspring relationships were reconfirmed; for the other four cultivars, questionable parent–offspring relationships were identified (Kimura et al., 2003b). Twenty-four major Japanese pear cultivars can be differentiated by using 10 SSR markers with tetra- and penta-nucleotide motifs (Yamamoto et al., 2012) (Fig. 1). These SSR markers, which were developed from genomic NGS data on ‘Hosui’, generate clear amplified fragments with no stutter bands, making them very suitable for DNA profiling (Fig. 1). After intra-laboratory validation of the above markers, Narita et al. (2014) established a “DNA profiling method for 24 Japanese pear cultivars”, which has contributed greatly to protecting breeders’ rights.
Clear amplified SSR fragments with no stutter bands obtained from the TsuGNH111 locus in eight Japanese pear cultivars in the pedigree of ‘Hosui’. The SSR locus TsuGNH111, which has a tetra-nucleotide motif of CTCC, had five alleles, with observed heterozygosity (HO) and expected heterozygosity (HE) both equal to 0.71, in 79 Japanese pear accessions. Nucleotide sequences of TsuGNH111 are registered as AB733230 in GenBank <https://www.ncbi.nlm.nih.gov/>.
The parentage of 16 peach cultivars (two bud sport mutants, five chance seedlings, and nine cultivars produced by controlled hybridization) has been analyzed using 17 SSR markers (Yamamoto et al., 2003a, b) and the parent-offspring relationships were confirmed for the nine crossbreeding cultivars, but one of the bud sport mutants did not appear to be a mutant. SSR analysis revealed that all peaches cultivated in Japan are derived from a specific Chinese cultivar ‘Shanhai Suimitsuto’ (Yamamoto et al., 2003a).
SSR markers developed in pear and apple have been applied across genera to other Rosaceae species. For instance, the parentage and origin of quince (Cydonia oblonga) cultivars could be identified by using pear and apple SSR markers (Yamamoto et al., 2004b). Watanabe et al. (2008) established DNA fingerprinting for loquat cultivars by using pear and apple SSR markers, and confirmed the parentages of 24 loquat cultivars commercially grown in Japan, including 15 diploid, six triploid, and three tetraploid cultivars. The genetic diversity and relatedness of 94 loquat accessions in Japan was also characterized by using pear and apple SSRs (Fukuda et al., 2013).
Intergeneric hybrids between Japanese pear and apple were clearly identified for the first time by using SSR markers derived from pear and apple and flow cytometry (Gonai et al., 2006). Because mature hybrids could not be generated by conventional breeding due to hybrid lethality, these viable intergeneric hybrids between Japanese pear and apple were produced by gamma irradiating the shoots from immature hybrid embryos and culturing them under normal temperature.
Genome-wide molecular markers combined with reference genetic linkage maps are very useful for fundamental and applied genetic research, and for marker-assisted selection (MAS) in breeding programs. In the past two decades, genetic linkage maps have been reported for the European pear, Japanese pear, and Chinese pear. Current genetic linkage maps for pear are sufficiently dense to cover all regions of the genome; the number of LGs corresponds to the basic chromosome number (x = 17). Furthermore, several molecular markers associated with genes or traits of interest have been identified. Iketani et al. (2001) reported the first RAPD-based genetic linkage maps of the Japanese pears ‘Kinchaku’ and ‘Kosui’ and these maps cover about half of the pear genome. The ‘Kinchaku’ map includes loci for resistance to pear scab disease (Vn) and susceptibility to black spot disease (A). Yamamoto et al. (2002c) established genetic linkage maps of the European pear ‘Bartlett’ and the Japanese pear ‘Hosui’ based on AFLPs and SSRs (from pear, apple, and Prunus) in the F1 progenies of the interspecies cross between these cultivars. The ‘Bartlett’ map (total length, 949 cM) consisted of 226 loci (175 AFLPs, 49 SSRs, one isozyme locus, and one self-incompatibility locus) on 18 LGs. Dondini et al. (2004) constructed two genetic linkage maps of the European pears ‘Passe Crassane’ and ‘Harrow Sweet’. The ‘Passe Crassane’ map (total length, 912 cM) consisted of 155 loci on 18 LGs. Partial genetic linkage maps of the European pears ‘Passe Crassane’, ‘Harrow Sweet’, ‘Abbe Fetal’, and ‘Max Red Bartlett’ established three LGs (LGs 10, 12, and 14) were constructed by using apple SSRs (Pierantoni et al., 2004).
More recently, reference genetic linkage maps have been constructed for the European pears ‘Bartlett’ and ‘La France’, and the Japanese pear ‘Hosui’ by using SSRs from pear, apple, and Prunus, and AFLPs, isozymes and phenotypic traits (Terakami et al., 2009; Yamamoto et al., 2007). The ‘Bartlett’ map (spanning >1000 cM) consists of 447 loci including 58 pear-derived SSRs, 60 apple-derived SSRs, and 322 AFLPs and the ‘La France’ map (spanning 1156 cM) consists of 414 loci including 66 pear-derived SSRs, 68 apple-derived SSRs, and 279 AFLPs. The ‘Hosui’ map (spanning 1174 cM) contains 335 loci (224 AFLPs, 105 SSRs, and six others) (Terakami et al., 2009; Yamamoto et al., 2004a). The ‘Bartlett’, ‘La France’, and ‘Hosui’ maps all cover 17 LGs, corresponding to the basic chromosome number of pear (x = 17). Three genomic regions (LGs 4, 5, and 12) have been found to be homozygous in ‘Hosui’, perhaps due to biased crossing and particular selection during Japanese pear breeding programs (Terakami et al., 2009).
Several high-density SNP- and SSR-based consensus maps have been constructed in pear. An updated reference genetic linkage map of ‘Hosui’ consists of 1033 loci, including 609 SNPs from EST and genome analyses (Terakami et al., 2014), 61 SNPs from potential intron polymorphism markers (Terakami et al., 2013), 202 SSRs from pear, 141 SSRs from apple, and 20 other markers (Yamamoto and Terakami, 2016). Montanari et al. (2013) evaluated 1096 pear SNPs and 7692 apple SNPs, and then mapped a total of 857 pear and 1031 apple SNPs onto the pear genetic map. Chen et al. (2015) constructed a high-density genetic map consisting of 734 SSR loci derived from 1341 newly designed SSRs obtained from the whole-genome sequence of P. bretschneideri. Wu et al. (2014) mapped 3143 SNPs on linkage maps of Chinese pear by using RADseq. A total of 905 SNPs obtained from genotyping-by-sequencing data (GBS-SNPs) and 69 SSRs were anchored in 17 LGs with a total genetic distance of 1760.1 cM by using a pear pseudo-BC1 population (Oh et al., 2020).
MAS has particular benefits for the breeding of fruit trees rather than annual crops, because the breeding of fruit trees is greatly limited by the large tree size, long generation cycle, and long juvenile phase (Luby and Shaw, 2001; Rikkerink et al., 2007). In Japanese pear, responsible genes or tightly linked DNA markers for several traits of interest—e.g., resistance to black spot disease, resistance to pear scab disease, self-compatibility, fruit skin color, and harvest time (fruit storage potential)—have been identified in genetic linkage maps and then used for MAS in practical Japanese pear breeding programs at the National Agriculture and Food Research Organization (NARO), Japan (Saito, 2016) (Fig. 2). The MAS system in NARO has more than tripled the efficiency of obtaining target individuals compared with conventional breeding systems (Saito, 2016).
Six important traits identified in the pear reference genetic linkage map and used in marker-assisted selection in the NARO Japanese pear breeding program. Positions of Mendelian trait loci (resistance or susceptibility to black spot disease, resistance to pear scab disease, and self-compatibility) and QTLs (fruit skin color and two loci for harvest time) in the pear reference genetic linkage map are indicated by lines and closed circles, respectively.
The location of the gene(s) responsible for resistance (or susceptibility) to the most important fungal disease, black spot caused by the Alternaria alternata Japanese pear pathotype, has been identified (Banno et al., 1999; Iketani et al., 2001; Terakami et al., 2007). Terakami et al. (2016) finely mapped the gene for susceptibility to black spot disease to the top region of LG 11 in the Japanese pear genome (Fig. 2), which corresponds to a 107-kbp region in the Chinese pear genome. Terakami et al. (2016) also revealed that black spot susceptibility genes Aki in ‘Kinchaku’, Ani in ‘Osa Nijisseiki’, and Ana in ‘Nansui’ were located in very similar positions at the top of LG 11. DNA markers associated with Vnk, the gene for resistance to pear scab disease caused by Venturia nashicola, have been identified in Japanese pear ‘Kinchaku’ (Gonai et al., 2012; Iketani et al., 2001; Terakami et al., 2006). Terakami et al. (2006) mapped Vnk to the middle region of LG 1 in ‘Kinchaku’ (Fig. 2) and mapped the SSR marker CH-Vf2 closely linked to the apple scab gene Vf (Belfanti et al., 2004; Maliepaard et al., 1998) to the bottom of LG 1, suggesting that the Vnk and Vf loci reside in different genomic regions of the same homologous LG.
Self-incompatibility, which prevents self-fertilization and generates outcrossing in Japanese pear, is controlled by a single multi-allelic S-locus. Identification of the S-genotype is important for commercial fruit production and cross breeding. Several identification systems for rapid and reliable S-genotyping have been established, such as PCR–restriction fragment length polymorphism (PCR-RFLP) analysis (Ishimizu et al., 1999) and allele-specific PCR amplification (Nashima et al., 2015). A self-compatible mutant ‘Osa-Nijisseiki’ derived from a self-incompatible cultivar ‘Nijisseiki’ possesses the S4sm haplotype controlling self-compatibility. Okada et al. (2008) demonstrated that self-compatibility is caused by the lack of a 236-kbp genomic region that includes the S4-RNase coding region. Therefore, detection of this 236-kbp genomic region has been applied in pear breeding programs to select for the self-compatibility trait (Okada, 2015; Okada et al., 2008).
Fruit-related traits have also been mapped to the pear genome by using molecular markers. Itai et al. (2003) reported that fruit storage potential is controlled by ethylene production via 1-aminocyclopropane-1-carboxylate (ACC) synthase. Analysis of the F1 population from a cross between Japanese pear cultivars showed that two major quantitative trait loci (QTLs), one located at the top of LG 15 and another at the bottom of LG 3, control harvest time (or fruit ripening day), preharvest fruit drop, and fruit storage potential (Yamamoto et al., 2014) (Fig. 2). The PPACS2 gene, a member of the ACC synthase gene family, is located within the QTL. The association of these two QTLs with fruit ripening day was validated in six Japanese pear populations by using variance components (Nishio et al., 2016). The russet skin of Japanese pear “protects the fruit against external stress caused by disease, insects, bad weather, and shipping” (Inoue et al., 2006). Yamamoto et al. (2014) showed that a major QTL at the top of LG 8 is associated with skin color (classified into five types according to the area of suberin deposited on the fruit surface) (Fig. 2), and that RAPD markers linked to fruit skin color (Inoue et al., 2006) map to this same region of LG 8. More recently, QTLs associated with total and individual sugar contents were mapped to LGs 1 and 7, while the genes encoding acid invertases PPAIV1 and PPAIV3, which cleave sucrose into glucose and fructose, were located in these regions and are therefore good candidate genes responsible for the QTLs (Nishio et al., 2018).
In Pyrus, a total of 45 QTLs and qualitative trait loci (Mendelian trait loci, MTLs) are described in the Genome Database for Rosaceae (<http://www.rosaceae.org>; Jung et al., 2019): e.g., resistance to pear psylla (Cacopsylla pyri), resistance to pear scab disease, leaf color, fruit weight, soluble solid content, flesh color, fruit seed number, and fruit skin texture. These QTLs and MTLs can be used for MAS in pear breeding programs.
The basic chromosome number in Rosaceae members is x = 7, 8, 9, 15, or 17 (Dirlewanger et al., 2009; Evans and Campbell, 2002; Potter et al., 2007). The subfamily Rosoideae, which contains raspberry, rose, and strawberry, usually has the chromosome number x = 7. The tribe Amygdaleae of the subfamily Spiraeoideae, known for almond, apricots, cherries, peaches, and plums, has the chromosome number x = 8. The tribe Spiraeeae of the subfamily Spiraeoideae has x = 9. As mentioned above, the basic chromosome number of x = 17 is observed for the tribe Pyreae of the subfamily Spiraeoideae, which includes apple, loquat, quince, and pear. Although Challice (1974, 1981) suggested that the Pyreae tribe (x = 17) was produced by allopolyploidization between Amygdaleae (x = 8) and Spiraeeae (x = 9), the latest molecular genetic studies support allopolyploidization between closely related members of Spiraeeae (Evans and Campbell, 2002). Velasco et al. (2010) reported that a draft genome sequence of apple showed a relatively recent genome-wide duplication (~50 million years ago), resulting in 17 chromosomes from nine ancestral chromosomes.
SSR markers derived from apple have been used across genera to characterize several Pyrus species (Japanese pear, European pear and the two Chinese pears P. bretschneideri and P. ussuriensis) (Yamamoto et al., 2001). Both sequencing and Southern blot analyses detected nucleotide repeats in amplified fragments of both pear and apple, with the inter-species differences in fragment sizes being mainly due to the differences in the number of repeats. When the genetic linkage maps of ‘Bartlett’ and ‘La France’ pear were compared with the apple reference maps of ‘Discovery’ and ‘Fiesta’ (Liebhard et al., 2002, 2003), 66 apple-derived SSR loci could be located on the homologous LGs of pear (Yamamoto et al., 2007). Furthermore, the SSR loci within LGs showed almost identical positions in pear and apple, indicating good co-linearity in all 17 LGs (Fig. 3).
Co-linearity of the genetic linkage maps (LGs 10) in loquat, pear and apple. SSR loci from pear and apple are underlined and italicized, respectively. Numbers to the left side indicate genetic distances (cM). LG Loquat10 was obtained by using a three-way cross of loquat ‘Mogi’ × 78-51 (loquat ‘Shiromogi’ × bronze loquat ‘Taiwan loquat No. 1’) (Fukuda et al., 2016). LG Pear10 was obtained from the European pear ‘Bartlett’ (Fukuda et al., 2016; Yamamoto et al., 2007). LG Apple10 was obtained from the apple ‘Akane’ (Kunihisa et al., 2014).
There are numerous examples of the use of SSR markers across genera within the tribe Pyreae (apple, pear, quince, and loquat) (Silfverberg-Dilworth et al., 2006; Soriano et al., 2005; Yamamoto et al., 2001, 2004a, b). In more recent examples, Gisbert et al. (2009) used SSR markers developed from apple and pear to construct genetic linkage maps of the loquat cultivars ‘Algerie’ and ‘Zaozhong-6’, indicating that the loquat maps showed a high synteny with apple maps. Fukuda et al. (2014, 2016) identified co-linearity of all LGs among apple, pear, loquat (its wild relative bronze loquat Eriobotrya deflexa), and in particular almost perfect co-linearity around the loquat canker resistance locus at the top of LG 10. These findings suggest that all chromosomes of the genera in the tribe Pyreae show good co-linearity despite considerable differences in genome size (range, 1.11 pg/2C to 1.57 pg/2C) (Dickson et al., 1992; Dirlewanger et al., 2009) (Fig. 3).
Whole-genome sequences of apple (Velasco et al., 2010), Chinese pear (Wu et al., 2013), and European pear (Chagné et al., 2014) showed that pear and apple diverged from each other about 5.4 to 21.5 million years ago. Comparison of these genome sequences showed that the genome size differences are mainly due to differences in repetitive sequences, most of which are transposable elements, whereas genic regions are very similar between species.
Comparative genomics in Rosaceae fruit crops can be used to identify homologous genes and functional synteny across species and genera: e.g., synteny in molecular markers associated with traits of interest and QTLs, and in candidate genes controlling fruit quality and texture. Synteny of functional genes is observed between pear and apple. Genes responsible for resistance to Alternaria diseases, black spot in Japanese pear and Alternaria blotch in apple, are located on the top of chromosome 11 in pear and apple, respectively (Moriya et al., 2019; Terakami et al., 2016). The same SSR markers developed from apple contigs are closely linked to both genes (Moriya et al., 2019; Terakami et al., 2016), suggesting that these genes are located in a homologous genome region, and may have the same origin. A self-incompatibility locus exists at the bottom of LG 17 in apple (Maliepaard et al., 1998; Moriya et al., 2012) and pear (Yamamoto et al., 2002c, 2007). Significant QTLs controlling harvest time (preharvest fruit drop) were observed at the top of LG 15 in apple (Kunihisa et al., 2014) and pear (Yamamoto et al., 2014). Members of the ACC synthase gene family are located in this region of LG 15 and are the likely responsible genes.
In contrast, transferability of SSR markers across tribes (e.g., between Amygdaleae [Prunus species such as peach] and Pyreae [pear and apple]) is very low. Cipriani et al. (1999) found that only 18% of peach SSRs showed amplified bands in apple. Similarly, Yamamoto et al. (2004a) observed that only 10% of Prunus SSRs could be transferred to the genetic linkage maps of pears ‘Bartlett’ and ‘Hosui’. Liebhard et al. (2002) reported that only one out of the 15 apple SSR markers they tested was transferable to Prunus.
The draft genome sequences of several Rosaceae fruit species have been produced: apple (Velasco et al., 2010), Chinese pear (Wu et al., 2013), European pear (Chagné et al., 2014), peach (Verde et al., 2013), wild strawberry (Fragaria vesca, Shulaev et al., 2011), and cultivated strawberry (Fragaria × ananassa, Hirakawa et al., 2014). The draft genome of the Chinese pear ‘Dangshansuli’ (P. bretschneideri) consists of a total of 2,103 scaffolds spanning 512.0 Mb, corresponding to 97.1% of the estimated genome size (Wu et al., 2013). In this draft genome, a total of 42,812 protein-coding genes, 28.5% of which encode multiple isoforms, and repetitive sequences of total length 271.9 Mb (53.1% of the genome) were identified. The assembly of the genome of the European pear ‘Bartlett’ (Chagné et al., 2014), contains 142,083 scaffolds and covers a total of 577.3 Mb; from this assembly, a total of 43,419 putative genes were predicted, of which 1,219 were unique to European pear compared with other plants with known genome sequences. It is expected that the genome sequences of Chinese and European pears will be assigned to 17 pseudo-chromosomes, which will greatly help us to conduct genetics and genomics studies in pears.
A web resource of Japanese pear omics information, TRANSNAP <http://plantomics.mind.meiji.ac.jp/nashi>, has recently been developed (Koshimizu et al., 2019). To exhaustively collect information on gene expression, RNA samples from various organs and stages of Japanese pear ‘Hosui’ were reverse-transcribed and then sequenced by three technologies: SMRT (Single-molecule Real-time) sequencing, 454 pyrosequencing, and Sanger sequencing. Using all the reads from these three methods, comprehensive reference sequences of Japanese pear transcripts were determined, protein sequences were predicted using TransDecoder, and biological functional annotations were assigned. Out of the 44,098 predicted protein-coding sequences (from 38,687 loci), 23,239 protein-coding sequences (from 20,060 loci) that begin with start codons and end at stop codons were identified. TRANSNAP will aid molecular research and breeding in Japanese pear, and comparative analysis among pear species and other members of the Rosaceae family. Recent omics studies of major fruit trees, including transcriptomics, proteomics, metabolomics, hormonomics, ionomics, and phenomics studies of Rosaceae fruit trees, are reviewed in Shiratake and Suzuki (2016).
MAS can accelerate and reduce the cost of breeding programs compared with conventional breeding. This is because MAS allows selection of genotype rather than phenotype, thereby reducing the number of progeny required and avoiding the need to cultivate individuals to maturity in the field (Luby and Shaw, 2001; Rikkerink et al., 2007). However, in fruit tree breeding programs, attempts to conduct MAS have been rather limited for some simply inherited traits (e.g., Mendelian trait loci), because marker development for MAS through bi-parental mapping is hindered by the need to determine the phenotypes of numerous mature individuals. Novel high-throughput genotyping techniques such as SNP array and NGS-based genotyping have enabled genome-wide association studies (GWAS) and genomic selection (GS; Meuwissen et al., 2001) to be developed as alternatives to bi-parental QTL mapping (Iwata et al., 2016). Genome-wide markers combined with reference genetic linkage maps have facilitated GWAS and GS for breeding programs in pear (Iwata et al., 2013a, b; Kumar et al., 2019; Minamikawa et al., 2018), apple (Kumar et al., 2012, 2013), and forest trees (Grattapaglia and Resende, 2011).
Iwata et al. (2013b) examined the potential of GWAS and GS by using 76 Japanese pear cultivars and 162 markers for nine agronomic traits. In GWAS, significant associations with markers were detected for harvest time, black spot resistance, and the number of spurs. In GS, the genome-wide predictions of breeding values were very high for harvest time (0.75), and moderately accurate (0.38–0.61) for five other traits. These results indicated that GWAS and GS could potentially be efficiently used in Japanese pear breeding programs. To further evaluate the use of GWAS and GS in pear breeding, Minamikawa et al. (2018) used a pear parental population of 86 accessions and breeding populations of 765 trees from 16 full-sib families, which were phenotyped for 18 traits and genotyped for 1506 SNPs. The results indicated that the power of GWAS and accuracy of GS were improved when the data from the breeding populations and the parental population were combined. Recently, interspecific pear (Pyrus spp.) hybrid populations (550 hybrid seedlings) were evaluated for 10 pear fruit phenotypes by using genotyping-by-sequencing. The results showed that the average GS accuracy varied from 0.32 (for crispness) to 0.62 (for sweetness), with an across-trait average of 0.42 (Kumar et al., 2019).
Iwata et al. (2013b) proposed a method for predicting the segregation of target traits and for selecting promising parental combinations based on genome-wide markers and phenotype data of parental cultivars. This method combines segregation simulation and Bayesian modeling for GS. When applied to Japanese pear data, the method predicted the segregation of target traits with reasonable accuracy, especially in highly heritable traits. Genomic prediction is useful for choosing a parental combination and the breeding population size.
DNA markers, molecular genetics, genome sequencing, comparative genomics (collinearity and functional synteny), and molecular breeding have greatly progressed in pear and other Rosaceae fruit trees in the last two to three decades. In Japanese pear, genomic regions associated with several phenotypic characteristics have been located on genetic linkage maps, and MAS for different forms of disease resistance, self-compatibility, and other phenotypic traits has achieved more than three times the selection efficiency compared with conventional selection protocols in practical breeding (Saito, 2016). As pointed out by Iwata et al. (2016), due to the increased throughput and the decreased cost of genome-wide SNP genotyping, as well as the improved accuracy and power of recent statistical methods, GWAS and GS will become of major importance in future fruit tree breeding and genetics research.
New breeding techniques (NBTs) are attractive alternative approaches that could accelerate the development of new traits in plant breeding. Although no trials of NBTs have been reported in pear, they are very promising approaches. NBTs involve “genome editing” with the intent to modify DNA at specific location(s) within a gene or genes to introduce new traits and properties in crop plants. In apple, Nishitani et al. (2016) presented the first study showing efficient genome editing using the CRISPR/Cas9 system. In this study, an endogenous phytoene desaturase gene was precisely modified in a transgenic apple. Using another form of NBT, a fast-track breeding system was developed to shorten the juvenile phase in fruit trees such as apple and citrus; this system controls the juvenile to adult transition by inducing a flowering gene or silencing a floral repressor (Endo et al., 2005; Flachowsky et al., 2011; Wenzel et al., 2013). Furthermore, simultaneous induction of Arabidopsis thaliana FLOWERING LOCUS T gene and silencing of apple TERMINAL FLOWER 1 gene using the Apple latent spherical virus vector has been used to stably induce flowering in apple. Using this technique, apple plants reached fruiting maturity within a year (Yamagishi et al., 2011). Such plant virus vector–induced transient induction could potentially be applied to other fruit crops, including pear, to accelerate generation time.
