Invited Review

DNA Markers and Molecular Breeding in Pear and Other RosaceaeFruit Trees

Toshiya Yamamoto1,2*

1Department of Intellectual Property, National Agriculture and Food Research Organization, Tsukuba 305-8517, Japan2Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan

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 fewdecades. The development of reliable DNA markers, such as simple sequence repeats and single nucleotidepolymorphisms, has allowed DNA profiling of pear accessions, assessment of the genetic diversity within pearspecies, and analyses of phylogenetic relationships among pear species. Reference genetic linkage maps andgenome-wide molecular markers have enabled practical marker-assisted selection for resistance to black spotand/or pear scab diseases, self-compatibility, harvest time, and fruit skin color in Japanese pear breedingprograms. Molecular breeding has been shown to more than triple the selection efficiency of practical breedingcompared with conventional breeding. Furthermore, breeding programs using two novel genomics-basedapproaches—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 identifiedbetween pear and apple (Malus × domestica Borkh), and have been used to efficiently predict the function of agene of interest and develop selection markers in related species.

Key Words: Japanese pear, marker-assisted selection, Pyrus, SSR marker, synteny.

IntroductionThe 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 andJanick, 2009). The family includes 2,500 to 3,000diverse species from 90 genera, which are primarilynative to temperate climate regions (Hummer andJanick, 2009; Potter et al., 2007). Traditionally, the fam‐ily has been classified into several subfamilies: e.g.,Amygdaloideae, Maloideae, Rosoideae, Spiraeoideae(Hummer and Janick, 2009). However, it has been sug‐gested that the family comprises three subfamilies—Dryadoideae, Rosoideae, and Spiraeoideae—based onnucleotide sequence data from six nuclear and fourchloroplast regions (Potter et al., 2007); all genera pre‐viously assigned to Amygdaloideae and Maloideae areincluded in the subfamily Spiraeoideae in this classifi‐

Received; September 30, 2020. Accepted; November 11, 2020.First Published Online in J-STAGE on December 22, 2020.* Corresponding author (E-mail: toshiya@affrc.go.jp).

cation system.The most economically important members of the

Rosaceae are pear (Pyrus spp.) and apple (Malus × domestica Borkh.), both of which belong to the sub‐family Spiraeoideae, tribe Pyreae (or Maleae). Anothereconomically important fruit tree, loquat (Eriobotryajaponica (Thunb.) Lindl.), also belongs to this tribe.Several Prunus species that bear “stone fruit” are im‐portant fruit trees; these include peaches and nectarines(Prunus persica (L.) Batsch), cherries (P. avium L.,P. cerasus L.), plums (P. domestica L., P. salicinaLindl.), and apricots (P. armeniaca L., P. mume Sieboldet Zucc.), which all belong to the subfamilySpiraeoideae, tribe Amygdaleae (Potter et al., 2007).

Global annual fruit production of apples exceeds 86million tons (FAOSTAT, 2018), making it the third mostimportant fruit after bananas and citrus. Annual fruitproduction of peaches and nectarines is 24.5 milliontons, and that of pears is 23.7 million tons (FAOSTAT,2018). Four important Pyrus species, i.e., Europeanpear (P. communis L.), Japanese pear (P. pyrifoliaNakai), and Chinese pear (P. bretschneideri Rehd. andP. ussuriensis Maxim.), have been commercially grownfor edible fruit production for at least two to three

The Horticulture Journal 90 (1): 1–13. 2021.doi: 10.2503/hortj.UTD-R014

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thousand years (Bell, 1990; Bell et al., 1996). Themajor edible pear species, European pear, is cultivatedin Europe, North America, South America, Africa, andAustralia (Bell et al., 1996), whereas Japanese pear andChinese pear are cultivated in East Asian countries(Bell et al., 1996).

Breeding of perennial Rosaceae fruit trees is oftenhampered by many disadvantages compared with annu‐al crops, such as lengthy breeding cycles, a long juve‐nile period, high cost of raising individuals to maturityin the field, and high heterozygosity. The advent of ge‐nomics and biotechnology has opened new opportuni‐ties to overcome these disadvantages in fruit breeding.Here, recent progress in genetics and genomics is re‐viewed for pear and other Rosaceae fruit trees, focusingon highly reliable molecular markers, diversity of ge‐netic resources, DNA profiling systems, genetic linkagemaps, marker-assisted selection in breeding programs,functional synteny, whole-genome sequences, transcrip‐tome analysis, and data-driven breeding. Subsequently,attractive new breeding approaches are discussed interms of future perspectives including genome-wide as‐sociation studies, genomic selection, omics studies, andnew breeding techniques.

Genome-wide Simple Sequence Repeat and SingleNucleotide Polymorphism Markers

Simple sequence repeat (SSR, also referred to asmicrosatellite or short tandem repeat) markers are geno‐mic polymorphic loci that consist of repeating DNAmotifs that are usually 1–6 bp in length. They are typi‐cally co-dominant and show high polymorphism andsuitability for automated use (Weber and May, 1989).Compared with other molecular markers, SSR markersprovide a more reliable method for DNA fingerprinting,constructing genetic maps, and evaluating genetic di‐versity because of their co-dominant inheritance and thelarge number of alleles per locus. Since SSR analysis isbased on a polymerase chain reaction (PCR), the proce‐dure is simple and requires only a small amount ofDNA. In the last couple of decades, over 1000 SSRmarkers in Japanese and European pears have been de‐veloped from genomic DNA sequences (Fernandez-Fernandez et al., 2006; Inoue et al., 2007; Sawamuraet al., 2004; Yamamoto et al., 2002a, b, c), expressedsequence tags (ESTs) (Nishitani et al., 2009; Zhanget al., 2014), and next-generation sequencing (NGS)data (Yamamoto et al., 2013). Numerous SSR markershave been developed from the whole-genome sequenceof the Chinese pear ‘Dangshansuli’ (Chen et al., 2015;Fan et al., 2013; Wu et al., 2013). SSR markers devel‐oped in several Pyrus species have been used as anchorloci for pear reference genetic linkage maps (Chenet al., 2015; Yamamoto et al., 2007).

Although SSR markers seem to be the best choice forstudies of genetics and genomics, single nucleotidepolymorphisms (SNPs) derived from whole-genome

sequencing data or EST data can be used as a high-throughput marker system. An SNP is a substitution ofa single nucleotide at a specific position in the genome.Montanari et al. (2013) developed 1096 SNPs for threeEuropean pear cultivars. Among them, 857 SNP mark‐ers showed polymorphism and were mapped in the seg‐regating populations of European pear and interspecificfamilies. Data from a large-scale EST analysis of theJapanese pear ‘Hosui’ (synonym ‘Housui’) was used togenerate a 1536 SNP array (Terakami et al., 2014). Byanalyzing progeny of an interspecific cross, a total of756 SNPs were genotyped, and 609 SNP loci weremapped to linkage groups (LGs) on a genetic linkagemap of ‘Hosui’ (Terakami et al., 2014). Usingrestriction-associated DNA sequencing (RADseq), Wuet al. (2014) genotyped and mapped 3143 SNPs derivedfrom Chinese pear. Montanari et al. (2019) recentlyidentified the most robust and informative SNPs to in‐clude on the Axiom Pear 70 K Genotyping Array. Eval‐uation of this array in 1416 diverse pear accessionsfrom the USDA repository identified 66,616 SNPs(more than 90% of all SNPs) as being high quality andpolymorphic. Li et al. (2019) developed a large-scaleSNP genotyping array, 200K Axiom PyrSNP, based ona diverse panel of 113 re-sequenced pear genotypes;83% of the 200,000 SNPs on this array were of highquality. The high density and uniform distribution ofthe SNPs on this array facilitated prediction of the cen‐tromeric regions on all 17 pear chromosomes.

In apple, in the related genus Malus, thousands ofSSR 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 diver‐sity analyses, and DNA fingerprinting in both appleand pear. The 8K apple Infinium SNP array has beendeveloped by an international research program,RosBREED (Chagné et al., 2012), and a 20K SNP arrayhas been developed by a European research program,FruitBreedomics (Bianco et al., 2014); both these pro‐grams focus on bridging the gap between genomics andbreeding. More recently, the Axiom apple 480K SNPgenotyping array has been developed and validated(Bianco et al., 2016).

Genetic Diversity in PyrusThe genus Pyrus contains at least 22 widely recog‐

nized primary species, all of which are native to mildlytemperate regions of Europe, North Africa, and Asia.Some Pyrus species are commercially cultivated inmore than 50 countries around the world (Bell, 1990;Bell et al., 1996). Despite the wide geographic distribu‐tion, all Pyrus species are intercrossable, and thereseems to be no incompatibility with regards to inter‐specific hybridization (Westwood and Bjornstad, 1971).Genetic resources have not been fully identified due to

2 T. Yamamoto

the low morphological diversity, lack of non-morphological characteristics that can be used to differ‐entiate species, and widespread crossability. During thepast few decades, genetic diversity of Asian pears,European pears, and other Pyrus has been evaluated byusing several types of DNA markers: i.e., random am‐plified polymorphic DNA (RAPD; Williams et al.,1990), amplified fragment length polymorphisms(AFLP; Vos et al., 1995), SSRs, and inter-simple se‐quence repeats (ISSRs; Zietkiewicz et al., 1994).

These DNA marker systems have been used to exam‐ine the genetic diversity and genetic relatedness ofAsian pears. RAPD analysis allowed successful evalua‐tion of 19 Japanese pear cultivars (Kim et al., 2000a, b),33 Asian pear accessions (Kim and Ko, 2004), and l18Pyrus accessions that are native mainly to East Asia(Teng et al., 2001, 2002). Species-specific RAPD mark‐ers were identified, and the grouping of the species andcultivars by RAPD agreed with morphological taxono‐my (Teng et al., 2001, 2002). Kimura et al. (2002) iden‐tified 58 Asian pear accessions from six Pyrus speciesby using nine SSR markers, and Bao et al. (2007) iden‐tified 98 pear cultivars that are native mainly to EastAsia by using six SSR markers.

The genetic diversity of a total of 145 wild relativesof European pear and cultivated individuals ofEuropean pear maintained in the National PlantGermplasm System (USA) was evaluated at 13 SSRloci (Volk et al., 2006) by Bayesian cluster analysis.The cultivated pears were closely related to each otherand were most closely related to wild relatives thatshowed a genotype intermediate between theP. communis ssp. pyraster and P. communis ssp.caucasica groups. Many studies on genetic diversity ofEuropean pears have been reported; e.g., wild and semi-wild pears (P. pyraster) in Poland were evaluated byusing AFLP markers (Dolatowski et al., 2004); severalcultivars of European pear and Japanese pear and sever‐al wild species were evaluated by using RAPD markers(Oliveira et al., 1999); 24 European pear cultivars wereevaluated by using several markers (Monte-Corvoet al., 2001); 25 cultivars of European pear andJapanese pear were evaluated by using RAPD and 18SrDNA markers (Lee et al., 2004); 31 Tunisian pearaccessions (P. communis L.) were evaluated by using7 SSR markers (Brini et al., 2008); 95 Italian pearlandraces were evaluated by using 9 SSR markers andchloroplast DNA (cpDNA) (Ferradini et al., 2017); 48pear accessions native to the Indian Himalayan regionwere evaluated by using 20 SSR markers and 23 mor‐phological traits (Rana et al., 2015); and 94 Slovenianpear accessions were evaluated by using SSR andAFLP markers (Sisko et al., 2009).

cpDNA usually shows maternal inheritance in an‐giosperms. Although cpDNA evolves very slowly rela‐tive to nuclear and mitochondrial DNA, structuralalterations in cpDNA, such as insertions, deletions, in‐

versions, and translocations, have been found in relatedplants (Palmer et al., 1985). Structural mutationalevents in cpDNA provide useful tools for reconstruct‐ing the plant phylogeny and thereby tracing the courseof evolution (Downie and Palmer, 1992). Iketani et al.(1998) examined cpDNA polymorphisms in 106 EastAsian Pyrus accessions; they observed four haplotypeswith a combination of three independent restriction sitemutations. Kimura et al. (2003a) identified nucleotidesequences at six noncoding regions of cpDNA (5.7 kbpin total) that were polymorphic among eight pear acces‐sions from five species: a total of 38 nucleotide substi‐tutions, deletions, and insertion mutations were found.The complete sequence (159,922 bp) of the Japanesepear chloroplast genome has been reported (Terakamiet al., 2012); the genome includes a pair of invertedrepeats separated by a small single-copy region and alarge single-copy region and a total of 130 predictedgenes including 79 protein-coding genes, four ribo‐somal RNA genes, and 30 tRNA genes.

DNA Profiling in Pear and Other Rosaceae FruitTrees

SSR markers have been widely used in forensic in‐vestigations of human parentage (Roewer, 2013), andhave been shown to display high reliability and highdiscriminative ability. In plants, DNA identificationtechniques have played important roles in protectingbreeders’ rights: e.g., in preventing the false labeling offruit tree cultivars, preventing illegal fruit imports fromforeign countries, and solving problems relevant to cul‐tivar registration. The large number of SSR markers de‐veloped in pear and other Rosaceae fruit species couldlead to the identification of true pedigrees and exactparentages. The parentage of the Japanese pear ‘Hosui’,the second-most common pear produced in Japan, wassuccessfully ascertained by DNA analysis: ‘Kosui’(synonym ‘Kousui’) and ‘I-33’ were found to be thefemale parent and the male parent, respectively, about50 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, comprisingeight and six cultivars derived from intraspecific andinterspecific crosses, respectively, has been analyzedusing 20 SSR markers. In 10 out of 14 cultivars, theparent–offspring relationships were reconfirmed; forthe other four cultivars, questionable parent–offspringrelationships were identified (Kimura et al., 2003b).Twenty-four major Japanese pear cultivars can bedifferentiated by using 10 SSR markers with tetra-and penta-nucleotide motifs (Yamamoto et al., 2012)(Fig. 1). These SSR markers, which were developedfrom genomic NGS data on ‘Hosui’, generate clear am‐plified fragments with no stutter bands, making themvery suitable for DNA profiling (Fig. 1). After intra-

Hort. J. 90 (1): 1–13. 2021. 3

laboratory validation of the above markers, Narita et al.(2014) established a “DNA profiling method for 24Japanese pear cultivars”, which has contributed greatlyto protecting breeders’ rights.

The parentage of 16 peach cultivars (two bud sportmutants, five chance seedlings, and nine cultivars pro‐duced by controlled hybridization) has been analyzedusing 17 SSR markers (Yamamoto et al., 2003a, b) andthe parent-offspring relationships were confirmed forthe nine crossbreeding cultivars, but one of the budsport mutants did not appear to be a mutant. SSR analy‐sis revealed that all peaches cultivated in Japan arederived from a specific Chinese cultivar ‘ShanhaiSuimitsuto’ (Yamamoto et al., 2003a).

SSR markers developed in pear and apple have beenapplied across genera to other Rosaceae species. For in‐stance, the parentage and origin of quince (Cydoniaoblonga) cultivars could be identified by using pearand apple SSR markers (Yamamoto et al., 2004b).Watanabe et al. (2008) established DNA fingerprintingfor loquat cultivars by using pear and apple SSR mark‐ers, and confirmed the parentages of 24 loquat cultivarscommercially grown in Japan, including 15 diploid, sixtriploid, and three tetraploid cultivars. The genetic di‐versity and relatedness of 94 loquat accessions in Japanwas also characterized by using pear and apple SSRs(Fukuda et al., 2013).

Intergeneric hybrids between Japanese pear andapple were clearly identified for the first time by usingSSR markers derived from pear and apple and flow cy‐

Hosui

Kosui

I-33

Nijisseiki

Ishiiwase

Kikusui

Wasekouzou

Kinchaku

(bp)

Fig. 1. Clear amplified SSR fragments with no stutter bands ob‐tained from the TsuGNH111 locus in eight Japanese pear culti‐vars 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. Nu‐cleotide sequences of TsuGNH111 are registered as AB733230in GenBank <https://www.ncbi.nlm.nih.gov/>.

tometry (Gonai et al., 2006). Because mature hybridscould not be generated by conventional breeding due tohybrid lethality, these viable intergeneric hybrids be‐tween Japanese pear and apple were produced bygamma irradiating the shoots from immature hybridembryos and culturing them under normal temperature.

Genetic Linkage Maps in PearGenome-wide molecular markers combined with ref‐

erence genetic linkage maps are very useful for funda‐mental and applied genetic research, and for marker-assisted selection (MAS) in breeding programs. In thepast two decades, genetic linkage maps have been re‐ported for the European pear, Japanese pear, andChinese pear. Current genetic linkage maps for pear aresufficiently dense to cover all regions of the genome;the number of LGs corresponds to the basic chromo‐some number (x = 17). Furthermore, several molecularmarkers associated with genes or traits of interest havebeen identified. Iketani et al. (2001) reported the firstRAPD-based genetic linkage maps of the Japanesepears ‘Kinchaku’ and ‘Kosui’ and these maps coverabout half of the pear genome. The ‘Kinchaku’ map in‐cludes loci for resistance to pear scab disease (Vn) andsusceptibility to black spot disease (A). Yamamoto et al.(2002c) established genetic linkage maps of theEuropean pear ‘Bartlett’ and the Japanese pear ‘Hosui’based on AFLPs and SSRs (from pear, apple, andPrunus) in the F1 progenies of the interspecies crossbetween these cultivars. The ‘Bartlett’ map (totallength, 949 cM) consisted of 226 loci (175 AFLPs, 49SSRs, one isozyme locus, and one self-incompatibilitylocus) on 18 LGs. Dondini et al. (2004) constructed twogenetic linkage maps of the European pears ‘PasseCrassane’ and ‘Harrow Sweet’. The ‘Passe Crassane’map (total length, 912 cM) consisted of 155 loci on 18LGs. Partial genetic linkage maps of the Europeanpears ‘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 havebeen constructed for the European pears ‘Bartlett’ and‘La France’, and the Japanese pear ‘Hosui’ by usingSSRs 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 AFLPsand the ‘La France’ map (spanning 1156 cM) consistsof 414 loci including 66 pear-derived SSRs, 68 apple-derived SSRs, and 279 AFLPs. The ‘Hosui’ map (span‐ning 1174 cM) contains 335 loci (224 AFLPs, 105SSRs, and six others) (Terakami et al., 2009; Yamamotoet al., 2004a). The ‘Bartlett’, ‘La France’, and ‘Hosui’maps all cover 17 LGs, corresponding to the basic chro‐mosome number of pear (x = 17). Three genomic

4 T. Yamamoto

regions (LGs 4, 5, and 12) have been found to behomozygous in ‘Hosui’, perhaps due to biased crossingand particular selection during Japanese pear breedingprograms (Terakami et al., 2009).

Several high-density SNP- and SSR-based consensusmaps have been constructed in pear. An updated refer‐ence genetic linkage map of ‘Hosui’ consists of 1033loci, including 609 SNPs from EST and genome analy‐ses (Terakami et al., 2014), 61 SNPs from potentialintron polymorphism markers (Terakami et al., 2013),202 SSRs from pear, 141 SSRs from apple, and20 other markers (Yamamoto and Terakami, 2016).Montanari et al. (2013) evaluated 1096 pear SNPs and7692 apple SNPs, and then mapped a total of 857 pearand 1031 apple SNPs onto the pear genetic map. Chenet al. (2015) constructed a high-density genetic mapconsisting of 734 SSR loci derived from 1341 newlydesigned SSRs obtained from the whole-genome se‐quence of P. bretschneideri. Wu et al. (2014) mapped3143 SNPs on linkage maps of Chinese pear by usingRADseq. A total of 905 SNPs obtained fromgenotyping-by-sequencing data (GBS-SNPs) and 69SSRs were anchored in 17 LGs with a total genetic dis‐tance of 1760.1 cM by using a pear pseudo-BC1 popu‐lation (Oh et al., 2020).

MAS in Japanese Pear Breeding ProgramsMAS has particular benefits for the breeding of fruit

trees rather than annual crops, because the breeding offruit trees is greatly limited by the large tree size, longgeneration cycle, and long juvenile phase (Luby andShaw, 2001; Rikkerink et al., 2007). In Japanese pear,responsible genes or tightly linked DNA markers forseveral traits of interest—e.g., resistance to black spot

disease, resistance to pear scab disease, self-compatibility, fruit skin color, and harvest time (fruitstorage potential)—have been identified in genetic link‐age maps and then used for MAS in practical Japanesepear breeding programs at the National Agriculture andFood Research Organization (NARO), Japan (Saito,2016) (Fig. 2). The MAS system in NARO has morethan tripled the efficiency of obtaining target individu‐als compared with conventional breeding systems(Saito, 2016).

The location of the gene(s) responsible for resistance(or susceptibility) to the most important fungal disease,black spot caused by the Alternaria alternata Japanesepear pathotype, has been identified (Banno et al., 1999;Iketani et al., 2001; Terakami et al., 2007). Terakamiet al. (2016) finely mapped the gene for susceptibility toblack spot disease to the top region of LG 11 in theJapanese pear genome (Fig. 2), which corresponds to a107-kbp region in the Chinese pear genome. Terakamiet al. (2016) also revealed that black spot susceptibilitygenes Aki in ‘Kinchaku’, Ani in ‘Osa Nijisseiki’, andAna in ‘Nansui’ were located in very similar positionsat the top of LG 11. DNA markers associated with Vnk,the gene for resistance to pear scab disease caused byVenturia nashicola, have been identified in Japanesepear ‘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 (Belfantiet al., 2004; Maliepaard et al., 1998) to the bottom ofLG 1, suggesting that the Vnk and Vf loci reside in dif‐ferent genomic regions of the same homologous LG.

Self-incompatibility, which prevents self-fertilization

CH03g12-2

NH013a

TsuGNH175TsuENH184

TsuENH142-m2

Mdo.chr1.24

TsuENH101-m1

RLG-1

Mdo.chr1.27-m2Mdo.chr1.25

Mdo.chr1.35

Mdo.chr1.34

Mdo.chr1.39

Hi02c07-m1

TsuENH157-m1Mdo.chr1.36

STS-OPAW13

TsuENH174

MEST-120

TsuGNH095

MEST-193TsuENH156b

MEST-160

TsuSNP1114

MEST-157

TsuSNP1112

TsuSNP1010*

TsuENH197-m1*KA4b*

MEST-199

TsuENH207

TsuENH229

TsuENH179b

CH-Vf2TsuENH049

NZmsCN879773

TsuSNP006

NH010a

CH05g08

TsuGNH241TsuENH003

TsuSNP1102

TsuGNH194

LG1Hi22d06

TsuSNP1057

CH04e12b

CH02f06

MEST87NB124b-2

Hi03e04-m1

TsuGNH173

NH046a

TsuENH017

MEST28

CN493139SSR

TsuENH001

TsuENH243

MEST67

NH033b

NH212aTsuENH062

CN444636SSR

NZmsEB106592

TsuSNP007

Hi24f04

BGT23b

TsuENH216

NZmsEB149808

CH03d10

Hi01c11-m2

CH02b10

TsuSNP1169

TsuSNP001

NH002b

NZmsEB107305-m1TsuGNH239

LG2Mdo.chr11.30-m2

TsuGNH130MEST76

Mdo.chr11.35-m1

TsuENH208

Mdo.chr11.5-m2

CH02d08

TsuSNP016TsuGNH153

TsuENH246

NH023a

TsuENH127

TsuSNP1001

TsuSNP1182

TsuGNH234-m1

NB113aMEST60

NH203a

Mdo.chr1.28-m2

NB119b-1

CH01c08

NB109a

MS14h03MEST80

CH03g12-1

LG3NZ05g8

TsuSNP1005TsuSNP015

TsuSNP1072

TsuGNH121

NH011a

TsuENH121b-m1

Mdo.chr1.28-m1

TsuSNP1121

CH01d03

TsuENH019Hi23g08

TsuSNP1092

TsuGNH023

TsuENH131

NB127b

TsuGNH244-m1

TsuENH004-1NZmsDR999337

CH02h11b-1

CH01d07

NB141b

TsuENH014

TsuENH171b-m2

TsuGNH063

MEST91-m2

AT000420SSR-m1

CH02c02bTsuGNH076

LG4TsuSNP1119

TsuSNP1097

TsuENH135

NB101a

CH05e06*

CH04g09-1TsuENH018

NZmsEB134379

EMPc106

TsuGNH189

MEST089

TsuENH242

Hi04d02-m1TsuSNP1071

TsuSNP1069

TsuGNH187

NZmsCN898349

NH020a-1NB103a

TsuGNH042

CH02a08-2

CH02b12

MEST065

TsuSNP1055

NB124b-1

MEST-109

LG5TsuENH183b

TsuGNH213

TsuGNH043

TsuSNP020

TsuSNP002

TsuGNH235-m1TsuGNH227

TsuENH121b-m2

MEST-171

CH01b11

TsuENH046

TsuENH177

CH03d12

TsuENH012

TsuENH150-m1TsuENH168-m1

U78949SSR-m1

NZmsEB132582

MEST056

TsuENH126

CH05a05

TsuSNP1089

Hi03a03-m1

Hi07b06-m1

LG6NZmsEB107305-m2

TsuENH155TsuENH165

MEST83

TsuSNP1109

NH041a

CH04e05

Mdo.chr1.30

Mdo.chr1.31

TsuGNH082

Mdo.chr11.29-m1

TsuSNP1020TsuENH130b

Mdo.chr1.9

TsuENH157-m2

Mdo.chr1.41-m1

NZmsCN943067

TsuSNP1012

TsuSNP1019EMPc117

MS06c09

TsuENH173b

TsuENH067

EMPc111

TsuGNH159

TsuSNP019

LG7TsuSNP1077

TsuSNP1068TsuENH217-m1

Mdo.chr11.10-m1

NH036b

TsuENH204-m1

MEST-101

CH04g12TsuGNH116

TsuSNP004

TsuSNP011

Z71980SSR

Mdo.chr1.1

TsuGNH113

IPPN19

MEST-128

MEST19-m1

MEST-106

LG8CH04d11

NB110a-1IPPN13

MEST034

EMPc01

NH031a

NH029a

MEST097

MEST063

TsuENH116

TsuENH008TsuENH097

CH05c07

KA20

TsuSNP1066

Mdo.chr1.41-m2

NB106a

NB130b

TsuSNP005

NB134a

TsuSNP1014TsuGNH111

Hi03b03-m2

TsuENH064

TsuSNP1031CH05a03*

CH01h02*

TsuENH005*

EMPc101-m1**

Mdo.chr1.29**

TsuENH107-m2***

LG9CH04c06-2

TsuENH159EMPc115

TsuENH181

TsuENH180

CH02a08-1

AF057134SSR

MEST043

Hi03e04-m2NH039a

CH04f03

Hi03f06

TsuSNP1070

TsuSNP1075

EMPc114CH01f12

NH045a

AU223548SSR

CH02c11

EMPc105

TsuENH043

TsuSNP1083

TsuENH235

CH04g09-2

TsuGNH208

TsuGNH203

TsuENH172

Hi22f04Hi08h12

Hi05b02

TsuENH009

CH02b03b

MS02a01

MS06g03TsuSNP1176

CH01f07a

TsuENH029

TsuENH161-m1

LG10Mdo.chr11.27

CH04h02

Mdo.chr11.35-m2

Mdo.chr11.45

Mdo.chr11.30-m1Mdo.chr11.25

Mdo.chr11.7

IPPN02

Mdo.chr11.44-m1

Mdo.chr11.5-m1

Mdo.chr11.11TsuENH102

TsuSNP1094

TsuENH186-m1

CH03d02

NB111a-2

MEST44

TsuENH044

CH05c02

Hi02a09

IPPN14

Hi04g11

TsuGNH164

TsuSNP1048

TsuENH120

Mdo.chr11.17

MEST-117

NB105a

CH04g07

LG11TsuGNH080

CH05d04

NZmsAB052994TsuSNP1004

TsuSNP1052

NZ28f4CH05g07-2

CH05d11

KA16

TsuSNP1175

Hi03b03-m1

TsuENH124b

MEST-154CN496913SSR

TsuGNH215

TsuENH004-2

CH02h11b-2

CH04d02

MEST084CH01f02

CH03c02

MEST011

TsuENH110

MEST30

TsuSNP1027TsuENH171b-m1

MEST91-m1

LG12NH044b

CH05h05

TsuENH209

TsuSNP1148

TsuENH025

MEST3

MEST-168

TsuENH198

NH009bMEST052

TsuENH042-1

NB133aMEST-153

TsuGNH212

NB120a

CH03a08

MEST-139

Hi07e08-m1

TsuSNP1007

CH05f04

NH021a-1

LG13NH004a

TsuSNP1002

EMPc108**

CH05g07-1**

CH01a09***

TsuENH234**

TsuGNH029-m1**

CH04f06***

CH01g05***

CH03d08***

TsuSNP1155***NB119b-2***

MS01a05***

NH001c***

CH05g11***

Hi02d11***

CH04c07***TsuENH031***

TsuENH240***

NH035a***

TsuENH123***

CH05d03***

TsuSNP1166***TsuENH150-m2***

TsuENH168-m2***

TsuSNP1090***

Hi03a03-m2***

CH03g06***

TsuENH199***TsuENH011***

U78949SSR-m2***

TsuGNH240***

TsuGNH228***

LG14NH027a

TsuENH204-m2

TsuENH093

TsuENH217-m2

CH02d10bTsuENH096

NZ02b1

MEST62

TsuENH133

TsuENH194

TsuSNP1103TsuSNP1170

TsuENH128

NH025a

PpACS2

TsuSNP1107

TsuGNH081Hi02d02

IPPN08

CH01d08

TsuGNH013

NB129a

EMPc104

Mdo.chr1.27-m1

TsuENH143

TsuSNP1135

CH03h06

CH02d11

TsuENH232TsuENH201b

TsuENH142-m1

Hi09f01

Hi11a01

TsuENH219

TsuSNP003TsuENH040

CH02c09

TsuGNH050

TsuSNP1008

TsuENH016

TsuSNP1126TsuENH035

LG15TsuSNP1116

KA14Hi15a13

TsuENH054

TsuGNH191

Hi15g11

TsuSNP1160

CH05c06-1CH01f03a

NH007b

Hi04e04

AU301431SSR

TsuGNH167

Hi08d09

TsuGNH120NZmsEB147967

TsuENH042-2

TsuENH214

NB123a

NB116b

Hi01c11-m1

LG16CH04c10

TsuGNH061

CH04c06-1

TsuENH163

NB110a-2NH015a

MEST69

TsuENH106

AT000174SSR

TsuENH112b

TsuENH026NZmsEE663955

AF527800SSR

TsuENH033

AJ001681SSR

TsuENH140CH01h01

NB125a

NZmsMDAJ1681

TsuSNP1042

MEST20

TsuGNH045

Hi01c11-m3TsuSNP1101

CH01b12

MEST29

TsuSNP1032

NH006b-1

NB126a

TsuENH028

TsuSNP1016

NH014a

TsuENH107-m1

CN444542SSR

AY187627SSRSlocus

LG17

resistance (susceptibility) to black spot disease

self-compatibility

●

fruit skin color

●

●

resistance to pear scab disease

harvest time harvest time

Fig. 2. Six important traits identified in the pear reference genetic linkage map and used in marker-assisted selection in the NARO Japanese pearbreeding program. Positions of Mendelian trait loci (resistance or susceptibility to black spot disease, resistance to pear scab disease, andself-compatibility) and QTLs (fruit skin color and two loci for harvest time) in the pear reference genetic linkage map are indicated by linesand closed circles, respectively.

Hort. J. 90 (1): 1–13. 2021. 5

and generates outcrossing in Japanese pear, is con‐trolled by a single multi-allelic S-locus. Identification ofthe S-genotype is important for commercial fruit pro‐duction and cross breeding. Several identification sys‐tems for rapid and reliable S-genotyping have beenestablished, such as PCR–restriction fragment lengthpolymorphism (PCR-RFLP) analysis (Ishimizu et al.,1999) and allele-specific PCR amplification (Nashimaet al., 2015). A self-compatible mutant ‘Osa-Nijisseiki’derived from a self-incompatible cultivar ‘Nijisseiki’possesses the S4

sm haplotype controlling self-compatibility. Okada et al. (2008) demonstrated thatself-compatibility is caused by the lack of a 236-kbpgenomic region that includes the S4-RNase codingregion. Therefore, detection of this 236-kbp genomicregion has been applied in pear breeding programsto select for the self-compatibility trait (Okada, 2015;Okada et al., 2008).

Fruit-related traits have also been mapped to the peargenome by using molecular markers. Itai et al. (2003)reported that fruit storage potential is controlled byethylene production via 1-aminocyclopropane-1-carboxylate (ACC) synthase. Analysis of the F1 popula‐tion from a cross between Japanese pear cultivarsshowed that two major quantitative trait loci (QTLs),one located at the top of LG 15 and another at the bot‐tom of LG 3, control harvest time (or fruit ripeningday), preharvest fruit drop, and fruit storage potential(Yamamoto et al., 2014) (Fig. 2). The PPACS2 gene, amember of the ACC synthase gene family, is locatedwithin the QTL. The association of these two QTLswith fruit ripening day was validated in six Japanesepear populations by using variance components (Nishioet al., 2016). The russet skin of Japanese pear “protectsthe fruit against external stress caused by disease, in‐sects, bad weather, and shipping” (Inoue et al., 2006).Yamamoto et al. (2014) showed that a major QTL at thetop of LG 8 is associated with skin color (classified intofive types according to the area of suberin deposited onthe fruit surface) (Fig. 2), and that RAPD markerslinked to fruit skin color (Inoue et al., 2006) map to thissame region of LG 8. More recently, QTLs associatedwith total and individual sugar contents were mapped toLGs 1 and 7, while the genes encoding acid invertasesPPAIV1 and PPAIV3, which cleave sucrose into glu‐cose and fructose, were located in these regions and aretherefore good candidate genes responsible for theQTLs (Nishio et al., 2018).

In Pyrus, a total of 45 QTLs and qualitative trait loci(Mendelian trait loci, MTLs) are described in theGenome Database for Rosaceae (<http://www.rosaceae.org>; Jung et al., 2019): e.g., resistance to pear psylla(Cacopsylla pyri), resistance to pear scab disease, leafcolor, fruit weight, soluble solid content, flesh color,fruit seed number, and fruit skin texture. These QTLsand MTLs can be used for MAS in pear breeding pro‐grams.

Co-linearity and Functional Synteny betweenPyrus and Malus

The basic chromosome number in Rosaceae mem‐bers is x = 7, 8, 9, 15, or 17 (Dirlewanger et al., 2009;Evans and Campbell, 2002; Potter et al., 2007). Thesubfamily Rosoideae, which contains raspberry, rose,and strawberry, usually has the chromosome numberx = 7. The tribe Amygdaleae of the subfamilySpiraeoideae, known for almond, apricots, cherries,peaches, and plums, has the chromosome number x = 8.The tribe Spiraeeae of the subfamily Spiraeoideae hasx = 9. As mentioned above, the basic chromosomenumber of x = 17 is observed for the tribe Pyreae of thesubfamily Spiraeoideae, which includes apple, loquat,quince, and pear. Although Challice (1974, 1981) sug‐gested that the Pyreae tribe (x = 17) was produced byallopolyploidization between Amygdaleae (x = 8) andSpiraeeae (x = 9), the latest molecular genetic studiessupport allopolyploidization between closely relatedmembers of Spiraeeae (Evans and Campbell, 2002).Velasco et al. (2010) reported that a draft genome se‐quence of apple showed a relatively recent genome-wide duplication (~50 million years ago), resulting in17 chromosomes from nine ancestral chromosomes.

SSR markers derived from apple have been usedacross genera to characterize several Pyrus species(Japanese pear, European pear and the two Chinesepears P. bretschneideri and P. ussuriensis) (Yamamotoet al., 2001). Both sequencing and Southern blot analy‐ses detected nucleotide repeats in amplified fragmentsof both pear and apple, with the inter-species differ‐ences in fragment sizes being mainly due to the differ‐ences in the number of repeats. When the geneticlinkage maps of ‘Bartlett’ and ‘La France’ pear werecompared with the apple reference maps of ‘Discovery’and ‘Fiesta’ (Liebhard et al., 2002, 2003), 66 apple-derived SSR loci could be located on the homologousLGs of pear (Yamamoto et al., 2007). Furthermore, theSSR loci within LGs showed almost identical positionsin pear and apple, indicating good co-linearity in all 17LGs (Fig. 3).

There are numerous examples of the use of SSRmarkers 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 andpear to construct genetic linkage maps of the loquat cul‐tivars ‘Algerie’ and ‘Zaozhong-6’, indicating that theloquat maps showed a high synteny with apple maps.Fukuda et al. (2014, 2016) identified co-linearity of allLGs among apple, pear, loquat (its wild relative bronzeloquat Eriobotrya deflexa), and in particular almost per‐fect co-linearity around the loquat canker resistancelocus at the top of LG 10. These findings suggest thatall chromosomes of the genera in the tribe Pyreae show

6 T. Yamamoto

good co-linearity despite considerable differences ingenome 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 Europeanpear (Chagné et al., 2014) showed that pear and applediverged from each other about 5.4 to 21.5 millionyears ago. Comparison of these genome sequencesshowed that the genome size differences are mainly dueto differences in repetitive sequences, most of whichare transposable elements, whereas genic regions arevery similar between species.

Comparative genomics in Rosaceae fruit crops canbe used to identify homologous genes and functionalsynteny across species and genera: e.g., synteny inmolecular markers associated with traits of interest andQTLs, and in candidate genes controlling fruit qualityand texture. Synteny of functional genes is observed be‐tween pear and apple. Genes responsible for resistanceto Alternaria diseases, black spot in Japanese pear andAlternaria blotch in apple, are located on the top ofchromosome 11 in pear and apple, respectively (Moriyaet al., 2019; Terakami et al., 2016). The same SSRmarkers developed from apple contigs are closelylinked to both genes (Moriya et al., 2019; Terakamiet al., 2016), suggesting that these genes are located in ahomologous genome region, and may have the sameorigin. A self-incompatibility locus exists at the bottom

SSR0254SSR0599SSR0791PseASSR1764CH04c06TsuENH159TsuENH181ssrEJ-329RAPD-R-13aRAPD-E-03RAPD-AF-04MEST043RAPD-AO-05MEST038RAPD-Q-7aCH04f03RAPD-AB-17CH03d08AU223548SSRRAPD-BA-03b

RAPD-AA-16bRAPD-AX-03

Hi05b02TsuENH009MEST023Hi08h12

MS06g03

Loquat10

CH04c06TsuENH159EMPc115TsuENH181TsuENH180CH02a08AF057134SSRMEST043

Hi03e04NH039aCH04f03Hi03f06EMPc114CH01f12NH045aAU223548SSRCH02c11EMPc105TsuENH043TsuENH235CH04g09TsuENH172Hi22f04CH01f07aMS02a01TsuENH009CH02b03bHi05b02Hi08h12MS06g03TsuENH029TsuENH161

Pear10

Hi02d04

CH04c06MEST111CH02b07NB124bTsuENH180AF057134SSR

NH039aCH02a10TsuENH109Hi03f06CH01f12CH02c11NH045a

TsuENH151EMPc105Hi01b01Hi01a03TsuENH008

Hi22f04Hi08g06COLaU50187SSRNZmsCN899300MS02a01Hi05b02Hi08h12

MS06g03MS01a03

Apple10

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70(cM)

Fig. 3. Co-linearity of the genetic linkage maps (LGs 10) in loquat,pear and apple. SSR loci from pear and apple are underlinedand italicized, respectively. Numbers to the left side indicategenetic distances (cM). LG Loquat10 was obtained by using athree-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 wasobtained from the apple ‘Akane’ (Kunihisa et al., 2014).

of LG 17 in apple (Maliepaard et al., 1998; Moriyaet al., 2012) and pear (Yamamoto et al., 2002c, 2007).Significant QTLs controlling harvest time (preharvestfruit 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 arelocated in this region of LG 15 and are the likely re‐sponsible genes.

In contrast, transferability of SSR markers acrosstribes (e.g., between Amygdaleae [Prunus species suchas peach] and Pyreae [pear and apple]) is very low.Cipriani et al. (1999) found that only 18% of peachSSRs showed amplified bands in apple. Similarly,Yamamoto et al. (2004a) observed that only 10% ofPrunus SSRs could be transferred to the genetic linkagemaps of pears ‘Bartlett’ and ‘Hosui’. Liebhard et al.(2002) reported that only one out of the 15 apple SSRmarkers they tested was transferable to Prunus.

Whole-genome Sequences and TranscriptomeAnalysis in Pear

The draft genome sequences of several Rosaceaefruit 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), wildstrawberry (Fragaria vesca, Shulaev et al., 2011), andcultivated strawberry (Fragaria × ananassa, Hirakawaet al., 2014). The draft genome of the Chinese pear‘Dangshansuli’ (P. bretschneideri) consists of a total of2,103 scaffolds spanning 512.0 Mb, corresponding to97.1% of the estimated genome size (Wu et al., 2013).In this draft genome, a total of 42,812 protein-codinggenes, 28.5% of which encode multiple isoforms, andrepetitive sequences of total length 271.9 Mb (53.1% ofthe genome) were identified. The assembly of thegenome of the European pear ‘Bartlett’ (Chagné et al.,2014), contains 142,083 scaffolds and covers a total of577.3 Mb; from this assembly, a total of 43,419 putativegenes were predicted, of which 1,219 were unique toEuropean pear compared with other plants with knowngenome sequences. It is expected that the genome se‐quences of Chinese and European pears will be as‐signed to 17 pseudo-chromosomes, which will greatlyhelp us to conduct genetics and genomics studies inpears.

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 ex‐pression, RNA samples from various organs and stagesof Japanese pear ‘Hosui’ were reverse-transcribed andthen sequenced by three technologies: SMRT (Single-molecule Real-time) sequencing, 454 pyrosequencing,and Sanger sequencing. Using all the reads from thesethree methods, comprehensive reference sequences ofJapanese pear transcripts were determined, proteinsequences were predicted using TransDecoder, and

Hort. J. 90 (1): 1–13. 2021. 7

biological functional annotations were assigned. Out ofthe 44,098 predicted protein-coding sequences (from38,687 loci), 23,239 protein-coding sequences (from20,060 loci) that begin with start codons and end at stopcodons were identified. TRANSNAP will aid molecularresearch and breeding in Japanese pear, and compara‐tive analysis among pear species and other members ofthe Rosaceae family. Recent omics studies of majorfruit trees, including transcriptomics, proteomics,metabolomics, hormonomics, ionomics, and phenomicsstudies of Rosaceae fruit trees, are reviewed inShiratake and Suzuki (2016).

Genome-Wide Association Studies and GenomicSelection in Japanese Pear Breeding

MAS can accelerate and reduce the cost of breedingprograms compared with conventional breeding. This isbecause MAS allows selection of genotype rather thanphenotype, thereby reducing the number of progeny re‐quired and avoiding the need to cultivate individuals tomaturity in the field (Luby and Shaw, 2001; Rikkerinket al., 2007). However, in fruit tree breeding programs,attempts to conduct MAS have been rather limited forsome simply inherited traits (e.g., Mendelian trait loci),because marker development for MAS through bi-parental mapping is hindered by the need to determinethe phenotypes of numerous mature individuals. Novelhigh-throughput genotyping techniques such as SNParray and NGS-based genotyping have enabledgenome-wide association studies (GWAS) and genomicselection (GS; Meuwissen et al., 2001) to be developedas alternatives to bi-parental QTL mapping (Iwata et al.,2016). Genome-wide markers combined with referencegenetic linkage maps have facilitated GWAS and GSfor 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 GWASand GS by using 76 Japanese pear cultivars and 162markers for nine agronomic traits. In GWAS, signifi‐cant associations with markers were detected for har‐vest time, black spot resistance, and the number ofspurs. In GS, the genome-wide predictions of breedingvalues were very high for harvest time (0.75), and mod‐erately accurate (0.38–0.61) for five other traits. Theseresults indicated that GWAS and GS could potentiallybe efficiently used in Japanese pear breeding programs.To further evaluate the use of GWAS and GS in pearbreeding, Minamikawa et al. (2018) used a pearparental population of 86 accessions and breeding pop‐ulations of 765 trees from 16 full-sib families, whichwere phenotyped for 18 traits and genotyped for 1506SNPs. The results indicated that the power of GWASand accuracy of GS were improved when the data fromthe breeding populations and the parental populationwere combined. Recently, interspecific pear (Pyrus

spp.) hybrid populations (550 hybrid seedlings) wereevaluated for 10 pear fruit phenotypes by usinggenotyping-by-sequencing. The results showed that theaverage GS accuracy varied from 0.32 (for crispness) to0.62 (for sweetness), with an across-trait average of0.42 (Kumar et al., 2019).

Iwata et al. (2013b) proposed a method for predictingthe segregation of target traits and for selecting promis‐ing parental combinations based on genome-wide mark‐ers and phenotype data of parental cultivars. Thismethod combines segregation simulation and Bayesianmodeling for GS. When applied to Japanese pear data,the method predicted the segregation of target traitswith reasonable accuracy, especially in highly heritabletraits. Genomic prediction is useful for choosing aparental combination and the breeding population size.

PerspectivesDNA markers, molecular genetics, genome sequenc‐

ing, comparative genomics (collinearity and functionalsynteny), and molecular breeding have greatly pro‐gressed in pear and other Rosaceae fruit trees in the lasttwo to three decades. In Japanese pear, genomic regionsassociated with several phenotypic characteristics havebeen located on genetic linkage maps, and MAS for dif‐ferent forms of disease resistance, self-compatibility,and other phenotypic traits has achieved more thanthree times the selection efficiency compared with con‐ventional selection protocols in practical breeding(Saito, 2016). As pointed out by Iwata et al. (2016), dueto the increased throughput and the decreased cost ofgenome-wide SNP genotyping, as well as the improvedaccuracy and power of recent statistical methods,GWAS and GS will become of major importance infuture fruit tree breeding and genetics research.

New breeding techniques (NBTs) are attractive alter‐native approaches that could accelerate the develop‐ment of new traits in plant breeding. Although no trialsof NBTs have been reported in pear, they are verypromising 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 prop‐erties in crop plants. In apple, Nishitani et al. (2016)presented the first study showing efficient genome edit‐ing using the CRISPR/Cas9 system. In this study, anendogenous phytoene desaturase gene was preciselymodified in a transgenic apple. Using another form ofNBT, a fast-track breeding system was developed toshorten the juvenile phase in fruit trees such as appleand citrus; this system controls the juvenile to adulttransition by inducing a flowering gene or silencing afloral repressor (Endo et al., 2005; Flachowsky et al.,2011; Wenzel et al., 2013). Furthermore, simultaneousinduction of Arabidopsis thaliana FLOWERINGLOCUS T gene and silencing of apple TERMINALFLOWER 1 gene using the Apple latent spherical virusvector has been used to stably induce flowering in

8 T. Yamamoto

apple. Using this technique, apple plants reached fruit‐ing maturity within a year (Yamagishi et al., 2011).Such plant virus vector–induced transient inductioncould potentially be applied to other fruit crops, includ‐ing pear, to accelerate generation time.

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