Breeding Science
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Research Papers
Development of an SSR marker set for efficient selection for resistance to black spot disease in pear breeding
Shingo Terakami, Yoshihiko Adachi, Yukie Takeuchi, Norio Takada, Sogo Nishio, Toshihiro Saito, Toshiya Yamamoto
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Keywords: Alternaria alternata, black spot disease, Pyrus pyrifolia, Pyrus ussuriensis, marker-assisted selection
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2021 Volume 71 Issue 2 Pages 240-252

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Abstract

Black spot disease, which is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. Because of the potential harm of fungicides to consumers and the environment, resistant cultivars are desired. In this study, to enable efficient marker-assisted selection in pear breeding, we conducted comprehensive inoculation tests and genotyping with 207 pear cultivars. We identified a marker set (Mdo.chr11.27 and Mdo.chr11.34) suitable for selection for black spot resistance. In most susceptible cultivars, Mdo.chr11.27 amplified a 220-bp band and Mdo.chr11.34 amplified a 259-bp band. The genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease. Using linkage analysis, we identified the positions of the gene for susceptibility to black spot disease in Chinese pear. Mdo.chr11.27 and Mdo.chr11.34 were tightly linked to susceptibility in Chinese pear, and the susceptibility gene was mapped at the top of linkage group 11, similar to that in Japanese pear. This marker set and the accumulation of phenotypic data will enable efficient marker-assisted breeding for black spot resistance in pear breeding.

Introduction

Pears (Pyrus spp.) belong to the family Rosaceae, subfamily Spiraeoideae, tribe Pyreae. Pears have been grown in East Asia, Europe, and North America for more than 3000 years and are among the most important fruit trees in more than 50 temperate regions worldwide (Bell 1990, Bell et al. 1996). This genus is believed to have originated during the Paleocene (65–55 million years ago) in what is now the mountainous area of western and southwestern China and spread east and west from there. The Japanese pear (Pyrus pyrifolia Nakai), the European pear (P. communis L.), and the Chinese pear (P. bretschneideri Rehd. and P. ussuriensis Maxim.) are the major edible species grown commercially for fruit production (Bell et al. 1996). The Japanese and Chinese pears are grown in East Asia, while European pears are grown in Europe, North America, and temperate regions of the Southern Hemisphere. All Pyrus species are inter-crossable, and there are no major incompatibility barriers to interspecific hybridization in this genus (Westwood and Bjornsta 1971).

Japanese pear is vulnerable to many bacterial and fungal diseases, such as pear scab induced by the fungus Venturia nashicola and fire blight induced by the proteobacterium Erwinia amylovora. Black spot disease is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype (previously, A. kikutiana Tanaka). Infected pears suffer from leaf and fruit necrosis and early defoliation, resulting in reduced productivity and fruit quality. This disease is one of the most severe diseases of Japanese pears, such as ‘Nijisseiki’, grown in Asia, but has not been reported in North America or Europe (Sanada et al. 1988). Spraying fungicides to prevent infection by this pathogen is very costly (Kozaki 1973), and fungicides pose potential harm to consumers and the environment (Donald et al. 2002, Reis et al. 2007). As in many other crops, the breeding of disease-resistant cultivars is the most effective and economical method of control. A single dominant gene, designated A, controls susceptibility to black spot disease (Kozaki 1973). Susceptible cultivars are heterozygous (A/a), but no homozygous (A/A) cultivars have been identified (Kozaki 1973). Inactivation of A has been attempted to obtain resistant mutants, and moderately resistant cultivars, ‘Gold Nijisseiki’, ‘Osa Gold’, and ‘Kotobukishinsui’, were selected after chronic γ-ray irradiation (Kitagawa et al. 1999, Masuda et al. 1998, Sanada et al. 1993).

Traditional plant breeding methods are based on phenotypic selection, but phenotypic evaluation is environmentally dependent, unreliable, and labor-intensive. Plant breeding using molecular markers avoids these problems because molecular markers appear to be independent of the environment, unaffected by plant growth conditions, and detectable at all stages of plant growth. This approach, referred to as “marker-assisted breeding”, uses genetic selection instead of phenotypic selection. The development of easy-to-use marker selection systems and the accumulation of genotype data of cultivars are essential for its success. Several groups have reported the development of DNA markers linked to A. The first markers, random amplified polymorphic DNA (RAPD) markers, were reported in Japanese pears ‘Osa Nijisseiki’ and ‘Kinchaku’ (Banno et al. 1999, Iketani et al. 2001). However, these reports did not identify the position of the susceptibility gene on genetic linkage maps; therefore, little information on molecular markers linked to the gene was available. In our previous studies (Terakami et al. 2007, 2016), we constructed linkage group (LG) 11 of each of the Japanese pear cultivars ‘Osa Nijisseiki’, ‘Nansui’, and ‘Kinchaku’ and mapped the susceptible gene at the top of each cultivar LG. Fine mapping localized the susceptibility gene of ‘Kinchaku’ within a 1.5-cM region between the simple sequence repeat (SSR) markers Mdo.chr11.28 and Mdo.chr11.34 (Terakami et al. 2016). The physical size of this region was estimated to be 107 kb in the draft Chinese pear genome (Wu et al. 2013). SSR markers tightly linked to and co-segregating with the gene were identified (Terakami et al. 2016), but the polymorphism of these markers, i.e., their suitability for marker-assisted selection (MAS), has not been tested.

The aim of the present study was to develop an efficient system for MAS of seedlings resistant to black spot disease. We conducted spore inoculation tests on large-scale pear genetic resources and collected genotype data for markers linked to the susceptibility gene. We constructed a genetic map of LG 11 and identified the exact position of the susceptibility gene in Chinese pears. Lastly, we discuss the susceptibility to A. alternata in pear and apple.

Materials and Methods

Plant materials and DNA extraction

For black spot inoculation tests and genotyping by DNA markers (Table 1), we used 207 pear cultivars: 165 Japanese pears (P. pyrifolia Nakai), 35 Chinese pears (P. bretschneideri Rehd. and P. ussuriensis Maxim.), 4 interspecific hybrid cultivars, and 3 other cultivars. ‘Babaucchiaginashi’ and ‘Iwate Tanenashi’ are native cultivar and wild species collected in Japan, respectively. ‘Cheung Dang No Ri’ is introduced from Korea. The name and the species of each cultivar are given in Table 1 according to the registration with the Genebank project, NARO (https://www.gene.affrc.go.jp/index_en.php). All trees were propagated by grafting and maintained in the orchard at the Institute of Fruit Tree and Tea Science, NARO (NIFTS; Tsukuba, Ibaraki, Japan). Five F1 mapping populations were used for genetic linkage analysis of susceptibility to black spot disease (Table 2). Four of them were derived from interspecific crosses between the Japanese pear ‘Kousui’ (synonym ‘Kosui’; resistant to black spot) and Chinese pears (‘Xiang Ya Li’, ‘Mi Li Cui’, ‘Tai Huang Li’, and ‘Huang Li’; susceptible to black spot). The other population was derived from a cross between Japanese pear ‘Housui’ (synonym ‘Hosui’; resistant) and ‘Kinchaku’ (susceptible). Ungrafted seedlings were grown in plastic pots (18 cm diameter × 16 cm height) and maintained at NIFTS (Tsukuba).

Table 1. Response to black spot disease and genotypes of SSR markers linked to Aki in pear cultivars
Cultivar Species JP accession number in NARO Genebank Putative genotypea Susceptibility to black spot diseaseb SSR genotypec
Mdo.chr11.27 Mdo.chr11.34
Abumi P. pyrifolia Nakai 113559 a/a R 246/256 256/272
Aikansui P. pyrifolia Nakai 118522 R 230/230 284/284
Aiuchi P. pyrifolia Nakai 113560 a/a R 230/230 286/286
Akaho P. pyrifolia Nakai 113561 a/a R 230/230 284/284
Akiakari P. pyrifolia Nakai 118536 R 230/230 284/284
Akibae P. pyrifolia Nakai 238254 R 230/230 284/284
Akizuki P. pyrifolia Nakai 118538 R 230/230 284/284
Amanogawa P. pyrifolia Nakai 113562 a/a R 228/230 274/283
Aoyagi P. pyrifolia Nakai 113567 a/a R 230/230 284/284
Asahi P. pyrifolia Nakai 113568 a/a R 230/230 272/284
Asahiryuu P. pyrifolia Nakai 113569 A/a S 220/230 259/284
Atago P. pyrifolia Nakai 113570 R 230/230 284/284
Awayuki P. pyrifolia Nakai 113572 S 220/230 259/284
Azumanishiki P. pyrifolia Nakai 113573 a/a R 230/230 284/284
Cheong Sil Ri P. pyrifolia Nakai 113693 R 216/234 276/282
Chikusui P. pyrifolia Nakai 113716 R 230/230 284/284
Chizu P. pyrifolia Nakai 118524 S 220/230 259/284
Chouju P. pyrifolia Nakai 113575 R 230/230 284/284
Choujuurou P. pyrifolia Nakai 113574 a/a R 230/230 272/284
Chousen P. pyrifolia Nakai 113576 a/a R 230/230 284/284
Doitsu P. pyrifolia Nakai 113577 A/a S 220/220 259/284
Echigonishiki P. pyrifolia Nakai 115740 R 230/230 274/284
Edoya P. pyrifolia Nakai 113578 A/a S 220/258 259/272
Fukushima P. pyrifolia Nakai 113579 S 234/258 259/272
Geishun P. pyrifolia Nakai 113581 a/a R 230/230 274/284
Gion P. pyrifolia Nakai 113582 a/a R 230/230 284/284
Gold Nijisseiki P. pyrifolia Nakai 110823 S 220/230 259/284
Gozennashi P. pyrifolia Nakai 113583 a/a R 230/230 282/282
Hakataao P. pyrifolia Nakai 113584 A/a S 220/230 259/284
Hakkou P. pyrifolia Nakai 113585 a/a R 230/230 284/284
Hakuteiryuu P. pyrifolia Nakai 113586 a/a R 230/230 282/284
Han Henung Li Kou P. pyrifolia Nakai 113727 a/a R 216/230 264/276
Han Heung Li Otsu P. pyrifolia Nakai 113728 a/a R 230/230 284/284
Harikonatsu P. pyrifolia Nakai 113587 a/a R 230/234 272/275
Hatsuaki P. pyrifolia Nakai 113588 a/a R 230/230 284/284
Hatsumaru P. pyrifolia Nakai – R 230/230 284/284
Hatsushimo P. pyrifolia Nakai 113589 a/a R 228/230 274/284
Hattatsu P. pyrifolia Nakai 113590 a/a R 230/230 284/284
Hayatama P. pyrifolia Nakai 113591 A/a S 220/230 259/284
Heishi P. pyrifolia Nakai 113592 A/a S 220/230 259/284
Heiwa P. pyrifolia Nakai 113593 a/a R 230/230 272/284
Higashino P. pyrifolia Nakai 113594 A/a S 220/220 259/284
Hoe Ryng Saibai P. pyrifolia Nakai 113729 R 216/230 264/276
Hokkainashi P. pyrifolia Nakai 113596 a/a R 230/234 286/286
Hokkan P. pyrifolia Nakai 143889 S 220/230 259/284
Hokushin P. pyrifolia Nakai 238257 R 230/230 284/284
Hoshiakari P. pyrifolia Nakai – R 230/230 284/284
Hougetsu P. pyrifolia Nakai 113720 R 230/230 284/284
Hougyoku P. pyrifolia Nakai 113595 R 230/236 275/279
Housui (Hosui) P. pyrifolia Nakai 113598 a/a R 230/230 284/284
Ichihara Wase P. pyrifolia Nakai 113599 a/a R 228/258 272/274
Imamuraaki P. pyrifolia Nakai 113600 a/a R 228/230 274/284
Imamuranatsu P. pyrifolia Nakai 113601 a/a R 228/230 274/283
Inagi P. pyrifolia Nakai 113602 R 230/230 284/284
Inugoroshi P. pyrifolia Nakai 113607 R 228/228 274/282
Isai P. pyrifolia Nakai 118528 S 220/230 259/284
Ishii Wase P. pyrifolia Nakai 113603 a/a R 230/230 284/284
Ishinashi P. pyrifolia Nakai 239688 R 224/234 272/274
Iyohikari P. pyrifolia Nakai 113604 A/a S 220/220 259/284
Izunohomare P. pyrifolia Nakai 113605 a/a R 230/230 272/284
Jouhana P. pyrifolia Nakai 113606 A/a S 220/230 259/284
Kamenashi P. pyrifolia Nakai 113608 S 220/228 259/282
Kansai Asaryuu P. pyrifolia Nakai 113609 a/a R 230/230 272/284
Kansai Ichi P. pyrifolia Nakai 113610 a/a R 230/230 282/284
Kanta P. pyrifolia Nakai – R 230/230 284/284
Kikusui P. pyrifolia Nakai 113611 R 230/230 284/284
Kimizukawase P. pyrifolia Nakai 113612 A/a S 220/230 259/284
Kinchaku P. pyrifolia Nakai 113613 A/a S 220/230 259/284
Kiraseiki P. pyrifolia Nakai 118529 R 230/230 284/284
Kisui P. pyrifolia Nakai 238258 S 220/230 259/284
Kiyosumi P. pyrifolia Nakai 113614 S 220/230 259/284
Kokuchou P. pyrifolia Nakai 113621 a/a R 230/230 284/284
Konpeitou P. pyrifolia Nakai 113617 a/a R 228/230 274/284
Kotobukishinsui P. pyrifolia Nakai 110824 S 220/230 259/284
Kougetsu P. pyrifolia Nakai 113615 A/a S 220/230 259/284
Kougiku P. pyrifolia Nakai 116285 R 230/230 284/284
Kounowatashi P. pyrifolia Nakai 113616 a/a R 230/230 282/284
Koushuu P. pyrifolia Nakai 113618 a/a R 230/230 272/284
Kousui (Kosui) P. pyrifolia Nakai 113619 a/a R 230/230 284/284
Kouzan P. pyrifolia Nakai 118530 R 234/258 272/272
Kouzou P. pyrifolia Nakai 113620 a/a R 230/230 284/284
Koyuki P. pyrifolia Nakai 113622 R 230/230 274/284
Kumoi P. pyrifolia Nakai 113623 a/a R 230/230 284/284
Kunitomi P. pyrifolia Nakai 113624 A/a S 220/230 259/284
Kuroki P. pyrifolia Nakai 113625 a/a R 230/242 284/284
Kwankinbe P. pyrifolia Nakai 118531 R 230/230 284/284
Meigetsu P. pyrifolia Nakai 113626 A/a S 220/230 259/284
Mishirazu P. pyrifolia Nakai 113627 R 230/234 286/286
Musashi P. pyrifolia Nakai 221165 a/a R 230/230 284/284
Nangetsu P. pyrifolia Nakai 238261 R 230/230 284/284
Nansei Chabo P. pyrifolia Nakai 115741 R 230/230 272/284
Nansui P. pyrifolia Nakai 115742 S 220/230 259/284
Narumi P. pyrifolia Nakai – R 230/230 284/284
Natsushizuku P. pyrifolia Nakai 230439 R 230/230 284/284
Nekogoroshi P. pyrifolia Nakai 113628 R 228/234 256/282
Niigatanashi P. pyrifolia Nakai 113629 a/a R 230/230 270/283
Niitaka P. pyrifolia Nakai 113630 a/a R 230/230 284/284
Nijisseiki P. pyrifolia Nakai 113631 A/a S 220/230 259/284
Nikkori P. pyrifolia Nakai 118540 R 230/230 284/284
Okukouzou P. pyrifolia Nakai 113632 a/a R 230/230 284/284
Okuroku P. pyrifolia Nakai 113633 a/a R 230/230 284/284
Okusankichi P. pyrifolia Nakai 113634 R 230/230 284/284
Onba P. pyrifolia Nakai 113636 R 230/230 272/284
Oohiromaru P. pyrifolia Nakai 113637 a/a R 230/230 284/284
Ookoga P. pyrifolia Nakai 113638 R 230/230 284/284
Ootani P. pyrifolia Nakai 113639 A/a S 220/230 259/284
Osa Gold P. pyrifolia Nakai 110825 S 220/230 259/284
Osa Nijisseiki P. pyrifolia Nakai 113640 S 220/230 259/284
Oushuu P. pyrifolia Nakai 118539 R 230/230 282/284
Rikiya P. pyrifolia Nakai 113641 a/a R 230/230 284/284
Rinka P. pyrifolia Nakai – R 230/230 284/284
Rokugatsu P. pyrifolia Nakai 113642 S 220/230 259/284
Ruisannashi P. pyrifolia Nakai 113643 R 230/260 266/282
Sagami P. pyrifolia Nakai 113644 a/a R 230/230 284/284
Saizounashi P. pyrifolia Nakai 113645 a/a R 230/230 274/284
Segawa P. pyrifolia Nakai 113646 A/a S 220/258 259/272
Seigyoku P. pyrifolia Nakai 113647 a/a R 230/230 284/284
Seika P. pyrifolia Nakai 113648 A/a S 220/248 259/292
Seiryuu P. pyrifolia Nakai 113649 a/a R 228/260 272/274
Sekaiichi P. pyrifolia Nakai 113650 A/a S 220/230 259/284
Sekiryuu P. pyrifolia Nakai 113651 a/a R 230/230 274/284
Senryou P. pyrifolia Nakai 113652 a/a R 230/234 286/286
Shihyakume P. pyrifolia Nakai 113653 a/a R 230/230 284/284
Shikishima P. pyrifolia Nakai 113654 a/a R 230/230 284/284
Shimokatsuginashi P. pyrifolia Nakai 113662 R 230/234 284/284
Shimonashi P. pyrifolia Nakai 113661 R 224/228 274/274
Shinchuu P. pyrifolia Nakai 113656 a/a R 230/230 284/284
Shinkou P. pyrifolia Nakai 113657 a/a R 230/230 274/284
Shinsei P. pyrifolia Nakai 113694 a/a R 228/230 274/284
Shinseiki P. pyrifolia Nakai 113658 R 230/230 284/284
Shinsetsu P. pyrifolia Nakai 113659 R 230/230 284/284
Shinsui P. pyrifolia Nakai 113660 A/a S 220/230 259/284
Shirayuki P. pyrifolia Nakai 113663 a/a R 228/230 274/283
Shuugyoku P. pyrifolia Nakai 113707 R 230/230 284/284
Shuurei P. pyrifolia Nakai 118537 R 230/230 284/284
Shuusui P. pyrifolia Nakai 116286 R 230/230 284/284
Sotoorihime P. pyrifolia Nakai 113664 a/a R 234/240 272/274
Suisei P. pyrifolia Nakai 113665 a/a R 230/230 284/284
Suishuu P. pyrifolia Nakai 118541 R 230/230 284/284
Taihaku P. pyrifolia Nakai 113666 R 230/230 284/284
Taihei P. pyrifolia Nakai 113667 R 228/256 272/282
Tama P. pyrifolia Nakai 113668 R 230/230 284/284
Tamotoyabure P. pyrifolia Nakai 113695 R 230/230 284/284
Tanponashi P. pyrifolia Nakai 113669 R 228/230 282/284
Tanzawa P. pyrifolia Nakai 116287 a/a R 230/230 274/284
Tenyuu P. pyrifolia Nakai 113670 S 220/230 259/284
Tosajou P. pyrifolia Nakai 113672 a/a R 230/230 282/284
Tosajounishiki P. pyrifolia Nakai 113673 a/a R 228/230 274/284
Tosanashi P. pyrifolia Nakai 113674 R 230/230 272/284
Tosanishiki P. pyrifolia Nakai 113675 a/a R 228/230 274/284
Touhou P. pyrifolia Nakai 113671 S 220/234 259/272
Tsugaruao P. pyrifolia Nakai 113676 R 216/216 276/276
Tsukutounashi P. pyrifolia Nakai 113677 R 228/228 272/274
Wase Kouzou P. pyrifolia Nakai 113682 a/a R 230/230 284/284
Wase Taichou P. pyrifolia Nakai 113684 A/a S 220/230 259/284
Waseaka P. pyrifolia Nakai 113678 a/a R 228/230 274/284
Yabase P. pyrifolia Nakai 118542 S 220/230 259/284
Yachiyo P. pyrifolia Nakai 113686 a/a R 230/230 284/284
Yagoemon P. pyrifolia Nakai 113687 R 230/234 272/284
Yahatanishiki P. pyrifolia Nakai 113690 a/a R 230/230 284/284
Yakumo P. pyrifolia Nakai 113688 a/a R 230/230 284/284
Yasato P. pyrifolia Nakai 113718 R 230/230 284/284
Yokogoshi P. pyrifolia Nakai 113691 S 220/228 259/274
Yoshikaori P. pyrifolia Nakai 118543 R 230/230 284/284
Yoshino P. pyrifolia Nakai 113692 R 230/230 284/284
Bai Li P. bretschneideri Rehd. 113536 R 216/230 275/284
Agenoshou Shinanashi P. ussuriensis Maxim. 113730 R 224/230 274/284
Ba Li Xiang P. ussuriensis Maxim. 113749 R 222/246 274/282
Baozhuli P. ussuriensis Maxim. 118544 R 240/244 274/277
Bei Jin Bai Li P. ussuriensis Maxim. 113731 a/a R 230/236 275/284
Cang Xi Li P. ussuriensis Maxim. 113752 R 248/274 274/282
Chang Xi Li P. ussuriensis Maxim. 113751 R 236/236 272/310
Da Tou Huang Li P. ussuriensis Maxim. 113747 R 222/222 264/272
En Li P. ussuriensis Maxim. 113732 a/a R 228/248 282/292
Hong Li P. ussuriensis Maxim. 113733 a/a R 216/216 276/276
Hong Xiao Li P. ussuriensis Maxim. 113734 R 216/226 272/276
Huang Li P. ussuriensis Maxim. 113750 S 214/230 280/284
Huang Shi Li P. ussuriensis Maxim. 113748 R 216/234 276/282
Jian Ba Li P. ussuriensis Maxim. 113735 R 224/236 276/276
Lai Yang Ci Li P. ussuriensis Maxim. 113736 a/a R 228/248 282/292
Lunanhuangli P. ussuriensis Maxim. 118545 R 228/246 282/282
Ma Ke Zao Li P. ussuriensis Maxim. 113744 R 228/260 266/282
Ma Ti Huang P. ussuriensis Maxim. 113746 R 222/222 264/272
Mi Li P. ussuriensis Maxim. 113753 R 216/226 272/276
Mi Li Cui P. ussuriensis Maxim. 113759 S 228/230 280/282
Ping Li P. ussuriensis Maxim. 113754 R 230/230 280/284
Qiu Bai Li P. ussuriensis Maxim. 113737 a/a R 228/248 282/292
Su Hyang Ri P. ussuriensis Maxim. 113738 a/a R 216/230 264/276
Tai Huang Li P. ussuriensis Maxim. 113760 S 216/230 276/280
Wo Wo Li P. ussuriensis Maxim. 113739 a/a R 224/228 280/282
Xiang Ya Li P. ussuriensis Maxim. 113756 S 228/230 280/282
Xie Hua Tian P. ussuriensis Maxim. 113755 R 230/230 280/284
Xuehua Li P. ussuriensis Maxim. 245604 R 228/230 280/282
Ya Gua Li P. ussuriensis Maxim. 113740 a/a R 230/248 284/292
Ya Li P. ussuriensis Maxim. 113741 a/a R 230/230 284/284
Yang Nai Xiang P. ussuriensis Maxim. 116297 R 230/236 274/278
Yin Bai Li P. ussuriensis Maxim. 113757 R 222/230 284/296
Yuan Ba Li P. ussuriensis Maxim. 113742 a/a R 230/246 268/284
Zaosu Li P. ussuriensis Maxim. 245605 R 230/230 264/284
Zhu Zui Li P. ussuriensis Maxim. 113743 a/a R 216/248 256/276
Ninomiya P. pyrifolia × P. communis 113781 R 228/230 284/284
Ooharabeni P. pyrifolia × P. communis 113780 R 230/230 284/284
Taiheiyou P. pyrifolia × P. communis 113782 R 230/248 272/284
Ninomiya Bai Li P. ussuriensis × P. pyrifolia 113784 a/a R 230/230 284/284
Babaucchiaginashi P. babauttiaginashi Koidz. 113763 R 228/228 274/274
Cheung Dang No Ri Pyrus sp. 113828 R 216/234 272/276
Iwate Tanenashi Pyrus sp. 113802 R 234/260 272/272

a Proposed in Kozaki (1973). A/a: heterozygote susceptible to black spot; a/a: resistant; blank cell: not tested.

b S, necrotic symptoms (susceptible); R, no disease symptoms (resistant).

c Numbers separated by “/” indicate the estimated size (bp) of the alleles of the same locus.

Table 2. Mapping populations used in this study and segregation of susceptibility and resistance to black spot disease
Female (Resistant) Male (Susceptible) Number of progeny Expected ratio χ2 P-valuea
Susceptible Resistant
Hosui Kinchaku 526 535 1:1 0.076 0.782
Kosui Xiang Ya Li 72 50 1:1 3.967 0.046*
Kosui Mi Li Cui 31 28 1:1 0.153 0.696
Kosui Tai Huang Li 40 43 1:1 0.108 0.742
Kosui Huang Li 20 24 1:1 0.364 0.546

a P-values indicate fit to the expected ratio (1:1). Distorted segregation is indicated by a significant p-value of the χ2 test: *, p < 0.05.

Frozen young leaves (30–40 mg) were homogenized by strong shaking for 30 s in a Shake Master Auto (Bio Medical Science). Genomic DNA was extracted using a NucleoMag Plant (Macherey-Nagel) according to the manufacturer’s instructions with a slight modification in the lysis buffer MC1 (2-mercaptoethanol was added to a final concentration of 2%). The purified genomic DNA was quantified with a Qubit 3.0 fluorometer (Thermo Fisher Scientific) and Qubit dsDNA BR assay kit (Thermo Fisher Scientific) and then diluted to 2.5 ng/μL for PCR analysis.

Evaluation of black spot susceptibility or resistance

Responses to black spot disease were evaluated using the spore inoculation test (Hayashi et al. 1990, Terakami et al. 2016). All cultivars and F1 plantlets were inoculated with spores of the virulent isolate No. 15A of A. alternata, which was kindly provided by Dr. T. Tsuge (Chubu University). The isolate was cultured in potato dextrose broth without shaking for 10–14 days at 25°C. Mycelial mats were washed with tap water to remove culture medium and maintained at 25°C in the dark. The spores formed were collected, suspended in distilled water, and diluted to approximately 1 × 105/mL. The spore suspension was sprayed onto three young leaves with a glass atomizer. The second or third young leaves of each plant were detached and used for inoculation test. The inoculated leaves were incubated in a moist chamber at 25°C for 66 h in the dark. Leaves were classified into two groups: resistant (no disease symptoms) and susceptible (necrotic symptoms). Black necrotic spots occur only in susceptible cultivars, and black spots does not occur in resistant cultivars (Nishimura and Kohmoto 1983). All inoculation tests with all cultivars and F1 progeny were performed in duplicate.

SSR analysis and evaluation of the usefulness of the markers for MAS

We determined the genotypes of 4 SSR markers (Terakami et al. 2016; Mdo.chr11.27, Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34) around A and examined the relationship between marker genotypes and susceptibility to black spot disease in the 207 cultivars (Fig. 1). SSR-PCR analysis was performed using the one-tube, single-reaction nested PCR method (Schuelke 2000), in a total volume of 5 μL containing 2.5 μL of 2 × GoTaq G2 Hot Start Green Master Mix (Promega), 0.3 μM forward primer with a tail at the 5ʹ-end, 0.5 μM reverse primer, 0.2 μM 6-FAM-labeled universal primer (Thermo Fisher Scientific), and 2.5 ng of genomic DNA. The original M13(-21) tail sequence was modified to the 20-bp (5ʹ-GCTACGGACTGACCTCGGAC-3ʹ) universal sequence. A 7-bp pigtail sequence (5ʹ-GTTTCTT-3ʹ) (Brownstein et al. 1996) was added at the 5ʹ-end of each reverse primer to improve genotyping accuracy. DNA was amplified in a GeneAmp PCR system 9700 (Thermo Fisher Scientific) with an initial denaturation step at 95°C for 2 min; 40 cycles at 95°C for 30 sec (denaturation), 55°C for 30 sec (annealing), and 72°C for 45 sec (extension); and a final extension at 72°C for 5 min.

Fig. 1.

Comparison of linkage maps of Japanese and Chinese pears. Linkage group (LG) 11 in the Japanese pear ‘Kinchaku’ (Terakami et al. 2016; Kin.LG11a) is shown on the left. Aki is the black spot susceptibility gene of ‘Kinchaku’. The scale shows genetic distance (cM) from top marker of the linkage group. Kin.LG11b shows fine mapping of Aki and flanking regions. The numbers between markers indicate genetic distance. C-int.LG11 is the integrated LG 11 of black spot–susceptible Chinese pears; Ac, the black spot susceptibility gene in Chinese pears. In Kin.LG11b and C-int.LG11, numbers indicate genetic distance (cM) between markers.

Amplified DNA fragments were separated and detected using an Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific) with a 36 cm-capillary array, POP-7 polymer, and an internal size standard (GeneScan HD 400 ROX; Thermo Fisher Scientific). Data were collected and analyzed in GeneMapper v. 5.0 software (Thermo Fisher Scientific).

Fine mapping of Aki and linkage analysis of the susceptibility gene of Chinese pear

For fine mapping of the black spot susceptibility gene of ‘Kinchaku’, Aki, 1061 F1 plants obtained from a cross between ‘Hosui’ and ‘Kinchaku’ were genotyped with the four markers (Fig. 1). Because A were located in the same region of LG 11 in Japanese pear (Terakami et al. 2007, 2016), we suspected that the susceptibility gene of Chinese pear might also be in that region. To examine this possibility, we analyzed all F1 plantlets derived from four different mapping populations (‘Kosui’ × ‘Xiang Ya Li’, ‘Kosui’ × ‘Mi Li Cui’, ‘Kosui’ × ‘Tai Huang Li’, and ‘Kosui’ × Huang Li’) using Mdo.chr11.27 and Mdo.chr11.34 (Fig. 1), which show significant linkage to A. SSR-PCR analyses were performed as described above.

Statistical analysis (χ2 test) was performed in R v. 3.5.1 software using the chisq.test function (R Core Team 2018). Linkage analysis was performed in JoinMap v. 4.1 software (Van Ooijen 2006, 2011), and a pseudo-testcross strategy was used to create genetic linkage maps (Grattapaglia and Sederoff 1994). An independence logarithm of odds (LOD) threshold of 10.0 was used to define linkage groups. To construct a linkage group, the regression mapping algorithm was selected with the following parameters: recombination frequency ≤0.40, a LOD ≥1.0, goodness-of-fit jump threshold for removal of loci = 5.0, number of added loci after which to perform a ripple = 1, and third round = “No”. Map distances were calculated according to Kosambi’s mapping function (Kosambi 1944). The linkage map was drawn in MapChart v. 2.3 software (Voorrips 2002).

Map integration

An integrated linkage map was constructed in JoinMap v. 4.1 software assuming that the candidate genes of the four Chinese pear cultivars (‘Xiang Ya Li’, ‘Mi Li Cui’, ‘Tai Huang Li’, and ‘Huang Li’) were the same and using combined data from the four mapping populations. First, a map of each population was constructed to determine the coupling phase linked to the susceptibility gene. Then the groups were combined by applying the “Combine Groups for Map Integration” function from the JoinMap menu.

Results

Fine mapping of Aki and precise determination of the marker position

In our previous study, genetic linkage analysis of Aki was conducted using 621 F1 progeny (Terakami et al. 2016). Here, we performed fine mapping of Aki and constructed a more detailed linkage map using 1061 F1 plantlets of a ‘Hosui’ × ‘Kinchaku’ cross. In the tests for susceptibility or resistance to black spot, 526 plantlets showed necrotic symptoms and 535 showed no symptoms (Table 2); identical results were obtained in all duplicate tests. The segregation ratio of resistant to susceptible plants fitted the expected ratio of 1:1 in the chi-squared test (Table 2; χ2 = 0.076, p = 0.782).

Fine mapping of Aki was performed with four SSR markers (Mdo.chr11.27, Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34) developed from the apple genome sequence (Terakami et al. 2016). These four markers show scorable polymorphism, i.e., a heterozygous genotype in ‘Kinchaku’ and polymorphic band patterns between ‘Hosui’ and ‘Kinchaku’, and show significant linkage to Aki (Terakami et al. 2016). Aki was located within a 1.1-cM region between Mdo.chr11.28 and Mdo.chr11.34 (Fig. 1). Previously we mapped Mdo.chr11.27 and Mdo.chr11.28 to the same position (Terakami et al. 2016), but a more accurate mapping with more progeny showed that the distance between Aki and Mdo.chr11.27 was 0.4 cM and that between Aki and Mdo.chr11.28 was 0.3 cM (Fig. 1). In the mapping population, we detected recombination between Mdo.chr11.27 and Aki in four plantlets, between Mdo.chr11.28 and Aki in three, and between Mdo.chr11.34 and Aki in nine. No double recombination events between Mdo.chr11.27 and Mdo.chr11.34 were detected. Mdo.chr11.30 co-segregated with Aki in all 1061 F1 plantlets. The segregation ratio was not distorted at any locus.

Evaluation of black spot susceptibility or resistance

Of the 207 cultivars tested, 43 were susceptible (39 Japanese and 4 Chinese) and 164 were resistant to black spot disease (Table 1). Susceptible and resistant cultivars could be clearly distinguished because no cultivars showed an intermediate response. The results of the 101 cultivars that had been previously tested (Kozaki 1973) were consistent with those previous results (Table 1); 22 susceptible cultivars (18 Japanese and 4 Chinese) were newly identified. Disease symptoms were observed on the entire surface of inoculated leaves, with no differences among the susceptible cultivars, including ‘Gold Nijisseiki’, ‘Osa Gold’, and ‘Kotobukishinsui’, which reportedly have medium disease resistance (Kitagawa et al. 1999, Masuda et al. 1998, Sanada et al. 1993).

Relationship between SSR genotype and susceptibility to black spot disease

We genotyped the 207 cultivars to investigate the polymorphism of the four Aki-linked SSR markers and their correspondence with phenotypes (Table 1). Mdo.chr11.27 and Mdo.chr11.34 showed amplification of specific bands in most Japanese cultivars that were susceptible in the inoculation test.

Mdo.chr11.27 showed a 220-bp band in most susceptible cultivars (Tables 1, 3, Fig. 2). Most of the susceptible Japanese cultivars were heterozygous, whereas ‘Doitsu’, ‘Higashino’, and ‘Iyohikari’ were homozygous for the 220-bp band. ‘Fukushima’ was susceptible, but no 220-bp allele was detected. For Mdo.chr11.34, the presence or absence of the 259-bp band in Japanese cultivars was completely consistent with the results of the inoculation test (Tables 1, 3, Fig. 2). All susceptible Japanese cultivars were heterozygous for the 259-bp allele. However, no amplification of specific bands that could be related to the inoculation test results was observed in the Chinese cultivar. The 230-bp and 280-bp bands were commonly amplified in susceptible Chinese cultivars with Mdo.chr11.27 and Mdo.chr11.34, respectively, but were also observed in several resistant Chinese pear cultivars.

Table 3. Summary of pear cultivars and band pattern of Mdo.chr11.27 and Mdo.chr11.34 markers
Species Susceptibility to black spot disease Band pattern of Mdo.chr11.27 and Mdo.chr11.34 markersa
P_P N_P N_N
P. pyrifolia Nakai Resistant Abumi, Aikansui, Aiuchi, Akaho, Akiakari, Akibae, Akizuki, Amanogawa, Aoyagi, Asahi, Atago, Azumanishiki, Cheong Sil Ri, Chikusui, Chouju, Choujuurou, Chousen, Echigonishiki, Geishun, Gion, Gozennashi, Hakkou, Hakuteiryuu, Han Henung Li Kou, Han Heung Li Otsu, Harikonatsu, Hatsuaki, Hatsumaru, Hatsushimo, Hattatsu, Heiwa, Hoe Ryng Saibai, Hokkainashi, Hokushin, Hoshiakari, Hougetsu, Hougyoku, Hosui, Ichihara Wase, Imamuraaki, Imamuranatsu, Inagi, Inugoroshi, Ishii Wase, Ishinashi, Izunohomare, Kansai Asaryuu, Kansai Ichi, Kanta, Kikusui, Kiraseiki, Kokuchou, Konpeitou, Kougiku, Kounowatashi, Koushuu, Kosui, Kouzan, Kouzou, Koyuki, Kumoi, Kuroki, Kwankinbe, Mishirazu, Musashi, Nangetsu, Nansei Chabo, Narumi, Natsushizuku, Nekogoroshi, Niigatanashi, Niitaka, Nikkori, Okukouzou, Okuroku, Okusankichi, Onba, Oohiromaru, Ookoga, Oushuu, Rikiya, Rinka, Ruisannashi, Sagami, Saizounashi, Seigyoku, Seiryuu, Sekiryuu, Senryou, Shihyakume, Shikishima, Shimokatsuginashi, Shimonashi, Shinchuu, Shinkou, Shinsei, Shinseiki, Shinsetsu, Shirayuki, Shuugyoku, Shuurei, Shuusui, Sotoorihime, Suisei, Suishuu, Taihaku, Taihei, Tama, Tamotoyabure, Tanponashi, Tanzawa, Tosajou, Tosajounishiki, Tosanashi, Tosanishiki, Tsugaruao, Tsukutounashi, Wase Kouzou, Waseaka, Yachiyo, Yagoemon, Yahatanishiki, Yakumo, Yasato, Yoshikaori, Yoshino
Susceptible Asahiryuu, Awayuki, Chizu, Doitsu, Edoya, Gold Nijisseiki, Hakataao, Hayatama, Heishi, Higashino, Hokkan, Isai, Iyohikari, Jouhana, Kamenashi, Kimizukawase, Kinchaku, Kisui, Kiyosumi, Kotobukishinsui, Kougetsu, Kunitomi, Meigetsu, Nansui, Nijisseiki, Ootani, Osa Gold, Osa Nijisseiki, Rokugatsu, Segawa, Seika, Sekaiichi, Shinsui, Tenyuu, Touhou, Wase Taichou, Yabase, Yokogoshi Fukushima
P. bretschneideri Rehd. Resistant Bai Li
P. ussuriensis Maxim. Resistant Agenoshou Shinanashi, Ba Li Xiang, Baozhuli, Bei Jin Bai Li, Cang Xi Li, Chang Xi Li, Da Tou Huang Li, En Li, Hong Li, Hong Xiao Li, Huang Shi Li, Jian Ba Li, Lai Yang Ci Li, Lunanhuangli, Ma Ke Zao Li, Ma Ti Huang, Mi Li, Ping Li, Qiu Bai Li, Su Hyang Ri, Wo Wo Li, Xie Hua Tian, Xuehua Li, Ya Gua Li, Ya Li, Yang Nai Xiang, Yin Bai Li, Yuan Ba Li, Zaosu Li, Zhu Zui Li
Susceptible Huang Li, Mi Li Cui, Tai Huang Li, Xiang Ya Li
P. pyrifolia × P. communis Resistant Ninomiya, Ooharabeni, Taiheiyou
P. ussuriensis × P. pyrifolia Resistant Ninomiya Bai Li
P. babauttiaginashi Koidz. Resistant Babaucchiaginashi
Pyrus sp. Resistant Cheung Dang No Ri, Iwate Tanenashi

a P_N, with 220-bp band of Mdo.chr11.27 and 259-bp band of Mdo.chr11.34; N_P, without 220-bp band of Mdo.chr11.27 and with 259-bp band of Mdo.chr11.34; N_N, without 220-bp band of Mdo.chr11.27 and 259-bp band of Mdo.chr11.34.

Fig. 2.

Peak profiles of capillary gel electrophoresis output of fluorescently labeled SSR marker fragments. The target peaks are highlighted.

Markers Mdo.chr11.28 and Mdo.chr11.30 amplified multiple loci, as 1–6 bands were identified (Supplemental Table 1). Therefore, we examined the presence of the 255-bp band for Mdo.chr11.28 and the 186-bp band for Mdo.chr11.30, both of which were linked to Aki of ‘Kinchaku’. The 255-bp band was detected in all susceptible cultivars, but also in many resistant cultivars. The presence of the 186-bp band in the Japanese cultivars was completely consistent with susceptibility in the inoculation test. There was no correlation between the amplification of the 186-bp band and the results of the inoculation test in Chinese pear.

Inheritance mode of the susceptibility gene in Chinese pear

As a prerequisite to mapping the susceptibility gene to black spot disease in Chinese pear, we evaluated the inheritance mode of susceptibility. We obtained four F1 segregating populations from crosses between resistant and susceptible cultivars and evaluated plantlets for resistance or susceptibility to black spot (Table 2). Resistant and susceptible plantlets could be clearly differentiated; no plantlets showed an intermediate response. Identical results were obtained in all duplicate tests.

Of 122 F1 plantlets of the ‘Kosui’ × ‘Xiang Ya Li’ cross, 72 showed necrotic symptoms and were judged as susceptible (Table 2). The other 50 were resistant (no symptoms). The ratio of susceptible to resistant plants (72:50) showed a slight distortion from the expected 1:1 ratio at the 5% level in the chi-squared test (χ2 = 3.967, p = 0.046). Of 59 F1 ‘Kosui’ × ‘Mi Li Cui’ plantlets, 31 were susceptible and 28 were resistant (Table 2; χ2 = 0.153, p = 0.696). Of 83 F1 ‘Kosui’ × ‘Tai Huang Li’ plantlets, 40 were susceptible and 43 were resistant (Table 2; χ2 = 0.108, p = 0.742). Of 44 F1 ‘Kosui’ × ‘Huang Li’ plantlets, 20 were susceptible and 24 were resistant (Table 2; χ2 = 0.364, p = 0.546).The segregation ratio of susceptible to resistant plants fitted the expected ratio of 1:1 in three mapping populations (‘Kosui’ × ‘Mi Li Cui’, ‘Kosui’ × ‘Tai Huang Li’, and ‘Kosui’ × ‘Huang Li’). These results indicate that a single dominant gene might control susceptibility to black spot disease in Chinese pears, and that the four Chinese pears are heterozygous for this gene. We designated the genes responsible for susceptibility in the Chinese cultivars as follows: ‘Xiang Ya Li’, Axi; ‘Mi Li Cui’, Ami; ‘Tai Huang Li’, Ata; and ‘Huang Li’, Ahu, because the pedigree of each cultivar was unknown.

Mapping of the loci conferring susceptibility to black spot disease in Chinese pear

To identify the loci conferring susceptibility in Chinese pear, we tested Mdo.chr11.27 and Mdo.chr11.34, which showed significant linkage to susceptibility to black spot disease in Japanese pear, in the four mapping populations. Both SSRs showed scorable polymorphisms in all four populations, i.e., a heterozygous genotype in ‘Xiang Ya Li’, ‘Mi Li Cui’, ‘Tai Huang Li’, and ‘Huang Li’, and polymorphic band patterns in ‘Kosui’ (Table 1). From linkage analysis, the 230-bp allele of Mdo.chr11.27 showed significant linkage to Axi (genetic distance, 2.5 cM; LOD score, 30.39), Ami (5.1 cM; 12.66), Ata (1.2 cM; 22.89), and Ahu (9.2 cM; 7.45) (Fig. 3). The 280-bp allele of Mdo.chr11.34 co-segregated with Axi (LOD score, 35.86), Ami (17.73), and Ahu (13.17), and showed significant linkage to Ata (genetic distance, 1.2 cM; LOD score, 22.92) (Fig. 3). Markers Mdo.chr11.28 and Mdo.chr11.30 were excluded from the analysis because these markers amplified multiple loci (Supplemental Table 1),

Fig. 3.

Linkage groups 11 of Chinese pear cultivars ‘Xiang Ya Li’ (Xi.LG11), ‘Mi Li Cui’ (Mi.LG11), ‘Tai Huang Li’ (Ta.LG11), and ‘Huang Li’ (Hu.LG11). The respective genes for susceptibility to black spot are denoted as Axi, Ami, Ata, and Ahu. The numbers on the left of each marker show genetic distance (cM) from Mdo.chr11.27.

An integrated linkage map was constructed, assuming that the candidate genes of the four Chinese cultivars (‘Xiang Ya Li’, ‘Mi Li Cui’, ‘Tai Huang Li’, and ‘Huang Li’) were the same gene, denoted as Ac. Ac was located within a 3.9-cM region between Mdo.chr11.27 and Mdo.chr11.34 (Fig. 1). Thus, the susceptibility gene of Chinese pear, Ac, was located at the top of LG 11, very similar to that of Aki in Japanese pear.

Useful marker set for the breeding of pear resistant to black spot disease

We conclude that the marker set Mdo.chr11.27 and Mdo.chr11.34 would be useful for MAS. Both markers amplified a single locus, and the correspondence of the alleles to resistance and susceptibility was clear. Except in ‘Fukushima’, the 220-bp and 259-bp bands were amplified in susceptible Japanese pear cultivars with Mdo.chr11.27 and Mdo.chr11.34, respectively (Table 1). In Chinese pears, no specific bands were found in susceptible cultivars, but linkage analysis determined a coupling phase to the susceptibility gene. Non-specific amplification, e.g., a 215-bp band in ‘Hosui’ (Fig. 2), was no longer detected after we switched to a pre-labeled marker (data not shown). Although some cultivars had overlapping size ranges for each marker, multiplex analysis is possible by switching to the pre-labeled dye.

Discussion

In this study, we conducted comprehensive inoculation tests and genotyping on large-scale pear genetic resources. We newly identified 18 Japanese and 4 Chinese cultivars susceptible to black spot disease. For the 101 cultivars previously tested by Kozaki (1973), the results of our inoculation test were consistent with the reported data, indicating that the evaluation was stable and accurate. Four SSR markers tightly linked to the gene for susceptibility were used to investigate the genotypes. All markers amplified specific bands in most of the susceptible cultivars (Table 1, Supplemental Table 1). In particular, Mdo.chr11.27 and Mdo.chr11.34 amplified a single locus and were highly consistent with the phenotype, so we consider these markers useful for MAS (Table 3). Progeny test showed that the susceptible cultivars were all heterozygous (Kozaki 1973). Crosses between susceptible cultivars showed a 1:3 ratio of resistant to susceptible seedlings, so susceptible homozygous plantlets (A/A) were present at the early seedling stage (Kozaki 1973). We also obtained dominant homozygous (A/A) seedlings from two crosses (‘Doitsu’ × ‘Nansui’ and ‘Shinsui’ × ‘Nijisseiki’). Each seedling was genotyped with two SSRs (Mdo.chr11.27 and Mdo.chr11.34), confirming the presence of dominant homozygous (A/A) seedlings. Dominant homozygous seedlings showed no difference in appearance from other seedlings but died within a few months (data not shown). We conclude that the extant susceptible cultivars are heterozygous (A/a), indicating that the genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease.

Kozaki (1973) inoculation-tested 11 Chinese pear cultivars, all of which proved resistant to black spot disease (Table 1). Here, we inoculated 35 Chinese pear cultivars, 4 of which were found to be susceptible (Table 1). This is the first report of susceptible Chinese pear cultivars. We crossed these four cultivars with the resistant Japanese cultivar ‘Kosui’ to create four populations for genetic linkage analysis and to confirm the inheritance mode, and mapped the loci of the susceptibility genes in Chinese pear. The segregation ratio of resistant and susceptible progeny fitted the expected ratio of 1:1 in the chi-squared test. This result is in good agreement with the report by Kozaki (1973) that a single dominant gene controls susceptibility to black spot in pear. Linkage analysis revealed that Chinese pears’ susceptibility genes are strongly linked to Mdo.chr11.27 and Mdo.chr11.34 (Fig. 3). In an integrated linkage map, the susceptibility gene of Chinese pear, Ac, was located within a 3.9-cM region between Mdo.chr11.27 and Mdo.chr11.34 (Fig. 1). The order of the markers and the susceptibility gene was the same in the original maps and integrated linkage map, indicating the accuracy of the inoculation tests and SSR analysis. These two markers have been mapped at the top of LG 11 and are strongly linked to the susceptibility gene of Japanese pear (Terakami et al. 2007, 2016). The current study is the first to identify the inheritance mode and to map the position of the gene conferring susceptibility to black spot disease in Chinese pear.

Different pathotypes of A. alternata, which produce host-selective toxins, cause similar diseases among the Rosaceae, e.g., black spot disease of Japanese and Chinese pears, Alternaria blotch of apple, and black spot disease of strawberry (Akimitsu et al. 2014, Tsuge et al. 2013). Susceptibility of those host plants to the disease is controlled by a dominant gene (Kozaki 1973, Saito and Takeda 1984, Yamamoto et al. 1985). In apple, the susceptibility gene Alt has been mapped on chromosome 11 between markers Mdo.chr11.30 and Mdo.chr11.34, and the candidate genes have been identified (Moriya et al. 2019). In three different linkage maps, the susceptibility gene of Japanese pear have been mapped to LG 11 between Mdo.chr11.28 and Mdo.chr11.34 (Terakami et al. 2007, 2016). The black spot susceptibility gene of Chinese pear have also been mapped at the top of LG 11, indicating that these genes are orthologous to those of apple and Japanese pear. Although detailed analysis has not been carried out in strawberry, these results suggest that the genes for susceptibility to A. alternata are conserved among Rosaceous hosts. Cloning of the susceptibility genes of Japanese and Chinese pears would further elucidate the mechanism of susceptibility in the Rosaceae.

Genotypes of old native and present cultivars derived from them show that the Mdo.chr11.27 and Mdo.chr11.34 marker set would be useful for breeding pears resistant to black spot disease. Susceptible ‘Kinchaku’ and ‘Osa Nijisseiki’ have very useful traits in pear breeding. Pear scab is one of the most harmful diseases of pears, especially Japanese and Chinese pears. It is pathogenic to the major commercial Japanese pear cultivars (Bell et al. 1996, Ishii et al. 1992), but no scab symptoms have been observed on ‘Kinchaku’ or indigenous Japanese pear (Abe and Kotobuki 1998, Ishii et al. 1992). To achieve stable fruit set without the need for artificial pollination, self-compatibility, which is controlled by multiple S haplotypes at a single locus, has become an important objective in Japanese pear breeding programs (Saito 2016). ‘Osa Nijisseiki’ (a mutant of the self-incompatible cultivar ‘Nijisseiki’) is self-compatible and is used as a parent for the breeding of self-compatible cultivars in Japan (Saito 2016). When ‘Kinchaku’ or ‘Osa Nijisseiki’ is used as a parent for breeding, about half of the progeny are susceptible to black spot disease. The Mdo.chr11.27 and Mdo.chr11.34 marker set could efficiently and accurately select black spot–resistant seedlings. MAS is more efficient when the marker set is combined with DNA markers linked to pear scab resistance and self-compatibility (Okada et al. 2008, Terakami et al. 2006). Pear genetic resources that have useful traits for breeding but are susceptible to black spot disease may be found in the future. Phenotypic data from the spore inoculation test and the genotyping data of markers linked to the susceptibility gene will be useful for pear breeding by MAS.

Author Contribution Statement

ST conducted genetic experiments and inoculation test, analyzed data, and wrote the initial draft of the manuscript. YA and YT performed inoculation test. NT and SN provided the experimental materials. TS and TY contributed to the preparation of the final version of the manuscript. All authors reviewed and approved the manuscript.

Acknowledgments

We are grateful to Mss. N. Yagihashi, H. Takahashi, M. Tsukamoto, and N. Minagawa for their technical assistance. The virulent isolate No. 15A of A. alternata was provided by Dr. Takashi Tsuge, Chubu University, Japan. This work was partially supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Innovation, HOR-2001).

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