Open Access

Estimation of loss of genetic diversity in modern Japanese cultivars by comparison of diverse genetic resources in Asian pear (Pyrus spp.)

  • Sogo Nishio1Email author,
  • Norio Takada1,
  • Toshihiro Saito1,
  • Toshiya Yamamoto1 and
  • Hiroyuki Iketani2
BMC GeneticsBMC series – open, inclusive and trusted201617:81

https://doi.org/10.1186/s12863-016-0380-7

Received: 22 January 2016

Accepted: 24 May 2016

Published: 14 June 2016

Abstract

Background

Pears (Pyrus spp.) are one of the most important fruit crops in temperate regions. Japanese pear breeding has been carried out for over 100 years, working to release new cultivars that have good fruit quality and other desirable traits. Local cultivar ‘Nijisseiki’ and its relatives, which have excellent fruit texture, have been repeatedly used as parents in the breeding program. This strategy has led to inbreeding within recent cultivars and selections. To avoid inbreeding depression, we need to clarify the degree of inbreeding among crossbred cultivars and to introgress genetic resources that are genetically different from modern cultivars and selections. The objective of the present study was to clarify the genetic relatedness between modern Japanese pear cultivars and diverse Asian pear genetic resources.

Results

We genotyped 207 diverse accessions by using 19 simple sequence repeat (SSR) markers. The heterozygosity and allelic richness of modern cultivars was obviously decreased compared with that of wild individuals, Chinese pear cultivars, and local cultivars. In analyses using Structure software, the 207 accessions were classified into four clusters (K = 4): one consisting primarily of wild individuals, one of Chinese pear cultivars, one of local cultivars from outside the Kanto region, and one containing both local cultivars from the Kanto region and crossbred cultivars. The results of principal coordinate analysis (PCoA) were similar to those from the Structure analysis. Wild individuals and Chinese pears appeared to be distinct from other groups, and crossbred cultivars became closer to ‘Nijisseiki’ as the year of release became more recent.

Conclusions

Both Structure and PCoA results suggest that the modern Japanese pear cultivars are genetically close to local cultivars that originated in the Kanto region, and that the genotypes of the modern cultivars were markedly biased toward ‘Nijisseiki’. Introgression of germplasm from Chinese pear and wild individuals that are genetically different from modern cultivars seems to be key to broadening the genetic diversity of Japanese pear. The information obtained in this study will be useful for pear breeders and other fruit breeders who have observed inbreeding depression.

Keywords

Simple sequence repeat (SSR) markers Pyrus pyrifolia Nakai Population structure Principal coordinate analysis (PCoA)

Background

Inbreeding is a common problem in fruit breeding programs [19]. It reduces vigor trait, such as tree vigor [2, 3, 5, 8], viability [1, 2, 5] and fruit weight [4]. Selection of local cultivars from wild populations during domestication has increased productivity but narrowed genetic diversity [10, 11]. Moreover, an organized breeding program is generally started with limited genetic resources that have been already domesticated, such as local cultivars. Introducing materials from foreign countries is not easy on account of biosecurity concerns, and introgression of superior traits from wild individuals may require several generations to reach performance comparable to that of modern cultivars. In addition, fruit trees have a long juvenile period before fruit set and seed production. Thus, most fruit breeders are likely to improve genotypes by crossing well-known cultivars [6, 12]. As a result, the genetic diversity of modern cultivars has decreased dramatically over time.

Pears (Pyrus spp.) belong to the subtribe Pyrinae (formerly subfamily Maloideae) of the Rosaceae and are one of the most important fruit crops in temperate regions. In East Asia, major cultivated pears are traditionally classified into three species: P. ussuriensis Maxim., P. bretschneideri Rehder, and P. pyrifolia (Burm. f.) Nakai [13, 14]. Many taxonomists and horticulturists have tried to classify these species using their own criteria, according to morphological characters [13, 1517] or molecular markers [1821]. However, these species readily produce interspecific hybrids [22], and some cultivars are admixtures of different species [1821]. Also, the species themselves seem to be genetically continuous [15, 2325]. As a result, genetic classification based on these three species is obscure. Iketani et al. [25] proposed a new cultivar classification system based on the population structure of these species and historical enumeration, i.e., Pyrus Ussurian Pear Group (Pyrus ussuriensis), Pyrus Chinese White Pear Group (P. bretschneideri or P. pyrifolia), Pyrus Chinese Sand Pear Group (P. pyrifolia), and Pyrus Japanese Pear Group (P. pyrifolia). Many pear genetic diversity studies have been conducted using these various systems to classify species, groups, and cultivars. However, few studies have focused on comparison of modern cultivars with diverse genetic resources such as foreign cultivars, wild individuals, and local cultivars.

The Japanese pear breeding program began in 1909 [26] and continues to aim at developing new cultivars that ripen at various times and have high productivity and fruit quality, low production costs, high disease resistance, self-compatibility, and freedom from physiological disorders. In particular, breeding for soft fruit texture has been key to improving fruit quality [27]. Local cultivar ‘Nijisseiki’, which originated in the Kanto region and has been one of the leading cultivars in Japan, has excellent fruit texture. ‘Nijisseiki’ and its relatives have been repeatedly used as parents in the breeding program. In addition, ‘Osanijisseiki’, which arose as a natural mutant from ‘Nijisseiki’, was released as a self-compatible cultivar and introduced into the breeding program as a means of developing additional self-compatible cultivars [28, 29]. For these reasons, inbreeding due to repeated use of ‘Nijisseiki’-biased genotypes has become a problem for Japanese pear breeding [8, 9]. It was reported that both the pedigree-based inbreeding coefficient (F) and the marker-based inbreeding coefficient increased in Japanese pear cultivars as the year of the initial cross became more recent [9]. The tree height of 1-year-old seedlings decreased by 20 % for F = 0.25 and by 40 % for F = 0.5 [8]. Also, the decrease in number of S-genotypes among modern cultivars is problematic with respect to mating design. These days, more and more cultivars and selections have identical S-genotypes, with the result that some combinations among modern cultivars are incompatible. A possible solution may lie in the diverse genetic resources (including cultivars introduced from China, local cultivars collected from all over Japan, and wild individuals) that have been preserved at Institute of Fruit Tree and Tea Science, NARO (NIFTS). We are interested in using these genetic resources to avoid inbreeding depression.

So far, the use of foreign cultivars and wild individuals in Japanese pear breeding has been limited. It is extremely difficult to obtain elite genotypes in a short period of time while at the same time broadening genetic diversity using foreign cultivars and wild individuals instead of well-adapted genotypes. To overcome this difficulty, it is necessary to genetically characterize these materials so as to determine which cultivars or individuals would be most effective for broadening genetic diversity and how much genetic diversity has been lost among modern cultivars. The objective of the present study was to clarify the degree of inbreeding among modern cultivars and to estimate the genetic relatedness between modern cultivars and diverse genetic resources. On the basis of our results, we discuss the potential to broaden genetic diversity in pear breeding programs and the trend toward loss of genetic diversity in modern pear cultivars.

Methods

Plant materials

The nine groups (207 accessions) used in this study are shown in Table 1 and Additional file 1: Table S1. Several of the materials in this study are similar to those used in Iketani et al. [25]: wild individuals of P. ussuriensis collected from the Hayasaka-Kogen high plateau in Iwate Prefecture (IWA), which were unaffected by the genetic influence of cultivated trees [25]; Chinese pear cultivars generally considered to be P. bretschneideri (BRE) and P. ussuriensis (USS); and Japanese pear local cultivars that originated in the Kanto region of Japan (KAN), near the Sea of Japan (NSJ), and in western Japan (WJ). We also included Japanese pear crossbred cultivars and breeding lines from the first half of the 20th century (CFH), the latter half of the 20th century (CLH), and the 21st century (MDC).
Table 1

List of the 207 pear accessions used in this study

ID

Cultivar/selection

Type

Code

ID

Cultivar/selection

Type

Code

(group number)

(group number)

1

Hs-1

Wild

IWA (1)

106

Babaucchiaginashi

Local cultivar

WJ (6)

2

Hs-2

Wild

IWA (1)

107

Ichihara Wase

Local cultivar

WJ (6)

3

Hs-3

Wild

IWA (1)

108

Imamuraaki

Local cultivar

WJ (6)

4

Hs-4

Wild

IWA (1)

109

Imamuranatsu

Local cultivar

WJ (6)

5

Hs-5

Wild

IWA (1)

110

Nansei Chabo

Local cultivar

WJ (6)

6

Hs-6

Wild

IWA (1)

111

Nekogoroshi

Local cultivar

WJ (6)

7

Hs-7

Wild

IWA (1)

112

Sawairiyamanashi

Local cultivar

WJ (6)

8

Hs-8

Wild

IWA (1)

113

Segawa

Local cultivar

WJ (6)

9

Hs-9

Wild

IWA (1)

114

Shimokatsuginashi

Local cultivar

WJ (6)

10

Hs-10

Wild

IWA (1)

115

Shoumyoujinashi

Local cultivar

WJ (6)

11

Hs-11

Wild

IWA (1)

116

Tosajou

Local cultivar

WJ (6)

12

Hs-12

Wild

IWA (1)

117

Tosajounishiki

Local cultivar

WJ (6)

13

Hs-14

Wild

IWA (1)

118

Tosanashi

Local cultivar

WJ (6)

14

Hs-15

Wild

IWA (1)

119

Tsukushiinunashi

Local cultivar

WJ (6)

15

Hs-16

Wild

IWA (1)

120

Waseaka Ouryuu

Local cultivar

WJ (6)

16

Hs-18

Wild

IWA (1)

121

Atago

Crossbred cultivar

CFH (7)

17

Hs-19

Wild

IWA (1)

122

Ishii Wase

Crossbred cultivar

CFH (7)

18

Baozhuli

Cultivar

BRE (2)

123

Higashino

Crossbred cultivar

CFH (7)

19

Chang Xi Li

Cultivar

BRE (2)

124

Heiwa

Crossbred cultivar

CFH (7)

20

Hong Li

Cultivar

BRE (2)

125

Gion

Crossbred cultivar

CFH (7)

21

Hong Xiao Li

Cultivar

BRE (2)

126

Kikusui

Crossbred cultivar

CFH (7)

22

Huang Li

Cultivar

BRE (2)

127

Sagami

Crossbred cultivar

CFH (7)

23

Mi Li

Cultivar

BRE (2)

128

Seiryuu

Crossbred cultivar

CFH (7)

24

Mi Li Cui

Cultivar

BRE (2)

129

Yakumo

Crossbred cultivar

CFH (7)

25

Tai Huang Li

Cultivar

BRE (2)

130

Niitaka

Crossbred cultivar

CFH (7)

26

Ya Gua Li

Cultivar

BRE (2)

131

Asahi

Crossbred cultivar

CFH (7)

27

Ya Li

Cultivar

BRE (2)

132

Yachiyo

Crossbred cultivar

CFH (7)

28

Kuerren Xiang Li

Cultivar

BRE (2)

133

Hatsuaki

Crossbred cultivar

CFH (7)

29

Lunanhuangli

Cultivar

BRE (2)

134

Kimizukawase

Crossbred cultivar

CFH (7)

30

Ma Ke Zao Li

Cultivar

BRE (2)

135

Kougetsu

Crossbred cultivar

CFH (7)

31

Man Yuan Xiang

Cultivar

BRE (2)

136

Hattatsu

Crossbred cultivar

CFH (7)

32

Ping Li

Cultivar

BRE (2)

137

Shinkou

Crossbred cultivar

CFH (7)

33

Seuri Li

Cultivar

BRE (2)

138

Shinseiki

Crossbred cultivar

CFH (7)

34

Xie Hua Tian

Cultivar

BRE (2)

139

Seigyoku

Crossbred cultivar

CFH (7)

35

Yin Bai Li

Cultivar

BRE (2)

140

Yanaga

Crossbred cultivar

CFH (7)

36

Yuan Ba Li

Cultivar

BRE (2)

141

Shinsetsu

Crossbred cultivar

CLH (8)

37

Wo Wo Li

Cultivar

BRE (2)

142

Hiratsuka 7

Breeding line

CLH (8)

38

Suan Li

Cultivar

BRE (2)

143

Hiratsuka 1

Breeding line

CLH (8)

39

Tang Li

Cultivar

BRE (2)

144

Hiratsuka 11

Breeding line

CLH (8)

40

Dang Shan Fu Su Li

Cultivar

BRE (2)

145

Kumoi

Crossbred cultivar

CLH (8)

41

Ba Li Xiang

Cultivar

USS (3)

146

Suisei

Crossbred cultivar

CLH (8)

42

Bei Jin Bai Li

Cultivar

USS (3)

147

Kousui

Crossbred cultivar

CLH (8)

43

Cang Xi Li

Cultivar

USS (3)

148

Hiratsuka 10

Breeding line

CLH (8)

44

Dang Shan Jin Gai Su

Cultivar

USS (3)

149

Hiratsuka 17

Breeding line

CLH (8)

45

Dang Shan Mian Li

Cultivar

USS (3)

150

Hiratsuka 24

Breeding line

CLH (8)

46

Dang Shan Zi Su Li

Cultivar

USS (3)

151

Shinsui

Crossbred cultivar

CLH (8)

47

Huang Shan Li

Cultivar

USS (3)

152

Hayatama

Crossbred cultivar

CLH (8)

48

Hui Zhou Xue Li

Cultivar

USS (3)

153

Tama

Crossbred cultivar

CLH (8)

49

Jian Ba Li

Cultivar

USS (3)

154

Housui

Crossbred cultivar

CLH (8)

50

Lai Yang Ci Li

Cultivar

USS (3)

155

Hiratsuka 25

Breeding line

CLH (8)

51

Manshuu Yaseinashi

Cultivar

USS (3)

156

Hiratsuka 29

Breeding line

CLH (8)

52

Niao Li

Cultivar

USS (3)

157

Hiratsuka 27

Breeding line

CLH (8)

53

Ping Guo Li

Cultivar

USS (3)

158

Hakkou

Crossbred cultivar

CLH (8)

54

Su Hyang Ri

Cultivar

USS (3)

159

Chouju

Crossbred cultivar

CLH (8)

55

Zao Su

Cultivar

USS (3)

160

Hokukan

Crossbred cultivar

CLH (8)

56

Zhu Zui Li

Cultivar

USS (3)

161

Tsukuba 34

Breeding line

CLH (8)

57

Doitsu

Local cultivar

KAN (4)

162

Tsukuba 35

Breeding line

CLH (8)

58

Choujuurou

Local cultivar

KAN (4)

163

Tsukuba 37

Breeding line

CLH (8)

59

Nijisseiki

Local cultivar

KAN (4)

164

Tsukuba 39

Breeding line

CLH (8)

60

Yoshino

Local cultivar

KAN (4)

165

Shinsei

Crossbred cultivar

CLH (8)

61

Edoya

Local cultivar

KAN (4)

166

Shuugyoku

Crossbred cultivar

CLH (8)

62

Rokugatsu

Local cultivar

KAN (4)

167

Chikusui

Crossbred cultivar

CLH (8)

63

Okuroku

Local cultivar

KAN (4)

168

Yasato

Crossbred cultivar

CLH (8)

64

Jouhana

Local cultivar

KAN (4)

169

Nansui

Crossbred cultivar

CLH (8)

65

Heishi

Local cultivar

KAN (4)

170

Tsukuba 41

Breeding line

CLH (8)

66

Wase Kouzou

Local cultivar

KAN (4)

171

Tsukuba 42

Breeding line

CLH (8)

67

Kouzou

Local cultivar

KAN (4)

172

Tsukuba 43

Breeding line

CLH (8)

68

Shikishima

Local cultivar

KAN (4)

173

Tsukuba 44

Breeding line

CLH (8)

69

Shinchuu

Local cultivar

KAN (4)

174

Wakahikari

Crossbred cultivar

CLH (8)

70

Rikiya

Local cultivar

KAN (4)

175

Hougetsu

Crossbred cultivar

CLH (8)

71

Chousen

Local cultivar

KAN (4)

176

Natsuhikari

Crossbred cultivar

CLH (8)

72

Shirayuki

Local cultivar

KAN (4)

177

Nikkori

Crossbred cultivar

CLH (8)

73

Kokuchou

Local cultivar

KAN (4)

178

Akibae

Crossbred cultivar

CLH (8)

74

Taihei

Local cultivar

KAN (4)

179

Akemizu

Crossbred cultivar

CLH (8)

75

Sekiryuu

Local cultivar

KAN (4)

180

Nangetsu

Crossbred cultivar

CLH (8)

76

Taihaku

Local cultivar

KAN (4)

181

Hokushin

Crossbred cultivar

CLH (8)

77

Sekaiichi

Local cultivar

KAN (4)

182

Inagi

Crossbred cultivar

CLH (8)

78

Asahiryuu

Local cultivar

KAN (4)

183

Aikansui

Crossbred cultivar

CLH (8)

79

Kinchaku

Local cultivar

KAN (4)

184

Kisui

Crossbred cultivar

CLH (8)

80

Koyuki

Local cultivar

KAN (4)

185

Yoshikaori

Crossbred cultivar

CLH (8)

81

Saitama 2-1

Local cultivar

KAN (4)

186

Tsukuba 52

Breeding line

MDC (9)

82

Saitama 8

Local cultivar

KAN (4)

187

Tsukuba 53

Breeding line

MDC (9)

83

Amanogawa

Local cultivar

NSJ (5)

188

Tsukuba 49

Breeding line

MDC (9)

84

Ruisannashi

Local cultivar

NSJ (5)

189

Tsukuba 51

Breeding line

MDC (9)

85

Okusankichi

Local cultivar

NSJ (5)

190

Akizuki

Crossbred cultivar

MDC (9)

86

Hakuteiryuu

Local cultivar

NSJ (5)

191

Nashi Chukanbohon Nou 1 Gou

Crossbred cultivar

MDC (9)

87

Abumi

Local cultivar

NSJ (5)

192

Akiakari

Crossbred cultivar

MDC (9)

88

Yokogoshi

Local cultivar

NSJ (5)

193

Oushuu

Crossbred cultivar

MDC (9)

89

Awayuki

Local cultivar

NSJ (5)

194

Shuurei

Crossbred cultivar

MDC (9)

90

Hachibuse No Nashi

Local cultivar

NSJ (5)

195

Natsushizuku

Crossbred cultivar

MDC (9)

91

Oohiromaru

Local cultivar

NSJ (5)

196

Shinkansen

Crossbred cultivar

MDC (9)

92

Kounowatashi

Local cultivar

NSJ (5)

197

Kanta

Crossbred cultivar

MDC (9)

93

Miyadani

Local cultivar

NSJ (5)

198

Rinka

Crossbred cultivar

MDC (9)

94

Onba

Local cultivar

NSJ (5)

199

Hatsumaru

Crossbred cultivar

MDC (9)

95

Shimane Yamanashi

Local cultivar

NSJ (5)

200

Hoshiakari

Crossbred cultivar

MDC (9)

96

Hakataao

Local cultivar

NSJ (5)

201

Tsukuba 59

Breeding line

MDC (9)

97

Kunitomi

Local cultivar

NSJ (5)

202

Tsukuba 60

Breeding line

MDC (9)

98

Nishitonami 1

Local cultivar

NSJ (5)

203

Tsukuba 61

Breeding line

MDC (9)

99

Ookoga

Local cultivar

NSJ (5)

204

Tsukuba 62

Breeding line

MDC (9)

100

Shihyakume

Local cultivar

NSJ (5)

205

Tsukuba 63

Breeding line

MDC (9)

101

Tanponashi

Local cultivar

NSJ (5)

206

Tsukuba 64

Breeding line

MDC (9)

102

Touhou

Local cultivar

NSJ (5)

207

Narumi

Crossbred cultivar

MDC (9)

103

Tottori 4

Local cultivar

NSJ (5)

    

104

Waseaka

Local cultivar

NSJ (5)

    

105

Yagoemon

Local cultivar

NSJ (5)

    

IWA Wild individuals from Iwate, BRE P. bretschneideri cultivar, USS P. ussuriensis cultivar, KAN Local cultivar from Kanto region, NSJ Local cultivar from near the sea of Japan, WJ Local cultivar from western Japan, CFH Cultivar released in the first half of the 20th century, CLH Cultivar released in the latter half of the 20th century or breeding line developed during that time, MDC Modern cultivar (released in the 21st century) or breeding line developed during that time

SSR marker analysis

The 207 pear accessions were genotyped for 19 simple sequence repeat (SSR) markers (Additional file 2: Table S2). PCR amplification was performed in 10 μL containing 5 μL of 2× Green GoTaq reaction buffer (0.4 mM each dNTP, 3 mM MgCl2, and 1 U Taq polymerase, pH 8.5, Promega, Madison, USA), 20 pmol of each forward primer labeled with a fluorescent chemical (FAM or HEX) and unlabeled reverse primer, and 2.5 ng of genomic DNA. Amplification was performed in 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. PCR products were separated and detected with a 3130 xl genetic analyzer (Life Technologies Co., Carlsbad, CA, USA). The size of each amplified band was determined by comparison with an internal DNA standard (400HD-ROX, Life Technologies) in GeneScan software (Life Technologies).

Data analyses

The observed heterozygosity (H O) and expected heterozygosity (H E) were calculated in GenAlEx v. 6.5 software [30], and allelic richness (AR, n =15) was calculated in FSTAT v. 2.9.3 software [31]. Bayesian statistical inference on the population structure was performed in Structure v. 2.3.3 software [32] with the admixture model for ancestry and both independent and correlated models for allele frequency, without any prior information about the origin of each individual. After a burn-in period of 100,000 iterations, the analysis was run 10 times for each value of K (number of inferred ancestral populations) from 2 to 10 for 1,000,000 iterations. We used Evanno et al.’s [33] criterion of |L″(K)| = |L′(K + 1) – L′(K)| = |lnP (X|K + 1) – 2lnP(X|K + 1) + lnP(X|K + 1)| and ∆K = mean (|L″(K)|)/s[L(K)] and values of plateaued lnP(X|K) to estimate the optimal value of K. Simulation studies have shown that once the real value of K has been reached, lnP(X|K) will typically plateau or continue to increase slightly [33]. Principal coordinate analysis (PCoA) was calculated in GenAlEx 6.5 from the pairwise genetic distances obtained with the covariance-standardized method. Simple allele-sharing distances among the 207 accessions were calculated as described [34]. All data were calculated from the genotypes of the 207 accessions based on the 19 SSR markers.

Results

Basic genetic characteristics of pear accession groups

We genotyped nine groups, consisting of 207 accessions, by using 19 SSR markers. Heterozygosity of the nine groups was H O = 0.42–0.74 and H E = 0.39–0.80 (Table 2). Crossbred cultivar groups released after 1950 (CLH and MDC) had lower values than the other 7 groups. AR of the nine groups ranged from 3.2 to 9.6. AR of the Chinese pear groups (BRE and USS) showed the highest values (8.0 and 9.6) among the nine groups. On the other hand, AR of the crossbred cultivar groups (CFH, CLH, and MDC) decreased as the year of release became more recent (4.6, 3.5, and 3.2, respectively). The AR values of MDC were about half those of the local cultivar groups (KAN, NSJ, WJ).
Table 2

Genetic characteristics of group analyzed using 19 SSRs

Group number

Code

H O

H E

AR

1

IWA

0.69

0.71

6.4

2

BRE

0.72

0.75

8.0

3

USS

0.74

0.80

9.6

4

KAN

0.72

0.66

5.1

5

NSJ

0.68

0.71

6.1

6

WJ

0.66

0.71

6.3

7

CFH

0.69

0.61

4.6

8

CLH

0.54

0.47

3.5

9

MDC

0.42

0.39

3.2

H O observed heterozygosity, H E expected heterozygosity, AR allelic richness

Bayesian statistical inference of the population structure

To estimate the optimal number of genetic clusters (K) in Structure, we calculated ∆K values (Table 3). The ∆K values were highest at K = 2 in both the independent and correlated models. In both models, the two clusters corresponded to (1) wild individuals and Chinese pear groups (IWA, BRE, and USS) and (2) local and crossbred cultivar groups (KAN, NSJ, WJ, CFH, CLH and MDC), similar to the classification obtained by Iketani et al. [24]. In the independent model, the second-highest ∆K value occurred at K = 4 (Table 3). Moreover, the value of lnP(X|K) seemed to plateau at K = 4 (Fig. 1). Consequently we adopted K = 4 as the optimal classification in the independent model. With K = 4, the 207 accessions could be classified into four groups corresponding to (1) wild individuals from Iwate Prefecture (green cluster), (2) Chinese pear cultivars (yellow cluster), (3) local cultivars from the Kanto region and crossbred cultivars (red cluster), and (4) local cultivars from outside the Kanto region (blue cluster; Fig. 2a). We were not able to separate the local cultivars from the Kanto region from the crossbred cultivars by increasing the value of K in the independent model. The new clusters that appeared at K = 6 to 10 were distributed mainly in the Chinese pear cultivars.
Table 3

Values of ΔK for K = 1 to 10 in independent and correlated models

K

ΔK

Independent

Correlated

1

2

1646.7

1610.6

3

14.3

6.9

4

102.3

2.4

5

53.5

4.1

6

2.4

1.5

7

4.1

2.0

8

1.6

1.9

9

2.6

2.1

10

Fig. 1

Values of lnP(X|K) for values of K from 1 to 10

Fig. 2

A detailed bar plot diagram for (a) K = 4 in the independent model and (b) K = 6 in the correlated model. The first number under each bar represents the individual accession ID number (1–207); the second number (in parentheses) represents the group number (1–9). ID numbers and groups are defined in Table 1

On the other hand, no prominent ∆K was observed in the correlated model other than for K = 2 (Table 3), and the value of lnP(X|K) seemed to plateau somewhere between K = 6 and K = 10, although it did not plateau as clearly as for the independent model (Fig. 1). However, we confirmed that the bar plot diagrams at K = 6 (Fig. 2b) were similar across ten repetitions, suggesting that the clustering at K = 6 in the correlated model has high reliability. The difference in genetic structure between K = 4 in the independent model and K = 6 in the correlated model was the appearance of a new cluster in the Chinese pear groups (cyan) and in the local cultivar group from the Kanto region (magenta; Fig. 2). At K = 6, some accessions showed admixtures of different clusters (suggesting contributions from different populations), which was observed much less frequency in the K = 4 classification. In particular, some crossbred cultivars had characteristics of both the red and magenta clusters when K = 6, which we attribute to cross-hybridization between genotypes from different clusters in the pear breeding program. Among the local cultivars from the Kanto region (KAN), only ‘Nijisseiki’ (ID = 59) was dominated by the red cluster at K = 6, probably because cultivars released in the 20th century and later are based on ‘Nijisseiki’ and its relatives.

PCoA of the 207 accessions

The first two informative PCo components (Fig. 3) explained 23.15 % of the total variation. The results of PCoA were similar to those of the Structure analysis. The wild individual group (IWA) and Chinese pear groups (BRE, USS) appeared to be distinct from the other groups. BRE and USS showed similar distribution, as did NSJ and WJ. Crossbred cultivars (CFH, CLH, MDC) were plotted closer to ‘Nijisseiki’ (ID = 59) as the year of release became more recent. In particular, all of the MDC cultivars except ‘Oushuu’ (ID = 193) were distributed near ‘Nijisseiki’. Some cultivars showed unexpected distributions; for example, Chinese pear cultivars ‘Ya Gua Li’ (ID = 26) and ‘Manshuu Yaseinashi’ (ID = 51) were plotted between the Japanese local cultivars and the Chinese pear cultivars. ‘Saitama 2-1’ (ID = 81) and ‘Saitama 8’ (ID = 82), both of which belong to the group of local cultivars from the Kanto region, were close to the Chinese pear groups.
Fig. 3

Principal coordinate analysis (PCoA) plot generated from genetic distance calculations among the 207 accessions in GenAlEx software. Group codes are as shown in Table 1

We also calculated simple allele-sharing distances to estimate the genetic relationship between each pair of accessions (Additional file 3: Table S3). The average simple allele-sharing distances were 0.22, 0.30, 0.54 and 0.73 between MDC and wild P. ussuriensis (IWA), Chinese pear cultivars (BRE and USS), local cultivars (KAN, NSJ, and WJ), and crossbred cultivars (CFH, CLH, and MDC), respectively.

Discussion

Genetic relationships between modern Japanese pear cultivars and diverse genetic resources

We evaluated diverse genetic resources of Asian pear using Structure analysis, PCoA, and simple allele-sharing distance. The results of Structure analysis and PCoA were consistent in that wild individuals and Chinese pear cultivars were classified as genetically distinct from Japanese pear cultivars. This result is also in good agreement with previous studies [24, 25]. The modern cultivars seemed to be genetically close to local cultivars that originated in the Kanto region, especially ‘Nijisseiki’ (Figs. 2 and 3). Because modern cultivars were selected from ‘Nijisseiki’ and its relatives, this result was not unexpected. Both low heterozygosity and low AR were observed in Japanese pear modern cultivars, suggesting that the genetic diversity of modern cultivars has decreased over time. In the Structure result with K = 4, almost all crossbred cultivars and modern cultivars showed “red” genetic background (i.e., were part of the cluster indicated by red shading in Fig. 2), which was characteristic of local cultivars from the Kanto region. When K = 6, the “red” genetic background was shared by only ‘Nijisseiki’ and modern cultivars. In addition, almost all of the modern cultivars were plotted around ‘Nijisseiki’ in PCoA, and the average allele-sharing distance between modern cultivars and ‘Nijisseiki’ was high (0.76; Additional file 3: Table S3). These results suggest a marked biased in the genotypes of modern cultivars. Crossing among modern cultivars containing only the “red” genetic background would not generate a genotype that greatly exceeds the current performance of modern cultivars. To broaden genetic diversity and obtain superior genotypes in modern cultivars, we need to introduce genes from wild individuals and Chinese pear cultivars that are genetically different from modern Japanese pear cultivars.

Breeding history of ‘Oushuu’, a cultivar with a diverse genetic background

Among the modern cultivars, only ‘Oushuu’ (ID = 193) seemed to be distinct from the others in both Structure analysis and PCoA. This cultivar, released in 2003, is an offspring of a cross between C2 (an offspring of a cross between ‘Lai Yang Ci Li’ (ID = 50) and ‘Nijisseiki’) and ‘Shinsetsu’ (ID = 141, an offspring of a cross between ‘Imamuraaki’ (ID = 108) and ‘Okusankichi’ (ID = 85)). ‘Oushuu’ showed both strong tree vigor and desirable fruit texture characteristics (e.g., soft flesh firmness) preferred in the Japanese market [35]. In the Structure result with K = 6, ‘Oushuu’ had not only “red” genetic background (presumably from ‘Nijisseiki’) but also “cyan” (presumably from ‘Lai Yang Ci Li’), “blue” (presumably from ‘Imamuraaki’), and “magenta” (presumably from ‘Okusankichi’) genetic backgrounds. Its strong tree vigor may be caused by heterosis. The successful breeding history of ‘Oushuu’ indicates that it is possible to release new cultivars that have good fruit quality without repeatedly using ‘Nijisseiki’ and its relatives as parents.

Cultivars showing unexpected genetic structure

Some cultivars showed unexpected genetic structure and genetic relationships. ‘Ya Gua Li’ (ID = 26) and ‘Manshuu Yaseinashi’ (ID = 51) were classified into the Chinese pear group [25], but were plotted between Japanese local cultivars and Chinese pear cultivars in PCoA. ‘Ya Gua Li’ appears to have the genetic structure of both Chinese pear and local cultivars from the Kanto region. In fact, ‘Ya Gua Li’ and ‘Nijisseiki’ shared at least one allele at each of the 19 SSR markers, suggesting that ‘Ya Gua Li’ is a hybrid between Chinese pear and ‘Nijisseiki’. This hybrid could have been mislabeled during genetic resource preservation. ‘Manshuu Yaseinashi’ had admixed genetic structure, but the results with K = 4 and K = 6 were different, i.e., it contained “blue” genetic background (found in Japanese local cultivars) when K = 4 and “yellow” genetic background (found in Chinese pear cultivars) when K = 6. Because the word “Manshuu” is the old name of a place in northeastern China and “Yaseinashi” means “wild pear” in Japanese, wild individuals from northeastern China will be key to clarifying the true genetic structure of ‘Manshuu Yaseinashi’. Although ‘Tang Li’ (ID =39) was classified as BRE, it has an almost totally “blue” genetic background (characteristic of Japanese local cultivars) at both K = 4 and K = 6. Pear genetic resources are generally grafted onto seedling rootstocks of Japanese materials such as strain ‘Mamenashi’, the collective name of wild pear with small fruit. Thus, it is possible that ‘Tang Li’ had been mislabeled or mishandled during genetic resource preservation and is actually a Japanese rootstock genotype. We need to take care because these cultivars showing unexpected genetic structure are not appropriate for broadening the genetic diversity of Japanese pear cultivars. ‘Saitama 2-1’ (ID = 81) and ‘Saitama 8’ (ID = 82) are considered to be local cultivars from the Kanto region, because “Saitama” is the name of a prefecture in the Kanto region, but these cultivars were genetically close to the Chinese cultivar group in PCoA. As with ‘Manshuu Yaseinashi’, the Structure results for ‘Saitama 2-1’ and ‘Saitama 8’ were inconsistent between K = 4 and K = 6. Further analyses including verified Chinese materials are needed to identify the origin of these cultivars.

Putative spreading patterns of local cultivars

Local cultivars that originated outside the Kanto region seem to have been important contributors to the genetic diversity of cultivated pears. It has been reported that cultivars that originated near the Sea of Japan or Kyushu island tend to show late ripening and long fruit, whereas cultivars that originated in the Kanto region show early ripening and oblate fruit [36, 37]. However, some local cultivars that originated outside the Kanto region (‘Awayuki’ (ID = 89), ‘Kounowatashi’ (ID = 92), ‘Kunitomi’ (ID = 97), ‘Touhou’ (ID = 102), and ‘Waseaka’ (ID = 104)) showed genetic background similar to those that originated in the Kanto region at K = 4, suggesting that these cultivars might have been introduced from the Kanto region into other regions. In fact, it was reported that ‘Waseaka’ was introduced into Niigata Prefecture from the Kanto region, and ‘Kunitomi’ was discovered from offspring of ‘Taihaku’ (ID = 76), which originated in the Kanto region [26]. Similarly, these other cultivars might have been introduced from the Kanto region into other regions. In fruit tree species, it is common that local cultivars with traits of interest are vegetatively propagated and carried to other regions [3841]. We need to take into account the spread of cultivars when classifying local cultivars by geographical origin.

Possible origin of local cultivars in Japan

Iketani et al. [24] showed that local cultivars in Japan are genetically closer to Chinese cultivars than to wild individuals of P. ussuriensis collected from high plateaus in Iwate Prefecture. In the Structure result at K = 6, some local cultivars in Japan showed “yellow” genetic background, characteristic of Chinese pear cultivars. The history of pear breeding before the early modern period in Japan is still unclear, but some local cultivars may have been domesticated from Chinese materials. Jiang et al. [42] suggested that there were opportunities for ancient cultivar exchange between Japan and eastern China. Thus, the “yellow” genetic background might have been introduced by genetic exchange with Chinese cultivars.

Future breeding strategy for the Japanese pear breeding program

Our data strongly support the assumption that introgression of germplasm from Chinese pear cultivars and wild individuals into modern cultivars is an effective way to broaden genetic diversity. Thus, it might be easier to develop new cultivars using Chinese pear cultivars, which are already domesticated and bear large fruit. In fact, ‘Oushuu’ was selected from the first backcross of a Chinese cultivar to several Japanese cultivars and has good fruit quality. On the other hand, introgression of germplasm from indigenous species into modern cultivars seems to be challenging; for example, their fruit size is smaller than that of recent cultivars [43]. However, there are many hybrids between P. pyrifolia and P. ussuriensis, some of which bear fruits larger than those of wild individuals of the latter species [44]. In particular, local cultivar ‘Natsunashi’ would be good material for a pear breeding program because it shows early ripening and has high concentrations of ethyl and methyl esters, which are desirable flavor components.

Trends in genetic diversity during organized breeding of annual and perennial crops

Owing to the progress of organized breeding (scientific breeding), the genetic diversities of several annual crops, including maize [45, 46], rice [47], sorghum [48], tobacco [49], and wheat [50], decreased at one point during the latter part of the 20th century. According to a meta-analysis of genetic diversity trends in annual crops during the 20th century, diversity was reduced significantly in the 1960s compared with the 1950s, than then recovered from the 1970s to the 1990s [51]. Breeders probably averted the narrowing of the germplasm base and subsequently increased the genetic diversity in these crops through the introgression of novel materials. However, very few genetic studies have focused on genetic diversity trends during the organized breeding of perennial crops, although inbreeding depression has been a concern of fruit breeders [6, 51]. Our results clarify that loss of genetic diversity has occurred in a fruit crop, as has been reported in annual crops. However, the genetic diversity in perennial crops seems to have declined more slowly than that in annual crops, possibly because perennial crops have a longer juvenile phase and fewer sexual cycles than annual crops. Japanese pear breeding may have just reached the stage reached by annual crop breeding in the 1960s or 1970s. Given the availability of genetic resources and molecular tools, we now have the capability to work toward increasing the genetic diversity of pear cultivars.

Conclusions

In this study, we clarified the genetic relationship between Japanese pear modern cultivars and diverse genetic resources including wild individuals, Chinese pear cultivars, and local cultivars. The genetic diversity of modern cultivars decreased as the year of release became more recent. The modern cultivars were genetically close to local cultivar ‘Nijisseiki’, which had been repeatedly used as a parent in our breeding program, confirming that Japanese pear breeding has been carried out within a narrow gene pool. On the basis of these findings, we plan to broaden the genetic diversity in the NIFTS pear breeding program by introgressing germplasm from Chinese pear and wild individuals that are genetically distinct from modern cultivars. We also determined that Structure analysis and PCoA are effective for evaluating the degree of inbreeding and genetic relationships among accessions. The information obtained in this study will be useful for pear breeders as well as other fruit breeders who have problems with inbreeding depression in their breeding programs.

Abbreviations

AR, allelic richness; BRE, Chinese pear cultivars generally considered to be P. bretschneideri; CFH, Japanese pear crossbred cultivars and breeding lines from the first half of the 20th century; CLH, Japanese pear crossbred cultivars and breeding lines from the latter half of the 20th century; H E, expected heterozygosity; H O, observed heterozygosity; IWA, wild individuals of P. ussuriensis collected from the Hayasaka-Kogen high plateau in Iwate Prefecture; KAN, Japanese pear local cultivars that originated in the Kanto region of Japan; MDC, Modern cultivar released in the 21st century or breeding line developed during that time; NIFTS, Institute of Fruit Tree and Tea Science, NARO; NSJ, Japanese pear local cultivars that originated near the Sea of Japan; PCoA, principal coordinate analysis; SSR, simple sequence repeat; USS, Chinese pear cultivars generally considered to be P. ussuriensis; WJ, Japanese pear local cultivars that originated in western Japan

Declarations

Acknowledgments

We are deeply indebted to all the people involved in the Japanese pear breeding program at the Institute of Fruit Tree and Tea Science, NARO, Japan.

Funding

No funding was obtained for this study.

Availability of supporting data

The datasets supporting the conclusions of this article are included within the article and its additional files.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SN participated in the design of the experiments, genotyped SSRs, interpreted the data, and wrote the paper. HI participated in the design of the experiments, genotyped SSRs, and revised the manuscript. TY provide SSR markers and genotyped SSRs. NT and TS participated in the design of the experiments and provided the plant materials. All the authors read and approved the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Fruit Tree and Tea Science, NARO (NIFTS)
(2)
Department of Biosphere-Geosphere Science, Faculty of Biosphere-Geosphere Science, Okayama University of Science

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Copyright

© The Author(s). 2016