Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China.

The Chinese chestnut (Castanea mollissima Bl.) was introduced into Japan about 100 years ago. Since then, a number of Chinese chestnut cultivars and Japanese-Chinese hybrid cultivars have been selected by farmers and plant breeders, but little information has been available about their origins and genetic relationships. A classification based on simple sequence repeat markers was conducted using 230 cultivars including Japanese chestnut (Castanea crenata Sieb. et Zucc.) cultivars originated in Japan, Japanese-Chinese hybrid cultivars, and Chinese chestnut cultivars originated in both Japan and China. First, a search for synonyms (cultivars with identical genotypes) revealed 23 synonym groups among the Chinese chestnut cultivars, and all but one cultivar from each synonym group was omitted from further analyses. Second, genetic structure analysis showed a clear division between Japanese and Chinese chestnut, and most of the Japanese and Chinese cultivars had a simple genetic structure corresponding to the expected species. On the other hand, most Japanese-Chinese hybrid cultivars had admixed genetic structure. Through a combination of parentage and chloroplast haplotype analyses, 16 of the 18 hybrid cultivars in this study were inferred to have parent-offspring relationships with other cultivars originated in Japan. Finally, Bayesian clustering and chloroplast haplotype analysis showed that the 116 Chinese chestnut cultivars could be divided into two groups: one originated in the Hebei region of China and the other originated in the Jiangsu and Anhui regions of China. The Chinese chestnut cultivars selected in Japan showed various patterns of genetic structure including Hebei origin, Jiangsu or Anhui origin, and admixed. The chestnut cultivar genetic classifications obtained in this study will be useful for both Japanese and Chinese chestnut breeding programs.

Introduction

Japanese chestnut (Castanea crenata Sieb. et Zucc.) and Chinese chestnut (C. mollissima Bl.) are naturally distributed throughout Japan and China, respectively. Both species are reported to have been prehistorically domesticated [1-3] and are still economically important for production of edible nuts. These two species have large differences in both genetic and morphological properties. Japanese chestnut has large fruit size, adherent pellicle, and comparatively good yield, whereas Chinese chestnut has small nut size, easy-to-peel pellicle, and low yield [4]. In addition, the yellow brown shoot and pubescence at the nut tip are characters specific to Chinese chestnut and can be used to distinguish between the two species.

Interspecific hybridization has been used in chestnut breeding programs all over the world [4,5]. The breeding objectives of such programs have varied depending on the species. In the United States, the chestnut blight resistance gene from Chinese chestnut has been introduced into American chestnut (C. dentata [Marsh.] Borkh.) [6,7]. To improve European chestnut (C. sativa Mill.), quantitative trait loci for resistance to Phytophthora cinnamomic and chestnut gall wasp were introduced from Japanese chestnut [8,9]. In Japan, Chinese chestnut has been applied in Japanese chestnut breeding programs to improve nut quality and pellicle peelability [10,11]. Thus, it is important to clarify the genetic relationships among chestnut cultivars of different species.

According to Tanaka [12] and Isaki [13], Chinese chestnut was introduced into Japan about 100 years ago and attempts were made to cultivate it in Japan. However, this species was not suited to the Japanese climate [12] and had high susceptibility to the chestnut gall wasp [13]. Some local farmers in Japan planted nuts of Chinese chestnut and selected for adaptation to the local climate and conditions, leading to the development of Chinese chestnut cultivars such as ‘Houji’, ‘Miyagawa’, ‘Hinoharu’, and ‘Aioi’ in the early 20th century [13] Subsequently, the Japanese–Chinese hybrid cultivar ‘Riheiguri’ was selected by a local farmer and became one of the leading cultivars in Japan, accounting for 7% of total chestnut cultivation area in Japan in 2016. Even though this cultivar does not give high yields, its nut quality is highly valued by consumers and producers.

A number of Chinese chestnut and Japanese–Chinese hybrid cultivars have been collected and preserved at the NARO (National Agriculture and Food Organization) Genebank (www.gene.affrc.go.jp) and can be readily used for research and breeding purposes. However, information on the origin and genetic structure of these cultivars has been unclear or limited, since most of them were developed by local farmers and local agricultural experiment stations more than 50 years ago. Also, it is quite difficult to distinguish between pure Chinese chestnut and Japanese–Chinese hybrids by nut appearance. Although Japanese and Chinese chestnut readily produce interspecific hybrids, the extent of admixing from other species has not been clarified by molecular marker analyses.

The Chinese chestnut has the widest distribution among the chestnut species [14]. The northern range reaches 41°N, following the ranges of the Yanshan Mountains, and the southern range extends to 18°N on the Wuzhi Mountain of Hainan Island. Due to its large distribution, the Chinese chestnut is presumed to have higher genetic diversity than other chestnut species [15]. Several reports have suggested that central China, possibly the Shengnongia region near the Chang Jiang River, is the center of genetic diversity of Chinese chestnut and is one of the candidates for a refugium of this species [15-17]. On the other hand, Zhang and Liu [18] suggested that the southwest area of China is the center of diversity. So far, no obvious genetic structure corresponding to geographical location has been found by clustering analyses, suggesting that human-mediated transportation might have affected the wild chestnut population structure [17]. While a large number of chloroplast haplotypes were identified within wild chestnut populations, only two haplotypes were identified in a collection of cultivars [19]. Ovesná et al. [20] also suggested that the genetic variability of Chinese chestnut cultivars was less than that of wild populations. Thus, several studies have examined the genetic diversity of wild chestnut and the genetic relationship between wild populations and cultivar collections. However, classifications based on clustering analyses using a large number of chestnut cultivars collected from diverse regions have not previously been conducted. In addition, the genetic relationship between Chinese chestnut cultivars within and outside of China has been unclear.

The classification and clustering of chestnut cultivars in Europe and Japan have been assessed using simple sequence repeats (SSRs) [21-25]. Because SSRs are highly reliable markers, they have been used to conduct Bayesian structure analyses and to identify synonyms (differently named cultivars with identical genotypes), homonyms (different genotypes with the same cultivar name), and parent–offspring relationships. The Bayesian clustering analyses usually correspond to region-based classification or prevalent nut use. On the other hand, humans have carried scions between different locations, resulting in some cultivars showing unexpected cultivar origin and population structure [25]. In fact, many synonym groups contain local cultivars from different regions [21,24] and parentage analyses have clarified that traditional cultivars have contributed to the appearance of many local cultivars [23,25].

The main objective of the present study was to clarify the origin and genetic characteristics of Chinese chestnut cultivars in Japan. We used a three-step strategy for cultivar classification. The first step was to identify synonym groups and eliminate duplicate genotypes prior to further analyses. The second was to clarify the genetic relationship between Japanese–Chinese hybrids and Chinese chestnut cultivars in Japan by using highly reliable materials as references, i.e., Japanese and Chinese chestnut cultivars originated in Japan and China, respectively. The third was to clarify the genetic relationship between Chinese chestnut cultivars selected in Japan and those originated in China. Identification of synonyms, parentage, and genetic relationships among cultivars would be useful for chestnut breeding programs and further genetic classification studies.

Materials and methods

Plant materials and DNA extraction

The 12 groups (230 cultivars) used in this study are shown in Tables 1 and S1. The Japanese chestnut, Japanese–Chinese hybrid, and Chinese chestnut cultivars from Japan are preserved at the NARO Genebank (www.gene.affrc.go.jp). These include Japanese chestnut local cultivars that originated in the Kanto region of Japan (designated J_KA), the Tanba region of Japan (J_TA), other regions of Japan (J_OJ), and Korea (KOR); Japanese–Chinese chestnut hybrids developed in Japan (HYB); and Chinese chestnut cultivars selected in Japan (C_SJ) and introduced from other countries (C_IO). The Chinese chestnut cultivars originated in China were provided by the Liaoning Province Economic Forest Research Institute, Shandong Institute of Pomology, and Hebei Agriculture and Forestry Academy of Sciences Changli Guoshu Institute. Chinese chestnut cultivar groups in China were defined by region of origin, i.e., Hebei (C_HE), Shandong (C_SH), Anhui (C_AN), Jiangsu (C_JI), and other regions of China (C_OR). To avoid using duplicate genotypes in the analysis, only one cultivar was used from each synonym group identified by Nishio et al. [24]. Genomic DNA was extracted from young leaves with a DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions.

Table 1

Names, accession numbers, and genotype information for the 230 cultivars used in this study.

Genotype	Cultivar	Origin	Code (group number)
1	Arima	Kanto (Japan)	J_KA (1)
2	ChuutanA	Kanto (Japan)	J_KA (1)
3	Gosha	Kanto (Japan)	J_KA (1)
4	Hassaku	Kanto (Japan)	J_KA (1)
5	Moriwase	Kanto (Japan)	J_KA (1)
6	Nakatetanba	Kanto (Japan)	J_KA (1)
7	Odai	Kanto (Japan)	J_KA (1)
8	Osaya	Kanto (Japan)	J_KA (1)
9	Senri	Kanto (Japan)	J_KA (1)
10	Shichifukuwase	Kanto (Japan)	J_KA (1)
11	Taishouwase	Kanto (Japan)	J_KA (1)
12	Toyotamawase	Kanto (Japan)	J_KA (1)
13	Tsunehisa	Kanto (Japan)	J_KA (1)
14	Yamatowase	Kanto (Japan)	J_KA (1)
15	Choubei	Tanba (Japan)	J_TA (2)
16	Choukouji	Tanba (Japan)	J_TA (2)
17	Daihachi	Tanba (Japan)	J_TA (2)
18	Fukunami	Tanba (Japan)	J_TA (2)
19	Fukunishi	Tanba (Japan)	J_TA (2)
20	Ginyose	Tanba (Japan)	J_TA (2)
21	Higan	Tanba (Japan)	J_TA (2)
22	Ichiemon	Tanba (Japan)	J_TA (2)
23	Imakita	Tanba (Japan)	J_TA (2)
24	Kanotsume	Tanba (Japan)	J_TA (2)
25	Kenagaginyose	Tanba (Japan)	J_TA (2)
26	Kinseki	Tanba (Japan)	J_TA (2)
27	Kinyoshi	Tanba (Japan)	J_TA (2)
28	Konishiki	Tanba (Japan)	J_TA (2)
29	Matabei	Tanba (Japan)	J_TA (2)
30	Ogawa teteuchi	Tanba (Japan)	J_TA (2)
31	Otomune	Tanba (Japan)	J_TA (2)
32	Shimokatsugi	Tanba (Japan)	J_TA (2)
33	Shuuhouwase	Tanba (Japan)	J_TA (2)
34	Tajiriginyose	Tanba (Japan)	J_TA (2)
35	Yakko	Tanba (Japan)	J_TA (2)
36	Akachiu	Other regions in Japan	J_OJ (3)
37	Banseki	Other regions in Japan	J_OJ (3)
38	Buzen	Other regions in Japan	J_OJ (3)
39	Dengorou	Other regions in Japan	J_OJ (3)
40	Ganne	Other regions in Japan	J_OJ (3)
41	Hataya oguri	Other regions in Japan	J_OJ (3)
42	Ichikawawase	Other regions in Japan	J_OJ (3)
43	Kasaharawase	Other regions in Japan	J_OJ (3)
44	Katayama	Other regions in Japan	J_OJ (3)
45	Kinshuu	Other regions in Japan	J_OJ (3)
46	Ninomiya	Other regions in Japan	J_OJ (3)
47	Obiwase	Other regions in Japan	J_OJ (3)
48	Obuse 3	Other regions in Japan	J_OJ (3)
49	Okoma	Other regions in Japan	J_OJ (3)
50	Ooharaguri	Other regions in Japan	J_OJ (3)
51	Saimyouji 1	Other regions in Japan	J_OJ (3)
52	Tanabata	Other regions in Japan	J_OJ (3)
53	Tanoue 1	Other regions in Japan	J_OJ (3)
54	Terai	Other regions in Japan	J_OJ (3)
55	Togenashi	Other regions in Japan	J_OJ (3)
56	Tsuchidawase	Other regions in Japan	J_OJ (3)
57	Waseginzen	Other regions in Japan	J_OJ (3)
58	Yamaguchiwase	Other regions in Japan	J_OJ (3)
59	Yamaguchiwase 2	Other regions in Japan	J_OJ (3)
60	Yourou	Other regions in Japan	J_OJ (3)
61	Buyu 3	Korea	KOR (4)
62	Hamjung 3	Korea	KOR (4)
63	Jungbu 26	Korea	KOR (4)
64	Jungbu 8	Korea	KOR (4)
65	Jungbu 9	Korea	KOR (4)
66	Pochun B-1	Korea	KOR (4)
67	Ikaba	Hyogo	HYB (5)
68	Hayashi 1	Gifu	HYB (5)
69	Hayashi 3	Gifu	HYB (5)
70	Hayashi amaguri	Gifu	HYB (5)
71	Hyogo 493	Hyogo	HYB (5)
72	Kurakata amaguri	Tokyo	HYB (5)
73	Nishiharima	Hyogo	HYB (5)
74	Omatsuguri	Ehime	HYB (5)
75	Riheiguri	Gifu	HYB (5)
76	Senshu amaguri	Akita	HYB (5)
77	Shimaki 1	Ibaraki	HYB (5)
78	Shimaki 2	Ibaraki	HYB (5)
79	Shimaki 3	Ibaraki	HYB (5)
80	Shimaki 4	Ibaraki	HYB (5)
81	Shimaki 5	Ibaraki	HYB (5)
82	Shimaki 6	Ibaraki	HYB (5)
83	Wasetenshin	Unknown	HYB (5)
84	Yamewase	Fukuoka	HYB (5)
85	Aioi	Aichi	C_SJ (6)
86	C-4	Kanagawa	C_SJ (6)
87	Gifu 1	Gifu	C_SJ (6)
88	Hakuri	Nagano	C_SJ (6)
89	Hinoharu	Yamanashi	C_SJ (6)
90	Hinoharu 2	Yamanashi	C_SJ (6)
91	Houji 354	Kochi	C_SJ (6)
92	Houji 445	Kochi	C_SJ (6)
93	Houji 350	Kochi	C_SJ (6)
94	Houji 446	Kochi	C_SJ (6)
95	Houji 480	Kochi	C_SJ (6)
96	Hyogo shinaguri	Hyogo	C_SJ (6)
97	Iwate amaguri	Unknown	C_SJ (6)
98	Kahoku 10	Tsukuba	C_SJ (6)
99	Kanan 56	Tsukuba	C_SJ (6)
100	Konan 22	Tsukuba	C_SJ (6)
101	Konan 36	Tsukuba	C_SJ (6)
102	Konan 52	Tsukuba	C_SJ (6)
103	Kousei 2	Tsukuba	C_SJ (6)
104	Miyagawa 100	Yamanashi	C_SJ (6)
105	Miyagawa 18	Yamanashi	C_SJ (6)
106	Miyagawa 84	Yamanashi	C_SJ (6)
107	Miyagawa 85	Yamanashi	C_SJ (6)
108	Miyagiguri	Unknown	C_SJ (6)
109	Houji 360	Kochi	C_SJ (6)
110	Tsuchida Amaguri	Gifu	C_SJ (6)
111	Yunba2	Yamanashi	C_SJ (6)
112	Connecticut Yankee	U.S.A.	C_IO (7)
113	Hamden	U.S.A.	C_IO (7)
114	Nepal chestnut	Nepal	C_IO (7)
115	Sleeping Giant	U.S.A.	C_IO (7)
116	Tokuganri A	North Korea	C_IO (7)
117	2399	Hebei (China)	C_HE (8)
118	Dabanhong	Hebei (China)	C_HE (8)
119	Donglingmingzhu	Hebei (China)	C_HE (8)
	Xigou 7	Hebei (China)	C_HE (8)
120	Guanting 10	Hebei (China)	C_HE (8)
121	Houhanzhuang 20	Hebei (China)	C_HE (8)
122	Qiananli	Hebei (China)	C_HE (8)
123	Yanchangli	Hebei (China)	C_HE (8)
124	Yanfeng	Hebei (China)	C_HE (8)
125	Yankui	Hebei (China)	C_HE (8)
	Yancheng 3	Unknown (China)	C_OR (12)
126	Yanshanduanzhi	Hebei (China)	C_HE (8)
	Dahongpao	Unknown (China)	C_OR (12)
	Laiyangduanzhi	Shandong (China)	C_SH (9)
	Laizhouduanzhi	Shandong (China)	C_SH (9)
	Shuheduanzhi	Shandong (China)	C_SH (9)
127	Yanshanzaofeng	Hebei (China)	C_HE (8)
	Xinzhuang 2	Beijing (China)	C_OR (12)
128	Zaofeng	Hebei (China)	C_HE (8)
129	Zundali	Hebei (China)	C_HE (8)
130	Chuixhili 2	Shandong (China)	C_SH (9)
131	Fulaiwuhuali	Shandong (China)	C_SH (9)
132	Haifeng	Shandong (China)	C_SH (9)
133	Hongguang-LPEFRI	Shandong (China)	C_SH (9)
134	Hongli 1	Shandong (China)	C_SH (9)
135	Hongli 3	Shandong (China)	C_SH (9)
	Hongli	Unknown (China)	C_OR (12)
136	Huafeng	Shandong (China)	C_SH (9)
	Taianboke-HAAFS	Unknown (China)	C_OR (12)
137	Huagai	Shandong (China)	C_SH (9)
138	Huaguang	Shandong (China)	C_SH (9)
139	Jinfeng	Shandong (China)	C_SH (9)
	Hongguang-HAAFS	Shandong (China)	C_SH (9)
	Lianxujieguo	Unknown (China)	C_OR (12)
140	Junandagongshu	Shandong (China)	C_SH (9)
141	Mengshankuili	Shandong (China)	C_SH (9)
142	Shandongchuizhi 3	Shandong (China)	C_SH (9)
143	Shandongchushuhong	Shandong (China)	C_SH (9)
	Shimenzaoshuo	Shandong (China)	C_SH (9)
144	Shifeng	Shandong (China)	C_SH (9)
	Huaifeng	Unknown (China)	C_OR (12)
145	Songjiazao-HAAFS	Shandong (China)	C_SH (9)
	Wuhua	Shandong (China)	C_SH (9)
146	Songjiazao-SIP	Shandong (China)	C_SH (9)
	Guangxiyouli	Unknown (China)	C_OR (12)
147	Taianaisheng	Shandong (China)	C_SH (9)
148	Taianboke-SIP	Shandong (China)	C_SH (9)
149	Tancheng 207	Shandong (China)	C_SH (9)
150	Tanchengyouguangli	Shandong (China)	C_SH (9)
151	Weifeng	Shandong (China)	C_SH (9)
152	Yanming	Shandong (China)	C_SH (9)
153	Yimengduanzhi	Shandong (China)	C_SH (9)
	Duanzhiboke	Unknown (China)	C_OR (12)
154	Yimengkuili	Shandong (China)	C_SH (9)
155	Chali	Jiangsu (China)	C_JI (10)
156	Chongyangpu	Jiangsu (China)	C_JI (10)
157	Dadiqing	Jiangsu (China)	C_JI (10)
	Shuhe 10	Shandong (China)	C_SH (9)
	Shuhe 11	Shandong (China)	C_SH (9)
	Shuhe 14	Shandong (China)	C_SH (9)
158	Duanmaojiaozha	Jiangsu (China)	C_JI (10)
159	Guihuali	Jiangsu (China)	C_JI (10)
160	Jiujiazhong	Jiangsu (China)	C_JI (10)
161	Paoche 7	Jiangsu (China)	C_JI (10)
162	Qingmaoruanci-HAAFS	Jiangsu (China)	C_JI (10)
163	Qingzha	Jiangsu (China)	C_JI (10)
164	Yixingdahongpao	Jiangsu (China)	C_JI (10)
165	Ershuizao	Anhui (China)	C_AN (11)
166	Hefeichushuhong	Anhui (China)	C_AN (11)
	Shuangjili	Jiangxi (China)	C_OR (12)
167	Mifengqiu	Anhui (China)	C_AN (11)
	Paoche 2	Jiangxi (China)	C_JI (10)
168	Niandiban	Anhui (China)	C_AN (11)
169	Shuanghedahongpao	Anhui (China)	C_AN (11)
	Tedazaoyou	Unknown (China)	C_OR (12)
170	Shuchengdahongpao	Anhui (China)	C_AN (11)
	Liyang 1302	Fujian (China)	C_OR (12)
	Qingmaoruanci-SIP	Jiangsu (China)	C_JI (10)
171	Shuizao 2–11	Anhui (China)	C_AN (11)
172	Yebianza	Anhui (China)	C_AN (11)
173	Yelicang	Anhui (China)	C_AN (11)
	Taili 1	Shandong (China)	C_SH (9)
174	214	Unknown (China)	C_OR (12)
175	Duanzhatou	Unknown (China)	C_OR (12)
176	Guangxili	Unknown (China)	C_OR (12)
	Zajiao 35	Unknown (China)	C_OR (12)
177	Hongyouli	Guangxi (China)	C_OR (12)
178	Houzhuang 2	Unknown (China)	C_OR (12)
179	Huaiduanhua	Unknown (China)	C_OR (12)
	Huaiwuhua	Unknown (China)	C_OR (12)
180	Huaihuang	Beijing (China)	C_OR (12)
181	Huaiyanhong	Unknown (China)	C_OR (12)
182	Huangpeng	Unknown (China)	C_OR (12)
183	Jiandingyouli	Unknown (China)	C_OR (12)
184	Jinpingduanchui	Jiangxi (China)	C_OR (12)
185	Juhong	Unknown (China)	C_OR (12)
	Zhongming	Unknown (China)	C_OR (12)
186	Kui 1–3	Unknown (China)	C_OR (12)
187	Kuili	Zhejiang (China)	C_OR (12)
	Duancidaqing	Unknown (China)	C_OR (12)
188	Laokuili	Unknown (China)	C_OR (12)
189	Linfeng	Unknown (China)	C_OR (12)
190	Luotianzaoli	Hubei (China)	C_OR (12)
191	Panzhuang 1	Unknown (China)	C_OR (12)
192	Qianci	Hubei (China)	C_OR (12)
193	Qiannanyu 3	Unknown (China)	C_OR (12)
194	Shandongwuming	Unknown (China)	C_OR (12)
195	Shangguang	Zhejiang (China)	C_OR (12)
196	Taiboke	Unknown (China)	C_OR (12)
197	Tali	Hunan (China)	C_OR (12)
198	Yanchang	Beijing (China)	C_OR (12)
	Yanhong	Beijing (China)	C_OR (12)
199	Yebanli	Unknown (China)	C_OR (12)
200	Yinxuan 3	Guangxi (China)	C_OR (12)

Genetic markers

The 230 cultivars were genotyped for 31 nuclear SSRs [26-28] (S2 Table) and 5 chloroplast SSRs (cpSSRs) (Cmcs1–3, Cmcs5, and Cmcs7) [29]. PCR amplification was performed in a 10-μL solution containing 5 μL of 2× Green GoTaq G2 Hot Start Master Mix (0.4 mM each dNTP, Taq DNA polymerase, and 4 mM MgCl2, pH 8.5; Promega, Madison, WI, USA), 20 pmol of each forward primer labeled with a fluorescent dye (5-FAM or 5-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 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). The size of each amplified band was determined by comparison with a set of internal-standard DNA fragments (400HD ROX, Life Technologies) in GeneMapper software v. 5.0 (Life Technologies).

Data analysis

Prior to analysis of population structure and parent–offspring relationships, synonym groups were identified using the 31 nuclear SSR markers to analyze the 230 cultivars. After eliminating duplicate genotypes, a set of 200 unique cultivars (see Results) was used for further analyses. The probability of identity (PI) for each locus and for the whole SSR set (Cumulative PI) was calculated using the software Gimlet v1.3.3 [30] to check the power of discrimination. Chloroplast haplotypes were determined by using the 5 cpSSRs (S1 Table). Cultivars that had the identical combination of alleles for all 5 cpSSRs were considered to carry the same haplotype. The positions of the 5 cpSSRs within the complete Castanea mollissima chloroplast genome are shown in S1 Table.

Bayesian statistical inference on the population structure was performed by using Structure 2.3.4 software [31] with the independent model for allele frequency, without any prior information about the origin of each cultivar. First, the 200 cultivars representing unique genotypes were used to clarify the genetic structure of Japanese and Chinese chestnut and their hybrids. Next, only the pure Chinese chestnut cultivars were used in a second structure analysis to examine genetic structures within that species. The analysis was run 10 times for each value of K (number of inferred ancestral populations) from 2 to 8 for 1,000,000 iterations after a burn-in period of 100,000 iterations. Evanno et al.’s [32] criterion of ΔK was used to estimate the appropriate K value. Principal coordinate analysis (PCoA) was performed in GenAlEx 6.5 from the pairwise genetic distances obtained with the covariance-standardized method [33].

For the Japanese–Chinese hybrid cultivar group, putative parent–offspring relationships were calculated with the parent calculation program MARCO [34], which identifies possible parents from among the genotypes in a set of cultivars. Genotypes were considered to have a parent–offspring relationship if they shared at least one allele per SSR locus, with the exception that a discrepancy at a single SSR locus was accepted to allow for possible genotyping errors, presence of null alleles, or mutation, as previously proposed [25,35-38]. To determine whether a parent was the seed parent or pollen parent, chloroplast haplotype data were used.

For the Chinese chestnut groups that had more than 8 cultivars each (C_SJ, C_HE, C_SH, C_AN, C_JI, and C_OR), the observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (F) were calculated using GenAlEx v. 6.5 software [33], and allelic richness (AR, n = 9) was calculated using the R package Hierfstat [39].

Evaluation of phenotypic traits

Nut harvest date and nut weight were recorded in 2001–2003 for cultivars preserved at the NARO Genebank, which include Japanese chestnut cultivars, Japanese–Chinese hybrid cultivars, and Chinese chestnut cultivars preserved in Japan (S3 Table). To evaluate nut harvest date and nut weight, each bur was harvested when it had changed from green to brown and had begun to split open or had dropped. The nuts were removed from the burs and the number of nuts harvested on a given day was recorded for each tree. Burs and nuts were harvested every three or four days from late August to October. The harvest date for each nut was expressed as the number of days after July 31 (i.e., August 1 = day 1), and the average value of nut harvest date for each tree was used as its score for this trait. Nut weight (g) per nut was measured on a digital scale on each harvest date. The average nut weight was calculated as the total nut weight divided by the total number of intact nuts.

For Chinese chestnut cultivars originated in China, nut harvest date and nut weight could not be obtained because only DNA samples were available.

Results

Identification of synonym groups

We could differentiate all but 53 of the 230 chestnut cultivars with the 31 nuclear SSR markers. These 53 cultivars were divided into 23 synonym groups, each consisting of 2 to 5 cultivars having the same genotypes at all SSR loci (Table 2). Consequently, we identified 200 unique genotypes from the 230 cultivars. The probability of identity (PI) for each locus ranged from 0.016 for PRG79 to 0.269 for PRD83 (mean = 0.099), whereas the total PI was 6.56× 10−35. Synonym groups were found only within Chinese chestnut cultivars originated in China: none of the synonym groups contained cultivars originated in Japan. Some of the cultivars showing identical genotypes originated in different regions of China; however, most of them originated in regions adjacent to one another. Examples of synonym groups include ‘Yanshanduanzhi’ from Hebei and ‘Laiyangduanzhi’, ‘Laizhouduanzhi’, and ‘Shuheduanzhi’ from Shandong (Syn-10); ‘Yanshanzaofeng’ from Hebei and ‘Xinzhuang 2’ from Beijing (Syn-6); ‘Mifengqiu’ from Anhui and ‘Paoche 2’ from Jiangsu (Syn-2); and ‘Hefeichushuhong’ from Jiangsu and ‘Shuangjili’ from Anhui (Syn-1).

Table 2

Chinese chestnut cultivar groups with identical SSR genotypes for all 31 SSR markers.

Group	Cultivars
Syn-1	Hefeichushuhong	Shuangjili
Syn-2	Mifengqiu	Paoche 2
Syn-3	Shuanghedahongpao	Tedazaoyou
Syn-4	Shuchengdahongpao	Qingmaoruanci-SIP	Liyang 1302
Syn-5	Yelicang	Taili 1
Syn-6	Yanshanzaofeng	Xinzhuang 2
Syn-7	Yanchang	Yanhong
Syn-8	Donglingmingzhu	Xigou 7
Syn-9	Yankui	Yancheng 3
Syn-10	Yanshanduanzhi	Laiyangduanzhi	Laizhouduanzhi	Shuheduanzhi	Dahongpao
Syn-11	Dadiqing	Shuhe 10	Shuhe 11	Shuhe 14
Syn-12	Hongguang-HAAFS	Jinfeng	Lianxujieguo
Syn-13	Hongli 3	Hongli
Syn-14	Huafeng	Taianboke-HAAFS
Syn-15	Shifeng	Huaifeng
Syn-16	Songjiazao-HAAFS	Wuhua
Syn-17	Songjiazao-SIP	Guangxiyouli
Syn-18	Yimengduanzhi	Duanzhiboke
Syn-19	Duancidaqing	Kuili
Syn-20	Guangxili	Zajiao 35
Syn-21	Huaiduanhua	Huaiwuhua
Syn-22	Juhong	Zhongming
Syn-23	Shandongchushuhong	Shimenzaoshuo

Chloroplast haplotype frequency

In total, 6 chloroplast haplotypes were identified among the 200 unique cultivars by using 5 cpSSRs (Table 3). The three Japanese chestnut cultivar groups (J_KA, J_TA, and J_OJ) carried only HAP1, whereas the Chinese chestnut cultivar groups mainly carried HAP3 and HAP4. HAP2 was only found in the Korean cultivar ‘Pochun B-1’ (ID#66). HAP5 and HAP6 were only found in Chinese chestnut cultivars ‘Kousei 2’ (ID#103) and ‘Duanzhatou’ (ID#175), respectively. Among the Chinese chestnut cultivars, HAP3 was the only haplotype found in C_JI and C_AN (East central regions), whereas HAP4 dominated in C_HE (Northern region). The cultivars originated in Shandong (C_SH), which is located between Hebei and Jiangsu, carried both HAP3 and HAP4. Chinese chestnut cultivars from other regions of China (C_OR) and those from Japan (C_SJ) and other world areas (C_IO) also carried both HAP3 and HAP4.

Table 3

Haplotype frequencies in the set of 200 cultivars.

	HAP1	HAP2	HAP3	HAP4	HAP5	HAP6
J_KA	14
J_TA	21
J_OJ	25
KOR	4	1		1
HYB	6		10	2
C_SJ			19	7	1
C_IO			4	1
C_HE			1	12
C_SH			18	7
C_JI			10
C_AN			9
C_OR			17	9		1

Genetic relationship between Japanese and Chinese chestnut cultivars and their hybrids

To clarify the genetic relationships between Japanese and Chinese chestnut cultivars and their hybrids, we performed Bayesian clustering analyses (Fig 1). The values of Δ(K) were much higher at K = 2 than at K = 3 to K = 8, so we constructed bar plot diagrams at K = 2. The “red” and “light green” shading indicate the Japanese and Chinese chestnut clusters, respectively. Most of the Japanese and Chinese chestnut cultivars had membership in a single cluster corresponding to their respective species. On the other hand, most Japanese–Chinese hybrid cultivars (HYB) had admixed structure, i.e., had approximately equal membership in both the “red” and “light green” clusters. Among the HYB cultivars, only ‘Ikaba’ (ID#67) had membership predominantly in the “light green” (Chinese) cluster. In both the Japanese and Chinese chestnut cultivar groups, a few cultivars had admixed structures (ratio of red:light green = 0.78:0.22 for ‘Obiwase’, 0.24:0.76 for ‘Hamjung 3’, and 0.16:0.84; for ‘Miyagawa 18’). Some Chinese chestnut cultivars had very low membership in the “red” (Japanese) cluster, but the amount was too small to declare that they were hybrids (for example, red:light green = 0.04:0.96 for ‘Tokuganri A’ and 0.03:0.97 for ‘2399’).

Fig 1

Detailed bar plot diagram for K = 2 in the independent model using 66 Japanese chestnut cultivars, 18 Japanese–Chinese hybrids, and 116 Chinese chestnut cultivars.

The first number under each bar represents the individual accession ID number (1–200); the second number (in parentheses) represents the group number (1–12). ID numbers and groups are defined in Table 1.

Putative parentage of Japanese–Chinese hybrid cultivars

Because some Japanese–Chinese hybrid cultivars had records indicating that they had been selected from crosses between Japanese and Chinese chestnut cultivars in Japan, we used MARCO software to perform parent–offspring relationship analysis using cultivars originated in Japan (Table 4). Out of the 18 Japanese–Chinese cultivars in this study, we could infer both parents for 6 cultivars (‘Ikaba’, ‘Hayashi 1’, ‘Hayashi 3’, ‘Hayashi amaguri’, ‘Hyogo 493’, and ‘Yamewase’; ID#67–71, 84) and one parent for 10 cultivars (‘Kurakata amaguri’, ‘Nishiharima’, ‘Omatsuguri’, ‘Senshu amaguri’, ‘Shimaki 1’, ‘Shimaki 2’, ‘Shimaki 3’, ‘Shimaki 4’, ‘Shimaki 5’, and ‘Shimaki 6’; ID#72–74, 76–82). Out of the six cultivars for which we were able to infer both parents, five cultivars were F1 hybrids between Japanese and Chinese chestnut and one cultivar, ‘Ikaba’ (ID#67), was presumed to be an offspring between Japanese–Chinese hybrid cultivar ‘Riheiguri’ (ID#75) and Chinese chestnut cultivar ‘Gifu 1’ (ID#87).

Table 4

Putative parent–offspring relationships for Japanese–Chinese hybrid cultivars.

Offspring	Seed parent	Pollen parent
Ikaba	Riheiguri (H)	Gifu 1 (C)
Hayashi 1	Houji 480 (C)	Kasaharawase (J)
Hayashi 3	Houji 360 (C)	Kasaharawase (J)
Hayashi amaguri	Houji 360 (C)	Kanotsume (J)
Hyogo 493	Kinseki (J)	Hyogo shinaguri (C)
Kurakata amaguri	Houji 480 (C)	Unknown
Nishiharima	Hyogo shinaguri (C)	Unknown
Omatsuguri	Unknown	Nakatetanba (J)
Senshu amaguri	Unknown	Tsuchidawase (J)
Shimaki 1	Ganne (J)	Unknown
Shimaki 2	Ganne (J)	Unknown
Shimaki 3	Choubei (J)	Unknown
Shimaki 4	Houji 446 (C)	Unknown
Shimaki 5	Ganne (J)	Unknown
Shimaki 6	Ganne (J)	Unknown
Yamewase	Houji 480 (C)	Nakatetanba (J)
Riheiguri	Unknown	Unknown
Wasetenshin	Unknown	Unknown

"J", "C", and "H" in parentheses represent Japanese chestnut, Chinese chestnut and a hybrid, respectively.

Phenotypic trait evaluation of Japanese chestnut cultivars

Nut harvest date ranged from August 19 [‘Hassaku’ (ID#4)] to October 15 [‘Shimokatsugi’ (ID#32)] (average, September 18) for Japanese chestnut cultivars and from August 31 [‘Hayashi 3’ (ID#69)] to October 1 [‘Shimaki 1’ (ID#77)] (average, September 17) for Japanese–Chinese chestnut cultivars (S3 Table). The average nut harvest date of the Chinese chestnut cultivars preserved in Japan (September 29) was later than those of the Japanese and Japanese–Chinese chestnut hybrid cultivars and ranged from September 10 [‘Kahoku 10 (ID#98) and’‘Miyagawa 84’ (ID#106)] to October 17 [‘Konan 22’ (ID#100) and ‘Kousei 2’ (ID#103)]).

The average nut weight was large (24.7 g) in Japanese chestnut cultivars, intermediate (20.0 g) in Japanese–Chinese chestnut hybrids, and small (10.9 g) in Chinese chestnut cultivars preserved in Japan. The range was 11.9–40.9 g for Japanese chestnut cultivars, 11.9–29.5 g for Japanese–Chinese chestnut hybrids, and 5.2–17.6 g for Chinese chestnut cultivars in Japan. The average nut weight of Chinese chestnut cultivars that carried HAP3 (12.6 g) was larger than that of those carrying HAP4 (7.5g).

Genetic relationships among Chinese chestnut cultivars in Japan and China

To determine the genetic relationships among Chinese chestnut cultivars from different areas, Bayesian clustering analysis was performed using only the 116 Chinese chestnut cultivars (Fig 2). The values of Δ(K) were highest at K = 2: values of Δ(K) at K = 3 to K = 8 were less than one-thousandth that at K = 2. The two Bayesian clusters strongly corresponded to the results of chloroplast haplotype analysis (Fig 3). Membership in the “red” cluster was dominant in cultivars from northern China (C_HE), all but one of which were HAP4, whereas membership in the “light green” cluster was dominant in cultivars from east central China (C_JI and C_AN), all of which were HAP3. The C_SJ, C_SH, and C_OR cultivars had membership in both the “red” and “light green” clusters. Although most of the C_SH cultivars were admixed, many of the C_SJ cultivars had membership in a single cluster (ID#92–98, 105–109, 111). In C_SJ, cultivars that carried HAP3 (S1 Table) belonged to the “light green” or admixed clusters (Fig 2; ID#85–95, 97, 99–102, 108–110), while most of the cultivars that carried HAP4 belonged to the “red” cluster (ID#98, 105–107, 111). To validate the results of the Bayesian clustering analysis, PCoA was conducted after classifying each cultivar as predominantly part of the “red” cluster (ratio of “red” > 0.8), the “light green” cluster (ratio of “light green” > 0.8), or admixed (0.8 > ratio of “red” > 0.2). The results of PCoA were similar to those of the Structure analysis, and the first informative PCo component corresponded to the separation between the “red” and “green” clusters (S1 Fig).

Fig 2

Detailed bar plot diagram for K = 2 in the independent model using 117 Chinese chestnut cultivars.

The first number under each bar represents the individual accession ID number (1–200); the second number (in parentheses) represents the group number (6–12). ID numbers and groups are defined in Table 1. Red and green are used to indicate each of the K = 2 populations in this analysis and do not correspond to the same colors in Fig 1.

Fig 3

Geographic locations of Chinese chestnut cultivar groups and genetic structures.

Donut and pie charts indicate composition of chloroplast haplotypes and clusters identified in STRUCTURE, respectively. The green, orange, and blue in the donut charts indicate HAP3, HAP4, and HAP5, respectively. The colors for the pie charts are based on the results shown in Fig 2.

Genetic parameters were calculated to clarify the genetic diversity of Chinese chestnut cultivar groups that had more than 8 cultivars (Table 5). HO was the lowest for C_HE (0.457), whereas HO for C_AN (0.642) was slightly higher than for the other groups. HE and AR for C_HE were both lower than for the other groups. On the other hand, AR in C_SJ, C_SH, and C_OR, all of which had both HAP3 and HAP4 cultivars (Table 3), was higher than in the other groups. The inbreeding coefficient (F) was highest for C_SJ (0.085), whereas those for C_JI and C_AN were negative (−0.118 and −0.138, respectively).

Table 5

Genetic characteristics of Chinese chestnut cultivar groups analyzed using 31 SSRs.

Group	Number of cultivars	HO	HE	AR	F
C_SJ	27	0.544	0.599	4.35	0.085
C_HE	13	0.457	0.496	3.60	0.050
C_SH	25	0.570	0.603	4.40	0.066
C_JI	10	0.619	0.557	3.94	−0.118
C_AN	9	0.642	0.567	4.00	−0.137
C_OR	27	0.562	0.594	4.26	0.054

HO = Observed heterozygosity

HE = Expected heterozygosity

AR = Allelic richness

F = inbreeding coefficient

Discussion

Our set of 31 nuclear SSR loci proved to have an high discriminative power (total probability of identity: 6.56× 10−35) for the 200 unique cultivars. This value of the total probability of identity is quite low compared to those in other studies related to identification of synonyms (3.73× 10−12–2.99× 10−8) [40-42]. It is highly unlikely to detect false synonyms with the 31 nuclear SSR markers. We identified 23 synonym groups among the Chinese chestnut cultivars originated in China. A previous study using Japanese and Chinese chestnut cultivars in Japan had already identified two synonym groups for Chinese chestnut cultivars selected in Japan and one for Japanese–Chinese hybrid cultivars [24]. Synonyms have been commonly identified in chestnut cultivar collections [21-23]. One reason that chestnut cultivars may have many synonyms is that nut appearance is quite similar among cultivars. For most major fruit crops, fruit color would be a good characteristic to distinguish cultivars, but color differences are not helpful for distinguishing chestnut cultivars in most cases. Interestingly, synonyms were found only among cultivars originated in China, not between cultivars from Japan and China. Most Chinese chestnut genotypes introduced into Japan were assumed to be from seeds from China or Korea [13]. Because seeds can be produced via outcrossing, their progeny would be genetically diverse. Clonal propagation would have been difficult compared with seed propagation because of graft incompatibility between Japanese and Chinese chestnut. In addition, some of the Chinese chestnut cultivars might not have been suited to Japanese climates, reducing the chance of introducing the Chinese chestnut cultivars by clonal propagation. The cultivars in C_SJ would have been selected either directly from seed introduced from China and Korea or from successive generations of hybridization among the introduced genotypes.

Genetic relationships among Chinese chestnut, Japanese chestnut, and Japanese–Chinese hybrid cultivars were clarified by Bayesian clustering analysis. Most of the Chinese and Japanese chestnut cultivars had simple genetic structure (Fig 1), indicating that those cultivars were pure Chinese chestnut or Japanese chestnut, respectively. The cultivar collections from Japan and China have been present in those nations for a long time, limiting the chances for interspecific hybridization. According to Isaki [13], introduction of nuts of Chinese chestnut into Japan began in the 20th century. On the other hand, Korean native chestnut had been considered as an intermediate between Japanese and Chinese chestnut on the basis of its morphological characteristics [43]. Consistent with that previous report, our study showed that the Korean cultivar ‘Hamjung 3’ (ID#62) was an admixture between Chinese and Japanese chestnut (Fig 1).

In addition to ‘Hamjung 3’, definitive introgressions of Chinese chestnut into Japanese chestnut and vice versa were identified in Japanese chestnut ‘Obiwase’ (ID#47) and Chinese chestnut ‘Miyagawa 18’ (ID#105). In a previous study, however, ‘Obiwase’ was presumed to have a genetic structure derived from a wild chestnut population distributed on Kyusyu Island; its structure was different from that of other native cultivars but it was still considered to be a pure Japanese chestnut cultivar [25]. Since this study did not include wild populations, this cultivar may have been miscategorized. Likewise, ‘Miyagawa 18’ carried both Chinese and Japanese chestnut genetic structure. The percentage of Japanese chestnut genetic structure was about 16%; thus, this cultivar would be a first backcross (BC1) or a second backcross (BC2), not an F1 hybrid. For Japanese–Chinese chestnut hybrid cultivars, both parentage and chloroplast haplotype analyses were conducted to determine the putative seed and parent cultivars. Out of 18 Japanese–Chinese cultivars, we were able to presume both parents for six cultivars and one parent for 10 cultivars (Table 4). Because Chinese chestnut cultivars were relatively uncommon in Japan and the hybridizations had been done relatively recently (within 100 years), many parent–offspring relationships were identified. The results of the analyses were compared with those of Isaki [13], who reported the parentages of some of the same cultivars. ‘Shimaki 1’, ‘Shimaki 2’, ‘Shimaki 3’, ‘Shimaki 4’, and ‘Shimaki 5’ (ID#77–81) were reported by Isaki [13] to be selected from seedlings derived from ‘Ganne’ and Chinese chestnut accessions. Here, the parentage of ‘Shimaki 1’, ‘Shimaki 2’, and ‘Shimaki 5’ was reconfirmed. Also, the parentage of ‘Ikaba’, listed in a plant variety protection database in Japan (http://www.hinshu2.maff.go.jp/en/en_top.html), matched our results. On the other hand, ‘Hayashi 1’, ‘Hayashi 3’, and ‘Hayashi amaguri’ were reported by Isaki [13] to be offspring of ‘Kasashi 1’, an F1 hybrid between ‘Kasaharawase’ (ID#43) and a Chinese chestnut accession; however, in the present study they were presumed to be offspring of ‘Kasaharawase’ or ‘Kanotsume’ (ID#24).

The Japanese–Chinese chestnut hybrid cultivars showed nut size intermediate (20.0 g) between those of Japanese (24.7 g) and Chinese chestnut cultivars (10.9 g). The average nut harvest date of the Japanese–Chinese chestnut hybrid cultivars was the similar to that of Japanese chestnut and earlier than that of Chinese chestnut (S3 Table). Thus, for Chinese chestnut breeding, Japanese chestnut cultivars have the potential to increase nut weight and shorten the time to harvest. On the other hand, introducing Chinese chestnut cultivars into Japanese chestnut breeding programs would be not an effective way to shorten the time to harvest or increase nut weight. Today, some Japanese–Chinese cultivars are highly valued by Japanese farmers and consumers because of the high nut quality and moderate nut pellicle peelability. QTL analyses of interspecific backcross populations are necessary to identify interesting species-specific genes and accelerate breeding programs.

The origin of hybrid cultivar ‘Riheiguri’ (ID#75), one of the major cultivars in Japan, is an interesting example of interspecies hybridization giving rise to a desirable new cultivar. ‘Riheiguri’ was assumed to be an F1 hybrid between Japanese and Chinese chestnut since it had been developed in a Tsuchida orchard that contained both species [13]. Although we were unable to identify its parents by parentage analysis, Bayesian structure analysis revealed it had approximately equal membership in both the “red” and “light green” clusters, supporting the assumption that it was an F1 hybrid. In addition, ‘Riheiguri’ had HAP3, which originated in Jiangsu and Anhui. This cultivar might have been selected from seeds of other cultivars originated in these regions. It has relatively large nut size like Japanese chestnut (23.9 g; S3 Table), mealy texture like Chinese chestnut [13], and moderate pellicle peelability [44], which was likely inherited from Chinese chestnut. Because of its good nut quality, ‘Riheiguri’ and its relatives have been used in breeding programs, resulting in the release of new hybrid cultivars such as ‘Shuhou’ and ‘Mikuri’. On the other hand, trials to release Japanese chestnut cultivars with the easy-peeling pellicle trait from Chinese chestnut have not yet succeeded [4].

We identified four chloroplast haplotypes (HAP3–HAP6) among the Chinese chestnut cultivars. HAP4 was mainly found in cultivars from Hebei, Shandong, and Japan, whereas HAP3 dominated in most groups except for C_HE. HAP5 and HAP6 were each identified in only one cultivar. At least 38 chloroplast haplotypes were identified from wild populations by Chen and Huang [19] and Liu et al. [17] using cpSSRs. However, only two haplotypes [19] and four haplotypes (this study) were identified from Chinese chestnut cultivar collections, suggesting that cultivars have limited genetic diversity compared to wild populations. Because the numbers and types of markers were quite similar in those studies and ours, it is reasonable to compare the results. On the other hand, it is possible that more chloroplast haplotypes would be found if we detected a larger number of polymorphisms by sequencing the whole chloroplast genome of several cultivars. The finding that cultivars showed less genetic diversity than wild populations corresponds to the suggestions of Mattioni et al. [45] and Ovesná et al. [20], i.e., that because traits and genes useful for chestnut cultivation were artificially selected, the domestication process would typically reduce genetic diversity.

Both chloroplast haplotype and Bayesian clustering analyses showed that the Chinese chestnut cultivars used in the present study could be divided into two groups (Fig 3): one originated in the Hebei region and the other originated in Jiangsu and Anhui. Most of the cultivars from Shandong had admixed genetic structure (Fig 2), whereas cultivars selected in other regions of China had various patterns of genetic structure. The cultivar groups that had admixed structure showed higher H and AR, which is not unexpected because hybridization between cultivars from different clusters would increase genetic diversity. Since we had no information about cultivars selected in other regions of China, it was quite difficult for us to clarify the breeding history of these cultivars. Some cultivars from Shandong might have been selected from crosses between cultivars derived from Hebei and from Jiangsu or Anhui. Alternatively, cultivars might have been selected from wild chestnuts with an admixed genetic structure growing in Shandong. Although HE and AR were low in C_HE, C_JI, and C_AN, the values of F were positive in C_HE but negative in C_JI and C_AN, indicating that artificial selection pressure was higher in the Hebei region. According to Kikuchi [46], nuts of cultivars from Hebei are small and sweet, whereas cultivars from central China have comparatively large nut size and low flavor, and cultivars from Shandong have intermediate characteristics. Consistent with this previous report, the average nut weight of Chinese chestnut cultivars that carried HAP3, the only haplotype found in the cultivars from Jiangsu and Anhui, was larger than those carried HAP4, the most common haplotype found in the cultivars from Hebei (Tables 3 and S3).Thus, breeders could use cultivars showing differences in genetic structure according to the objectives of their breeding programs.

The Chinese chestnut cultivars selected in Japan showed various patterns of genetic structure. More than half of those cultivars showed a “light green”-dominated structure presumed to have originated in Jiangsu or Anhui, while some cultivars showed admixed structure or “red”-dominated structure presumed to have originated in Hebei. The cultivars selected by Houji in Kochi prefecture (‘Houji 354’, ‘Houji 445’, ‘Houji 350’, ‘Houji 446’, and ‘Houji 480’; ID#91–95) had high membership in the “light green” cluster, and the cultivars selected by Miyagawa in Yamanashi prefecture (‘Miyagawa 100’, ‘Miyagawa 18’, ‘Miyagawa 84’, and ‘Miyagawa 85’; ID#104–107) had high membership in the “red” cluster. According to PCoA analysis, the cultivars selected by Houji were closest to those originated in Jiangsu or Anhui, while the cultivars selected by Miyagawa were closest to those originated in Hebei (S1 Fig), suggesting that Houji cultivars were selected from seeds originated in Jiangsu or Anhui and that Miyagawa cultivars were selected from seeds originated in Hebei. Geographical data support this hypothesis because Hebei is located in the northern part of China, and Hakushu in Yamanashi, where Miyagawa cultivars were selected, is located in a cold, high-altitude part of Japan. According to one account, Miyagawa cultivars were introduced from Songchong in North Korea, which is relatively close to Hebei [13]. The nut sizes of Miyagawa cultivars (which had genetic structure originated in Hebei) were 5.2–9.2 g, whereas those of cultivars that had genetic structure originated in Jiangsu or Anhui averaged around 12 g (S3 Table), corresponding to previous reports that cultivars in northern China have smaller nut size than those in central China [13]. In this study, we analyzed only Chinese chestnuts from Japan and from the seaside of China, not from western or southern China. Fortunately, chloroplast haplotype analysis clarified that the Chinese chestnut materials from Japan and China were similar (Fig 3), suggesting that the Chinese chestnut cultivars collected in this study were sufficient basic materials to classify Chinese chestnut cultivars in Japan. However, we are sure that there are many other genetic structures in Chinese chestnuts in other regions. Further analyses, including cultivars and wild populations from all over China, would clarify the detailed genetic relationships and domestication process of Chinese chestnut.

Conclusions

We used SSRs to genotype chestnut cultivars preserved in both Japan and China and to determine the genetic structure of Chinese and Japanese chestnut cultivars. The synonym groups and putative parentages of some Chinese chestnut cultivars were identified here for the first time. Most of the Chinese and Japanese chestnut cultivars had a simple genetic structure corresponding to their respective species, whereas Japanese–Chinese hybrid cultivars had admixed structures. The Chinese chestnut cultivars could be divided into two groups: one that originated in Hebei and one that originated in Jiangsu and Anhui. The Chinese chestnut cultivars selected in Japan also carried the genetic structures originated in these two divergent regions, suggesting that their ancestral genotypes originated in those two groups. The information obtained in this study will be useful for population genetic studies for those species and for Japanese and Chinese chestnut breeding programs.

(PDF)

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List of the 31 SSR nuclear markers used in the present study.

The linkage group (LG) and position (pos) of each marker are based on an integrated map of a Kunimi × 709–34 population (Kx709CP; Nishio et al., 2018).

(XLSX)

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(XLSX)

Click here for additional data file.

Principal coordinate analysis (PCoA) plot generated from genetic distance calculations among the 116 Chinese chestnut cultivars in GenAlEx software.

Cultivars were classified as predominantly part of the “red” cluster (ratio of “red” > 0.8), the “light green” cluster (ratio of “light green” > 0.8), or admixed (0.8 > ratio of “red” > 0.2; shown here in black).

(TIF)

Click here for additional data file.

22 Jan 2020

PONE-D-19-32891

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China

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Reviewer #1: Overall it is well written manuscript. Clustering patterns and genetic structure were not surprising given that the study population comprised accessions from two different species. I have following suggestions to further investigate the genetic relationships between accessions from different species and/or regions.

1. Figure 1: The group C_SJ is clustered (100% green cluster) with the Chinese accessions. It’s not clear if all the Chinese chestnuts selected in Japan were originated from the first introduction of seeds. Could some accession haven been selected from successive generation of natural hybridisation with Japanese genepool?

2. Lin 357-366: The argument presented to support the origin of C_SJ group of accessions is not strong. I suggest authors to present neighbour-joining (NJ) and principal component analysis (PCA) of nSSR genotypes to further support their hypothesis.

3. Line 150: Authors should describe the steps for constructing chloroplast haplotypes. Some comments on the extent of linkage disequilibrium would be helpful in order to check the integrity of haplotypes.

4. Could a relatively lower chloroplast diversity (hence a simpler genetic structure) be a result of only 5 cpSSR used in this study?

5. It would be useful if authors could provide information on the distribution of 31 SSRs across different chromosomes. Perhaps using 31 SSRs would only detect genetic diversity of limited regions of the whole genome, and could lead to biased inferences about population genetics. Perhaps authors could discuss this point.

Reviewer #2: Authors tried to clarify the origin and genetic characteristics of Chinese chestnut cultivars in Japan by using 31 nuclear SSR markers and 5 cpDNA SSR markers in this research. They said results obtained by this research would be useful for both Japanese and Chinese chestnut breeding program.

However objectives of this research remain unclear, please consider following points,

1. What is the purpose (or significance) to reveal the origin of Chinese chestnut cultivars introduced into Japan? If authors would like to understand the origin of Japanese chestnut cultivars in relation to Chinese chestnut, materials used in this study were not enough. More Chinese chestnut cultivars in China should be involved.

2. Nuclear SSR markers were applied to reveal the genetic structure of Chinese and Japanese cultivars. But the resolution of SSR marker seems too high to apply for different species i.e., Castanea mollissima and C. crenata. Therefore only three clusters such as Chinese cultivar cluster, Japanese cultivar cluster and hybrid cluster were obtained. Is it possible to use other molecular markers such as the sequence of single nuclear gene or gene encoding chloroplast genome?

3. Authors say this research is useful for chestnut breeding program. But it is too general. Is there any information concerning agricultural traits for used cultivars? For example, is there any excellent or desirable trait inherited from putative parent to offspring?

4. This manuscript seems suitable to submit to more specific journal focused on the plant breeding or woody plants genetics.

**********

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Reviewer #1: No

Reviewer #2: No

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25 Feb 2020

Response to the Associate Editor

We thank the Editor and both Reviewers very much for taking time to review our manuscript and for your comments. We have revised our manuscript accordingly.

Responses to the comments from Reviewer 1

Comment

1. Figure 1: The group C_SJ is clustered (100% green cluster) with the Chinese accessions. It’s not clear if all the Chinese chestnuts selected in Japan were originated from the first introduction of seeds. Could some accession haven been selected from successive generation of natural hybridisation with Japanese genepool?

Response

There were some cultivars in C_SJ that had originated from the first introduction of seeds into Japan: for example, ‘Houji’ and ‘Miyagawa’ had records of breeding history indicating that they were directly selected from seeds introduced from China into Japan. But others would have been selected from successive generations of the introduced genotypes, as this reviewer suggested. To clarify this point, we revised several sentences in the Discussion (Line 314, 327-329)

.

Comment

2. Lin 357-366: T The argument presented to support the origin of C_SJ group of accessions is not strong. I suggest authors to present neighbour-joining (NJ) and principal component analysis (PCA) of nSSR genotypes to further support their hypothesis.

Response

We performed principal coordinate analysis (PCoA; S1 Fig) and describe the results in the indicated paragraph (Line 419-422).

Comment

3. Line 150: Authors should describe the steps for constructing chloroplast haplotypes. Some comments on the extent of linkage disequilibrium would be helpful in order to check the integrity.

Response

According to this suggestion, we now describe in the Materials and Methods how we classified the haplotypes (Line 159-162). We also added the positions of the 5 cpSSRs in the complete Castanea mollissima chloroplast genome (KY951992) to S1 Table.

Comment

4. Could a relatively lower chloroplast diversity (hence a simpler genetic structure) be a result of only 5 cpSSR used in this study?

Response

Only two haplotypes (Chen et al., 2009) and four haplotypes (this study) were identified from Chinese chestnut cultivar collections because small numbers of cpSSRs (4 and 5, respectively) developed by Sebastiani et al. (2004) were used in those studies. Liu et al. (2013) also used a small number of cpSSRs (7) to clarify the genetic relationship between wild populations, in which they identified 38 chloroplast haplotypes. It is reasonable to compare the results of our study and of those previous studies because the numbers and types of markers were quite similar. On the other hand, it is possible that more chloroplast haplotypes would be found if we sequenced the whole chloroplast genomes of several cultivars and identified SNPs. We revised the text to clarify this point (Line 382-389).

Comment

5. It would be useful if authors could provide information on the distribution of 31 SSRs across different chromosomes. Perhaps using 31 SSRs would only detect genetic diversity of limited regions of the whole genome, and could lead to biased inferences about population genetics. Perhaps authors could discuss this point.

Response

The information on distribution of the 31 SSRs is provided in S2 Table and shows that the markers were mapped on different chromosome regions. The number of SSRs in this study was larger than that in most population genetic studies. Since we applied reliable SSRs sampled from among more than 500 developed in several previous studies, we have confidence in our results. Using a large number of SNPs from next-generation sequencing (NGS) might increase the amount of information; on the other hand, genotyping using NGS is not always precise because genomes contain much duplication.

Responses to Reviewer 2

Comment

1. What is the purpose (or significance) to reveal the origin of Chinese chestnut cultivars introduced into Japan? If authors would like to understand the origin of Japanese chestnut cultivars in relation to Chinese chestnut, materials used in this study were not enough. More Chinese chestnut cultivars in China should be involved.

Response

We revised several sentences in the Introduction to clarify the objective of this study (Line 52-60). Interspecific hybridization has been done by chestnut breeders all over the world to introduce disease resistance genes, an easy-peeling gene, and QTLs associated with nut quality into their breeding materials. Therefore, classification of cultivars in Japanese, Chinese, and their hybrids is quite important for breeders.

At the present time, materials from China are difficult to introduce into other countries because the Nagoya protocol (2017) prohibits exchange of cultivars. We examined 84 Chinese chestnut cultivars from 4 different provinces in China, that are moderate numbers and quite valuable for the first report of Chinese chestnut cultivar classification by SSRs. In addition, Chinese chestnut cultivars already in Japan have been preserved at the NARO (National Agriculture and Food Organization) Genebank (www.gene.affrc.go.jp) and can be readily used for research and breeding purposes all over the world. Thus, it is useful to clarify the genetic structure of these cultivars.

Comment

2. Nuclear SSR markers were applied to reveal the genetic structure of Chinese and Japanese cultivars. But the resolution of SSR marker seems too high to apply for different species i.e., Castanea mollissima and C. crenata. Therefore only three clusters such as Chinese cultivar cluster, Japanese cultivar cluster and hybrid cluster were obtained. Is it possible to use other molecular markers such as the sequence of single nuclear gene or gene encoding chloroplast genome?

Response

As the reviewer suggests, applying nuclear SSRs is not the best way to determine the genetic distance between different species. In this study, we applied SSRs only to identify synonym groups (first step) and to classify Japanese, Chinese, and Japanese–Chinese hybrid cultivars and determine the parentage of the hybrids (second step) not to determine genetic distances between cultivars of different species. The high level of SSR polymorphism was no problem for these analyses. We then performed additional classification within the group of Chinese chestnut cultivars (third step), which revealed two divergent genetic structures, i.e., Hebei origin and Jiangsu or Anhui origin. The values of Δ(K) were much higher at K = 2 than at K = 3 to K = 8 in Bayesian structure analysis, suggesting that there would be no more than 2 clusters in the Chinese cultivar collection.

Comment

3. Authors say this research is useful for chestnut breeding program. But it is too general. Is there any information concerning agricultural traits for used cultivars? For example, is there any excellent or desirable trait inherited from putative parent to offspring?

Response

To provide an example, we added the story of the hybrid cultivar Riheiguri, which is one of the major cultivars in Japan and has good nut quality and moderate peelability derived from Chinese chestnut (Line 362-375).

Comment

This manuscript seems suitable to submit to more specific journal focused on the plant breeding or woody plants genetics.

Response

Thank you for your comment. We considered our manuscript appropriate for submission because we have seen a number of similar population genetic studies reported in PLoS One, and we have attempted to follow the journal’s policies and guidelines.

Submitted filename: 200225Response to Reviewers.docx

Click here for additional data file.

25 Mar 2020

PONE-D-19-32891R1

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China

PLOS ONE

Dear Dr. Nishio,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I'd like to ask you to consider to add data of wild accessions and/or agronomic traits as suggested by the Reviewer 2.

We would appreciate receiving your revised manuscript by May 09 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Hidenori Sassa

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have addressed all my suggestions. However, I could not find Supporting Information S1 Fig. Can you please upload this information?

Reviewer #3: This manusript showed how cultivars are and how the relation among cultivars. It is important data for breeders. However, as for this level of journal (PLOSone), I feel wild accessions should be included or trait data. Particularly, author estimated the offspring and how many crosses had been done. Then, I am very interested in how their agronomical traits. And also in synonymous strains whether they share same kinds of traits.

Kinship is another issue. Even small number of SSRs, phylogenetic relationship is important data to consider breeding program. In addition, further research like GWAS, the preliminary data are required to conduct further research. In this meaning, authors can add these data and supposed to do. By the other hand, if not, authors are recommended to try other journals.

Minor comments

L220

Obiwase (#ID is required)

Some parts of cultivars are referred ID #, however, some are not. It is hard to follow the cultivar in Figure of STRUCTURE analysis to know how they are in data.

L240 "The sixth" is hard to get the meaning. One the sixth cultivars you mentioned?

Then, Just "Ikaba" is enough. If there are other Ikaba cultivars, please show ID#.

**********

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Reviewer #1: No

Reviewer #3: Yes: Ryuji Ishikawa

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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26 Apr 2020

Response to the comment from Reviewer 1

Comment

However, I could not find Supporting Information S1 Fig.

Response

We added S1 Fig to the revised manuscript.

Responses to the comments from Reviewer 3

Comment

However, as for this level of journal (PLOSone), I feel wild accessions should be included or trait data. Particularly, author estimated the offspring and how many crosses had been done. Then, I am very interested in how their agronomical traits.

Response

We do not agree with Reviewer 3 on this point. We found a number of studies including only cultivars and accessions (i.e., not including wild accessions or phenotypic data) published in PLoS ONE within the past three years. Here are some examples:

Arnau, G., Bhattacharjee, R., Sheela, M. N., Chair, H., Malapa, R., Vincent Lebot, A. K., ... & Pavis, C. (2017). Understanding the genetic diversity and population structure of yam (Dioscorea alata L.) using microsatellite markers. PLoS ONE, 12(3), e0174150.

Manechini, J. R. V., da Costa, J. B., Pereira, B. T., Carlini-Garcia, L. A., Xavier, M. A., de Andrade Landell, M. G., & Pinto, L. R. (2018). Unraveling the genetic structure of Brazilian commercial sugarcane cultivars through microsatellite markers. PLoS ONE, 13(4), e0195623.

Urrestarazu, J., Errea, P., Miranda, C., Santesteban, L. G., & Pina, A. (2018). Genetic diversity of Spanish Prunus domestica L. germplasm reveals a complex genetic structure underlying. PLoS ONE, 13(4), e0195591.

Bernard, A., Barreneche, T., Lheureux, F., & Dirlewanger, E. (2018). Analysis of genetic diversity and structure in a worldwide walnut (Juglans regia L.) germplasm using SSR markers. PLoS ONE, 13(11), e0208021.

Zhu, S., Zhang, X., Liu, Q., Luo, T., Tang, Z., & Zhou, Y. (2018). The genetic diversity and relationships of cauliflower (Brassica oleracea var. botrytis) inbred lines assessed by using SSR markers. PLoS ONE, 13(12), e0208551.

Atnaf, M., Yao, N., Martina, K., Dagne, K., Wegary, D., & Tesfaye, K. (2017). Molecular genetic diversity and population structure of Ethiopian white lupin landraces: Implications for breeding and conservation. PLoS ONE, 12(11), e0188696.

Nevertheless, we added phenotypic data for nut harvest date and nut weight of cultivars preserved at the NARO Genebank in Japan. Such data were not available for the Chinese chestnut cultivars from China, for which we only had DNA samples.

Comment

And also in synonymous strains whether they share same kinds of traits.

Response

All of the synonym groups we identified were Chinese chestnut cultivars from China. As described above, only DNA samples were available for these cultivars, so we could not perform phenotypic analysis. However, in a previous study (Nishio et al. 2011), cultivars that had identical genotypes showed similar phenotypic traits.

Nishio S, Yamamoto T, Terakami S, Sawamura Y, Takada N, Saito T. Genetic diversity of Japanese chestnut cultivars assessed by SSR markers. Breeding Sci. 2011;61(2):109-20. doi: Doi 10.1270/Jsbbs.61.109.

Comment

Kinship is another issue. Even small number of SSRs, phylogenetic relationship is important data to consider breeding program. In addition, further research like GWAS, the preliminary data are required to conduct further research. In this meaning, authors can add these data and supposed to do. By the other hand, if not, authors are recommended to try other journals.

Response

The results obtained using phylogenetic trees sometimes fluctuate depending on the parameters selected or the method of calculation. Instead of showing phylogenetic trees, we present the results of principal coordinate analysis (PCoA; S1 Fig) to clarify the genetic relationships between cultivars. The Bayesian structure and PCoA results will be useful for both chestnut breeding programs and further genetic studies including GWAS and population genetics.

Comment

Obiwase (#ID is required)

Some parts of cultivars are referred ID #, however, some are not. It is hard to follow the cultivar in Figure of STRUCTURE analysis to know how they are in data.

Response

In response to this suggestion, we now include both cultivar names and ID numbers in the text.

Comment

“The sixth" is hard to get the meaning. One the sixth cultivars you mentioned?

Then, Just "Ikaba" is enough. If there are other Ikaba cultivars, please show ID#.

Response

In response to this suggestion, we combined and revised the sentences mentioned here.

Submitted filename: 200426Response to Reviewers.docx

Click here for additional data file.

26 May 2020

PONE-D-19-32891R2

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China

PLOS ONE

Dear Dr. Nishio,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The last point raised by the reviewer would be most important. Please carefully examine all the comments and revise the manuscript

Please submit your revised manuscript by 30 June 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Hidenori Sassa

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: There some comments should be substituted or corrected;

Table 1

In cases of synonymous groups, authors omitted country name originated. Please use same notation.

199 Yanchang Beijing (China) C_OR (12)

Yanhong Beijing “no description of country here” C_OR (12)

Use alphabetical numeral letters in a case of less than 10.

253 we were able to infer both parents, 5 cultivars were F1 hybrids between Japanese and Chinese

254 chestnut and 1 cultivar, ‘Ikaba’ (ID#67), was presumed to be an offspring between Japanese–

353 18 Japanese–Chinese cultivars, we were able to presume both parents for 6 cultivars and one

354 parent for 10 cultivars (Table 4).

Five cultivars, a (or one ) cultivars

Table 4 does it require top bar to adjust format of PLOSone? Please confirm

Insert a space “),m”.

387 Table),mealy

387 Table), mealy

Is there a chance authors to misclassify synonymous or different types based on restricted number of SSRs. Because no other detail characteristics you can add to this data. Only a single evidence is not enough. You should refer the possible case in discussion section.

Authors mentioned Japanese case in the following reference but they are restricted genetic resources. In china, probably more numbers of resources and there might be similar but not the same landraces. At least need to reveal the possibility.

Nishio S, Yamamoto T, Terakami S, Sawamura Y, Takada N, Saito T. Genetic diversity of

Japanese chestnut cultivars assessed by SSR markers. Breeding Sci. 2011;61(2):109-20. doi: Doi

10.1270/Jsbbs.61.109.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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9 Jun 2020

Responses to the comments from the Editor

We thank the Editor and both Reviewers very much for taking time to review our manuscript and for your comments. We have revised our manuscript accordingly. During the revision, we found that there was a set of duplicated genotypes in the C_IO. Therefore, we revised all of the tables and figures. Because we only discarded one genotype, these changes had no effect on result and conclusion.

Comment

The last point raised by the reviewer would be most important. Please carefully examine all the comments and revise the manuscript.

Response

We take the last point is the possibility of the misclassification of the synonyms. We added the probability of identity, that is quite low (6.56× 10−35). It is unlikely to detect false synonyms with the 31 SSRs. We would like to show you the number of markers which can distinguish in the combination of all the two varieties (N_of_markers_distinguish.xlsx). Except for synonyms, cultivars can be distinguished with small numbers of markers.

Responses to the comments from Reviewer 3

Comment

In cases of synonymous groups, authors omitted country name originated. Please use same notation.

199 Yanchang Beijing (China) C_OR (12)

Yanhong Beijing “no description of country here” C_OR (12)

Response

In response to this suggestion, we add the country names.

Comment

Use alphabetical numeral letters in a case of less than 10.

253 we were able to infer both parents, 5 cultivars were F1 hybrids between Japanese and Chinese

254 chestnut and 1 cultivar, ‘Ikaba’ (ID#67), was presumed to be an offspring between Japanese–

353 18 Japanese–Chinese cultivars, we were able to presume both parents for 6 cultivars and one

354 parent for 10 cultivars (Table 4).

Response

In response to this suggestion, we revised the sentences.

Comment

Is there a chance authors to misclassify synonymous or different types based on restricted number of SSRs. Because no other detail characteristics you can add to this data. Only a single evidence is not enough. You should refer the possible case in discussion section.

Authors mentioned Japanese case in the following reference but they are restricted genetic resources. In china, probably more numbers of resources and there might be similar but not the same landraces. At least need to reveal the possibility.

Response

We do not think there are misclassification of synonymous based on the 31 SSRs. We added the probability of identity, that is quite low (6.56× 10−35). It is unlikely to detect false synonyms with the 31 SSRs.

For fruit and nut species, it is typical that cultivars were spread by clonal propagation and the new names were given by local breeders and farmers. Because the cultivars in tree species have high heterozygosity and number of alleles in each locus are high, it is not difficult to distinguish cultivars using small numbers of SSRs. In most cases, the number of the SSRs to identify the synonyms were less than ours.

Submitted filename: 200609Response to Reviewers.docx

Click here for additional data file.

15 Jun 2020

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China

PONE-D-19-32891R3

Dear Dr. Nishio,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hidenori Sassa

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Reviewer #3: (No Response)

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Reviewer #3: Yes: Ryuji Ishikawa

18 Jun 2020

PONE-D-19-32891R3

Genetic evidence that Chinese chestnut cultivars in Japan are derived from two divergent genetic structures that originated in China

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