Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers.

Sea buckthorn (Hippophae rhamnoides) is an ecologically and economically important species. Here, we assessed the diversity of 78 accessions cultivated in northern China using 8 agronomic characteristics, oil traits (including oil content and fatty acid composition) in seeds and fruit pulp, and SSR markers at 23 loci. The 78 accessions included 52 from ssp. mongolica, 6 from ssp. sinensis, and 20 hybrids. To assess the phenotypic diversity of these accessions, 8 agronomic fruit traits were recorded and analyzed using principal component analysis (PCA). The first two PCs accounted for approximately 78% of the variation among accessions. The oil contents were higher in pulp (3.46-38.56%) than in seeds (3.88-8.82%), especially in ssp. mongolica accessions. The polyunsaturated fatty acid (PUFA) ratio was slightly lower in the seed oil of hybrids (76.06%) than that of in ssp. mongolica (77.66%) and higher than that of in ssp. sinensis (72.22%). The monounsaturated fatty acid (MUFA) ratio in the pulp oil of ssp. sinensis (57.00%) was highest, and that in ssp. mongolica (51.00%) was equal to the ratio in the hybrids (51.20%). Using canonical correspondence analysis (CCA), we examined the correlation between agronomic traits and oil characteristics in pulp and seeds. Oil traits in pulp from different origins were correlated with morphological groupings (r = 0.8725, p = 0.0000). To assess the genotypic diversity, 23 SSR markers (including 17 loci previously reported) were used among the 78 accessions with 59 polymorphic amplified fragments obtained and an average PIC value of 0.2845. All accessions were classified into two groups based on the UPGMA method. The accessions of ssp. sinensis and ssp. mongolica were genetically distant. The hybrid accessions were close to ssp. mongolica accessions. The 8 agronomic traits, oil characteristics in seed and pulp oils, and 23 SSR markers successfully distinguished the 78 accessions. These results will be valuable for cultivar identification and genetic diversity analysis in cultivated sea buckthorn.

Introduction

Sea buckthorn (Hippophae rhamnoides L.) is a hardy winter shrub that is naturally distributed throughout Asia and Europe. It is an economically valuable species, divided into eight subspecies. Of these subspecies, ssp. sinensis and mongolica are mainly distributed in Asia, where they are abundant and commercially cultivated [1-2]. The fruits of sea buckthorn are rich in a variety of phytochemicals with physiological properties, such as lipids, carotenoids, ascorbic acid, tocopherols, and flavonoids [3-5]. The main applications for the fruits include food, cosmetics, and pharmaceutical products [6-7]. One of the most requested products for therapeutic practices is sea buckthorn oil, which is extracted from both seeds and fruit pulp. The applications of sea buckthorn oil include healing of the skin, mucosa, and immune systems, especially in cancer and cardiovascular disease therapy [8-9].

Two important parameters in analyzing oil yield and quality are oil content and fatty acid (FA) composition (referred to here as ‘oil traits’ for simplicity). Sea buckthorn seed and pulp oils are considered the most valuable products of the berries with a unique FA composition [10]. The seed oil contains omega-3 (α-linolenic acid) and omega-6 (linoleic acid) FAs, and the pulp oil is characterized by a high concentration of FAs from the omega-7 group (e.g., palmitoleic acid). Seed oil is rich in unsaturated FAs (commonly 30–40% linoleic acid and 20–35% linolenic acid) [10]. The soft parts (pulp and peel) of the berries have an FA composition that differs from the seeds that is characterized by a high level of palmitoleic acid (16–54%), which is very uncommon in plants. The oil traits of sea buckthorn berries vary greatly according to their origin, based on the climatic and geological conditions of the growing areas [11].

Sea buckthorn adapts well to extreme conditions, including drought, salinity, alkalinity, and extreme temperatures [12]. Its vigorous vegetative reproduction and strong, complex root system with nitrogen-fixing nodules make it an optimal pioneer plant for soil and water conservation. For these reasons, sea buckthorn is cultivated widely in arid and semiarid areas of China [13]. However, the fruits of native cultivars are small and thorny and of low economic value, which encourages the breeding of sea buckthorn has undergone different stages of development in China, such as introduction, domestication, seedling selection and artificial hybridization for elite accessions. The cultivars of ssp. mongolica (introduced from Russia and Mongolia), ssp. sinensis (China origin) and hybrids (ssp. mongolica × ssp. sinensis) are abundant in northern China [14]. However, as a perennial woody plant, traditional cross breeding that takes long time and has low efficiency cannot meet the needs of modern production in sea buckthorn. It is essential for economic production to utilize molecular marker-assisted breeding (MAB) in sea buckthorn, especially to breed accessions with desirable oil traits. An essential step in this process is the genetic analysis of sea buckthorn germplasm. At present, molecular markers are mainly used for the analysis of genetic diversity, the taxonomic and geographic origin of cultivars, sex determination and population genetic structure in sea buckthorn [14-16]. SSR (simple sequence repeat, microsatellite) markers, with 1- to 6-bp DNA regions repeated in tandem, have been used in these analyses for their advantages of codominance, random distribution throughout the genome, easy detection, and high polymorphism and reproducibility [17]. Currently, an increasing number of microsatellite markers are being developed in sea buckthorn using high-throughput sequencing techniques for transcriptome datasets (RNA-Seq), which have become valuable resources for SSR discovery [14, 18]. In our previous study, 17 RNA-Seq SSR markers (SB1-SB17) were developed and validated on 31 accessions, which were utilized in the present study for genetic diversity assessment of larger set of accessions [14].

Diversity analysis helps clarify the relationships between germplasm characteristics and genotype and will improve our understanding of sea buckthorn germplasm to achieve greater production with higher quality regarding the important traits correlated with germplasm [19].

In the present study, 78 accessions of sea buckthorn with variation in fruit traits were selected as materials. The aim of this study is to report the phenotypic characteristics and oil traits in fruit pulp and seeds and the genetic diversity of the 78 sea buckthorn accessions in northern China, providing a foundation for MAB in sea buckthorn.

Materials and methods

Plant materials

Berries and leaves of 78 sea buckthorn accessions belonging to ssp. mongolica (52 accessions), ssp. sinensis (6 accessions) and hybrids (ssp. mongolica × ssp. sinensis, 20 accessions) were collected from the end of July to mid-September in 2015. Table 1 summarizes information on the plant materials. Three research institutes located in northern China, the Institute of Selection and Breeding of Hippophae (42°26′N, 121°28′E; 380 m) in Fuxin, the Research Institute of Berry (47°14′N, 127°06′E; 202 m) in Suiling and the Jiuchenggong Breeding Base of Sea Buckthorn (39°40′N, 110°09′E; 1400 m) in Dongsheng, provided 76 accessions of sea buckthorn samples (Fig 1, S1 Table). The other two accessions, Quyisike and Zhongguoshaji, were harvested from cultivated fields in Qinghe (46°40′N, 90°22′E; 1218 m) and Datong (36°53′N, 101°35′E; 2800 m) (Fig 1, S1 Table, S2 Table). These areas have various geographical and climatic conditions (S3 Table).

Table 1

Accessions of sea buckthorn used for the study.

No.	Accession name	Abbrev.a	Collection site	ssp. b	No.	Accession name	Abbrev.a	Collection site	ssp.b
1	Zhuangyuanhuang	ZYH	Fuxin	M	40	E13-10	E13-10	Suiling	M
2	Wucifeng	WCF	Fuxin	M	41	E13-11	E13-11	Suiling	M
3	Liusha-1	LS1	Fuxin	M	42	E13-14	E13-14	Suiling	M
4	Siberia rumianes	SR	Fuxin	M	43	HS-1	HS1	Suiling	M
5	Fangxiang	FX	Fuxin	M	44	HS-4	HS4	Suiling	M
6	Yalishanda-12	YLSD12	Fuxin	M	45	HS-9	HS9	Suiling	M
7	Jiuyuehuang	JYH	Fuxin	M	46	HS-10	HS10	Suiling	M
8	Nanren	NR	Fuxin	M	47	HS-12	HS12	Suiling	M
9	Botanical garden	BG	Fuxin	M	48	HS-14	HS14	Suiling	M
10	Zajiao-1	ZJ1	Fuxin	H	49	HS-18	HS18	Suiling	M
11	Zajiao-2	ZJ2	Fuxin	H	50	HS-20	HS20	Suiling	M
12	Zajiao-3	ZJ3	Fuxin	H	51	HS-22	HS22	Suiling	M
13	MZ-14	MZ14	Suiling	M	52	Xin’e-1	XE1	Suiling	M
14	Shoudu	SD	Suiling	M	53	Xin’e-2	XE2	Suiling	M
15	Fenlan	FL	Suiling	M	54	Xin’e-3	XE3	Suiling	M
16	Aertai	AET	Suiling	M	55	Zhongguoshaji	ZGSJ	Suiling	S
17	Chengse	CS	Suiling	M	56	EZ-4	EZ4	Suiling	H
18	Chuyi	CY	Suiling	M	57	Za-56	Za56	Suiling	H
19	Hunjin	HJ	Suiling	M	58	Za1-2	Za1-2	Suiling	H
20	Jinse	JS	Suiling	M	59	Za05-6	Za05-6	Suiling	H
21	Juren	JR	Suiling	M	60	Za05-20	Za05-20	Suiling	H
22	Xiangyang	XY	Suiling	M	61	Za05-21	Za05-21	Suiling	H
23	Yousheng	YS	Suiling	M	62	Za4	Za4	Suiling	H
24	Katuni	KTN	Suiling	M	63	Za13-19	Za13-19	Suiling	H
25	Wulangemu	WLGM	Suiling	M	64	Za13-25	Za13-25	Suiling	H
26	TF1	TF1	Suiling	M	65	Juda	JD	Dongsheng	S
27	TF2-13	TF2-13	Suiling	M	66	Jianpingdahuang	JPDH	Dongsheng	S
28	TF2-23	TF2-23	Suiling	M	67	Manhanci	MHC	Dongsheng	S
29	TF2-24	TF2-24	Suiling	M	68	Zhongxiongyou	ZXY	Dongsheng	S
30	TF2-36	TF2- 36	Suiling	M	69	Liaofuza	LFZ	Dongsheng	H
31	Suiji-1	SJ1	Suiling	M	70	Zaciyou-1	ZCY1	Dongsheng	H
32	Suiji-3	SJ3	Suiling	M	71	Zaciyou-10	ZCY10	Dongsheng	H
33	Suiji-4	SJ4	Suiling	M	72	Zaciyou-12	ZCY12	Dongsheng	H
34	HD-3	HD3	Suiling	M	73	Xinzaci-26	XZC26	Dongsheng	H
35	E10-06	E10-06	Suiling	M	74	Shiciyou-2	SCY2	Dongsheng	H
36	E10-34	E10-34	Suiling	M	75	Shiciyou-5	SCY5	Dongsheng	H
37	E10-42	E10-42	Suiling	M	76	Shiciyou-30	SCY30	Dongsheng	H
38	E10-47	E10-47	Suiling	M	77	Zhongguoshajiwild	ZGSJwild	Datong	S
39	E13-00	E13-00	Suiling	M	78	Qiuyisike	QYSK	Qinghe	M

a Abbrev., abbreviation.

b ssp., subspecies; M, ssp. mongolica; S, ssp. sinensis; H, hybrid (ssp. mongolica ♀ × ssp. sinensis ♂).

Fig 1

The 78 sea buckthorn accessions from five cultivated lands used in this study.

The young leaves of each plant were kept at −80°C for use. The berries of each accession were pooled and frozen as quickly as possible at −20°C. When all plant materials were harvested, the berries were transferred to –50°C for storage until analysis.

Morphological characteristics of fruit

Hundred berry weight (HBW) was the weight of 100 fresh berries after they were picked from bushes. Hundred seed weight (HSW) was the weight of 100 seeds after air drying at room temperature (25°C) for 2 weeks [20]. There were three biological replicates for each measurement. The transverse and longitudinal diameters of berries (BTD and BLD) and the length, width and thickness of seeds (SL, SW and ST) were measured over 20 times each (on average) by micrometer calipers. The berry shape indices (BSIs) were estimated by the ratio of BLD to BTD. The minimum (Min), maximum (Max), mean ± standard deviation (SD), and coefficient of variation (CV%) were reported.

Oil extraction and FA analysis in seeds and pulp

The methods of lipid extraction, transesterification (methylation) and purification of methyl esters of the lipid extracts were described by Yang and Kallio [11]. Briefly, samples (1 g) of seeds and fruit pulp were isolated from freeze-dried berries and lipids from the samples were extracted with chloroform/methanol (2:1, v/v) with mechanical homogenization of the tissues. The purified oils were filtered before the solvent was removed on a rotary evaporator. The lipids were weighed, and the oil contents (percentages) in seeds and fruit pulp were calculated. Three biological replicates were taken for analysis. Lipids were stored in chloroform at −20°C until analysis.

The oil (10 mg) was transesterified by sodium methoxide catalysis [11, 21]. It was dissolved in sodium-dried diethyl ether (1 ml) and methyl acetate (20 μl). Then, 1 M sodium methoxide in dry methanol (20 μl) was added, and the solution was agitated briefly and incubated for 5 min at room temperature. The reaction was stopped by adding a saturated solution of oxalic acid in diethyl ether (30 μl) with brief agitation. The mixture was centrifuged at 1500 g for 2 min, and the supernatant was dried in a gentle stream of nitrogen. Fresh hexane (1 ml) was added and the solution was filtered with microporous filtering films (0.22 μm) for analysis.

Fatty acid methyl esters (FAMEs) were analyzed with a gas chromatography-tandem mass spectrometry (GC/MS/MS) system (model AxION® iQTTM, PerkinElmer, Shelton, CT, USA). Chromatographic separation was achieved using a DB-23 capillary column (60 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA) with the following temperature program: initial temperature 50°C, hold for 1 min, heat to 175°C at 25°C/min, then heat to 215°C at 3°C/min and hold for 10 min, heat to 230°C at 3°C/min and hold for 5 min. The inlet was operated in split mode (1:20) at a temperature of 250°C with helium as the carrier gas at constant flow of 1.0 ml/min. The transfer line temperature was 215°C, and the MS ion source was set to 230°C. MS detection was carried out in electron impact (EI) ionization mode, scanning all masses from 45–400 amu. FAME components were identified based on mass spectral comparison with an external standard (Supelco 37 Component FAME Mix, Sigma-Aldrich, St. Louis, MO, USA) and previous studies [10-11]. The main FA composition was expressed as a weight percentage of the total FAs from three replicates. The minimum, maximum, mean ± SD, and CV% were reported.

Statistical analysis

The data analysis for morphological traits and oil characteristics was performed with SPSS® 24.0 (IBM®). The following parameters were evaluated: mean, minimum value, maximum value, SD and CV%. One-way analysis of variance (ANOVA) was used in the comparison of all traits among subsp. of sinensis, subsp. of mongolica and hybrids. Pearson correlation coefficients were calculated to analyze the relationship between pairs of 8 agronomic traits. Principal component analysis (PCA) was used to determine relationships among the accessions. In addition, a canonical correspondence analysis (CCA) was applied to the data between morphological characteristics and oil traits in different tissues (seeds and pulp).

DNA extraction and SSR analysis

Total genomic DNA was extracted from young leaves using the TaKaRa MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, Beijing, China) based on the manufacturer's protocol. The purity and quantity of extracted DNA were evaluated by gel electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-three polymorphic microsatellite loci (SB1-SB23) developed using RNA-Seq were evaluated. Of these, 17 (SB1-SB17) had been deployed in a previous study by the group [14] (S4 Table). PCR amplification was performed in 20 μl volumes containing 40 ng of DNA template, 1× PCR buffer, 1.5 mM MgCl2, 0.15 mM of each dNTP (Takara, Dalian, China), 1.5 U of Taq polymerase (Takara, Dalian, China) and 0.5 μM of each primer. The PCR conditions included an initial denaturation at 94°C for 2 min and 35 cycles of 30 s at 94°C for denaturation, 30 s at 54–60°C for annealing and 45 s at 72°C for extension, with a final extension for 7 min at 72°C using a C1000 Touch™ Thermal Cycler (Bio-Rad, Berkeley, CA, USA). PCR products were electrophoresed on 8% nondenaturing polyacrylamide gels using an SE 600 Ruby Standard Dual Cooled Vertical Unit (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and visualized by silver staining.

The microsatellites were scored as codominant markers for genetic diversity analysis. The number of alleles (Na), effective number of alleles (Ne), observed and expected heterozygosity (Ho and He), Shannon’s information index (Is) and polymorphic information content (PIC) for each of the genic SSR markers were calculated using GenAlEx 6.5 [22-23] and PowerMarker version 3.25 [24] software packages. A genetic similarity matrix based on the proportion of shared alleles was generated, and a UPGMA tree was constructed using PowerMarker. The dendrogram was displayed using MEGA 6 software [25] to reveal genetic relationships between the 78 sea buckthorn accessions.

Results

Morphological characterization of berries and seeds

Descriptive statistical analysis of 8 agronomic fruit traits for the 78 sea buckthorn accessions is shown in Table 2, S5 and S6 Tables. Relatively high CV values were observed for the HBW, BLD, and HSW (> 20%). The highest CV% was observed for the HBW (39.12%), which varied from 8.52 to 69.74 g. ANOVA (p < 0.05) showed that the HBW of ssp. mongolica berries was 47.69 ± 11.03 g, which was much higher than those of ssp. sinensis berries (10.73 ± 1.54 g) and hybrids (31.44 ± 13.84 g). In hybrids, the HBW values were high in EZ4, Za56, Za1-2, Za05-6 and Za05-21(> 45 g), which were approximately the size of those in ssp. mongolica berries on average (S6 Table). The BTD varied from 5.54 to 10.80 mm, and the BLD varied from 4.83 to 14.25 mm. In addition, the BLD of berries from ssp. mongolica was higher than the BTD, and this relationship was the opposite in berries of ssp. sinensis. According to BSI values, the berry shapes of the three groups were significantly different (p = 0.000): oblong berries for ssp. mongolica (1.35 ± 0.20), oblate berries for ssp. sinensis (0.90 ± 0.05) and circular berries for the hybrids (1.08 ± 0.11). The HSW varied from 0.61 to 2.19 g with an average of 1.45 g. Similar to the HBW, there were significant differences in the HSW among seeds from ssp. mongolica, ssp. sinensis, and hybrids (p = 0.000). The SL varied from 2.00 to 3.49 mm, and the SW varied from 2.98 to 7.43 mm. The ST varied from 1.54 to 2.73 mm, with an average of 1.93 mm. Overall, the agronomic characteristics of seeds (HSW, SL, SW, and ST) showed relatively low coefficients of variation, ranging from 11.50–24.33%; however, the berries (HBW, BTD, BLD, and BSI) had high CV%s.

Table 2

Fruit traits of sea buckthorn berries of two different subspecies and hybrid accessions.

Trait name	Abbrev.b	ssp. mongolica	ssp. sinensis	Hybrid
Hundred berry weight (g)	HBW (g)	47.69 ±11.03a	10.73 ± 1.54c	31.44 ±13.84b
Berry transverse diameter (mm)	BTD (mm)	8.17 ± 0.99a	5.84 ± 0.23b	7.61 ± 1.24a
Berry longitudinal diameter (mm)	BLD (mm)	10.90 ± 1.48a	5.20 ± 0.19c	8.15 ± 1.18b
Berry shape index	BSI	1.35 ± 0.20	0.90 ± 0.05	1.08 ± 0.11
Hundred seed weight (g)	HSW (g)	1.60 ± 0.28a	0.79 ± 0.23c	1.28 ± 0.25b
Seed length (mm)	SL (mm)	5.91 ± 0.68a	3.31 ± 0.27c	4.64 ± 0.56b
Seed width (mm)	SW (mm)	2.76 ± 0.27a	2.18 ± 0.18c	2.52 ± 0.22b
Seed thickness (mm)	ST (mm)	1.98 ±0.18a	1.67 ± 0.16 b	1.86 ± 0.26a

a Values with different lowercase letters (a–c) are significantly different at p < 0.05.

b Abbrev., Abbreviation.

In previous multilocation trials in Suiling (47°14′N, 127°06′E; 202 m) and Dengkou (40°43′N, 106°30′E; 1053 m, Inner Mongolia), the fruit characteristics of 11 large berry accessions (AET, CS, CY, HJ, JS, JR, XY, YS, KTN, WLGM and SJ1) were comparatively analyzed (S7 Table). The HBWs values in Suiling (38.33–67.59 g) were higher than those in Dengkou (32.87–63.85 g). For all the introduced cultivars, the HBW values in the two experimental fields were lower than those in their country of origin, Russia. The phenotypic characteristics of sea buckthorn berries showed differences due to their origins, different parts of fruit analyzed, climatic and growing conditions. In this study, 78 accessions were selected for their good adaptabilities to growth sites.

PCA was performed using fruit characteristics (Fig 2). The first two PCs explained 78.11% of the total morphological variance. The first PC accounted for 41.74% of the variance. It was associated with BTD, HBW, ST, HSW, and SW in descending order. Therefore, these traits were important attributes for the classification of sea buckthorn accessions. The second PC accounted for 36.37%, which were correlated with BSI, SL, and BLD in descending order. The plot shows the distribution of 78 sea buckthorn accessions on PC1 and PC2 (Fig 2). The ssp. mongolica accessions with larger berries tended to cluster together, mainly positive on PC2. Six accessions of ssp. sinensis with the smallest berries were negative on both PC1 and PC2. The hybrids were largely distributed between the above two groups. Some hybrids (including ZCY1, ZCY10, ZCY12, XZC26, SCY2, and SCY5) were close to the accessions from ssp. sinensis.

Fig 2

Two-dimensional scatter plot for the first two principal components (PC1 and PC2) based on the agronomic fruit characteristics of 78 sea buckthorn accessions.

Numbers associated with symbols are the variety codes listed in Table 1. ▲ = ssp. mongolica; ● = ssp. sinensis; ◇ = hybrid.

Oil characterization in seeds and seedless parts

The oil characteristics of seeds and seedless parts (pulp and peel) among the 78 accessions are summarized in Table 3 and Table 4. One special feature of sea buckthorn fruit was the high oil content in the pulp and peel (20.41%), in contrast to the oil content in the seeds (8.82%). A higher CV% was observed in pulp oil (42.72%) and varied over a wide range, from 3.46 to 38.56%. The pulp fraction of berries of ssp. mongolica had the highest oil content (24.68%) based on dry weight. The lowest pulp oil content (7.10%) on average was found in the berries of ssp. sinensis. In hybrids, the berries of ZJ2 contained 27.22% pulp oil, which slightly exceeded that of ssp. mongolica on average (S6 Table). The seed oil content varied from 3.88 to 12.75% with an average of 8.82%. The seeds of ssp. mongolica had the highest oil contents, with an average of 9.46%, and those of the other two groups did not differ significantly.

Table 3

Oil characteristics of pulp and seeds of 78 sea buckthorn accessions (weight percentages).

Character	Pulp				Seed
	Mina	Maxb	Mean ± SDc	CVd (%)	Mina	Maxb	Mean ± SDc	CVd(%)
oil content	3.46	38.56	20.41 ± 8.72	42.72	3.88	12.75	8.82 ± 1.86	21.08
Palmitic acid (16:0)	24.52	53.08	36.26 ± 4.83	13.32	3.84	11.77	6.55 ± 1.39	21.16
Palmitoleic acid (16:1n7)	17.93	57.75	35.12 ± 7.64	21.76	tre	tre	tre
Stearic acid (18:0)	0.38	5.12	1.26 ± 0.70	55.58	1.41	4.58	2.16 ± 0.43	20.11
Oleic acid (18:1n9)	1.44	23.43	8.72 ± 4.72	54.13	3.05	25.95	13.25 ± 4.04	30.50
Vaccenic acid (18:1n7)	3.51	24.24	7.68 ± 4.09	53.28	0.45	2.38	1.20 ± 0.47	39.17
Linoleic acid (18:2n6)	3.02	17.40	9.97 ± 3.18	31.91	34.22	52.75	42.17 ± 3.60	8.54
α-Linolenic acid (18:3n3)	0.12	7.16	1.00 ± 1.03	102.83	21.37	47.16	34.67 ± 4.42	12.75

a Minimum value.

b Maximum value.

c Standard deviation.

d Coefficient of variation expressed as a percentage.

e tr, trace (< 0.5%).

Table 4

Oil content and fatty acid composition in the seeds and fruit pulp of sea buckthorn berries of different origins (weight percentages).

	Pulp oil			Seed oil
Character	ssp. mongolica	ssp. sinensis	Hybrid	ssp. mongolica	ssp. sinensis	Hybrid
oil content	24.68 ± 6.79 a	7.10 ± 3.28c	13.34 ± 4.85b	9.46 ± 1.56a	6.70 ± 1.32b	7.78 ±1.84b
Palmitic acid (16:0)	37.68 ± 4.64a	29.39 ± 3.71b	34.62 ± 3.14a	6.52 ± 1.16	7.41 ± 1.55	6.38 ± 1.82
Palmitoleic acid (16:1n7)	37.43 ±7.09a	23.65 ± 4.16b	32.55 ± 5.84a	trb	trb	trb
Stearic acid (18:0)	1.08 ±0.69b	1.73 ± 0.64a	1.59 ± 0.57ab	2.13 ± 0.29	2.19 ± 0.44	2.23 ± 0.69
Oleic acid (18:1n9)	7.56 ±3.97b	16.67 ± 6.84a	9.33 ± 3.40b	12.62 ± 3.75b	16.37 ± 3.77a	13.96 ± 4.46ab
Vaccenic acid (18:1n7)	6.01 ±1.79c	16.68 ± 6.20a	9.32 ± 3.63b	1.07 ± 0.37b	1.80 ± 0.39a	1.37 ± 0.55b
Linoleic acid (18:2n6)	9.55 ±2.76ab	8.34 ± 5.54b	11.53 ± 2.92a	42.10 ± 3.08	40.44 ± 4.06	42.87 ± 4.62
α-Linolenic acid (18:3n3)	0.69 ±0.41b	3.54 ± 2.09a	1.07 ± 0.64b	35.56 ± 4.13a	31.78 ± 2.91b	33.20 ± 4.89 ab
MUFA	51.00 ±5.38b	57.00 ± 9.46a	51.20 ± 3.52b	13.69 ± 3.93b	18.18 ± 4.09a	15.33 ± 4.90ab
PUFA	10.24 ±2.98	11.89 ± 7.54	12.60 ±3.37	77.66 ± 4.31a	72.22 ±5.54b	76.06 ± 6.23ab

a Values with different lowercase letters (a–c) are significantly different at p < 0.05.

b tr, trace (< 0.5%).

For sea buckthorn, the FA composition in the seed oil differed significantly from that in the pulp oil. The proportions of FAs ranked from high to low as linoleic (18:2n6), α-linolenic (18:3n3), oleic (18:1n9), palmitic (16:0), stearic (18:0) and vaccenic (18:1n7) acids in the seed oil of most accessions (Table 4). Linoleic acid varied from 34.22 to 52.75% with an average of 42.17%. The proportion of α-linolenic acid varied from 21.37 to 47.16% with an average of 34.67%. High CV values were observed in oleic (30.50%) and vaccenic (39.17%) acids. Furthermore, the level of palmitoleic acid (16:1n7, < 0.5%) was extremely low in the seed oil. The FA composition of sea buckthorn seeds was similar among berries of the two different subspecies and hybrid accessions. Small variations were found in the proportion of linoleic acid in seed oil (40.44–42.87%). Its proportion in hybrids was slightly higher than in ssp. mongolica (42.87% vs 42.10%), and showed the highest mean value among the two different subspecies and hybrid accessions. α-Linolenic acid showed little variation, with a larger proportion in ssp. mongolica than in ssp. sinensis (35.56% vs 31.78%). A higher proportion of palmitic (7.41% vs 6.38%) and oleic (16.37% vs 13.96%) acids and a lower proportion of stearic acid (2.19% vs 2.23%) were discovered between the accessions of ssp. sinensis and hybrids. The polyunsaturated fatty acids (PUFA) ratio in hybrids (76.06%) was slightly lower than that in ssp. mongolica (77.66%) and higher than that in ssp. sinensis (72.22%). Some hybrids (including ZJ1, Za1-2, Za13-25, Za05-6, LFZ, and ZCY12) contained a high proportion of PUFAs (> 80%) in seed oil, which was more than the average level of ssp. mongolica accessions (S6 Table).

In pulp oil, the dominant FAs were palmitoleic, palmitic, linoleic, oleic, and vaccenic acids (Table 3). Major differences were observed in the proportion of palmitoleic (17.93–57.75%), oleic (1.44–23.43%) and vaccenic (3.51–24.24%) acids. The special feature of pulp oil is high proportions (> 35%) of palmitoleic and palmitic acids. Compared to ssp. sinensis, ssp. mongolica contained a higher proportion of palmitoleic and palmitic acids in the berry pulp (p < 0.05) (Table 4). In particular, the proportions of oleic and vaccenic acids were highest in ssp. sinensis, much higher than those in ssp. mongolica and hybrid accessions. The relative levels of α-linolenic and stearic acids in pulp of ssp. sinensis were higher than ssp. mongolica (p < 0.05) (Table 4). For hybrids, the proportions of most FAs were between ssp. mongolica and ssp. sinensis accessions, except for linoleic acid. Similar to the results in seed oils, the hybrids had the highest proportions of linoleic acid (11.53%) and PUFA (12.60%). The monounsaturated fatty acid (MUFA) ratio in the pulp oil of ssp. sinensis (57.00%) was highest and that of ssp. mongolica (51.00%) was almost equal to that of the hybrids (51.20%). In the hybrids, the pulp oil of SCY2 contained 39.16% palmitoleic acid, and the content of MUFAs was 60.77%, which was higher than that in ssp. sinensis (S6 Table).

Correlations among the agronomic traits and oil characteristics

Canonical analyses allow direct comparisons of two data matrices. All sea buckthorn accessions were represented in a two-dimensional space using CCA between phenotypic traits and oil characteristics (Fig 3). For berries of the two different subspecies and hybrid accessions, phenotypic characters (BLD, HBW, BSI, and BTD) of berries and oil traits in pulp showed a close correlation (r = 0.8725, p = 0.0000). Based on CCA, accessions of ssp. mongolica were clustered on the upper side (mainly positive on D1 and D2), those of ssp. sinensis on the other, and the hybrids in the middle in Fig 3A. The positioning of samples in the first dimension was mostly related to differences in their berry characteristics that were primarily provided by the phenotypic character of BLD. The second dimension indicated differences in the oil contents and FA compositions of pulp oil among sea buckthorn accessions. Differences between pulp oil traits were primarily related to percentages of oil content, 16:0 and 16:1n7, which were highest in ssp. mongolica, followed by hybrids, and lowest in ssp. sinensis. For seeds of 78 accessions, phenotypic characteristics (SL, SW, ST, and HSW) and seed oil traits were correlated (r = 0.7482, p = 0.0000). The positioning of samples was staggered (Fig 3B), which reflected that all seed samples had relatively little variation among phenotypic traits and oil characteristics. These results verified the previous analysis (Table 2 and Table 3).

Fig 3

Canonical correspondence analysis of phenotypic traits (A. berry; B. seed) and oil characteristics (A. pulp oil; B. seed oil) of sea buckthorn germplasms. D1, Dimension 1; D2, Dimension 2. ▲ = ssp. mongolica; ● = ssp. sinensis; ◇ = hybrid.

SSR diversity

Twenty-three pairs of RNA-Seq SSR primers with good amplification and band stability were used among 78 accessions of sea buckthorn. A total of 69 bands were amplified using the 23 primer pairs, of which 59 were polymorphic, accounting for 85.51% of all bands. The number of amplified bands per locus ranged from 2 to 5, averaging 3, and Ne ranged from 1.0392 to 3.1049, averaging 1.6602 (Table 5). SB2, SB3, SB5, SB6, SB8, SB13, SB16 and SB23 were informative SSR loci, each revealing more than four effective alleles distributed among all of the accessions. Compared with Na, Ne and their average values were lower, which was caused by the uneven distribution of gene frequencies in SSR loci. In the genetic diversity analysis, Ho ranged from 0.0385 to 0.7949, with an average of 0.2965; He ranged from 0.0377 to 0.6779, with an average of 0.3291; and Is ranged from 0.0950 to 1.2152, with an average of 0.5681. The PIC value, regarded as discriminating power, varied from 0.0370 to 0.6174, with an average of 0.2845. Loci SB6 (PIC = 0.6174) and SB8 (PIC = 0.5820) showed higher effectiveness because of their high informativity and could be used to construct the fingerprint map of sea buckthorn germplasm. The characteristics of 23 polymorphic SSR markers in sea buckthorn accessions are shown in Table 5.

Table 5

Genetic diversity analyses of 78 accessions of sea buckthorn germplasm using 23 SSR markers.

Loci code	Na	Ne	Ho	He	PIC	Is
SB1	3	1.2745	0.2436	0.2154	0.2025	0.3956
SB2	4	1.1382	0.1282	0.1214	0.1166	0.2791
SB3	4	2.2372	0.4615	0.5530	0.4627	0.9090
SB4	2	1.5006	0.2692	0.3336	0.2779	0.5160
SB5	4	2.1129	0.3333	0.5267	0.4735	0.9288
SB6	4	3.1049	0.7051	0.6779	0.6174	1.2152
SB7	2	1.0799	0.0769	0.0740	0.0712	0.1630
SB8	5	2.8490	0.3846	0.6490	0.5820	1.1890
SB9	2	1.1509	0.1410	0.1311	0.1225	0.2550
SB10	3	1.5350	0.2949	0.3485	0.3114	0.6253
SB11	2	1.9287	0.1667	0.4815	0.3656	0.6745
SB12	3	1.2430	0.2179	0.1955	0.1753	0.3687
SB13	4	2.1644	0.4231	0.5380	0.4392	0.8687
SB14	2	1.9987	0.3077	0.4997	0.3750	0.6928
SB15	2	1.0662	0.0641	0.0620	0.0601	0.1418
SB16	4	1.4567	0.1923	0.3135	0.2956	0.6427
SB17	2	1.4175	0.3590	0.2945	0.2512	0.4706
SB18	2	1.0392	0.0385	0.0377	0.0370	0.0950
SB19	3	1.0804	0.0641	0.0744	0.0724	0.1804
SB20	2	1.1803	0.1667	0.1528	0.1411	0.2868
SB21	3	1.9123	0.7308	0.4771	0.3802	0.7318
SB22	3	1.2905	0.2564	0.2251	0.2025	0.4084
SB23	4	2.4239	0.7949	0.5874	0.5102	1.0284

Na, observed number of alleles; Ne, effective number of alleles; Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphism information content; Is, Shannon’s information index.

Genetic relationships among sea buckthorn germplasm

Using 23 polymorphic SSR markers, the UPGMA dendrogram based on the proportion of shared alleles was constructed to assess the genetic relationships between the 78 accessions (Fig 4). The results showed that all the accessions could be divided into two groups (I and II). The accessions of ssp. sinensis (JD, ZGSJ, MHC, ZGSJ, JPDH and ZXY) were clustered into group I. These accessions had closer relationships, despite great geographic differences. The second group was divided into 3 subgroups, namely, IIa, IIb, and IIc. The 20 hybrid accessions were all clustered into IIa. Subgroups IIb and IIc contained all the accessions of ssp. mongolica (introduced from Russia and Mongolia). Subgroup IIb included 6 accessions, namely WCF, LS1, QYSK, FX, SR, and MZ14. The remaining accessions of ssp. mongolica were clustered into IIc. Among them, KTN, WLGM, HS4, HS9, HS10, HS12, HS14, HS18, HS20, HS22, WCF, FX and MZ14 composed one sub-subgroup. SJ3, ZYH, SD, NR, FL, XE2, XE3, JYH and YLSD12 showed close relationships. The other 23 accessions clustered into the third sub-subgroup. Overall, the relationship between ssp. mongolica and ssp. sinensis was relatively distant. The hybrids are close to ssp. mongolica, to which their female parents belonged.

Fig 4

UPGMA dendrogram of sea buckthorn germplasm based on SSR data (sample abbreviations described in Table 1).

▲ = ssp. mongolica; ● = ssp. sinensis; ◇ = hybrid.

Discussion

Morphological characteristics, biochemical traits, and microsatellite markers have been used for germplasm identification and genetic diversity analysis in many horticultural plants [26-27]. The diversity at the morphological, biochemical, and molecular levels of 78 sea buckthorn accessions, composed of 52 from ssp. mongolica, 6 from ssp. sinensis, and 20 hybrids, was investigated.

The morphological characterization of plant materials with desired traits is an essential step for the effective use of germplasm [28]. Here, 8 important agronomic traits were measured among 78 sea buckthorn accessions, and a considerable amount of variation in morphological traits was found. The sizes of berries from the two different subspecies and hybrid accessions were significantly different according to the HBW value (p = 0.000). Compared to ssp. sinensis berries, ssp. mongolica berries were much larger on average. The berry size of hybrid accessions was between the two subspecies. In the PCA, we plotted 2D plots with PC1 and PC2 scores of phenotypes (Fig 2). PC1 was mainly related to BTD and HBW, which explained the largest portion of the variance in 78 accessions. The distribution of 78 accessions on PC1 and PC2 was consistent with their agronomic characteristics (Fig 2). These results estimating morphological traits are valuable tools for identifying variation among plant germplasms [26].

For biochemical traits, oil content and FA composition in the seeds and seedless parts were selected for their important roles in human health. The oil of sea buckthorn seems to be a good source of unsaturated FAs. Seed oil is rich in PUFAs, including linoleic and α-linolenic acids. The proportion of PUFAs did not differ significantly among berries from three origins, despite the differences in some morphological characteristics and in growth conditions. These results were consistent with previous studies [10]. The results of the present study and previous investigations also suggested that the berries of ssp. mongolica were a good source of palmitic and palmitoleic acids in pulp oil and that those of ssp. sinensis were a good source of oleic acid in both seeds and fruit pulp [29]. Although carefully selected for intersubspecies crosses, some hybrids displayed elite oil traits. For example, the proportion of MUFAs in the pulp of SCY2 and of PUFAs in the seeds of 6 accessions (including ZJ1, Za1-2, Za13-25, Za05-6, LFZ, and ZCY12) exceeded the average level of ssp. mongolica accessions, the subspecies that one of their parents belonged to. These results demonstrate the effectiveness of traditional cross breeding in the improvement of native accessions (ssp. sinensis), even though it is time-consuming and has low efficiency.

Previous studies found that berry size is a useful indicator of Vc, sugars and acids in population identification [19, 30]. The nutrients in the seedless fraction were more concentrated in the small berries of ssp. sinensis than in the large berries of ssp. mongolica [29]. In the present study, we analyzed the correlation between agronomic characteristics and oil traits at different levels (seed and pulp) by CCA. The results showed that the phenotypic characteristics (BLD, HBW, BSI, and BTD) of berries and the oil traits in pulp were positively correlated (r = 0.8725, p = 0.0000). The BLD, as a promising marker, provided the primary difference in CCA. Our results illustrated that berry size had different correlations with various biochemical characteristics in sea buckthorn.

Variation in phenotypic traits among germplasms may be attributed to differences in genetic backgrounds, geographical location, climate, harvest period and berry maturity, while molecular markers are independent of environmental condition and growth stage [31]. Twenty-three polymorphic SSR markers were used to investigate the genetic relationships among 78 sea buckthorn accessions. The 23 selected SSR markers detected 2–5 alleles, and their PIC values ranged from 0.1166 to 0.6155 and had an average of 0.3249. The PIC mean value was significantly lower than that of RAPD, ISSR and SRAP markers previously reported [15–16, 32], suggesting that the gene sequences of these SSR markers were conserved in sea buckthorn germplasm.

Based on UPGMA, the 78 accessions were classified into two groups. There is a large genetic distance between accessions of ssp. sinensis and ssp. mongolica. The hybrids were in between and rather close to ssp. mongolica accessions. Coincidentally, these hybrids were also between accessions of ssp. sinensis and ssp. mongolica on the PCA plot based on 8 agronomic characteristics. This uniformity indicated that the diversity of morphological characteristics could reflect genetic diversity and be used as markers in agronomy. Ruan et al. [15] assessed 14 Chinese, Russian and Mongolian sea buckthorn accessions using RAPD markers and obtained similar results. In a previous publication, the genetic relationship of 31 sea buckthorn accessions (also contained in this study) was analyzed based on 17 RNA-Seq SSRs [14]. However, the accessions of ssp. mongolica clustered in one group and those of ssp. sinensis and hybrids were divided in the other. This revealed that genetic relationships mainly relied on the diversity of genotypes and genetic backgrounds.

With the continuous development of high-throughput sequencing technology, transcriptome databases have become a powerful resource for SSR mining. An increasing number of RNA-Seq SSRs have been developed and applied to the study of species genetic diversity and population genetic structure [33-34]. The SSRs obtained by transcriptomes are associated with many important quantitative traits [35].

The results in the present study yielded useful knowledge regarding the diversity and genetic relationships of sea buckthorn germplasm in northern China, and could therefore facilitate further studies, including the selection of mapping populations and promising candidates, marker-trait association analysis based on establishing the consistency of the traits, and characterization of parents used in future breeding programs.

Conclusion

In the present study, 8 phenotypic characteristics, oil traits in seeds and seedless parts, and 23 SSR markers successfully distinguished all 78 sea buckthorn accessions. In PCA, BTD and HBW in the first PC were the most important characteristics for distinguishing the accessions. The agronomic traits of berries were closely correlated with the oil content and FA composition in the pulp by CCA. This information will be valuable for germplasm identification and genotypic diversity analysis in H. rhamnoides.

78 berry samples used in this study.

Numbers are the variety codes listed in Table 1.

(TIF)

Click here for additional data file.

Total ion flow chromatography of 37 FAMEs Mix (A) and FAMEs in pulp oil in MHC (B).

(TIF)

Click here for additional data file.

Samples of sea buckthorn grouped according to different genetic backgrounds.

(DOCX)

Click here for additional data file.

Characterization of the hybrids of sea buckthorn accessions studied.

(DOCX)

Click here for additional data file.

Climatic conditions at different growth sites of sea buckthorn samples in China.

(DOCX)

Click here for additional data file.

Primer sequences, annealing temperature, and estimated allelic size of 23 SSR markers.

(DOCX)

Click here for additional data file.

Descriptive statistics for morphological traits of berries and seeds among the sea buckthorn accessions studied.

(DOCX)

Click here for additional data file.

The morphological characteristics and oil traits of pulp and seeds of the 78 sea buckthorn accessions studied.

(XLSX)

Click here for additional data file.

Fruit traits and Vc contents of large berry accessions of sea buckthorn in two experimental fields (located in Suiling and Dengkou).

(DOCX)

Click here for additional data file.

Allele combinations obtained at the 23 microsatellite loci in 78 sea buckthorn accessions.

(TXT)

Click here for additional data file.

23 Aug 2019

PONE-D-19-17567

Diversity in sea buckthorn (Hippophae rhamnoides L.) varieties with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Tian

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 changes required for the revision might take longer then provided. If you need additional time, please let us know and we will extend the period for required revision.

We would appreciate receiving your revised manuscript by 15 October, 2019. 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Additional Editor Comments (if provided):

The manuscript “Diversity in sea buckthorn (Hippophae rhamnoides L.) varieties with different origins based on morphological characteristics, oil traits, and microsatellite markers” has been reviewed by two reviewers. It took me a while to secure reviewers for this manuscript, hence the delay, I apologies for it. Both the reviewers have submitted their comments, and one of them has also provided a sanitized version of the manuscript which can be seen by the authors. Both the reviewers have various concerns regarding the manuscript, but more importantly both have shown concern about the plant material and data generated. Since they raised a concern on the data, it will be better that manuscript is submitted along with answers to both the reviewer’s comments.

I will like to point out some important points:

1) The use of term varieties, cultivars, subspecies and hybrids have been without much explanation. For example, what is the basis of assigning hybrid status to a particular cultivar? More clarity is required in explanation of the material. How these varieties were assigned varietal status?

2) Generation of morphological dataset is also not mentioned clearly. You have 76 varieties growing at three locations. You need to provide environmental parameters for each location. Are all 76 growing at each location? If all the varieties are not growing at same location, many of morphological traits will be influenced by environmental factors. Did you do any multilocation trials to see the influence of environment on these traits? Did you try to collect data during different years and see if the data is consistent or showing variation. A statistical analysis of such data only will generate confidence in morphological data. Even a multilocation trial of a subset will provide information on reliability of data. Please include such data.

3) The sequencing data has been published earlier and 17 of SSR are coming from that data. Only 3 new markers have been used in the present study. This undermines the amount of data presented in this MS. You have to clearly mention these facts in the MS and the abstract. In my opinion more data needs to be generated. I suggest another 25-30 SSRs should be used for analysing the diversity.

4) There is no comparison given between the varieties used in previous publication and the present one. Are you using common varities? If you are than SSR data must be same and must have been presented in previous MS already. This has not been mentioned in the MS.

I hope you will appreciate comments made by two reviewers and will appreciate the time and efforts spent by them in reviewing the manuscript. The comments are positive and are an effort to improve the quality of manuscript further.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. 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 #2: No

**********

4. 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: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Reviewer #1: General comments

The present study was conducted on 78 so called “varieties” of Hippophae rhamnoides, out of which two sets belonged to two sub-species and the third one is supposedly the hybrids. The study is aimed to decipher the genetic diversity and their oil content (in “berries”) of the “varieties”. With the given results and methodology authors are attempting to generated the data on two different aspects - first assessment of agronomic traits, especially the oil content and to relate it to a reliable phenotypic marker (among the eight selected morphological traits), and genetic diversity of species by using 20 SSR markers. An attempt has been made to link these traits together, which needs to be highlighted in the Introduction pertinently in the light of earlier attempts on related/unrelated taxa.

In general the MS needs to be modified for technical reasons and usage of sources in the study. A major revision of the MS is suggested before acceptance.

Specific comments

1. I think that in such bio-prospection studies sampling strategy is very crucial. The sampling method needs to explain that how these accessions were sourced. The MS needs elaboration on -

• How many individuals of a “variety” from each site were collected?

• Are these the random collections of registered varieties from the cultivated field in the five regions OR sampled from the wild?

• It is also not clear that how the hybrids were distinguished from parents while making collections.

• Do these sites differ in climatic conditions?

• What is the link of “origin” with oil content? Did you expect that there are bound to be differences because of differences in the climatic conditions of area of collection/cultivation of the same “variety/hybrid”?

Importantly instead of the term varieties the term accessions would be appropriate, as the authors have mentioned it in Table 1. According to the definition by The International Union for the Protection of New Varieties of Plants, "a variety must be recognizable by its characteristics, recognizably different from any other variety and remain unchanged through the process of propagation".

Do these two subspecies hybridize freely in nature and such hybrids have been characterized? This needs some population analysis like by using STRUCTURE, or at least there should be a note on the characterization of hybrids (including the features), even if they are procured form some Research Institute.

2. I don’t understand the usage of term pulp/peel in the MS (also see page 15, line 251). As the entire fleshy region was separately used for extraction of oil from the "berries" (see Methods), the use of term pulp would be appropriate. One cannot expect to remove the epidermal peel especially during the mechanical homogenization process.

3. How the present study for the genetic diversity analysis of 78 cultivars is different from other previous studies? May be highlighted in the introduction. Authors may also highlight that trait: i.e. Oil yield was correlated with the “promising” accessions.

4. Although attempt has been made of possible use of MAB in future, but it has not been justified with the discussion. For example, do the authors will depend on the same plants in the cultivated lands across the region or some mapping populations will be established. In former case GPS tagging of the individuals will be required for sourcing the material on regular basis and to establish the consistency of the trait.

Materials and Methods

5. Need to mention whether hundred-berry weight, hundred-seed weight and other dimensions were taken from mature or immature berries? In Supplementary figure 1 some samples are showing immature berries e.g. sample 65, 68 etc.

6. What do the ‘Berry Shape Indices’ refer to and what are its implications on the results/oil trait/ with genetic diversity. Provide any suitable reference if possible. (Page: 8, subsection: Morphological….)

7. The usage of phrase ‘8 agronomic traits’ seems to be superfluous as these are the traits of berries itself. How the seed width is different from the seed thickness? The difference is not apparent. Table 2 and 3; as well as in text.

8. The usage of abbreviation has not been followed see table 2 and 3. Table 2 is not necessary, may be omitted or shifted to Supplementary Data. In Tables SD is not mentioned.

9. The reference is missing for the SB18-SB20 SSRs; in the text (Page 10, line 181).

Results

10. Results should be given in the format mean ± SD. Minimum and maximum can be given in supplementary tables.

11. It is not clear from the table caption and content that whether values in the Table 4 is the minimum, maximum and mean values are representing the cumulative results of 78 varieties e.g. minimum in variety… and maximum in variety…. Need to mention in the results.

12. The results of CCA are driving a correlation between phenotypic traits and oil characteristics. The authors may use the information for total oil content (pulp+seed) or oil content in pulp and seeds separately for drawing any correlation. That would possibly help as a descriptor for the potential crop in identifying the elite/superior “variety” and further can be linked to genetic diversity.

Discussion

13. Page:28, Line:449-453. The link of this part of discussion is lacking with the previous text.

14. In conclusion part authors are concluding that this information may be useful for cultivar identification but initially they started their work for the varieties. Taxonomically these two are different entities.

Some suggestion:

1. The sequence of S1 and S2 table can be reversed as per the citation in the text.

2. Page:3, Line:54. Reference 1 is incorrect. The lead author here is Bartish I.V.

3. Page:3, Line:56-57. ….flavonoids [3-7]; ….products [8-10]. Here over-citation may be avoided.

4. Page:3, Line:59. ‘Sea buckthorn oil’ instead of ‘sea buckthorn oils’

5. Page: 4, Line 74. Add a reference to the statement. The plant is able to avoid cold and is not resistant, because the leaves are shed under extreme cold condition in this plant. Even the species is not resistant to alkali too.

6. Page:4. Line:85. Use full form at first place ‘MAB’.

7. Page:5. Line:110. What was the premise of including two known elite varieties in the study? Any supportive reference(s) for the statement, and also mention the context in which these varieties are elite.

8. Page:12. Line:204-205. May be included in Material and Methods.

Reviewer #2: The publication can be accepted post minor reviews.

Some of the comments to authors have been listed below. Some changes required are highlighted in the manuscript attached.

1. The authors mention that 76 varieties were used. There is no mention of the different species they belonged to in M&M, although it has been mentioned later in the text and table. Incorporate that information in the M&M.

2. Are these 76 different varieties or just different accessions? At many places they are being referred to as ‘cultivars’ also. Please correct accordingly in the text wherever mentioned.

3. How variable are the climatic conditions of the three research institutes?

4. Line 109: ‘………provided 76 varieties’. Does this mean that all the 76 were grown at all the 3 fields? There is no clarity on this aspect in the M&M. Most quantitative traits exhibit a huge variation across environments. To study the phenotypic variations it would have been much informative if all the 76 varieties were grown together across all the three fields. Why was that not considered?

5. There is no mention of how these varieties were grown in the field, and data from how many plants were considered for the morphological and oil analysis. For eg. for hundred berry weight (HBW), berries were collected from how many different plants?

6. Line 137: For the oil extraction and FA analysis, the authors mention that ‘each sample was analyzed three times’. Why weren’t three biological replicates taken for this analysis?

7. Line 180-181: The authors have used 17 previously developed SSR markers and 3 newly developed SSR markers using RNA-Seq. What was the basis of selection of just 3 new markers from the RNA-Seq. Why weren’t more markers deployed for the genetic characterization?

8. Line 180: Please reframe the sentence. It appears that the authors have done RNA-seq to generate the 3 new SSR markers. Although, the RNA-seq had been done in previous study from where the 17 SSR were also developed (Reference 17).

9. Instead of ‘different origins’ that has been used repeatedly by authors throughout the text and tables, I suggest use the two different species and hybrid accessions.

10. Line 340: ‘All the primers’. Reframe this line. All primers did not give 59 bands. A total of 59 bands were amplified.

11. Line 341: ‘accounting for 86.44%’ . Incomplete sentence, 86.44% of what??

12. Line 372: the 3 subgroups have been referred incorrectly. They are IIa, IIb and IIc.

13. Line 421: ‘in comparison of populations’. Statement not clear. Please reframe.

14. Line 436: ‘gene sequences’. Are all the SSR markers used genic in nature?

15. Table 1: Could just be described as the ‘Accessions of sea buckthorn used for the study’

**********

6. 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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: 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 to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-19-17567_reviewer.pdf

Click here for additional data file.

16 Nov 2019

Response to Reviewer' Comments letter to PLOS ONE

The authors thank the additional editor and two reviewers for their careful reading, comments and suggestion. We revised our manuscript in the best way as we could. Revised portions are marked in red in the revised manuscript. For the individual comments see our reply below.

Additional Editor Comments:

1) The use of term varieties, cultivars, subspecies and hybrids have been without much explanation. For example, what is the basis of assigning hybrid status to a particular cultivar? More clarity is required in explanation of the material. How these varieties were assigned varietal status?

Response: The Reviewer 1 gave the definition of ‘variety’ that "a variety must be recognizable by its characteristics, recognizably different from any other variety and remain unchanged through the process of propagation". The ‘cultivar’ refers to a variety of a plant developed from a natural species and maintained under cultivation. The authors accepted the reviewers’ advice that the term ‘accessions’ would be appropriate according to the plant materials in present study.

The hybrid accessions in this study generated by hybridization experiment in control between ssp. mongolica and ssp. sinensis at specialized experimental fields and selected for their desirable traits. After a complex process of identification of experts, some hybrids may became a new ‘cultivar’.

2) Generation of morphological dataset is also not mentioned clearly. You have 76 varieties growing at three locations. You need to provide environmental parameters for each location. Are all 76 growing at each location? If all the varieties are not growing at same location, many of morphological traits will be influenced by environmental factors. Did you do any multilocation trials to see the influence of environment on these traits? Did you try to collect data during different years and see if the data is consistent or showing variation. A statistical analysis of such data only will generate confidence in morphological data. Even a multilocation trial of a subset will provide information on reliability of data. Please include such data.

Response: The environmental parameters for each location were provided in the S2 Table of revised manuscript. All the accessions are not growing at same location. However, they could adapt to the environment of their cultivated lands well. We had performed some multi-location trials to see the influence of environment on berry characteristics before that was supplemented in the results of revised manuscript (S4 Table).

3) The sequencing data has been published earlier and 17 of SSR are coming from that data. Only 3 new markers have been used in the present study. This undermines the amount of data presented in this MS. You have to clearly mention these facts in the MS and the abstract. In my opinion more data needs to be generated. I suggest another 25-30 SSRs should be used for analysing the diversity.

Response: We have mentioned it in the MM and the abstract of the revised manuscript. We screened 3 new SSR loci (SB21-23) with polymorphism from 20 SSR primer pairs during the revision of the manuscript. These information has been supplemented in revised manuscript. It is difficult to develop more RNA-Seq SSRs. On one hand, the genic sequences used for developing SSR markers were highly conserved in sea buckthorn germplasm. On the other hand, the species and subspecies of sea buckthorn germplasm used in this study are limited to facilitate more polymorphism at SSR loci.

4) There is no comparison given between the varieties used in previous publication and the present one. Are you using common varities? If you are than SSR data must be same and must have been presented in previous MS already. This has not been mentioned in the MS.

Response: In previous publication, 31 accessions (common in the present one) were used for the validation of developed SSR markers. They included 6 accessions of ssp. sinensis, 14 accessions of ssp. mongolica and 11 hybrid accessions. They were selected according to their genetic origins and cultivated lands. In present study, the accessions were selected based on various fruit traits. The results of genetic relationship were different from that in the previous publication. That was supplemented in the discussion of revised manuscript.

‘In previous publication, the genetic relationship of 31 sea buckthorn accessions (also contained in the 78 accessions) were analyzed based on 17 RNA-Seq SSRs [14]. However, the accessions of ssp. mongolica clustered in one group and those of ssp. sinensis and hybrid were in the other one. That revealed the genetic diversity is related on the genotypes and genetic backgrounds.’

Reviewer #1:

Specific comments

1. I think that in such bio-prospection studies sampling strategy is very crucial. The sampling method needs to explain that how these accessions were sourced. The MS needs elaboration on –

• How many individuals of a “variety” from each site were collected?

• Are these the random collections of registered varieties from the cultivated field in the five regions OR sampled from the wild?

• It is also not clear that how the hybrids were distinguished from parents while making collections.

• Do these sites differ in climatic conditions?

• What is the link of “origin” with oil content? Did you expect that there are bound to be differences because of differences in the climatic conditions of area of collection/cultivation of the same “variety/hybrid”?

Importantly instead of the term varieties the term accessions would be appropriate, as the authors have mentioned it in Table 1. According to the definition by The International Union for the Protection of New Varieties of Plants, "a variety must be recognizable by its characteristics, recognizably different from any other variety and remain unchanged through the process of propagation".

Do these two subspecies hybridize freely in nature and such hybrids have been characterized? This needs some population analysis like by using STRUCTURE, or at least there should be a note on the characterization of hybrids (including the features), even if they are procured form some Research Institute.

Response: The part of ‘Plant materials’ in original text was revised according to above advice.

• 235 individuals (2−5 ramet plants each accession) of 5−8 years in 5 growth sites were collected.

• These are registered accessions from the cultivated field and adapt to local environment.

• For the identification of the hybrid accessions, they are labelled and recorded with documents. Furthermore, most hybrid accessions and their parents are not in the same growth site. The parents of them are cultivated in the experimental field for hybridization.

• The growth sites differ in climatic conditions which are described in S2 Table.

• According to the results in this study, the oil contents in pulp and seeds are highest in ssp. mongolica accessions on average. That is the link of origin with oil content. In this study, we ignored the difference in the climatic conditions of cultivated fields for the sea buckthorn accessions we selected adapted local environment well.

• The authors agreed the opinion that the term accessions would be appropriate and all the term varieties were revised to the term accessions.

• These two subspecies hybridized by experiments in control which were performed in specialized experimental fields, And the hybrid accessions are characterized in the Research Institutes.

2. I don’t understand the usage of term pulp/peel in the MS (also see page 15, line 251). As the entire fleshy region was separately used for extraction of oil from the "berries" (see Methods), the use of term pulp would be appropriate. One cannot expect to remove the epidermal peel especially during the mechanical homogenization process.

Response: The authors accepted the advice and the phrase ‘pulp/peel’ in the original text was revised to ‘pulp’ in the revised manuscript.

3. How the present study for the genetic diversity analysis of 78 cultivars is different from other previous studies? May be highlighted in the introduction. Authors may also highlight that trait: i.e. Oil yield was correlated with the “promising” accessions.

Response: The related content has been supplemented in the introduction of the revised manuscript.

‘The diversity analysis helps understand the relationships between germplasm characters and genotype will improve the sea buckthorn germplasm to achieve higher production of higher quality for the important traits were correlated with the promising germplasm [19].

In present study, 78 accessions of sea buckthorn with large variation of fruit traits were selected as materials.’

4. Although attempt has been made of possible use of MAB in future, but it has not been justified with the discussion. For example, do the authors will depend on the same plants in the cultivated lands across the region or some mapping populations will be established. In former case GPS tagging of the individuals will be required for sourcing the material on regular basis and to establish the consistency of the trait.

Response: The results in present study yielded useful knowledge regarding the diversity and genetic relationships of sea buckthorn germplasm in northern China, and could therefore facilitates further studies, including selection of mapping populations and promising candidates, marker-trait association analysis based on establishing the consistency of the traits , and characterizing parents used in future breeding programs. The above information on possible use of MAB in future has been supplemented in the discussion of the revised manuscript.

Materials and Methods

5. Need to mention whether hundred-berry weight, hundred-seed weight and other dimensions were taken from mature or immature berries? In Supplementary figure 1 some samples are showing immature berries e.g. sample 65, 68 etc.

Response: The hundred-berry weight, hundred-seed weight and other dimensions should be taken from mature berries. So the berries of all accessions were collected from the end of July to mid-September, according to their ripening stages. But it is difficult to collected ripening fruits of 78 sea buckthorn accessions. The berries of several accession were harvested when they are approaching maturity. So the data error existed in the dimensions of several accessions. The authors admitted it and hope be understood at this point.

6. What do the ‘Berry Shape Indices’ refer to and what are its implications on the results/oil trait/ with genetic diversity. Provide any suitable reference if possible. (Page: 8, subsection: Morphological….)

Response: The berry shape index (BSI) is estimated by the ratio of BLD to BTD, also called length/width ratio in some studies, which indicates berry shape. According to the results in present study, the phenotypic characters (BLD, HBW, BSI, and BTD) of berries and oil traits in pulp showed close correlation (r = 0.8725, p = 0.0000) using CCA. The relevant literature is below. The results of it showed that the morphological traits established were consistent with those derived from the SSR markers in olive plant materials. The length/width ratio was one of the morphological traits of endocarp in that study.

Patricia RR, Carmen GB, Beatriz CG, Jesús SG, Isabel T. Genotypic and phenotypic identification of olive cultivars from northwestern Spain and characterization of their extra virgin olive oils in terms of fatty acid composition and minor compounds. Sci Hort. 2018; 232:269-279.

7. The usage of phrase ‘8 agronomic traits’ seems to be superfluous as these are the traits of berries itself. How the seed width is different from the seed thickness? The difference is not apparent. Table 2 and 3; as well as in text.

Response: For sea buckthorn, the traits of berries (including seeds) are very important for their economic value. The seed thickness could be regarded as the ‘height’ of seeds, which is a parameter of oilseed, e.g. olive.

8. The usage of abbreviation has not been followed see table 2 and 3. Table 2 is not necessary, may be omitted or shifted to Supplementary Data. In Tables SD is not mentioned.

Response: The authors accepted the advice. Table 2 was shifted to S4 Table. The data of ‘Mean

9. The reference is missing for the SB18-SB20 SSRs; in the text (Page 10, line 181).

Response: The SB18-SB20 SSRs were firstly reported in this study and no reference could be given for them.

Results

10. Results should be given in the format mean ± SD. Minimum and maximum can be given in supplementary tables.

Response: The authors accepted the advice and the results have been given in the format mean ± SD.

11. It is not clear from the table caption and content that whether values in the Table 4 is the minimum, maximum and mean values are representing the cumulative results of 78 varieties e.g. minimum in variety… and maximum in variety…. Need to mention in the results.

Response: The authors accepted the advice. The table caption in the Table 4 of original text is not clear because we want to use the abbreviation of ‘minimum, maximum’ but the notes were forgotten to give bellow the table. And these data have been mentioned in the results in the revised manuscript.

12. The results of CCA are driving a correlation between phenotypic traits and oil characteristics. The authors may use the information for total oil content (pulp+seed) or oil content in pulp and seeds separately for drawing any correlation. That would possibly help as a descriptor for the potential crop in identifying the elite/superior “variety” and further can be linked to genetic diversity.

Response: For the difference in the FAs composition between pulp oil and seed oil, the total oil content was not be used for drawing any correlation in this study. In practical production, the seed oil and pulp oil are separately extracted for their different functions. During the course of CCA, the factors in each data matrix would be analyzed by pairwise correlation analysis. So oil content in pulp and seeds separately for drawing any correlation is not necessary.

Discussion

13. Page:28, Line:449-453. The link of this part of discussion is lacking with the previous text.

Response: In the part of ‘Introduction’, the superiority of SSR markers was mentioned. The significance of developing SSR markers with RNA-Seq technique was also mentioned in it. The SSR markers used in this study are developed by RNA-Seq. All these description was the link of this part of discussion.

14. In conclusion part authors are concluding that this information may be useful for cultivar identification but initially they started their work for the varieties. Taxonomically these two are different entities.

Response: The authors agreed this opinion. The phrase ‘cultivar identification’ was revised to ‘germplasm identification’ and all the word ‘varieties’ were changed into ‘accessions’ in the revised manuscript according to the taxonomical definition.

Some suggestion:

1. The sequence of S1 and S2 table can be reversed as per the citation in the text.

Response: The good advice mentioned above is accepted by the authors. The tables were reversed in the revised manuscript.

2. Page:3, Line:54. Reference 1 is incorrect. The lead author here is Bartish I.V.

Response: The authors in reference 1 were corrected in the revised manuscript.

3. Page:3, Line:56-57. ….flavonoids [3-7]; ….products [8-10]. Here over-citation may be avoided.

Response: The authors accepted the advice and the references cited in the two sentences were cut down in the revised manuscript.

4. Page:3, Line:59. ‘Sea buckthorn oil’ instead of ‘sea buckthorn oils’

Response: The phrase was corrected in the revised manuscript.

5. Page: 4, Line 74. Add a reference to the statement. The plant is able to avoid cold and is not resistant, because the leaves are shed under extreme cold condition in this plant. Even the species is not resistant to alkali too.

Response: The authors agreed this opinion and this sentence was revised to ‘Sea buckthorn adapts well to extreme conditions, including drought, salinity, alkalinity, and temperatures [12]’ in the revised manuscript.

12. Ruan CJ, Li H, Mopper S. Characterization and identification of ISSR markers associated with resistance to dried-shrink disease in sea buckthorn. Mol. Breeding. 2009; 24:255−268.

6. Page:4. Line:85. Use full form at first place ‘MAB’.

Response: The sentence was corrected in the revised manuscript and the full form ‘molecular marker-assisted breeding’ was used at first place ‘MAB’.

7. Page:5. Line:110. What was the premise of including two known elite varieties in the study? Any supportive reference(s) for the statement, and also mention the context in which these varieties are elite.

Response: The premise of elite varieties include high yield, good agronomic traits and strong adaptability to environment, etc. Some Chinese references support that Quyisike and Zhongguoshajiwild are elite cultivars. The word ‘elite’ in the sentence was deleted in the revised manuscript for no English reference supported it.

8. Page:12. Line:204-205. May be included in Material and Methods.

Response: The authors accepted the advice and the sentence ‘Minimum, maximum, mean, standard deviation (SD), and coefficient of variation (CV%) were recorded.’ was added in Material and Methods of the revised manuscript.

Reviewer #2:

1. The authors mention that 76 varieties were used. There is no mention of the different species they belonged to in M&M, although it has been mentioned later in the text and table. Incorporate that information in the M&M.

Response: The good advice mentioned above is accepted by the authors. The related information has been added in M&M of the revised manuscript.

2. Are these 76 different varieties or just different accessions? At many places they are being referred to as ‘cultivars’ also. Please correct accordingly in the text wherever mentioned.

Response: After careful consideration, the authors thought ‘accessions’ would be appropriate. The 'varieties' has been replaced into ‘accessions’ in the revised manuscript.

3. How variable are the climatic conditions of the three research institutes?

Response: The climatic conditions of different growth sites of sea buckthorn samples has been added in S2 Table of the revised manuscript, with the caption ‘Geographical and climatic conditions at different sample collection sites of sea buckthorn in northern China’.

4. Line 109: ‘………provided 76 varieties’. Does this mean that all the 76 were grown at all the 3 fields? There is no clarity on this aspect in the M&M. Most quantitative traits exhibit a huge variation across environments. To study the phenotypic variations it would have been much informative if all the 76 varieties were grown together across all the three fields. Why was that not considered?

Response: Among the 76 accessions of sea buckthorn samples, 12 were grown in the Institute of Selection and Breeding of Hippophae, 52 were grown in the Research Institute of Berry and 12 were grown in the Jiuchenggong Breeding Base of Sea Buckthorn. These accessions are able to adapt to local climate and screened to be excellent germplasm.

The authors agreed the opinion that most quantitative traits exhibit a huge variation across environments. We did the comparative analysis on fruit morphological traits of the same cultivars grown in different cultivated fields in our early studies and the data was complemented in the results (S4 Table) of the revised manuscript. The aim in this study is to further screen the elite accessions from the 78 accession with good adaption to the environments of cultivated fields and prepare for the next step of MAB. In the follow-up study, the continuous observation of the environmental factors would be considered.

5. There is no mention of how these varieties were grown in the field, and data from how many plants were considered for the morphological and oil analysis. For eg. for hundred berry weight (HBW), berries were collected from how many different plants?

Response: The information has been supplemented in the introduction of the revised manuscript. The sea buckthorn samples in this study are collected from 235 individuals (2−5 ramet plants each accession) of 5−8 years in 5 growth sites. The berries of each accession were pooled and frozen as quickly as possible at −20 °C. When all plant materials were harvested, the berries were transferred to –50 °C for storage until analysis. The related information has been supplemented in the M&M and Table 1.

6. Line 137: For the oil extraction and FA analysis, the authors mention that ‘each sample was analyzed three times’. Why weren’t three biological replicates taken for this analysis?

Response: The authors are sorry for the incorrect expression. In this study, three biological replicates were taken for every analysis. The sentence is corrected in the revised manuscript.

7. Line 180-181: The authors have used 17 previously developed SSR markers and 3 newly developed SSR markers using RNA-Seq. What was the basis of selection of just 3 new markers from the RNA-Seq. Why weren’t more markers deployed for the genetic characterization?

Response: The authors obtained many SSR sequences using RNA-Seq method and designed the primers to screen those SSR loci with polymorphism in sea buckthorn cultivars. We reported 17 developed SSR markers at first. In subsequent experiments, we screened 3 new SSR markers which also showed polymorphic amplification in sea buckthorn germplasm. RNA-Seq SSR loci with polymorphism in sea buckthorn germplasm were difficult to develop for that SSR markers derived from expressed region of genome showed high conservation to some extent in our study. That’s why no more markers deployed for the genetic characterization in sea buckthorn at now.

8. Line 180: Please reframe the sentence. It appears that the authors have done RNA-seq to generate the 3 new SSR markers. Although, the RNA-Seq had been done in previous study from where the 17 SSR were also developed (Reference 17).

Response: The authors accepted the advice and the sentence has been changed into ‘The Twenty polymorphic microsatellite loci (SSR) developed using RNA-Seq were evaluated and loci SB1-SB17 were previously published [17]’ in the revised manuscript.

9. Instead of ‘different origins’ that has been used repeatedly by authors throughout the text and tables, I suggest use the two different species and hybrid accessions.

Response: The authors accepted the good advice. Some ‘different origins’ were changed into the ‘two different subspecies and hybrid accessions’ and the others were deleted in the revised manuscript.

10. Line 340: ‘All the primers’. Reframe this line. All primers did not give 59 bands. A total of 59 bands were amplified.

Response: The sentence has been revised according to the advice in the revised manuscript.

11. Line 341: ‘accounting for 86.44%’ . Incomplete sentence, 86.44% of what??

Response: The sentence has been revised according to the advice in the revised manuscript.

12. Line 372: the 3 subgroups have been referred incorrectly. They are IIa, IIb and IIc.

Response: The names of 3 subgroups were corrected in the revised manuscript.

13. Line 421: ‘in comparison of populations’. Statement not clear. Please reframe.

Response: The phrase has been changed to ‘in population identification’ in the revised manuscript.

14. Line 436: ‘gene sequences’. Are all the SSR markers used genic in nature?

Response: SSR can be divided into genomic SSRs and genic SSRs because of the resource of sequences used for SSR identification. Genic SSRs derived from transcriptome or expressed sequence tag sequences are located in expressed genes. These markers can be linked with important phenotypic characteristics through quantitative trait loci analysis. In this study, all SSR markers are genic SSRs.

15. Table 1: Could just be described as the ‘Accessions of sea buckthorn used for the study’

Response: The authors accepted the good advice and the title of Table 1 was revised into the ‘Accessions of sea buckthorn used for the study’.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Dec 2019

PONE-D-19-17567R1

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Ruan,

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.

We would appreciate receiving your revised manuscript by Feb 10 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Both the reviews for revision have been received. One of the reviewer is mostly satisfied with the response from authors but other still has certain queries. Both the reviewers have pointed out queries regarding presentation of data, especially the inconsistencies in the SSR data. One of the reviewer has provided a sanitised version of MS. The biggest problem Is the quality of written English. The MS requires substantial improvement in English quality. I suggest authors have to take help from a professional. They also have to address the inconsistencies in MS. Authors also need to answer all the questions raised about data.

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #2: (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 #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #2: No

**********

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 #2: No

**********

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: Most of the queries have been answered and incorporated in text appropriately. However, some of them need quick attention.

1. In the abstract authors have mentioned they have used 23 SSR markers out of which 17 were previously reported. Did they assessed 6 new SSRs while in response/text the number of new makers tested is 3. Query:9 (revision 1)

2. The query 7 related to seed width and seed thickness is not answered suitably, though reference is given. Seed thickness can not be regarded as seed height. Seed thickness may be similar to seed width/diameter.

3. Answer/justification for query 12 need to be recheck and if so…..it must be mentioned in materials and methods of the manuscript.

Reviewer #2: General Comments:

The manuscript has a lot of issues with the language. Many such sentences have been highlighted in the document attached. Some of these appear as two half statements fused. At other places, the sentences lack clarity. The authors need to re-frame all such statements.

The Materials & Methods needs to be revised at places (please see suggestions). Most trait measures in various tables lack the unit of measurements. Please incorporate that.

Specific comments:

1. The text inconsistently mentions the deployment of 20 SSR primer pairs at certain places (line 357, 450, Header of Table 5) and at other places (Line 27, 41, 46, 197, 358, 373, 383, 451, 482) the use of 23 SSR primer pairs has been mentioned. The supplementary table (S4) gives sequence information for 23, while its header says 20 SSR primers. Table 5, gives information for 23 markers although the Header says 20. Please ensure that all these ambiguities are taken care of.

2. Abstract says 69 polymorphic bands, while in results 59 polymorphic bands are mentioned. This ambiguity also needs to be addressed.

Introduction:

1. Line 62: ‘Two important parameters in……..oil quantity are oil content’. Oil content cannot be a parameter of oil quality. So, this statement needs modification.

2. Line 78: ‘Due to small berries………………….artificial hybridization for elite accessions.’ The statement needs to be reframed.

3. Line 98: ‘The diversity analysis…………………………germplasm’. The authors appear to have have fused to incomplete sentences. This needs to be re-wrtitten.

All other such statements have been highlighted in the document attached.

Materials & Methods

1. Line 139: ‘There were three biological replicates………measurement’.

Do the authors mean that 300 berries were taken for the analysis? 100 berries from 2-5 plants/ accession is a good enough number for the analysis.

2. Line142: ‘…with over 20 measurements…for each’

This is not clear. Do the authors mean 20 berries per accession?? And how many plants did these berries belong to?

3. For the oil extraction, were the seeds and fruit pulp weighed prior to oil extraction to maintain some uniformity. This has not been mentioned in the M&M.

The oil contents in both seeds and fruit pulp as mentioned in Line 153 is expressed as percentage. Percentage of what? Seed/pulp weight? The authors need to clearly mention that in the M&M.

In the results (Line 278), the authors mention ‘….highest oil content (24.68%) based on dry weight.’ This means that the weight of the pulp/seed was considered. But, this has not been clearly mentioned either in the M&M or in the Table 3. The units for oil characteristic (min and max) have not been mentioned in the table.

4. Line 197: ‘Twenty-three polymorphic microsatellite loci (SSR) developed using RNA-Seq was evaluated and loci SB1-SB17 were previously reported’.

Please mention here the names of the SSR markers (SB1-SB23). Nowhere in the text have they been mentioned except for tables. Then it can be mentioned that SB1-17 were previously deployed (Ref. 14).

The authors need to clearly mention in the introduction itself that in a previous study, RNA seq analysis was done to generate SSR markers and these were tested on 31 accessions. The 17 SSR markers developed in that study have been utilized in the present endeavor for genetic diversity assessment of larger set of accessions. This description in the ‘introduction’ will bring more clarity in the text. This previous study and its outcomes should be mentioned clearly in the ‘Introduction’ so that its extension in the present study can be deciphered.

Results:

1. Line 246: ‘In previous mutilocation trials in Suiling (47°14′N, 127°06′E; 202 m) and Dengkou

(40°43′N, 106°30′E; 1053m, Inner Mongolia), the fruit characteristics of 11 large……’.

How many berries per accession were taken for this analysis? The data should be represented as + SD in Table S7.

2. Line 302: ‘Small variations were found in the proportion of linoleic acid in seed oil (40.44 – 42.87%). Its proportion in hybrids were slightly higher than in ssp. mongolica (42.87% vs 42.10%.....’

Are these differences significant?

3. Table 4: How is the oil content being measured? Total oil per gram weight of seeds and pulp or some other measure?

Tables and Figures

1. Table 1: Since the authors have already mentioned that 2-5 ramet plants were collected per accession. The columns indicating the number of plants taken per accession can be removed from the Table.

2. Table 3: The units for the min. and max values of the oil characteristics have not been mentioned in the table. Similarly mention the units of measurement for each of the component in Table 4.

3. Table 3 & 4: The different fatty acid names should be included in the first column. Example: Oleic (18:1), Palmitic acid (16:0) etc.

4. Table S1: This table again classifies all the lines used as ‘cultivars’. Are these accessions or cultivars? Please check.

5. Table S3 carries a different header than the one that has been listed at the end of the manuscript. Please change that.

6. Table S7: The header for this table has been titled as Table S5. Please correct. Also it mentions ‘two experimental fields’ although it has data from three places. So, please correct.

**********

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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: 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 to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-19-17567_R1-comments.pdf

Click here for additional data file.

Submitted filename: Comments.docx

Click here for additional data file.

24 Jan 2020

General Comments:

The manuscript has a lot of issues with the language. Many such sentences have been highlighted in the document attached. Some of these appear as two half statements fused. At other places, the sentences lack clarity. The authors need to re-frame all such statements.

Response: Thank you for the valuable suggestion. The sentences mentioned above are all re-framed with clarity. And the revised manuscript was professionally edited by American Journal Experts (AJE, ID: HS1GCXH7) for the improvement in English quality.

The Materials & Methods needs to be revised at places (please see suggestions). Most trait measures in various tables lack the unit of measurements. Please incorporate that.

Response: Thank you for the valuable suggestion. The places mentioned above in the Materials & Methods have been revised according to the reviewers’ suggestions. And the unit of measurements has been added in the tables of the revised manuscript.

Specific comments:

1. The text contradictorily mentions the deployment of 20 SSR primer pairs at certain places (line 357, 450, Header of Table 5) and at other places (Line 27, 41, 46, 197, 358, 373, 383, 451, 482) the use of 23 SSR primer pairs has been mentioned. The supplementary table (S4) gives sequence information for 23, while its header says 20 SSR primers. Table 5, gives information for 23 markers although the Header says 20. Please ensure that all these ambiguities are taken care of.

Response: Twenty-three SSR primer pairs were used in this study. The number ‘20’ at certain places (line 357, 450, Headers of Table 5 and S4 Table) have been replaced by 23 in the revised manuscript.

2. Abstract says 69 polymorphic bands, while in results 59 polymorphic bands are mentioned. This ambiguity also needs to be addressed.

Response: The number of polymorphic bands is 59. It has been revised in the abstract of the revised manuscript.

Introduction:

1. Line 62: ‘Two important parameters in……..oil quantity are oil content’. Oil content cannot be a parameter of oil quality. So, this statement needs modification.

Response: The authors agreed with this opinion. The oil content is a parameter of oil yield. The sentence has been revised as bellow.

‘Two important parameters in analyzing oil yield and quality are oil content and fatty acid (FA) composition (referred to here as ‘oil traits’ for simplicity).’

2. Line 78: ‘Due to small berries………………….artificial hybridization for elite accessions.’ The statement needs to be reframed.

Response: Thank you for the valuable suggestion. The statement is reframed as bellow.

‘Due to the small berries and thorns of native cultivars (ssp. sinensis), which result in little economic value, the breeding of sea buckthorn has undergone different stages of development in China, such as introduction, domestication, seedling selection and artificial hybridization for elite accessions.’

3. Line 98: ‘The diversity analysis…………………………germplasm’. The authors appear to have fused to incomplete sentences. This needs to be re-written.

All other such statements have been highlighted in the document attached.

Response: Thank you for the valuable suggestion. All such statements are re-written in the revised manuscript.

Materials & Methods

1. Line 139: ‘There were three biological replicates………measurement’.

Do the authors mean that 300 berries were taken for the analysis? 100 berries from 2-5 plants/ accession is a good enough number for the analysis.

Response: Yes. 300 berries were taken for the analysis. We collected more than 300 berries per accession and the analyses of other nutrients were performed in our research work, e.g. vitamin C, vitamin E and carotenoids.

2. Line142: ‘…with over 20 measurements…for each’

This is not clear. Do the authors mean 20 berries per accession?? And how many plants did these berries belong to?

Response: The authors agreed with this view. It means averaged 20 determinations were done for each character. These berries were selected from the berry samples randomly collected from 2-5 ramet plants per accession. This sentence mentioned above has been re-framed for clarity in the revised manuscript as bellow.

‘The transverse and longitudinal diameters of berries (BTD and BLD) and the length, width and thickness of seeds (SL, SW and ST) were measured over 20 times each (on average) by micrometer calipers.’

3. For the oil extraction, were the seeds and fruit pulp weighed prior to oil extraction to maintain some uniformity. This has not been mentioned in the M&M.

The oil contents in both seeds and fruit pulp as mentioned in Line 153 is expressed as percentage. Percentage of what? Seed/pulp weight? The authors need to clearly mention that in the M&M.

In the results (Line 278), the authors mention ‘….highest oil content (24.68%) based on dry weight.’ This means that the weight of the pulp/seed was considered. But, this has not been clearly mentioned either in the M&M or in the Table 3. The units for oil characteristic (min and max) have not been mentioned in the table.

Reponse: Thank you for the valuable suggestion. The method of lipid extraction was described by Yang and Kallio (2001). Samples (1 g) of seeds and fruit pulp were isolated from freeze-dried berries and lipids from the samples were extracted with chloroform/methanol (2:1, v/v) with mechanical homogenization of the tissues. The oil contents (percentages) in seeds and fruit pulp were calculated (oil % in seeds and lyophilized fruit pulp). The fatty acid composition was also expressed as a weight percentage of the total fatty acids. The units (weight percentages) for oil characteristics in Table 3 and Table 4 have been added in the revised manuscript.

4. Line 197: ‘Twenty-three polymorphic microsatellite loci (SSR) developed using RNA-Seq was evaluated and loci SB1-SB17 were previously reported’.

Please mention here the names of the SSR markers (SB1-SB23). Nowhere in the text have they been mentioned except for tables. Then it can be mentioned that SB1-17 were previously deployed (Ref. 14).

The authors need to clearly mention in the introduction itself that in a previous study, RNA seq analysis was done to generate SSR markers and these were tested on 31 accessions. The 17 SSR markers developed in that study have been utilized in the present endeavor for genetic diversity assessment of larger set of accessions. This description in the ‘introduction’ will bring more clarity in the text. This previous study and its outcomes should be mentioned clearly in the ‘Introduction’ so that its extension in the present study can be deciphered.

Reponse: Thank you for the valuable suggestion. The sentences mentioned above have been re-framed for clarity in the revised manuscript as bellow.

‘Twenty-three polymorphic microsatellite loci (SB1-SB23) developed using RNA-Seq were evaluated. Of these, 17 (SB1-SB17) had been deployed in a previous study by the group [14].’

And the authors added the statements of 17 RNA-Seq SSR markers developed in our previous study and mentioned these SSR markers have been utilized in the present endeavor for genetic diversity assessment of larger set of accessions in the revised manuscript.

‘In our previous study, 17 RNA-Seq SSR markers (SB1-SB17) were developed and validated on 31 accessions, which were utilized in the present study for genetic diversity assessment of larger set of accessions [14].’

Results

1. Line 246: ‘In previous mutilocation trials in Suiling (47°14′N, 127°06′E; 202 m) and Dengkou (40°43′N, 106°30′E; 1053m, Inner Mongolia), the fruit characteristics of 11 large……’.

How many berries per accession were taken for this analysis? The data should be represented as + SD in Table S7.

Reponse: 300 berries of each cultivar were randomly sampled and divided into 3 groups (100 berries were divided into 1 group) to determine the hundred berry weight (HBW). 20 berries of each cultivar were randomly sampled to determine the transverse, longitudinal diameters of berries and berry shape indices (BTD, BLD and BSI). The data has been represented as + SD in S7 Table in the revised manuscript.

2. Line 302: ‘Small variations were found in the proportion of linoleic acid in seed oil (40.44 – 42.87%). Its proportion in hybrids were slightly higher than in ssp. mongolica (42.87% vs 42.10%.....’

Are these differences significant?

Reponse: These differences are significant despite small variations. The content of seed oil in hybrids is lower than that in ssp. mongolica. However, the proportion of linoleic acid (an important polyunsaturated fatty acid) in seed oil is higher in hybrids than that in ssp. mongolica, which showed high oil quality of seed oil in hybrids.

3. Table 4: How is the oil content being measured? Total oil per gram weight of seeds and pulp or some other measure?

Reponse: The method of lipid extraction was described by Yang and Kallio (2001). Samples (1 g) of seeds and fruit pulp were isolated from freeze-dried berries and lipids from the samples were extracted with chloroform/methanol (2:1, v/v) with mechanical homogenization of the tissues. The oil contents (percentages) in seeds and fruit pulp were calculated (oil % in seeds and lyophilized fruit pulp).

Tables and Figures

1. Table 1: Since the authors have already mentioned that 2-5 ramet plants were collected per accession. The columns indicating the number of plants taken per accession can be removed from the Table.

Reponse: The columns indicating the number of plants taken per accession have been removed from Table 1 in the revised manuscript.

2. Table 3: The units for the min. and max values of the oil characteristics have not been mentioned in the table. Similarly mention the units of measurement for each of the component in Table 4.

Reponse: The units of the oil characteristics have been added in the headers of Table 3 and Table 4.

3. Table 3 & 4: The different fatty acid names should be included in the first column. Example: Oleic (18:1), Palmitic acid (16:0) etc.

Reponse: Thank you for the valuable suggestion. The different fatty acid names are included in the first column of Table 3 and Table 4 according to the examples.

4. Table S1: This table again classifies all the lines used as ‘cultivars’. Are these accessions or cultivars? Please check.

Reponse: The ‘accession’ has replaced the ‘cultivar’ in S1 Table of revised manuscript.

5. Table S3 carries a different header than the one that has been listed at the end of the manuscript. Please change that.

Reponse: The header of S3 Table listed at the end of the manuscript has been changed in the revised manuscript.

6. Table S7: The header for this table has been titled as Table S5. Please correct. Also it mentions ‘two experimental fields’ although it has data from three places. So, please correct.

Reponse: The header for S7 Table has been corrected. The mutilocation trials were performed in two experimental fields (Suiling and Dengkou). Russia is the country of origin of those cultivars and the related data were provided by the units where they were introduced.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Feb 2020

PONE-D-19-17567R2

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Ruan,

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.

==============================

ACADEMIC EDITOR:

Both the reviewers have agreed with the changes made in Manuscript. One of the reviewer has suggested some changes in the MS and has provided a sanitized version of MS, the authors are requested to include these changes in MS. I think we will not require any further reviewing and as and when MS is submitted with required changes, the MS can be accepted.

==============================

We would appreciate receiving your revised manuscript by Mar 30 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #2: 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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #2: 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 #2: (No Response)

**********

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: As most of my queries have been attended, the MS may accepted for publication provided the other reviewer has also reached a similar decision.

Reviewer #2: The authors have made the suggested changes and the manuscript should be accepted for publication after few minor changes as suggested below. These have also been highlighted in the manuscript attached.

1. Some text changes are suggested: Line 36, 77, 78, 84, 87, 362, 375, 452, 461, 462, 489

2. Line 307, 345: Lacks clarity.

3. Suggest changing Table 5 title to: ‘Genetic diversity analyses of 78 accessions of sea buckthorn germplasm using 23 SSR markers’.

4. As previously suggested to the authors, since for each of the traits multiple measurements were taken, please include +/- SD values for each trait/accession in Table S6.

**********

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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: 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 to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-19-17567_R2-Comments.pdf

Click here for additional data file.

25 Feb 2020

The authors thank two reviewers for their careful reading, comments and suggestion. We revised our manuscript in the best way as we could. Revised portions are marked in red in the revised manuscript. For the individual comments see our reply below.

Reviewer #1: As most of my queries have been attended, the MS may accepted for publication provided the other reviewer has also reached a similar decision.

Response: The authors thank for your acceptance of the revised MS.

Reviewer #2: The authors have made the suggested changes and the manuscript should be accepted for publication after few minor changes as suggested below. These have also been highlighted in the manuscript attached.

1. Some text changes are suggested: Line 36, 77, 78, 84, 87, 362, 375, 452, 461, 462, 489.

Response: The authors thank for your suggestions. The text changes mentioned above have been corrected in the revised MS. The details are as follows.

Line 36: The word ‘approximately’ has been deleted.

Line 77,78: The sentence has been revised to ‘However, the fruits of native cultivars are small and thorny and of low economic value, which encourages the breeding of sea buckthorn has undergone different stages of development in China’.

Line 84: The word ‘a’ has been deleted.

Line 87: The word ‘associated’ has been deleted.

Line 362: The word ‘in’ has been revised to ‘among’.

Line 375: The sentence has been changed into ‘The characteristics of 23 polymorphic SSR markers in sea buckthorn accessions are shown in Table 5’.

Line 452: The word ‘identify’ has been revised to ‘investigate the genetic relationships among 78 sea buckthorn accessions’.

Line 461: The phrase ‘between ssp. sinensis and ssp. mongolica accessions’ has been changed into ‘between accessions of ssp. sinensis and ssp. mongolica’.

Line 462: The words ‘This result illustrated’ has been revised to ‘This uniformity indicated’.

Line 489: The word ‘Hippophae’ has been revised to ‘H.’.

2. Line 307, 345: Lacks clarity.

Response: The authors accepted the opinion and revised these sentences for clarity. The details are as follows.

Line 307: The sentence ‘and had the highest value of the samples from the two different subspecies and hybrid accessions’ has been revised to ‘and showed the highest mean value among the two different subspecies and hybrid accessions’.

Line 345: The sentence ‘that were primarily provided by a marker of BLD’ has been revised to ‘that were primarily provided by the phenotypic character of BLD’.

3. Suggest changing Table 5 title to: ‘Genetic diversity analyses of 78 accessions of sea buckthorn germplasm using 23 SSR markers’.

Response: The authors thank for your suggestion. The title of Table 5 has been changed into ‘Genetic diversity analyses of 78 accessions of sea buckthorn germplasm using 23 SSR markers’ in the revised MS.

4. As previously suggested to the authors, since for each of the traits multiple measurements were taken, please include +/- SD values for each trait/accession in Table S6.

Response: The authors thank for you suggestion. The SD values for each trait/accession have been added in Table S6.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Feb 2020

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PONE-D-19-17567R3

Dear Dr. Ruan,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Both the reviewers are now satisfied by the changes made in the MS. Henceforth, I recommend the publication of this MS in PLOSOne. I thank you for your patience during the process and hope that authors will appreciate all the efforts from the two reviewers in improving the manuscript.

Reviewers' comments:

3 Mar 2020

PONE-D-19-17567R3

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

Dear Dr. Ruan:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Shailendra Goel

Academic Editor

PLOS ONE