The ITS analysis and identification of Actinidia eriantha and its related species.

The dried plant material of medically important plant Actinidia eriantha especially when it remains in the form of powder often look morphologically similar to its related species. The lack of efficient methods to distinguish the authentic material from other similar species leads to chances of adulteration. The molecular authentication of herbal plant materials such as the internal transcribed spacer (ITS) sequences is considered as more reliable method compared to morphological traits. In this study, we aim to evaluate the potential of identification for roots of A. eriantha and its related species by ITS sequences. The lengths of ITS regions ranged from 624 to 636 bp with GC content ranging from 50.96% to 59.55%. A total of 194 variation sites and 46 haplotypes were formed in 185 samples. Among them, the roots of A. eriantha possessed specific sites at 85bp (C), 205bp (T), 493bp (C), 542bp (G), 574bp (C), 582bp (T) and 610bp (G), while A. hemsleyana, A. callosa, A. valvata and A. polygama have their own specific sites. The inter-specific genetic distance among 8 Actinidia species in the range 2.28% to 11.00%. The phylogenetic tree constructed with ITS, ITS1 and ITS2 region showed that the ITS sequences have higher potential for identification in 8 Actinidia species. However, as to A. eriantha, A. hemsleyana and A. valvata, these three barcodes have the same identification ability. The ITS regions indicated that different samples from same species can be grouped together, except for A. arguta and A. melanandrah. In conclusion, the ITS sequences can be used as an efficient DNA barcode for the identification of A. eriantha and its related species.

1 Introduction

The quality and safety of medical materials are important guarantees for the health and legal rights of patients. The roots of Actinidia Eriantha Benth. is a traditional Chinese medicine. It has been included in the Standard of TCM processing in Zhejiang Province in 2015 [1], and has the function of clearing heat and detoxifying, dampness and swelling in the Traditional Chinese Medicines. The roots A. Eriantha are often used to treat breast carbuncle, rheumatic pain, tumor and other diseases [2, 3].

The genus Actinidia (family: Actinidiaceae) includes approximately 66 species, about 62 of which are distributed in China. Among them, A. Eriantha, A. valvata, A. macrosperma, and A. arguta, are identified as important medicinal species by the Standard of TCM processing in Zhejiang Province in 2015 [1]. However, it is difficult to identify them by leaves due to the diverse variability in morphological characters and short flowering and fruiting period [4]. And the medicinally important A. Eriantha species need exhaustive taxonomic expertise for taxonomic identification especially to discriminate among A. Eriantha, A. valvata, A. macrosperma, and A. arguta. The dried plant material of A. Eriantha in the form of powder looks very similar to A. Eriantha, A. valvata, A. macrosperma, and A. arguta and therefore lead to chances of adulteration [5]. For example, the root of A. Eriantha had good inhibitory effect on liver cancer and colorectal cancer [3, 6], whereas the A. valvata and A. macrosperma had obvious inhibitory effect on gastric cancer and lung cancer, and the anti-tumor effect of A. arguta was seldom reported [7, 8]. So, the adulteration in medicinal plants affect the efficacy of the drug Fig 1.

Fig 1

Leaf image of 8 species of Actinidia.

Note: All pictures from Plant Photo Bank of China.

The DNA sequence based molecular authentication of herbal plant materials such as the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) is considered as a more reliable method as compared to the methods based on morphological traits and biochemical methods [9, 10]. Although some scholars used SSR and RADP techniques to analyze the genetic diversity of the root of A. Eriantha [11, 12], and some scholars also studied the phylogenetic development of the genus of Actinidia by using matK and rbcL sequences [13], no report hitherto has been found to use ITS sequences to conduct species identification for the root of A. Eriantha and its related species.

In this study, we attempted to use the ITS sequences for rapid identification of eight Actinidia species in China. This study will not only lay a foundation for phylogenetic analysis but also provides experimental and practical basis for the rapid authentication of A. Eriantha.

2 Materials and methods

2.1 Plant materials

All 8 Actinidia species were wild species, A. Eriantha were collected from Lishui city, Zhejiang Province and Nanping City, Fujian Province, in China, respectively, and other Actinidia species were collected from Lishui City, Zhejiang Province, in China. A total of 185 samples were collected, including 53 samples from Genbank gene database (Table 1). All voucher specimens are kept in Lishui Hospital of Traditional Chinese Medicine.

Table 1

Samples information.

Species Name	Locatiy information	Samples No.	Samples size	Haplotype(number)
Actinidia eriantha	Qingtian, Lishui, Zhejiang	DY(1~5)	5	MH1(4),MH2(1)
	Dagangtou,Lishui, Zhejiang	HK(1~5)	5	MH1(5)
	DAshanfeng,Lishui, Zhejiang	SF(1~12)	12	MH1(12)
	Wuyishan, Nanping, Fujian	WY(1~2)	2	MH1(1),MH3(1)
	Pucheng, Nanping, Fujian	PC(1~7)	7	MH1(4), MH3(2), MH4(1)
	Pucheng, Nanping, Fujian	FG	1	MH1(1)
	Longquan,Lishui, Zhejiang	AR(1~2)	2	MH1(2)
	Longquan,Lishui, Zhejiang	LQ	1	MH1(1)
	Shakengkou, Lishui, Zhejiang	SK(1~4)	4	MH1(2), MH3(1),MH5(1)
	Lishan,Lishui, Zhejiang	LL(1~13)	13	MH1(11), MH3(1), MH4(1)
	Genbank (AF323800)	Ae-1	1	MH1(1)
	Genbank (MG714287)	Ae-2	1	MH6(1)
	Genbank (MK425151)	Ae-3	1	MH1(1)
	Genbank (AF323801)	Ae-4	1	MH1(1)
	Genbank (KP314044)	Ae-5	1	MH7(1)
	Genbank (KP314039)	Ae-6	1	MH8(1)
	Genbank (KP314038)	Ae-7	1	MH9(1)
A. polygama	Lishan,Lishui, Zhejiang	GT(1~10)	10	GZ1(1)
	Genbank (KP314060)	Ap-1	1	GZ2(1)
	Genbank (KP314056)	Ap-2	1	GZ1(1)
	Genbank (KP314051)	Ap-3	1	GZ3(1)
	Genbank (KP314035)	Ap-4	1	GZ1(1)
	Genbank (KC519766)	Ap-5	1	GZ4(1)
	Genbank (KC519765)	Ap-6	1	GZ4(1)
	Genbank (AF323796)	Ap-7	1	GZ5(1)
	Genbank (MW234501)	Ap-8	1	GZ2(1)
	Genbank (MW234500)	Ap-9	1	GZ2(1)
A. melanandra	Lishan,Lishui, Zhejiang	HT(1~10)	10	HL1(1)
	Genbank (AF443211)	Am-1	1	HL2(1)
	Genbank (MG714285)	Am-2	1	HL1(1)
	Genbank (MG714282)	Am-3	1	HL3(1)
	Genbank (AF323808)	Am-4	1	HL4(1)
A. arguta	Lishan,Lishui, Zhejiang	RT(1~10)	10	WD1(1)
	Genbank (KP314061)	Aa-1	1	WD2(1)
	Genbank (KP314034)	Aa-2	1	WD3(1)
	Genbank (AF323836)	Aa-3	1	WD4(1)
	Genbank (AF323835)	Aa-4	1	WD5(1)
	Genbank (MW234506)	Aa-5	1	WD4(1)
	Genbank (KP314062)	Aa-6	1	WD6(1)
	Genbank (MW234481)	Aa-7	1	WD4(1)
	Genbank (MW234478)	Aa-8	1	WD4(1)
A. chinensis	Lishan,Lishui, Zhejiang	ZT(1~10)	10	ZH1(10)
	Shakengkou, Lishui, Zhejiang	ZK	10	ZH1(9), ZH2(1)
	Genbank (KC832305)	Ach-1	1	ZH3(1)
	Genbank (KC832307)	Ach-2	1	ZH4(1)
	Genbank (MH711099)	Ach-3	1	ZH1(1)
	Genbank (MH710876)	Ach-4	1	ZH1(1)
	Genbank (KC832316)	Ach-5	1	ZH5(1)
	Genbank (KC519784)	Ach-6	1	ZH6(1)
	Genbank (KC832302)	Ach-7	1	ZH6(1)
A. valvata	Lishan,Lishui, Zhejiang	DT(1~10)	10	DE1(10)
	Genbank (MG714283)	Av-1	1	DE2(1)
	Genbank (MG714281)	Av-2	1	DE3(1)
	Genbank (KC519764)	Av-3	1	DE4(1)
	Genbank (KC519763)	Av-4	1	DE4(1)
	Genbank (KC519762)	Av-5	1	DE4(1)
	Genbank (AF323842)	Av-6	1	DE5(1)
A. hemsleyana	Lishan,Lishui, Zhejiang	CT(1~10)	10	CY1(10)
	Genbank (MG714284)	Ah-1	1	CY1(1)
	Genbank (KC519783)	Ah-2	1	CY2(1)
	Genbank (KC519782)	Ah-3	1	CY2(1)
	Genbank (KC519781)	Ah-4	1	CY2(1)
	Genbank (AF323802)	Ah-5	1	CY3(1)
A. callosa	Lishan,Lishui, Zhejiang	YT(1~10)	10	YS1(10)
	Genbank (AF323803)	Aca-1	1	YS2(1)
	Genbank (MH808396)	Aca-2	1	YS3(1)
	Genbank (MH808395)	Aca-3	1	YS4(1)
	Genbank (KC519753)	Aca-4	1	YS5(1)
	Genbank (KC519739)	Aca-5	1	YS6(1)
	Genbank (AF323804)	Aca-6	1	YS7(1)
	Genbank (AF323828)	Aca-7	1	YS8(1)
Saurauia tristyla	Genbank (KP092594)	ST	1	ST1(1)

Note: Saurauia tristyla is the outgroup.

2.2 DNA extraction, amplification, and sequencing

Fresh young leaves of the collected samples were used for genomic DNA isolation, as previously reported. The pair of universal primers used to amplify nrDNA ITS sequences consist of P1 (5’- AGAAGTCGTAACAAGGTTTCCGTAGG- 3’) and P4 (5’- TCCTCCGCTTATTGATATGC-3’). PCR amplification was conducted using 50 μL volumes containing 5×PCR Buffer 5 μL, dNTPs (5 mmol/L) 5 μL, MgCL (25 mmol/L) 4 μL and each primer (0.5 μM) 2 μL, and 1 U Taq DNA polymerase. PCR amplification was conducted with the following parameter settings: holding at 94°C for 5 min followed by 35 cycles at 94°C for 30 s, at 56°C for 45 s, and at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were analyzed by 1.0% agarose gel electrophoresis, and then were sequenced in Shanghai Shenggong Biotechnology Co., Ltd, Shanghai, China.

2.3 DNA analysis

The ContingExpress and DNAman software were used to align the ITS sequences, analyze the sequence length, variation sites, and this was assisted by manual editing. The Editseq and Mega-X software were used to construct the Phylogenetic tree, calculate the inter-specific and intras-pecific genetic distances. Phylogenetic analysis was conducted according to the maximum likelihood (ML), with the following parameter: 1000 bootstrap replicates. The inter-specific and intras-pecific genetic distances were conducted according to the K2-P models.

2.4 additional information regarding the permits

The research material is plant leaves, it is not involve ethical protection and genetic information protection, so no additional information regarding the permits for the work we need.

3 Results

3.1 PCR efficiency and characteristics of ITS sequences

The success rates of PCR amplification and sequencing of ITS regions from the sampled specimens were 100%. The lengths of ITS regions were ranged from 624 to 636 bp with GC content ranging from 50.96% to 59.55%. The ITS sequence was divided into ITS1, 5.8S and ITS2 sequences, as shown in Table 2. The 5.8s sequences of the eight Actinidia species were highly conserved, with 164bp in length. The lengths of ITS1 regions were ranged from 240 to 246 bp with GC content ranging from 49.79% to 60.25%, and the ITS2 regions were ranged from 218 to 223 bp with GC content ranging from 49.77% to 62.73%.

Table 2

Composition of ITS sequences of 8 Actinidia species.

Species Name	Haplotype	Sequences lengthens of ITS1 (bp)	GC content of ITS1 (%)	Sequences lengthens of 5.8S (bp)	G+C%	Sequences lengthens of ITS2 (bp)	G+C content of ITS2 (%)	Sequences lengthens of ITS (bp)
A. eriantha	MH1	245	55.51	164	53.66	220	56.82	629
A. eriantha	MH2	244	55.33	164	53.66	220	56.82	628
A. eriantha	MH3	245	55.92	164	53.66	220	56.82	629
A. eriantha	MH4	245	55.92	164	53.66	220	57.27	629
A. eriantha	MH5	245	55.51	164	53.66	220	56.82	629
A. eriantha	MH6	245	55.10	164	53.66	220	56.82	629
A. eriantha	MH7	245	56.73	164	53.66	220	57.27	629
A. eriantha	MH8	245	56.33	164	53.66	220	56.82	629
A. eriantha	MH9	245	56.33	164	53.66	220	56.36	629
A. polygama	GZ1	244	51.64	164	52.44	219	55.71	627
A. polygama	GZ2	243	51.85	164	53.05	219	54.79	626
A. polygama	GZ3	243	51.44	164	53.05	219	54.79	626
A. polygama	GZ4	244	51.23	164	52.44	219	55.71	627
A. polygama	GZ5	242	51.65	164	53.05	219	54.79	625
A. melanandra	HL1	244	59.84	164	54.27	220	62.73	628
A. melanandra	HL2	246	58.13	168	52.98	222	61.26	636
A. melanandra	HL3	245	59.18	164	54.27	220	62.73	629
A. melanandra	HL4	245	58.78	164	54.27	220	60.45	629
A. arguta	WD1	245	58.78	164	54.27	220	61.82	629
A. arguta	WD2	244	59.84	164	54.27	220	62.73	628
A. arguta	WD3	244	58.61	164	54.88	220	62.73	628
A. arguta	WD4	244	60.25	164	54.27	220	62.73	628
A. arguta	WD5	240	59.17	164	54.27	220	62.73	624
A. arguta	WD6	243	58.02	164	54.88	220	62.73	627
A. chinensis	ZH1	243	49.79	164	54.27	219	49.77	626
A. chinensis	ZH2	243	50.62	164	54.27	219	51.60	626
A. chinensis	ZH3	243	50.62	164	54.27	219	51.60	626
A. chinensis	ZH4	243	50.21	164	54.27	219	52.05	626
A. chinensis	ZH5	243	50.62	164	54.27	219	52.05	626
A. chinensis	ZH6	243	50.62	164	54.27	219	52.05	626
A. valvata	DE1	244	55.74	164	53.66	221	61.09	629
A. valvata	DE2	244	55.74	164	53.66	221	60.63	629
A. valvata	DE3	243	55.56	164	53.66	221	61.09	628
A. valvata	DE4	244	54.92	164	53.66	223	60.99	631
A. valvata	DE5	244	55.74	164	53.66	221	60.63	629
A. hemsleyana	CY1	243	50.21	164	53.05	219	50.23	626
A. hemsleyana	CY2	244	50.00	164	53.05	219	50.68	627
A. hemsleyana	CY3	243	50.21	164	53.66	218	50.46	625
A. callosa	YS1	244	56.15	164	54.27	219	56.16	627
A. callosa	YS2	243	55.97	164	54.27	219	56.62	626
A. callosa	YS3	244	55.33	164	54.27	219	56.62	627
A. callosa	YS4	244	58.20	164	54.27	220	57.27	628
A. callosa	YS5	244	58.20	164	54.27	221	57.01	629
A. callosa	YS6	244	56.15	164	54.27	219	56.62	627
A. callosa	YS7	242	56.61	164	54.27	218	56.42	624
A. callosa	YS8	243	53.09	164	54.27	220	56.82	627

3.2 Analysis of haplotype and variable sites

A total of 46 haplotypes were formed in 185 samples (Table 1), among which 9 haplotypes were formed in A. eriantha, 5 in A. polygama, 4 in A. melanandra, 6 in A. arguta, 6 in A. chinensis, 5 in A. valvata, 3 in A. hemsleyana, and 5 in A. callosa. Analysis of Variation sites in all samples is shown in Table 3. There are 194 variation sites in 8 Actinidia species, except for A. melanandra, A. arguta and A. chinensis, the other 5 species have unique Variable sites. For example, bases at 85 bp, 205 bp, 493 bp, 542 bp, 574 bp, 582 bp, and 610 bp of A. eriantha are C, T, C, G, C, C, G, respectively. Bases at 28 bp, 79 bp, 83 bp, 109 bp, 128 bp, 448 bp, and 501 bp of A. hemsleyana are T, A, A, T, A, T, T, respectively. These mutated loci can be considered as the unique identification loci for the root of A. eriantha, A. polygama, A. valvata, A. hemsleyana and A. callosa.

Table 3

Analysis of base variation in 8 species of Actinidia.

Haplotype	base variation																																						GenbanK No.
	ITS1																	5.8S	ITS2
	2	4	7	7	8	8	9	9	9	1	1	1	1	1	1	2	2	4	4	4	4	4	4	4	4	5	5	5	5	5	6	6	6	6	6	6	6	6
	8	8	3	9	3	5	2	3	5	0	1	2	2	4	8	0	3	1	2	3	3	4	4	8	9	0	3	4	7	8	0	0	0	0	0	1	2	4
										9	5	2	8	7	4	5	2	6	2	5	6	6	8	7	3	1	0	2	4	2	2	3	4	5	6	0	0	0
MH1	C	C	C	C	T	C	C	C	T	C	G	G	/	G	T	T	C	G	T	A	C	A	C	T	C	C	A	G	C	C	T	T	A	C	T	G	G	/	OK036712
MH2	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	OK036713
MH3	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	OK036795
MH4	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	OK036796
MH5	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	OK036797
MH6	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	MG714287
MH7	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	KP314044
MH8	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	KP314039
MH9	*	*	*	*	*	*	*	*	*	*	*	*	/	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	*	/	KP314038
CY1	T	*	T	A	A	T	*	*	*	T	*	*	A	*	*	A	*	*	*	*	*	*	T	*	T	T	*	A	T	G	*	*	*	*	*	T	*	/	OK036804
CY2	T	*	T	A	A	T	*	*	*	T	*	*	A	*	*	A	*	*	*	*	*	*	T	*	T	T	*	A	T	G	*	*	*	*	*	T	*	/	KC519783
CY3	T	*	T	A	A	T	*	*	*	T	*	*	A	*	*	A	*	*	*	*	*	*	T	*	T	T	*	A	T	G	*	*	*	*	*	T	*	/	AF323802
DE1	*	G	A	*	*	T	T	*	C	*	*	C	/	*	C	A	A	*	*	C	*	*	*	C	T	*	G	A	T	G	C	*	G	*	*	C	T	T	OK036803
DE2	*	G	A	*	*	T	T	*	C	*	*	C	/	*	C	A	A	*	*	C	*	*	*	C	T	*	G	A	T	G	C	*	G	*	*	C	T	T	MG714283
DE3	*	G	A	*	*	T	T	*	C	*	*	C	/	*	C	A	A	*	*	C	*	*	*	C	T	*	G	A	T	G	C	*	G	*	*	C	T	T	MG714281
DE4	*	G	A	*	*	T	T	*	C	*	*	C	/	*	C	A	A	*	*	C	*	*	*	C	T	*	G	A	T	G	C	*	G	*	*	C	T	T	KC519764
DE5	*	G	A	*	*	T	T	*	C	*	*	C	/	*	C	A	A	*	*	C	*	*	*	C	T	*	G	A	T	G	C	*	G	*	*	C	T	T	AF323842
GZ1	*	T	T	*	*	T	*	T	*	*	A	*	/	T	*	A	*	A	*	*	T	*	*	*	T	*	*	A	T	G	*	G	C	T	C	T	*	/	OK036798
GZ2	*	T	T	*	*	T	*	T	*	*	A	*	/	T	*	A	*	A	*	*	T	*	G	*	T	*	*	A	T	G	*	G	C	T	C	T	*	/	KP314060
GZ3	*	T	T	*	*	T	*	T	*	*	A	*	/	T	*	A	*	A	*	*	T	*	G	*	T	*	*	A	T	G	*	G	C	T	C	T	*	/	KP314051
GZ4	*	T	T	*	*	T	*	T	*	*	A	*	/	T	*	A	*	A	*	*	T	*	*	*	T	*	*	A	T	G	*	G	C	T	C	T	*	/	KC519766
GZ5	*	T	T	*	*	T	*	T	*	*	A	*	/	T	*	A	*	A	*	*	T	*	G	*	T	*	*	A	T	G	*	G	C	T	C	T	*	/	AF323796
HL1	*	*	/	G	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	G	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	OK036799
HL2	*	*	T	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	T	*	*	C	*	/	AF443211
HL3	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	G	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	MG714282
HL4	*	*	/	G	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	T	*	*	C	*	/	AF323808
WD1	*	*	T	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	OK036800
WD2	*	*	/	G	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	KP314061
WD3	*	*	T	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	KP314034
WD4	*	*	/	G	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	AF323836
WD5	*	*	/	G	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	AF323835
WD6	*	*	T	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	G	*	*	C	*	/	KP314062
YS1	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	OK036805
YS2	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	AF323803
YS3	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	MH808396
YS4	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	T	*	*	*	*	*	C	*	/	MH808395
YS5	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	T	*	*	*	*	*	C	*	/	KC519753
YS6	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	KC519739
YS7	*	*	*	*	*	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	AF323804
YS8	*	T	T	T	C	T	*	*	*	*	*	*	/	*	*	A	*	*	C	*	*	G	*	*	T	*	*	A	T	T	*	*	*	*	*	C	*	/	AF323828
ZH1	*	T	T	T	C	T	*	*	*	*	*	*	/	A	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	OK036801
ZH2	*	T	T	T	C	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	OK036802
ZH3	*	*	T	T	*	T	*	*	*	*	*	*	/	A	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	KC832305
ZH4	*	*	T	T	C	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	KC832307
ZH5	*	*	T	T	*	T	*	*	*	*	*	*	/	*	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	KC832316
ZH6	*	*	T	T	*	T	*	*	*	*	*	*	/	A	*	A	*	*	*	*	*	*	*	*	T	*	*	A	T	G	*	*	*	*	*	T	*	/	KC519784

Note: M = A+C, R = A+G, /, base deletion.

3.3 K2P genetic distance analysis

The genetic distance of 8 Actinidia species based on ITS sequence is shown in Tables 4 and 5. The intra-specific genetic distance of A. callosa, A. arguta and A. melanandra is 2.18%, 2.13% and 2.26%, respectively. And then, the intra-specific genetic distance of A. eriantha and A. valvata is less than A. callosa, A. arguta and A. melanandra, which is 0.48% and 0.35%, respectively.

Table 4

Intra-specific genetic distance of 8 species of Actinidia.

Species name	YS	WD	ZH	HL	GZ	DE	CY	MH
Genetic distance	2.18%	2.13%	1.65%	2.26%	0.94%	0.35%	0.59%	0.48%

Note: DE = A. valvata, GZ = A. polygama, HL = A. melanandrah, CY = A. hemsleyana, MH = A. eriantha, ZH = A. chinensis, YS = A. callosa, WD = A. arguta.

Table 5

Genetic distances among 8 species of Actinidia.

Species name	YS	WD	ZH	HL	GZ	DE	CY	MH
YS
WD	9.86%
ZH	5.69%	10.15%
HL	10.11%	2.28%	10.28%
GZ	6.95%	9.73%	6.42%	9.94%
DE	9.01%	9.72%	9.60%	10.04%	7.51%
CY	5.99%	10.89%	2.88%	11.00%	6.53%	10.46%
MH	4.77%	9.56%	6.19%	9.87%	5.97%	7.77%	6.36%

Note: DE = A. valvata, GZ = A. polygama, HL = A. melanandrah, CY = A. hemsleyana, MH = A. eriantha, ZH = A. chinensis, YS = A. callosa, WD = A. arguta.

The inter-specific genetic distance among 8 Actinidia species ranged from 2.28% to 11.00%. The genetic distance between A. melanandra and A. hemsleyana was the highest (11.00%), while the genetic distance between A. melanandra and A. arguta was the lowest (2.28%). For A. eriantha, there was a largest genetic distance between A. eriantha and A. arguta (9.56%), and the smallest genetic distance between A. eriantha and A. callosa (4.77%).

3.4 Phylogenetic analysis

ITS1, ITS2 and ITS sequences were tested 1000 times by bootstrap method to build ML phylogenetic tree (Fig 2), with Saurauia tristyla as the outgroup. Phylogenetic tree of ITS sequences showed that all of the tested samples can be grouped into two main clusters. One cluster includes A. eriantha, A. callosa, A. chinensis and A. hemsleyana, and each species also clustered separate group respectively. The other cluster includes A. polygama, A. valvata, A. arguta and A. melanandrah, among which A. polygama and A. valvata clustered into each separate group respectively, and A. arguta and A. melanandrah clustered into one group. Phylogenetic tree of ITS1 sequences showed that A. arguta and A. melanandrah clustered into one group, and the other 6 species were group into another category. Moreover, in this category, A. eriantha, A. hemsleyana, A. polygama and A. valvata were separately distinguished, and A. callosa and A. chinensis scattered in the category. Phylogenetic tree of ITS2 sequences shows similar results with ITS sequence, except A. hemsleyana and A. chinensis which are clustered into one group. For ITS sequences, except A. arguta and A. melanandrah the other 6 species can be distinguished separately from others. For ITS1 sequences, A. eriantha, A. hemsleyana, A. polygama and A. valvata can be distinguished separately from the other 4 varieties. For ITS2 sequences, A. eriantha, A. callosa, A. polygama and A. valvata can be distinguished separately from the other 4 varieties.

Fig 2

Phylogenetic tree of 8 species of Actinidia.

(a) Analysis of ITS sequences fragments; (b) Analysis of ITS1 sequences fragments; (c) Analysis of ITS2 sequences fragments.

4 Discussion

As early as in the “compendium of materia medica”, there is a record of the medicinal plants of the Actinidia. Many Actinidia species with high commercial value have received considerable attention because of their edible fruits, medical value and ornamental values [14-16]. Therefore, their accurate identification is highly in demand. However, currently the identification of Actinidia mainly depends on the characteristics of the original plant, which requires a high degree of experience, especially when the plant is dry [17]. For example, A. eriantha and A. chinensis have a high degree of similarity, in terms of the body of the plant, the size of the fruit and the coat characteristics of the fruit, which are very closely. The only distinguishing feature is that the coat of A. eriantha is milky white, thick and dense [18]. The ITS region has been used as a DNA barcode to authenticate various plants with similar morphological traits [19]. In this study, the ITS sequences were used for barcoding Actinidia species for the first time.

The ITS sequence analysis of 8 Actinidia species showed that there were 194 mutants, including A. eriantha (85 bp C、205 bp T、493 bp C、542 bp G、574 bp C、582 bp T、610 bp G), A. hemsleyana (28 bp T、79 bp A、83 bp A、87 bp C、109 bp T、128 bp A、448 bp T、501 bp T), A. valvata (48bpG、73bpA、92bpT、95bpC、122bpC、184bpC、232bpA、435bpC、487bpC、530bpG、602bpC、620bpT、640bpT), A. polygama (93 bp T、115 bp A、147 bp T、416 bp A、436 bp T、603 bp G、605 bp T、606 bp C)and A. callosa (422 bp C、446 bp G). All of them have their own specific loci. These specific loci can be used as the basis for the identification of several Actinidia species.

Many researchers have reported that ITS region has the advantages of high intra-specific conservation, large inter-specific variation, small fragment, easy amplification, easy analysis and high success rate for species identification [20, 21]. Similar results were found in our study. We found that the genetic distance between the most samples from the same species in this study is less than 1% (A. eriantha, A. hemsleyana, A. valvata, A. polygama), and there were no specific loci differentiating among them. This feature is thus useful for identifying different Actinidia species and related species.

The phylogenetic tree which was constructed with ITS, ITS1 and ITS2 region showed that the ITS sequences have higher identification ability in 8 Actinidia species. However, these three barcodes have the same identification ability for A. eriantha, A. hemsleyana and A. valvata. The ITS regions indicated that different samples from same species can be grouped together, except for A. arguta and A. melanandrah. Previoues study reported two groups, one group includes A. eriantha, A. callosa, A. chinensis and A. hemsleyana, the other group includes A. polygama, A. valvata, A. arguta and A. melanandrah, which are based on matK gene. Whereas, A. eriantha, A. callosa, A. chinensis, A. hemsleyana and A. polygama clustered into one group based on rbcL gene. Our study is consistent with the results indicated by matK gene. Therefore, the ITS sequences are useful for species identification, as well as contribute to the phylogenetic analysis of Actinidia and its related species.

5 Conclusion

Our study demonstrates that the ITS sequences possess high species discriminability and it could be a useful DNA barcode for Actinidia species. However, more Actinidia species should be collected in the future to verify whether ITS sequences that could be used to identify all species of Actinidia. In addition, ML tree analyses provided a solid evidence that the ITS sequences have the potential in the phylogenetic analysis of Actinidia and its related species.

Mutation loci analysis of 8 Actinidia.

(XLS)

Click here for additional data file.

6 Jun 2022

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Additional Editor Comments:

Dear authors,

based on reviewer's comments and also from my expertise your article has many deficiencies and needs further improvement. So please answer reviewers's somments and additionaly please answer to the following comments:

1. why did you prefer ITS although discriminations power of SSR are more accurate? is it less expensive to do a sequencing analysis rather genotyping?

2. please provide some photos also for the difficult part of morphological description

3. finally the manuscript needs to be critically proof read before resubmitted.

With kind regards

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Reviewer #1: Partly

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Reviewer #1: I Don't Know

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Reviewer #1: No

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Reviewer #1: 1. The authors suggested ITS as a reliable marker for species identification of Actinidia eriantha and its Related Species (n=185 samples which includes 53 samples from Genbank). It is not clear from the methodology whether these are their own samples or from others, thus questioning the reliability of species vouchering. This is crucial to be clarified due to: “However, it is difficult to identify them due to the diverse variability in morphological characters[4].”, as stated by the authors. And it's also not clear if the new barcodes generated from the other haplotypes listed in Table 1 have been submitted and verified by Genbank.

2. Furthermore, the methods lack details and are only vaguely described, without proper citation. Eg: "Fresh young leaves of the collected samples were used for genomic DNA isolation, as previously reported". A supplementary data of the characteristics of all 8 species would be extremely helpful to convince the readers on the morphological species identification.

3. The authors generated a good dataset but the way it was reported and discussed is really confusing and only partly supports the overall conclusion.

4. Overall, the manuscript is poorly written and difficult to follow. The manuscript needs to be critically proof read before resubmitted for further review. Obvious language mistakes are observed throughout the manuscript.

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4 Aug 2022

1. Dr. Bin Wu of Zhejiang University thoroughly copyedit manuscript for language usage, spelling, and grammar.

2. The minimal data set underlying the results described in your manuscript can be found in Genbank(accession numbers see S1 Table).

3. Ms. Xiaoqin Zhang was funded by [Zhejiang Provincial Natural Science Foundation of China] grant number [LGF20H280005], Mr. Houxing Lei was funded by[development project in Lishui city] grant number [2020ZDYF 15] and Ms. Yan Lan was funded by [Lishui Science and Technology project] grant number [2021SJZC039].

Ms. Xiaoqin Zhang – Methodology and Writing–original draft

Mr. Houxing Lei –Conceptualization

Ms. Yan Lan –Software

4. We add Leaf image of 8 species of Actinidia in manuscript for the difficult part of morphological description.

5. The research material is plant leaves , it is not involve ethical protection and genetic information protection, so no additional information regarding the permits for the work we need.

6. SSR has some shortcomings: 1) the detection of SSR polymorphism largely depends on the effect of PCR amplification; 2) The existing SSR markers are limited in number, and can not mark all functional genes; 3) The development and synthesis of primers are expensive, difficult and time-consuming.

Submitted filename: Response to Reviewers.docx

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26 Aug 2022

The ITS Analysis and Identification of Actinidia eriantha and its Related Species

PONE-D-22-07942R1

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7 Sep 2022

PONE-D-22-07942R1

The ITS Analysis and Identification of Actinidia eriantha and its Related Species

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