Reliability and Utility of Standard Gene Sequence Barcodes for the Identification and Differentiation of Cyst Nematodes of the Genus Heterodera

Figure 1

Figure 2

Figure 3

Species pairs of Heterodera which are inadequately or poorly distinguished from one another using one or more of the standard molecular markers evaluated (18S, ITS, 28S, coxI), including minimum base pair differences observed between sequences of problematic species pairs_

Species group	Species pair	Gene region	Minimum base pair difference	Delineation power	Notes
Avenae	H. avenae–H. filipjevi	18S	1	Inadequate
Avenae	H. avenae–H. hordecalis	18S	4	Weak	H. hordecalis not monophyletic
					in NJ/ML analyses; potentially
					distinguishable from closely
					related species in isolated
					analyses.
Avenae	H. avenae–H. mani	18S	1	Inadequate
Avenae	H. filipjevi–H. hordecalis	18S	1	Inadequate
Avenae	H. filipjevi–H. mani	18S	2	Inadequate
Avenae	H. hordecalis–H. mani	18S	3	Weak	See above note.
Avenae	H. arenaria–H. avenae	ITS	0	Inadequate
Avenae	H. avenae–H. pratensis	ITS	0	Inadequate
Avenae	H. avenae–H. australis	ITS	2	Weak	Although only two bp different,
					sequences of H. australis form
					clade with some sequences
					of “H. avenae” which have
					been shown to be H. australis
					(see Subbotin et al., 2018).
Avenae	H. avenae–H. mani	ITS	2	Weak	Despite only two bp difference,
					sequences of H. mani form
					monophyletic clade to
					exclusion of other closely
					related sequences.
Avenae	H. aucklandica–H.	28S	1	Inadequate	Only one 28S sequence of
	avenae				H. aucklandica available for
					comparison.
Avenae	H. aucklandica–H.	28S	1	Inadequate	Two 28S sequences of
	hordecalis				H. hordecalis 9 bp different
Avenae	H. aucklandica–H.	28S	1	Inadequate	Only one 28S sequence of
	pratensis				H. pratensis available for
					comparison.
Avenae	H. avenae–H. pratensis	28S	0	inadequate	See above note.
Avenae	H. avenae–H. hordecalis	28S	0	Inadequate	Two 28S sequences of
					H. hordecalis 9 bp different
Avenae	H. arenaria–H. avenae	cox1	2	Weak	Despite only two bp
					difference, H. arenaria forms
					monophyletic clade within
					H. avenae group.
Cyperi	H. elachista–H. oryzae	ITS	1	Inadequate
Cyperi	H. elachista–H. oryzae	28S	0	Inadequate
Goettingiana	H. carotae–H. cruciferae	ITS	1	Inadequate
Goettingiana	H. goettingiana–H.	28S	0	Inadequate	Two 28S sequences of
	microulae				H. goettingiana are 19 bp
					different from one another; one
					potentially misidentified
Goettingiana	H. carotae–H. urticae	28S	2	Weak	Only one 28S sequence
					of H. urticae available for
					comparison.
Goettingiana	H. carotae–H. cruciferae	28S	2	Weak	Only one 28S sequence of
					H. cruciferae available for
					comparison (two on GenBank
					but one appears to actually be
					H. schachtii).
Goettingiana	H. carotae–H. cruciferae	cox1	1	Inadequate	Three cox1 sequences
					available for H. cruciferae, one
					15 bp different from others.
Schachtii	H. betae–H. glycines	18S	2	Weak	H. glycines forms
					monophyletic clade to
					exclusion of H. betae,
					H. schachtii & H. trifolii in ML,
					but not NJ, analyses.
Schachtii	H. betae–H. schachtii	18S	0	Inadequate
Schachtii	H. betae–H. trifolii	18S	0	Inadequate
Schachtii	H. schachtii–H. glycines	18S	2	Weak	See above note.
Schachtii	H. schachtii–H. trifolii	18S	0	Inadequate
Schachtii	H. trifolii–H. glycines	18S	2	Weak	See above note.
Schachtii	H. betae–H. daverti	ITS	0	Inadequate
Schachtii	H. betae–H. schachtii	ITS	0	Inadequate
Schachtii	H. betae–H. trifolii	ITS	0	Inadequate
Schachtii	H. ciceri–H. schachtii	ITS	2	Inadequate
Schachtii	H. ciceri–H. trifolii	ITS	1	Inadequate
Schachtii	H. daverti–H. schacthii	ITS	0	Inadequate
Schachtii	H. daverti–H. trifolii	ITS	0	Inadequate
Schachtii	H. glycines–H.	ITS	1	Inadequate
	medicaginis
Schachtii	H. schachtii–H. trifolii	ITS	0	Inadequate
Schachtii	H. betae–H. daverti	28S	1	Inadequate
Schachtii	H. betae–H. schachtii	28S	2	Inadequate
Schachtii	H. betae–H. trifolii	28S	1	Inadequate
Schachtii	H. daverti–H. schachtii	28S	3	Weak
Schachtii	H. daverti–H. trifolii	28S	1	Inadequate
Schachtii	H. schachtii–H. trifolii	28S	1	Inadequate

Numbers of species of Heterodera for which one or more gene sequences of one or more molecular markers (18S, ITS, 28S, and coxI) are available on GenBank_

	Marker
Species of Heterodera	18S	ITS	28S	coxI	All
Species with at least one sequence	18	55	36	38	13
Species with more than one sequence	13	38	22	29	11

Species of Heterodera, and associated number of sequences, recorded from 59 countries based on data from the NCBI database GenBank, up to 10 August 2021_

		Total number of sequences recorded
Species	Country	18S	ITS	28S	coxI
Heterodera arenaria	Italy		1		3
	Netherlands				2
	United Kingdom		3		1
Heterodera aucklandica	Belgium		1
	New Zealand		1	1	3
	United Kingdom		1		2
Heterodera australis	Australia		7		5
	China		11
Heterodera avenae	Algeria	4		6
	Australia			1
	Azerbaijan		9
	China	4	359	57	4
	Czech Republic	7	7	7
	Egypt		5		3
	France		9		7
	Germany		3		4
	India		4		1
	Iran		2	1	4
	Iraq		3
	Israel		3		4
	Morocco		27		5
	Norway				4
	Pakistan		1
	Saudi Arabia		23	1	1
	Serbia		1		1
	South Korea		1	2	1
	Spain		3		3
	Switzerland				1
	Syria		3		6
	Tunisia		1		6
	Turkey		39		26
	United Kingdom		1
	USA	1	10	3	14
	Unknown	4
Heterodera betae	Belgium		1
	Germany		3
	Netherlands		3	2	2
	Unknown	2
Heterodera bifenestra	Belgium		2
	Sweden	1	1
Heterodera cajani	India		12	13
Heterodera cardiolata	Pakistan		1	1
	United Kingdom				1
Heterodera carotae	Belgium				1
	Algeria		2
	Canada		5	6	5
	France		1		2
	Italy		3	1	5
	Mexico		2		1
	South Africa		1	1	1
	Switzerland				1
Heterodera ciceri	Syria		3		1
Heterodera circeae	Germany		1
Heterodera cruciferae	Algeria		5
	Iran		1	1
	Italy		1	1
	Netherlands		1
	Russia		1		1
	Serbia		1
	USA	1			2
Heterodera cyperi	Spain		1
	USA		1		1
Heterodera daverti	Germany		3		2
	Italy		1	1	1
	Netherlands		1		1
Heterodera dunensis	Spain	3	3	3	8
Heterodera elachista	China	1	27	10
	Iran		2	1	1
	Italy	2	4	4	2
	Japan		11	3
Heterodera fici	Canada		2
	Georgia		1		1
	Greece				4
	Iran		1		3
	Italy		2	1
	Qatar		1
	USA	2	1		1
Heterodera filipjevi	Algeria		2
	Azerbaijan		20
	China		58	7	2
	Germany		2		1
	Greece				1
	India		1
	Iran		5		19
	Italy		1
	Kazakhstan		25
	Kyrgyzstan		31
	Russia		3		7
	Serbia		1		2
	Slovakia		1
	Spain		1		2
	Sweden	1			1
	Syria				6
	Tajikistan		1		3
	Turkey		80		20
	Ukraine				3
	United Kingdom		1		3
	USA		3	1	5
	Unknown	2
Heterodera glycines	Canada	1	12	1
	Chile			1
	China	5	275	29
	Iran		2	1
	Japan		6	10
	South Korea USA	1	4 21	7 7	81
	Unknown		1	1
Heterodera goettingiana	China	1	10	1	3
	Germany		2	1
	Iran		1
	Ireland		1
	Italy		2
	Netherlands	4
	USA		1
Heterodera goldeni	Egypt	2
	Iran	2
	Israel	1
Heterodera graminis	India		1
Heterodera graminophila	USA	1
Heterodera guangdongensis	China		3	3
	Myanmar			2
	Vietnam			2	1
Heterodera hainanensis	China		1	1
Heterodera hordecalis	Algeria	2	21	2
	Estonia		1
	Germany		1		2
	Iran		4		3
	Israel		3		2
	Italy		2
	Netherlands	1	2
	Slovakia		1		2
	Sweden		1		1
	Tunisia		1
	United Kingdom		1
Heterodera humuli	Belgium				1
	Germany		1		2
	Iran		3	1	8
	Kyrgyzstan		1		1
	Portugal		1
	Russia		1		2
	USA		6		5
Heterodera koreana	China	1	2	2
	Iran		1	1
	Japan		20	41	41
	South Korea		3	3	3
	USA	2	3	1
Heterodera latipons	Cyprus		21
	Iran		3		3
	Israel				2
	Jordan		2	3
	Kazakhstan		28
	Morocco		3
	Russia		1		1
	Syria		9	1	7
	Turkey		87		3
Heterodera litoralis	New Zealand		1	1	4
Heterodera mani	Germany		4		1
	United Kingdom		1
	USA		1		4
	Unknown	1
Heterodera medicaginis	Russia		1
	USA		13	1	14
Heterodera mediterranea	Italy	2	2
	Spain		2
	Tunisia	1	2	2
Heterodera microulae	China		1	1	1
Heterodera mothi	Azerbaijan		1		1
	Iran		1		1
Heterodera orientalis	Guatemala		2
	Russia		1
	USA		2	1
Heterodera oryzae	South Korea		1	1	1
Heterodera oryzicola	India		1	1
Heterodera persica	Iran		1
Heterodera pratensis	Belgium				1
	Germany		8		8
	Iran				5
	Netherlands		1		1
	Russia		1		2
	South Korea		2	1	2
	USA		1		1
Heterodera ripae	Belgium Germany		1		5 4
	Greece		1		2
	Russia Serbia		1 1		4
	Slovakia				2
	Sweden		1
	United Kingdom				2
Heterodera sacchari	Ghana		2
	Ivory Coast		1
Heterodera salixophila	Belgium		1	1	3
	Germany				4
	Iran		1	1
	Russia				5
	Ukraine		3		4
Heterodera schachtii	Algeria		1
	Australia		4
	Belgium	1	15
	China	1	1	8	3
	France		1	3
	Germany		4
	Iran		2		1
	Ireland	2
	Japan		1	1	1
	Mexico		1		2
	Morocco		3
	Netherlands		8	1	1
	Poland		1		1
	Serbia		3
	South Africa		1
	South Korea		3	4	7
	Turkey		12
	USA		2	3	22
	Unknown	6	1
Heterodera scutellariae	Germany		1
Heterodera sinensis	China		1
Heterodera skohensis	India		1
Heterodera sojae	China		1	1
	Japan		1	1
	South Korea		3	8	12
Heterodera sorghi	India		1
Heterodera sturhani	China				7
Heterodera trifolii	Costa Rica		1	1	1
	Estonia		1
	Iran		1	1	2
	Italy				1
	Japan		8	8	8
	South Korea		8	8	9
	United Kingdom	1	2	1
	USA		2		5
	Unknown	4
Heterodera turcomanica	Iran		1		1
Heterodera urticae	Belgium		1	1	3
Heterodera ustinovi	Belgium		1
	Germany		1
	Slovakia		1
	United Kingdom		1
	USA		1		2
Heterodera vallicola	Russia		1		1
Heterodera zeae	Afghanistan			1
	China		5
	Greece	1	5	1
	India		4
	Portugal			1
	USA		2	1

Total number of sequences of species of Heterodera per country available on the NCBI database GenBank, up to 10 August 2021_

	Number of sequences recorded
	ITS	18S	28S	coxI	Total
Afghanistan			1		1
Algeria	62	7	8		77
Australia	11		1	6	18
Azerbaijan	36			1	37
Belgium	21	1	2	14	38
Canada	19	1	7	5	32
Chile			1		1
China	761	16	131	21	937
Costa Rica	1		1	1	3
Cyprus	21				21
Czech Republic	7	7	7		21
Egypt	7			3	10
Estonia	2				2
France	11			9	20
Georgia	1			1	2
Germany	35		1	28	64
Ghana	2				2
Greece	6	1	1	7	15
Guatemala	2				2
India	26		15	1	42
Iran	41		16	51	108
Iraq	3			8	11
Ireland	1	2			3
Israel	7				7
Italy	19	6	8	12	45
Ivory Coast	1				1
Japan	47		64	50	161
Jordan	2		3		5
Kazakhstan	53				53
Kyrgyzstan	32			1	33
Madagascar	2				2
Mexico	3			3	6
Morocco	34			5	39
Myanmar			2		2
Netherlands	16	2	3	7	28
New Zealand	2		2	7	11
Norway				4	4
Pakistan	2		1		3
Poland	1			2	3
Portugal	1		1		2
Qatar	1				1
Russia	12			23	35
Saudi Arabia	23		1	1	25
Serbia	7			3	10
Slovak Republic	3			4	7
South Africa	3		1	1	5
South Korea	30		38	36	104
Spain	10	3	3	13	29
Sweden	4	2		2	8
Switzerland				3	3
Syria	15		1	21	37
Tajikistan	1			3	4
Tunisia	4	1	2	6	13
Turkey	219			53	272
Ukraine	3		1	7	11
United Kingdom	11		1	9	21
USA	71	7	18	158	254
Vietnam			2	1	3
Unknown	2	21	1	1	25
Total	1724	77	346	592	2739

Intraspecific differences observed in the 18S, ITS, 28S, and coxl gene regions for those species of Heterodera for which more than one sequence of one or more of the respective gene regions are available, excluding problematic sequences_

	18S		ITS		28S		cox1
Species	n	bp (%)	n	bp (%)	n	bp (%)	n	bp (%)
Heterodera arenaria			4	0–1; 0.50 (0–0.29; 0.15)			6	0–2; 2 (0–0.48; 0.48)
Heterodera aucklandica			3	0(0)			5	0–1; 0 (0–0.24; 0)
Heterodera australis			18	0–9; 3 (0–0.93; 0.31)			5	0(0)
Heterodera avenae	17	0–11; 1 (0–1.09; 0.06)	542	0–80; 8 (0–11.89; 0.89)	76	0–5; 0 (0–0.67; 0)	91	0–34; 9 (0–8.17; 2.40)
Heterodera betae	2	1 (0.06)	7	0–12; 3 (0–1.29; 0.37)	2	0(0)	2	0(0)
Heterodera bifenestra			3	2–10; 8 (0.20–1.02; 0.81)
Heterodera cajani			12	0–12; 4 (0–1.18; 0.39)	13	0–11; 2 (0–1.46; 0.26)
Heterodera carotae			13	0–13; 6 (0–1.35; 0.62)	8	0–6; 3 (0–0.83; 0.41)	17	0–20; 3 (0–4.77; 0.82)
Heterodera ciceri			3	7–14; 11 (0.74–1.49; 1.18)
Heterodera cruciferae			11	0–10; 3 (0–1.21; 0.36)	2	52 (9.49)	3	1–16; 16 (0.24–4.01; 3.77)
Heterodera cyperi			2	0(0)
Heterodera daverti			5	0–2; 1 (0–0.24; 0.11)			3	0–2; 2 (0–0.51; 0.51)
Heterodera dunensis	3	0(0)	3	0(0)	3	0(0)	8	0–1; 0 (0–0.28; 0)
Heterodera elachista	3	3–4; 3 (0.25–0.36; 0.36)	43	0–48; 11 (0–4.52; 1.05)	18	0–4; 1 (0–0.54; 0.15)	2	0(0)
Heterodera fici	2	2 (0.34)	8	0–3; 1 (0–0.31; 0.10)			9	0–2; 1 (0–0.47; 0.24)
Heterodera filipjevi	3	1–2; 1 (0.12–0.17; 0.17)	256	0–43; 2 (0–4.46; 0.21)	10	0–2; 1 (0–0.30; 0.13)	75	0–44; 10 (0–10.58; 2.42)
Heterodera glycines	6	0–1; 0 (0–0.16; 0)	319	0–112; 3 (0–11.89; 0.32)	56	0–43; 2 (0–5.79; 0.26)	81	0–9; 0 (0–1.04; 0)
Heterodera goettingiana	4	0–10; 5 (0–1.21; 0.59)	15	0–81; 37 (0–11.34; 5.08)	2	19 (2.92)	3	0–17; 17 (0–4.84; 4.84)
Heterodera goldeni			5	0–6; 2 (0–0.59; 0.23)
Heterodera guangdongensis			3	0–3; 3 (0–0.26; 0.26)	7	1–6; 4 (0.15–0.91; 0.58)
Heterodera hordecalis	2	5 (0.31)	37	0–68; 7 (0–8.89; 0.84)	2	9 (1.33)	10	0–47; 14 (0–11.4; 3.37)
Heterodera humuli			13	0–4; 1 (0–0.57; 0.17)			19	0–9; 2 (0–5.92; 0.59)
Heterodera koreana	3	1–22; 2 (0.17–1.27; 0.34)	29	0–28; 7 (0–3.08; 0.73)	48	0–21; 2 (0–2.88; 0.27)	44	0–18; 5 (0–4.57; 1.27)
Heterodera latipons			154	0–122; 13 (0–13.32; 1.46)	2	1 (0.15)	16	0–50; 27 (0–12.05; 6.52)
Heterodera litoralis							4	0(0)
Heterodera mani			6	1–9; 3 (0.10–0.93; 0.31)			6	0–1; 1 (0–0.27; 0.24)
Heterodera medicaginis			14	0–9; 3 (0–0.95; 0.42)			14	0–2; 0 (0–0.49; 0)
Heterodera mediterranea	2	1 (0.17)	6	1–9; 7 (0.11–0.95; 0.74)	2	3 (0.38)
Heterodera mothi			2	3 (0.30)			2	2 (0.44)
Heterodera orientalis			5	6–18; 12 (0.66–1.94; 1.28)
Heterodera pratensis			13	0–10; 6 (0–1.08; 0.62)			20	0–8; 2 (0–1.93; 0.53)
Heterodera ripae			5	0–4; 2 (0–0.46; 0.22)			19	0–11; 1 (0–2.59; 0.24)
Heterodera sacchari			3	0–1; 1 (0–0.09; 0.09)
Heterodera salixophila			5	0–20; 15 (0–2.10; 1.58)	2	1 (0.15)	16	0–18; 2 (0–4.57; 0.51)
Heterodera schachtii	9	1–9; 4 (0.06–0.99; 0.31)	64	0–31; 12 (0–3.04; 1.29)	20	0–4; 1 (0–0.58; 0.13)	38	0–7; 1 (0–1.88; 0.12)
Heterodera sojae			5	0–12; 4.5 (0–1.36; 1.03)	10	0–8; 4 (0–1.06; 0.53)	12	0–6; 1 (0–1.59; 0.24)
Heterodera sturhani							7	0–3; 0 (0–0.72; 0)
Heterodera trifolii	5	0–8; 3 (0–0.47; 0.29)	23	0–14; 3 (0–1.48; 0.43)	19	0–1; 0 (0–0.15; 0)	26	0–5; 0 (0–1.24; 0)
Heterodera ustinovi			5	1–8; 3.5 (0.10–0.88; 0.36)			5	0–1; 1 (0–0.24; 0.24)
Heterodera zeae			16	0–39; 14 (0–5.65; 2.19)	4	0–2; 1 (0–0.31; 0.15)