Intragenomic Polymorphism of the Internal Transcribed Spacer Region of Ribosomal DNA in Camellia hongkongensis (Theaceae) and Species Identification

doi:10.3724/SP.J.1259.2015.00217

Abstract

Abstract: The internal transcribed spacer (ITS) region of ribosomal DNA has been selected as an important barcoding region in plants, but its use in Camellia species is difficult. In this study, we amplified, cloned and sequenced the ITS region of a Camellia plant from Vietnam that was similar to C. hongkongensis and obtained 74 sequences from an individual. The ITS region showed high polymorphism within the individual, and 76% of the sequences were pseudogenes. Phylogenetic analysis showed that more than half of the pseudogenes originated from a common ancestor. These older rDNA pseudogenes differentiated into at least 5 lineages after repeated gene duplication, and sequences in each lineage were similar to each other, which suggests that some pseudogenes had not been deleted but had recently undergone rapid duplication events. The high polymorphism of the ITS region within an individual in Camellia species would likely result in faulty identities when using this region as a barcode. However, by comparing the species-specific rDNA pseudogenes in C. hongkongensis, we identified the sample from Vietnam that belongs to C. hongkongensis.

Wen Fan, Ying Xu, Ting Xu, Jing Xu, Takahiro Yonezawa, Jiyin Gao, Wenju Zhang. Intragenomic Polymorphism of the Internal Transcribed Spacer Region of Ribosomal DNA in Camellia hongkongensis (Theaceae) and Species Identification[J]. Chinese Bulletin of Botany, 2015, 50(2): 217-226.

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Type^a	rDNA region	Haptotypes^b	Genetic distance	h±SD	π	Lengths (bp)	GC contents	ΔG (kcal∙mol^-1)^c
All	ITS1-5.8S-ITS2	47	0.210 5±0.011 7	0.991±0.008	0.166 72	589-687	0.557
	ITS1		0.297 4±0.023 0	0.958±0.015	0.242 21	221-291	0.550
	5.8S		0.083 1±0.011 5	0.961±0.013	0.065 27	139-163	0.501
	ITS2		0.240 1±0.020 1	0.962±0.012	0.171 55	198-233	0.610
F	ITS1-5.8S-ITS2	10	0.010 3±0.001 9	0.978±0.054	0.009 74	674-687	0.686
	ITS1		0.007 0±0.002 1	0.667±0.163	0.006 47	281-291	0.719
	5.8S		0.008 4±0.003 6	0.667±0.163	0.008 32	163	0.543	-36.90, -47.42
	ITS2		0.015 7±0.004 2	0.933±0.077	0.014 69	230-233	0.751
P	ITS1-5.8S-ITS2	37	0.212 2±0.012 0	0.994±0.009	0.164 71	589-632	0.531
	ITS1		0.283 5±0.023 0	0.964±0.015	0.225 14	221-258	0.515
	5.8S		0.095 8±0.013 3	0.959±0.014	0.073 47	139-163	0.491
	ITS2		0.248 3±0.020 9	0.953±0.016	0.176 47	198-223	0.581
Pa1^d	ITS1-5.8S-ITS2	5	0.003 6±0.001 6	1.000±0.126	0.003 58	614	0.523
	ITS1		0.002 6±0.002 6	0.600±0.175	0.002 61	230	0.523
	5.8S		0.004 9±0.003 5	0.700±0.218	0.004 91	163	0.461	-24.90, -36.00
	ITS2		0.003 6±0.002 6	0.700±0.218	0.003 62	221	0.567
Pa2	ITS1-5.8S-ITS2	8	0.006 1±0.002 0	1.000±0.063	0.006 06	607	0.541
	ITS1		0.005 8±0.003 0	0.857±0.108	0.005 77	223	0.535
	5.8S		0.005 1±0.003 7	0.679±0.122	0.005 04	163	0.512	-30.22, -35.70
	ITS2		0.007 2±0.003 7	0.750±0.139	0.007 11	221	0.568
Pa3	ITS1-5.8S-ITS2	5	0.005 8±0.002 1	1.000±0.126	0.005 77	589	0.529
	ITS1		0.003 5±0.002 5	0.700±0.218	0.003 51	228	0.528
	5.8S		0.009 9±0.005 0	0.900±0.161	0.009 82	163	0.490	-20.80, -23.62
	ITS2		0.005 1±0.003 6	0.800±0.164	0.005 05	198	0.564
Pa4	ITS1-5.8S-ITS2	9	0.011 3±0.002 9	0.972±0.064	0.011 11	610-611	0.577
	ITS1		0.006 7±0.003 6	0.833±0.098	0.006 70	224-225	0.584
	5.8S		0.020 0±0.008 3	0.806±0.120	0.019 43	163	0.501	-23.10, -34.50
	ITS2		0.009 6±0.004 6	0.694±0.147	0.009 47	223	0.624
Pb	ITS1-5.8S-ITS2	9	0.151 4±0.009 5	1.000±0.052	0.121 31	590-632	0.582
	ITS1		0.174 4±0.017 4	1.000±0.052	0.139 17	221-258	0.587
	5.8S		0.088 8±0.013 5	0.917±0.092	0.076 94	139-163	0.499	-15.22, -42.12
	ITS2		0.181 0±0.019 4	0.917±0.092	0.136 33	206-221	0.644

Type^a	rDNA region	Haptotypes^b	Genetic distance	h±SD	π	Lengths (bp)	GC contents	ΔG (kcal∙mol^-1)^c
All	ITS1-5.8S-ITS2	47	0.210 5±0.011 7	0.991±0.008	0.166 72	589-687	0.557
	ITS1		0.297 4±0.023 0	0.958±0.015	0.242 21	221-291	0.550
	5.8S		0.083 1±0.011 5	0.961±0.013	0.065 27	139-163	0.501
	ITS2		0.240 1±0.020 1	0.962±0.012	0.171 55	198-233	0.610
F	ITS1-5.8S-ITS2	10	0.010 3±0.001 9	0.978±0.054	0.009 74	674-687	0.686
	ITS1		0.007 0±0.002 1	0.667±0.163	0.006 47	281-291	0.719
	5.8S		0.008 4±0.003 6	0.667±0.163	0.008 32	163	0.543	-36.90, -47.42
	ITS2		0.015 7±0.004 2	0.933±0.077	0.014 69	230-233	0.751
P	ITS1-5.8S-ITS2	37	0.212 2±0.012 0	0.994±0.009	0.164 71	589-632	0.531
	ITS1		0.283 5±0.023 0	0.964±0.015	0.225 14	221-258	0.515
	5.8S		0.095 8±0.013 3	0.959±0.014	0.073 47	139-163	0.491
	ITS2		0.248 3±0.020 9	0.953±0.016	0.176 47	198-223	0.581
Pa1^d	ITS1-5.8S-ITS2	5	0.003 6±0.001 6	1.000±0.126	0.003 58	614	0.523
	ITS1		0.002 6±0.002 6	0.600±0.175	0.002 61	230	0.523
	5.8S		0.004 9±0.003 5	0.700±0.218	0.004 91	163	0.461	-24.90, -36.00
	ITS2		0.003 6±0.002 6	0.700±0.218	0.003 62	221	0.567
Pa2	ITS1-5.8S-ITS2	8	0.006 1±0.002 0	1.000±0.063	0.006 06	607	0.541
	ITS1		0.005 8±0.003 0	0.857±0.108	0.005 77	223	0.535
	5.8S		0.005 1±0.003 7	0.679±0.122	0.005 04	163	0.512	-30.22, -35.70
	ITS2		0.007 2±0.003 7	0.750±0.139	0.007 11	221	0.568
Pa3	ITS1-5.8S-ITS2	5	0.005 8±0.002 1	1.000±0.126	0.005 77	589	0.529
	ITS1		0.003 5±0.002 5	0.700±0.218	0.003 51	228	0.528
	5.8S		0.009 9±0.005 0	0.900±0.161	0.009 82	163	0.490	-20.80, -23.62
	ITS2		0.005 1±0.003 6	0.800±0.164	0.005 05	198	0.564
Pa4	ITS1-5.8S-ITS2	9	0.011 3±0.002 9	0.972±0.064	0.011 11	610-611	0.577
	ITS1		0.006 7±0.003 6	0.833±0.098	0.006 70	224-225	0.584
	5.8S		0.020 0±0.008 3	0.806±0.120	0.019 43	163	0.501	-23.10, -34.50
	ITS2		0.009 6±0.004 6	0.694±0.147	0.009 47	223	0.624
Pb	ITS1-5.8S-ITS2	9	0.151 4±0.009 5	1.000±0.052	0.121 31	590-632	0.582
	ITS1		0.174 4±0.017 4	1.000±0.052	0.139 17	221-258	0.587
	5.8S		0.088 8±0.013 5	0.917±0.092	0.076 94	139-163	0.499	-15.22, -42.12
	ITS2		0.181 0±0.019 4	0.917±0.092	0.136 33	206-221	0.644