FISH Map** of Telomeric and Non-Telomeric (AG₃T₃)₃ Reveal the Chromosome Numbers and Chromosome Rearrangements of 41 Woody Plants

Abstract

Data for the chromosomal FISH map** localization of (AG₃T₃)₃ are compiled for 37 species belonging 27 families; for 24 species and 14 families, this is the first such report. The chromosome number and length ranged from 14–136 and 0.56–14.48 μm, respectively. A total of 23 woody plants presented chromosome length less than 3 μm, thus belonging to the small chromosome group. Telomeric signals were observed at each chromosome terminus in 38 plants (90.5%) and were absent at several chromosome termini in only four woody plants (9.5%). Non-telomeric signals were observed in the chromosomes of 23 plants (54.8%); in particular, abundant non-telomeric (AG₃T₃)₃ was obviously observed in Chimonanthus campanulatus. Telomeric signals outside of the chromosome were observed in 11 woody plants (26.2%). Overall, ten (AG₃T₃)₃ signal pattern types were determined, indicating the complex genome architecture of the 37 considered species. The variation in signal pattern was likely due to chromosome deletion, duplication, inversion, and translocation. In addition, large primary constriction was observed in some species, probably due to or leading to chromosome breakage and the formation of new chromosomes. The presented results will guide further research focused on determining the chromosome number and disclosing chromosome rearrangements of woody plants.

1. Introduction

Most eukaryotes possess chromosomal termini made up of 5–8 bp simple tandem DNA repeats—commonly called telomeric repeats [1,2,3]—which, in all eukaryotes, share similarities. Telomeric DNA repeats typically follow the formula (T_xA_yG_z)_n. Furthermore, they are well-conserved, and previous findings have revealed their diversity. There exist at least 17 variable telomeric sequences: TAG₃ [4], TA₂G₂ [5], TA₂G₃ [5], T₂AG₂ [6], T₂AG₃ [7], T₃AG₃ [8,9,10], AG₃T₃ [11], T₄AG₃ [12], A₂TG₆ [13], T₂AT₂AG₃ [7], T₆AG₃ [14,15], TCAG₂ [16], T₂AG₂C [17], T₂CAG₂ [18], T₃CAG₂ [18], C₃TA₃ [19], T₂N₄AG₃ [20], and T₂ATG₃CTCG₂ [21]. Undoubtedly, the most widespread telomeric repeat is T₂AG₃ in animals [22], while that in most plants is T₃AG₃ [23].

Telomeric repeats are not often localized at chromosomal termini and have also been found in multiple intercalary sites of chromosomes in many species [3,23,24,25]. Interstitial telomeric sequences may represent a significant part of the telomeric DNA [3]. Cytogenetic analysis has found two major types of interstitial telomeric sequences: one is heterochromatic and large, found in centromeric or pericentromeric regions, while the other type is short and distributed at various positions in chromosomes [3]. Unlike telomeric repeats located at termini, interstitial telomeric sequences confer karyotype plasticity. Their unstable and high-length polymorphisms may increase chromosomal fragility and contribute to chromosomal reorganization [3,23,24]. Interstitial telomeric sequences have been shown to cause a change in the chromosome number in Cardamine cordifolia A. Gray [26], as well as in Asteraceae and Brassicaceae [27]. Interstitial telomeric sequences may be derived from centric fusions of Robertsonian translocation [28,29,30], generated through pericentric inversions [31,32], or be the product of equilocal dispersion of telomeric DNA to interstitial regions of the chromosome through transposition or heterologous recombination [33,34].

Many studies have reported on the T₃AG₃ distribution in woody plants. T₃AG₃ were observed at each chromosome end in Populus trichocarpa Torr. and A. Gray ex Hook. [35], Picea abies (L.) H. Karst. and Larix decidua Mill. [36], Abies alba Mill. [37,38], Cycas revoluta Thunb. [39], Strobus spp. [40], Aralia elata (Miq.) Seem., Dendropanax morbiferus H. Lév., Eleutherococcus sessiliflorus (Rupr. Et Maxim.) Seem. and Kalopanax septemlobus (Thunb. ex A.Murr.) Koidz [41]. Furthermore, T₃AG₃ has been observed at the interstitial regions and at all chromosome ends in Pinus spp. [42,43,44,45] and Podocarpus spp. [46]. No T₃AG₃ signal was found in species of Sessea, Vestia, and Cestrum, but the T₃AG₃ sequence was dispersed in the Cestrum genome [14,15].

AG₃T₃ is similar to but different from T₃AG₃ and has only been found to be used in 22 woody plants, 14 species, and 11 genera. AG₃T₃ have been observed at each chromosome end in Hibiscus mutabilis L. [47]; Juglans regia L. ‘Chuanzao1’, J. regia L. ‘Yanyuanzao’, Juglans sigillata Dode ‘Maerkang’, and J. sigillata Dode ‘Muzhilinhe’ [48]; Fraxinus pennsylvanica Marsh., Syringa oblata Ait., Ligustrum lucidum Lindl. and Ligustrum × vicaryi Rehder [49]; and Berberis diaphana Maxim. and Berberis soulieana Schneid [50]. Telomeric AG₃T₃ ensures integrated and easily countable chromosomes. Furthermore, T₃AG₃ has been observed in the interstitial regions and at all chromosome ends in Hippophaë rhamnoides ‘Wucifeng’, H. rhamnoides ‘Shenqiuhong’, H. rhamnoides ‘Zhuangyuanhuang’, cultural H. rhamnoides ssp. sinensis, wild H. rhamnoides ssp. sinensis [11], Robinia pseudoacacia L., R. pseudoacacia ‘idaho’, R. pseudoacacia f. decaisneana (Carr.) Voss, Styphnolobium japonicum (L.) Schott, Amorpha fruticose L. [51], and C. campanulatus R.H. Chang and C.S. Ding [52]. Non-telomeric AG₃T₃ may indicate chromosomal variation. There is still a great need to explore the telomeric and non-telomeric AG₃T₃ chromosome distribution in other woody plants. To determine the chromosome number and disclose the chromosome rearrangements in woody plants, in this study, we carried out fluorescent in situ hybridization (FISH) map** to reveal highly abundant (AG₃T₃)₃, established an ideogram to describe the complex genome architecture and, finally, discussed the proposed origin of (AG₃T₃)₃ diversity in woody plants.

2. Materials and Methods

The species chosen for these experiments were firstly considered due to the occurrence of karyotype rearrangements [11,47,48,49,50,51,52,53], and secondly for investigation of species in which (AG₃T₃)₃ has not yet been explored. Zea mays L. was chosen as it possesses the telomeric repetitive unit AGGGTTT conserved in plant chromosome telomeres. It is contained in the sequence named M8-2D, a B chromosome-specific sequence in Z. mays, which has low homology to clones from Z. mays chromosome 4 centromere. M8-2D is localized in B chromosome centromeric and telomeric regions [53]. Hence, we used Z. mays to test the used probe and as a control.

Details of the seeds or seedlings of Z. mays and the 41 woody plants (belonging to 37 species, 27 genera, 18 families) used in the present work are provided in

Table 1. Details of Z. mays and the 41 woody plants used in this study.

Family	No.	Species	Collection Location
Poaceae	1	Z. mays L.	Wenjiang, Sichuan
Fabaceae	2	Cercis chinensis Bunge	Wenjiang, Sichuan
	3	R. pseudoacacia L.	Wenjiang, Sichuan
	4	Erythrina crista-galli L.	Wenjiang, Sichuan
Euphorbiaceae	5	Croton tiglium L.	Yaan, Sichuan
Cupressaceae	6	Platycladus orientalis (L.) Franco	Wenjiang, Sichuan
	7	Cupressus funebris Endl.	Wenjiang, Sichuan
	8	Cryptomeria japonica (L. f.) D. Don	Wenjiang, Sichuan
	9	Cryptomeria fortune Hooibrenk ex Otto et Dietr.	Wenjiang, Sichuan
Cycadaceae	10	Cycas revolute Thunb.	Wenjiang, Sichuan
Calycanthaceae	11	C. campanulatus R.H.Chang, C.S.Ding	**niu, Sichuan
Taxaceae	12	Taxus chinensis (Pilger) Rehd.	Wenchuan, Sichuan
	13	Taxus yunnanensis W.C.Cheng and L.K.Fu	Yanan, Sichuan
	14	Taxus media	Yanan, Sichuan
	15	Taxus cuspidate Sieb. et Zucc.	Dujiangyan, Sichuan
	16	Taxus wallichiana Zucc.	Yanan, Sichuan
Fagaceae	17	Quercus semecarpifolia Smith	Wenchuan, Sichuan
Elaeagnaceae	18	wild H. rhamnoides ssp. sinensis Rousi	Wenchuan, Sichuan
	19	H. rhamnoides L. ‘Wucifeng’	Wenchuan, Sichuan
	20	H. rhamnoides L. ‘Shenqiuhong’	Wenchuan, Sichuan
	21	H. rhamnoides L. ‘Zhuangyuanhuang’	Wenchuan, Sichuan
	22	cultural H. rhamnoides ssp. sinensis Rousi	Wenchuan, Sichuan
	23	Elaeagnus lanceolate Warb. apud Diels	Wenchuan, Sichuan
Lauraceae	24	Persea Americana Mill	**chang, Sichuan
	25	Litsea baviensis Lec.	**niu, Sichuan
	26	Litsea elongate Lec.	**niu, Sichuan
Berberidaceae	27	B. diaphana Maxim.	Wenchuan, Sichuan
Sapindaceae	28	Koelreuteria paniculate Laxm.	Wenjiang, Sichuan
Juglandaceae	29	J. regia L. ‘Chuanzao1’	Qingbaijiang, Sichuan
	30	J. regia L. ‘Yanyuanzao’	Yanyuan, Sichuan
	31	J. sigillata Dode ‘Maerkang’	Maerkang, Sichuan
	32	J. sigillata Dode ‘Muzhilinhe’	Gulin, Sichuan
Podocarpaceae	33	Podocarpus macrophyllus (Thunb.) Sweet	Wenjiang, Sichuan
Salicaceae	34	Idesia polycarpa Maxim.	Wenjiang, Sichuan
Oleaceae	35	S. oblata Lindl.	Wenjiang, Sichuan
	36	L. lucidum Ait.	Wenjiang, Sichuan
	37	L. × vicaryi Rehder	Wenjiang, Sichuan
	38	F. pennsylvanica Marsh.	Wenjiang, Sichuan
Malvaceae	39	Firmiana simplex (Linnaeus) W. Wight	Wenjiang Sichuan
	40	H. mutabilis L.	**niu, Sichuan
Rutaceae	41	Zanthoxylum armatum DC.	**yang, Sichuan
	42	Zanthoxylum bungeanum Maxim.	Hanyuan, Sichuan

. All 42 plants were collected from 12 Counties or Districts of Sichuan Province, China.

2.1. Probe and Chromosome Preparation

The 21 bp oligo-probe of (AG₃T₃)₃, 5′-AGGGTTTAGGGTTTAGGGTTT-3′, was first reported in Z. mays [53] and has been further applied in Berberidaceae [50], Calycanthaceae [52], Elaeagnaceae [11], Fabaceae [51], Juglandaceae [48], Malvaceae [47], and Oleaceae [49]. This oligo-probe was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and tested simultaneously in a single round of FISH. The oligo-probe was 5′-labeled with 6-carboxy-fluorescein (6-FAM; absorption/emission wavelengths 494 nm/518 nm; green).

Collected seeds were germinated in culture dishes with wet filter paper and kept at 25 °C in the daytime and at 18 °C in the night until the roots were ~2 cm in length, which were then cut. The collected seedlings were cultured in soil at room temperature (15–25 °C) until many new roots grew out, which were then cut again. The cut roots were treated with nitrous oxide (N₂O) gas for 2–6 h, with treatment time depending on chromosome length and cell wall lignification. Next, the samples were fixed in glacial acetic acid for 5–10 min, then kept in 75% ethyl alcohol. Chromosome preparation was carried out according to the procedure described by Luo et al. [51]. As these techniques have been described elsewhere, they will be detailed briefly here. Approximately 1 mm of the meristematic zone of the root tip was enzymolyzed at 37 °C for 45 min by using cellulase and pectinase (1 mL buffer + 0.04 g cellulase + 0.02 g pectinase, the buffer 50 mL was included 0.5707 g trisodium citrate + 0.4324 g citric acid), which were produced by Yakult Pharmaceutical Ind. Co., Ltd. (Tokyo, Japan) and Kyowa Chemical Products Co., Ltd. (Osaka, Japan), and then mixed into suspension for drop** onto slides. These slides were air dried then examined using an Olympus CX23 microscope (Olympus, Tokyo, Japan).

2.2. FISH Hybridization

Slides with well-spread samples were used for hybridization. Chromosomes were first subjected to a series of fixation (10 min, 4% paraformaldehyde, room temperature), dehydrated (5 min, 75%, 95%, 100% ethanol, room temperature), denatured (2 min, deionized formamide, 80 °C), and a second dehydration (5 min, 75%, 95%, 100% ethanol, −20 °C), and then hybridized (10 μL hybridization mixture of 0.375 μL of (AG₃T₃)₃, 4.675 μL of 2× SSC, and 4.95 μL of ddH₂O) for 1–2 h at 37 °C. Subsequently, hybridized chromosomes were washed with 2× SSC and ddH₂O twice for 5 min at room temperature, and air-dried. Finally, they were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA, USA) for 5 min, referring to the procedure described by Luo et al. [51]. Slides were checked using an Olympus BX-63 microscope (Olympus Corporation, Tokyo, Japan), and FISH images were captured by a DP-70 CCD camera connected to the microscope.

2.3. Karyotype Analysis

Raw images were processed using the DP Manager (Olympus Corporation, Tokyo, Japan) and Photoshop CC 2015 (Adobe Systems Incorporated, San Jose, CA, USA) software. At least ten slides of each plant were observed, and at least fifteen cells with good chromosome spread were used for chromosome counting and length measurement. All chromosomes were aligned from longest to shortest. The chromosome ratio was determined by comparing the length of the longest chromosome to that of the shortest chromosome. Further karyotype analysis could not be carried out due to the small chromosome size and unclear centromere location of many of the species.

3. Results

3.1. Karyotype Analysis Revealed Differences among 37 Species

For 24 species and 14 families, this is the first time that (AG₃T₃)₃ testing has been reported. To visualize the chromosomal distribution of (AG₃T₃)₃ in Z. mays and the 41 woody plants, we performed FISH analysis, as shown in Figure 1(A1–A11), Figure 2(B1–B16), and Figure 3(C1–C15). To clearly display each chromosome distribution of the (AG₃T₃)₃, we cut those in Figure 1, Figure 2 and Figure 3 and aligned them, as shown in Figure 4(A1–A11), Figure 5(B1–B16), and Figure 6(C1–C15).

The chromosome number and length for the considered species were sorted in

Table 2. Chromosome number and length of the 42 plants used in this study.

Accession	Species	Chromosome Number	Chromosome Length	Karyotype Asymmetry
A1	Z. mays	2n = 20	2.79–5.58 μm	2.00
A2	C. chinensis	2n = 14	1.67–2.55 μm	1.53
A3	R. pseudoacacia	2n = 22	1.12–1.74 μm	1.56
A4	E. crista-galli	2n = 42	1.57–2.51 μm	1.60
A5	C. tiglium	2n = 20	2.09–3.70 μm	1.77
A6	P. orientalis	2n = 22	4.71–7.67 μm	1.63
A7	C. funebris	2n = 22	1.67–7.15 μm	4.28
A8	C. japonica	2n = 22	4.01–6.70 μm	1.67
A9	C. fortune	2n = 22	7.85–8.79 μm	1.12
A10	C. revolute	2n = 22	8.06–14.48 μm	1.80
A11	C. campanulatus	2n = 22	1.60–2.52 μm	1.58
B1	T. chinensis	2n = 24	2.16–5.76 μm	2.68
B2	T. yunnanensis	2n = 24	2.48–6.56 μm	2.65
B3	T. media	2n = 24	3.14–7.33 μm	2.33
B4	T. cuspidate	2n = 24	3.07–7.09 μm	2.31
B5	T. wallichiana	2n = 24	3.31–8.37 μm	2.53
B6	Q. semecarpifolia	2n = 24	1.57–3.21 μm	2.04
B7	wild H. rhamnoides ssp. sinensis	2n = 24	1.15–2.41 μm	2.10
B8	H. rhamnoides L. ‘Wucifeng’	2n = 24	1.40–2.48 μm	1.77
B9	H. rhamnoides L. ‘Shenqiuhong’	2n = 24	1.36–2.44 μm	1.79
B10	H. rhamnoides L. ‘Zhuangyuanhuang’	2n = 24	1.47–2.72 μm	1.84
B11	cultural H. rhamnoides ssp. sinensis	2n = 24	1.60–2.69 μm	1.68
B12	E. lanceolate	2n = 28	1.05–3.84 μm	3.66
B13	P. americana	2n = 24	1.26–4.36 μm	3.46
B14	L. baviensis	2n = 24	2.09–4.99 μm	2.39
B15	L. elongate	2n = 24	1.74–3.28 μm	1.89
B16	B. diaphana	2n = 28	2.09–2.93 μm	1.40
C1	K. paniculate	2n = 32	0.56–1.12 μm	2.00
C2	J. regia L. ‘Chuanzao1’	2n = 34	0.63–2.60 μm	4.13
C3	J. regia L. ‘Yanyuanzao’	2n = 34	1.01–2.55 μm	2.52
C4	J. sigillata Dode ‘Maerkang’	2n = 34	0.66–1.88 μm	2.85
C5	J. sigillata Dode ‘Muzhilinhe’	2n = 34	0.87–2.79 μm	3.21
C6	P. macrophyllus	2n = 36	1.92–7.01 μm	3.65
C7	I. polycarpa	2n = 38	0.94–2.23 μm	2.37
C8	S. oblata	2n = 46	1.64–2.20 μm	1.34
C9	L. lucidum	2n = 46	0.98–1.36 μm	1.39
C10	L. × vicaryi	2n = 46	1.37–3.07 μm	2.24
C11	F. pennsylvanica	2n = 46	0.98–2.06 μm	2.10
C12	F. simplex	2n = 36	1.08–2.37 μm	2.19
C13	H. mutabilis	2n = 90	1.22–2.90 μm	2.38
C14	Z. armatum	2n = 98	0.98–1.99 μm	2.03
C15	Z. bungeanum	2n = 136	0.94–2.62 μm	2.79

. The chromosome number in the 42 plants ranged from 14 (C. chinensis, A3) to 136 (Z. bungeanum, C15). A total of 14 woody plants possessed 24 chromosomes (one third), whereas seven woody plants possessed 22 chromosomes (one sixth). The longest chromosome length of each plant ranged from 1.12 μm (K. paniculata, C1) to 14.48 μm (C. revoluta, A10), while the shortest chromosome length of each plant ranged from 0.56 μm (K. paniculate, C1) to 8.06 μm (C. revoluta, A10). A total of 23 woody plants (nearly one third) had chromosome length less than 3 μm, thus falling into the small chromosome category. Due to the indistinct location of centromeres and small size of chromosomes in many of the considered woody plants, further karyotype analysis—such as long/short arm length and karyotype formula—was not carried out. Karyotype asymmetry was assessed using the ratio of longest to shortest chromosome length. The largest ratio was 4.28 in C. funebris (A7), while the smallest ratio was 1.12 in C. fortunei (A9). The ratio for 17 plants ranged from 1 to 2 (40.5%), while that of 19 plants ranged from 2 to 3 (45.2%). The ratio was greater than 3 for six plants: C. funebris (A7), E. lanceolata (B12), P. americana (B13), J. regia ‘Chuanzao1’ (C2), J. sigillata ‘Muzhilinhe’ (C5), and P. macrophyllus (C6). These results indicated that abundant differences exist among 37 of the considered species.

3.2. The Diverse Signal Patterns of (AG₃T₃)₃ Reveal the Complex Genome Architecture

To better investigate diversity of (AG₃T₃)₃, different types of ideograms for the 42 plants were drawn based on the FISH karyograms shown in Figure 4, Figure 5 and Figure 6, which are illustrated in Figure 7, Figure 8 and Figure 9. Telomeric signals were observed at each chromosome terminus in 38 plants (90.5%, the first class): Z. mays (A1), C. chinensis (A2), R. pseudoacacia (A3), E. crista-galli (A4), C. tiglium (A5), P. orientalis (A6), C. japonica (A8), C. fortune (A9), C. campanulatus (A11), T. chinensis (B1), T. media (B3), T. cuspidata (B4), T. wallichiana (B5), Q. semecarpifolia (B6), wild H. rhamnoides ssp. sinensis (B7), H. rhamnoides ‘Wucifeng’ (B8), H. rhamnoides ‘Shenqiuhong’ (B9), H. rhamnoides ‘Zhuangyuanhuang’ (B10), cultural H. rhamnoides ssp. sinensis (B11), E. lanceolata (B12), P. americana (B13), L. baviensis (B14), L. elongate (B15), B. diaphana (B16), J. regia ‘Chuanzao1’ (C2), J. regia ‘Yanyuanzao’ (C3), J. sigillata ‘Maerkang’ (C4), J. sigillata ‘Muzhilinhe’ (C5), P. macrophyllus (C6), I. polycarpa (C7), S. oblata (C8), L. lucidum (C9), L. × vicaryi (C10), F. pennsylvanica (C11), F. simplex (C12), H. mutabilis (C13), Z. armatum (C14), and Z. bungeanum (C15). Meanwhile, telomeric signals were absent at several chromosome termini in only four woody plants (9.5%, the fourth class): C. funebris (A7), C. revoluta (A10), T. yunnanensis (B2), and K. paniculata (C1). Non-telomeric signals were observed at chromosome termini in 23 plants (54.8%, the second class): Z. mays (A1), R. pseudoacacia (A3), C. funebris (A7), C. revoluta (A10), and C. campanulatus (A11), T. chinensis (B1), T. yunnanensis (B2), T. media (B3), T. cuspidata (B4), T. wallichiana (B5), wild H. rhamnoides ssp. sinensis (B7), H. rhamnoides ‘Wucifeng’ (B8), H. rhamnoides ‘Shenqiuhong’ (B9), H. rhamnoides ‘Zhuangyuanhuang’ (B10), cultural H. rhamnoides ssp. sinensis (B11), J. sigillata ‘Muzhilinhe’ (C5), P. macrophyllus (C6), L. lucidum (C9), F. pennsylvanica (C11), F. simplex (C12), H. mutabilis (C13), Z. armatum (C14), and Z. bungeanum (C15). Telomeric signals outside of the chromosome were observed in 11 woody plants (26.2%, the third class): E. crista-galli (A4), C. tiglium (A5), C. campanulatus (A11), P. americana (B13), L. baviensis (B14), K. paniculata (C1), J. sigillata ‘Muzhilinhe’ (C5), S. oblata (C8), L. × vicaryi (C10), F. pennsylvanica (C11), and F. simplex (C12).

Furthermore, we summarized the results shown in Figure 7, Figure 8 and Figure 9 in order to produce the (AG₃T₃)₃ signal pattern presented in Figure 10. The results for Z. mays and 41 woody plants, belonging to 18 families, are shown, including six plants in Elaeagnaceae (yellow), five in Taxaceae (red), four in Cupressaceae (dark blue), four in Juglandaceae (light green), four in Oleaceae (orange), three in Fabaceae (pink), three in Lauraceae (light blue), two in Malvaceae (dark green), two in Rutaceae (grey), and one in each of Berberidaceae, Calycanthaceae, Cycadaceae, Euphorbiaceae, Fagaceae, Poaceae, Podocarpaceae, Salicaceae, and Sapindaceae, respectively. Except for Lauraceae and Rutaceae, which each presented a single signal pattern type, the other families (Elaeagnaceae, Cupressaceae, Juglandaceae, Oleaceae, Lauraceae, and Malvaceae) all presented at least two signal pattern types.

As shown in Figure 10, there were ten (AG₃T₃)₃ signal pattern types in total: Type I, chromosome only includes signal at both ends; Type II, chromosome not only includes signal at both ends but also includes a non-telomeric signal location; Type III, chromosome includes single end signal, and the other telomeric signals outside of the chromosome; Type IV, chromosome not only includes signal at both ends, but also includes telomeric signal deviating from chromosome; Type V, chromosome not only includes signal at both ends, but also includes a large primary constriction; Type VI: chromosome not only includes signal at both ends signal, but also includes a large primary constriction, as well as a non-telomeric signal location; Type VII, chromosome only includes telomeric signal outside of the chromosome; Type VIII, chromosome only includes single end signal; Type IX, chromosome only includes non-telomeric signal location; and Type X, chromosome includes no signals. These types of signal pattern indicate that there is an abundant diversity in (AG₃T₃)₃ signal arrangement.

All 42 plants possessed the 12 signal pattern types or type combinations shown in Figure 10. Ten woody plants only possessed signal pattern type I; Z. mays and 11 woody plants possessed the combination of type I + type II; eight woody plants possessed the combination of type I + type III; P. macrophyllus possessed the combination of type I + type II + type III; C. campanulatus possessed the combination of type I + type II + type IV; J. sigillata ‘Muzhilinhe’ possessed the combination of type I + type II + type III + type IV; B. diaphana possessed the combination of type I + type V; T. media and T. yunnanensis possessed the combination of type I + type II + type V; T. chinensis, T. cuspidata, and T. wallichiana possessed the combination of type I + type II + type VI; K. paniculate possessed the combination of type I + type III + type VII + type VIII; C. revolute possessed the combination of type VIII + type IX; and C. funebris possessed the combination of type VIII + type IX + type X.

There were diverse signal patterns of (AG₃T₃)₃ among 37 species, indicating a complex genome architecture. For example, considering (i) Elaeagnaceae, five plants of H. rhamnoides possessed type I + type II, but E. lanceolata possessed type I; (ii) in Taxaceae, T. media and T. yunnanensis possessed the combination type I + type II + type V, but T. chinensis, T. cuspidata, and T. wallichiana possessed the combination type I + type II + type VI; (iii) in Cupressaceae, C. fortune, C. japonica, and P. orientalis possessed type I, but C. funebris possessed the combination type VIII + type IX + type X; (iv) in Juglandaceae, J. regia ‘Chuanzao1’, J. regia ‘Yanyuanzao’, and J. sigillata ‘Maerkang’ possessed type I, but J. sigillata ‘Muzhilinhe’ possessed the combination type I + type II + type III + type IV; (v) in Oleaceae, L. lucidum possessed the combination type I + type II, L. × vicaryi and S. oblata possessed the combination type I + type III, and F. pennsylvanica possessed the combination type I + type II + type III; (vi) in Fabaceae, C. chinensis possessed type I, R. pseudoacacia possessed the combination type I + type II, and E. crista-galli possessed the combination type I + type III; and (vii) in Malvaceae, H. mutabilis possessed the combination type I + type II, but F. simplex possessed the combination type I + type III.

The number shown after the species name in Figure 10 represents the ratio of longest to shortest chromosome length, indicating karyotype asymmetry. Type I included ten plants with ratio ranging from 1.12–4.13, with variance of 0.94. Type I + Type II included 12 plants with ratio ranging from 1.39–3.65, with variance of 0.38. Type I + Type II + Type III included eight plants with ratio ranging from 1.34–3.46, with variance of 0.48. These results indicate that chromosomes with conserved telomeric signal (Type I), in fact, have a wider range of karyotype asymmetry (VAR 0.94), while chromosomes with non-telomeric signals (Type II, III) have relatively concentrated karyotype asymmetry (VAR 0.38 and 0.48, respectively). In addition, no correlation between non-telomeric signals and karyotype asymmetry was observed.

3.3. Proposed Origin of (AG₃T₃)₃ Signal Diversity

Based on Figure 7, Figure 8, Figure 9 and Figure 10, the proposed origin of (AG₃T₃)₃ signal diversity is illustrated in Figure 11. There are three major groups. (i) Signal number: Increase signal number is likely caused by chromosome duplication, inversion, translocation, and/or sequence changes; a constant signal number indicates chromosome conservation and a decreased signal number is likely caused by chromosome deletion and/or sequence changes. (ii) Signal location: End signals on chromosomes indicate chromosome conservation; non-end signals are likely caused by chromosome deletion, duplication, inversion, translocation, and/or sequence changes; end signals deviating from the chromosome are probably caused by chromosome satellites, while end signal loss is probably caused by chromosome end deletion and/or sequence changes. (iii) Primary constriction: Normal primary constriction indicates chromosome conservation, while primary constriction likely becomes large due to chromosome breakage and the formation of new chromosomes (e.g., in Figure 8, T. media chromosome 9 and B. diaphana chromosome 9, T. cuspidata chromosome 9 and T. wallichiana chromosome 9 possibly indicate the formation of new chromosomes, while T. cuspidata chromosome 12 and T. wallichiana chromosome 12 were possibly formed in a reverse manner).

In brief, the variations in signal number and signal location were probably caused by chromosome deletion, duplication, inversion, translocation, and sequence changes, as well as chromosome satellites. It is likely that large primary constriction was due to chromosome breakage and the formation of new chromosomes.

4. Discussion

4.1. Karyotype Analysis of Z. mays and 41 Woody Plants

Chromosome number, size, centromere location, long/short arm ratio, and satellites are basic characteristics of karyotype. The presence of telomeric (AG₃T₃)₃ in both chromosome ends may guarantee the accuracy of chromosome counting. The chromosome number in the 42 plants ranged from 14 (C. chinensis) to 136 (Z. bungeanum). There were six woody plants that presented chromosome numbers different to that reported in previous studies: J. regia ‘Chuanzao1’, J. regia ‘Yanyuanzao’, J. sigillata ‘Maerkang’, and J. sigillata ‘Muzhilinhe’ presented 2n = 34, a result supported by the work of Luo and Chen [48] but contradicted by Mu and **, described the diverse distribution of (AG₃T₃)₃, and disclosed the complex genome architecture of woody plants. We inferred that the observed non-telomeric signals were probably caused by chromosome arrangements. We intend to continue our research by testing more woody plants, such as species of Calycanthaceae, to explore the abundant non-telomeric (AG₃T₃)₃, as well as Rutaceae, to determine the chromosome number.