Oligo-FISH Can Identify Chromosomes and Distinguish Hippophaë rhamnoides L. Taxa

Oligo-fluorescence in situ hybridization (FISH) facilitates precise chromosome identification and comparative cytogenetic analysis. Detection of autosomal chromosomes of Hippophaë rhamnoides has not been achieved using oligonucleotide sequences. Here, the chromosomes of five H. rhamnoides taxa in the mitotic metaphase and mitotic metaphase to anaphase were detected using the oligo-FISH probes (AG3T3)3, 5S rDNA, and (TTG)6. In total, 24 small chromosomes were clearly observed in the mitotic metaphase (0.89–3.03 μm), whereas 24–48 small chromosomes were observed in the mitotic metaphase to anaphase (0.94–3.10 μm). The signal number and intensity of (AG3T3)3, 5S rDNA, and (TTG)6 in the mitotic metaphase to anaphase chromosomes were nearly consistent with those in the mitotic metaphase chromosomes when the two split chromosomes were integrated as one unit. Of note, 14 chromosomes (there is a high chance that sex chromosomes are included) were exclusively identified by (AG3T3)3, 5S rDNA, and (TTG)6. The other 10 also showed a terminal signal with (AG3T3)3. Moreover, these oligo-probes were able to distinguish one wild H. rhamnoides taxon from four H. rhamnoides taxa. These chromosome identification and taxa differentiation data will help in elucidating visual and elaborate physical mapping and guide breeders’ utilization of wild resources of H. rhamnoides.


Introduction
Hippophaë rhamnoides L. (Elaeagnaceae), also known as sea buckthorn, is a spiny deciduous shrub or small tree [1]. This species originated and migrated from the Qinghai-Tibet Plateau and adjacent regions [2]. Its natural habitats include severe environments with excessive salinity, drought, cold, and heat [3]. H. rhamnoides is known for its nutritional, medicinal, and ecological values [4]; it has been shown to improve the health of consumers. Moreover, its berries, which are edible, are used as a general body-toning agent [3]. H. rhamnoides, and its processed products, are potentially nontoxic when consumed by humans as a food or as a dietary supplement [5]. Thus, the ecological and commercial values of H. rhamnoides have drawn the attention of researchers for centuries [6]. Furthermore, an increase in its demand has prompted the fine breeding of various cultivars with genetic improvements to achieve high productivity and quality.
The systematic treatment of H. rhamnoides has been controversial. Studies have reported inconsistent findings with respect to the number of H. rhamnoides subspecies, for example, two subspecies [7], three subspecies [8], six subspecies [9], eight subspecies [10], and nine subspecies [11]. The treatment of Hippophaë rhamnoides ssp. sinensis Rousi has been supported by the findings of Rousi [10] and Bartish et al. [12]. To date, the WFO [13] has shown that H. rhamnoides comprises three accepted subspecies, four unresolved subspecies, and one accepted variety. H. rhamnoides ssp. sinensis is an unresolved subspecies with one of the largest distribution ranges. Moreover, considering that abundant morphological variations have been described within the subspecies [12,14], it is critical to identify the Genes 2022, 13,195 2 of 16 genetic basis of these variations to facilitate the selection of superior cultivars from wild H. rhamnoides ssp. sinensis.
Hippophaë rhamnoides taxa are often misidentified owing to similarities in their vegetative morphology. Furthermore, the fruits of different species are labeled with the same name and are primarily sold or used in dried form or as powders. Therefore, different taxa cannot be identified based on only morphological characteristics, and accurate identification methods are needed to avoid misidentification and misuse. All Hippophaë species have been successfully identified by DNA barcoding, and four H. rhamnoides subspecies have also been differentiated using ITS2 and psbA-trnH [15]. The male/female plants of H. rhamnoides have been identified using inter-simple sequence repeat [16] and fluorescence in situ hybridization (FISH) [17]. However, none of the other molecular cytogenetic technologies can be used to identify H. rhamnoides, thus limiting investigations on its identification and characterization.
Oligos designed from conserved DNA sequences from one species, particularly from part/whole/multiple chromosomes, can be precisely identified from genetically related species, thereby allowing comparative cytogenetic mapping of these species. These oligonucleotide sequences can then be readily produced and tagged with fluorescent markers for use as oligo-probes in FISH [18]. Species identification based on such oligo-probes has been reported in an increasing number of plant species, such as Avena L. species [19], Arachis hypogaea L. [20], Saccharum spontaneum L. [21], Citrus L. species [22], Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf., CC [23], Populus L. species [24], Strobus Opiz species [25], and Pinus L. species [26]. However, information regarding H. rhamnoides is limited. Chromosome identification remains a major challenge in H. rhamnoides with small chromosomes. In the present study, we aimed to use three oligo-probes--(AG 3 T 3 ) 3 , 5S rDNA, and (TTG) 6 -to identify H. rhamnoides chromosomes simultaneously in a single round of FISH.

Materials and Methods
The seeds of five H. rhamnoides taxa were used in this study; three H. rhamnoides cultivars ('Shenqiuhong', 'Zhuangyuanhuang', and 'Wucifeng') were collected from Hebei Province in China, one cultural H. rhamnoides ssp. sinensis was collected from Liaoning Province in China, and one wild H. rhamnoides ssp. sinensis was collected from Sichuan Province in China.

FISH and Karyotype Analysis
Root tips were cut from H. rhamnoides seedlings and treated with nitrous oxide gas for 3 h, fixed in acetic acid for approximately 10 min, and finally preserved in 75% ethanol Genes 2022, 13, 195 3 of 16 for further chromosome preparation. The root tip slides were prepared according to the method described by Luo et al. [33]. The meristematic zone (~1 mm) of the root tip was digested with pectinase and cellulase (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) and then suspended; this suspension was used for slide preparation using the drop method. Chromosomes were denatured for 2 min at 80 • C and hybridized with oligo-probes for 2 h at 37 • C using the method described by Luo et al. [33]. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI) containing VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Inc., Burlingame, CA, USA) and covering with a coverslip, the slides were observed under an Olympus BX-63 microscope (Olympus Corporation, Tokyo, Japan). FISH photomicrographs were obtained using a DP-70 CCD camera connected to the BX-63 microscope. Chromosome spreads in raw images were processed with DP Manager (Olympus Corporation, Tokyo, Japan) and Photoshop CC 2015 (Adobe Systems Incorporated, San Jose, CA, USA). Approximately 90 mitotic metaphases or mitotic metaphase to anaphases from 30 slides of 15 H. rhamnoides root tips were observed. More than 10 cells in the mitotic metaphase or mitotic metaphase to anaphase with good chromosome spread were used to count the chromosomes. Three high-quality spreads were used for karyotype analysis. All chromosomes were aligned by length, from the longest to shortest. The chromosome ratio was determined as the length of the longest chromosome to that of the shortest chromosome.

FISH-Enabled Visualization of H. rhamnoides Chromosomes
The mitotic metaphase of five H. rhamnoides taxa detected using (AG 3 T 3 ) 3 , 5S rDNA, and (TTG) 6 is illustrated in Figure 1. To visualize FISH signal distribution, each chromosome was cut from Figure 1 and aligned in Figure 2 based on its length and signal pattern. A total of 24 chromosomes were observed in each taxon of H. rhamnoides 'Wucifeng' (Figures 1A and 2A sinensis. The size ranged from 0.89 to 3.03 µm, which is similar to that of small chromosomes. The ratio of the longest to shortest chromosomes in the mitotic metaphase was 3.40, indicating karyotype asymmetry in H. rhamnoides. Owing to the unclear centromeres of most chromosomes and their small size, the short and long arms of the chromosomes were not well characterized for further karyotype analysis. (AG 3 T 3 ) 3 was located not only at the end of each chromosome but also at four chromosomally proximal regions (chromosomes 3/4/11/12); it was even dissociated from one end of chromosome 19 (satellite bodies) in five H. rhamnoides taxa ( Figure 1). Two strong signals of (AG 3 T 3 ) 3 were observed in the proximal region of chromosome 3/4, whereas the other chromosomes showed minor differences in (AG 3 T 3 ) 3 signal intensity in five H. rhamnoides taxa ( Figure 2). (TTG) 6 was observed at six chromosomally proximal regions (chromosome 1/2/7/8/23/24) in three cultivars H. rhamnoides 'Wucifeng' (Figures 1A and 2A), H. rhamnoides 'Shenqiuhong' (Figures 1B and 2B), and H. rhamnoides 'Zhuangyuanhuang' (Figures 1C and 2C) and one cultural H. rhamnoides ssp. sinensis ( Figures 1D and 2D), but only at two chromosomally proximal regions (chromosome 1/2) in wild H. rhamnoides ssp. sinensis ( Figures 1E and 2E). Two strong signals of (TTG) 6 Figures 3E and 4E). The size ranged from 0.94 to 3.10 μm, which is similar to that of small chromosomes. The ratio of the longest to shortest chromosomes in the mitotic metaphase to anaphase was 3.30, indicating karyotype asymmetry in H. rhamnoides. Due to chromosome segregation in the mitotic metaphase to anaphase, chromosome numbers in each taxon in Figure 3 ranged from 24 to 48. Several of them have been split into two separate chromosomes and far away at a certain distance (to make them easy to count, e.g., in Figure 3A,B,D, shown by the dotted line), whereas most of them were closely matched to each other (which makes it difficult to determine whether there is one or two chromosomes) in Figure 3. The signal number and intensity of (AG3T3)3, 5S rDNA, and (TTG)6 mitotic metaphase to anaphase chromosomes were nearly consistent with those of The mitotic metaphase to anaphase chromosomes of five H. rhamnoides taxa detected using (AG 3 T 3 ) 3 , 5S rDNA, and (TTG) 6 Figures 3D and 4D), and 1.20-2.74 µm for wild H. rhamnoides ssp. sinensis ( Figures 3E and 4E). The size ranged from 0.94 to 3.10 µm, which is similar to that of small chromosomes. The ratio of the longest to shortest chromosomes in the mitotic metaphase to anaphase was 3.30, indicating karyotype asymmetry in H. rhamnoides. Due to chromosome segregation in the mitotic metaphase to anaphase, chromosome numbers in each taxon in Figure 3 ranged from 24 to 48. Several of them have been split into two separate chromosomes and far away at a certain distance (to make them easy to count, e.g., in Figure 3A,B,D, shown by the dotted line), whereas most of them were closely matched to each other (which makes it difficult to determine whether there is one or two chromosomes) in Figure 3. The signal number and intensity of (AG 3 T 3 ) 3 , 5S rDNA, and (TTG) 6 mitotic metaphase to anaphase chromosomes were nearly consistent with those of mitotic metaphase chromosomes if the two split chromosomes were integrated as one unit ( Figure 4). Owing to the cryptic centromeres of several chromosomes and their small size, the short and long arms of the chromosomes were not well characterized for further karyotype analysis.   Figure 3B (red dotted line), and 2 chromosomes in Figure 3D (red dotted line). The blue chromosomes were counterstained by DAPI. Scale bar = 3 μm.

Physical Map Distinguished Chromosomes
Next, as shown in Figures 5 and 6, the chromosomes were further eliminated with a common signal. As a result, the chromosomes of H. rhamnoides identified by (AG3T3)3, (TTG)6, and 5S rDNA were aligned into a simplified version of Figures 3 and 4. To better exhibit the centromere location, each chromosome was visualized in a black-white version (Figures 7 and 8). The signal pattern ideograms were constructed based on the above black-white visualization of the chromosomes and their signal patterns in Figures 5 and  6. A clear centromere location was observed in chromosomes 1/2, 3/4 in all five H. rhamnoides taxa. Generally, chromosome 3 of H. rhamnoides 'Wucifeng' was seen as a dicentric chromosome (Figure 7). The chromosome 1/2, 3/4 arm ratio ranged from 1 to 1.7; hence, the two chromosomes have been designated as median region (m, 1 < r < 1.7). The symmetry of chromosome 1/2 was higher than that of chromosome 3/4. The centromere location was also observed for a few other chromosomes, such as chromosome 7/8, 19/20 of H. rhamnoides 'Zhuangyuanhuang', albeit not as clearly as that of chromosomes 1/2, 3/4. It was difficult to determine the centromere location of other chromosomes as they were small in size and had lightly stained centromeres, which also made it difficult to count their arm ratios and construct a karyotype formula.

Physical Map Distinguished Chromosomes
Next, as shown in Figures 5 and 6, the chromosomes were further eliminated with a common signal. As a result, the chromosomes of H. rhamnoides identified by (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA were aligned into a simplified version of Figures 3 and 4. To better exhibit the centromere location, each chromosome was visualized in a black-white version (Figures 7 and 8). The signal pattern ideograms were constructed based on the above blackwhite visualization of the chromosomes and their signal patterns in Figures 5 and 6. A clear centromere location was observed in chromosomes 1/2, 3/4 in all five H. rhamnoides taxa. Generally, chromosome 3 of H. rhamnoides 'Wucifeng' was seen as a dicentric chromosome (Figure 7). The chromosome 1/2, 3/4 arm ratio ranged from 1 to 1.7; hence, the two chromosomes have been designated as median region (m, 1 < r < 1.7). The symmetry of chromosome 1/2 was higher than that of chromosome 3/4. The centromere location was also observed for a few other chromosomes, such as chromosome 7/8, 19/20 of H. rhamnoides 'Zhuangyuanhuang', albeit not as clearly as that of chromosomes 1/2, 3/4. It was difficult to determine the centromere location of other chromosomes as they were small in size and had lightly stained centromeres, which also made it difficult to count their arm ratios and construct a karyotype formula.    Figure 5 only exhibits chromosomes with (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA signals, exclusively identified chromosomes, whereas Figure 5 does not present chromosomes with no diagnostic chromosome signals, such as chromosomes only with (AG 3 T 3 ) 3 end signal. Therefore, Figure 5 is a simplified version of Figure 3.    Figure 5 only exhibits chromosomes with (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA signals, exclusively identified chromosomes, whereas Figure 6 does not present chromosomes with no diagnostic chromosome signals, such as chromosomes only with (AG 3 T 3 ) 3 end signal. Therefore, Figure 6 is a simplified version of Figure 4.
Owing to the lack of effective discernment, (AG 3 T 3 ) 3 located at the end of each chromosome was ignored here. Three (AG 3 T 3 ) 3 signal types identified six chromosomes of H. rhamnoides (Figures 5-8). Type I (AG 3 T 3 ) 3 discerned chromosome 3/4 by two strong signals in the proximal region. Type II (AG 3 T 3 ) 3 discerned chromosome 11/12 by two small signals in the proximal region. Type III (AG 3 T 3 ) 3 discerned chromosome 19 by a signaldissociated chromosome end (satellite body). Chromosome 20 could not be discerned well based on its match with chromosome 19 (chromosome length, arm, centromere, and common signal).
Overall, (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA may discern 14 chromosomes in five H. rhamnoides taxa. More importantly, the combination of the three oligo-probes may identify one wild H. rhamnoides taxon from four H. rhamnoides cultivars. mosome was ignored here. Three (AG3T3)3 signal types identified six chromosomes of H. rhamnoides (Figures 5-8). Type I (AG3T3)3 discerned chromosome 3/4 by two strong signals in the proximal region. Type II (AG3T3)3 discerned chromosome 11/12 by two small signals in the proximal region. Type III (AG3T3)3 discerned chromosome 19 by a signal-dissociated chromosome end (satellite body). Chromosome 20 could not be discerned well based on its match with chromosome 19 (chromosome length, arm, centromere, and common signal).   Figure 5. The red dotted line indicates centromere location. Small chromosomes with dim centromere location were aligned by the subtle clues and traces of chromosome white/black contrast. Therefore, determination of their centromere location is difficult. The signal pattern ideograms were constructed based on the above black-white chromosome and signal patterns of chromosomes in Figure 5. The numbers on the upper side represent chromosome number, and the (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA signal types at the bottom are consistent with H. rhamnoides in Figure 5.
by the subtle clues and traces of chromosome white/black contrast. Therefore, determination of their centromere location is difficult. The signal pattern ideograms were constructed based on the above black-white chromosome and signal patterns of chromosomes in Figure 5. The numbers on the upper side represent chromosome number, and the (AG3T3)3, (TTG)6, and 5S rDNA signal types at the bottom are consistent with H. rhamnoides in Figure 5. In order to better exhibit the centromere location, each chromosome in black-white was another version of the chromosome in blue in Figure 6. The red dotted line indicates centromere location. Small chromosomes with dim centromere location were aligned by the subtle clues and traces of chromosome white/black contrast. Therefore, determination of their centromere location is difficult. The signal pattern ideograms were constructed based on the above black-white chromosome and signal patterns of chromosomes in Figure 6. The numbers on the upper side represent chromosome number and the (AG3T3)3, (TTG)6, and 5S rDNA signal types at the bottom are consistent with H. rhamnoides in Figure 6.
Satellite bodies, as hereditary features, may be used to identify chromosomes and distinguish species [47,48]. One pair of H. rhamnoides taxon satellite chromosomes was observed in previous studies [36][37][38], whereas Liang et al. [40] observed three pairs of H. rhamnoides subsp. sinensis satellite chromosomes. However, Li et al. [41] did not observe satellite chromosomes in H. rhamnoides taxon. Interestingly, only one satellite body was clearly observed in the present study. The possible reasons are as follows: (1) the other satellite body was too close to the chromosome arm to be well discovered; (2) the other satellite body was lost during slide preparation; (3) the H. rhamnoides chromosome was small in size, causing the satellite body to be smaller; (4) the satellite body is a fickle structure; hence, translocation and transfer of the satellite body occurs readily; and (5) the inconsistent evolution of two satellite bodies caused the other one to lack the portion that is visualized by oligo-probes. These possibilities may cause a change in the number of satellite bodies.
(TTG) 6 , as a useful non-chromosome end marker, has demonstrated abundant variation in 16 Avena species [35], F. pennsylvanica, S. oblata, L. lucidum, and L. × vicaryi [29]. The signal location moved from the subterminal region to the proximal region, whereas the signal intensity ranged from weak and small to strong and large. The signal band on one chromosome ranged from one to more. Research on (TTG) n as an oligo-FISH marker is scarce. However, (TTG) 10 has also emerged as an important microsatellite for genetic marker characterization in Capsicum annuum L. [58], Triticum aestivum L. [59], and Nicotiana tabacum L. [60]. In the present study, (TTG) 6 sites in H. rhamnoides were relatively stable and were only located in the proximal region; nevertheless, the signal strength changed from weak to strong, similar to that in Avena species and Oleaceae species. Moreover, our results revealed variability in the number of (TTG) 6 among H. rhamnoides taxa that showed divergence (two sites in wild H. rhamnoides ssp. sinensis, but six sites in the other four H. rhamnoides cultivars), which also agreed with the varied (TTG) 6 distribution among Avena species and Oleaceae species. Therefore, (TTG) 6 is an effective oligo-FISH marker for detecting species or subspecies. 5S rDNA has been used extensively as a chromosome marker and exhibits substantial conservation and stability in woody plants Annona cherimola L. [61], C. sinensis × P. trifoliata [23], A. elata, D. morbiferus, E. sessiliflorus, K. septemlobus [51], Ch. campanulatus [30], G. biloba and P. densiflora [52], H. rhamnoides [17], R. wichurana [53], Passiflora species [62], Cestrum species [56], and V. foetida [57]. However, 5S rDNA has also showed high diversity in other plants, including A. hypogaea [20], Fragaria L. species [63], Crocus sativus L., Crocus vernus (L.) Hill [64,65], and P. concolor [33].
In the current study, 5S rDNA nearly colocalized with (AG 3 T 3 ) 3 at the two chromosome ends. Similar colocalization has been found in B. diaphana [28] and Chrysanthemum zawadskii (Herb.) Tzvel. [66]. Puterova et al. [17] also found two 5S rDNA terminal signals in H. rhamnoides chromosome, which supports the results of the present study. The 5S rDNA distribution in the termini has also been reported in F. pennsylvanica, S. oblata, L. lucidum, L. × vicaryi [29], and P. foetida [62]. The FISH results presented herein confirm a substantial conservation in the number and location of 5S rDNA among H. rhamnoides taxa. As a consequence, the present study results indicate that 5S rDNA cannot clearly distinguish H. rhamnoides taxa.

Conclusions
To the best of our knowledge, this is the first study to assess (AG 3 T 3 ) 3 , (TTG) 6 , and 5S rDNA in H. rhamnoides. This study was conducted to identify the chromosomes of H. rhamnoides and compare cultural/wild H. rhamnoides ssp. sinensis with three varieties of H. rhamnoides. Information on chromosome identification, as well as the identification of taxa, will not only help elucidate visual and elaborate physical mapping but will also guide breeders' utilization of wild resources of H. rhamnoides. The use of the oligo-FISH system will enable, for the first time in the genomics era, a comprehensive cytogenetic analysis in H. rhamnoides. The results of this study will help identify chromosomes and establish physical maps of other Hippophaë taxa and close genera. We are committed to developing additional oligos (such as detection centromeres) to generate a high-resolution and informative cytogenetic map of the genome regions of H. rhamnoides.