Karyotype and Phylogenetic Relationship Analysis of Five Varieties and Cultivars of Zanthoxylum armatum Based on Oligo-FISH

Green prickly ash (Zanthoxylum armatum) has edible and medicinal value and is an economically significant plant in many countries. Z. armatum has many cultivars and varieties with similar phenotypes that are difficult to distinguish via traditional methods. In this study, we utilized oligo-FISH to distinguish five varieties and cultivars of Z. armatum on the basis of three oligonucleotide probes of 5S rDNA, (AG3T3)3, and (GAA)6. Karyotype analysis of the five varieties and cultivars of Z. armatum showed that the Z. armatum ‘Tengjiao’ karyotype formula was 2n = 2x = 98m with karyotype type 1C and an arm ratio of 4.3237, including two pairs of 5S rDNA signals and five pairs of (GAA)6 signals. The karyotype formula of Z. armatum ‘Youkangtengjiao’ was 2n = 2x = 128m + 8sm with karyotype type 2B and an arm ratio of 3.5336, including three pairs of 5S rDNA signals and 17 pairs of (GAA)6 signals. The karyotype formula of Z. armatum var. novemfolius was 2n = 2x = 134m + 2sm with karyotype type 1C and an arm ratio of 5.5224, including two pairs of 5S rDNA signals and eight pairs of (GAA)6 signals. The karyotype formula of Z. armatum ‘YT-03’ was 2n = 2x = 2M + 128m + 4sm + 2st with karyotype type 2C and an arm ratio of 4.1829, including three pairs of 5S rDNA signals and nine pairs of (GAA)6 signals. The karyotype formula of Z. armatum ‘YT-06’ was 2n = 2x = 126m + 10sm with cytotype 2B and an arm ratio of 3.3011, including three pairs of 5S rDNA signals and two pairs of (GAA)6 signals. The five varieties and cultivars of Z. armatum had (AG3T3)3 signals on all chromosomes. The chromosomal symmetry of Z. armatum ‘Tengjiao’ was high, whereas the chromosomal symmetry of Z. armatum 'YT-03' was low, with the karyotypes of the five materials showing a trend toward polyploid evolution. The phylogenetic relationship between Z. armatum ‘Tengjiao’ and Z. armatum var. novemfolius was the closest, while that between Z. armatum ‘YT-03’ and Z. armatum ‘YT-06’ was closer than with Z. armatum ‘Youkangtengjiao’ according to oligo-FISH. The results provided a karyotype profile and a physical map that contributes to the distinction of varieties and cultivars of Z. armatum and provides strategies for distinguishing other cultivated species.


Introduction
Zanthoxylum is a genus of economically important aromatic plants in the family Rutaceae, commonly referred to as Sichuan pepper [1,2], which has over 200 species [3]. Currently, there are 45 species and 13 varieties of wild Zanthoxylum in China [4]. Zanthoxylum has been bred for more than 2000 years in China [5]. In addition to being used in cooking, Zanthoxylum is used as an insect repellent and as a medicinal treatment for fever, These materials in the cultivar garden were managed at the same level and sampled at the same time. Five seedlings were transplanted to the Chengdu Academy of Agriculture and Forestry Sciences and photographed with a stereomicroscope after normal growth. When the root tip grew to 1.5-2 cm, the root-tip meristem was removed, placed in nitrous oxide for 2.5 h, soaked in acetic acid for 5 min, and then stored in 75% ethanol at −20 • C.

FISH
The root-tip meristems were dispersed in cellulose and pectinase (2:1) and then maintained in this mixture at 37 • C for 45 min, as described by Luo et al. [29]. The enzyme mixture was subsequently washed using ddH 2 O twice. The ddH 2 O was then removed from the mixture, and the mixture was subsequently washed in ethyl alcohol twice. After it air-dried completely, 20 µL of glacial acetic acid was added; then, the enzyme mixture was dropped onto a clean slide and examined using an Olympus CX23 microscope (Olympus Corporation, Tokyo, Japan). This study used (AG 3 T 3 ) 3 [31], 5S rDNA [14], and (GAA) 6 [3] as probes; they were produced by Sangon Biotechnology Co., Ltd. (Shanghai, China). The 5' ends of the probe were labeled using 6-carboxyfluorescein (FAM) or 6-carboxytetramethylrhodamine (TAMRA). The synthetic probes were dissolved in 1× Tris-ethylenediaminetetraacetic acid (TE) at a concentration of 10 µM and stored at −20 • C.
The slides were subjected to chromosome fixation, dehydration, and denaturation before being dehydrated again and then hybridized using the denatured probe mixture at 37 • C for 1.5 h [3]. After hybridization, 10 µL of 4,6-diamidino-2-phenylindole (DAPI) was dropped onto slides for observation using an Olympus BX63 fluorescence microscope combined with a Photometric SenSys Olympus DP70 CCD camera (Olympus Corporation, Tokyo, Japan). Signal patterns were analyzed using the three best spreads. The length of each chromosome was calculated using Photoshop version 2021 (Adobe Systems Inc., San Jose, CA, USA); each spread was measured three times to obtain an average value and consistent chromosome data. Karyotype data (karyotype formula, arm ratio, and karyotype type) were determined using NucType 2013; the analysis was based on the length of each chromosome. The physical map was generated on the basis of the signals of 5S rDNA and (GAA) 6 using Photoshop version 2021.

Genetic Relationships
The study utilized 5S rDNA-and (GAA) 6 -specific oligonucleotide probes to analyze the genetic relationships among five Z. armatum materials. The 5S rDNA signals were used as the primary reference basis for phylogenetic analysis; these were previously used in various species. To determine the genetic relationship, the number of 5S rDNA signals was screened; close numbers indicated a close relationship. If the number of 5S rDNA signals was consistent, (GAA) 6 was used as an auxiliary reference. The physical map of Z. armatum was created using Photoshop version 2021.

Flower Bud and Fruit Morphology of Z. armatum
The phenotypic similarity of the flower buds and fruits of the five varieties and cultivars of Z. armatum was evaluated. Compared with the flower bud stage, the bud at the bottom of Z. armatum 'Tengjiao' had little morphological change, and the bud at the top of Z. armatum 'Tengjiao' became round and long in shape. In the early quilt differentiation stage of flower bud differentiation of Z. armatum, the flower bud development degree of Z. armatum 'Tengjiao' was slightly lower than that of other varieties and cultivars of Z. armatum. The upper end of the flower bud of Z. armatum var. novemfolius became round and large, and the young leaves covering the flower bud began to stretch while the bud became spherical, reaching the late stage of ovary differentiation of Z. armatum with the fastest development degree. The other varieties and cultivars of Z. armatum were in the early stage of ovary differentiation, and the flower buds became slightly round to spherical in shape. The fruit morphologies of the five varieties and cultivars of Z. armatum were similar and difficult to distinguish. The flower bud and fruit morphologies of the five materials of Z. armatum are shown in Figure 1.
The phenotypic similarity of the flower buds and fruits of the five varieties and tivars of Z. armatum was evaluated. Compared with the flower bud stage, the bud a bottom of Z. armatum 'Tengjiao' had little morphological change, and the bud at the to Z. armatum 'Tengjiao' became round and long in shape. In the early quilt differentia stage of flower bud differentiation of Z. armatum, the flower bud development degr Z. armatum 'Tengjiao' was slightly lower than that of other varieties and cultivars armatum. The upper end of the flower bud of Z. armatum var. novemfolius became ro and large, and the young leaves covering the flower bud began to stretch while the became spherical, reaching the late stage of ovary differentiation of Z. armatum with fastest development degree. The other varieties and cultivars of Z. armatum were in early stage of ovary differentiation, and the flower buds became slightly round to sp cal in shape. The fruit morphologies of the five varieties and cultivars of Z. armatum similar and difficult to distinguish. The flower bud and fruit morphologies of the five terials of Z. armatum are shown in Figure 1.

Karyotype of Z. armatum
The metaphase chromosomes of Z. armatum were analyzed using FISH, as shown in Figures 2 and 3. The chromosome number of Z. armatum 'Tengjiao' was 98, while the chromosome number of Z. armatum 'Youkangtengjiao', Z. armatum var. novemfolius, Z. armatum 'YT-03', and Z. armatum 'YT-06' was 136. To better characterize these chromosomes, individual chromosomes were aligned according to length from the longest to the shortest, as illustrated in Figures 4 and 5.

Inconsistent Chromosome Number of Five Z. armatum Materials
Chromosomal data are fundamental to cytological studies [32] and widely documented in most families and genera [15]. Chromosomal data are useful for solving traditional plant classifications [31]; moreover, because they are relatively stable in plants, they can also be used to study chromosome aberration, cell function, and inference of genomic events [14,16].
Previously, Z. armatum was reported to have 2n = 66 [17], ~128 [3], 132 [20], and 136 chromosomes [16], which differed from our results. Our chromosome number results for the five varieties and cultivars of Z. armatum were inconsistent with those of Zhang and Hartley [17]; this may be because Zhang and Hartley [17] adopted the traditional production method. Their process for obtaining chromosomes was rough, and some chromosomes may have been washed out. Additionally, chromosomes overlapped during production, which may have caused the chromosome numbers to differ. Our results for Z. armatum 'YT-03', Z. armatum 'YT-06', Z. armatum 'Youkangtengjiao', and Z. armatum var. novemfolius were inconsistent with Luo et al.'s results for two possible reasons. First, different research materials were used. Luo et al. [3] used Z. armatum 'Hanyuanputaoqing' The phylogenetic relationship between Z. armatum 'Tengjiao' and Z. armatum var. novemfolius was the closest, while that between Z. armatum 'YT-03' and Z. armatum 'YT-06' was closer than with Z. armatum 'Youkangtengjiao' according to 5S rDNA and (GAA) 6 signals. The phylogenetic relationships of the five Z. armatum materials were also confirmed by the karyotype analysis ( Table 2). The strong symmetry of Z. armatum 'Tengjiao' and Z. armatum var. novemfolius indicated that their divergence was minimal.

Inconsistent Chromosome Number of Five Z. armatum Materials
Chromosomal data are fundamental to cytological studies [32] and widely documented in most families and genera [15]. Chromosomal data are useful for solving traditional plant classifications [31]; moreover, because they are relatively stable in plants, they can also be used to study chromosome aberration, cell function, and inference of genomic events [14,16].
Previously, Z. armatum was reported to have 2n = 66 [17],~128 [3], 132 [20], and 136 chromosomes [16], which differed from our results. Our chromosome number results for the five varieties and cultivars of Z. armatum were inconsistent with those of Zhang and Hartley [17]; this may be because Zhang and Hartley [17] adopted the traditional production method. Their process for obtaining chromosomes was rough, and some chromosomes may have been washed out. Additionally, chromosomes overlapped during production, which may have caused the chromosome numbers to differ. Our results for Z. armatum 'YT-03', Z. armatum 'YT-06', Z. armatum 'Youkangtengjiao', and Z. armatum var. novemfolius were inconsistent with Luo et al.'s results for two possible reasons. First, different research materials were used. Luo et al. [3] used Z. armatum 'Hanyuanputaoqing' instead of our research cultivars of Z. armatum. Thus, Luo et al.'s [3] research results might indirectly prove that different cultivars of Z. armatum have different chromosome numbers. Their Z. armatum cultivars were ambiguous, and the materials themselves were problematic [33]. Second, Luo et al. [3] did not use telomere (AG 3 T 3 ) 3 probes to ensure chromosome integrity and counting accuracy, which may have resulted in counting errors. Hu et al. [20] used telomere probes, but some chromosomes were superimposed in the division phase, which could have easily led to miscounting. Secondly, their research did not specify the name of the Z. armatum cultivars, which may have differed from the materials we used.
Z. armatum 'Youkangtengjiao' was bred from Z. armatum 'Tengjiao'; they belong to the same species and should have the same number of chromosomes. However, we found an interesting phenomenon; i.e., their chromosome numbers were inconsistent. We hypothesize that the reason for this might be related to the apomictic reproduction of Z. armatum. Apomictic reproduction does not require parental gametophytes to produce zygotes; offspring are produced directly from the mother [34]. This method of reproduction could fix the mother's economic traits, which would be beneficial for breeding [35]. It is possible that apomictic reproduction led to similar phenotypic characteristics in Z. armatum, although there were many varieties and cultivars. Apomixis may have formed nested chromosome fusions (NCF), leading to changes in chromosome numbers. The vast majority of cases were polyploid, which resulted in abnormal chromosome numbers [36]. This phenomenon was previously found in wheat [37], Coccinia grandis (L.) Voigt [38], and grapevine varieties [39]. The Z. armatum genus genome is large; extensive transposable element (TEs) and events of whole-genome duplication (WGD) led to changes in the plant genome [40][41][42]. Active TEs might have triggered fission and fusion events of Z. armatum chromosomes [43], which might have led to changes in the chromosome number [44]. Understanding chromosome numbers is beneficial for future Z. armatum gene assembly and would allow a physical map of oligonucleotides to be drawn. In the future, we will identify the chromosomes of other varieties and cultivars of Z. armatum.

Divergence Stages of Five Z. armatum Materials
Karyotype analysis can not only study the phylogenetic relationship but also provide evidence for the cell research of this species [35]. In this study, the karyotype data of five varieties and cultivars of Z. armatum were inconsistent, which may be related to the region and growing environment [45]. Z. armatum 'Tengjiao' grows in the rainy zone of West China, while Z. armatum var. novemfolius grows in a subtropical monsoon humid climate [46]. Plants living in different environments for a long time will not only have divergent adaptation but will also undergo genetic changes [47], thus influencing chromosome data. The inconsistent karyotype analysis data may also be related to the difficulty of karyotype analysis due to the small chromosome size of Z. armatum [3]. In the karyotype analysis of five varieties and cultivars of Z. armatum, some centromeric points were not obvious, and there was difficultly in the karyotype analysis. We conducted the karyotype analysis on the basis of the clearest centromeric points. The inconsistent karyotype data of the five varieties and cultivars of Z. armatum may also be related to the apomictic reproduction of Z. armatum, which resulted in nested chromosome fusion, leading to changes in the number of chromosome karyotypes. At the same time, Z. armatum has a large genome, and the occurrence of TEs and WGD can also lead to gene changes, which may alter the chromosome karyotype data. The genome of Z. armatum is still undetermined despite some studies [8,10]; thus, further studies are needed to determine how genetic changes affect the chromosome.
Stebbins et al. [48] believed that a higher chromosome symmetry indicates earlier divergence. The evolutionary trend of karyotype ranges from symmetrical to asymmetrical. The chromosomes of the five varieties and cultivars of Z. armatum were mainly m and sm chromosomes with a small number of M and st chromosomes. Overall, the five varieties and cultivars of Z. armatum had high symmetry, possibly indicating early divergence. The m chromosome of Z. armatum 'Tengjiao' was the least evolved. Z. armatum 'YT-03' had lower symmetry and was more evolved. Some plant karyotypes can evolve from diploid to polyploid [49]. The evaluation of the five materials showed that the karyotype of varieties and cultivars of Z. armatum also potentially evolved from diploid to polyploid. Z. armatum 'Tengjiao' can maintain a relatively primitive state, which may be related to the stability of the current environment [45]. At present, the climate is relatively stable and there is no extreme climate; hence, rattan pepper has adapted to the local climate environment with a low degree of evolution.
The chromosome of Z. armatum can be considered small [3], complicating karyotype analysis. Although our study could not accurately describe the karyotype data of Z. armatum, it could reflect the genetic differences at the chromosome level among the five varieties and cultivars of Z. armatum. The evolutionary relationship of the different varieties and cultivars of Z. armatum requires further study.

Oligonucleotide Signal Differences of Five Z. armatum Materials
Using single-copy and repetitive DNA sequences of plants and chromosome-specific probes [50], FISH is a valuable molecular cytogenetic mapping tool, especially for species with small chromosome numbers and similar morphologies [51]. The numbers and distribution of chromosomes according to FISH can reveal genetic composition patterns and karyotype variations [52]. Oligo-FISH can be used to analyze karyotypes, chromosome rearrangements, and meiotic pairing [53], and it has been widely used in determining genetic relationships among plant species [25,26], as well as in phylogenetic reconstruction [27] and molecular cytology [28], with rapid and efficient results [32].
It is known that 5S rDNA may be present at the middle, near middle, and end of each chromosome [14,54]. There is a strong relationship between signal number and ploidy [55]. To date, FISH has identified the number and location of rDNA loci in more than 1600 plant species [56]. The quantity and location of 5S and 45S rDNA are often characteristic of a particular species or genus [57][58][59]. As a powerful cytogenetic marker, 5S rDNA [60] is helpful in the study of reproductive segregation and speciation [61], chromosome characterization [62], and genome differentiation [27]. Using FISH technology to locate repetitive DNA sequences helps correct the karyotypes of small chromosomes and insignificant morphological characteristics, which can reflect the degree of differentiation between species and genera at the chromosome level [54,63]. Luo et al. [3] obtained two pairs of 5S rDNA signal sites, which was consistent with Z. armatum's 5S rDNA signal sites identified in this study but inconsistent with the three pairs of 5S rDNA sites of Z. armatum 'Youkangtengjiao', Z. armatum 'YT-03', and Z. armatum 'YT-06'. We speculated that this may be because Z. armatum 'Youkangtengjiao', Z. armatum 'YT-03', and Z. armatum 'YT-06' had new chromosomes containing more genetic information. The signal locations of Z. armatum 'Tengjiao' and Z. armatum var. novemfolius' were the same as those found by Luo et al. [3]; this may indicate that the two cultivars share similar genetic information.
The (AG 3 T 3 ) 3 signals may be present in the proximal, distal, and dissociative parts of chromosomes [29]. The (AG 3 T 3 ) 3 probe was used to determine the integrity and number of chromosomes because it is often present at both ends of chromosomes [29]. The (AG 3 T 3 ) 3 probe was used herein for the first time in Z. armatum. The probe signal sites appeared at both ends of the chromosome, which is convenient for counting chromosomes. GAA signals may appear in the proximal telomeric, sub-telomeric, and interstitial regions [64][65][66][67]; this is beneficial for cytogenetics and genome organization research. Luo et al. [3] found five pairs of (GAA) 6 signals in Z. armatum, which was consistent with our results for Z. armatum 'Tengjiao' but inconsistent with the 17 pairs of (GAA) 6 signals in Z. armatum 'Youkangtengjiao', the two pairs of (GAA) 6 signals in Z. armatum 'YT-03', the nine pairs of (GAA) 6 signals in Z. armatum 'YT-06', and the eight pairs of (GAA) 6 signals in Z. armatum var. novemfolius.
It is difficult to distinguish intra-and interspecific differences using only FISH [30]. To completely distinguish intra-and interspecific differences, it is necessary to design sufficient probes and draw oligonucleotide maps. However, because of the large number of chromosomes and unclear genome sequences, this work will be extremely challenging. Some chromosomal changes (deletions, duplications, inversions, or translocations) [30] and B chromosome generation [15,50] can result in changes in the number of chromosomes and more complex genetic information. However, woody plant genomes are highly heterozygous and have a complex genetic background [68]. In the Rutaceae family, complete genome sequences have been studied only in Citrus [1,2,69]. Although there have been some studies of the Zanthoxylum genome [8,10,36], they had pre-existing limitations. Therefore, more in-depth research is needed.

Conclusions
There were differences in the karyotypes of the five varieties and cultivars of Z. armatum, showing a trend toward polyploid evolution. Our research method can be used to distinguish different varieties and cultivars of Z. armatum as well as to provide a reference for the future cultivation of different species.