Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH

International Journal of Molecular Sciences Article Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH Fan Yu 1,2, Jin Chai 1,2, Xueting Li 1,2, Zehuai Yu 3, Ruiting Yang 1,2, Xueer Ding 1,2, Qiusong Wang 1,2, Jiayun Wu 1,4, *, Xiping Yang 3 and Zuhu Deng 1,2,3, * 1 National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, China; yufanky@163.com (F.Y.); CJ1152648@163.com (J.C.); lxt19910226@163.com (X.L.); Angelinating2021@163.com (R.Y.); 15294800931@163.com (X.D.); 13110898682@163.com (Q.W.) 2 Key Lab of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China 3 State Key Laboratory for Protection and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China; ArHuay_Yu@163.com (Z.Y.); xipingyang@gxu.edu.cn (X.Y.) 4 Institute of Nanfan & Seed Industry, Guangdong Academy of Sciences, Guangzhou 510316, China * Correspondence: jiayunng@163.com (J.W.); dengzuhu@163.com (Z.D.) Citation: Yu, F.; Chai, J.; Li, X.; Yu, Z.; Yang, R.; Ding, X.; Wang, Q.; Wu, J.; Yang, X.; Deng, Z. Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH. Int. J. Mol. Sci. 2021, 22, 8539. https://doi.org/10.3390/ ijms22168539 Academic Editors: Natalia Borowska-Zuchowska, Ewa Robaszkiewicz and Robert Hasterok Abstract: Sugarcane is of important economic value for producing sugar and bioethanol. Tripidium arundinaceum (old name: Erianthus arundinaceum) is an intergeneric wild species of sugarcane that has desirable resistance traits for improving sugarcane varieties. However, the scarcity of chromosome markers has hindered the cytogenetic study of T. arundinaceum. Here we applied maize chromosome painting probes (MCPs) to identify chromosomes in sorghum and T. arundinaceum using a repeated fluorescence in situ hybridization (FISH) system. Sequential FISH revealed that these MCPs can be used as reliable chromosome markers for T. arundinaceum, even though T. arundinaceum has diverged from maize over 18 MYs (million years). Using these MCPs, we identified T. arundinaceum chromosomes based on their sequence similarity compared to sorghum and labeled them 1 through 10. Then, the karyotype of T. arundinaceum was established by multiple oligo-fish. Furthermore, FISH results revealed that 5S rdna and 35S rdna are localized on chromosomes 5 and 6, respectively, in T. arundinaceum. Altogether, these results represent an essential step for further cytogenetic research of T. arundinaceum in sugarcane breeding. Keywords: T. arundinaceum; sugarcane; sorghum; maize chromosome painting probes; oligo-fish; ribosomal DNA; chromosome identification; karyotype Received: 3 July 2021 Accepted: 7 August 2021 Published: 9 August 2021 Publisher s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction Sucrose is produced from two major crops, sugarcane (Saccharum spp.) and sugar beet (Beta vulgaris). Sugarcane, however, accounts for the vast majority of global sugar production and provides feedstocks for bio-energy production. For sugarcane breeding, interspecific hybridization is a powerful way to enhance resistance but also provides unexpected benefits in increasing yield and improving ratooning ability and adaptability [1]. S. spontaneum (2n = 40 128), which for more than a century has played a major role in sugarcane breeding, has been widely applied to improve the resistance of sugarcane cultivars. Simultaneously, E. arundinaceum (2n = 60, x = 10) is also used for sugarcane breeding due its high biomass productivity, superior ratooning ability, and exceptional adaptability to biotic and abiotic stresses [2]. Most recently, Lloyd Evans et al. placed E. arundinaceum to the Tripidium genus based on the whole chloroplast genome [3]. Thus, E. arundinaceum was referred to T. arundinaceum accordingly. Currently, many studies have reported cytogenetic research on T. arundinaceum, especially on the chromosome inheritance of the hybrids between sugarcane and T. arundinaceum [4 6]. However, basic cytogenetic information such as the karyotype, and the precise chromosome contribution Int. J. Mol. Sci. 2021, 22, 8539. https://doi.org/10.3390/ijms22168539 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 8539 2 of 10 No. of Oligo of T. arundinaceus in interspecific hybridization, could not be addressed due to lack of effective cytogenetic markers to identify the individual chromosomes. FISH is a powerful tool that has widely answered the cytogenetics issues, such as karyotype, chromosome recombination, genetic relationship of the related species, and chromosome transmission [7]. Successful and efficient FISH needs a reliable probe, such as genomic DNA (gdna), repetitive sequences, bacterial artificial chromosome (BAC) clones, and oligonucleotides (oligos) [7 9]. Of these, oligos are a recent development and application of a probe that can be computationally identified according to sequencing genome data [7,10]. Oligo probes now are classified as repetitive oligonucleotide (repetitive DNA sequence), chromosome barcode (a specific chromosomal region), and chromosome painting (an entire chromosome region), and have been successfully applied in many plants, i.e., rice [11], wheat [12], citrus [13], maize [14], etc. Oligos designed from genome sequences based on low-copy sequences are better conserved than the repetitive DNA, which can be used for chromosome identification in related species that have diverged for several million years (MYs), or even more than 15 MYs [13,15,16]. In the present study, we tested maize chromosome painting probes (MCPs) in sorghum and T. arundinaceum using multiple rounds of FISH. Based on the oligo-fish results, for the first time, we reported that MCPs can distinctly detect chromosomes 1 10 of T. arundinaceum. Then, the karyotype of T. arundinaceum was established according to the individual chromosome identification combining MCPs, 5S rdna, and 35S rdna probes. Altogether, these results will be useful for further understanding the chromosome inheritance of T. arundinaceum and improving the efficiency of sugarcane breeding. 2. Results 2.1. Sequence Alignment Analysis between MCP Sequences and Sorghum Genome We aimed to align 10 MCP sequences to 10 pseudomolecules of sorghum. Then, the sequence comparison was performed between MCP sequences and sorghum genome (see Methods section). The results showed that the number of oligos that align to the sorghum genome ranged from 277 to 27,169 (Table 1), which implied that these MCPs will produce various signals on the chromosomes of sorghum. To further understand the distribution of MCP sequences, we selected the MCP sequences that have the potential to produce obvious signals (the number of oligos > 1000) for displaying their locations on ten sorghum chromosomes (Figure 1); for example, MCP1 sequences aligned with sorghum chromosomes 1, 7, and 8 (Figure 1a and Figure S1). This implies that the MCP1 probe may produce hybridization signals on these three sorghum chromosomes. Although the MCP sequences showed an uneven distribution in sorghum, the number of the aligned MCP oligos was up to 10,000 on half of the sorghum chromosomes (chromosomes 1, 2, 3, 4, and 9, Table 1), and even 27,169 (chromosome 1, Table 1). These results indicated that the MCPs may be valid chromosome markers for sorghum chromosome identification. Table 1. The number of maize CP sequences aligned to each of the sorghum chromosomes. Sorghum1 Sorghum2 Sorghum3 Sorghum4 Sorghum5 Sorghum6 Sorghum7 Sorghum8 Sorghum9 Sorghum10 MCP1 27,169 746 892 820 657 689 4812 2772 700 791 MCP2 701 9016 564 521 2613 11,784 405 446 525 524 MCP3 711 546 20,574 550 495 415 463 3761 435 513 MCP4 836 531 600 9900 5120 449 4999 564 484 543 MCP5 7236 539 552 15,991 382 468 411 490 530 2548 MCP6 625 416 507 404 424 322 1390 659 10,918 6910 MCP7 488 17,672 421 422 316 316 387 347 367 415 MCP8 466 402 11,297 420 294 282 313 372 5725 419 MCP9 8544 434 412 411 277 304 296 300 329 9614 MCP10 648 471 501 479 466 9307 2888 3983 1136 481

Int. J. Mol. Sci. 2021, 22, 8539 3 of 10 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 11 Figure 1. Maize CP sequences distribution on sorghum chromosomes. Arabic numerals indicate the chromosome of sorghum; (a j) indicate the MCP1-10 sequence distributions on sorghum chromosomes in each 500 kb window, respectively. Figure 1. Maize CP sequences distribution on sorghum chromosomes. Arabic numerals indicate the chromosome of sorghum; (a j) indicate the MCP1-10 sequence distributions on sorghum chromosomes in each 500 kb window, respectively. Table 1. The number of maize CP sequences aligned to each of the sorghum chromosomes. 2.2. Chromosome Painting Using MCPs in Sorghum No. of Oligghumghum2 MCP10, ghum3 that were ghum4 sufficient ghum5 to distinguish ghum6 ten sorghum ghum7 chromosomes ghum8 ghum9 in a few ghum10 rounds of Sor- Sor- Based Sor- on the Sor- distributions Sor- of thesor- ten MCPsSor- above, we Sor- selected Sor- six probes, Sor- MCP5 MCP1 27,169 746 oligo-fish. 892 These 820 six MCPs657 were labeled 689 by digoxigenin 4812 or 2772 biotin, and 700 conjugated 791 with MCP2 701 9016 anti-dig 564 or anti-bio 521 antibodies, 2613 respectively. 11,784 Pairs405 of probes 446 were sequentially 525 hybridized 524 MCP3 711 546 to the20,574 same metaphase 550 chromosomes 495 prepared 415 from 463 the root3761 tips of sorghum. 435 For example, 513 MCP4 836 531 MCP9600 and MCP10 9900 probes were 5120 hybridized 449 to the4999 metaphase564 cell (Figure484 2a). The slide 543 was MCP5 7236 539 then washed 552 and15,991 re-probed with 382 MCP7468 and MCP8411 probes (Figure 490 2b). Finally, 530 all six 2548 MCPs MCP6 625 416 probes 507 were applied 404 after three 424 sequential 322 FISH experiments 1390 659 (Figure 10,918 2a c). 6910 Expectedly, our FISH results suggested that the MCPs signal patterns were well MCP7 488 17,672 421 422 316 316 387 347 367 415 correlated with the sequence alignment distribution between MCP sequences and sorghum MCP8 466 402 11,297 420 294 282 313 372 5725 419 genome (Figures 1 and 2). For example, MCP10 produced variously distinct signals on MCP9 8544 434 412 411 277 304 296 300 329 9614 chromosomes 6, 7, and 8, which presented unique signal types for each of these chromosomes MCP10 648 471 501 (Figure 2a). 479 However, 466 MCP10 did 9307 not produce 2888 an observed 3983 signal 1136 on chromosome 481 9 (Figure 2a). Furthermore, MCP6 (Figures 1f and 2c) and MCP5 (Figures 1e and 2c) did 2.2. not produce Chromosome an observed Painting Using signalmcps on chromosome in Sorghum 7 and chromosome 10, respectively. These results Based suggested on the that distributions the unobservable of the signal ten MCPs of these above, probes we may selected be caused six probes, by themcp5 limited MCP10, oligo numbers that were on sufficient the chromosomes to distinguish (Figure ten S1). sorghum In addition, chromosomes MCP5 did in a not few produce rounds an of oligo-fish. expected strong These signal six MCPs on chromosome were labeled 4 by (Figure digoxigenin 2c), which or biotin, impliedand that conjugated the sequential with anti-dig FISH may or affect anti-bio theantibodies, efficiency of respectively. hybridization. Pairs of probes were sequentially hybridized to the same metaphase chromosomes prepared from the root tips of sorghum. For example, MCP9 and MCP10 probes were hybridized to the metaphase cell (Figure 2a). The slide was then washed and re-probed with MCP7 and MCP8 probes (Figure 2b). Finally, all six MCPs probes were applied after three sequential FISH experiments (Figure 2a c).

Int. J. Mol. Sci. 2021, 22, 8539 4 of 10 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 11 Figure 2. Sequential FISH identifying each of the ten chromosomes in sorghum. (a) First round of Figure 2. Sequential FISH identifying each of the ten chromosomes in sorghum. (a) First round of FISH using MCP10 (digoxigenin-red) and MCP9 (biotin-green) probes. (b) Second round of FISH FISH using MCP10 (digoxigenin-red) and MCP9 (biotin-green) probes. (b) Second round of FISH using MCP8 (digoxigenin-red) and MCP7 (biotin-green) probes. (c) Third round of FISH using using MCP6 MCP8 (digoxigenin-red) (digoxigenin-red) and MCP5 and MCP7 (biotin-green) (biotin-green) probes. probes. Arabic (c) numerals Third round denote of the FISHchromosome using MCP6 (digoxigenin-red) number in sorghum. and Bar MCP5 = 10 (biotin-green) μm. probes. Arabic numerals denote the chromosome number in sorghum. Bar = 10 µm. Expectedly, our FISH results suggested that the MCPs signal patterns were well correlated 2.3. Chromosome with the Identification sequence alignment in T. arundinaceum distribution Using between MCPs, MCP 5S sequences rdna, and and 35S sorghum rdna Probes genome (Figures 1 and 2). For example, MCP10 produced variously distinct signals on chromosomes So far, the 6, T. 7, arundinaceum and 8, which genome presented dataunique are still signal unavailable. types for Iteach is difficult of these tochromo- somes karyotype (Figure using 2a). However, repetitivemcp10 sequences did not or BAC produce clones. an observed Hence, we signal attempted on chromosome to use the establish the same 9 (Figure metaphase 2a). Furthermore, cell for chromosome MCP6 (Figures identification 1f and 2c) in and T. MCP5 arundinaceum. (Figures 1e The and sequential 2c) did FISH not produce prevented an cross-hybridization observed signal on signals chromosome from non-target 7 and chromosome chromosomes. 10, respectively. We selected athese putative results hexaploid suggested T. arundinaceum that the unobservable (Hainan92-77, signal of 2n these = 60, probes x = 10) may forbe chromosome caused by painting the limited analysis oligo using numbers MCPs. on the We chromosomes performed five(figure roundss1). of sequential In addition, FISH MCP5 using did MCPs not on produce the same an metaphase expected strong platesignal of T. arundinaceum. on chromosome Based 4 (Figure on the2c), distribution which implied of MCP that onthe the sorghum sequential genome, FISH may weaffect named the the efficiency chromosomes of hybridization. 1 10 of T. arundinaceum according to the sequential FISH results (Figure 3 and Figure S2). For example, MCP2 and MCP1 were labeled 2.3. Chromosome with digoxigenin Identification (redin signal) T. arundinaceum or biotin Using (green MCPs, signal), 5S rdna, respectively and 35S (Figure rdna S2a and Probes Figure 3c). FISH showed that MCP2 produced differential signals on chromosomes 2, 5. and So far, 6 of the T. T. arundinaceum arundinaceum (Figure genome S2a). data are We still named unavailable. them as It chromosomes is difficult to establish 2, 5 and 6 the of karyotype T. arundinaceum using repetitive according sequences to the sorghum or BAC chromosome clones. Hence, nomenclature we attempted (Figure to use the 1b). The same MCP1 metaphase probe produced cell for chromosome distinct signals identification on six chromosomes in T. arundinaceum. of T. arundinaceum The sequential and we FISH identified prevented them cross-hybridization as chromosome signals 1 in T. from arundinaceum non-target (Figure chromosomes. 3c). Altogether, We selected all the a T. putative arundinaceum hexaploid chromosomes T. arundinaceum were identified (Hainan92-77, using 2n the = 60, eight x = MCPs, 10) for chromosome although thepaint- ing analysis on different using chromosomes. MCPs. We performed Additionally, five rounds the above of sequential FISH results FISH between using MCPs sorghum on signals varied and the T. same arundinaceum metaphase showed plate of that T. arundinaceum. the synteny ofbased all tenon chromosomes the distribution wasof conserved MCP the over more sorghum thangenome, 9 MYs ofwe divergence named the among chromosomes these two1 10 species of T. [17]. arundinaceum according to the sequential We then FISH performed results (Figures FISH using 3 and 5SS2). andfor 35Sexample, rdnas probes MCP2 and in the MCP1 fifthwere rounds labeled of the sequential with digoxigenin FISH after (red MCP. signal) FISH or biotin results (green suggested signal), that respectively 5S and 35S (Figures rdnas S2a sites and were 3c). located FISH showed on chromosome that MCP2 5 and produced chromosome differential 6 (Figure signals S2c), on respectively. chromosomes However, 2, 5. and 5S6 rdna of T. was arundinaceum close to the (Figure centromeric S2a). We region named andthem 35Sas rdna chromosomes was mapped 2, 5 and on the 6 of distal T. arundinaceum region. according to the sorghum chromosome nomenclature (Figure 1b). The MCP1 probe produced distinct signals on six chromosomes of T. arundinaceum and we identified them as chromosome 1 in T. arundinaceum (Figure 3c). Altogether, all the T. arundinaceum chromosomes were identified using the eight MCPs, although the signals varied on different chromosomes. Additionally, the above FISH results between sorghum and T. arundinaceum showed that the synteny of all ten chromosomes was conserved over more than 9 MYs of divergence among these two species [17]. We then performed FISH using 5S and 35S rdnas probes in the fifth rounds of the sequential FISH after MCP. FISH results suggested that 5S and 35S rdnas sites were located on chromosome 5 and chromosome 6 (Figure S2c), respectively. However, 5S rdna was close to the centromeric region and 35S rdna was mapped on the distal region.

Int. J. Mol. Sci. 2021, 22, 8539 5 of 10 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 11 Figure 3. Sequential oligo-fish identifying all 10 chromosomes on the same metaphase plate of T. arundinaceum. (a) Second round of oligo-fish using MCP5 (biotin-green) and MCP3 (digoxigenin-red) probes. (b) Fourth round of oligo-fish Figure 3. Sequential oligo-fish identifying all 10 chromosomes on the same metaphase plate of T. arundinaceum. (a) Second round using of MCP9 oligo-fish (biotin-green) using MCP5 and MCP10 (biotin-green) (digoxigenin-red) and MCP3 probes. (digoxigenin-red) (c) Statistical probes. results of (b) different Fourth round probes. of Arabic oligo-fish numerals using MCP9 denote (biotin-green) the chromosome and MCP10 numbers. (digoxigenin-red) The dotted box probes. denotes (c) the Statistical weak signals results of of chromosomes different probes. 8 and Arabic 7. The numerals symbol * indicates chromosomes numbers. 5 and 9 The were dotted both missing box denotes one copy thein weak this particular signals ofcell chromosomes due to the preparation 8 and 7. The of the symbol slides. * indicates The six denote the chromosomes copies of chromosomes 5 and 9 were 5 and both9 on missing metaphase one copy plate inare this provided particular in cell Figure dues3. tobar the= preparation 10 μm. of the slides. The six copies of chromosomes 5 and 9 on metaphase plate are provided in Figure S3. Bar = 10 µm. 2.4. Standard Karyotype Analysis of T. arundinaceum Based on Sequential Oligo-FISH 2.4. Standard In order Karyotype to identify Analysis the T. of arundinaceum T. arundinaceum chromosomes Based on Sequential quickly, Oligo-FISH we selected seven probes, In order namely tomcp3, identify MCP5, the T. MCP6, arundinaceum MCP7, MCP9, chromosomes 5S rdna, quickly, and 35S we rdna. selected The combined probes namely could MCP3, be used MCP5, to identify MCP6, MCP7, T. arundinaceum MCP9, 5Schromosomes rdna, and 35S with rdna. three The rounds com- seven probes, bined of sequential probes FISH could(figure be used4). tofor identify example, T. arundinaceum we used MCP3 chromosomes (red) and MCP5 with(green) three rounds probes of sequential to identify FISH chromosomes (Figure 4). 1, For 3, 4, example, and 8 (Figure we used 4a). Among MCP3 (red) them, and chromosome MCP5 (green) 8 produced probes to identify a weak signal. chromosomes Then MCP6 1, 3, (red) 4, and and 8 MCP9 (Figure(green) 4a). Among were selected them, chromosome to identify chromosomes 8 produced a weak 1, 9, and signal. 10 (Figure Then MCP6 4b). MCP7 (red) and (red), MCP9 5S rdna (green) (red), were and selected 35S rdna to identify (green) chromosomes probes were 1, 9, used andto 10 classify (Figurechromosomes 4b). MCP7 (red), 2, 5, 5S and rdna 6 (Figure (red), 4c). and Finally, 35S rdna chromosome (green) probes 7 was classified were used to by classify excluding chromosomes nine nonhomologous 2, 5, and 6 chromosomes (Figure 4c). Finally, already chromosome identified above. 7 was classified by excluding nine nonhomologous chromosomes already identified above. Identification of chromosomes on the same metaphase plate provided us a standard karyotype based on the individually identified chromosome. After identification of chromosomes 1 10, the centromere probe was also located on T. arundinaceum chromosomes for further chromosome measurement (Figure S4). We measured each of ten chromosomes on the metaphase cells for T. arundinaceum, then the karyotype was established accordingly (Table 2 and Figure 5). According to the traditional chromosome classification [18], most chromosomes of T. arundinaceum are metacentric with the arm ratio ranging from 1.14 ± 0.17 to 1.45 ± 0.21 (Table 2). The idiogram of T. arundinaceum was also constructed

Int. J. Mol. Sci. 2021, 22, 8539 6 of 10 based on the measured data (Figure 5). Chromosome 1 was the longest (3.58 ± 0.19 µm, Int. J. Mol. Sci. 2021, 22, x FOR PEER Table REVIEW 2 and Figure 5) and chromosome 8 was the shortest one (2.50 ± 0.23 µm, Table 62 of and 11 Figure 5). Figure Figure 4. 4. Sequential Sequential oligo-fish oligo-fish identifying identifying all all 10 10 chromosomes chromosomes on on the the same same metaphase metaphase plate plate of of T. T. arundinaceum. arundinaceum. (a) (a) First First round of FISH using MCP3 (digoxigenin-red) and MCP5 (biotin-green) probes. (b) Second round of FISH using MCP6 round of FISH using MCP3 (digoxigenin-red) and MCP5 (biotin-green) probes. (b) Second round of FISH using MCP6 (digoxigenin-red) and MCP9 (biotin-green) probes. (c) Third round of FISH using MCP7 (digoxigenin-red), 5S rdna (digoxigenin-red), and 35S rdna (biotin-green) probes. Arabic numerals denote the chromosome numbers. Bar = 10 μm. (digoxigenin-red) and MCP9 (biotin-green) probes. (c) Third round of FISH using MCP7 (digoxigenin-red), 5S rdna (digoxigenin-red), and 35S rdna (biotin-green) probes. Arabic numerals denote the chromosome numbers. Bar = 10 µm. TableIdentification 2. Chromosomeof length chromosomes and arm ratio on the of mitotic same metaphase chromosomes plate provided of T. us arundinaceum. a standard karyotype based on the individually identified chromosome. After identification of chromosomes Chromosome 1 10, the centromere Long Arm probe was Short also Armlocated on Chromosome T. arundinaceum Arm chromosomes Ratio for further Number chromosome (µm) measurement (Figure (µm) S4). We Length measured (µm) each of ten chromosomes Chr.1 on the metaphase 2.02 cells (±0.19) for T. arundinaceum, 1.56 (±0.10) then the 3.58 karyotype (±0.19) was established 1.30 (±0.18) accordingly Chr.2(Table 2 and 2.03 Figure (±0.06) 5). According 1.54 (±0.10) to the traditional 3.57 (±0.11) chromosome 1.32 classification (±0.11) Chr.3 2.08 (±0.21) 1.49 (±0.21) 3.57 (±0.30) 1.42 (±0.24) [18], most chromosomes of T. arundinaceum are metacentric with the arm ratio ranging Chr.4 1.84 (±0.16) 1.29 (±0.15) 3.13 (±0.22) 1.45 (±0.21) from 1.14 ± 0.17 to 1.45 ± 0.21 (Table 2). The idiogram of T. arundinaceum was also constructed Chr.6 based on the 1.80 measured (±0.90) data (Figure 0.90 (±0.10) 5). Chromosome 2.69 (±0.22) 1 was the longest 2.03 (±0.37) (3.58 ± Chr.5 1.76 (±0.21) 1.49 (±0.13) 3.25 (±0.26) 1.19 (±0.17) 0.19 μm, Chr.7 Table 2 and 1.70 Figure (±0.19) 5) and chromosome 1.39 (±0.09) 8 was the 3.10 shortest (±0.24) one (2.50 1.22 (±0.12) ± 0.23 μm, Table 2 Chr.8 and Figure 5). 1.40 (±0.13) 1.10 (±0.14) 2.50 (±0.23) 1.29 (±0.15) Chr.9 1.42 (±0.16) 1.19 (±0.11) 2.61 (±0.20) 1.20 (±0.18) Table 2. Chromosome length Chr.10 and arm ratio 1.38 of (±0.08) mitotic metaphase 1.21 (±0.09) chromosomes 2.59 of T. (±0.12) arundinaceum. 1.14 (±0.10) Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 11 Chromosome Long Arm Short Arm Chromosome Number (μm) (μm) Length(μm) Arm Ratio Chr.1 2.02 (±0.19) 1.56 (±0.10) 3.58 (±0.19) 1.30 (±0.18) Chr.2 2.03 (±0.06) 1.54 (±0.10) 3.57 (±0.11) 1.32 (±0.11) Chr.3 2.08 (±0.21) 1.49 (±0.21) 3.57 (±0.30) 1.42 (±0.24) Chr.4 1.84 (±0.16) 1.29 (±0.15) 3.13 (±0.22) 1.45 (±0.21) Chr.5 1.76 (±0.21) 1.49 (±0.13) 3.25 (±0.26) 1.19 (±0.17) Chr.6 1.80 (±0.90) 0.90 (±0.10) 2.69 (±0.22) 2.03 (±0.37) Chr.7 1.70 (±0.19) 1.39 (±0.09) 3.10 (±0.24) 1.22 (±0.12) Chr.8 1.40 (±0.13) 1.10 (±0.14) 2.50 (±0.23) 1.29 (±0.15) Chr.9 1.42 (±0.16) 1.19 (±0.11) 2.61 (±0.20) 1.20 (±0.18) Chr.10 1.38 (±0.08) 1.21 (±0.09) 2.59 (±0.12) 1.14 (±0.10) Figure 5. 5. Idiogram of of the the karyotype in T. in arundinaceum. T. The different The different colors colors denote denote the different the different probes or probes combined or combined probes. probes. 3. Discussion The development of the FISH method based on oligo probes provided a powerful tool for understanding the structure, organization, and evolution of plants [7]. However, karyotype analysis is still a huge challenge in non-model plants. Although there are vari-

Int. J. Mol. Sci. 2021, 22, 8539 7 of 10 3. Discussion The development of the FISH method based on oligo probes provided a powerful tool for understanding the structure, organization, and evolution of plants [7]. However, karyotype analysis is still a huge challenge in non-model plants. Although there are various available DNA probes for FISH in plants, such as repetitive sequences [19] and bacterial artificial chromosome (BAC) clones [20], etc., application in related species that diverged a few MYs ago has always shown an unsatisfactory result. For example, BAC probes are not suitable for FISH in some plant species with large complex genomes [21]. Repetitive DNA probes are the FISH probes used widely in plant genome research, however, such probes always show a varied signal among different species as a result of the instability of genome, so that they cannot be used for cytogenetics research [22]. In T. arundinaceum, Yu et al. [23] screened many repetitive sequences; unfortunately, these repeats showed a diversity site that cannot be used as a stable marker for chromosome identification. By comparison, recently, the FISH probe based on single copy sequences designed from genome sequences has provided us a more universal and stable marker for cytological study in plants [15,24]. Previous researchers have shown that oligo probes can be applied to chromosome identification in related species that diverged ~12 MYs ago, such as in Cucumis [24], or even as long ago as ~15 MYs in Solanum species [15]. In our study, MCPs were used to identify all sorghum chromosomes successfully, even though sorghum and maize diverged from the common ancestor about 11.9 MYs ago [25]. Braz et al. [26] tested the maize barcode probes in sorghum, however, it did not produce a sufficient number of signals to identify all sorghum chromosomes. In this case, it could be due to the insufficient number of oligos used. Altogether, these results suggest that chromosome painting probes should have greater potential for related species chromosome identification. T. arundinaceum is an important wild resource for sugarcane breeding. As T. arundinaceum is a ployploid plant with 2n = 60 chromosomes (basic chromosome number x = 10), the genome sequences are still unavailable, which has greatly hindered the development of cytogenetics research. Although many repetitive sequences were obtained in T. arundinaceum [27], none of them have been successfully applied to identify individual chromosomes in T. arundinaceum. Here we tested the MCPs derived from maize, suggesting an unexpected signal in T. arundinaceum. We demonstrated that the MCPs can be used for chromosome identification in T. arundinaceum, even though the divergence time between T. arundinaceum and maize is approximately 18 MYs [17]. Furthermore, 5S rdna was located on chromosome 5 of T. arundinaceum, which is quite different from sorghum, in which the locus of 5S rdna was mapped on chromosome 9 [28]. This result suggests an unidentified chromosome rearrangement between sorghum and T. arundinaceum, although the synteny of the ten chromosomes has been conserved based on the oligo-fish patterns. There are also many reports about chromosome inheritance between sugarcane and T. arundinaceum [29]. Notably, Babil et al. found that there were significant positive correlations between E. arundinaceus chromosome and agronomic characterization [4]. However, these results are just based on the counted number of T. arundinaceum chromosomes. It is necessary for us to explore the exact chromosome inheritance or significant positive correlation according to the individual chromosome identification in T. arundinaceum. In this study, we identified all T. arundinaceum chromosomes for the first time using MCPs and classified them 1 through 10 according to the sorghum genome data. These MCPs will be powerful tools for further understanding the chromosome inheritance in the hybrids between sugarcane and T. arundinaceum. In addition, individual chromosome identification will dramatically accelerate the research about the exact chromosome, which will contribute to trait improvement in sugarcane breeding. 4. Materials and Methods 4.1. Plant Material and the Preparation of Metaphase Plates T. arundinaceum (Hainan92-77, 2n = 60, x = 10) was maintained at Fujian Agriculture and Forestry University and the root tips were collected from healthy plants. The seeds

Int. J. Mol. Sci. 2021, 22, 8539 8 of 10 of Sorghum bicolor inbred line BTx623 were used to generate roots at room temperature. Then, the root tips were treated and the slides were prepared according to Braz et al. s protocol [30] with minor adjustments. Treated root tips were washed in water, then the section containing dividing cells was dissected and digested in enzyme mixture (1% pectolyase Y23, 2% pectinase, 2% RS, and 4% cellulase Onozuka R-10) for 4 h at 37 C. After digestion, the root sections were washed in water and then washed in Carnoy s fixative two times briefly. The root sections were carefully broken by using a pipette tip. The suspension cells were dropped onto glass slides and another 10 µl acetic acid were dropped onto them when the slide had almost dried. 4.2. Sequence Alignment and Analysis The sequence of maize CPs is available in the published paper [14] (https://www.pnas. org/content/suppl/2019/01/15/1813957116.dcsupplemental, accessed on 17 January 2019). The MCP sequences evenly cover the entire chromosome sequence of maize with an average oligo density of 0.25 oligo per kb. The sorghum genome was downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/genome/?term=sorghum%20bicolor, accessed on 7 April 2017). TBtools software [31] was used to sequence alignment between MCP sequences and the sorghum genome with default parameters. The chromosome location of the ten MCP sequences was drawn by RIdeogram software [32]. We discarded the aligned sequences that were smaller than 32 bp, meaning that at least a 32 bp (70% homology) match with the sorghum genome was required for sequences to be retained and counted. 4.3. Oligo-FISH and Karyotype Analysis 5S and 35S rdnas were labeled with digoxigenin-11-dutp or biotin-16-dutp (Roche Diagnostics, Mannheim, Germany) using a Nick Translation Kit (Roche Diagnostics, Mannheim, Germany). The centromere probe was prepared according to Huang et al. [33]. The So1 probe was used to localize the centromeric region, as it has the highest genome proportion and is located on all chromosomes in sugarcane. MCPs were amplified and labeled according to published protocols [30] using a T7 in vitro transcription method. The first round of FISH was performed as described by Braz et al. [30]. All biotin-labeled (~500 ng) probes were detected by anti-biotin fluorescein (Vector Laboratories, Burlingame, CA, USA) and digoxigenin-labeled probes (~400 ng) were detected by antidigoxigenin rhodamine (Roche Diagnostics, Indianapolis, IN, USA). Chromosomes were counterstained with DAPI (4, 6-diamidino-2-phenylindole). An AxioScope A1 Imager fluorescent microscope (Carl Zeiss, Gottingen, Germany) was used for capturing images. The final image contrast was processed using Adobe Photoshop 21.0.0 software. Measurement of the short arm and long arm of the individual chromosomes was conducted in the DRAWID software [34]. Arm ratio = the long arm/the short arm; 10 metaphase cells were used for measurement on each chromosome. Slides with high-quality metaphases were retained for sequential FISH. After the first round of FISH and image capture, the slides were washed three times in 4 SSC (10 min each). The slides were then washed three times in 2 SSC at room temperature (5 min each). Finally, the slides were continuously dehydrated in 70% and 100% ethanol series (room temperature, 3 min each), denatured again in 70% formamide at 70 C for 2 min, dehydrated in a second ethanol series (pre-cooled at 20 C, 5 min each) and further hybridized with different probes. 5. Conclusions In this study, the MCPs were applied in sorghum and T. arundinaceum using stable oligo-fish. Our results suggest that MCPs can be used as reliable markers for chromosome identification in T. arundinaceum. Using this system, for the first time, we were able to identify all chromosomes of T. arundinaceum though chromosomes 7 and 8 had a weak signal. The tested MCPs may be a useful FISH marker for further cytogenetics research in

Int. J. Mol. Sci. 2021, 22, 8539 9 of 10 References the hybrids between T. arundinaceum and sugarcane, since genomic DNA probes have been used to distinguish these species chromosomes separately. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/ijms22168539/s1. Author Contributions: Z.D. designed the research. F.Y., J.C., X.L., Z.Y., R.Y., X.D. and Q.W. performed the experiments. F.Y., X.Y., J.W. and Z.D. analyzed the results and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (31771863). This project was also supported by independent fund of Guangxi Key Laboratory of sugarcane biology, Scientific Research Foundation of Graduate School of Fujian Agriculture and Forestry University (324-1122yb056), Guangdong Provincial Team of Technical System Innovation for Sugarcane Sisal Hemp Industry (2019KJ104-04), grants from the State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources and the project supported by China Agriculture Research System of MOF and MARA (No. CARS-20-1-5). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Sorghum genome originated from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/genome/?term=sorghum%20bicolor, accessed on 7 April 2017) database. Acknowledgments: We thank Jiming Jiang for providing the maize oligo library. Conflicts of Interest: The authors declare no conflict of interest. 1. Roach, B.T. Nobilisation of sugarcane. Proc. Int. Soc. Sugar Cane Technol. 1972, 14, 206 216. 2. Anzoua, K.G.; Yamada, T.; Henry, R.J. Wild Crop Relatives: Genomic and Breeding Resources; Kole, C., Ed.; Springer: Berlin/Heidelberg, Germany, 2011. [CrossRef] 3. Lloyd Evans, D.; Joshi, S.V.; Wang, J. Whole chloroplast genome and gene locus phylogenies reveal the taxonomic placement and relationship of Tripidium (Panicoideae: Andropogoneae) to sugarcane. BMC Evol. Biol. 2019, 19, 33. [CrossRef] [PubMed] 4. Pachakkil, B.; Terajima, Y.; Ohmido, N.; Ebina, M.; Irei, S.; Hayashi, H.; Takagi, H. Cytogenetic and agronomic characterization of intergeneric hybrids between Saccharum spp. hybrid and Erianthus arundinaceus. Sci. Rep. 2019, 9, 1748. [CrossRef] 5. Piperidis, N.; Chen, J.W.; Deng, H.H.; Wang, L.P.; Jackson, P.; Piperidis, G. GISH characterization of Erianthus arundinaceus chromosomes in three generations of sugarcane intergeneric hybrids. Genome 2010, 53, 331 336. [CrossRef] [PubMed] 6. Yang, S.; Zeng, K.; Chen, K.; Wu, J.; Wang, Q.; Li, X.; Deng, Z.; Huang, Y.; Huang, F.; Chen, R.; et al. Chromosome transmission in BC 4 progenies of intergeneric hybrids between Saccharum spp. and Erianthus arundinaceus (Retz.) Jeswiet. Sci. Rep. 2019, 9, 2528. [CrossRef] 7. Jiang, J. Fluorescence in situ hybridization in plants: Recent developments and future applications. Chromosome Res. 2019, 27, 153 165. [CrossRef] 8. Jiang, J.; Gill, B.S. Nonisotopic in situ hybridization and plant genome mapping: The first 10 years. Genome 1994, 37, 717 725. [CrossRef] 9. Singh, R.S.; Jiang, J.; Gill, B.S. Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 2006, 49, 1057 1068. 10. Zhang, T.; Liu, G.; Zhao, H.; Braz, G.T.; Jiang, J. Chorus2: Design of genome-scale oligonucleotide-based probes for fluorescence in situ hybridization. Plant Biotechnol. J. 2021. [CrossRef] 11. Liu, X.; Sun, S.; Wu, Y.; Zhou, Y.; Gu, S.; Yu, H.; Yi, C.; Gu, M.; Jiang, J.; Liu, B.; et al. Dual-color oligo-fish can reveal chromosomal variations and evolution in Oryza species. Plant J. 2020, 101, 112 121. [CrossRef] 12. Song, X.; Song, R.; Zhou, J.; Yan, W.; Zhang, T.; Sun, H.; Xiao, J.; Wu, Y.; Xi, M.; Lou, Q.; et al. Development and application of oligonucleotide-based chromosome painting for chromosome 4D of Triticum aestivum L. Chromosome Res. 2020, 28, 171 182. [CrossRef] 13. He, L.; Zhao, H.; He, J.; Yang, Z.; Jiang, J. Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome: Pecific painting. Plant J. 2020, 103, 2225 2235. [CrossRef] 14. Albert, P.S.; Zhang, T.; Semrau, K.; Rouillard, J.M.; Kao, Y.H.; Wang, C.J.R.; Danilova, T.V.; Jiang, J.; Birchler, J.A. Wholechromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships. Proc. Natl. Acad. Sci. USA 2019, 116, 1679 1685. [CrossRef] [PubMed]

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