ND-FISH-positive oligonucleotide probes for detecting specific segments of rye (Secale cereale L.)chromosomes and new tandem repeats in rye

Wei Xi, Shuyao Tang, Haimei Du, Jie Luo, Zongxiang Tang,*,Shulan Fu,*

aCollege of Agronomy, Sichuan Agricultural University,Wenjiang 611130,Sichuan,China

bInstitute of Ecological Agriculture,Sichuan Agricultural University,Wenjiang 611130,Sichuan,China

cProvincial Key Laboratory for Plant Genetics and Breeding, Wenjiang 611130,Sichuan,China

A B S T R A C T

1. Introduction

Rye(Secale cereale L.)is a source of elite genes for improvement of wheat (Triticum aestivum L.) cultivars. The effective deployment of elite rye genes in wheat breeding programs depends on the accurate identification and characterization of rye chromatin in wheat backgrounds. Many rye-specific or rye chromosome-specific markers have been developed for this purpose [1-5]. Although these markers can be used to precisely identify rye chromosomal segments that have been transferred into wheat, they cannot verify the translocation between wheat and rye chromosomes[6].Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH)technologies are often used to identify rye chromosomes in wheat backgrounds [1,4,7-13], and can compensate for the defects of DNA markers. However, the need to label probe sequences and to denature probes and chromosomes make GISH and FISH inconvenient [6,14].

Since Cuadrado et al. [15] used non-denaturing FISH (NDFISH)to investigate plant telomeres,this technology combined with oligonucleotide(oligo)probes has often been used to study the chromosomes of wheat and its relatives because of its convenience [16-23]. Oligo probes Oligo-1162, Oligo-Ku, OligopSc200, and Oligo-pSc250 can be used for ND-FISH and can replace GISH and FISH for identifying rye chromosomes in wheat backgrounds [5,6,24-26]. Although the ND-FISH-positive oligo probes Oligo-pSc119.2-1 and (AAC)6are often used to distinguish individual rye chromosomes[5,6,24-26],it is difficult to recognize broken segments of rye chromosomes using these probes because their signals are not chromosome-or chromosome segment-specific. Although 2R-specific repetitive sequences are known[27],and the ND-FISH-positive oligo probes Oligo-pTa71-2, Oligo-pTa71A-1, and Oligo-pTa71A-2 can be used to distinguish 1R from the other rye chromosomes [6,25],rye chromosome-specific probes are still limited. Probes that can identify specific segments of rye chromosomes are needed.Given the homoeologous relationship between wheat and rye chromosomes [28] and the ability of the rye-origin tandem repeat pSc119.2[29]to produce FISH signals on wheat chromosomes,repetitive DNA sequences from wheat might be used as probes for FISH analysis of rye chromosomes. Rye 4R chromosomes are worth studying because they contain elite genes that may be used in improving wheat cultivars[5,9,11,30-32].

In the present study, oligo probes derived from tandem repeats of T. aestivum cv. Chinese Spring were used to hybridize rye chromosomes by ND-FISH, and this aims to develop rye chromosome-specific probes and construct an improved FISH map of chromosome 4R.

2. Materials and methods

2.1. Plant materials

The materials used in this study and their pedigrees are listed in Table 1. MK is an octoploid triticale line that was derived from the wheat cultivar Mianyang 11 (T. aestivum L.) × rye Kustro (S. cereale L.). A wheat-rye 4RLKu(long arm of chromosome 4R of Kustro) monotelosomic addition line(MTA4RLKu) was obtained by the procedure described by Duan et al. [5]. Seeds of the progeny of MTA4RLKuwere irradiated following the same author. The irradiated MTA4RL Kuwas crossed with the wheat cultivar Chuannong 27 and the F1plants were crossed with cultivar Chuanmai 62. From this cross combination, lines 17T164-1, 17T189-20, 17T180-7, and 17T159-45 containing respectively wheat-rye 3BS.3BL-4RLKu,4BL.4BS-4RLKu, 5BS.5BL-4RLKu, and 7BS.7BL-4RLKutranslocation chromosomes, were obtained (Table 1). Chinese Spring,Mianyang 11, Kustro rye, Imperial rye, and King II rye were used in this study. Kustro, Mianyang 11, and Chinese Spring are maintained in our laboratory. Imperial and King II were supplied by J. Perry Gustafson, USDA-ARS, University of Missouri, Columbia,Missouri, USA.

Table 1-Information of materials used in this study.

2.2. Development of oligo probes

The genomic DNA sequences IWGSC WGA v0.4 and IWGSC RefSeq v1.0 of Chinese Spring were retrieved from the IWGSC(http://www.wheatgenome.org). Tandem repeat searching and the design of new oligo probes were performed following Tang et al. [33], resulting in 192 probes. The 120 oligo probes developed by Tang et al.[33]were also used.

2.3. ND-FISH

The oligo probes derived from tandem repeats of Chinese Spring were first used to hybridize root tip metaphase chromosomes of Kustro. Probes that produced signals on rye chromosomes were used to hybridize the root tip metaphase chromosomes of Imperial and King II for testing whether these oligo probes produce similar signal patterns in different rye backgrounds. Probes that generated signals on 4R chromosomes were used to hybridize root tip metaphase chromosomes of wheat-rye 4RLKutranslocation lines.Probes that produced signals on rye chromosomes are listed in Table 2. Oligo probes Oligo-pSc119.2-1, Oligo-pTa535-1[34], Oligo-pSc200, Oligo-pSc250 [6], Oligo-Ku, OligopTa71A-2 [25], and (AAC)6were used to identify individual wheat and rye chromosomes. Chromosome spreads were prepared following Han et al. [35]. Oligonucleotide sequences were 5′-end-labeled with 6-carboxyfluorescein (6-FAM), 6-carboxytetramethylrhodamine (TAMRA) or Cyanine dye 5 (Cy5). ND-FISH followed Xiao et al. [25]. Each of the seven probes Oligo-5BL.46, Oligo-5A8080, Oligo-5A8080.1, Oligo-44, Oligo-45, Oligo-1AL.73, and Oligo-0R3 was combined with (AAC)6and Oligo-pSc119.2-1 to determine their locations on rye chromosomes. Each of the five probes Oligo-5BL.46, Oligo-5A8080, Oligo-5A8080.1, Oligo-1AL.73, and Oligo-0R3 was combined with Oligo-pTa535-1 and Oligo-pSc119.2-1 to determine their locations on wheat chromosomes.The signal patterns of Oligo-44 and Oligo-45 on wheat chromosomes have been reported[33].Each of the three probes Oligo-5BL.46,Oligo-5A8080.1,and Oligo-45 was combined with Oligo-pTa71A-2 to determine their satellite location on the 1RS arm. The probe combination (AAC)6,Oligo-pSc119.2-1, Oligo-pSc200, Oligo-pSc250, and Oligo-1AL.73 was used to construct a FISH map of 4RKu. The probe combination Oligo-Ku, Oligo-pSc119.2-1, and Oligo-1AL.73 was used to determine the breakpoints of 4RLKuarms in wheat-rye 4RLKutranslocation chromosomes.

2.4. Similarity searches of the original sequences of probes

Table S1 lists the original consensus sequences used for designing the seven oligo probes. These consensus sequences were obtained following Tang et al.[33],and they were used for nucleotide BLAST searching against sequences in the nucleotide(nr/nt)database of NCBI(https://blast.ncbi.nlm.nih.gov/Blast.cgi).The rye Lo7 scaffold database in GrainGenes (https://wheat.pw.usda.gov/cgi-bin/seqserve/blast_rye.cgi)was also used for nucleotide BLAST searching.

3. Results

3.1. Oligo probes generating signals on rye chromosomes

Among the 312 (192 + 120) oligo probes derived from tandem repeats of Chinese Spring, seven probes produced signals on rye chromosomes (Table 2). Individual rye chromosomes can be distinguished according to the signal patterns of probes Oligo-pSc119.2-1 and (AAC)6(Figs. 1A, C, E, 2A, C, E, 3A, C).Probes Oligo-5BL.46, Oligo-5A8080 and Oligo-44 produced signals only in intercalary regions of 1RSKu,5RSKu, and 5RLKu,respectively (Fig. 1). Oligo-5A8080.1 produced signals in the intercalary regions of 1RSKuand 5RSKu(Fig. 2A, B), and Oligo-45 produced signals in the intercalary regions of 1RSKuand 6RSKu(Fig.2C,D).Oligo-0R3 produced signals in the telomeric and subtelomeric regions of 1RKu, 2RLKu, 3RSKu, 4RLKu, 5RLKu,6RLKu, and 7RKu(Fig. 2E, F). The signals of Oligo-1AL.73 appeared in the pericentromeric regions of 4RSKu, 4RLKu, and 7RLKuand in the intercalary region of 4RLKu(Fig.3).The signal patterns of the seven oligo probes in Imperial and King II ryes were the same as those in Kustro rye (Figs. S1-S7). Different signal strengths of Oligo-5A8080.1 on the two 5R chromosomes of Kustro,Imperial,and King II were observed(Fig. 2B,Fig. S3B, D). Different signal strengths of this probe were also observed on the two 5RKichromosomes of King II (Fig. S2D).The 1RS, 5RS, and 6RS arms could be distinguished by the combination of Oligo-5A8080.1 and Oligo-45 (Fig. S8). The signal site of Oligo-5BL.46 was the same as the one of OligopTa71A-2 (Fig. S9). The signal sites of both Oligo-5A8080.1 and Oligo-45 were located in the satellite regions of 1RS arms and differed from that of Oligo-pTa71A-2(Fig.S10).The sites of Oligo-45 also differed from those of Oligo-5A8080.1 (Fig. S8). Based on the FISH map of the 4RKuchromosome constructed by Duan et al.[5], a more denser FISH map of chromosome 4RKuwas constructed(Fig.3D),and the 4RKuchromosome was divided into ten regions by the signal sites of probes Oligo-1AL.73,Oligo-pSc119.2-1,Oligo-pSc200,Oligo-pSc250,and(AAC)6(Fig.3D).

Table 2-Oligonucleotide sequences of oligo probes producing signals on rye and wheat chromosomes.

Fig.1-ND-FISH of root tip metaphase chromosomes of Kustro rye using probes Oligo-5BL.46,Oligo-5A8080,Oligo-44,(AAC)6,and Oligo-pSc119.2-1.Individual rye chromosome can be recognized from the signal patterns of probes(AAC)6 and OligopSc119.2-1.(A)and(B)are the same cells.Oligo-5BL.46 produces signals only on the short arms of 1RKu chromosomes.(C)and(D)are the same cells.Oligo-5A8080 produces signals only on the short arms of 5RKu chromosomes.(E)and (F)are the same cells.Oligo-5BL.46 produces signals only on the long arms of 5RKu chromosomes.Chromosomes were counterstained with DAPI(blue).Scale bar,10 μm.

3.2. Breakpoints of 4RLKu arms in the four wheat-rye 4RLKu translocation chromosomes

The number of wheat chromosomes in these wheat-rye 4RLKutranslocation lines was disregarded because the breakpoints of the 4RLKuarms were the focus.The breakpoints of the 4RLKuarms in these translocation chromosomes could be determined by the signal sites of Oligo-Ku, Oligo-pSc119.2-1,and Oligo-1AL.73 (Fig. 5E). Signals of Oligo-Ku helped to distinguish 4RLKusegments from wheat, although its signals in the subtelomeric and telomeric regions were weak(Figs. 4,5). No signals of probe Oligo-1AL.73 appeared on chromosomes 3B and 5B or on chromosome arms 4BS and 7BL (Fig.5C-E, Fig. S11). In the 3BS.3BL-4RLKu, 4BL.4BS-4RLKu, 5BS.5BL-4RLKu, and 7BS.7BL-4RLKutranslocation chromosomes, the breakpoints in the wheat segments occurred on arms 3BL,4BS,5BL,and 7BL,respectively(Figs.4,5).Thus,the signals of Oligo-1AL.73 can be used to determine the breakpoints of 4RLKuarms. Both the breakpoints of 4RLKuarms in 3BS.3BL-4RLKuand 4BL.4BS-4RLKutranslocation chromosomes were located between sites L.4 and L.6(Fig.5E).The 4RLKuarm was broken at the L.6 site in the 5BS.5BL-4RLKutranslocation chromosome(Fig.5E).The breakpoint of the 4RLKuarm in the 7BS.7BL-4RLKutranslocation chromosome was located at the L.4 site(Fig.5E).

Fig.2- ND-FISH of root tip metaphase chromosomes of Kustro rye using probes Oligo-5A8080.1, Oligo-45,Oligo-0R3,(AAC)6,and Oligo-pSc119.2-1.Individual rye chromosomes can be recognized by the signal patterns of probes(AAC)6 and OligopSc119.2-1.(A)and (B)are the same cells.Oligo-5A8080.1 produces signals only on the short arms of 1RKu and 5RKu chromosomes.(C)and(D)are the same cells.Oligo-45 produces signals only on the short arms of 1RKu and 6RKu chromosomes.(E) and(F)are the same cells.Oligo-0R3 produces signals on the telomeric and subtelomeric regions of 14 chromosomes.Chromosomes were counterstained with DAPI(blue). Scale bar,10 μm.

Fig.3- ND-FISH of root tip metaphase chromosomes of rye Kustro.(A,C)Hybridization of probes Oligo-1AL.73, (AAC)6,and Oligo-pSc119.2-1 with the same cells of Kustro rye. (B)Using Oligo-1AL.73, Oligo-pSc200,and Oligo-pSc250 as probes on chromosomes of Kustro rye. (D)Cut-and-pasted 4RKu chromosomes with the signal patterns of probes Oligo-1AL.73,OligopSc200,Oligo-pSc250,(AAC)6,and Oligo-pSc119.2-1,and a schematic diagram of the improved FISH map of chromosome 4RKu.In the schematic diagram,the signal sites of these probes are numbered,and green-Oligo-pSc119.2-1,red-Oligo-pSc200,red-Oligo-pSc250, yellow-(AAC)6,and blue-Oligo-1AL.73 are indicated.Cen represents the centromere. Chromosomes were counterstained with DAPI (blue).Scale bar,10 μm.

3.3. The signal patterns of seven oligo probes on wheat chromosomes

It has been reported [33] that Oligo-44 produced signals on Chinese Spring chromosomes 3A,5A,7A,5B,and 5D and that Oligo-45 produced signals on chromosomes 3A,5A,6A,4D,5D,and 7D. The signals of Oligo-1AL.73 occurred on 11 pairs of wheat chromosomes (Table 2, Fig. 5D, Fig. S11H). Oligo-5A8080, Oligo-5A8080.1, and Oligo-0R3 also generated signals on some wheat chromosomes(Table 2,Fig.S11).No signals of Oligo-5BL.46 were observed on wheat chromosomes.

3.4. Similar sequences with the original consensus sequences of oligo probes

For convenience,the consensus sequences of Oligo-5BL.46,Oligo-44, Oligo-5A8080/5A8080.1, Oligo-45, Oligo-1AL.73, and Oligo-0R3 were named 5BL.46, 5AL.44, 5A8080, 6AS.1, 1AL.73,and 0R3,respectively(Table S1).The nucleotide BLAST search in NCBI indicated that the consensus sequences 5BL.46,6AS.1,1AL.73, and 0R3 are tandem repeats (Table S1). The sequence 5A8080 is similar with 5S rDNA.No sequence similar to 5AL.44 was found in the nucleotide (nr/nt) database in NCBI. The similarities between the original consensus sequences of the seven oligo probes and the genome sequences of S.cereale Lo7 are also described in Table S1. All the S. cereale Lo7 scaffolds:Lo7_v2_scaffold_843128 0R, Lo7_v2_scaffold_979396 0R,Lo7_v2_scaffold_373733 5R, Lo7_v2_scaffold_1227647 0R,Lo7_v2_scaffold_616093 0R, and Lo7_v2_scaffold_706276 0R,contained tandem repeats.

4. Discussion

4.1. Rye chromosomal segment-specific oligo probes

Individual rye chromosomes can be easily distinguished when they are intact.However,it is difficult to identify broken segments of rye chromosomes because of lacking rye chromosome-specific probes. For example, when chromosomes 4R,5R,and 6R are broken,their short arms are difficult to discriminate. In the present study, some new rye chromosome-specific oligo probes were developed. These oligo probes are accordingly useful for distinguishing specific chromosomal segments of rye, and when they are combined with probe Oligo-Ku, segments of rye chromosomes can also be identified in wheat backgrounds (Figs.4, 5).

4.2. Breakpoints in wheat-alien translocation chromosomes determined by FISH map

In the present study, the FISH map of 4RKuchromosome is useful for confirming the breakpoints of 4RLKuin wheat-rye 4RKutranslocation chromosomes. Based on the more denser FISH map of 4RKuchromosome,it can be clearly seen that the breakpoints of 4RLKuin the 3BS.3BL-4RLKu, 4BL.4BS-4RLKu,5BS.5BL-4RLKu, and 7BS.7BL-4RLKutranslocation

Fig.4- ND-FISH of root tip metaphase chromosomes of wheat-rye 4RLKu translocation lines using probes Oligo-Ku, Oligo-1AL.73,and Oligo-pSc119.2-1.(A,B)The same cells of the 3BS.3BL-4RLKu translocation line.(C,D)The same cells of the 4BL.4BS-4RLKu translocation line.(E,F)The same cells of the 5BS.5BL-4RLKu translocation line.Chromosomes were counterstained with DAPI(blue).Scale bar,10 μm.

Fig.5-ND-FISH of root tip metaphase chromosomes of wheat-rye 4RLKutranslocation lines and wheat cultivar Mianyang 11.(A,B) Using Oligo-Ku,Oligo-1Al.73, and Oligo-pSc119.2-1 as probes on the same cells of the 7BS.7BL-4RLKutranslocation line.(C,D)Using Oligo-1AL.73,Oligo-pTa535-1,and Oligo-pSc119.2-1 as probes on the same cells of Mianyang 11.(E)Cut-andpasted 4RKuchromosome, wheat-rye 4RLKutranslocation chromosomes and some wheat chromosomes with the signal patterns of probes Oligo-pTa535-1,Oligo-pSc119.2-1,Oligo-Ku,and Oligo-1AL.73.According to the signal sites of these probes,the breakpoints of the 4RLKuon these translocation chromosomes can be determined.Chromosomes were counterstained with DAPI(blue).Scale bar,10 μm.chromosomes are different. A line 16T75-24 that carried one 5BS.5BL-4RLKutranslocation chromosome has been described,and the breakpoint of 4RLKuwas located at site L.4[5].In fact,the 5BS.5BL-4RLKutranslocation chromosome in line 16T75-24 was the same as those in line 17T180-7.Thus,the 4RLKuarms in lines 16T75-24 and 17T180-7 have the same breakpoints,but L.4 in 16T75-24 became L.6 in 17T180-7 because of the addition of Oligo-1AL.73 signal sites. Probe Oligo-3A1 was used to confirm the physical breakpoints on the 3AL arms of the T3AL-7Js.7JsL and T3DS-3AS.3AL-7JsS translocation chromosomes[36].The breakpoints determined by Oligo-3A1 were consistent with those determined by DNA markers [36]. The results of the previous [36] and present study indicate that a fine FISH map of chromosomes can be used to confirm the breakage points of wheat-alien translocation chromosomes.

Probes Oligo-pSc119.2-1 and(AAC)6could be used not only to identify individual rye chromosome [5,6,24-26], but also to construct FISH maps of rye chromosomes [4,5,37]. The rye chromosome segment-specific oligo probes developed in this study will be useful for the improvement of FISH maps of rye chromosomes. These FISH maps will be helpful for physical location of rye chromosome-specific markers [4,5,37]. However,these FISH maps await further improvement because of the low density of FISH signal bands on rye chromosomes.More rye chromosome segment-specific FISH probes are needed, and FISH maps should be combined with DNA markers for the effective deployment of rye genes in wheat breeding programs.

4.3. Tandem repeats in rye

In rye, tandemly repeated sequence families including 480,610,630,120,pSc200(350 family),pSc250(550 family),JNK,and TaiI have been described,with monomer sizes of longer than 100 bp [27,38-41]. Microsatellites with repeat elements AAC,AAG [42] and AC, AG, AGG, CAC, ACG, and CAG [43] are also present in the rye genome.In the present study,the monomer sizes of sequences 5BL.46, 5AL.44, 6AS.1 and 1AL.73 were shorter than 100 bp.The FISH signal patterns of Oligo-5BL.46,Oligo-44, Oligo-45, Oligo-1AL.73, and Oligo-0R3 on rye chromosomes differed from those of the reported tandem repeats[27,38-41]. No sequences similar to the five tandem repeats have been reported. Although the FISH signal sites of Oligo-5BL.46 were the same as those of Oligo-pTa71A-2 and probe Oligo-pTa71A-2 was derived from the rDNA pTa71 [25], the nucleotide sequence of 5BL.46 differs from that of pTa71.Nor was a 45S rDNA sequence to 5BL.46 found in the NCBI nucleotide (nr/nt) database. Thus, 5BL.46 is not a 45S rDNA,and the five tandemly repeated sequences in this study are new tandem repeats in the rye genome. Tandemly repeated DNA sequences can be classified into three groups:microsatellites,minisatellites,and satellite DNA[44].According to the lengths of the repeated monomer [44], 5BL.46,5AL.44, 6AS.1, and 1AL.73 represent minisatellites and 0R3 represents satellite DNA.

4.4. Why the tandem repeats in wheat could produce FISH signals on rye chromosomes

Wheat and rye have a common ancestor and wheat and rye chromosomes show homoeology [28,45]. Although they evolved and differentiated, many genes and other sequences are present in both genera. Chromosomal rearrangements have also occurred in wheat and rye. For example, chromosomes 4A, 5A, and 7B of wheat have been involved in translocations and inversions [46], and chromosomal rearrangements have also occurred in both wheat and rye genomes [47]. It is thus possible that tandem repeats in wheat could produce FISH signals on rye chromosomes, and these signals might also reflect rearrangements in wheat and rye chromosomes.

Modifications of rye chromosomes, such as translocation between 4R and 7R,are known[48].The signals of Oligo-1AL.73 on chromosomes 4R and 7R also display the relationship between the two chromosomes. It is noteworthy that Oligo-5BL.46 did not produce signals on wheat chromosomes. Explanations could be the low copy number or dispersed distribution of 5BL.46 sequences in the wheat genome.

4.5.Probes Oligo-5A8080 and Oligo-5A8080.1 reflect different distributions of 5S rDNA sequences

Wheat chromosomes 1A, 1B, 1D, and 5D and rye chromosomes 1R and 5R contain 5S rDNA sequences [49,50]. In the present study, signals of Oligo-5A8080 were absent from wheat chromosomes 1A,1B and rye chromosome 1R,whereas signals of Oligo-5A8080.1 were absent only from wheat chromosome 1A. Oligo-5A8080 and Oligo-5A8080.1 produced different signal strengths on the two 5R chromosomes of Kustro, Imperial, and King II (Fig. 2B, Fig. S2D, Fig. S3B, D).These findings suggest that Oligo-5A8080 and Oligo-5A8080.1 target diverse chromosome regions, each with different copy number.Oligo probes and ND-FISH reveal the distribution and structural status of different segments of tandem repeats in different chromosome regions [25,51]. The results of the present study also reveal different distribution patterns of Oligo-5A8080 and Oligo-5A8080.1 nucleotide sequences on wheat and rye chromosomes, although they were derived from the same original sequence.

5. Conclusion

Five new tandemly repeated sequences of rye were discovered and revealed evolutionary relationships between wheat and rye. The short arms of chromosomes 1R, 5R, and 6R and the long arms of chromosomes 4R and 7R were easily discriminated using the oligo probes developed in this study when these chromosomes were broken. Oligo-1AL.73 was used to construct an improved FISH map of chromosome 4RKuand to confirm the physical breakpoints of 4RLKuin wheat-rye 4RLKutranslocation chromosomes.Oligo-0R3 can be used to identify subtelomeric regions of rye chromosomes.These oligo probes may be helpful for the use of rye chromosomes in wheat breeding programs.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.10.003.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2016YFD0102001) and the National Natural Science Foundation of China (31770373).