Dissection of rye chromosomes by the gametocidal system

Chromosome mutations occur in common wheat carrying a monosome of gametocidal (Gc) chromosomes 2C and 3C. These Gc chromosomes have been known to induce chromosomal breakage in a rye chromosome 1R added to common wheat. We attempted to introduce the two Gc chromosomes into the other six rye chromosome (2R to 7R) addition or substitution lines of common wheat to establish a set of chromosomal rearrangement-inducing lines for rye chromosomes. We obtained critical plants that had a pair of rye chromosomes and one Gc chromosome for 2R, 3R, 4R and 6R, and semi-critical plants that were monotelodisomic and monosomic for 5R. Chromosomal aberrations are expected to occur in the progeny of these plants. Besides we established self-fertile disomic 2C addition lines of common wheat that were disomic substitution for 3R, disomic addition for 6R, monotelodisomic for 5R, and monosomic for 7R. We can produce the critical plants of the respective rye chromosomes by crossing above lines to the respective wheat-rye disomic addition or substitution lines. During the cytological screening in this study, we found Gc-induced chromosomal aberrations for every rye chromosome. The stocks reported here can be used to produce dissection lines for each of the rye chromosomes in common wheat by the Gc system.


INTRODUCTION
Rye (Secale cereale L., 2n = 2x = 14, genome formula RR) is a diploid outbreeding species, belonging to the tribe Triticeae (Poaceae).Rye is an important genetic resource for the breeding of wheat because of its outstanding biotic and abiotic stress tolerance (Mohammadi et al., 2003;Bartoš et al., 2008), rye makes an important genetic resource in the breeding programs of wheat, and its genomic analysis is in progress.However, due to the huge size (1C = 7917 Mbp) (Bartoš et al., 2008) and rearranged complex structure of the rye genome containing highly repetitive sequences DNA (about 70 to 75%) (Ranjekar et al., 1974;Appels et al., 1978;Hutchinson and Lonsdale, 1982;Vershinin et al., 1995), physical maps are mandatory for the development of whole genome reference sequences of rye (Stein, 2009).
In the present study, we applied the Gc system to dissect the rye chromosomes other than 1R in common wheat.We report the breeding process of introducing two Gc chromosomes into the wheat-rye addition or substitution lines of 2R to 7R, and also on the occurrence of Gc-induced rye aberrant chromosomes found during this study.

MATERIALS AND METHODS
Plant materials and a crossing scheme We used six rye addition and substitution lines of a common wheat cultivar Chinese Spring (CS) (T.aestivum L, 2n = 6x = 42, genome formula AABBDD) that were disomic for individual chromosomes (2R to 7R) of a rye cultivar Imperial (Imp) (S. cereale L, 2n = 2x = 14, RR).We obtained all these lines from Dr. Adam J. Lukaszewski, University of California, USA.To dissect the rye chromosomes by the Gc system, we used two Gc chromosomes, 2C from Ae. cylindrica (2n = 4x = 28) and 3C SAT from Ae. triuncialis (2n = 4x = 28).There are two types of the 2C chromosome, acrocentric and telocentric ones with the same Gc effect (in this study both 2C chromosomes were used), and 3C SAT is a satellited chromosome (Endo, 1996(Endo, , 2007)).We also used the euploid Imp rye as probing DNA for fluorescence in situ hybridization (FISH) and genomic in situ Fig. 1.Breeding scheme for the genetic induction of structural changes in rye chromosomes added to common wheat.CS, R and 2C/3C SAT stand for Chinese Spring wheat, rye chromosomes, and chromosomes 2C or 3C SAT , respectively.
3) We searched for semi-critical plants only when we found no critical plants for a rye chromosome.
Chromosome constitutions are described individually.hybridization (GISH).We obtained the 2C and 3C SAT disomic addition lines of CS from National BioResource Project-Wheat (NBRP-wheat), Japan (Accession numbers LPGKU2153 and LPGKU2165, respectively), and used the euploid lines of CS and Imp that had been maintained in our laboratory.
Figure 1 shows a crossing scheme to produce common wheat plants that are disomic for the respective rye chromosomes and monosomic for the Gc chromosomes 2C or 3C SAT .Chromosome aberrations are expected to occur in the progeny of the above plants.
Cytology We made chromosome spreads by the squash method using root tips that were immersed in ice-cold distilled water for ca.20 h and fixed in 3:1 (99% ethanol: glacial acetic acid) fixative for ca.one week at room temperature, and stained in a 1% acetocarmine solution for 1-2 h.We conducted FISH and GISH as described by Endo (2011), using the probes of a rye subtelomeric pSc200 sequence (amplified by PCR) (Vershinin et al., 1995(Vershinin et al., , 1996)), a NOR sequence (Koebner, 1995) (amplified by PCR) and the total rye genomic DNA.Also, we employed the C-banding method (Gill et al., 1991) to identify the wheat chromosomes for which rye chromosome 3R substituted.

RESULTS
We crossed the five wheat-rye disomic addition lines (2n = 44) and one substitution line (3R, 2n = 42) as female to the 2C and 3C SAT addition lines, and then we backcrossed the hybrids to the respective parental rye addition or substitution lines to obtain BC 1 progeny (Fig. 1).Since no disomic 5R addition plants were available at the time of backcrossing, we used monotelodisomic (5R'/ 5RS'; 'S' stands for the short arm of the chromosome) plants for the backcrossing.We also self-pollinated some of the F 1 hybrids involving 5R and 7R.
We cytologically screened the progeny of the F 1 hybrids to obtain critical plants that were disomic for the respective rye chromosomes and monosomic for the Gc chromosome, 2C or 3C SAT .As summarized in Table 1, we obtained critical plants for 2R (2n = 45) (Fig. 2A) and 3R (2n = 43) carrying each of the Gc chromosomes, critical plants for 4R (2n = 45) carrying 3C SAT (Fig. 2D), and a critical plant for 6R (2n = 45) carrying 2C.We found semi-critical monotelodisomic 5R/5RL ('L' stands for the long arm of the chromosome) plants carrying one dose of the Gc chromosome; the semi-critical plant carrying 2C was self-sterile but the one carrying 3C SAT was selffertile.We also obtained self-fertile disomic 2C -monotelodisomic 5R/5RS plants.We found a disomic 2Cmonosomic 7R plant from which we will be able to obtain 2n = 45 plants by crossing to disomic 7R addition line and also 2n = 46 plants by self-pollination.(Fig. 2G, Table 1).
In the backcrossed and selfed progeny of the critical and semi-critical plants, we attempted to reselect critical plants and also to select double disomic addition or sub- 2) Disomic for each rye chromosome and monosomic for the Gc chromosome.
3) Chromosome constitutions are described individually.4) We did not count telocentric chromosomes, isochromosomes and Robertsonian translocations as Gc-induced aberrations.Rye chromosome dissection stitution plants for the rye and Gc chromosomes (Table 2).We reselected the 2R critical plants; those with 3C SAT were self-fertile but those with 2C were all too sterile to leave offspring.We reselected many of the 3R critical plants with 2C and besides established a disomic 3R substitution and disomic 2C addition line (2n = 44) in which 3R substituted for wheat chromosome 3D (Fig. 2, B and  C).Since the 3R critical plants with 2C were fully fertile and generated 3R aberrant chromosomes with a high frequency (Table 2), we did not take the trouble to check the progeny of the 3R critical plants with 3C SAT to avoid redundancy.We reselected the self-fertile 4R critical plants with 3C SAT from the selfed progeny.In the selfed progeny of the disomic 2C -monotelodisomic 5R/5RS addition plants, we found disomic 2C plants that were disomic for 5RS, monotelodisomic for 5R/5RS and monotelodisomic for 5R/5RL (Fig. 2E).We found neither critical nor double disomic plants in the progeny of the monotelodisomic 5R/5RL carrying 3C SAT .We obtained the 6R critical plants with 2C with high frequencies both in the backcrossed and selfed progeny and also established double disomic addition line carrying two pairs of the 2C and 6R chromosomes (Fig. 2F).We failed to obtain neither critical nor double disomic plants in the progeny of the 7R monotelodisomic plant carrying 3C SAT .

Cytological maps
are not yet available for the rye chromosomes except for chromosome 1R (Gyawali et al., 2009(Gyawali et al., , 2010)).This is simply due to the absence of dissection stocks of those rye chromosomes, i.e. common wheat stocks carrying fragments of rye chromosomes.Although Friebe et al. (2000) produced some rye deletion stocks except for 7R, their numbers were far from sufficient for chromosome mapping and genomic studies.
The rye genome, except 1R, is highly rearranged relative to that of wheat (Naranjo and Fernández-Rueda, 1991;Devos et al., 1993;Martis et al., 2012).During the development of rye specific PLUG markers, we found that the rye genome had suffered more complex rearrangements (Li et al., 2013).To reveal exact chromosomal rearrangement in the rye genome, we need to dissect the rye chromosomes individually.
In this study, we obtained self-fertile critical plants that were disomic for the rye chromosome and monosomic for the Gc chromosome for 2R, 3R, 4R and 6R, but no critical plants for 5R and 7R.For 5R we obtained self-fertile plants that were monotelodisomic for 5R/5RL and monosomic for 3C SAT ; we can use this semi-critical plants to induce aberrations in 5R, with somewhat reduced efficiency.
We established self-fertile disomic 2C addition lines of common wheat that were disomic each for 3R (2n = 44) and 6R (2n = 46) and monotelodisomic for 5R (2n = 45 + t), and found a disomic 2C and monosomic 7R plant (2n = 45).We can produce the critical plants of the respective rye chromosomes by crossing these lines to the respective wheat-rye disomic addition or substitution lines.Thus we demonstrated that at least one of the Gc chromosomes could be introduced into all rye addition and substitution lines of common wheat.
For the six rye chromosomes 2R to 7R, we found structural changes, other than telosomes, isochromosomes and Robertsonian translocations, in the course of the cytological screening in this study.We are sure that these rye chromosome aberrations were induced by the Gc chromosomes because the breakpoints were not in the centromere (see Supplementary Fig. S1).Although we did not intend to detect aberrant rye chromosomes in this study, the frequencies were comparable to those reported for chromosome 1R (Endo et al., 1994) and for barley chromosomes (Schubert et al., 1998;Shi and Endo, 1999).In the backcrossed and selfed progeny of the 2R critical plants with 2C, the 2R critical plants (four out six plants examined) and plants with 2R aberrations (15 out 30) appeared with extremely high frequencies.This fact suggested that the Gc action became too severe for the survival of the egg cells without 2C and that pollen was more tolerant of chromosomal aberrations than egg cells because aberrant 2R chromosomes in many of the selfed progeny must have transmitted from the pollen of the critical plants.Most of the deletions and translocations had single rye fragments, with two exceptions (2R-12 and 3R-4).This fact suggests that the dissection of rye chromosomes by the Gc system is suitable for the deletion mapping of the rye chromosomes.As mentioned above, we demonstrated that the Gc system using 2C and 3C SAT was effective in inducing structural changes in the rye chromosomes (2R to 7R), as well as in 1R, in the wheat background.
By PCR analysis using rye-chromosome specific markers located in the distal regions of the chromosomes, we would be able to detect rearrangements of the rye chromosomes more easily than the cytological screening.Joshi et al. (2013) conducted a PCR selection using DNA samples from leaves in the progeny of a common wheat line disomic for barley chromosome 2H and monosomic for 2C, and found six 2H aberrations among 81 plants carrying a cytologically-normal 2H chromosome in root-tip cells.This fact suggests that the Gc-induced chromosomal mutations also occur after fertilization.In this study we have shown that we can produce wheat-rye disomic addition and substitution plants carrying one dose of the Gc chromosome for all rye chromosomes, and that the Gc chromosome induces structural changes in every rye chromosome in common wheat.Now we are ready to establish dissection lines for the individual rye chromosomes by the Gc system, and these dissection lines would become valuable experimental material for the genetic and genomic research of rye.

Fig. 2 .
Fig. 2. Chromosome constitutions of the critical and semi-critical plants carrying rye and Gc chromosomes (A-G) and representative Gc-induced aberrant rye chromosomes (H).(A) A FISH/GISH image of a 45-chromosome cell carrying a pair of 2R chromosomes and one 3C SAT chromosome.Five prominent 45S rDNA FISH signals appear on two pairs of chromosomes 1B and 6B and on one 3C SAT chromosome (indicated with an arrow).(B) A C-banding image of a 44-chromosome cell carrying a pair of 3R chromosomes, substituting for a pair of wheat 3D chromosomes, and a pair of 2C chromosomes (indicated with arrows).A C-banding image of chromosome 3D is shown in the inset.(C) A FISH/GISH image of the same cell as shown in (B).Take notice that the 2C chromosomes have faint GISH signals.(D) A FISH/GISH image of a 45-chromosome cell carrying a pair of 4R chromosomes and one 3C SAT chromosome.Five prominent 45S rDNA FISH signals appear on two pairs of chromosomes 1B and 6B and on one 3C SAT chromosome (indicated with an arrow).(E) A FISH/GISH image of a 46-chromosome cell carrying one normal 5R and one telocentric 5RL chromosomes and a pair of 2C chromosomes (indicated with arrows).Take notice that the 2C chromosomes have faint GISH signals.(F) A FISH/GISH image of a 46-chromosome cell carrying a pair of 6R chromosomes and a pair of 2C chromosomes (indicated with arrows).Take notice that the 2C chromosomes had faint GISH signals.(G) A FISH/GISH image of a 45-chromosome cell carrying one 7R chromosome and a pair of 2C chromosomes (indicated with arrows).Take notice that the 2C chromosomes are acrocentric and have faint GISH signals.(H) Representative structural changes of rye chromosomes 2R to 7R.In all figures the pink GISH signals show the rye chromatin, and the green FISH signals show the pSc200 repetitive sequences and 45S rDNA.Bars = 10 μm.

Table 2 .
Cytological screening of the backcrossed and selfed progeny of the critical and semi-critical plants 1) 21" and 20" stand for the normal and nullisomic-3D chromosome complements of CS wheat, respectively.