Male sterility of triticale lines generated through recombination of triticale and rye maintainers

The Triticum timopheevi cytoplasmic male sterility (cms) system in triticale (×Triticosecale Wittmack) suffers from a low frequency of maintainers and environmental instability of the male sterility. On the other hand, the Pampa cms system in rye (Secale cereale) exhibits strong male sterility and a low frequency of restorers. Here, we report generating hybrids between maintainers of the T. timopheevi cms system in triticale and maintainers of the rye Pampa cms system. Ten hybrids were obtained. Their hybridity was verified by PCR (polymerase chain reaction) using ISSR (inter simple sequence repeats) primers. The cms maintaining ability of F2 individuals and their progeny was tested. The F2 plants were crossed to male sterile lines of triticale carrying the T. timopheevi cytoplasm. Among 180 G1 offspring of these crosses, 71 (39.4%) were completely male sterile. Fourteen F2 individuals (7.8%), as well as their F2S1 and progeny, generated stable male sterility in G1, G1BC1 and G1BC2 generations after the crosses. Our results suggest that it is possible to produce a more stable cms system in triticale based on the T. timopheevi cytoplasm as compared to the existing one. Additional key words: cms systems; cytoplasm; Triticum timopheevi; Pampa; ISSR. * Corresponding author: rrwarzec@cyf-kr.edu.pl Received: 24-04-14. Accepted: 04-11-14 This work has one supplementary figure that does not appear in the printed article but that accompanies the paper online Abbreviations used: AFLP (amplified fragment length polymorphism); BC13, BC17 (backcross, digit indicates number of backcross); cms (cytoplasmic male sterility); DH (doubled haploids); F1 (hybrid generation); F2 (f irst segregating generation); F2S1, F2S2 (subsequent self-pollinated generations of F2 generation); G1 (generation originated from crosses of cms triticale line to F2 individuals); G1BC1, G1BC2 (subsequent generations of backcrossing cms G1 to male fertile progenies of F2 generation); ISSR (inter simple sequence repeats); PCR (polymerase chain reaction); RAPD (random amplified polymorphic DNA); S9, S13, S17, S24 (selfpollinated generation, digit indicates number of generation); SSR (single sequence repeat ). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(4): 1124-1130 http://dx.doi.org/10.5424/sjar/2014124-6144 ISSN: 1695-971X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS Male sterility of triticale 1125 of the T. timopheevi cytoplasm (Góral & Spiss, 2005; Góral et al., 2007). Because of the low frequency of maintainers, a large number of cultivars and lines must be screened and germplasm development must be carefully planned. Since wheat lines with T. timopheevi cytoplasm have a low frequency of restorer lines but triticale shows a high frequency of genotypes containing restorer genes, it has been suggested that genes restoring male fertility in triticale originate from the rye genome. This hypo thesis comes from the Curtis & Lukaszewski (1993) study, which found that genes restoring male fertility in F1 hybrids of hexaploid wheat possessing T. timo pheevi cytoplasm are located on the long arm of rye chromosomes 6R and 4R. Nevertheless, the Pampa cms system (Geiger & Schnell, 1970) can produce complete and stable male sterility in rye. It was hypothesized that non-restoring genes functioning in rye in the Pampa cms system may act in a similar way when trans mitted to triticale with the T. timopheevi cyto plasm. The ISSR-PCR markers offer several advantages over other marker types, such as high polymorphism levels, relatively low demands on the quality and quantity of DNA, and no requirements for special PCR or electrophoresis conditions. Additionally, higher temperatures, compared for example to the random amplified polymorphic DNA (RAPD) technique that utilizes lower annealing temperatures, work better to reduce nonspecific amplification. Many authors who compared RAPD and ISSR markers concluded that ISSR markers showed higher polymorphism and reproducibility (Nagaoka & Ogihara, 1997; Carvalho et al., 2005; Liu et al., 2007). As suggested by Naresh et al. (2009) sometimes SSR markers are preferred over the dominant markers like RAPD, ISSR, and amplified fragment length polymorphism (AFLP) due to its co-dominant nature. Therefore, the dominant nature of ISSR markers is definitely a disadvantage of this type of markers. But the ISSR markers were also used in the studies of Bianco et al. (2011) concerning purity of F1 popu lations generated by crossing different forms of arti choke including male sterile artichoke (MS6) and in ter specific hybrids from the Triticeae tribe (Carvalho et al., 2005). Moreover ISSR markers reveal polymor phism of non-coding regions associated with micro sa tellite sequences but located between them. It means we get at the same time information about the presence or absence of microsatellite sequences as well as polymorphism between microsatellite sequences. The objectives of this study were (i) to obtain hy brids between triticale maintainers of the T. timopheevi cms system (used as female) and rye maintainers of the Pampa cms system (used as male); (ii) to verify that the F1 generation is indeed of hybrid origin using ISSR (inter simple sequence repeats) markers; and (iii) to examine if it is possible to obtain stable non-restorer lines for the T. timopheevi cms system through crossing triticale and rye. Material and methods Six crosses were made between two hexaploid triti ca le maintainers of the T. timopheevi cms system, which were used as female (Salvo, which was in the S17 generation and 19 in the S13 generation) and three rye (Secale cereale L.) maintainers of the Pampa cms system (541-6 in S24, 585/92-6-1 in S9, and 585/92-12 in S9), which were used as male. Several fertile F1 hybrids were obtained and their hybrid nature was verified with ISSR-PCR. Leaves of four F1 hybrids and parental lines (Table 1) were used to isolate the geno mic DNA utilizing the Plant Mini AX kit (A and A Bio technology). The analyses were conducted with four ISSR primers: ISSR01 (TC)8C, ISSR03 (GGGTG)3, ISSR06 (AC)8G and ISSR07 (AC)8T. Their sequence and amplification conditions came from the publica tion by Stepansky et al. (1999). The ISSR markers that we utilized are considered to give higher poly mor phism and reproducibility since the higher temperatu res that we used, compared to the RAPD PCR method, reduce nonspecific amplification when compared for example to RAPD markers (Nagaoka & Ogihara, 1997; Carvalho et al., 2005; Liu et al., 2007). PCR was carried out using a Gene Amp 2400 thermal cycler Table 1. Number of F1 hybrids generated from crosses between male sterility maintainers of triticale and rye Triticale Rye No. of No. of No. of line line ears flowers F1 grains Salvo 15/1* 541-6 21 876 3* 585/92-6-1 65 2912 2* 585/92-1-2 21 924 0* 19* 541-6* 15 840 1* 585/92-6-1* 12 722 1* 585/92-1-2* 17 908 3* Sum 151 7182 10* * Individuals for which molecular analyses were performed 1126 T. Warzecha et al. / Span J Agric Res (2014) 12(4): 1124-1130 (Applied Biosystems). ISSR-PCR products were separated in a 1% agarose gel in TBE buffer. A DNA marker of 100 to 1000 bp and a concentration of 0.5 mg mL (Fermentas) were used to determine the length of the ISSR-PCR fragments. The gels were ima ged using ImageMaster VDS gel reader (Amersham Pharmacia Biotech). Gel analysis was performed using GelScan ver. 1.45 (Kucharczyk Electrophoretic Tech ni ques), the software enables precise determination of PCR fragments. With the application of the software it is possible to detect differences with the accuracy of ten nucleotides. F1 hybrid plants were self-pollinated to obtain the F2 generation. One hundred eighty F2 hybrid indivi duals were tested for the presence of non-restoring genes. To do this, hybrid plants were used as pollinators in crosses to male sterile triticale lines: cms Salvo 15/1 and cms 19 (in BC17 and BC13 generations, respecti vely). G1 progenies generated from these crosses were evaluated for male fertility. Subsequently, G1BC1 and G1BC2 backcross generations were obtained by using plants originating from fully male sterile progenies of the preceding generation (G1 and G1BC1) as females, and inbred progenies of F2 individuals (F2S1 and F2S2) as males. Male fertility of each generation was eva lua ted by examining seed set in heads, covered with isola tion bags prior flowering, of 10-20 plants grown at wide spacing (20 × 40 cm) at the Experimental Station in Prusy, Poland near Krakow. Plants were considered male sterile if no seeds were recovered. The total procedure of crosses is presented in Fig. 1.


Introduction
Cultivars of triticale (×Triticosecale Wittmack) are pure line cultivars, in terms of their genetic structure, obtained by the pedigree breeding method.However, there has been a growing interest in breeding hybrid triticale.Breeding of hybrid cultivars requires controlled crossing of hybrid components in the last stage of the process.In triticale, similar to other small grain cereals, it is possible to use chemical emasculation of female lines or transform them into male sterile lines.The male sterility system to be used in triticale must include fully male sterile lines, as female and pollina -tors effective in fertility restoration in F 1 hybrids.Among several alien cytoplasm sources that cause pollen sterilizing effect in triticale, only Triticum timopheevi (Zhuk.)and Aegilops sharonensis cytoplasms appear useful for male sterility systems, since they do not compromise important agronomic traits (Nalepa, 1990).The T. timopheevi cytoplasmic male sterility (cms) system in triticale is characterized by a low frequency of maintainers (Warzecha et al., 1998;Góral & Spiss, 2005;Góral et al., 2007).Additionally, the male sterility phenotype is not stable across environments (Góral et al., 2006).Most triticale lines and cultivars substantially, albeit not fully, restore fertility

Abstract
The Triticum timopheevi cytoplasmic male sterility (cms) system in triticale (×Triticosecale Wittmack) suffers from a low frequency of maintainers and environmental instability of the male sterility.On the other hand, the Pampa cms system in rye (Secale cereale) exhibits strong male sterility and a low frequency of restorers.Here, we report generating hybrids between maintainers of the T. timopheevi cms system in triticale and maintainers of the rye Pampa cms system.Ten hybrids were obtained.Their hybridity was verified by PCR (polymerase chain reaction) using ISSR (inter simple sequence repeats) primers.The cms maintaining ability of F 2 individuals and their progeny was tested.The F 2 plants were crossed to male sterile lines of triticale carrying the T. timopheevi cytoplasm.Among 180 G 1 offspring of these crosses, 71 (39.4%) were completely male sterile.Fourteen F 2 individuals (7.8%), as well as their F 2 S 1 and progeny, generated stable male sterility in G 1 , G 1 BC 1 and G 1 BC 2 generations after the crosses.Our results suggest that it is possible to produce a more stable cms system in triticale based on the T. timopheevi cytoplasm as compared to the existing one.
of the T. timopheevi cytoplasm (Góral & Spiss, 2005;Góral et al., 2007).Because of the low frequency of maintainers, a large number of cultivars and lines must be screened and germplasm development must be carefully planned.Since wheat lines with T. timopheevi cytoplasm have a low frequency of restorer lines but triticale shows a high frequency of genotypes containing restorer genes, it has been suggested that genes restoring male fertility in triticale originate from the rye genome.This hypothesis comes from the Curtis & Lukaszewski (1993) study, which found that genes restoring male fertility in F 1 hybrids of hexaploid wheat possessing T. timopheevi cytoplasm are located on the long arm of rye chromosomes 6R and 4R.Nevertheless, the Pampa cms system (Geiger & Schnell, 1970) can produce complete and stable male sterility in rye.It was hypothesized that non-restoring genes functioning in rye in the Pampa cms system may act in a similar way when trans mitted to triticale with the T. timopheevi cyto plasm.
The ISSR-PCR markers offer several advantages over other marker types, such as high polymorphism levels, relatively low demands on the quality and quantity of DNA, and no requirements for special PCR or electrophoresis conditions.Additionally, higher temperatures, compared for example to the random amplified polymorphic DNA (RAPD) technique that utilizes lower annealing temperatures, work better to reduce nonspecific amplification.Many authors who compared RAPD and ISSR markers concluded that ISSR markers showed higher polymorphism and reproducibility (Nagaoka & Ogihara, 1997;Carvalho et al., 2005;Liu et al., 2007).As suggested by Naresh et al. (2009) sometimes SSR markers are preferred over the dominant markers like RAPD, ISSR, and amplified fragment length polymorphism (AFLP) due to its co-dominant nature.Therefore, the dominant nature of ISSR markers is definitely a disadvantage of this type of markers.
But the ISSR markers were also used in the studies of Bianco et al. (2011) concerning purity of F1 populations generated by crossing different forms of artichoke including male sterile artichoke (MS6) and inter specific hybrids from the Triticeae tribe (Carvalho et al., 2005).Moreover ISSR markers reveal polymorphism of non-coding regions associated with micro satellite sequences but located between them.It means we get at the same time information about the presence or absence of microsatellite sequences as well as polymorphism between microsatellite sequences.
The objectives of this study were (i) to obtain hybrids between triticale maintainers of the T. timopheevi cms system (used as female) and rye maintainers of the Pampa cms system (used as male); (ii) to verify that the F 1 generation is indeed of hybrid origin using ISSR (inter simple sequence repeats) markers; and (iii) to examine if it is possible to obtain stable non-restorer lines for the T. timopheevi cms system through crossing triticale and rye.

Material and methods
Six crosses were made between two hexaploid tritica le maintainers of the T. timopheevi cms system, which were used as female (Salvo, which was in the S 17 generation and 19 in the S 13 generation) and three rye (Secale cereale L.) maintainers of the Pampa cms system (541-6 in S 24 , 585/92-6-1 in S 9 , and 585/92-1-2 in S 9 ), which were used as male.Several fertile F 1 hybrids were obtained and their hybrid nature was verified with ISSR-PCR.Leaves of four F 1 hybrids and parental lines (Table 1) were used to isolate the genomic DNA utilizing the Plant Mini AX kit (A and A Biotechnology).The analyses were conducted with four ISSR primers: ISSR01 (TC) 8 C, ISSR03 (GGGTG) 3 , ISSR06 (AC) 8 G and ISSR07 (AC) 8 T. Their sequence and amplification conditions came from the publication by Stepansky et al. (1999).The ISSR markers that we utilized are considered to give higher poly morphism and reproducibility since the higher temperatures that we used, compared to the RAPD PCR method, reduce nonspecific amplification when compared for example to RAPD markers (Nagaoka & Ogihara, 1997;Carvalho et al., 2005;Liu et al., 2007).PCR was carried out using a Gene Amp 2400 thermal cycler (Applied Biosystems).ISSR-PCR products were separated in a 1% agarose gel in TBE buffer.A DNA marker of 100 to 1000 bp and a concentration of 0.5 mg mL -1 (Fermentas) were used to determine the length of the ISSR-PCR fragments.The gels were imaged using ImageMaster VDS gel reader (Amersham -Pharmacia Biotech).Gel analysis was performed using GelScan ver.1.45 (Kucharczyk -Electrophoretic Techni ques), the software enables precise determination of PCR fragments.With the application of the software it is possible to detect differences with the accuracy of ten nucleotides.F 1 hybrid plants were self-pollinated to obtain the F 2 generation.One hundred eighty F 2 hybrid individuals were tested for the presence of non-restoring genes.To do this, hybrid plants were used as pollinators in crosses to male sterile triticale lines: cms Salvo 15/1 and cms 19 (in BC 17 and BC 13 generations, respectively).G 1 progenies generated from these crosses were evaluated for male fertility.Subsequently, G 1 BC 1 and G 1 BC 2 backcross generations were obtained by using plants originating from fully male sterile progenies of the preceding generation (G 1 and G 1 BC 1 ) as females, and inbred progenies of F 2 individuals (F 2 S 1 and F 2 S 2 ) as males.Male fertility of each generation was eva luated by examining seed set in heads, covered with isolation bags prior flowering, of 10-20 plants grown at wide spacing (20 × 40 cm) at the Experimental Station in Prusy, Poland near Krakow.Plants were considered male sterile if no seeds were recovered.The total procedure of crosses is presented in Fig. 1.

Results
In our study, only ten putative hybrid grains (0.14%) were obtained after pollinating over seven thousand flowers (Table 1).One hybrid was phenotypically simi lar to a male rye line and sterile, while the remaining hybrids, similar to female triticale lines, exhibited full male fertility and were used to generate the F 2 gene ration through selfing.
The hybrid nature of the F 1 plants from crosses of tri ti cale and rye was conf irmed using the ISSR01, ISSR02, ISSR06, ISSR07 primers.Our primers produced 24 to 49 amplification fragments of different lengths.Among them, several fragments were monomor phic, i.e. common to maintainers of triticale, maintainers of rye and their F 1 hybrids (Table 2).In most primer-genotype combinations, one to four fragments were specific to the rye maintainers and the F 1 hybrids.These data provide evidence for the hybrid character of the F 1 plants (Table 3, Suppl.Fig. S1 [pdf online]).These fragments were not observed in female triticale (19 and Salvo maintainers).In all F 1 hybrids, PCR fragments were detected that were absent from both parents (e.g. a 510 bp fragment generated with the ISSR01 primer in the hybrid derived from triticale and rye maintainers (19 × 585/92-1-2; Suppl.Fig. S1 [pdf online]).
In the set of hybrids, we obtained seven fragments using ISSR01 primer that were present in the male parents and the hybrids, and seven fragments that were present in the females and the hybrids.The ISSR03 primer generated the largest number of fragments, confirming the hybrid nature of the obtained hybrids.We identified 7 fragments that were present in the male parents and the hybrids, and 16 fragments that were present in the female parents and the hybrids (Table 3).This primer also proved to be the most useful for hybrid identification, since it enabled amplifying marker fragments for all analyzed genotypes.For example, three fragments (590, 420, 260 bp) produced with the ISSR01 primer were common to the rye maintainers, Figure 1.A diagram of procedures for developing G 1 and crossing with F 2 (maintainers of triticale × maintainers of rye) and inbred progenies of F 2 used as males (585/92-1-2, S 9 ) and their F 1 hybrids (19 × 585/92-1-2, S 9 ), proving the hybrid origin of the F 1 plants (Table 3, Suppl.Fig. S1 [pdf online]).Two fragments (810, 310 bp) generated by the ISSR01 primer were common to triticale maintainers used as females (line 19) and their F 1 hybrids (19 × 585/92-1-2, S 9 ), proving the hybrid origin of the F 1 (Table 3, Suppl.Fig. S1 [pdf online]).All primer-genotype combinations are presented in Table 3, which is based on Suppl.Fig. S1 [pdf online].Our research shows that in the case of the 19 S 13 × 585/92-1-2, S 9 and the 19 S 13 × 585/92-6-1, S 9 hybrids, it was possible to confirm the hybrid nature of the F 1 plants with all primers.
Male fertility of the 180 G 1 , obtained from crosses of male sterile lines of triticale to individuals of the F 2 generation, measured by seed set, ranged from 0 to 37 grains per head.Seed sets within offspring varied from 0 and 10 seeds per head.Most progenies formed 0 to 2 per head (Fig. 2).Among the 180 G 1 progenies, 71 offspring (39.4%) were completely male sterile (Table 4).In subsequent generations (G 1 BC 1 and G 1 BC 2 ), obtained by pollination of completely male sterile plants from G 1 and G 1 BC 1 offspring of F 2 S 1 and F 2 S 2 individuals (that were, in turn, generated by inbreeding of F 2 segregants), the proportion of male sterile individuals increased as a result of increasing homozygosity of males and selection of completely male sterile females.Fourteen F 2 segregants, as well  as F 2 S 1 and F 2 S 2 individuals obtained from the F 2 plants (7.8%), exhibited consistent male sterility in G 1 , G 1 BC 1, and G 1 BC 2 generations.

Discussion
At present only on a limited scale cytoplasmic male sterility is utilized to produce hybrid seeds in triticale (Nalepa, 1990;Góral, 2002;Ammar et al., 2006;Longin et al., 2012) because of limited number of restorer and maintainer lines in applied sterility sys tems based on T. timopheevi and Ae.sharonensis cytoplasms.The maintenance of male sterility in the Ae.sharonensis system is not problematic, since only 1-2% of cultivars restore male fertility in F 1 hybrids (Nalepa, 2003).Another system, cms-T.timopheevi is characterised by a low frequency of maintainers (Warzecha et al., 1998;Góral, 2002;Góral & Spiss, 2005;Góral et al., 2007).
Additionally, male sterility is not stable in different environments (Nalepa, 2003;Góral et al., 2006).Most strains and cultivars largely, albeit not fully, restore fertility of the T. timopheevi cytoplasm (Góral, 2002;Góral & Spiss, 2005;Góral et al., 2007).A frequency of over a 19% and 14% of maintainers and restorers respectively, for the cms-T.timopheevi system was detected in Mexican germ plasm of spring triticale (Ammar et al., 2006).Because of the low frequency of maintainers in Polish germ plasm, a great number of culti vars and lines must be screened, and new germplasm must be intentionally generated.New maintainers were obtained from the F 1 hybrids (non-restoring line × culti var/strain) as a result of transferring maintaining genes to lines and cultivars from existing maintainers through recombination and homozygotic process with the application of a doubled haploid (DH) line tech nique.Over 9% of the DH lines generated that way sustained male sterility, and 1.5% fully restored ferti lity (Góral et al., 2007).In this study, we present an effort to implement non-restoring genes from rye to tri ticale through recombination and exami nation of their function.
In triticale breeding, genetic variation among exis ting breeding germplasm, as well as variation intro du ced from its progenitors, i.e. wheat and rye, can be uti li zed through genomic, chromosomal or gene recom bi nation.In the present study, crosses conducted among maintainers of triticale and rye showed low efficiency.This contrasts with studies of other authors (Tarkowski  & Otl -owska, 1968; Guedes-Pinto et al., 2001; Hills et  al., 2007), who obtained higher seed set, although crossability in these studies depended on the parental genotype and environmental conditions (Guedes-Pinto et al., 2001).
In the case of wide crossing, it is crucial to confirm the hybrid nature of the obtained plants.The hybrid na ture can be verified based on morphological features revealed during the vegetative phase (Naresh et al., 2009) and after the transition to the generative phase (Ladizinsky, 2000).However, techniques allowing iden tification of hybrids in the early stages of plant development, for example in the seeds or seedlings stage, offer a huge advantage and provide the opportuni ty to eliminate very early the plants produced through self-pollination.
Molecular markers are frequently used for the identification of hybrid forms.The ISSR primers utilized in this work allowed the determination of the PCR profile specific to the parental forms and their segregation in the studied hybrids (Table 4, Suppl.Fig. S1 [pdf online]).Grądzielewska et al. (2012) identified the presence of Aegilops juvenalis genetic material in seven triticale breeding lines using the ISSR-PCR method.Using 14 ISSR primers, they generated 240 DNA fragments, of which 72 (55%) were polymorphic.In our study, 4 primers generated 141 ISSR fragments, and 62 of them (43%) were polymorphic.Grądzielewska et al. (2012) reported that a single oligonucleotide generated 3 to 9 amplification fragments unique to hybrids, and one of the parent.Thirteen primers amplified Ae. juvenalis specific fragments that were also present in the hy brids.
In addition to the fragments from parents in the genomes of hybrids, unique fragments were reported missing in any parental forms.Similar results were observed by Feldman et al. (1997Feldman et al. ( , 2012) ) in his research on hybrid wheat.He stated that in hybrids, especially those originated from wide crosses, there is a rearrangement of genomes consisting largely of deletions, methylation and gene silencing.Research by Feldman et al. (1997Feldman et al. ( , 2012) ) has shown that deletion primarily undergo non-coding DNA regions including satellite, but also to coding regions.Moreover, he observed that this phenomenon occurs particularly in the F 1 generation.A similar phenomenon was described by Han et al. (2005), who also analyzed wheat hybrids.ISSR markers utilized in this study allowed us to observe this phenomenon in triticale -rye hybrids.According to the above-cited authors, this phenomenon is associated with stabilization of hybrid genomes at the DNA level necessary for establishing such homeology, which enables the correct conjugations of chromosomes in meiosis.
The maintaining ability of F 2 recombinants and the F 2 -derived lines was tested in crosses to male sterile lines of triticale with T. timopheevi cytoplasm.Among 180 progenies of these test crosses, 71 (39.4%) were completely male sterile and 14 F 2 segregants (7.8%) as well as their descendant, consistently sustained male sterility in G 1 , G 1 BC 1 and G 1 BC 2 generations.These re sults suggest a possibility of obtaining new non-restorers lines for triticale of the T. timopheevi cms system through recombination of triticale and rye maintainers representing two distinct male sterility systems.Consequently, a more stable cms system, compared to the existing one, could be developed in triticale based on the T. timopheevi cytoplasm.The results do not prove directly that new maintainers possess non-restoring genes originating from rye genome, but they suggest such a possibility.

Table 1 .
Number of F 1 hybrids generated from crosses between male sterility maintainers of triticale and rye * Individuals for which molecular analyses were performed

Table 3 .
Fragments observed in F 1 hybrids and their parental lines (female triticale lines and male rye lines) generated with ISSR primers (length of fragments in bp)

Table 2 .
PCR fragment numbers obtained in all studied genotypes (triticale lines, rye lines, F 1 hybrids)

Table 4 .
Male sterile offspring (%) in consecutive generations of crosses of male sterile triticale lines to F 2 segregants